Patent Publication Number: US-11655447-B2

Title: Global gene regulators (GGR) as vaccine candidates against paratuberculosis

Description:
CROSS-REFERENCE TO RELATED APPLICATION(S) 
     This application is a continuation of U.S. patent application Ser. No. 16/718,892, filed Dec. 18, 2019, which is issuing as U.S. Pat. No. 10,968,425 on Apr. 6, 2021, which is a divisional of U.S. patent application Ser. No. 15/492,553, filed Apr. 20, 2017, now U.S. Pat. No. 10,544,389, issued Jan. 28, 2020, which is a continuation of U.S. patent application Ser. No. 14/193,818 filed Feb. 28, 2014, now U.S. Pat. No. 9,663,758, issued May 30, 2017, which claims priority to U.S. Patent Application 61/777,907, filed Mar. 12, 2013, each of which is incorporated by reference in its entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with government support under 14-CRHF-0-6055 awarded by the USDA/NIFA. The government has certain rights in the invention. 
    
    
     REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB 
     The content of the ASCII text file of the sequence listing named “960296_01676_ST25.txt” which is 6.05 kb in size was created on Feb. 28, 2014 and electronically submitted via EFS-Web herewith the application is incorporated herein by reference in its entirety. 
     BACKGROUND 
       Mycobacterium avium  subspecies  paratuberculosis  (a.k.a.  M. paratuberculosis ) is the etiological agent of Johne&#39;s disease, a chronic enteritis of domestic and wild animals, especially ruminants. Johne&#39;s disease (JD) has been reported on every continent (1-3), and is considered one of the greatest causes of economic hardship to the ruminant industries (4). More than two thirds of the U.S. dairy herds are infected with JD (5) and this wide distribution of the disease, reduced milk production and premature culling of infected animals together causes severe economic losses estimated to be over $200 million a year for the dairy industry (6,7). 
     Majority of the  M. paratuberculosis  infection occurs through fecal-oral route and the mycobacteria are endocytosed by enterocytes and M cells in the Peyer&#39;s patches of the ileum (8,9). After subsequent internalization by subepithelial and intraepithelial macrophages,  M. paratuberculosis  is able to survive and persist within the cells (10) using mechanisms that are not completely understood. Several studies examined gene expression patterns and host defense mechanisms of bovine macrophages from naturally infected cows (11), peripheral blood mononuclear cells (PBMC) (12) or monocytes-derived macrophages (MDMs) (13,14) following infection with  M. paratuberculosis . Alternatively, our group characterized the general and specific stress responses of  M. paratuberculosis  under various in vitro conditions as well as the transcriptomes of  M. paratuberculosis  in fecal samples from diseased cows (15). 
     Survival of  M. paratuberculosis  in environmental samples (16), macrophages (17) and animal models (18,19) is well-documented, however, the genetic basis for this survival remains unknown. Reports employing a large-scale screening of  M. paratuberculosis  mutants in relevant animal models (20,21) provided some insights into virulence of this organism with the identification of novel virulence factors associated with biofilm formation (22) and epithelial cell invasion (23). Recently, Zhu, et al. analyzed intracellular  M. paratuberculosis  gene expression patterns in bovine MDMs using SCOTS (selective capture of transcribed sequences), identifying similar patterns of responses to oxidative stress, metabolic activity, and cell survival among  M. paratuberculosis  with distinct host origins (24). The same group further analyzed the expression profiles of  M. paratuberculosis  isolated from naturally infected bovine tissues, identifying tissue-specific pathways (25). However, no comprehensive study has been conducted to clarify the relationship between  M. paratuberculosis  gene expression and specific host microenvironments following macrophage infection. 
     The current vaccine has a limited use to farmers in some regions (e.g. a few European countries) because of its inability to reduce  M. ap  shedding in feces of infected animals, the main source for spreading JD. There is only one vaccine (Mycopar®, Boehringer Ingelheim) approved for limited use in the USA. This vaccine causes significant granuloma formation at the site of inoculation 21, which persists throughout the animal&#39;s life, increasing the possibility for tissue condemnation at the slaughterhouse. 
     Despite the ability of this vaccine to induce cell mediated immunity in animals 17 , shedding of  M. ap  from vaccinated animals continues to cause a problem for transmitting the disease to naïve animals (5,22). In sheep, some animals both shed  M. ap  and died from multi-bacillary form of JD despite being vaccinated (23). In a long-term study of the effect of killed vaccine on dairy herds to reduce the transmission of the disease, no significant difference in prevalence was found between vaccinated and non-vaccinated herds (22). In another study of commercial JD vaccines, cross reactivity to bovine tuberculosis was prominent, further hampering efforts for controlling tuberculosis in farm animals (24,25). More efforts are needed to better understand the pathogenesis of JD and to plan an effective control strategy. 
     The present invention starts with a goal to gain insights into how  M. paratuberculosis  respond to the intracellular microenvironments of macrophages, the primary site for mycobacterial persistence within the host, using targeted mutagenesis and an array of transcriptome analyses. In this study, the inventors took advantage of analytical microscopy to define the phagosome environment of  M. paratuberculosis -containing macrophages in association with the expression profile of mycobacterial bacilli using DNA microarrays. The analysis suggested key changes in the metabolic pathways of  M. paratuberculosis  once the bacteria encounter active macrophages and the activation of various alternative sigma factors (Global Gene Regulators, GGRs) that could help  M. paratuberculosis  survive the hostile intracellular environment of macrophages. One such alternative GGR, sigH, has been shown to contribute to the resistance encountered during variable environmental stress conditions, such as temperature and oxidative stress in  M. tuberculosis  (26,27). However, the basis of transcriptional regulation of sigH remains elusive in  M. paratuberculosis.    
     The inventors therefore sought to define the gene regulatory network under control of GGRs, for example, sigH, sigL, sigE and ECF-1 in  M. paratuberculosis . Accordingly, they confirmed a role for these key GGRs activated inside macrophages in defending  M. paratuberculosis  against thiol-specific oxidative stress and characterized the effect of these GGRs on global transcriptome in  M. paratuberculosis . Based on the results, the inventor envisions that the GGR mutants could play an important role in designing effective vaccines against mycobacterial infections. 
     BRIEF SUMMARY OF THE INVENTION 
     In its first aspect, the present invention relates to a  Mycobacterium  mutant, comprising at least one mutation in at least one gene sequence encoding global gene regulators (GGRs) selected from the group consisting of sigH, sigL, sigE, ECF-1, and mixtures thereof, wherein the GGR gene is at least partially inactivated. 
     In some embodiments, the  Mycobacterium  is selected from the group consisting of  Mycobacterium avium  subspecies  paratuberculosis  ( M. ap ),  Mycobacterium bovis  ( M. bovis ),  Mycobacterium tuberculosis  ( M. tuberculosis ),  Mycobacterium avium  subsp.  avium  ( M. avium ), and mixtures thereof. Preferably, the  Mycobacterium  is  M. ap, M. bovis , or  M. tuberculosis.    
     In some embodiments, the GGR is  M. ap  sigH (SEQ ID NO:1) or a sequence substantially identical to SEQ ID NO: 1. In some embodiments, the GGR is  M. ap  sigL (SEQ ID NO:2) or a sequence substantially identical to SEQ ID NO:2. In some embodiments, the GGR is  M. ap  sigE (SEQ ID NO:3) or a sequence substantially identical to SEQ ID NO:3. In some embodiments, the GGR is  M. ap  ECF-1 (SEQ ID NO:4) or a sequence substantially identical to SEQ ID NO:4. In other embodiments, the GGR is  M. ap  sigB (SEQ ID NO:5) or a sequence substantially identical to SEQ ID NO:5. 
     In some embodiments, the inactivation of the GGR genes is achieved by at least partial deletion of the gene sequence encoding GGRs. Preferably, the inactivation is achieved by a complete deletion of the gene sequence encoding GGRs. 
     In some embodiments, the inactivation of the GGR genes is achieved by at least partial transposon insertion of the gene sequence encoding GGRs. Preferably, the inactivation is achieved by a complete transposon insertion of the gene sequence encoding GGRs. 
     In some embodiments, the inactivation of the GGR genes is achieved by at least partial anti-sense construct of the gene sequence encoding GGRs. Preferably, the inactivation is achieved by a complete anti-sense construct of the gene sequence encoding GGRs. 
     In its second aspect, the present invention relates to an isolated mycobacterial organism comprising a  Mycobacterium  mutant described herein. 
     In its third aspect, the present invention relates a  Mycobacterium  vaccine comprising a  Mycobacterium  mutant described herein. In some embodiments, the vaccine may also comprise the  Mycobacterium  organism described above. 
     In some embodiments, the vaccine is physically inactivated. Preferably, the vaccine is heat inactivated. 
     In some embodiments, the vaccine is chemically inactivated. Preferably, the vaccine is formaldehyde inactivated. 
     In other embodiments, the vaccine is a live attenuated vaccine. 
     In its fourth aspect, the present invention relates to a method of differentiating between subjects that have been infected with  Mycobacterium  and subjects that have not been infected with  Mycobacterium  or have been vaccinated with a  Mycobacterium  vaccine. 
     In one embodiment, the method comprises the steps of identifying a sequence that is specific to a  Mycobacterium  mutant and is not found in the wild-type  Mycobacterium  strain; and detecting the presence of the sequence in the subject. In another embodiment, the method comprises the steps of identifying a sequence that is specific to a wild-type  Mycobacterium  strain and is not found in the  Mycobacterium  mutant; and detecting the presence or the quantity of the sequence in the subject. 
     In a preferred embodiment, the  Mycobacterium  mutant is a mutant described herein. In another preferred embodiment, the detection is made by using probes or primers complementary to the  Mycobacterium  mutant in an amplification reaction. More preferably, the amplification is the loop-mediated isothermal amplification (LAMP). 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1 A . Temporal expression of  M. avium  subsp.  paratuberculosis  sigma factors within macrophages using DNA microarrays. A selected list of the  M. avium  subsp.  paratuberculosis  sigma factor genes are shown under naïve macrophages. Note that sigH and other ECF-1 (ECF-1 on the chart for clarity) were upregulated immediately after infection, followed by expression of sigE and sigB. 
         FIG.  1 B . Temporal expression of  M. avium  subsp.  paratuberculosis  sigma factors within macrophages using DNA microarrays. A selected list of the  M. avium  subsp.  paratuberculosis  sigma factor genes are shown under activated macrophages. Note that sigH and other ECF-1 (ECF-1 on the chart for clarity) were upregulated immediately after infection, followed by expression of sigE and sigB. 
         FIG.  2 A . Survival of  M. avium  subsp.  paratuberculosis  strains in bovine macrophages. Naive MDM cells were infected with ΔsigH mutant and wild-type  M. avium  subsp.  paratuberculosis  strains. Cells were lysed at 1, 4, and 8 days postinfection, and numbers of viable bacilli were determined by CFU plating. The survival levels at 4 and 8 days were relative to the viable counts of bacterial strains at day 1. Survival data represent the average of macrophage infections collected from three different donor animals with significance levels in Student&#39;s t test (*, P&lt;0.05). Error bars represent the standard deviations. 
         FIG.  2 B . Survival of  M. avium  subsp.  paratuberculosis  strains in bovine macrophages. IFN-γ-pretreated MDM cells were infected with ΔsigH mutant and wild-type  M. avium  subsp.  paratuberculosis  strains. Cells were lysed at 1, 4, and 8 days postinfection, and numbers of viable bacilli were determined by CFU plating. The survival levels at 4 and 8 days were relative to the viable counts of bacterial strains at day 1. Survival data represent the average of macrophage infections collected from three different donor animals with significance levels in Student&#39;s t test (*, P&lt;0.05). Error bars represent the standard deviations. 
         FIG.  3 A . Virulence of  M. avium  subsp.  paratuberculosis  K10 and the ΔsigH mutant in mice. Mice groups (n=15) were inoculated with ˜2×10 8  CFU/mouse of  M. avium  subsp.  paratuberculosis  wild-type strain or the ΔsigH mutant via intraperitoneal injection. Intestines were collected at 3, 6, and 12 wpi (n=5 mice/group/time point) and cultured for bacterial counts. Colony counts for each group are represented by scattered plots accompanied with a median line. Organs with significant difference in bacterial load were denoted with * for P values of &lt;0.05 or ** for P values of &lt;0.01). 
         FIG.  3 B . Virulence of  M. avium  subsp.  paratuberculosis  K10 and the ΔsigH mutant in mice. Mice groups (n=15) were inoculated with ˜2×10 8  CFU/mouse of  M. avium  subsp.  paratuberculosis  wild-type strain or the ΔsigH mutant via intraperitoneal injection. Spleens were collected at 3, 6, and 12 wpi (n=5 mice/group/time point) and cultured for bacterial counts. Colony counts for each group are represented by scattered plots accompanied with a median line. Organs with significant difference in bacterial load were denoted with * for P values of &lt;0.05 or ** for P values of &lt;0.01). 
         FIG.  3 C . Pathology of spleen collected from mice infected with  M. avium  subsp.  paratuberculosis  K10. H&amp;E-stained sections with 100× magnification (scale bar=200 μm) are shown. Inset images (1,000× magnification, scale bar=10 μm) show the  M. avium  subsp.  paratuberculosis  bacilli in purple color (arrows). WP, white pulp; RP, red pulp. 
         FIG.  3 D . Pathology of spleen collected from mice infected with  M. avium  subsp.  paratuberculosis  K10 and its isogenic ΔsigL mutant. H&amp;E-stained sections with 100× magnification (scale bar=200 μm) are shown. Inset images (1,000× magnification, scale bar=10 μm) show the  M. avium  subsp.  paratuberculosis  bacilli in purple color (arrows). WP, white pulp; RP, red pulp. 
         FIG.  4 A . Construction of  M. avium  subsp.  paratuberculosis  ΔsigL using wild-type  M. avium  subsp.  paratuberculosis  strain. A Physical map displaying the deletion of sigL (MAP4201) gene with homologous recombination via pYUB854 cosmid shuttle cloning vector, which resulted in the deletion of ˜750 bp coding region and the insertion of a ˜2 kb region encoding a hygromycin resistance cassette. 
         FIG.  4 B . Construction of  M. avium  subsp.  paratuberculosis  ΔsigL using wild-type  M. avium  subsp.  paratuberculosis  strain. The  M. avium  subsp.  paratuberculosis  ΔsigL mutant was confirmed with PCR and sequence verification using genomic DNA from the wild-type and the mutant strains. Primer pairs were designed for the sigL region, hygromycin resistance gene (hyg r ), or the recombinant region after allelic exchange. A 1.5% agarose gel showed amplicons from the sigL region only when wild-type genomic DNA was used (lane 1), whereas hyg r  was amplified only from the  M. avium  subsp.  paratuberculosis  ΔsigL mutant genomic DNA (lane 4). Lane 6 showed amplicon from the recombinant region only when  M. avium  subsp.  paratuberculosis  ΔsigL mutant genomic DNA was used. 
         FIG.  4 C . Construction of  M. avium  subsp.  paratuberculosis  ΔsigL using wild-type  M. avium  subsp.  paratuberculosis  strain. The polarity of the  M. avium  subsp.  paratuberculosis  ΔsigL knockout mutant was assessed using reverse-transcriptase PCR analysis to check for transcription of its neighboring genes. In the wild-type (left) and complemented (right) strains, positive bands show that map′, sigL and the downstream gene MAP4202 are both encoded in the genome and transcribed (amplified from cDNA), with no amplification from RNA used as a negative control. In the  M. avium  subsp.  paratuberculosis  ΔsigL mutant (middle), the sigL coding region is absent in the genome or as cDNA, but transcripts for the neighboring genes are present. 
         FIG.  5 A . Analysis of immune responses in immunized mice before challenge. Scheme illustrating the immunization study. C57BL/6 mice received a total of 2 doses, each containing ˜2×10 6  CFU  M. avium  subsp.  paratuberculosis  ΔsigL by s.c injection. Mock group received PBS buffer. Following vaccination, both groups of mice were challenged with wild-type  M. avium  subsp.  paratuberculosis  strain as described above. After 6 weeks post-immunization (6PWI; week0 in the scheme) or 6 weeks post-challenge (WPC), mice (N=4-6) from each group were sacrificed for analysis of immune response. 
         FIG.  5 B . Analysis of immune responses in immunized mice before challenge. Splenocytes (6PWI) were isolated and re-stimulated in vitro with Johnin PPD to measure IFN-γ levels from culture supernatant by ELISA after 48 h. 
         FIG.  5 C . Analysis of immune responses in immunized mice before challenge.  M. avium  subsp.  paratuberculosis  specific antibody (anti-PPDj antibodies) in the mouse sera (6PWI) was detected by ELISA (OD 450 nm) using Horseradish peroxidase conjugated rabbit anti-mouse antibody. *p&lt;0.05. 
         FIG.  6 A . Protective efficacy of immunization. At 6 weeks following vaccination, mice received a challenge dose containing ˜7×10 8  CFU wild-type  M. avium  subsp.  paratuberculosis  strain i.p (see  FIG.  5 A ). Following challenge, mice groups (N=4) were sacrificed at 6 and 12 weeks and bacterial burden was analyzed in the spleen. 
         FIG.  6 B . Protective efficacy of immunization. At 6 weeks following vaccination, mice received a challenge dose containing ˜7×10 8  CFU wild-type  M. avium  subsp.  paratuberculosis  strain i.p (see  FIG.  5 A ). Following challenge, mice groups (N=4) were sacrificed at 6 and 12 weeks and bacterial burden was analyzed in the liver. 
         FIG.  6 C . Protective efficacy of immunization. At 6 weeks following vaccination, mice received a challenge dose containing ˜7×10 8  CFU wild-type  M. avium  subsp.  paratuberculosis  strain i.p (see  FIG.  5 A ). Following challenge, mice groups (N=4) were sacrificed at 6 and 12 weeks and bacterial burden was analyzed in the intestine. Horizontal lines indicate median value. Statistical analyses were done using student&#39;s t test and Mann-Whitney test to evaluate differences in bacterial organ load among mice groups vaccinated with the PBS (mock) or  M. avium  subsp.  paratuberculosis  ΔsigL mutant. 
         FIG.  6 D . Secretion of IFN-γ production (6WPC) from the cell supernatant was measured by ELISA. The histograms show mean values with error bars representing the standard deviation. *p&lt;0.05, **p&lt;0.01. 
         FIG.  7 A . Pathological analysis of mice organs following vaccination. Photographs shows haematoxylin and eosin staining liver. 
         FIG.  7 B . Pathological analysis of mice organs following vaccination. Photographs shows haematoxylin and eosin staining liver. 
         FIG.  7 C . Pathological analysis of mice organs following vaccination. Photographs shows haematoxylin and eosin staining intestine section (100× magnification, scale bar=200 μm) from mock and  M. avium  subsp.  paratuberculosis  ΔsigL vaccinated animals following challenge with wild-type  M. avium  subsp.  paratuberculosis  strain at 6WPC and 12WPC. Ziehl-Neelsen staining of both liver and intestine displayed higher acid-fast bacilli (inset images; 1000× magnification, scale bar=10 μm) in the mock vaccinated animals compared to the ones that received  M. avium  subsp.  paratuberculosis  ΔsigL vaccination. 
         FIG.  7 D . Pathological analysis of mice organs following vaccination. Photographs shows haematoxylin and eosin staining intestine section (100× magnification, scale bar=200 μm) from mock and  M. avium  subsp.  paratuberculosis  ΔsigL vaccinated animals following challenge with wild-type  M. avium  subsp.  paratuberculosis  strain at 6WPC and 12WPC. Ziehl-Neelsen staining of both liver and intestine displayed higher acid-fast bacilli (inset images; 1000× magnification, scale bar=10 μm) in the mock vaccinated animals compared to the ones that received  M. avium  subsp.  paratuberculosis  ΔsigL vaccination. 
         FIG.  8 A . Virulence of  M. ap  mutants in BALB/c mice (n=15/group) following IP. Spleen (circle). 
         FIG.  8 B . Virulence of  M. ap  mutants in BALB/c mice (n=15/group) following IP. Liver (square) were collected at 3, 6, and 12 WPI. Colonization levels were compared to  M. ap  K10 (Continued line). * denotes significant difference between groups in Student&#39;s t-test (p&lt;0.05). 
         FIG.  9 A . Pathology of spleen collected from mice infected with wild type  M. ap.    
         FIG.  9 B . Pathology of spleen collected from mice infected with isogenic mutant ΔsigH. H&amp;E stained sections with 100× (Bar=200 μm) are shown. Inset images (1000×, Bar=10 μm). WP, white pulp; RP, red pulp. 
         FIG.  10 A . Vaccine potential of  M. ap  a factors deleted mutants. Recovery of virulent  M. ap  (cfu/g tissue) from Intestine. of mice immunized with mock (PBS), ΔsigL or ΔsigH at 6 and 12 weeks following challenge. Colony forming unit (cfu) counts are shown as a scatter plot where the bar represents the median. *p&lt;0.05. 
         FIG.  10 B . Vaccine potential of  M. ap  a factors deleted mutants. Recovery of virulent  M. ap  (cfu/g tissue) from Liver of mice immunized with mock (PBS), ΔsigL or ΔsigH at 6 and 12 weeks following challenge. Colony forming unit (cfu) counts are shown as a scatter plot where the bar represents the median. *p&lt;0.05. 
     
    
    
     DESCRIPTION OF THE INVENTION 
     The present invention provides  Mycobacterium  mutants and a vaccine based on the mutants for successfully generating immune response to the infection by Mycobacteria, and in particular for generating immune response to infection caused by  Mycobacterium avium  subspecies  paratuberculosis  ( M. ap ). The present invention is based on the identification of the gene regulatory network under control of global gene regulators (GGRs) (e.g. sigma factors, transcriptional regulators). It is found that some of key GGRs can be genetically mutated to be activated inside macrophages in defending  M. paratuberculosis  against thiol-specific oxidative stress and characterized the effect of GGRs on global transcriptome in  M. paratuberculosis . It is envisioned that a mutant with the deletion, inactivation or reduction of GGRs gene could provide strains capable of replication in hosts to generate enough protective immunity. 
     Definitions 
     A “ Mycobacterium ” as used herein, refers to micro-organisms of the genus  Mycobacterium  (sometimes abbreviated as M. herein), from the family of Mycobacteriaceae. The particularly interested mycobacteria for the purpose of the present invention include members of  Mycobacterium avium  subspecies  paratuberculosis  ( M. ap ),  Mycobacterium bovis  ( M. bovis ),  Mycobacterium avium  subsp.  avium avium ),  M. bovis  BCG (the strain most often used for vaccination purposes),  Mycobacterium tuberculosis  ( M. tuberculosis ), which includes the species  M. tuberculosis  (the major cause of human tuberculosis),  M. africanum, M. microti, M. canetti , and  M. pinnipedii.    
     A “global gene regulator, GGR” refers to any protein needed for initiation of RNA synthesis, for example, “sigma factors” and “transcriptional regulators”. They are bacterial transcription initiation factors that enable specific binding of RNA polymerase to gene promoters. RNA polymerase holoenzyme complex consists of core RNA polymerase and a GGR executes transcription of a DNA template strand. The specific GGR used to initiate transcription of a given gene will vary, depending on the gene and on the environmental signals needed to initiate transcription of that gene. 
     A “mutation” as used herein refers to a mutation in the genetic material (typically DNA), in particular a non-naturally occurring mutation, obtained via genetic engineering techniques. Mutations may include insertions, deletions, anti-sense constructs, substitutions (e.g., transitions, transversion), transpositions, inversions and combinations thereof. Mutations also include mutations upstream of the start codon, such as in the promoter region, particularly mutations within 30 nucleotides upstream of the start codon, as long as these mutations affect the levels and/or function of the gene product. Mutations may involve only a single nucleotide (e.g., a point mutation or a single nucleotide polymorphism) or multiple nucleotides. 
     In some embodiments, the mutation is a partial or complete deletion of the gene. In some embodiments, the mutation is introduced by inserting heterologous sequences into the gene of interest. In other embodiments, the mutation is introduced by replacing a portion of the wild-type gene or allele, or a majority of the wild-type gene or allele, with a heterologous sequence, or an engineered (e.g., manually altered, disrupted, or changed), non-functional, copy of the wild-type sequence. In some embodiments, the mutation is introduced by a partial or complete anti-sense construct, which refers to a DNA sequence complementary to a particular gene. When such construct is introduced in the genome, it will be transcribed in RNA and will be paired with the RNA of the gene it is complementary to. This complex of two RNA (double stranded RNA) will then be degraded so that no protein of the gene will be expressed. 
     Preferably, the mutation is done by deleting at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70% or at least 90% of the wild-type gene sequence. In other embodiments, the mutation is done by deleting the full wild-type sequence. In some other embodiments, the mutation may even include a deletion of more than 100% of the wild-type gene sequence (e.g., the full wild-type gene sequence may be deleted but also include one or more heterologous and/or non-functional nucleic acid sequences attached thereto or inserted therein). 
     Mutations could be silent, that is, no phenotypic effect of the mutation is detected. On the other hand, mutations may also cause a phenotypic change. For example, the expression level of the encoded product is altered, or the encoded product itself is altered. It is also called as the gene with the genetically engineered mutation encodes a gene product that has a reduced (knock-down) or absent (knock-out) functionality. This may be either because there is none or less of the gene product present than in the corresponding wild type strain, and/or because the gene product is not or only partially functional. 
     Thus, the mutations may influence expression levels of the gene product, stability of the gene product, encode defunct or nonsense gene products (e.g. by insertion of a stop codon), encode a different gene product, or any combination of these. For example, a mutation may result in a disrupted gene with decreased levels of expression of a gene product (e.g., protein or RNA) as compared to the wild-type strain (e.g.,  M. ap ). A mutation may also result in an expressed protein with activity that is lower as compared to the activity of the expressed protein from the wild-type strain (e.g.,  M. ap ). In some embodiments, the activity of the protein is reduced by 10%, 30%, 50%, 70%, 90% or more. 
     In specific embodiments of the present invention, mutations are conducted in at least one gene sequence encoding global gene regulators (GGRs). Such mutations result in at least partial inactivation of the function of the GGR genes. The term “function” as used herein is intended to mean the function of GGR gene sequences directly and/or indirectly attributable to the expression of the GGR genes. The “function” also refers to the function and/or activity of the proteins encoded by GGR genes as to their indirectly and/or directly attribution to infectious and/or inflammasome activation of mycobacteria. 
     The term “at least partially inactivated” as used herein, is meant to indicate a loss, interruption, or alteration of GGR genes function directly and/or indirectly attributable to the expression of GGR genes. It is also meant to indicate a loss, interruption, alteration of the infectious and/or inflammasome activation of the protein products encoded by GGR genes in mycobacteria. Preferably, “at least partially inactivated” relates to a loss of more than 10%, preferably more than 30%, more preferably more than 50%, more preferably more than 70%, most preferably more than 90% of the function directly and/or indirectly attributable to the expression of a GGR gene in mycobacteria. The “at least partially inactivation” may also include a inactivation of the full (100%) function of a GGR gene. 
     Partial and/or full inactivation of genes in mycobacteria for the purpose of the present invention can be achieved by any conventional method known in the art of gene mutation, e.g., recombinant insertion, transposon insertion, replacement, deletion, frameshift, anti-sense construct and/or homologous recombination. Preferably, the specific methods for partially and/or fully inactivating a GGR gene in mycobacteria is to delete more than 10%, preferably more than 30%, more preferably more than 50%, more preferably more than 70%, most preferably more than 90% of a GGR gene sequence. 
     The mechanism thereof is not vital to the invention, but typically may involve reduction or loss of transcription and/or translation of the affected gene, or only transcription/translation of a dysfunctional gene product. Genetically engineered mutations in a gene need not necessarily be in an exon or open reading frame, they can be in an intron or even be upstream of the start codon, as long as it affects the levels of the functional gene product. This can easily be checked, e.g. by Q-PCR for gene transcription levels. Functionality of a gene product can also be checked, as one of skill in the art would know, e.g. by providing a natural substrate to the affected enzyme. Of course, for the purpose of the present invention, the inactivation and the extent of inactivation can best be determined in direct comparison to a wild-type  Mycobacterium  or a  Mycobacterium  of the same strain under identical experimental conditions but comprising a full functional and expressed GGR gene. 
     The term “contiguous portions of a sequence” as used herein refers to a non-interrupted sequence of nucleic acids or amino acids also occurring in the same order in the sequence referred to. Particularly envisaged are contiguous portions having a length of at least 25%, 50%, 70%, 75%, 80% or 90% of the length of the reference sequence, and contiguous portions are typically at least 25 nucleic acids or at least 8 amino acids. 
     The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Nucleic acid and protein sequence identities can be evaluated by using any method known in the art. For example, the identities can be evaluated by using the Basic Local Alignment Search Tool (“BLAST”). The BLAST programs identity homologous sequences by identifying similar segments between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from protein or nuclei acid sequence database. The BLAST program can be used with the default parameters or with modified parameters provided by the user. 
     The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. 
     The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 25% sequence identity. Alternatively, percent identity can be any integer from 25% to 100%. More preferred embodiments include at least: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. 
     The term “substantial identity” of amino acid sequences for purposes of this invention normally means polypeptide sequence identity of at least 40%. Preferred percent identity of polypeptides can be any integer from 40% to 100%. More preferred embodiments include at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.7%, or 99%. 
       Mycobacterium  Mutants 
     According to a first aspect, a  Mycobacterium  mutant is provided comprising at least one genetically engineered mutation in at lease one gene sequence encoding global gene regulators (GGRs), wherein the GGR gene is at least partially inactivated. 
     In some embodiments, the  Mycobacterium  is selected from the group consisting of  Mycobacterium avium  subspecies  paratuberculosis  ( M. ap ),  Mycobacterium bovis  ( M. bovis ),  Mycobacterium tuberculosis  ( M. tuberculosis ),  Mycobacterium avium  subsp.  avium  ( M. avium ), and mixtures thereof. Preferably, the  Mycobacterium  is  M. ap.    
     In preferred embodiments, the GGR gene is selected from the group consisting of sigH, sigL, sigE, ECF-1, and mixtures thereof. 
     To inactivate a GGR gene in mycobacteria, one may partially or completely delete the sequence of the GGR gene. For example, one may insert heterologous sequences into the GGR gene. One may also replace a portion of the GGR gene or allele, or a majority of the GGR gene or allele, with a heterologous sequence, or an engineered (e.g., manually altered, disrupted, or changed), non-functional, copy of the wild-type sequence. Preferably, the mutation is done by deleting at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70% or at least 90% of the wild-type GGR gene sequence. More preferably, the mutation is done by deleting the full GGR sequence. In some other embodiments, the mutation may even include a deletion of more than 100% of the wild-type GGR gene sequence (e.g., the full wild-type gene sequence may be deleted but also include one or more heterologous and/or non-functional nucleic acid sequences attached thereto or inserted therein). 
     While these genes across different species of mycobacteria are expected, not all genes in each species have the identical sequences, locations, or functions. But based on sequence homology, one skilled person would be able to readily identify the corresponding (or equivalent) gene in other species. In a particular embodiment, the  Mycobacterium  is  M. paratuberculosis  strain which has 19 GGRs, as listed in Table 1 below. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 List of 19 sigma factors (global gene regulators, GGR) in  M. paratuberculosis  genome. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Gene 
                 Start 
                 End 
                 Start 
                 End 
               
               
                   
                 Old locus 
                 Revised 
                 length 
                 nucleotide 
                 nucleotide 
                 nucleotide 
                 nucleotide 
               
               
                 Gene 
                 tag* 
                 locus tag** 
                 (bp) 
                 number* 
                 number* 
                 number** 
                 number** 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 sigA 
                 MAP2820 
                 MAPK_0948 
                 1530 
                 3148633 
                 3150162 
                 1062326 
                 1060797 
               
               
                 sigB 
                 MAP2826 
                 MAPK_0942 
                 999 
                 3153258 
                 3154256 
                 1057701 
                 1056703 
               
               
                 sigC 
                 MAP1814 
                 MAPK_1954 
                 1380 
                 1991733 
                 1993112 
                 2219261 
                 2217882 
               
               
                 sigD 
                 MAP4275 
                 MAPK_4277 
                 597 
                 4742530 
                 4743126 
                 4745339 
                 4745935 
               
               
                 
                   sigE 
                 
                 
                   MAP2557c 
                 
                   MAPK _1211 
                 
                   756 
                 
                 
                   2874942 
                 
                 
                   2875697 
                 
                 
                   1335260 
                 
                 
                   1336015 
                 
               
               
                 sigF 
                 MAP3406c 
                 MAPK_0362 
                 789 
                 1613298 
                 1614107 
                 428572 
                 429360 
               
               
                 sigF-like 
                 MAP1474c 
                 MAPK_2294 
                 810 
                 3781604 
                 3782392 
                 2596880 
                 2597689 
               
               
                 sigG 
                 MAP3621c 
                 MAPK_0147 
                 1101 
                 4018733 
                 4019833 
                 191134 
                 192234 
               
               
                 
                   sigH 
                 
                 
                   MAP3324c 
                 
                   MAPK _0444 
                 
                   807 
                 
                 
                   3692929 
                 
                 
                   3693735 
                 
                 
                   517229 
                 
                 
                   518035 
                 
               
               
                 sigI 
                 MAP0170 
                 MAPK_3598 
                 858 
                 173736 
                 174593 
                 4037249 
                 4036392 
               
               
                 sigJ 
                 MAP3446c 
                 MAPK_0322 
                 888 
                 3825108 
                 3825995 
                 384969 
                 385856 
               
               
                 
                   sigL 
                 
                 
                   MAP4201 
                 
                   MAPK _4203 
                 
                   546 
                 
                 
                   4670740 
                 
                 
                   4671285 
                 
                 
                   4673550 
                 
                 
                   4674095 
                 
               
               
                 sigM 
                 MAP4337 
                 MAPK_4339 
                 588 
                 4818485 
                 4819072 
                 4821294 
                 4821881 
               
               
                 
                   ECF-1 
                 
                 
                   MAP0946c 
                 
                   MAPK _2822 
                 
                   924 
                 
                 
                   975766 
                 
                 
                   976689 
                 
                 
                   3234298 
                 
                 
                   3235221 
                 
               
               
                 ECF-2 
                 MAP1770c 
                 MAPK_1998 
                 936 
                 1932783 
                 1933718 
                 2277276 
                 2278211 
               
               
                 ECF-3 
                 MAP2166 
                 MAPK_1602 
                 891 
                 2397944 
                 2398834 
                 1813051 
                 1812161 
               
               
                 ECF-4 
                 MAP1757c 
                 MAPK_2011 
                 630 
                 1919501 
                 1920130 
                 2290864 
                 2291493 
               
               
                 ECF-5 
                 MAP4114c 
                 MAPK_4116 
                 1263 
                 4584803 
                 4586065 
                 4588877 
                 4587615 
               
               
                 ECF-6 
                 MAP4217 
                 MAPK_4219 
                 540 
                 4688035 
                 4688574 
                 4690845 
                 4691384 
               
               
                   
               
               
                 *Annotations according to Li, et al., 2005 (35). 
               
               
                 **Improved annotations according to the revised genome sequence of  M. paratuberculosis  (GenBank Accession No. AE016958) (38). 
               
               
                 Bolded genes were up-regulated during macrophage survival and are potential vaccine mutants. 
               
            
           
         
       
     
     In the present invention, one of the preferred GGR genes is sigH. The term “sigH” as used herein refers to a gene which encodes a RNA polymerase sigma-H factor. In some embodiments, the sigH is the sigH (SEQ ID NO:1) of  M. paratuberculosis . It has a size of 807 base pairs (bp) and is located at positions 517229 to 518035 of the  M. paratuberculosis  genomic sequence. In one specific embodiment, a  Mycobacterium  mutant comprises at least one mutation in  M. ap  sigH (SEQ ID NO: 1) or contiguous portions thereof, or sequences at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1 or the contiguous portions thereof. Preferably, the mutation in sigH is at least partial deletion of the sigH gene, as characterized as “ M. paratuberculosis  ΔsigH”. As a result of the deletion, the sigH gene is expressed at levels lower than the corresponding wild-type gene, or not expressed at all. Preferably, the expression level of sigH gene is so low as to have no effect or the expressed protein is non-functional. 
     Another preferred GGR gene is sigL (SEQ ID NO:2). The term “sigL” as used herein refers to a gene which encodes a Sigma-L-dependent transcriptional regulator. In some embodiments, the sigL is the sigL (SEQ ID NO:2) of  M. paratuberculosis . It has a size of 546 base pairs (bp) and is located at positions 4673550 to 4674095 of the  M. paratuberculosis  genomic sequence. In one specific embodiment, a  Mycobacterium  mutant comprises at least one mutation in sigL (SEQ ID NO: 2) or contiguous portions thereof, or sequences at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 2 or the contiguous portions thereof. Preferably, the mutation in the sigL gene is at least partial deletion of the sigL gene, as characterized as “ M. paratuberculosis  ΔsigL”. As a result of the deletion, the sigL gene is expressed at levels lower than the corresponding wild-type gene, or not expressed at all. Preferably, the expression level of sigL gene is so low as to have no effect or the expressed protein is non-functional. 
     Another preferred GGR gene is sigE (SEQ ID NO:3). The term “sigE” as used herein refers to a gene which encodes a RNA polymerase sigma-E factor. In some embodiments, the sigE is the sigE (SEQ ID NO:3) of  M. paratuberculosis . It has a size of 756 base pairs (bp) and is located at positions 1335260 to 1336015 of the  M. paratuberculosis  genomic sequence. In one specific embodiment, a  Mycobacterium  mutant comprises at least one mutation in sigE (SEQ ID NO: 3) or contiguous portions thereof, or sequences at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 3 or the contiguous portions thereof. Preferably, the mutation in sigE gene is at least partial deletion of the sigE gene, as characterized as “ M. paratuberculosis  ΔsigE”. As a result of the deletion, the sigE gene is expressed at levels lower than the corresponding wild-type gene, or not expressed at all. Preferably, the expression level of sigE gene is so low as to have no effect or the expressed protein is non-functional. 
     Another yet preferred GGR gene is ECF-1. The term “ECF-1” as used herein refers to a gene which encodes a RNA polymerase ECF sigma factor. In some embodiments, the ECF-1 is the ECF-1 (SEQ ID NO:4) of  M. paratuberculosis . It has a size of 924 base pairs (bp) and is located at positions 3234298 to 3235221 of the  M. paratuberculosis  genomic sequence. In one specific embodiment, a  Mycobacterium  mutant comprises at least one mutation in ECF-1 (SEQ ID NO: 4) or contiguous portions thereof, or sequences at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 4 or the contiguous portions thereof. Preferably, the mutation in ECF-1 gene is at least partial deletion of the ECF-1 gene, as characterized as “ M. paratuberculosis  Δecf-1”. As a result of the deletion, the ECF-1 gene is expressed at levels lower than the corresponding wild-type gene, or not expressed at all. Preferably, the expression level of ECF-1 gene is so low as to have no effect or the expressed protein is non-functional. 
     Another preferred GGR gene is sigB. The term “sigB” as used herein refers to a gene which encodes a RNA polymerase sigma-B factor. In some embodiments, the sigB is the sigB (SEQ ID NO:5) of  M. paratuberculosis . It has a size of 999 base pairs (bp) and is located at positions 3153258 to 3154256 of the  M. paratuberculosis  genomic sequence. In one specific embodiment, a  Mycobacterium  mutant comprises at least one mutation in sigB (SEQ ID NO: 5) or contiguous portions thereof, or sequences at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 5 or the contiguous portions thereof. Preferably, the mutation in sigE gene is at least partial deletion of the sigB gene, as characterized as “ M. paratuberculosis  ΔsigB”. As a result of the deletion, the sigB gene is expressed at levels lower than the corresponding wild-type gene, or not expressed at all. Preferably, the expression level of sigB gene is so low as to have no effect or the expressed protein is non-functional. 
     Also, in some embodiments, a  Mycobacterium  mutant comprises a combination of two or more of these genes that are mutated. For instance, both sigH and sigL genes are mutated, or sigH gene and one or two of sigL, sigE, sigB and ECF-1 genes are mutated. Additionally and/or alternatively, one or more of these genes may contain more than one mutation. 
     In some other embodiments, a  Mycobacterium  mutant may have further non-GGR mutations. One example of these further mutations includes additional mutations in other genes that function independent or dependent from GGRs in directly or indirectly causing mycobacteria infections. Other non-limiting examples of the further mutation sites, as described in the United States Patent Application Publication Number 2007/0134274, include gcpE, pstA, kdpC, papA2, impA, umaA1, fabG2_2, aceAB, mbtH2, lpqP, map0834c, cspB, lipN, map1634 MAP-1, MAP-2, MAP-3, MAP-4, MAP-5, MAP-6, MAP-7, MAP-8, MAP-9, MAP-10, MAP-11, MAP-12, MAP-13, MAP-14, MAP-15, MAP-16, MAP-17, or MAP-18 of  M. paratuberculosis , or homologs of these genomic islands, and also include, as described in the United States Patent Application Publication Number 2007/0134708, MAV-1, MAV-2, MAV-3, MAV-4, MAV-5, MAV-6, MAV-7, MAV-8, MAV-9, MAV-10, MAV-11, MAV-12, MAV-13, MAV-14, MAV-15, MAV-16, MAV-17, MAV-18, MAV-19, MAV-20, MAV-21, MAV-22, MAV-23, or MAV-24, or homologs thereof. 
     According to the second aspect of the present invention, a  Mycobacterium  mutant may be defined at the protein (amino acid) level. Thus, the present invention provides an isolated mycobacterial protein, which is encoded by the gene sequence of a  Mycobacterium  mutant as described herein. The protein expressed by the mutant may have a lower activity and/or function as compared to the activity of the expressed protein from the wild-type strain (e.g.,  M. ap ). In some embodiments, the activity and/or the function of the protein is reduced by 10%, 30%, 50%, 70%, 90% or more. 
     Vaccine 
     The third aspect of the present invention provides a vaccine based on the  Mycobacterium  mutants described herein. Specifically, in some embodiments, the  Mycobacterium  mutant comprises at least one mutation in at lease one gene sequence encoding global gene regulators (GGRs) described herein, wherein the GGR gene is at least partially inactivated. Preferably, the  Mycobacterium  is selected from the group consisting of  Mycobacterium avium  subspecies  paratuberculosis  ( M. ap ),  Mycobacterium bovis  ( M. bovis ),  Mycobacterium tuberculosis  ( M. tuberculosis ), and mixtures thereof. More preferably, the  Mycobacterium  is  M. ap.    
     The GGR gene may be selected from the group consisting of a sequence of or sequence substantially identical to sigH (SEQ ID NO:1), sigL (SEQ ID NO:2), sigE (SEQ ID NO:3), ECF-1 (SEQ ID NO:4), sigB (SEQ ID NO:5) and mixtures thereof. 
     The vaccine of the present invention may be a suspension of live attenuated, or inactivated (killed) microorganisms comprising the mutants described herein. The types microorganisms may vary, such as viruses, bacteria, or rickettsiae of the microorganisms, or of other antigens such as antigenic proteins and other substances derived from them, administered for prevention, amelioration, or treatment of  Mycobacterium  infections. 
     In some embodiments, the vaccine is a live attenuated vaccine, wherein the  Mycobacterium  mutant or the organism thereof used for the invention is alive. By “alive”, we mean the mutant or the organism thereof is capable of propagation in a subject, in particular in a mammalian subject. To be an effective live attenuated vaccine, the live mycobacteria must be attenuated to a degree not harmful to a subject in need thereof. Hence, the  Mycobacterium  mutant used for the invention is preferably non-virulent, i.e. the genes responsible for virulence have been inactivated, and does not evoke or at least evokes minor disease symptoms of a mycobacterial infection in a subject. 
     In some embodiments, the vaccine is an inactivated vaccine, wherein the  Mycobacterium  mutant or the organism thereof used for the invention is killed or inactivated. The advantage of an inactive vaccine is safety. The vaccine may be inactivated by any art-known method. For example, it may be inactivated by using chemical methods such as beta-propiolactone, thimerosal or (another mercury donating agent), formaldehyde, etc. The vaccine may be inactivated by applying physical methods such as heat, UV-light, micro-waves etc. The vaccine may also be inactivated by using biological methods such as enzyme-based methods to kill or inactivate the bacteria and any other method as is commonly applied in the art. 
     In some other embodiments, the vaccine is an acellular vaccine, i.e., a cell-free vaccine prepared from the purified mycobacterial components of cell-free microorganisms, carrying less risk than whole-cell preparations. 
     Vaccination may be accomplished by administering nucleic acid sequence of the  Mycobacterium  mutant in a form of a DNA vaccine. A “DNA vaccine” or “immunogenic” or “immunological composition” is composed of at least one vector (e.g., plasmid) which may be expressed by the cellular machinery of the subject to be vaccinated or inoculated and of a pharmaceutically acceptable carrier, vehicle, or excipient. The nucleotide sequence of this vector encodes one or more immunogens, such as proteins or glycoproteins capable of inducing, in the subject to be vaccinated or inoculated, a cellular immune response (mobilization of the T lymphocytes) and an immune response. 
     The vaccine of the present invention may be administered to a subject at risk of being infected by mycobacteria, or to a subject who has been exposed to mycobacteria, or even to a subject known to be infected with mycobacteria. The typical subject is illustratively a living organism capable of mounting an immune response to challenge from the vaccine. Non-limiting examples of a subject include a human, any lower primate, cow, camel, dog, cat, rabbit, rat, mouse, guinea pig, pig, hamster, horse, donkey, cattle, opossum, badger, goat, sheep, or other mammals or non-mammals. 
     The vaccine of the present invention is suitable for the prophylaxis and/or treatment of a disease or medical condition affected by antigen and/or immunogen expression of the  Mycobacterium , more preferably the prophylaxis and/or treatment of mycobacterial infections, preferably an infection of  M. ap, M. avium, M. tuberculosis, M. bovis , or mixtures thereof. According to most specific embodiments, the vaccine is a vaccine against Johne&#39;s disease caused by mycobacterial infection. 
     The vaccine of the present invention may be administered in any conventional dosage form in any conventional manner. In some embodiments, routes of administration include, but are not limited to, intravenously, intramuscularly, subcutaneously, intranasally, intrasynovially, by infusion, sublingually, transdermal, orally, topically, or by inhalation. The preferred modes of administration are subcutaneous, intravenous and intranasal. 
     In some embodiments, the  Mycobacterium  mutant of the vaccine may be administered alone or in combination with adjuvants that enhance stability and/or immunogenicity of the mycobacteria, facilitate administration of pharmaceutical compositions containing them, provide increased dissolution or dispersion, increase propagative activity, provide adjunct therapy, and the like, including other active ingredients. 
     In some embodiments, the pharmaceutical dosage forms of the vaccine according to the present invention may include pharmaceutically acceptable carriers and/or adjuvants known to those of ordinary skill in the art. These carriers and adjuvants include, for example, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins, buffer substances, water, salts, electrolytes, cellulose-based substances, gelatine, water, petrolatum, animal or vegetable oil, mineral or synthetic oil, saline, dextrose or other saccharide and glycol compounds such as ethylene glycol, propylene glycol or polyethylene glycol, antioxidants, lactate, etc. Preferred dosage forms include tablets, capsules, solutions, suspensions, emulsions, reconstitutable powders and transdermal patches. Methods for preparing dosage forms are well known in the art. 
     In other embodiments, the dosage regimen of the vaccine may also be determined by the skilled person using his expertise (e.g. single administration, repeated administration (twice or more at regular or irregular intervals), etc. This will typically also depend on the disease to be treated and the subject receiving the treatment. In a typical animal vaccination, a dose of 10 5 -10 10  cfu/animal is given via parental or oral routes of vaccination. In one embodiment, the dose is in the range of 10 7 -10 10  cfu/animal. In another embodiment, the dose is in the range of 10 5 -10 9  cfu/animal. 
     Differentiate Infected from Vaccinated Animals (DIVA) 
     In its fourth aspect, the present invention provides a method of in vitro differentiating between subjects that have been infected with  Mycobacterium  and subjects that have not been infected with  Mycobacterium  or have been vaccinated with a  Mycobacterium  vaccine. In one embodiment, the method comprises the steps of (a) identifying a gene sequence that is specific to a  Mycobacterium  mutant and is not found in the wild-type  Mycobacterium  strain; and (b) detecting the presence or the quantity of the sequence in the subject. In other embodiments, the method comprises the steps of (a) identifying a gene sequence that is specific to a wild-type  Mycobacterium  strain and is not found in the  Mycobacterium  mutant; and (b) detecting the presence or the quantity of the sequence in the subject. The  Mycobacterium  mutant for the embodiments is any one  Mycobacterium  mutant described herein. These sequences are detectable by use of a complimentary probe or primer. 
     The gene or the polynucleotides of the invention may contain less than an entire microbial genome and can be single- or double-stranded nucleic acids. A polynucleotide can be RNA, DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA or combinations thereof. The polynucleotides can be purified free of other components, such as proteins, lipids and other polynucleotides. For example, the polynucleotide can be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% purified. The purified polynucleotides can comprise additional heterologous nucleotides (that is, nucleotides that are not from  Mycobacterium ). The purified polynucleotides of the invention can also comprise other nucleotide sequences, such as sequences coding for linkers, primer, signal sequences, TMR stop transfer sequences, transmembrane domains, or ligands. 
     The gene or the polynucleotides of the invention can be isolated. An isolated polynucleotide is a naturally occurring polynucleotide that is not immediately contiguous with one or both of the 5′ and 3′ flanking genomic sequences with which it is naturally associated. An isolated polynucleotide can be, for example, a recombinant DNA molecule of any length, provided that the nucleic acid sequences naturally found immediately flanking the recombinant DNA molecule in a naturally-occurring genome is removed or absent. Isolated polynucleotides also include non-naturally occurring nucleic acid molecules. 
     The gene or the polynucleotides of the invention can also comprise fragments that encode immunogenic polypeptides. Polynucleotides of the invention can encode full-length polypeptides, polypeptide fragments, and variant or fusion polypeptides. Polynucleotides of the invention can comprise coding sequences for naturally occurring polypeptides or can encode altered sequences that do not occur in nature. If desired, polynucleotides can be cloned into an expression vector comprising expression control elements, including for example, origins of replication, promoters, enhancers, or other regulatory elements that drive expression of the polynucleotides of the invention in host cells. 
     The gene or the polynucleotides of the invention can be detected by, for example, a probe or primer, for example, a PCR primer, or can be the basis for designing a complimentary probe or primer, to detect the presence and/or quantity of  Mycobacterium  in a subject, such as a biological sample. Probes are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example, through hybridization. Primers are a subset of probes that can support an specific enzymatic manipulation and that can hybridize with a target nucleic acid such that the enzymatic manipulation occurs. A primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art that do not interfere with the enzymatic manipulation. “Specific” means that a gene sequence recognizes or matches another gene of the invention with greater affinity than to other non-specific molecules. Preferably, “specifically binds” or “specific to” also means a gene sequence recognizes and matches a gene sequence comprised in a wild-type  Mycobacterium  or a  Mycobacterium  mutant described herein, with greater affinity than to other non-specific molecules. More preferably, the probe or the primer is complimentary to a  Mycobacterium  mutant with at least one mutation in the gene of sigH (SEQ ID NO:1), sigL (SEQ ID NO:2), sigE (SEQ ID NO:3), ECF-1 (SEQ ID NO:4), sigB (SEQ ID NO:5) and mixtures thereof. 
     The hybridization of nucleic acids is well understood in the art. Typically a primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art. The ability of such primers to specifically hybridize to  Mycobacterium  polynucleotide sequences will enable them to be of use in detecting the presence of complementary sequences in a given subject. The primers of the invention can hybridize to complementary sequences in a subject such as a biological sample, including, without limitation, saliva, sputum, blood, plasma, serum, urine, feces, cerebrospinal fluid, amniotic fluid, wound exudate, or tissue of the subject. Polynucleotides from the sample can be, for example, subjected to gel electrophoresis or other size separation techniques or can be immobilized without size separation. 
     The probes or the primers can also be labeled for the detection. Suitable labels, and methods for labeling primers are known in the art. For example, the label includes, without limitation, radioactive labels, biotin labels, fluorescent labels, chemiluminescent labels, bioluminescent labels, metal chelator labels and enzyme labels. The polynucleotides from the sample are contacted with the probes or primers under hybridization conditions of suitable stringencies. Preferably, the primer is fluorescent labeled. Also, the detection of the presence or quality of the gene sequence of interest can be accomplished by any method known in the art. For instance, the detection can be made by a DNA amplification reaction. In some embodiments, “amplification” of DNA denotes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixtures of DNA sequences. 
     Preferably, the amplification of DNA is done by the loop-mediated isothermal amplification (LAMP). LAMP was developed by Notomi, et al. (72), incorporated by reference herein in its entirety. Similar to PCR, LAMP utilizes a polymerization-based reaction to amplify DNA from examined samples, but the enzyme for LAMP, Bst DNA polymerase large fragment, possesses a DNA strand displacement activity. This makes the DNA extension step possible without having to fully denature DNA templates. Moreover, the primers are designed in a way that a hairpin loop structure is formed in the first cycle of amplification, and the following products are further amplified in an auto-cycling manner. Therefore, in about an hour, the repeated reactions can amplify by ˜10 9  copies of DNA molecules and can be done at a constant temperature in a single heat block, instead of at various cycles of temperature in a relatively expensive thermal cycler. 
     The detection of LAMP can be made by vary technologies known in the art. Preferably, the detection of LAMP can occur without gel electrophoresis through the addition of fluorophore (73,74). This detection method could readily be used in the field to detect positive samples. As a result, a complete diagnosis can be done and visualized in about one hour (72). Quantification and multiplexing techniques have also been developed which could help determine the bacterial load of a sample (73,74). In addition, variants of Bst polymerase and primer additions have increased Bst polymerase stability, decreased amplification time, and provided the polymerase with the ability to reverse transcribe and amplify RNA targets (73,74). Specifically, the increase in stability could help transfer the diagnostic process to the field. 
     Accordingly, the detection of the presence of the gene or the specific binding between the gene in  Mycobacterium  mutant and a gene that is not a component of a subject&#39;s immune response to a particular vaccine may indicate a natural or experimental  Mycobacterium  infection. For example, the absence of such binding or presence may indicate the absence of  Mycobacterium  infection. Or, a second, separate gene, such as a mutated  Mycobacterium  gene that specific to a component of a subject&#39;s immune response to a particular  Mycobacterium  vaccine, may be used to detect a corresponding antibodies produced in response to vaccination. Thus, if an antibody specific to a gene in  Mycobacterium  vaccine is detected, then the subject has been vaccinated and/or infected. The detection of neither genes indicates no infection and no vaccination. As such, various combinations can lead to a determination of the vaccination and/or infection status of the subject. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, useful methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description and from the claims. 
     EXAMPLES 
     The following examples set forth preferred markers and methods in accordance with the invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention. 
     Example 1 
     The disclosure of Example 1 has been published in  Infection and Immnunity , June 2013, 81(6): 2242-2257, entitled “Key Role for the Alternative Sigma Factor, SigH, in the Intracellular Life of  Mycobacterium avium  subsp.  paratuberculosis  during Macrophage Stress,” which is incorporated herein by reference in its entirety. 
     Bacteria. 
       Escherichia coli  DH5α and HB101 were used as host cells for cloning purposes in all experiments presented here.  M. avium  subsp.  paratuberculosis  K10 and  Mycobacterium smegmatis  mc 2 155 strains were grown in Middlebrook 7H9 broth and on Middlebrook 7H10 plates as previously described (1). 
     Construction of the ΔsigH Mutant. 
     A specialized transduction protocol was adopted with a few modifications to delete the sigH/MAP3324c gene using the  M. avium  subsp.  paratuberculosis  strain (12). Briefly, two ˜900-bp PCR fragments flanking each end of the sigH coding region were amplified and cloned into the pYUB854 shuttle vector. The resulting pYUB854::sigH allelic-exchange substrate (AES) was then digested with Pad and ligated to the Pad-digested concatemers of a temperature-sensitive phasmid, phAE87. The ligation mixture was then packaged into phage particles with an in vitro lambda-packaging system (GIGAPackIII; Stratagene, La Jolla, Calif.). Mid-log-phase  Escherichia coli  culture was transduced with the packaged phage particles, resulting in hygromycin-resistant colonies. From the mixture of these colonies, shuttle plasmid DNA was extracted and then electroporated into  M. smegmatis  competent cells. Lysate of plaques formed at 30° C. from the transformants was collected, propagated, and titrated in  M. smegmatis  to produce a high-titer recombinant phage stock. A mid-log-phase culture of  M. avium  subsp.  paratuberculosis  was transduced with the phage stock at nonpermissive temperature (37° C.) with a multiplicity of infection of 10. Individual hygromycin-resistant colonies were picked and grown in broth medium following gDNA isolation. The genotype of sigH-deletion mutants was confirmed with PCR and sequence analysis as outlined before (12). 
     Stress Treatments of  M. avium  Subsp.  paratuberculosis.    
     Wild-type and the ΔsigH mutant of  M. avium  subsp.  paratuberculosis  were grown to late-log phase (optical density at 600 nm [OD 600 ]=1.0), and 200 ul was spread on 7H10 agar plates (Difco, Sparks, Md.) supplemented with 0.5% glycerol, 2 ug/ml mycobactin J, and 10% ADC (2% glucose, 5% bovine serum albumin [BSA] fraction V, and 0.85% NaCl). For disc diffusion assay (DDA), 20 ul of diamide solution (0.5 M, 1 M, or 1.5 M) and H 2 O 2  solution (50 mM, 100 mM, or 0.5 M) was impregnated onto each 6-mm disc (Whatman, Piscataway, N.J.), and discs were placed on each of the spread plates. As a positive control, ethambutol discs (5 ug/disc, Sensi-Disc; BD Diagnostics) were used. Plates were incubated at 37° C. until a thick confluent lawn developed. The sustained effect of stressors (diamide and heat shock) on the viability of the wild type and ΔsigH mutant was monitored by determining their CFU counts. Aliquots of  M. avium  subsp.  paratuberculosis  cultures 1, 3, and 7 days following continuous exposure to 10 mM diamide or a 45° C. water bath were serially diluted and plated. In another experiment,  M. avium  subsp.  paratuberculosis  cultures from midlog phase were exposed to 10 mM diamide for 3 h. The cultures were centrifuged (3,000×g, 4° C., 10 min), and pellets were immediately stored at −80° C. until RNA extraction. 
     Mouse Infections. 
     For the animal infections with  M. avium  subsp.  paratuberculosis  strains, female BALB/c mice (Harlan Laboratories, Indianapolis, Ind.) were purchased at 4 weeks of age and housed in a pathogen free environment according to the protocol approved by the Institutional Animal Care and Use Committee, University of Wisconsin-Madison. Two groups of mice (n=15 per group) were challenged intraperitoneally with the wild-type and ΔsigH mutant strains of  M. avium  subsp.  paratuberculosis . Actual infection inoculum sizes (˜2×10 8  CFU per mouse) of these two strains were similar, as determined by plate count on the day of infection. Mouse groups (n=5) were sacrificed at 3, 6, and 12 weeks postinfection (wpi), and samples from livers, intestines, and spleens were collected for bacterial CFU enumeration and histopathological examinations as described before (17). Portions of livers, spleens, and intestines were fixed in 10% neutral buffered formalin before being sectioned and stained with hematoxylin and eosin (H&amp;E) and Ziehl-Neelsen stain. Student&#39;s t test and Mann-Whitney test were used to statistically evaluate differences in CFU counts among mouse groups infected with the wildtype and ΔsigH mutant strains of  M. avium  subsp.  paratuberculosis.    
     Bovine Blood Monocyte Isolation and Infection. 
     Blood was collected from a Johne&#39;s disease-free herd that we maintained at the University of Wisconsin-Madison. Three cows (36-month-old Holstein, designated animals 5695, 5970, and 6117) were bled by jugular venipuncture using blood collection bags (TERUFLEX, Somerset, N.J.) containing citrate phosphate dextrose adenine as an anticoagulant. Blood was transferred to 50-ml polypropylene tubes and centrifuged at 1,400×g for 20 min at 25° C. Buffy coat containing white blood cells was isolated and mixed with phosphate-buffered saline (PBS) (Ca 2+ - and Mg 2+ -free) to a final volume of 30 ml. The cell suspension was layered onto 58% isotonic PERCOLL (medium for density gradient centrifugation) (Sigma) at a 1:1 ratio and centrifuged at 2,000×g for 30 min at 25° C. Peripheral blood mononuclear cells (PBMC) were collected from the PERCOLL-PBS interface and washed three times with PBS to remove residual PERCOLL. To isolate bovine monocyte-derived macrophages (MDMs), PBMC were resuspended in RPMI 1640 (Sigma-Aldrich, St. Louis, Mo.) with 20% autologous serum and transferred to TEFLON nonstick coating jars followed by incubation for 4 days at 37° C. and 5% CO2. MDM cells were harvested, washed, and seeded with 2×10 6  cells/well in 24-well plates with 5% autologous serum. Immediately before MDM cell infection,  M. avium  subsp.  paratuberculosis  cultures grown to mid-log phase (OD 600  of 0.4 to 0.6) were pelleted and resuspended in an appropriate volume of cell culture medium to achieve a 50:1 multiplicity of infection (MOI). The cells were incubated at 37° C. with 5% CO2 for 3 h, and, subsequently, the monolayers were washed two times with warm PBS to remove extracellular bacteria, and RPMI 1640 medium containing 5% autologous serum was added. The plates were incubated at 37° C. for up to 8 days, and the culture medium was replaced with fresh medium at 4 days after infection. In another set of experiments, MDM cells were pretreated overnight (18 h) with 0.01 ug/ml recombinant bovine IFN-γ (Kingfisher Biotech, St. Paul, Minn.) before infection with  M. avium  subsp.  paratuberculosis  strains. This concentration of IFN-γ was adequate to activate bovine monocytes (23). Bovine MDM cells were lysed at 1, 4, and 8 days postinfection for CFU plating with serial dilutions. Student&#39;s t test was used for statistical analysis, where P values of 0.05 were considered to be significant to evaluate differences in CFU counts. 
     J744A.1 Cell Culture Infection. 
     The mouse macrophage cells (J774A.1) were maintained in RPMI 1640 (Sigma-Aldrich, St. Louis, Mo.) supplemented with 5 to 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich) in 75-cm 2  filter-cap tissue culture flasks (Techno Plastic Products, Trasadingen, Switzerland) in a water jacketed incubator (Thermo Scientific, Waltham, Mass.) at 37° C. with 5% CO 2 . When confluent, cells were detached with a cell scraper and resuspended, and 10% of the cell suspension was replenished with fresh culture medium every 3 to 4 days. 
     Macrophages were seeded at 1.5×10 7  cells per 15-mm cell culture dish (Techno Plastic Products) in 15 ml of culture medium as described above, 30 to 36 h prior to infection, and were incubated at 37° C. with 5% CO 2 . At least 5 dishes were seeded for each time point. For IFN-γ activation experiments, old culture medium was discarded and 15 ml of fresh medium with 100 U/ml recombinant murine IFN-γ (Pepro Tech, Rocky Hill, N.J.) was added to each dish, 16 to 20 h prior to infection. An approximate 109 CFU bacterial suspension from mid-log phase was mixed with 12 ml of cell culture medium (RPMI 1640-10% FBS, mycobactin J-free) and added to each decanted dish (MOI=50). The cells were incubated at 37° C. with 5% CO2 for 2 or 24 h before intracellular bacteria isolation. For the 24-h-time-point experiments, extracellular bacteria were washed away at 2 h postinfection with 15 ml of warm PBS at least five times or until no visible bacterial particles were observed under an inverted microscope at ×400 magnification. The washed cells were replenished with 15 ml of fresh cell culture medium and incubated until 24 h postinfection. Each condition was replicated at least twice until the quality of extracted RNA passed the criteria described below. 
     Immunofluorescent Staining for LAMP-1 Expression. 
     Culture cells grown on a circular coverslip were fixed in 2.5% paraformaldehyde for 10 min and permeated with cold methanol-acetone (1:1) at −20° C. for 5 min. A few drops of TB AuramineM (BD Diagnostics, Franklin Lakes, N.J.) were added and incubated at room temperature for 10 min to stain mycobacteria. The coverslip was washed with 95% EtOH three times and rinsed with PBS containing 0.2% saponin and 2% goat serum. Rat monoclonal antibody 1D4B against mouse LAMP-1 purchased from the Developmental Studies Hybridoma Bank at the University of Iowa was diluted to 20 ug/ml in PBS-saponin-goat serum and incubated with the fixed cells at room temperature for 1 h. The cells were washed with PBS-saponin-goat serum three times, each for 10 min. Goat antibody conjugated with ALEXA FLUOR 633 fluorophore dye against rat IgG (Invitrogen, Carlsbad, Calif.) was diluted to 10 ug/ml in PBS-saponin-goat serum and incubated with the cells for 1 h in the dark at room temperature. The cells were then washed in the same way as described in the last step. Finally, the coverslip was mounted on a microscope slide in Vectashield mounting medium (Vector Laboratories, Burlingame, Calif.) and observed with a Nikon Cl confocal microscope system. 
     Phagosome pH Measurements. 
     Phagosome pH measurement was slightly modified from previous studies (26, 27) based on ratiometric measurements. J774A.1 cells were seeded at 2×10 5  cells per well on a 24-well cell culture plate (Techno Plastic Products) in 0.5 ml of culture medium with or without 100 U/ml murine IFN-γ. A poly-L-lysine (Electron Microscopy Sciences, Hatfield, Pa.)-coated 12-mm circular coverslip was placed in each well before seeding. After overnight incubation, culture medium was replaced with 0.3 ml of prewarmed fresh medium with 5 uM LYSOSENSOR radiometric probe Yellow/Blue DND-160 (Invitrogen), and the cells were incubated for 5 min at 37° C. To generate an in situ pH gradient standard curve, each coverslip was then incubated with morpholineethanesulfonic acid (IVIES) buffer (25 mM MES, 5 mM NaCl, 115 mM KCl, and 1.2 mM MgSO4) of known pH (from 3.5 to 7.0 at a 0.5 interval), in the presence of 10 uM nigericin and 10 uM monensin, for 2 min. The coverslip was immediately mounted on a glass slide and observed under an OLYMPUS BX51 microscope with a reflected fluorescence system. Sixteen-bit grayscale images of two separate channels (excitation of 365/10 nm, emission of 460/50 nm, dichroic of 400 nm; excitation of 365/10 nm, emission of 540/20 nm, dichroic of 400 nm; Chroma, Bellows Falls, Vt.) from each field were taken. 
     The processing time from sample mounting to image acquisition was controlled so it took no longer than 10 min for each coverslip. Image processing was done with ImageJ 1.44 k (28). For each pH standard, at least 20 individual regions of interest (ROIs) were randomly chosen, and mean intensities of each ROI from both channels were recorded. Ratios of intensities of green (540 nm) to blue (460 nm) from the same pH standard were then averaged, excluding values of = or &gt;2 standard deviations (SD) from the mean. A standard curve of ratios was plotted against pH by applying a Boltzmann equation, y=A2+(A1−A2)/{1+exp [(x−x0)/dx]}, where A1 and A2 represent the limits of the fluorescent ratio at infinitely low and high pHs, respectively, x0 is the pH midpoint at (A1+A2)/2, x is the observed pH, and dx is the slope of the curve. When needed, cells were infected with  M. avium  subsp.  paratuberculosis  as described above, except the bacteria were prestained with 5 uM VYBRANT dye DiD cell-labeling solutions (Invitrogen) for 10 min (29). Intracellular bacteria could be observed with a third filter set (excitation of 535/50 nm, emission of 675/20 nm, dichroic of 565 nm). ROIs were chosen where the bacteria were colocalized with LYSOSENSOR radiometric probe-stained phagosomes. The average 540/460 ratio of ROIs was plugged into the equation to calculate phagosome pH. 
     Intracellular Bacterial Isolation and RNA Extraction. 
     Intracellular bacteria were isolated by a protocol described before (30) with modifications. At 2 or 24 h post infection, infected cells were washed with 15 ml ice-cold PBS at least five times or until no visible bacterial particles were observed under an inverted microscope. The washed cells on each dish were then lysed with 10 ml cell lysis buffer (4 M guanidine thiocyanate, 0.5% sodium N-lauryl sarcosine, 25 mM sodium citrate, 0.5% TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), and 0.1 M β-mercaptoethanol) and collected with a rubber cell scraper. To reduce viscosity and help dissolve cell debris, cell lysates from all dishes were pooled and passed through a 23-gauge needle five times. The lysate was then split into four 14-ml polypropylene centrifuge tubes (Falcon 352059; BD Biosciences, San Jose, Calif.) and centrifuged at 3,200×g and 4° C. for 25 min. Each pellet was washed in 1 ml of TRIZOL regent (monophasic solution of phenol and guanidine isothiocyanate) (Invitrogen) twice and subjected to RNA extraction. Total RNA was extracted by a protocol described before (12, 31). Briefly, bacterial pellets were resuspended in 2 ml of TRIZOL reagent (monophasic solution of phenol and guanidine isothiocyanate) and split into two 2-ml screw-cap tubes, each with 3.0 g of 0.1-mm zirconia/silica beads (BioSpect Products, Bartlesville, Okla.) and disrupted in a Mini-BeadBeater-8 (BioSpect Products) at top speed for three pulses of 60 s with 30-s intervals on ice. Following a 5-min incubation at room temperature, the supernatant was transferred to RNase-free tubes and centrifuged at 12,000×g for 15 min. RNA was then isolated according to the manufacturer&#39;s instruction. To remove genomic DNA contamination, RNA samples were treated with 10 U of Turbo DNase (Ambion, Austin, Tex.) at 37° C. for 30 min. An IS900 241-bp PCR was performed to confirm that no genomic DNA was detectable in the RNA samples (1). DNase treatments were repeated if needed. Quality of the extracted RNA was examined with a NANODROP 1000 spectrophotometer (Thermo Scientific). The ratios of A 260 /A 280  and A 260 /A 230  must be higher than 1.8 and 1.5, respectively, before proceeding to cDNA synthesis for transcriptome studies. 
     Transcriptome Studies. 
     The NimbleGen (NimbleGen System Inc., Madison, Wis.)  M. avium  subsp.  paratuberculosis  microarray was designed from the 4,350 open reading frame sequences in the genome of  M. avium  subsp.  paratuberculosis  (32). The whole genome was represented three times on each chip. In addition, each gene was represented by 20 probes of 60-mer oligonucleotides. As a result, each gene was represented by a total of 60 probes. Double-stranded cDNA synthesis from isolated RNA samples, microarray hybridization, and data analysis were performed as previously described (12, 33). Significantly expressed genes were selected by ±2-fold of change and P values of &lt;0.05 by Student&#39;s t test. The intensities were also exported to Gene Spring GX (Agilent Technologies, Santa Clara Calif.) for principal component analysis (PCA) on treatment conditions, which is a method to reduce dimensionality in multicondition microarray experiments and to find relevant patterns across conditions (34). Two biological replicates were included for each condition. 
     For RNA-seq studies, purified RNA samples were used for depletion of rRNA sequences to enrich mRNA using the MICROBEXPRESS bacterial mRNA enrichment kit (Ambion, Austin, Tex.). Approximately, 10 ug of total RNA for each sample was processed according to the manufacturer&#39;s instructions. For cDNA library preparation and sequencing, samples containing at least 1 ug of enriched mRNA were sent to the DNA Sequencing Facility at the University of Wisconsin-Madison Biotechnology Center. An Illumina HiSeq 2000 platform using one flow cell lane with 100-cycle paired-end chemistry (Illumina, San Diego, Calif.) was used to sequence the cDNA library clusters. Raw RNA-seq data files in FASTQ format were assembled against the  M. avium  subsp.  paratuberculosis  (35) using the CLC Genomics Workbench 4.8 (CLC bio, Aarhus, Denmark). Gene expression for each of the different sample conditions was calculated using “reads per kilobase million” (RPKM) expression values (36). The following formula was used to determine the RPKM values: RPKM=number of reads/(kilobase length of gene x millions of mapped reads). 
     This RPKM metric enables comparisons between data sets with a varying number of total reads. All reads mapping to annotated noncoding RNA (ncRNA) were removed from the data sets before determining RPKM values. Data sets were quantile normalized, and Kal&#39;s Z-test (37) was used for the comparative gene expression analysis. Genes were considered significantly, differentially expressed if they showed a ±2.5-fold change with FDR P values of &lt;0.05. 
     Quantitative Real-Time PCR. 
     Quantitative real-time PCR (qRT-PCR) was previously described (12, 38) for confirmation of transcript levels. A SYBR green-based reagent with ROX (Bio-Rad, Hercules, Calif.) was used with 50 ng of double-stranded cDNA in each reaction. Double-stranded cDNA synthesis is described in the microarray sample preparation session. No gDNA was detected from the RNA samples for cDNA synthesis. qRT-PCRs were performed with a 7300 real-time PCR system (Applied Biosystems, Foster City, Calif.). The threshold cycle (CT) of each gene was normalized to the CT of the 16S rRNA gene from the same cDNA sample. The expression fold changes were calculated by comparing the normalized CT of treated samples to the control sample as detailed before (39, 40). 
     Microarray Data Accession Number. 
     Data sets discussed in this report were deposited in NCBI&#39;s Gene Expression Omnibus (41) and are accessible through GEO Series accession number GSE43645. 
     Results 
     Characterization of  M. avium  Subsp.  paratuberculosis -Containing Phagosomes. 
     In our previous study, we defined the stressome of  M. avium  subsp.  paratuberculosis  under various in vitro conditions that mimicked the hostile host microenvironments, including low pH and oxidative stress (12). In the present study, we further examined the bacterial responses in the early stage of cell infection using a murine macrophage infection model. Both naive and IFN-γ-activated cells were used in our study. We monitored the expression of inducible nitric oxide synthase (iNOS), a marker for macrophage activation, with quantitative real-time PCR following IFN-γ treatment of J774A.1 cells. The transcription activity of iNOS in IFN-γ-treated cells was over 1,000 times higher than naive cells (data not shown). The temporal profile of iNOS expression indicated that naive macrophages were activated by 2 h postinfection and throughout the course of infection, with comparable mRNA levels regardless of the viability of  M. avium  subsp.  paratuberculosis  bacilli. Activated macrophages had a similar profile, but the iNOS expression levels were between 1.6 to 2.6 times greater than those of infected naive macrophages at each time point. 
     Additionally, we measured the phagosomal pH in both naive and activated macrophages using a dual-emission dye LYSOSENSOR radiometric probe DN-160 that emits fluorescent signals in a pH-dependent manner. Before infection, naive and activated macrophages had similar lysosomal pH levels ranging from 5.1 to 5.3. At 2 h postinfection, the pH in phagosomes containing heat-killed  M. avium  subsp.  paratuberculosis  decreased below 4.0 regardless of cell activation status. However, the pH in phagosomes containing live  M. avium  subsp.  paratuberculosis  bacilli decreased just below the preinfection level (i.e., 4.8 to 5.0), suggesting the ability of live bacteria to prevent phagosome acidification. Activated macrophages, but not naive ones, were able to continuously decrease the pH of phagosomes containing live bacilli up to 4 h of postinfection. As the infection progressed (24 h), activated macrophages exhibited a better ability to maintain a lower pH level than naive macrophages. 
     To examine the role of  M. avium  subsp.  paratuberculosis  on phagosome maturation, we examined the percentage of colocalization between intracellular  M. avium  subsp.  paratuberculosis  and the lysosome marker LAMP-1. While heat-killed bacteria showed over 85% colocalization with LAMP-1, live  M. avium  subsp.  paratuberculosis  significantly reduced the percentage of colocalization with LAMP-1 at 2 h postinfection (67.6%±5.5) in naive macrophages, suggesting live  M. avium  subsp.  paratuberculosis  is able to rapidly circumvent the hostile environment and to delay phagosome maturation. The percentage of colocalization did not significantly change in activated macrophages over the course of the experiment (87.92%±5.32 and 83.7%±9.5 at 2 h and 24 h, respectively), suggesting that preactivated host cells have a better ability to control invading intracellular pathogens by means of phagosome maturation. The reduced phagolysosome fusion of naive macrophages was restored to a level (78.4%±6.8) similar to that of the preactivated phagosome (81.6%±8.8) at 24 h postinfection, as also evidenced by the increased iNOS expression level of the naive macrophage infection compared to uninfected cells (data not shown). In general, both phagosomal pH and LAMP-1 colocalization indicated the ability of live, virulent  M. avium  subsp.  paratuberculosis  to prevent phagosome acidification and to delay lysosomal fusion by 2 h postinfection. 
     Transcriptional Profiling of  M. avium  Subsp.  paratuberculosis  Isolated from Infected Macrophages. 
     To profile changes in the levels of  M. avium  subsp.  paratuberculosis  transcripts within macrophages, we isolated intracellular bacilli at 2 and 24 h postinfection, with or without IFN-γ activation. Because the bacteria must stay in the cell culture medium (RPMI 1640-10% FBS, mycobactin J-free) before they can infect host macrophages, we compared the transcriptomes of intracellular bacteria to those incubated in vitro in cell culture medium for 2 h. Under all conditions tested, the correlation coefficients (r) between biological replicates ranged between 0.92 and 0.99. To examine the statistical distance between each biological replicate and among treatments, a three-dimensional principal component analysis plot was generated, indicating high correlations between biological replicates. Cluster analysis identified groups of genes active only during macrophage infection. Compared to the RPMI-incubated control sample, expression levels of 136 and 333  M. avium  subsp.  paratuberculosis  genes were significantly changed in naive macrophages at 2 and 24 h postinfection, respectively. On the other hand, in IFN-γ-activated macrophages, the numbers of genes with significantly changed expression levels were 284 and 328, respectively. Among those genes, 47 were common in all of the 4 examined macrophage conditions, representing a core set of genes responsible for interacting with the macrophage microenvironment. 
     In general,  M. avium  subsp.  paratuberculosis  transcriptomes in infected macrophages preactivated with IFN-γ were more similar to those observed under in vitro stressors reported earlier (12). Also, IFN-γ activation of macrophages resulted in significant induction of a group of genes (n=48), mostly those involved in energy production and conversion (e.g., icl, fdxA, sdhABCD, and ndh) or nutrient transport and metabolism (e.g., fad genes, dapA_1, and cysH_2), at 2 h postinfection in IFN-γ-activated macrophages compared to naive macrophages. 
     At 24 h postinfection, we started to see a significant change in  M. avium  subsp.  paratuberculosis  transcriptomes indicative of change of their microenvironment, especially in activated macrophages. For example, the mbt operon (mbtA to mbtE and MAP2172c) involved in iron metabolism was significantly upregulated at 24 h postinfection in IFN-γ-treated macrophages compared to the RPMI-incubated control samples. A similar observation was reported for 120 h postinfection of bovine monocyte-derived macrophages (MDMs) (21). MAP2172c was shown to be repressed in  M. avium  subsp.  paratuberculosis  cultures grown in mycobactin J-depleted medium over time, where expression levels of other mbt genes remained constant (42). This paradigm could suggest that an intracellular environment is not iron exochelin-free, or there are other intracellular factors that stimulate alternative iron metabolic pathways, at least in the examined times. On the other hand, a gene involved in iron storage, bfrA (43), was significantly downregulated at the same time point, while the mbt operon was activated, suggesting the lack of access to iron inside the mycobacterial phagosome. Interestingly, the expression of the iron-dependent regulator ideR (43) remained unchanged during the examined time course regardless of macrophage activation status, suggesting a lesser role of ideR in early stages of macrophage infection. Overall, a significant time-dependent shift in  M. avium  subsp.  paratuberculosis  transcriptomes was evident from examining  M. avium  subsp.  paratuberculosis  collected at 2 and 24 h after macrophage infection. 
     Microenvironment of  M. avium  Subsp.  paratuberculosis.    
     One of our goals is to gain more insights into the intracellular environment of  M. avium  subsp.  paratuberculosis  using transcriptome analysis. Schnappinger et al. reported that  M. tuberculosis  upregulates β-oxidation genes by 4 h postinfection in murine bone marrow macrophages, suggesting a transition of carbon source from carbohydrates to fatty acids (44). Similarly, our study indicated activation of  M. avium  subsp.  paratuberculosis  orthologues (fadA6_3, fadB_1, fadD9, fadE17, fadE21, fadE3_1, and fadE5) in the β-oxidation pathway starting from as early as 2 h postinfection, suggesting the transition of carbon source utilization is a common bacterial strategy between  M. tuberculosis  and  M. avium  subsp.  paratuberculosis . The id gene, previously known as aceA, was also among the highly upregulated genes involved in carbon metabolism. The gene product, an isocitrate lyase, bridges the β-oxidation pathway to glyoxylate cycle, an anabolic pathway with a net product of glucose. The contribution of id to  M. avium  subsp.  paratuberculosis  survival in macrophages remains to be analyzed. 
     Once entering host cell compartments, intracellular bacteria encounter host defense mechanisms such as reactive oxygen intermediates (ROIs), reactive nitrogen intermediates (RNIs), digestive enzymes, and, most importantly, lower pH. In addition, the phagosome is also a nutrient-depleted environment. Accordingly, we examined genes that are associated with stress response and intracellular bacterial survival. Several known oxidative stress-induced genes, such as oxyR, trxB, trxC, tpx, ahpC, and ahpD, were significantly upregulated in all intracellular conditions. OxyR is a redox sensor protein that, when oxidized, positively regulates a group of genes, including ahpC, katG, gorA, and furA (45). Among those genes, the ahpCD operon was highly upregulated (6.2- to 11.0-fold) in samples taken from naive or activated macrophages. In addition, TrxB, TrxC, and Tpx, proteins involved in reduction of thio-disulfide and resistance of hydroperoxide processes (46), were upregulated, suggesting active machinery for counteracting oxidative stress within host cells. However, other known oxidative responsive genes, such as katG, gorA, furA, sodA, and sodC, were not activated in these samples, possibly because those genes are indirectly regulated by oxyR or also under the control of other stress-response regulators. Overall,  M. avium  subsp.  paratuberculosis  deployed specific gene products to defend against the hostile microenvironment during this early stage of infection. 
     Changes in global gene regulators. Sigma factors play a central role in bacterial gene regulation (47) and pathogenesis as reviewed in  M. tuberculosis  (48) and other pathogens (49). Nineteen sigma factors were identified in  M. avium  subsp.  paratuberculosis  by sequence analysis (24), 13 of which are homologous to  M. tuberculosis  sigma factors. The sigA gene, though considered a constitutively expressed sigma factor gene in  M. tuberculosis  (50), was found downregulated to nearly 2-fold at 24 h in activated macrophages. The sigB gene, which is a dispensable sigma factor and partially responsive to some oxidative and heat shock stresses in  M. tuberculosis  (51), showed a slight increase (˜1.6-fold) at 24 h postinfection under both activated and naive states of macrophages. Genes of sigC, sigG, sigJ, sigM, and other extracytoplasmic sigma factors (ECF-2 through ECF-6) remained constantly expressed in all examined conditions. However, as shown in  FIG.  1   , sigH was the most induced among other sigma factors of  M. avium  subsp.  paratuberculosis , and the activation seems to be augmented by macrophage activation over time. sigH transcripts were upregulated under in vitro heat shock and oxidative stress treatments in  M. avium  subsp.  paratuberculosis  (12). Also, sigL was upregulated at the 2-h time point but downregulated by 24 h postinfection, suggesting a potential role for sigL in the very early stage of infection. On the contrary, sigE expression was significantly upregulated after 2 h postinfection and remained high at 24 h postinfection, suggesting a prolonged role during  M. avium  subsp.  paratuberculosis  persistence. Overall, a few sigma factors showed a dynamic and active gene regulation transition during the first 24 h postinfection within macrophages. It is possible that other regulators could play a similar role during later times of infection or in different host cells. 
     Role of sigH in  M. avium  Subsp.  paratuberculosis  to Variable Stress Conditions. 
     The sigH gene in  M. tuberculosis  has been shown to be upregulated upon heat shock, upon diamide treatment (25), and during survival in human macrophages (52), suggesting its importance in responding to extracellular stimuli and intracellular survival. To test the hypothesis that sigH could play an important role in  M. avium  subsp.  paratuberculosis  stress responses, we employed a specialized transduction protocol (53) to generate a sigH isogenic knockout mutant in the  M. avium  subsp.  paratuberculosis  K10 genetic background. Because sigH and its anti-sigma factor (MAP3323c) are likely encoded in an operon (54), the ΔsigH mutant was examined for possible polar effects on the downstream gene, MAP3323c. Using reverse transcriptase PCR, the presence of the MAP3323c transcript was confirmed in the ΔsigH mutant strain. 
     After construction, the resistance of the ΔsigH mutant was evaluated against various stressors. Analysis of the disc diffusion assay revealed that the ΔsigH mutant does not tolerate thiol-specific oxidation compared to the wild-type strain, as evidenced by the observed halo zones on plates. However, no other differential resistance was observed when a cell wall stressor (sodium dodecyl sulfate) or ethambutol discs were used (data not shown). To measure viability of the ΔsigH mutant after sustained exposure to diamide or heat stress, we cultured both wild-type and the mutant strains for an extended time period in the presence of diamide or a 45° C. water bath. In both stress conditions, there was significant reduction at each time point in the viability of the ΔsigH mutant compared to that of the wild-type strain. At day 7, the viability of the ΔsigH mutant was reduced by almost 2-log orders and more than 1-log order in CFU counts relative to the wild-type strain for diamide and heat stress, respectively. Unfortunately, replicative plasmid complementation of the ΔsigH mutant with a wild-type sigH under the control of the hsp65 promoter did not restore the diamide resistance phenotype (data not shown), most likely due to inefficient complementation in mycobacterial strains (25, 55, 56). 
     Intracellular Survival of the ΔsigH Mutant in Bovine MDM Cells. 
     Intracellular growth kinetics of  M. avium  subsp.  paratuberculosis  strains were analyzed using bovine MDM cells. MDM cells were infected with the ΔsigH mutant and its parental strain for a prolonged time up to 8 days after infection. The MDM monolayer in the culture wells was checked at a regular interval for cell confluence under an inverted light microscope. We first determined intracellular viability of both the ΔsigH mutant and wild-type strains within the naive MDM cells. The numbers of wild-type strain of  M. avium  subsp.  paratuberculosis  bacilli increased 2-fold, whereas the growth of the ΔsigH mutant was not supported within naive MDM cells as determined by CFU plating at 8 days after infection ( FIG.  2 A ). Next, we examined whether the ΔsigH mutant would be able to survive inside activated MDM cells pretreated with recombinant IFN-γ. At 8 days post infection, there was more than a 2-fold increase in the number of wild-type  M. avium  subsp.  paratuberculosis  bacilli, whereas in the IFN-γ pretreated MDM cells, viability of the ΔsigH mutant was significantly reduced almost by 50% ( FIG.  2 B ). These observations suggested an important role for sigH in defending  M. avium  subsp.  paratuberculosis  against IFN-γ activation. 
     Virulence Analysis of the  M. avium  Subsp.  paratuberculosis  ΔsigH Mutant. 
     To assess the role of SigH in  M. avium  subsp.  paratuberculosis  virulence, we investigated the persistence of the  M. avium  subsp.  paratuberculosis  ΔsigH mutant using the mouse model of  paratuberculosis . The initial growth kinetics of the wildtype and ΔsigH mutant strains were similar, with an equal burden of bacteria in both intestine and spleen up to 6 wpi ( FIG.  3   ). However, the colonization levels of the ΔsigH mutant compared to its parental strain were significantly reduced in spleen and intestine at 12 wpi, suggesting a role for sigH in the long-term survival of  M. avium  subsp.  paratuberculosis  in mice. Interestingly, when  M. tuberculosis  ΔsigH was used to challenge mice, no differences in bacterial load were observed in mouse organs compared to that of the wild-type strain (57). 
     Evaluation of the hematoxylin and eosin-stained spleen, liver, and intestine organs at 3, 6, and 12 wpi showed moderately similar tissue pathology when infected with the ΔsigH mutant or wildtype  M. avium  subsp.  paratuberculosis . Granulomatous inflammation was evident in the liver tissues by 12 wpi, with no visual differences in mycobacterial colonization among the mouse groups infected with wild-type or mutant strains. However, mouse spleen tissues infected with the wild-type strain displayed higher follicular atrophy than the spleen tissues infected with the ΔsigH mutant. Consistent with the bacterial colonization data, Ziehl-Neelsen staining showed higher numbers of acid-fast bacilli in the mouse spleen infected with the wild-type strain than the ΔsigH mutant at 12 wpi ( FIG.  3 C ). Taken together, our data indicated that the ΔsigH mutant was attenuated in the murine model of  paratuberculosis  compared to the wild-type  M. avium  subsp.  paratuberculosis  strain. 
     Transcriptional Regulation of sigH in  M. avium  Subsp.  paratuberculosis.    
     Our stress experiments showed that the ΔsigH mutant was hypersensitive to elevated temperature and diamide exposure, each resulting in impaired growth. On the basis of these findings, we hypothesized that sigH may play an important role in directing transcriptional control under unfavorable environmental conditions. To identify gene regulatory networks under the control of sigH, both wild-type  M. avium  subsp.  paratuberculosis  and ΔsigH mutant transcriptomes were profiled before and after diamide exposure using the next-generation RNA sequencing (RNA-seq). When the wild-type strain was compared to the ΔsigH mutant, approximately 15% of the  M. avium  subsp.  paratuberculosis  genes (˜307 induced and ˜344 repressed) were found to be differentially regulated at 3 h postexposure to diamide stress. This large number of gene perturbation was likely orchestrated by additional sigma factors (e.g., sigB, sigD, sigE) along with various transcriptional regulators that were differentially expressed in examined samples. Genes were grouped into different functional categories, and a large number of genes (e.g., hsp, clpB) belonging to the chaperonin functional category were significantly upregulated. Many induced genes were involved in detoxification and maintaining cellular redox homeostasis (e.g., trxB2, adhE) during oxidative stress as detailed before (46, 58). Many transcriptional factors and two-component systems were found to be upregulated (e.g., sigB, sigE, whiB4, MAPK_0206, mtrA) under the control of SigH. The expression of mycobacterial sigB and sigE was known to be linked with the presence of the sigH gene (59). WhiB-like proteins are redox-responsive DNA binding factors and could play a protective role against oxidative stress (60). In mycobacteria, the role of the MtrB-MtrA two-component system is not entirely understood, but it was found to be essential for bacterial viability, particularly involved in the regulation of cell wall permeability (61, 62). 
     A number of induced genes in the SigH regulon were related to virulence, and many of them were included in the mce gene family (e.g., mceA1, mceC, mceD). Several mce genes were shown to be upregulated during phagocytosis and oxidative stress exposure (52, 63), indicating that they are active during infection. Other key functional gene categories were associated with central intermediary/sulfate metabolism (e.g., rmlB, rmlC, cysQ_2, cysD), energy metabolism (e.g., rpi, tpi, nuoA), and cell processes/transport (e.g., fdxC_2, MAPK_4062) or were cell envelope related (e.g., mmpL4_1, mmpS3). Our results indicate that many of these genes were induced under intraphagosomal stresses inside macrophages. Genes belonging to functional categories, including lipid metabolism (e.g., fadE14, fadD33_2, MAPK_2213), polyketides (e.g., pks2, papA3_2), and biosynthesis of amino acids (e.g., leuC, metA, trpE2), were among the down regulated genes in  M. avium  subsp.  paratuberculosis  relative to the ΔsigH mutant following diamide stress. We also examined the differential expression profile of  M. avium  subsp.  paratuberculosis  in the absence of sigH during standard physiological growth conditions (mid-log phase). In this case, gene categories belong to lipid metabolism (e.g., fadD29), cell processes (e.g., kdpA, pstS), transcriptional regulation (e.g., MAPK_0788), and electron transport (e.g., fdxC 2). Additionally, we found that a large number of genes belong to the hypothetical functional category (˜30%). To verify the transcriptome results, a few upregulated genes were randomly selected, and qRTPCR was employed using the SYBR green method. The transcript levels of these genes analyzed by RNA-seq and qRT-PCR were in good agreement and corroborated with the transcriptome data. 
     Identification of sigH-Regulated Promoters in  M. avium  Subsp.  paratuberculosis.    
     For identification of promoters that were likely to be directly controlled by sigH, we analyzed a list of candidate transcripts with higher expression ratios (wild type/ΔsigH mutant). Since SigH of  M. avium  subsp.  paratuberculosis  is a very close homologue of  M. tuberculosis  SigH (59), we searched for the presence of the consensus sequence of  M. tuberculosis  SigH-dependent nondegenerate promoter motifs GGAA-N18-20-GTT in the 250-bp region upstream of start codons of  M. avium  subsp.  paratuberculosis  genes using the Genolist webserver (genodb.pasteur.fr/cgi-bin/WebObjects/GenoList). A total of 30 genes were found to be directly upregulated by SigH with GGAA and GTT core motifs at the −35 and −10 regions, respectively, in their promoter regions. Many of these targets of SigH in  M. avium  subsp.  paratuberculosis  were also found to be controlled in  Corynebacterium glutamicum  (64),  Streptomyces coelicolor  (65), and  M. tuberculosis  (59), suggesting a conserved regulon directly controlled by SigH across the high-percentage GC Gram-positive actinobacteria. 
     Discussion 
     The intracellular pathogen  M. avium  subsp.  paratuberculosis  is known to infect and persist within host macrophages with unclear mechanisms. To examine how  M. avium  subsp.  paratuberculosis  responds to intracellular environments, especially during the early stages of infection, we used a macrophage cell line coupled with DNA microarrays to profile macrophage-induced changes in  M. avium  subsp.  paratuberculosis  transcriptome. A clear advantage of this infection model is the flexibility to control the activation status of the host cells in addition to the availability of reagents and protocols for manipulation. By comparing the results of phagosome pH and phagosome colocalization markers, we found significant differences in intracellular environments of naive versus active macrophages consistent with earlier studies (44, 66). Activated macrophages, at the time of infection, showed much higher iNOS gene expression than naive macrophages. At 2 or 24 h postinfection, they showed higher phagosome colocalization with ingested  M. avium  subsp.  paratuberculosis  particles, which clearly exhibited a better cell defense mechanism than naive macrophages. However, during the course of infection up to 24 h, overall survival of intracellular  M. avium  subsp.  paratuberculosis  did not differ in either naive or activated macrophages. This phenotype could change later during persistent infection which we did not address in this study. Most of the differentially expressed genes between these states are core stress responsive genes involved in energy production, indicating  M. avium  subsp.  paratuberculosis  initiates stress responses to a higher level more rapidly in activated intracellular environments. On the host side, once activated, the host cells maintained their activation status throughout the course of infection. This suggests that virulent  M. avium  subsp.  paratuberculosis  has the ability to prevent phagosome maturation and subsequently circumvent detrimental low pH and oxidative stresses during the very early stages of infection, possibly without interfering with the host early signal transduction pathways responsible for macrophage activation. 
     When  M. avium  subsp.  paratuberculosis  transcriptomes in macrophages were compared to our previous study of the in vitro stressome (12), there were more common genes with the 24-h than 2-h-postinfection samples, indicating that during the early stage of infection,  M. avium  subsp.  paratuberculosis  is adjusting to more acidic and oxidizing environments. We also observed the metabolic shift of  M. avium  subsp.  paratuberculosis  to utilize fatty acids as the major carbon source, which has already been observed in  M. tuberculosis  (44) and  M. avium  subsp.  paratuberculosis  (21). The shift of metabolic activity at early infection may be a common theme employed by mycobacterial pathogens under nutrient-depleted conditions. By 24 h postinfection, securing iron for  M. avium  subsp.  paratuberculosis  became a significant quest, especially in activated macrophages, as suggested by the activation of the mbt operon. It is well established that the phagosome is an iron-depleted compartment (44) and intracellular pathogens have evolved ways to scavenge iron within mammalian cells. However, iron acquisition mechanisms of  M. avium  subsp.  paratuberculosis  remain unknown given that  M. avium  subsp.  paratuberculosis  possesses a truncated mtbA gene and thus is unable to produce mycobactin (32). 
     Because of the important role played by global gene regulators in bacterial pathogenesis, we focused our analysis on the expression profile of the 19 sigma factors encoded in the  M. avium  subsp.  paratuberculosis  genome (32). Accordingly, in the experiments reported here, we were able to capture active gene regulation of a set of sigma factors (e.g., sigH, ECF-1) during early macrophage infection with  M. avium  subsp.  paratuberculosis . Consistent with the  M. tuberculosis  infection studies, we found an immediate upregulation of sigH within macrophages. This trend continued through 24 h postinfection and indicated a crucial role of sigH to regulate stress-responsive genes, especially those activated during exposure to thiol oxidation, as indicated by the disc diffusion assay. Moreover, we have demonstrated that the ΔsigH mutant is very sensitive to sustained exposure to diamide or heat stress compared to the wild-type strain. The sigE gene, another stress-induced sigma factor, did not show higher expression levels until 24 h postinfection in either naive or activated macrophages. This delayed response of sigE as well as modest induction of sigB may indicate an indirect regulation by other immediate stress-responsive genes and support the important role played by sigma factors in mycobacterial pathogenesis (25, 59). The other sigma factor that was upregulated throughout the examined time course was ECF-1. This sigma factor may also play an important role immediately upon infection, which was not reported before. 
     We have also profiled the regulatory network under the control of sigH by studying the relative abundance of gene transcripts using RNA-seq. We found that a large number of  M. avium  subsp.  paratuberculosis  genes were directly or indirectly regulated by sigH after exposure to diamide stress. In fact, analysis of the upstream sequence of the upregulated genes revealed a set of genes that could be directly controlled by SigH. Among them, many genes are involved in the functional category of heat shock and protein processing. Heat shock proteins (e.g., Hsp, DnaJ2, ClpB) are found widely on prokaryotic cells and act as molecular chaperones helping to configure proteins correctly upon encountering an unfavorable milieu (67, 68). Such environments, i.e., oxidative stress, could result in nonrepairable protein structures which may necessitate full degradation by the ClpC protease (63). Oxidative stress scavengers induced following diamide stress include TrxB2, TrxC, and AdhE (46, 58). All of these genes likely play important roles in redox homeostasis under thiol oxidation and are found in high levels inside the macrophages (44, 63). Interestingly, the effect of diamide stress in  M. tuberculosis  also resulted in a transcriptional profile similar to that of  M. avium  subsp.  paratuberculosis  (63), indicating the pivotal role of sigH across mycobacterial species. 
     Consistent with the estimated large regulon of SigH, a significant difference in survival rates between the ΔsigH and wild-type strains was observed inside activated bovine macrophages. Intracellular growth of the wild-type  M. avium  subsp.  paratuberculosis  strain was not inhibited regardless of the activation state up to 8 days postinfection. This observation was in corroboration with the earlier findings which showed that activated bovine monocytes were inadequate to inhibit intracellular growth of  M. avium  subsp.  paratuberculosis  up to 9 days after infection as determined by the CFU method (69). In contrast, viability of the ΔsigH mutant was significantly impaired, indicating an important function of sigH for the intracellular growth of  M. avium  subsp.  paratuberculosis , possibly by blocking IFN-γ activity as suggested earlier (23). In recent studies, clues have been obtained on macrophage interaction with  M. avium  subsp.  paratuberculosis  that indicate the capacity of this pathogen to subvert host immune responses by blocking the ability of mononuclear phagocyte maturation (23, 70, 71). Although it is tempting to speculate that the ΔsigH mutant failed to interfere with macrophage maturation, especially when preactivated with IFN-γ, more experiments are needed to fully understand the mechanisms that sigH could play during macrophage infection. The survival profile of  M. avium  subsp.  paratuberculosis  constructs in MDM was further supported by the inability of the ΔsigH mutant to survive in mice. Both bacteriological and histological analyses displayed impaired organ colonization of the ΔsigH mutant with a low inflammatory response. However, we did not find any comparative differences in liver organs infected with the wild-type and ΔsigH mutant strains. A recent study showed that the  M. tuberculosis  ΔsigH mutant was completely attenuated in nonhuman primates (72), a better experimental model than mice for studying human tuberculosis (57). It will be interesting and important to examine the survival of the  M. avium  subsp.  paratuberculosis  ΔsigH mutant in a ruminant model of  paratuberculosis  (e.g., goat). 
     In conclusion, our analyses indicated significant changes in mycobacterial gene expression during macrophage survival, most likely under the control of sigH and other sigma factors. The activation status of macrophages also directs the mycobacterial response to a specific stress-responsive profile. We demonstrated that sigH offers a massive temporal response on the  M. avium  subsp.  paratuberculosis  transcriptome to cope with the adverse effects of oxidative stress. Our data indicate that sigH could play a critical role during infection, and activation of its regulon is required for replication and full virulence of  M. avium  subsp.  paratuberculosis . Further interrogation of these sigma factors and their regulatory networks should ultimately furnish a greater understanding of  M. avium  subsp.  paratuberculosis  pathogenesis and help design a better approach for controlling Johne&#39;s disease. 
     Example 2 
     References cited in this Example are listed in the section of References as “References cited in Example 2.” 
     Materials and Methods 
     Bacterial Strains. 
       M. avium  subsp.  paratuberculosis  K10 and  M. smegmatis  mc 2 155 strains were grown in Middlebrook 7H9 broth and on Middlebrook 7H10 plates as previously described (2,19). For cloning,  Escherichia coli  DH5α and HB101 were used as host cells. To generate an  M. avium  subsp.  paratuberculosis  mutant, a specialized transduction protocol was adopted to delete the sigL/MAP4201 gene using the  M. avium  subsp.  paratuberculosis  K10 strain (19,25). Primers were designed to amplify ˜750 bp PCR fragments flanking each end of the sigL coding region and cloned into pYUB854 (19). The resulting vector was used to generate sigL deleted mutant,  M. avium  subsp.  paratuberculosis  ΔsigL, according to the methods described elsewhere (26). Genotype of the  M. avium  subsp.  paratuberculosis  ΔsigL was confirmed by PCR and sequence analysis (19). To complement the  M. avium  subsp.  paratuberculosis  ΔsigL, a ˜4-kb fragment, encompassing sigL with its 5′ regulatory region and the distal genes (MAP4202-MAP4205), was amplified by PCR and cloned into pMV306 (24). The  M. avium  subsp.  paratuberculosis  ΔsigL strain was transformed with this recombinant construct and genotype of the complemented strain ( M. avium  subsp.  paratuberculosis  ΔsigL:sigL) was identified by PCR analysis. A similar approach was applied to complement  M. tuberculosis  mutant strain lacking such alternative sigma factor (27). 
     Stress Phenotype of  M. avium  Subsp.  paratuberculosis.    
       M. avium  subsp.  paratuberculosis  cultures were grown to log phase (OD 600 =0.5-1.0) and 200 μl spread on 7H10 plates. For disk diffusion assay (DDA), paper discs (6-mm, Whatman, Piscataway, N.J.) containing 20 μl of 0.5, 1, or 1.5M diamide (oxidative stressor) and 1, 2, or 3% sodium dodecyl sulfate (SDS;cell wall stressor) were placed on each of the spread plate. Plates were incubated at 37° C. until a thick confluent lawn developed. To determine sustained effect of stressor on the viability of bacilli, after washing with PBS,  M. avium  subsp.  paratuberculosis  cultures were exposed to the acidified 7H9 broth (pH 5.5 obtained by adding HCl) containing 0.3% bovine bile (cell wall stressor) and aliquots were collected at 0, 4, 15, 24, 48, and 72 h to monitor their viability by colony forming units (CFU) (19). 
     Cell Culture and Infection. 
     The mouse macrophage cells (J774A.1) were regularly maintained as described elsewhere (19). To activate macrophages, cells were pretreated overnight (18 h) with 100 U/ml recombinant murine IFN-γ (Pepro Tech, Rocky Hill, N.J.) before infection with  M. avium  subsp.  paratuberculosis  strains (19). For cell infection studies, wild-type and mutant strains were added to macrophage monolayers (multiplicity of infection [MOI], 20:1). Following incubation at 37° C. in 5% CO 2  for 3 h, macrophage monolayers were washed twice with warm PBS to remove extracellular bacteria and RPMI-1640 medium containing 5% fetal bovine serum was added. Cells were lysed at 1, and 8 days post-infection for bacterial CFU counts. To examine  M. avium  subsp.  paratuberculosis  ΔsigL survival in bovine monocyte-derived macrophages (MDM), MDM cells were isolated from peripheral blood of three cows and cell infection studies were performed as described in detail elsewhere (19). 
     Mouse Infections. 
     All animal experiments used in this study were performed according to the protocols approved by the Institutional Animal Care and Use Committee, UW-Madison. For the virulence study, two groups (N=15 per group) of female BALB/c mice (Harlan Laboratories, Indianapolis, USA) were challenged intraperitoneally (i.p) with the wild-type and mutant strains. Infection inocula (˜2×10 8  CFU/mouse) of the two strains were similar as determined by plate count on the day of infection. Mouse groups (N=5) were sacrificed at 3, 6 and 12 weeks post-infection (WPI), and organ samples were collected for bacterial CFU enumeration and histopathological examinations as described before (19). For the immunization studies, female C57BL/6 mice (Taconic, Hudson, N.Y.) were used. Mock group (N=12) was immunized with PBS buffer while  M. avium  subsp.  paratuberculosis  ΔsigL mice group (N=14) received ˜2×10 6  CFU in 0.2 ml PBS subcutaneously (s.c.) into the neck scruff twice, 2 weeks apart. Four weeks following booster dose, mice were challenged i.p. with ˜7×10 8  CFU wild-type  M. avium  subsp.  paratuberculosis  strain as determined by plate count on the day of infection. Mouse groups (N=4-6) were sacrificed at 6 weeks post-immunization and 6 and 12 weeks post-challenge (WPC), and organ samples were collected for bacterial CFU counts, histopathological examinations, and immune responses. 
     Evaluation of Immune Responses. 
     Mouse spleens were collected aseptically and homogenized by gentle mechanical disruption. Following spleen cell isolation, splenocytes were cultured in duplicate in round bottom 96-well tissue culture plates with 1×10 6  cells/well (28). Cells were re-stimulated in vitro with 10 μg/ml Johnin purified protein derivative (PPD) (NVSL, Ames, Iowa) for 48 and 72 h. Cell supernatants were collected and analyzed for cytokines by ELISA kit according to the manufacturer&#39;s instructions (BioLegend, San Diego, Calif.). To determine humoral immune response, sera were prepared from mouse blood and  M. avium  subsp.  paratuberculosis  specific antibody (anti-PPDj antibodies) was detected by ELISA using Horseradish peroxidase conjugated rabbit anti-mouse antibody (Pierce, Rockford, Ill.) (29). 
     Statistical Analysis. 
     Student&#39;s t test was performed to compare differences in mouse immune responses and bacterial CFU counts from in vitro stress treatments. Mann-Whitney U test was used to compare bacterial loads in mouse organs. A probability value of &lt;0.05 was considered significant. 
     Results 
     Effect of sigL Mutation on Viability of  M. avium  Subsp.  paratuberculosis  Under Stress. 
     Recent analysis of  M. avium  subsp.  paratuberculosis  transcriptome during macrophage infection suggested that sigL could be an important factor for  M. avium  subsp.  paratuberculosis  survival inside host macrophages (19). To test this hypothesis, we generated a sigL deletion mutant,  M. avium  subsp.  paratuberculosis  ΔsigL ( FIG.  4 A-B ) and examined survival of this mutant under different stress conditions. Because sigL and its anti-sigma factor (MAP4202) are likely encoded in an operon (24), we examined  M. avium  subsp.  paratuberculosis  ΔsigL strain for possible polarity on the downstream gene, MAP4202. By employing reverse-transcriptase PCR analysis, presence of the MAP4202 transcript was confirmed in the ΔsigL mutant ( FIG.  4 C ). 
     To examine a potential role for sigL in  M. avium  subsp.  paratuberculosis  response to unfavorable stress conditions, we analyzed the survival of  M. avium  subsp.  paratuberculosis  cultures under both oxidative (diamide) and cell wall stresses (SDS and bovine bile). Growth inhibition zones in disk diffusion assays indicated that  M. avium  subsp.  paratuberculosis  ΔsigL was susceptible to diamide oxidation. Such phenotypic differences also indicated the inability of  M. avium  subsp.  paratuberculosis  ΔsigL to survive under SDS stress when compared to the wild-type and complemented strains. Bile tolerance was also evaluated by culturing of the  M. avium  subsp.  paratuberculosis  strains in the presence of 0.3% bovine bile (oxgall). This concentration of bile is likely encountered by the bacteria within the intestinal content following oral infection (30). In addition, because of the ability of  M. avium  subsp.  paratuberculosis  to resist killing by acidic conditions (31), we made culture broths slightly acidic (pH 5.5) to partially mimic the physiological condition that  M. avium  subsp.  paratuberculosis  would encounter following infection in the gastrointestinal tract (e.g. abomasum of a cow and stomach of a human). Survival levels showed a significant drop in the viability of the  M. avium  subsp.  paratuberculosis  ΔsigL at 4 h post-exposure to bovine bile compared to the wild-type and complemented strains. This difference in bacterial survival for  M. avium  subsp.  paratuberculosis  ΔsigL was increased by more than 1.0 log at 24 h and the viability of the mutant continued to decline at later times suggesting that sigL is important in rendering resistance when bacteria experience initial bactericidal barriers in the host. Because complementation of the  M. avium  subsp.  paratuberculosis  ΔsigL restored wild-type phenotype under these stress conditions, the complemented strain was not included in further experiments. 
     Intracellular Survival within Macrophages. 
     Because sigL was up-regulated inside activated murine macrophages (19), we examined intracellular survival of  M. avium  subsp.  paratuberculosis  ΔsigL in the IFN-γ pretreated murine macrophages. Our analysis showed an increase in the number of wild-type  M. avium  subsp.  paratuberculosis  at 8 days relative to the numbers obtained at day 1 post-infection whereas viability of the  M. avium  subsp.  paratuberculosis  ΔsigL was significantly reduced at this time. To use a more relevant model for  M. avium  subsp.  paratuberculosis  infection, we evaluated the persistence of the  M. avium  subsp.  paratuberculosis  ΔsigL both in resting and IFN-γ-activated bovine monocyte-derived macrophages (MDM cells), the natural host cell for  M. avium  subsp.  paratuberculosis . At 8 days post-infection, the number of wild-type bacilli increased over twofold compared to the numbers obtained at day 1 in the resting MDM cells. Specifically, naive and IFN-γ pretreated MDM cells were infected with  M. avium  subsp.  paratuberculosis  ΔsigL and wild-type  M. avium  subsp.  paratuberculosis  strains. Cells were lysed at 1, and 8 days post-infection and numbers of viable bacilli were determined by serial dilutions for CFU plating. The survival level at 8 days was relative to the viable counts of bacterial strains at day 1. Survival data represent the average of macrophage infections collected from three different donor animals with significance levels in Student&#39;s t test (*p&lt;0.05). 
     In contrast, viability of the  M. avium  subsp.  paratuberculosis  ΔsigL was significantly reduced almost by half indicating a potential function for sigL in defending  M. avium  subsp.  paratuberculosis  against intracellular stress. A similar survival trend for the  M. avium  subsp.  paratuberculosis  ΔsigL was seen inside IFN-γ pretreated MDM cells whereas this activation status did not result in more inhibitory effect on the survival of wild-type bacilli. Collectively, survival assays indicated that deletion of sigL affected  M. avium  subsp.  paratuberculosis  viability following exposure to stress conditions suggesting a significant function for sigL in defending  M. avium  subsp.  paratuberculosis  against intracellular insults. 
     Virulence Analysis of  M. avium  Subsp.  paratuberculosis  ΔsigL Strain. 
     To evaluate the contribution of SigL to  M. avium  subsp.  paratuberculosis  virulence, we examined persistence of the  M. avium  subsp.  paratuberculosis  ΔsigL using the murine model of  paratuberculosis . The survival pattern indicated significant attenuation for  M. avium  subsp.  paratuberculosis  ΔsigL as early as 3 WPI in all of the examined organs. In the spleen, viability of  M. avium  subsp.  paratuberculosis  ΔsigL was reduced by more than 1-log and 2-log orders relative to the wild-type strain at 6 and 12 WPI, respectively. Similarly, colonization levels of the mutant strain in the liver were significantly lower compared to the parental strain at all examined time points. Interestingly,  M. avium  subsp.  paratuberculosis  ΔsigL did not persist in the intestines as we were unable to detect any bacteria (limit of detection 20 CFU) at 6 and 12 WPI. 
     The histological analysis revealed mild to moderate granulomatous inflammation in the liver tissues at both 3 and 6 WPI with either of the  M. avium  subsp.  paratuberculosis  strains with higher lymphocytic infiltration in the mice infected with  M. avium  subsp.  paratuberculosis  ΔsigL. At 12 WPI,  M. avium  subsp.  paratuberculosis  ΔsigL infected animals showed less granulomatous inflammation indicating reduced ability of  M. avium  subsp.  paratuberculosis  ΔsigL compared with wild-type bacilli to establish an infection in animals. In accordance with the bacterial organ burden data, Ziehl-Neelsen staining showed higher numbers of acid-fast bacilli in mice liver infected with the wild-type strain relative to the  M. avium  subsp.  paratuberculosis  ΔsigL at all examined time points. A similar observation was noticed for the spleen and intestine tissues (data not shown). Both bacterial organ colonization and histological data analyses suggested that  M. avium  subsp.  paratuberculosis  ΔsigL was attenuated for survival, compared to the wild-type strain in the mice model of infection. 
     Immunization with  M. avium  Subsp.  paratuberculosis  ΔsigL. 
     Because sigL encodes a mycobacterial GGR (32) and was critical for  M. avium  subsp.  paratuberculosis  survival in the present study, we investigated the vaccine potential of  M. avium  subsp.  paratuberculosis  ΔsigL in a challenge model of murine  paratuberculosis  ( FIG.  5 A ). To examine immunogenicity of the mutant strain, groups of mice were immunized twice with  M. avium  subsp.  paratuberculosis  ΔsigL and 6 weeks post-immunization (WPI) mice organs were analyzed for bacterial content. Two immunizations with this mutant resulted in low colonization (2×10 2  CFU) in the liver whereas no bacteria were detected (limit of detection 20 CFU) in the intestine or spleen (data not shown). To evaluate vaccine induced immune responses before challenge; ELISA was used to estimate levels of key cytokines in stimulated spleen cells. Statistical analysis revealed significantly (p&lt;0.05) high level of IFN-γ secretion in the  M. avium  subsp.  paratuberculosis  ΔsigL immunized mice compared to naïve animals ( FIG.  5 B ). Because of the importance of T-helper 17 cells (33) for intracellular bacterial infection, we examined IL-17A production in the immunized animals. However, we did not find any detectable levels for IL-17A at 6 WPI. Additionally, the mice group vaccinated with  M. avium  subsp.  paratuberculosis  ΔsigL had significantly (p&lt;0.05) higher anti-PPDj IgG level ( FIG.  5 C ). Thus, both IFN-γ and IgG data suggested ability of the  M. avium  subsp.  paratuberculosis  ΔsigL strain to induce enhanced immune responses. 
     Protection Against Challenge with  M. avium  Subsp.  paratuberculosis.    
     To examine the vaccine potential of sigL-based mutant, groups of mice were vaccinated with PBS (control) or  M. avium  subsp.  paratuberculosis  ΔsigL live strain and challenged with the virulent  M. avium  subsp.  paratuberculosis  K10 strain following 6 WPI ( FIG.  5 A ). At 6 weeks post challenge (WPC), the ΔsigL mice group had a significant reduction in the bacterial load in spleen and liver (˜0.5 log) compared to the PBS-vaccinated mice ( FIG.  6 A , B). More importantly, a higher level of bacterial load reduction (˜1 log) was observed in the intestine ( FIG.  6 C ), an important organ for  M. avium  subsp.  paratuberculosis  infection. A similar colonization pattern was observed for all examined organs (spleen, liver, intestine) at 12 WPC where the level of bacterial reduction reached &gt;1 log. 
     For histological examination, we focused our efforts on the liver because it is the most reflective organ for  M. avium  subsp.  paratuberculosis  infection (34). Liver sections from ΔsigL immunized animals displayed lower granulomatous scores and smaller size granulomas than the PBS-control group at 6 and 12 WPC ( FIG.  7 A-B ). In addition, low numbers of acid-fast bacilli were observed when liver sections were stained with Ziehl-Neelsen stain, another support for colonization data discussed above. Interestingly, sections from the intestines of the ΔsigL-immunized mice appeared normal compared to mock infected mice with no detectable acid-fast bacteria in Ziehl-Neelsen stained sections at both at 6 and 12 WPC ( FIG.  7 C-D ). Overall, reduction in  M. avium  subsp.  paratuberculosis  colonization levels combined with histological scores indicated the ability of the ΔsigL mutant to control tissue damage by a challenge with the virulent strain of  M. avium  subsp.  paratuberculosis.    
     Expansion of Immune Responses Following Challenge in Immunized Mice. 
     To evaluate expansion of the cellular immune response following challenge, splenocytes of immunized and challenged mice were analyzed for the production of key cytokines associated with protection against  paratuberculosis  (35,36). As shown in  FIG.  6 D , PPD-stimulated splenocytes from ΔsigL-immunized and challenged mice secreted significantly higher levels of IFN-γ than that of control animals at 6WPC, indicating increased levels of T cell activity (T-helper 1 cells) in the animals that received ΔsigL mutant. However, at 12 WPC there was no significant difference in IFN-γ response between these two groups of animals. Our data also showed a better ability, even though not significant, of the  M. avium  subsp.  paratuberculosis  ΔsigL vaccinated animals to induce PPD-specific IL-17A secretion compared to the mock challenged group at 6WPC. Taken together, the colonization, histological and immune response levels suggested that  M. avium  subsp.  paratuberculosis  ΔsigL induced protective immunity against challenge with virulent  M. avium  subsp.  paratuberculosis  strain. 
     Discussion 
     Infection with  M. avium  subsp.  paratuberculosis  represent a major threat (Johne&#39;s disease) to dairy animals (11,14) with the potential spread to human with the likely involvement in several diseases (7-10,12). Earlier reports indicated that  M. avium  subsp.  paratuberculosis  count on a large number of sigma factors (N=19) to establish the infection and survive diverse stress conditions (19,37). In this study, we have targeted sigL because of its activation during early macrophage infection suggesting a role to control important stage(s) of  M. avium  subsp.  paratuberculosis  pathogenesis following oral infection (19). Moreover, the orthologous sigL deletion mutant,  M. tuberculosis  ΔsigL, in  M. tuberculosis  was less lethal for mice relative to the wild-type strain (32). However, unlike  M. tuberculosis  ΔsigL (24), our analysis indicated that deletion of sigL affected ability of  M. avium  subsp.  paratuberculosis  to survive exposure to intracellular stimuli including oxidative stress and damaging the mycobacterial cell wall stresses (e.g. diamide and SDS) (38-40). This mutant was also unable to survive both naïve and activated macrophages to similar levels, an indication of the significant attenuation of this mutant. This intracellular survival defect of the mutant was further verified by the poor ability of  M. avium  subsp.  paratuberculosis  ΔsigL to replicate in mouse tissues. Both histological and bacteriological analyses revealed reduced organ colonization of the  M. avium  subsp.  paratuberculosis  ΔsigL with low inflammatory scores compared to the parental strain. Interestingly, this result was in contrast to the reports where orthologous  M. tuberculosis  ΔsigL survived in the mice organs to the same level as the wild-type strain suggesting a more comprehensive and dynamic role for sigL in  M. avium  subsp.  paratuberculosis  survival and pathogenesis (24,32). However, the nature and mechanisms employed by sigL to enable  M. avium  subsp.  paratuberculosis  virulence remain elusive. 
     The alternative sigma factor (e.g. sigE) mutant strains were targeted for the vaccine development and found to provide protection against infection with pathogenic bacteria including mycobacteria (41,42). In our study, observations gained from both in vitro and murine model experiments encouraged us to investigate the vaccine potential of  M. avium  subsp.  paratuberculosis  ΔsigL as a live attenuated vaccine against  M. avium  subsp.  paratuberculosis  infection in mice. The murine model of  paratuberculosis  represent an important screening tool (43,44) to examine vaccine candidates before testing in a more expensive goat model, despite the associated shortcomings of the murine model (no diarrhea or shedding of bacteria). In our hands, mice that received  M. avium  subsp.  paratuberculosis  ΔsigL were very efficient in producing IFN-γ (e.g. 6WPI), an important cytokine involved in controlling mycobacterial infection (45). The decline in IFN-γ is often associated with the onset of clinical JD in ruminants (35,46). Importantly, culturing tissue samples from immunized animals indicated the ability of  M. avium  subsp.  paratuberculosis  ΔsigL to persist in animals following immunization but to a low level which could be a critical factor in inducing protective immune responses. 
     Controlling  M. avium  subsp.  paratuberculosis  infection is dependent on developing vaccines that reduce bacterial colonization and shedding from infected animals. Earlier studies demonstrated the potential use of  M. avium  subsp.  paratuberculosis  mutants (e.g. WAg915,  M. avium  subsp.  paratuberculosis  ΔleuD) as live attenuated vaccine candidates in the murine model of  paratuberculosis  (44,47). WAg915 mutant strain ( M. avium  subsp.  paratuberculosis  ΔppiA), defective in the peptidyl-prolyl cis-trans-isomerase, showed mild attenuated phenotype relative to the wild-type strain and provided limited protection in mouse organ only at later stages after challenge with parental  M. avium  subsp.  paratuberculosis  strain (47), whereas the attenuated  M. avium  subsp.  paratuberculosis  ΔleuD, defective in leucine biosynthesis, exhibited some protection following challenge (44). In our experiment,  M. avium  subsp.  paratuberculosis  ΔsigL was attenuated in macrophages and in mice but persisted in murine tissues up to the time of challenge. Ability to persist following immunization could be responsible for the generated protective immunity, which was more efficient to reduce  M. avium  subsp.  paratuberculosis  colonization compared to the Wag915 candidate (47). However, it is not clear whether  M. avium  subsp.  paratuberculosis  ΔsigL could provide better protection compared to the  M. avium  subsp.  paratuberculosis  ΔleuD because the previous study examined  M. avium  subsp.  paratuberculosis  bacilli in mouse tissues by Ziehl-Neelsen staining which is less sensitive compared to the tissue culturing (48,49). We further evaluated longevity of immune responses in the mouse groups following challenge and these data suggest that  M. avium  subsp.  paratuberculosis  ΔsigL immunized mice maintained strong T-cell responses with secretion of higher IFN-γ and IL-17 at 6WPC, despite the later not being significant compared to the mock infected group. 
     Overall, an isogenic mutant of  M. avium  subsp.  paratuberculosis  lacking sigL had limited ability to survive in macrophages or mice, most likely because of a defective bacterial cell wall. Such an attenuated strain of  M. avium  subsp.  paratuberculosis  (ΔsigL) persisted in murine tissue following subcutaneous immunization and generated a robust immune response. The generated immune responses were sufficient to reduce tissue colonization and lesion scores in animals following a challenge with the wild-type strain of  M. avium  subsp.  paratuberculosis . Further vaccine testing in natural hosts of Johne&#39;s disease, e.g. goats or calves, will demonstrate the viability of developing an effective control strategy against  paratuberculosis  in both animals and humans. In general, approaches used to investigate sigL in  M. avium  subsp.  paratuberculosis  could be adopted to examine the role and potential use of other global gene regulators in pathogenesis and control of intracellular pathogens. 
     Example 3 
     References cited in this Example are listed in the section of References as “References cited in Example 3.” 
     Survival of GGR Mutants in the Murine Model of  paratuberculosis.    
     To assess the role of sigH and sigL in  M. ap  virulence, we investigated the persistence of the each mutant in mice. The initial growth kinetic of the wild-type and ΔsigH mutant strains was similar, with an equal burden of bacteria in liver, intestine and spleen up to 6 WPI ( FIG.  8   ). However, at 12 WPI, colonization levels were significantly reduced for ΔsigH compared to its parental strain only in spleen and intestine (p&lt;0.01 and p&lt;0.05, respectively), suggesting a role for sigH in the long term survival of  M. ap  in mice. For the ΔsigL mutant, bacterial colonization levels were significantly reduced in spleen, liver and intestine at all times post infection ( FIG.  8   ) compared to K10, suggesting a pivotal role for sigL in the pathogenesis of  M. ap.    
     Interestingly, when the ΔsigH mutant of  M. tuberculosis  was used to challenge mice, no differences in bacterial load were observed in mouse organs compared to the wild-type strain (54), suggesting a more significant role for sigH in  M. ap . Consistent with the colonization data, Ziehl-Neelsen staining of spleen showed higher acid fast bacilli in mice infected with wild-type strain compared to ΔsigH mutant at 12 WPI ( FIG.  9   ). 
     Taken together, our data indicated that both ΔsigH and ΔsigL mutants were attenuated in the murine model of  paratuberculosis  compared to the wild-type  M. ap  strain. 
     RNA Sequencing for Transcriptional Profiling. 
     During the last decade, it is evident that the use of DNA microarrays has greatly contributed to our understanding of the genetic basis of bacterial infections, including infection with  M. ap  (42,55-58). Recently, large scale sequencing approaches were developed for gene expression profiling to overcome problems associated with DNA microarrays (59). The use of Next-generation sequencers enabled us to sequence all transcripts in a given RNA sample, hence named RNA sequence (RNA-Seq) profiling. This naming reflects the ability of the high throughput sequencers (e.g. HighSeq2000, Roche 454) to be used for in-depth sequencing of millions of transcripts in a single run (59-61). The RNA-Seq approach has already been successfully applied in several systems, both mammalian and bacterial, (61) including  M. tuberculosis  and  M. ap  in our laboratory (62) (see below). Some key benefits of RNA-Seq over microarrays include less biased transcriptome data acquisition, actual sequences for transcripts that can show SNPs and antisense transcription, less complex data analysis; improved correlation between laboratories and increased sensitivity of low transcript numbers (63-65). 
     Because of the global nature of transcriptional regulation under control of a factors, we pursued the characterization of the sigH transcriptome using RNA-Seq profiling utilizing an Illumina-based sequencer operated by the University of Wisconsin Biotechnology Center (UWBC). Our stress experiments showed that the ΔsigH mutant was hypersensitive to elevated temperature and diamide exposure, each resulting in impaired growth. Accordingly, we hypothesize that sigH may play an important role in directing transcriptional control under unfavorable environmental conditions. To test this hypothesis, both wild-type  M. ap  K10 and ΔsigH mutant transcriptomes were profiled before and after diamide exposure. Briefly, cultures grown to mid-log phase (OD 600 =0.5) from both K-10 and its isogenic mutant, ΔsigH, were centrifuged (3,200×g) for 15 min at 4° C. for total RNA extraction as outlined before (32). Extracted RNA samples were treated with DNAse I (Ambion) to eliminate residual genomic DNA and then treated with MICROBExpress (Ambion, Austin) to enrich bacterial mRNA and reduce the amount of rRNA. Following a standard protocol for cDNA generation in our laboratory (42), all samples were sent to the UWBC for library construction and sequencing on the Illumina/GAIIx sequencing platform using 100-bp, paired end fragments. As a testament of the high resolution analysis of RNA-Seq profiling, we were not able to detect the presence of only 18 or 20 genes in both samples from  M. ap  K10 or ΔsigH, respectively (Table 2). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Sequencing run statistics on M. ap samples. 
               
            
           
           
               
               
               
            
               
                   
                 M. ap K10 
                 M. ap K10 ΔsigH 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Material Submitted 
                 2 μg mRNA 
                 2 μg mRNA 
               
               
                 Total Reads 
                 74, 747, 582 
                 53, 305, 460 
               
               
                 Read Length 
                 100 bp 
                 100 bp 
               
               
                 Total Reads Mapped 
                 57, 477, 920 
                 41, 101, 069 
               
               
                 Reads mapped in pairs 
                 53, 359, 286 
                 36, 887, 226 
               
               
                 Reads mapped to rRNA 
                 54, 209, 373 
                 37, 081, 401 
               
               
                 or tRNA 
                   
                   
               
               
                 Reads not mapped 
                 16, 903, 464 
                 11, 874, 445 
               
               
                 Most sequenced mRNA 
                 MAP 1975 
                 MAP 1975 
               
               
                 (reads) 
                 (1, 286, 560) 
                 (1, 390, 707) 
               
               
                 Transcripts not 
                 18         
                 20         
               
               
                 detected 
               
               
                   
               
            
           
         
       
     
     Examine the Vaccine Potential of GGR  M. ap  Mutants. 
     Because they control a large number of genes, we hypothesize that GGR mutants will be attenuated but able generate enough immune responses to serve as live attenuated vaccines that could control JD. To examine the vaccine potential of ΔsigH and ΔsigL mutants as live-attenuated vaccines, C57BL/6 mice groups (N=11) were vaccinated twice at 2 weeks interval with 10 6  CFU/mouse via subcutaneous (S/C.) injection. This route of immunization was chosen after evaluating other routes (oral and intra-peritoneal, I/P.) and was found to yield better immunity (data not shown). In addition, immunized mice via the S/C. showed no  M. ap  colonization in their organs when examined at 6 weeks post immunization (WPI). At 6 WPI, immunized mice (N=8/group) were challenged with 2×10 8  CFU/mouse of the wild-type strain  M. ap  K10 via I/P. Mock-immunized mice with (Phosphate Buffered Saline, PBS) showed the highest colonization levels of  M. ap  in their organs following challenge with  M. ap  K10. On the other hand, mice vaccinated with the attenuated mutants (ΔsigH and ΔsigL) showed a significant reduction in bacterial colony levels in the intestine at 6 weeks post challenge (WPC) ( FIG.  10   ). Only ΔsigL-vaccinated group showed significant reduction in both liver and spleen at this time point. By 12 WPC, only ΔsigL-vaccinated group showed more than 1 log reduction in intestine in all organs (intestine, liver, spleen) compared to the control group ( FIG.  10   ). Overall, ΔsigL immunization was more effective in inducing protection against virulent  M. ap  K10 strain compared to ΔsigH vaccination. 
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     REFERENCES CITED IN EXAMPLE 1 
     
         
         1. Ghosh, P., C. Hsu, E. J. Alyamani, M. M. Shehata, M. A. Al-Dubaib, A. Al-Naeem, M. Hashad, O. M. Mahmoud, K. B. Alharbi, K. Al-Busadah, A. M. Al-Swailem, and A. M. Talaat. 2012. Genome-wide analysis of the emerging infection with  Mycobacterium avium  subspecies  paratuberculosis  in the Arabian camels ( Camelus dromedarius ). PLoS. One. 7:e31947. 
         2. Yalo Ayele, W., M. Machackova, and I. Pavlik. 2001. The transmission and impact of  paratuberculosis  infection in domestic and wild ruminants. Vet. Med. 46:205-224. 
         3. Nielsen, S. r. S. and N. Toft. 2009. A review of prevalences of  paratuberculosis  in farmed animals in Europe. Preventive Veterinary Medicine 88:1-14. 
         4. Lombard, J. E. 2011. Epidemiology and economics of  paratuberculosis . Vet. Clin. North Am. Food Anim Pract. 27:525-35. 
         5. Lombard, J. E., I. A. Gardner, S. R. Jafarzadeh, C. P. Fossler, B. Harris, R. T. Capsel, B. A. Wagner, and W. O. Johnson. 2013. Herd-level prevalence of  Mycobacterium avium  subsp.  paratuberculosis  infection in United States dairy herds in 2007. Prev. Vet. Med. 108:234-238. 
         6. Ott, S. L., S. J. Wells, and B. A. Wagner. 1999. Herd-level economic losses associated with Johne&#39;s disease on US dairy operations. Prev. Vet. Med. 40:179-192. 
         7. Bermudez, L. E., M. Petrofsky, S. Sommer, and R. G. Barletta. 2010. Peyer&#39;s patch-deficient mice demonstrate that  Mycobacterium avium  subsp.  paratuberculosis  translocates across the mucosal barrier via both M cells and enterocytes but has inefficient dissemination. Infect. Immun. 78:3570-3577. 
         8. Chacon, O., L. E. Bermudez, and R. G. Barletta. 2004. Johne&#39;s disease, inflammatory bowel disease, and  Mycobacterium paratuberculosis . Annu. Rev. Microbiol. 58:329-363. 
         9. Coussens, P. M., C. J. Colvin, K. Wiersma, A. Abouzied, and S. Sipkovsky. 2002. Gene expression profiling of peripheral blood mononuclear cells from cattle infected with  Mycobacterium paratuberculosis . Infect. Immun. 70:5494-5502. 
         10. Coussens, P. M., A. Jeffers, and C. Colvin. 2004. Rapid and transient activation of gene expression in peripheral blood mononuclear cells from Johne&#39;s disease positive cows exposed to  Mycobacterium paratuberculosis  in vitro. Microbial. pathogenesis. 36:93-108. 
         11. Weiss, D. J., O. A. Evanson, M. Deng, and M. S. Abrahamsen. 2004. Gene expression and antimicrobial activity of bovine macrophages in response to  Mycobacterium avium  subsp.  paratuberculosis . Vet. Pathol. 41:326-337. 
         12. Wu, C. W., S. K. Schmoller, S. J. Shin, and A. M. Talaat. 2007. Defining the stressome of  Mycobacterium avium  subsp  paratuberculosis  in vitro and in naturally infected cows. Journal of Bacteriology 189:7877-7886. 
         13. Whittington, R. J., D. J. Marshall, P. J. Nicholls, I. B. Marsh, and L. A. Reddacliff. 2004. Survival and dormancy of  Mycobacterium avium  subsp.  paratuberculosis  in the environment. Appl. Environ. Microbiol. 70:2989-3004. 
         14. Bannantine, J. P. and J. R. Stabel. 2002. Killing of  Mycobacterium avium  subspecies  paratuberculosis  within macrophages. BMC Microbiol. 2:2. 
         15. Stabel, J. R., M. V. Palmer, B. Harris, B. Plattner, J. Hostetter, and S. Robbe-Austerman. 2009. Pathogenesis of  Mycobacterium avium  subsp.  paratuberculosis  in neonatal calves after oral or intraperitoneal experimental infection. Vet. Microbiol. 136:306-313. 
         16. Wu, C. W., M. Livesey, S. K. Schmoller, E. J. Manning, H. Steinberg, W. C. Davis, M. J. Hamilton, and A. M. Talaat. 2007. Invasion and persistence of  Mycobacterium avium  subsp.  paratuberculosis  during early stages of Johne&#39;s disease in calves. Infect. Immun. 75:2110-2119. 
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