bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Differentially Expressed Genes in Virulent and NonvirulentEntamoebaSpecies: Potential Implications for Amebic Pathogenesis
Authors: MacFarlane Ryan C. , Singh Upinder
Published: December 2005

Entamoeba histolyticais a protozoan parasite that causes colitis and liver abscesses. SeveralEntamoebaspecies and strains with differing levels of virulence have been identified.E. histolyticaHM-1:IMSS is a virulent strain,E. histolyticaRahman is a nonvirulent strain, andEntamoeba disparis a nonvirulent species. We used anE. histolyticaDNA microarray consisting of 2,110 genes to assess the transcriptional differences between these species/strains with the goal of identifying genes whose expression correlated with a virulence phenotype. We found 415 genes expressed at lower levels inE. disparand 32 genes with lower expression inE. histolyticaRahman than inE. histolyticaHM-1:IMSS. Overall, 29 genes had decreased expression in both the nonvirulent species/strains than the virulentE. histolyticaHM-1:IMSS. Interestingly, a number of genes with potential roles in stress response and virulence had decreased expression in either one or both nonvirulentEntamoebaspecies/strains. These included genes encoding Fe hydrogenase (9.m00419), peroxiredoxin (176.m00112), type A flavoprotein (6.m00467), lysozyme (6.m00454), sphingomyelinase C (29.m00231), and a hypothetical protein with homology to both aPlasmodiumsporozoite threonine-asparagine-rich protein (STARP) and a streptococcal hemagglutinin (238.m00054). The function of these genes inEntamoebaand their specific roles in parasite virulence need to be determined. We also found that a number of the non-long-terminal-repeat retrotransposons (EhLINEs andEhSINEs), which have been shown to modulate gene expression and genomic evolution, had lower expression in the nonvirulent species/strains than inE. histolyticaHM-1:IMSS. Our results, identifying expression profiles and patterns indicative of a virulence phenotype, may be useful in characterizing the transcriptional framework of virulence.
Human Recombinant Antimannan Immunoglobulin G1 Antibody Confers Resistance to Hematogenously Disseminated Candidiasis in Mice
Authors: Zhang Mason X. , Bohlman M. Charlotte , Itatani Carol , Burton Dennis R. , Parren Paul W. H. I. , St. Jeor Stephen C. , Kozel Thomas R.
Published: December 2005

Mannan is a major cell wall component found in
Candida
species. Natural antimannan antibody is present in sera from most normal adults, but its role in host resistance to hematogenously disseminated candidiasis is unknown. The purpose of this study was to develop recombinant human antimannan antibody and to study its protective function. A phage Fab display combinatorial library containing Fab genes from bone marrow lymphocytes was screened with
Candida albicans
yeast cells and chemically purified mannan. One antimannan Fab, termed M1, was converted to a full-length immunoglobulin G1 antibody, M1g1, and M1g1 was produced in CHO cells. The M1g1 epitope was found in
C. albicans
serotypes A and B,
Candida tropicalis
,
Candida guilliermondii
,
Candida glabrata
, and
Candida parapsilosis
. Its expression was active at both 23°C and 37°C and uniform over the cell surface. BALB/c mice passively immunized with M1g1 were more resistant than control mice to a lethal hematogenous infection by
C. albicans
, as evidenced by extension of survival in an M1g1 dose-dependent manner (
P
, 0.08 to <0.001) and by reduction in number of infection foci and their size in the kidney. In vitro studies found that M1g1 promoted phagocytosis and phagocytic killing of
C. albicans
yeast cells by mouse peritoneal macrophages and was required for activation of the mouse complement cascade. Thus, human antimannan antibody may have a protective role in host resistance to systemic candidiasis.
Glyceraldehyde-3-Phosphate Dehydrogenase ofParacoccidioides brasiliensisIs a Cell Surface Protein Involved in Fungal Adhesion to Extracellular Matrix Proteins and Interaction with Cells
Authors: Barbosa Mônica Santiago , Báo Sônia Nair , Andreotti Patrícia Ferrari , de Faria Fabrícia P. , Felipe Maria Sueli S. , dos Santos Feitosa Luciano , Mendes-Giannini Maria José Soares , de Almeida Soares Célia Maria
Published: December 2005

The pathogenic fungusParacoccidioides brasiliensiscauses paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ofP. brasiliensisas an adhesin, which can be related to fungus adhesion and invasion. TheP. brasiliensisGAPDH was overexpressed inEscherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase ofP. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. Of special note, the treatment ofP. brasiliensisyeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization ofP. brasiliensisto those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH inP. brasiliensiscould be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.
Immunological Correlates for Protection against Intranasal Challenge of
Bacillus anthracis
Spores Conferred by a Protective Antigen-Based Vaccine in Rabbits
Authors: Weiss Shay , Kobiler David , Levy Haim , Marcus Hadar , Pass Avi , Rothschild Nili , Altboum Zeev
Published: December 2005

Correlates between immunological parameters and protection against
Bacillus anthracis
infection in animals vaccinated with protective antigen (PA)-based vaccines could provide surrogate markers to evaluate the putative protective efficiency of immunization in humans. In previous studies we demonstrated that neutralizing antibody levels serve as correlates for protection in guinea pigs (S. Reuveny et al., Infect. Immun. 69:2888-2893, 2001; H. Marcus et al., Infect. Immun. 72:3471-3477, 2004). In this study we evaluated similar correlates for protection by active and passive immunization of New Zealand White rabbits. Full immunization and partial immunization were achieved by single and multiple injections of standard and diluted doses of a PA-based vaccine. Passive immunization was carried out by injection of immune sera from rabbits vaccinated with PA-based vaccine prior to challenge with
B. anthracis
spores. Immunized rabbits were challenged by intranasal spore instillation with one of two virulent strains (strains Vollum and ATCC 6605). The immune competence was estimated by measuring the level of total anti-PA antibodies, the neutralizing antibody titers, and the conferred protective immunity. The results indicate that total anti-PA antibody titers greater than 1 × 10
5
conferred protection, whereas lower titers (between 10
4
and 10
5
) provided partial protection but failed to predict protection. Neutralizing antibody titers between 500 and 800 provided partial protection, while titers higher than 1,000 conferred protection. In conclusion, this study emphasizes that regardless of the immunization regimen or the time of challenge, neutralizing antibody titers are better predictors of protection than total anti-PA titers.
Role of Hyaluronidase in Subcutaneous Spread and Growth of Group A Streptococcus
Authors: Rivera Starr Clarise , Engleberg N. Cary
Published: December 2005

Group A streptococcus (GAS) depends on a hyaluronic acid (HA) capsule to evade phagocytosis and to interact with epithelial cells. Paradoxically, GAS also produces hyaluronidase (Hyl), an enzyme that cleaves HA. A common assumption is that Hyl digests structurally identical HA in human tissue to promote bacterial spread. We inactivated the gene encoding extracellular hyaluronidase,
hylA
, in a clinical Hyl
+
isolate. Hyl
+
and an isogenic Hyl
−
mutant were injected subcutaneously into mice with or without high-molecular-weight dextran blue. The Hyl
−
strain produced small lesions with dye concentrated in close proximity. The Hyl
+
strain produced identical lesions, but the dye diffused subcutaneously. However, Hyl
+
bacteria were not isolated from unaffected skin stained by dye diffusion. Thus, Hyl digests tissue HA and facilitates spread of large molecules but is not sufficient to cause subcutaneous diffusion of bacteria or to affect lesion size. GAS capsule expression was assayed periodically during broth culture and was reduced in Hyl
+
strains relative to Hyl
−
strains at the onset and the end of active capsule synthesis but not during peak synthesis in mid-exponential phase. Thus, Hyl is not sufficiently active to remove capsule during peak synthesis. To demonstrate a possible nutritional role for Hyl, GAS was shown to grow with N-acetylglucosamine but not
d
-glucuronic acid (both components of HA) as a sole carbon source. However, only Hyl
+
strains could grow utilizing HA as a sole carbon source, suggesting that Hyl may permit the organism to utilize host HA or its own capsule as an energy source.
DNA Adenine Methyltransferase Influences the Virulence of
Aeromonas hydrophila
Authors: Erova Tatiana E. , Pillai Lakshmi , Fadl Amin A. , Sha Jian , Wang Shaofei , Galindo Cristi L. , Chopra Ashok K.
Published: December 2005

Among the various virulence factors produced by
Aeromonas hydrophila
, a type II secretion system (T2SS)-secreted cytotoxic enterotoxin (Act) and the T3SS are crucial in the pathogenesis of
Aeromonas
-associated infections. Our laboratory molecularly characterized both Act and the T3SS from a diarrheal isolate, SSU of
A. hydrophila
, and defined the role of some regulatory genes in modulating the biological effects of Act. In this study, we cloned, sequenced, and expressed the DNA adenine methyltransferase gene of
A. hydrophila
SSU (
dam
AhSSU
) in a T7 promoter-based vector system using
Escherichia coli
ER2566 as a host strain, which could alter the virulence potential of
A. hydrophila
. Recombinant Dam, designated as M.AhySSUDam, was produced as a histidine-tagged fusion protein and purified from an
E. coli
cell lysate using nickel affinity chromatography. The purified Dam had methyltransferase activity, based on its ability to transfer a methyl group from
S
-adenosyl-
l
-methionine to N
6
-methyladenine-free lambda DNA and to protect methylated lambda DNA from digestion with DpnII but not against the DpnI restriction enzyme. The
dam
gene was essential for the viability of the bacterium, and overproduction of Dam in
A. hydrophila
SSU, using an arabinose-inducible, P
BAD
promoter-based system, reduced the virulence of this pathogen. Specifically, overproduction of M.AhySSUDam decreased the motility of the bacterium by 58%. Likewise, the T3SS-associated cytotoxicity, as measured by the release of lactate dehydrogenase enzyme in murine macrophages infected with the Dam-overproducing strain, was diminished by 55% compared to that of a control
A. hydrophila
SSU strain harboring the pBAD vector alone. On the contrary, cytotoxic and hemolytic activities associated with Act as well as the protease activity in the culture supernatant of a Dam-overproducing strain were increased by 10-, 3-, and 2.4-fold, respectively, compared to those of the control
A. hydrophila
SSU strain. The Dam-overproducing strain was not lethal to mice (100% survival) when given by the intraperitoneal route at a dose twice that of the 50% lethal dose, which within 2 to 3 days killed 100% of the animals inoculated with the
A. hydrophila
control strain. Taken together, our data indicated alteration of
A. hydrophila
virulence by overproduction of Dam.
GroEL ofLactobacillus johnsoniiLa1 (NCC 533) Is Cell Surface Associated: Potential Role in Interactions with the Host and the Gastric PathogenHelicobacter pylori
Authors: Bergonzelli Gabriela E. , Granato Dominique , Pridmore Raymond D. , Marvin-Guy Laure F. , Donnicola Dominique , Corthésy-Theulaz Irène E.
Published: December 2005

Heat shock proteins of the GroEL or Hsp60 class are highly conserved proteins essential to all living organisms. Even though GroEL proteins are classically considered intracellular proteins, they have been found at the surface of several mucosal pathogens and have been implicated in cell attachment and immune modulation. The purpose of the present study was to investigate the GroEL protein of a gram-positive probiotic bacterium,Lactobacillus johnsoniiLa1 (NCC 533). Its presence at the bacterial surface was demonstrated using a whole-cell enzyme-linked immunosorbent assay and could be detected in bacterial spent culture medium by immunoblotting. To assess binding of La1 GroEL to mucins and intestinal epithelial cells, the La1 GroEL protein was expressed inEscherichia coli. We report here that La1 recombinant GroEL (rGroEL) binds to mucins and epithelial cells and that this binding is pH dependent. Immunomodulation studies showed that La1 rGroEL stimulates interleukin-8 secretion in macrophages and HT29 cells in a CD14-dependent mechanism. This property is common to rGroEL from other gram-positive bacteria but not to the rGroEL of the gastric pathogenHelicobacter pylori. In addition, La1 rGroEL mediates the aggregation ofH. pyloribut not that of other intestinal pathogens. Our in vitro results suggest that GroEL proteins from La1 and other lactic acid bacteria might play a role in gastrointestinal homeostasis due to their ability to bind to components of the gastrointestinal mucosa and to aggregateH. pylori.
Type IV Pili ofNeisseria gonorrhoeaeInfluence the Activation of Human CD4+T Cells
Authors: Plant Laura J. , Jonsson Ann-Beth
Published: December 2005

Neisseria gonorrhoeaeis the causative agent of the sexually transmitted disease gonorrhea, and infection with this organism is typically associated with an intense inflammatory response. In many individuals, however, the infection is asymptomatic and can progress to serious secondary complications. The type IV pili ofNeisseria gonorrhoeaemediate binding of the bacteria to host cells and are involved in cellular signal transduction. In these studies we have demonstrated that gonococcal pili influence human CD4+T cells by using isogenic strains ofN. gonorrhoeaewith piliated and nonpiliated phenotypes. To determine the impact of piliation on the cellular status, we examined the expression of activation markers, cellular proliferation, and the production of cytokines after infection. The activation marker CD69 showed significantly increased expression on cells infected with the piliated strain, and this expression was dependent on costimulation of the T-cell receptor. Infection with piliated gonococci also altered T-cell proliferation and influenced the production of the regulatory cytokine interleukin-10. PilC, the putative pilus adhesin, was also observed to influence cellular activation but had no impact on the proliferation of cells further indicating that pilus-mediated adhesion is important in gonococcal stimulation of CD4+T cells. These results show that the piliation status of gonococci influences CD4+T-cell activation and that the adhesion mediated by pilus components aids in the regulation of the T-cell response toN. gonorrhoeae.
Identification and Characterization of the Capsular Polysaccharide (K-Antigen) Locus of
Porphyromonas gingivalis
Authors: Aduse-Opoku Joseph , Slaney Jennifer M. , Hashim Ahmed , Gallagher Alexandra , Gallagher Robert P. , Rangarajan Minnie , Boutaga Khalil , Laine Marja L. , Van Winkelhoff Arie J. , Curtis Michael A.
Published: December 2005

Capsular polysaccharides of gram-negative bacteria play an important role in maintaining the structural integrity of the cell in hostile environments and, because of their diversity within a given species, can act as useful taxonomic aids. In order to characterize the genetic locus for capsule biosynthesis in the oral gram-negative bacterium
Porphyromonas gingivalis
, we analyzed the genome of
P. gingivalis
W83 which revealed two candidate loci at PG0106
-
PG0120 and PG1135-PG1142 with sufficient coding capacity and appropriate gene functions based on comparisons with capsule-coding loci in other bacteria. Insertion and deletion mutants were prepared at PG0106-PG0120 in
P. gingivalis
W50—a K1 serotype. Deletion of PG0109-PG0118 and PG0116-PG0120 both yielded mutants which no longer reacted with antisera to K1 serotypes. Restriction fragment length polymorphism analysis of the locus in strains representing all six K-antigen serotypes and K
−
strains demonstrated significant variation between serotypes and limited conservation within serotypes. In contrast, PG1135-PG1142 was highly conserved in this collection of strains. Sequence analysis of the capsule locus in strain 381 (K
−
strain) demonstrated synteny with the W83 locus but also significant differences including replacement of PG0109-PG0110 with three unique open reading frames, deletion of PG0112-PG0114, and an internal termination codon within PG0106, each of which could contribute to the absence of capsule expression in this strain. Analysis of the Arg-gingipains in the capsule mutants of strain W50 revealed no significant changes to the glycan modifications of these enzymes, which indicates that the glycosylation apparatus in
P. gingivalis
is independent of the capsule biosynthetic machinery.
Role of theluxSQuorum-Sensing System in Biofilm Formation and Virulence ofStaphylococcus epidermidis
Authors: Xu Lin , Li Hualin , Vuong Cuong , Vadyvaloo Viveka , Wang Jianping , Yao Yufeng , Otto Michael , Gao Qian
Published: December 2005

Nosocomial infections caused byStaphylococcus epidermidisare characterized by biofilm formation on implanted medical devices. Quorum-sensing regulation plays a major role in the biofilm development of many bacterial pathogens. Here, we describeluxS, a quorum-sensing system in staphylococci that has a significant impact on biofilm development and virulence. We constructed an isogenicΔluxSmutant strain of a biofilm-forming clinical isolate ofS. epidermidisand demonstrated thatluxSsignaling is functional inS. epidermidis. The mutant strain showed increased biofilm formation in vitro and enhanced virulence in a rat model of biofilm-associated infection. Genetic complementation and addition of autoinducer 2-containing culture filtrate restored the wild-type phenotype, demonstrating thatluxSrepressed biofilm formation through a cell-cell signaling mechanism based on autoinducer 2 secretion. Enhanced production of the biofilm exopolysaccharide polysaccharide intercellular adhesin in the mutant strain is presumably the major cause of the observed phenotype. Theagrquorum-sensing system has previously been shown to impact biofilm development and biofilm-associated infection in a way similar to that ofluxS, although by regulation of different factors. Our study indicates a general scheme of quorum-sensing regulation of biofilm development in staphylococci, which contrasts with that observed in many other bacterial pathogens.
The Cell-Specific Phenotype of the Polymorphic
vacA
Midregion Is Independent of the Appearance of the Cell Surface Receptor Protein Tyrosine Phosphatase β
Authors: Skibinski David A. G. , Genisset Christophe , Barone Silvia , Telford John L.
Published: December 2005

There are two alleles, m1 and m2, of the midregion of the vacuolating cytotoxin gene (
vacA
) of
Helicobacter pylori
which code for toxins with different cell specificities. Here we describe the construction of five chimeric strains in which regions of
vacA
were exchanged between the two genotypes. By analyzing the toxicity of these strains for HeLa and RK13 cells we have confirmed that a 148-amino-acid region determines the phenotypic differences between the two forms of the protein and that this entire region is important for cytotoxicity. Furthermore, we have used our chimeric strains to investigate whether variations in the midregion of VacA have an effect on phorbol 12-myristate 13-acetate (PMA)-induced VacA sensitivity in HL-60 cells. The PMA-induced VacA sensitivity of HL-60 cells has been previously associated with the appearance of the cell surface receptor protein tyrosine phosphatase beta (RPTPβ). Our data indicate that both the m1 and m2 forms of VacA are able to utilize RPTPβ, and the cell-specific phenotype of the midregion is independent of the presence of RPTPβ. It appears that another as-yet-unidentified receptor exists in HL-60 cells that accounts for the m2 phenotype in this cell line. Also, by studying the effect of PMA on levels of RPTPβ in other cell lines and toxicity of VacA in these cell lines we have shown that RPTPβ does not play a major role in the vacuolation of HeLa cells.
IdeS, a Highly Specific Immunoglobulin G (IgG)-Cleaving Enzyme from
Streptococcus pyogenes
, Is Inhibited by Specific IgG Antibodies Generated during Infection
Authors: Åkesson Per , Moritz Linnea , Truedsson Mikael , Christensson Bertil , von Pawel-Rammingen Ulrich
Published: December 2005

IdeS, a recently discovered cysteine proteinase secreted by the important human pathogen
Streptococcus pyogenes
, interferes with phagocytic killing by specifically cleaving the heavy chain of immunoglobulin G. The fact that the enzyme targets one of the key molecules of the adapted immune response raised the question of whether an antibody response against IdeS could inhibit, i.e., neutralize, enzyme activity. Paired acute- and convalescent-phase serum samples from patients with pharyngotonsillitis (
n
= 10), bacteremia (
n
= 7), and erysipelas (
n
= 4) were analyzed. Antibodies with the ability to neutralize IdeS enzymatic activity were already found in two-thirds of acute-phase sera. However, patients who seroconverted to IdeS, in particular patients with pharyngotonsillitis and erysipelas, developed specific antibodies during convalescence with an increased capability to efficiently neutralize the enzymatic activity of IdeS. Also, the presence of neutralizing antibodies decreased the ability of IdeS to mediate bacterial survival in human immune blood. In patients with bacteremia, several acute-phase sera contained neutralizing antibodies, but no correlation was found to severity or outcome of invasive infections. Still, the fact that the human immune response targets the enzymatic activity of IdeS supports the view that the enzyme plays an important role during streptococcal infection.
Urease Produced by
Coccidioides posadasii
Contributes to the Virulence of This Respiratory Pathogen
Authors: Mirbod-Donovan Fariba , Schaller Ruth , Hung Chiung-Yu , Xue Jianmin , Reichard Utz , Cole Garry T.
Published: December 2005

Urease activity during in vitro growth in the saprobic and parasitic phases of
Coccidioides
spp. is partly responsible for production of intracellular ammonia released into the culture media and contributes to alkalinity of the external microenvironment. Although the amino acid sequence of the urease of
Coccidioides posadasii
lacks a predicted signal peptide, the protein is transported from the cytosol into vesicles and the central vacuole of parasitic cells (spherules). Enzymatically active urease is released from the contents of mature spherules during the parasitic cycle endosporulation stage. The endospores, together with the urease and additional material which escape from the ruptured parasitic cells, elicit an intense host inflammatory response. Ammonia production by the spherules of
C. posadasii
is markedly increased by the availability of exogenous urea found in relatively high concentrations at sites of coccidioidal infection in the lungs of mice. Direct measurement of the pH at these infection sites revealed an alkaline microenvironment. Disruption of the urease gene of
C. posadasii
resulted in a marked reduction in the amount of ammonia secreted in vitro by the fungal cells. BALB/c mice challenged intranasally with the mutant strain showed increased survival, a well-organized granulomatous response to infection, and better clearance of the pathogen than animals challenged with either the parental or the reconstituted (revertant) strain. We conclude that ammonia and enzymatically active urease released from spherules during the parasitic cycle of
C. posadasii
contribute to host tissue damage, which exacerbates the severity of coccidioidal infection and enhances the virulence of this human respiratory pathogen.
A Recombinant Aspartyl Protease ofCoccidioides posadasiiInduces Protection against Pulmonary Coccidioidomycosis in Mice
Authors: Tarcha Eric J. , Basrur Venkatesha , Hung Chiung-Yu , Gardner Malcolm J. , Cole Garry T.
Published: December 2005

Coccidioidomycosis is a respiratory disease of humans caused by the desert soil-borne fungal pathogensCoccidioidesspp. Recurrent epidemics of this mycosis in the southwestern United States have contributed significantly to escalated health care costs. Clinical and experimental studies indicate that prior symptomatic coccidioidomycosis induces immunity against subsequent infection, and activation of T cells is essential for containment of the pathogen and its clearance from host tissue. Development of a human vaccine against coccidioidomycosis has focused on recombinant T-cell-reactive antigens which elicit a durable protective immune response against pulmonary infection in mice. In this study we fractionated a protective multicomponent parasitic cell wall extract in an attempt to identify T-cell antigens. Immunoblots of electrophoretic separations of this extract identified patient seroreactive proteins which were subsequently excised from two-dimensional polyacrylamide gel electrophoresis gels, trypsin digested, and sequenced by tandem mass spectrometry. The full-length gene which encodes a dominant protein in the immunoblot was identified using established methods of bioinformatics. The gene was cloned and expressed, and the recombinant protein was shown to stimulate immune T cells in vitro. The deduced protein was predicted to contain epitopes that bind to human major histocompatibility complex class II molecules using a TEPITOPE-based algorithm. Synthetic peptides corresponding to the predicted T-cell epitopes induced gamma interferon production by immune T lymphocytes. The T-cell-reactive antigen, which is homologous to secreted fungal aspartyl proteases, protected mice against pulmonary infection withCoccidioides posadasii. We argue that this immunoproteomic/bioinformatic approach to the identification of candidate vaccines against coccidioidomycosis is both efficient and productive.
The UDP
N
-Acetylgalactosamine 4-Epimerase Gene Is Essential for Mesophilic
Aeromonas hydrophila
Serotype O34 Virulence
Authors: Canals Rocío , Jiménez Natalia , Vilches Silvia , Regué Miguel , Merino Susana , Tomás Juan M.
Published: December 2005

Mesophilic
Aeromonas hydrophila
strains of serotype O34 typically express smooth lipopolysaccharide (LPS) on their surface. A single mutation in the gene that codes for UDP
N-
acetylgalactosamine 4-epimerase (
gne
) confers the O
−
phenotype (LPS without O-antigen molecules) on a strain in serotypes O18 and O34, but not in serotypes O1 and O2. The
gne
gene is present in all the mesophilic
Aeromonas
strains tested. No changes were observed for the LPS core in a
gne
mutant from
A. hydrophila
strain AH-3 (serotype O34). O34 antigen LPS contains
N-
acetylgalactosamine, while no such sugar residue forms part of the LPS core from
A. hydrophila
AH-3. Some of the pathogenic features of
A. hydrophila
AH-3
gne
mutants are drastically reduced (serum resistance or adhesion to Hep-2 cells), and the
gne
mutants are less virulent for fish and mice compared to the wild-type strain. Strain AH-3, like other mesophilic
Aeromonas
strains, possess two kinds of flagella, and the absence of O34 antigen molecules by
gne
mutation in this strain reduced motility without any effect on the biogenesis of both polar and lateral flagella. The reintroduction of the single wild-type
gne
gene in the corresponding mutants completely restored the wild-type phenotype (presence of smooth LPS) independently of the O wild-type serotype, restored the virulence of the wild-type strain, and restored motility (either swimming or swarming).
MyD88-Dependent Pathways Mediate Resistance toCryptosporidium parvumInfection in Mice
Authors: Rogers K. A. , Rogers A. B. , Leav B. A. , Sanchez A. , Vannier E. , Uematsu S. , Akira S. , Golenbock D. , Ward H. D.
Published: December 2005

Cryptosporidiumspp. cause diarrheal disease worldwide. Innate immune responses mediating resistance to this parasite are not completely understood. To determine whether MyD88-dependent pathways play a role in resistance toCryptosporidium parvum, we compared the course of infection in MyD88−/−mice to that in their wild-type (WT) littermate controls. Three- to 4-week-old mice were infected withC. parvum, and infection was monitored by quantifying fecal oocyst shedding. Twelve days postinfection, the histology of the intestines was examined to quantify intestinal parasite burden and to determine if there were any pathological changes. Fecal oocyst shedding and intestinal parasite burden were significantly greater in MyD88−/−mice than in littermate controls. Nonetheless, both WT and MyD88−/−mice cleared the infection within 3 weeks. These results indicate that MyD88-dependent pathways are involved in mediating initial resistance toC. parvum. Since gamma interferon (IFN-γ) is known to mediate resistance toC. parvum, we also studied infection in MyD88−/−mice and WT controls in which this cytokine was temporarily neutralized. Fecal oocyst shedding, as well as intestinal parasite burden, intestinal inflammation, and mortality, was significantly greater in MyD88−/−mice in which IFN-γ was neutralized than in IFN-γ-neutralized WT mice or in MyD88−/−mice in which this cytokine was active. These results suggest that MyD88 and IFN-γ had an additive effect in conferring protection fromC. parvuminfection. While this study confirms the importance of IFN-γ in conferring resistance to infection withC. parvum, it suggests that MyD88-mediated pathways also play a role in innate immunity to this parasite.
Iron and pH Homeostasis Intersect at the Level of Fur Regulation in the Gastric Pathogen
Helicobacter pylori
Authors: Gancz Hanan , Censini Stefano , Merrell D. Scott
Published: December 2005

Helicobacter pylori
persistently colonizes the stomach of the majority of the world's population and is a tremendous medical burden due to its causal role in diverse gastric maladies. Since the stomach is a constantly changing environment, successful colonization of
H. pylori
within this niche requires regulation of bacterial gene expression to cope with the environmental fluctuations. In
H. pylori
, the ferric uptake regulator (Fur) has been shown to play an intricate role in adaptation of the bacterium to two conditions known to oscillate within the gastric mucosa: iron limitation and low pH. To extend our knowledge of the process of regulation and adaptation in
H. pylori
, we show that Fur is required for efficient colonization of the Mongolian gerbil: the mutant strain exhibits a 100-fold increase in the 50% infectious dose, as well as a 100-fold defect in competitive colonization, when coinfected with wild-type bacteria. Furthermore, we used DNA microarrays to identify genes whose expression was altered in a Fur-deficient strain. We show that the Fur regulon of
H. pylori
consists of approximately 30 genes, most of which have been previously annotated as acid stress associated. Finally, we investigate the role of Fur in acid-responsive modulation of gene expression and show that a large number of genes are aberrantly expressed in the Fur mutant specifically upon acid exposure. This fact likely explains the requirement for this regulator for growth and colonization in the stomach.
Virulence of Broad- and Narrow-Host-RangeSalmonella entericaSerovars in the Streptomycin-PretreatedMouse Model
Authors: Suar Mrutyunjay , Jantsch Jonathan , Hapfelmeier Siegfried , Kremer Marcus , Stallmach Thomas , Barrow Paul A. , Hardt Wolf-Dietrich
Published: December 2005

Salmonella entericasubspecies I serovars are common bacterial pathogens causing diseases ranging from enterocolitis to systemic infections. Some serovars are adapted to specific hosts, whereas others have a broad host range. The molecular mechanisms defining the virulence characteristics and the host range of a givenS. entericaserovar are unknown. Streptomycin pretreated mice provide a surrogate host model for studying molecular aspects of the intestinal inflammation (colitis) caused by serovar Typhimurium (S. Hapfelmeier and W. D. Hardt, Trends Microbiol. 13:497-503, 2005). Here, we studied whether this animal model is also useful for studying otherS. entericasubspecies I serovars. All three tested strains of the broad-host-range serovar Enteritidis (125109, 5496/98, and 832/99) caused pronounced colitis and systemic infection in streptomycin pretreated mice. Different levels of virulence were observed among three tested strains of the host-adapted serovar Dublin (SARB13, SD2229, and SD3246). Several strains of host restricted serovars were also studied. Two serovar Pullorum strains (X3543 and 449/87) caused intermediate levels of colitis. No intestinal inflammation was observed upon infection with three different serovar Paratyphi A strains (SARB42, 2804/96, and 5314/98) and one serovar Gallinarum strain (X3796). A second serovar Gallinarum strain (287/91) was highly virulent and caused severe colitis. This strain awaits future analysis. In conclusion, the streptomycin pretreated mouse model can provide an additional tool to study virulence factors (i.e., those involved in enteropathogenesis) of variousS. entericasubspecies I serovars. Five of these strains (125109, 2229, 287/91, 449/87, and SARB42) are subject ofSalmonellagenome sequencing projects. The streptomycin pretreated mouse model may be useful for testing hypotheses derived from this genomic data.
The Global Regulator Ler Is Necessary for Enteropathogenic
Escherichia coli
Colonization of
Caenorhabditis elegans
Authors: Mellies Jay L. , Barron Alex M. S. , Haack Kenneth R. , Korson Andrew S. , Oldridge Derek A.
Published: December 2005

Enteropathogenic
Escherichia coli
(EPEC) is an important cause of infant diarrhea in developing countries and is useful for general investigations of the bacterial infection process. However, the study of the molecular pathogenesis of EPEC has been hampered by the lack of genetically tractable, convenient animal models. We have therefore developed the use of the nematode
Caenorhabditis elegans
as a small animal model of infection for this diarrheal pathogen. We found that nematodes died faster on nematode growth medium in the presence of EPEC pathogens than in the presence of the laboratory control strain MG1655. Increased numbers of pathogens in the gut, determined by standard plate count assays and fluorescence microscopy using green fluorescent protein-expressing bacteria, correlated with killing. Deletion of the gene encoding the global regulator Ler severely reduced the ability of EPEC to colonize the nematode gut and could be complemented by providing the
ler
gene on a multicopy plasmid in
trans
. Neither the type III secretion system nor the type IV bundle-forming pilus was required for colonization. Combined, the similarities and distinct differences between EPEC infection of nematodes and that of humans offer a unique opportunity to study several stages of the infection process, namely, attachment, colonization, and persistence, in a genetically tractable, inexpensive, and convenient in vivo system.
Invasion of the Placenta during Murine Listeriosis
Authors: Le Monnier Alban , Join-Lambert Olivier F. , Jaubert Francis , Berche Patrick , Kayal Samer
Published: December 2005

Feto-placental infections due toListeria monocytogenesrepresent a major threat during pregnancy, and the underlying mechanisms of placental invasion remain poorly understood. Here we used a murine model of listeriosis (pregnant mice, infected at day 14 of gestation) to investigate how this pathogen invades and grows within the placenta to ultimately infect the fetus. WhenL. monocytogenesis injected intravenously, the invasion of the placenta occurs early after the initial bacteremia, allowing the placental growth of the bacteria, which is an absolute requirement for vertical transmission to the fetus. Kinetically, bacteria first target the cells lining the central arterial canal of the placenta, which stain positively with cytokeratin, demonstrating their fetal trophoblast origin. Bacteria then disseminate rapidly to the other trophoblastic structures, like syncytiotrophoblast cells lining the villous core in the labyrinthine zone of placenta. Additionally, we found that an inflammatory reaction predominantly constituted of polymorphonuclear cells occurs in the villous placenta and participates in the control of infection. Altogether, our results suggest that the infection of murine placenta is dependent, at the early phase, on circulating bacteria and their interaction with endovascular trophoblastic cells. Subsequently, the bacteria spread to the other trophoblastic cells before crossing the placental barrier.
Genome Engineering in
Bacillus anthracis
Using Cre Recombinase
Authors: Pomerantsev Andrei P. , Sitaraman Ramakrishnan , Galloway Craig R. , Kivovich Violetta , Leppla Stephen H.
Published: December 2005

Genome engineering is a powerful method for the study of bacterial virulence. With the availability of the complete genomic sequence of
Bacillus anthracis
, it is now possible to inactivate or delete selected genes of interest. However, many current methods for disrupting or deleting more than one gene require use of multiple antibiotic resistance determinants. In this report we used an approach that temporarily inserts an antibiotic resistance marker into a selected region of the genome and subsequently removes it, leaving the target region (a single gene or a larger genomic segment) permanently mutated. For this purpose, a spectinomycin resistance cassette flanked by bacteriophage P1
loxP
sites oriented as direct repeats was inserted within a selected gene. After identification of strains having the spectinomycin cassette inserted by a double-crossover event, a thermo-sensitive plasmid expressing Cre recombinase was introduced at the permissive temperature. Cre recombinase action at the
loxP
sites excised the spectinomycin marker, leaving a single
loxP
site within the targeted gene or genomic segment. The Cre-expressing plasmid was then removed by growth at the restrictive temperature. The procedure could then be repeated to mutate additional genes. In this way, we sequentially mutated two pairs of genes:
pepM
and
spo0A
, and
mcrB
and
mrr
. Furthermore,
loxP
sites introduced at distant genes could be recombined by Cre recombinase to cause deletion of large intervening regions. In this way, we deleted the
capBCAD
region of the pXO2 plasmid and the entire 30 kb of chromosomal DNA between the
mcrB
and
mrr
genes, and in the latter case we found that the 32 intervening open reading frames were not essential to growth.
A Bacterial Flagellin,
Vibrio vulnificus
FlaB, Has a Strong Mucosal Adjuvant Activity To Induce Protective Immunity
Authors: Lee Shee Eun , Kim Soo Young , Jeong Byung Chul , Kim Young Ran , Bae Soo Jang , Ahn Ouk Seon , Lee Je-Jung , Song Ho-Chun , Kim Jung Mogg , Choy Hyon E. , Chung Sun Sik , Kweon Mi-Na , Rhee Joon Haeng
Published: December 2005

Flagellin, the structural component of flagellar filament in various locomotive bacteria, is the ligand for Toll-like receptor 5 (TLR5) of host cells. TLR stimulation by various pathogen-associated molecular patterns leads to activation of innate and subsequent adaptive immune responses. Therefore, TLR ligands are considered attractive adjuvant candidates in vaccine development. In this study, we show the highly potent mucosal adjuvant activity of a
Vibrio vulnificus
major flagellin (FlaB). Using an intranasal immunization mouse model, we observed that coadministration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific immunoglobulin A (IgA) responses in both mucosal and systemic compartments and IgG responses in the systemic compartment. The mice immunized with TT plus FlaB were completely protected from systemic challenge with a 200× minimum lethal dose of tetanus toxin. Radiolabeled FlaB administered into the nasal cavity readily reached the cervical lymph nodes and systemic circulation. FlaB bound directly to human TLR5 expressed on cultured epithelial cells and consequently induced NF-κB and interleukin-8 activation. Intranasally administered FlaB colocalized with CD11c as patches in putative dendritic cells and caused an increase in the number of TLR5-expressing cells in cervical lymph nodes. These results indicate that flagellin would serve as an efficacious mucosal adjuvant inducing protective immune responses through TLR5 activation.
Intercellular Spreading of
Porphyromonas gingivalis
Infection in Primary Gingival Epithelial Cells
Authors: Yilmaz Özlem , Verbeke Philippe , Lamont Richard J. , Ojcius David M.
Published: December 2005

Porphyromonas gingivalis
, an important periodontal pathogen, is an effective colonizer of oral tissues. The organism successfully invades, multiplies in, and survives for extended periods in primary gingival epithelial cells (GECs). It is unknown whether
P. gingivalis
resides in the cytoplasm of infected cells throughout the infection or can spread to adjacent cells over time. We developed a technique based on flow cytofluorometry and fluorescence microscopy to study propagation of the organism at different stages of infection of GECs. Results showed that
P. gingivalis
spreads cell to cell and that the amount of spreading increases gradually over time. There was a very low level of propagation of bacteria to uninfected cells early in the infection (3 h postinfection), but there were 20-fold and 45-fold increases in the propagation rate after 24 h and 48 h, respectively, of infection. Immunofluorescence microscopy of infected cells suggested that intercellular translocation of
P. gingivalis
may be mediated through actin-based membrane protrusions, bypassing the need for release of bacteria into extracellular medium. Consistent with these observations, cytochalasin D treatment of infected cells resulted in significant inhibition of bacterial spreading. This study shows for the first time that
P. gingivalis
disseminates from cell to cell without passing through the extracellular space. This mechanism of spreading may allow
P. gingivalis
to colonize oral tissues without exposure to the humoral immune response.
Differentially Expressed and Secreted Major Immunoreactive Protein Orthologs of
Ehrlichia canis
and
E. chaffeensis
Elicit Early Antibody Responses to Epitopes on Glycosylated Tandem Repeats
Authors: Doyle C. Kuyler , Nethery Kimberly A. , Popov Vsevolod L. , McBride Jere W.
Published: December 2005

Ehrlichia canis
major immunoreactive proteins of 36 and 19 kDa elicit the earliest detectable antibody responses during the acute phase of canine monocytic ehrlichiosis. Genes encoding the major immunoreactive 36-kDa protein of
E. canis
and the corresponding ortholog of
E. chaffeensis
(47 kDa) were identified and the proteins characterized. The molecular masses of the strongly immunoreactive recombinant proteins were larger than predicted (26.7 and 32.9 kDa, respectively) but were consistent with those of the corresponding native proteins (36 and 47 kDa). Similar to other reported ehrlichial immunoreactive glycoproteins, carbohydrate was detected on the recombinant expressed proteins, indicating that they were glycoproteins. Both glycoproteins (gp36 and gp47) have carboxy-terminal serine/threonine-rich tandem repeat regions containing repeats that vary in number (4 to 16 repeats) and amino acid sequence among different isolates of each species.
E. canis
gp36 was recognized by early acute-phase antibodies (day 14), and species-specific antibody epitopes were mapped to C-terminal nonhomologous repeat units of gp36 and gp47. Periodate treatment of recombinant gp36 reduced the antibody reactivity, and nonglycosylated synthetic peptide repeat units from
E. canis
gp36 and
E. chaffeensis
gp47 were substantially less immunoreactive than corresponding recombinant peptides, demonstrating that glycans are important epitope determinants that are structurally conserved on the recombinant proteins expressed in
Escherichia coli. E. canis
gp36 and
E. chaffeensis
gp47 were differentially expressed only on the surface of dense-cored ehrlichiae and detected in the
Ehrlichia
-free supernatants, indicating that these proteins are released extracellularly during infection.
Inhibition of Macrophage Apoptosis by
Neisseria meningitidis
Requires Nitric Oxide Detoxification Mechanisms
Authors: Tunbridge Anne J. , Stevanin Tania M. , Lee Margaret , Marriott Helen M. , Moir James W. B. , Read Robert C. , Dockrell David H.
Published: December 2005

Host-driven macrophage apoptosis contributes to innate immunity during bacterial infection.
Neisseria meningitidis
inhibits apoptosis in a variety of cells, but its impact on macrophage apoptosis is unknown. We demonstrate that
N. meningitidis
prevents macrophage apoptosis via genes encoding nitric oxide detoxification and a porin, PorB.
Contribution of Sialic Acid-Binding Adhesin to Pathogenesis of Experimental Endocarditis Caused by
Streptococcus gordonii
DL1
Authors: Takahashi Yukihiro , Takashima Eizo , Shimazu Kisaki , Yagishita Hisao , Aoba Takaaki , Konishi Kiyoshi
Published: December 2005

An insertional mutation in
hsa
, the gene encoding the sialic acid-binding adhesin of
Streptococcus gordonii
DL1, resulted in a significant reduction of the infection rate of the organism and an inflammatory reaction in the rat aortic valve with experimental endocarditis, suggesting that the adhesin contributes to the infectivity of the organism for heart valves.
The
pap
Operon of Avian Pathogenic
Escherichia coli
Strain O1:K1 Is Located on a Novel Pathogenicity Island
Authors: Kariyawasam Subhashinie , Johnson Timothy J. , Nolan Lisa K.
Published: December 2005

We have identified a 56-kb pathogenicity island (PAI) in avian pathogenic
Escherichia coli
strain O1:K1 (APEC-O1). This PAI, termed PAI I
APEC-O1
, is integrated adjacent to the 3′ end of the
pheV
tRNA gene. It carries putative virulence genes of APEC (
pap
operon), other
E. coli
genes (
tia
and
ireA
), and a 1.5-kb region unique to APEC-O1. The
kps
gene cluster required for the biosynthesis of polysialic acid capsule was mapped to a location immediately downstream of this PAI.
Listeria monocytogenes10403S HtrA Is Necessary for Resistance to Cellular Stress and Virulence
Authors: Wilson Rebecca L. , Brown Lindsay L. , Kirkwood-Watts Dana , Warren Travis K. , Lund S. Amanda , King David S. , Jones Kevin F. , Hruby Dennis E.
Published: December 2005

The HtrA serine protease has been shown to be essential for bacterial virulence and for survival after exposure to many types of environmental and cellular stresses. AListeria monocytogenes10403ShtrAmutant was found to be sensitive to oxidative and puromycin-induced stress at high temperatures, showed a reduced ability to form biofilms, and was attenuated for virulence in mice.
Adoptive Immunotherapy against Experimental Visceral Leishmaniasis with CD8+T Cells Requires the Presence of Cognate Antigen
Authors: Polley Rosalind , Stager Simona , Prickett Sara , Maroof Asher , Zubairi Soombul , Smith Deborah F. , Kaye Paul M.
Published: December 2005

CD8+T cells have a protective role in experimental visceral leishmaniasis. However, the observation that inflammatory cytokines induce bystander activation of CD8+T cells questions the need for antigen-dependent effector function. Here, we demonstrate that successful adoptive immunotherapy with CD8+T cells is strictly dependent upon the presence of cognate antigen.