Abstract:
The present invention relates to a  Klebsiella pneumoniae  gene-deleted mutant and its producing methods. The main objective of this invention is to construct a  Klebsiella pneumoniae  tonB deletion mutant used as an immunogenically effective amount of a live attenuated vaccine against the community-acquired pyogenic liver abscess, where the tonB gene was deleted. The tonB deletion mutant maintained the structure of the capsular polysaccharide on cell membrane surface, and induced no disease on the mouse after injection. In addition, the tonB deletion mutants induced the immunity against pyogenic liver abscess caused by  Klebsiella pneumoniae.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Field of the Invention 
         [0002]    The present invention relates to a  Klebsiella pneumoniae  tonB deletion mutant and its producing methods. More particularly, the present tonB deletion mutant is used as an immunogenically effective amount of a live attenuated vaccine against the community-acquired pyogenic liver abscess. 
         [0003]    2. Background of the Invention 
         [0004]      Klebsiella pneumoniae  belongs to the family of Enterobacteriaceae; which is Gram-negative, flagella-less and motionless bacterium with polysaccharide capsules. Strains of  Klebsiella pneumoniae  exist in the respiratory tract, digestive tract or urinary tract of healthy people. It&#39;s an opportunistic infectious pathogen. Infection results in pneumonia, urinary tract infections, or infections after surgical operation or intubation and the like.  Klebsiella pneumoniae  has become the predominant pathogen causing community-acquired pyogenic liver abscess (PLA) according to previous studies since 1986, which is an emerging endemic infectious disease particularly common in Asia and South Africa areas. Among the PLA patients, 10-13% combined with meningitis or endophthalmitis, and 40% to 78% of the patients had prior diabetes mellitus. 
         [0005]    The cases of community-acquired PLA caused by  Klebsiella pneumoniae  have increased year by year from recent epidemiologic studies in Korea, Taiwan and USA. Characterization studies using Ribotyping and Pulsed-Field gel electrophoresis have excluded the homology of these  Klebsiella pneumoniae  strains. It&#39;s shown that PLA was not caused by one or a few specified epidemic strain but predominate a capsular subtype of K1. And magA (mucoviscosityassociated gene) is one of the genetic determinants of capsular serotype K1. 
         [0006]    The known virulence factors of PLA-causing  Klebsiella pneumoniae  include not only K1 capsular associated antigens, magA, but also Allantoin metabolic gene, iron uptake gene kfu, and a regulatory gene for capsular exopolysaccharide synthesis, rmpA gene (regulator of the mucoid phenotype A). The general virulence factors include: (1) capsule, which prevents phagocytic destruction and complement serum lysis; and K1, K2 are the most virulent serotypes of the 77 recognized serotype strains; (2) lipopolysaccharide (endotoxin), elicits an immune reaction which is responsible for many of the harmful effects seen in septic shock, or provide serum resistance to avoid complement-mediated lysis; (3) adhesins, 5 types such as type 1, type 3 , Klebsiella pneumoniae  F-28 fimbriae, CF29K, and aggregative adhesion were identified, that are major factors in urinary tract and, respiratory tract infection since they facilitate the attachment of bacterium to the surface of host mucous; (4) siderophores, an iron chelating compound secreted by pathogenic bacteria to compete for iron with host iron-binding proteins since the concentration of free iron ions are very low in human bodies.  Klebsiella pneumoniae  acquires iron ion through synthesizing and secreting siderophores or hemophores to capture iron or heme, and using specialized outer membrane receptor to transport the irons. In addition, all siderophores, hemophores and outer membrane receptors shared the same characteristics: low similarity in N-terminal sequences, requiring a TonB complex which consists of 3 cytoplasmic membrane proteins: TonB, ExbB and ExbD. This TonB complex transduces energy to facilitate the iron internalization. The active transportation is coupled through gradients of concentration. 
         [0007]      Klebsiella pneumoniae  Studies toward community-acquired PLA or non-community PLA using transposon mutagenesis, full genome expression analysis and comparative genomic hybridization successfully identified virulence genes magA, allS and kfu. Further analysis by polymerase chain reaction (PCR) showed that these genes were mainly found in clinical isolates from patients of community-acquired PLA but not from non-community PLA. The induction of pathogenic genes of  Klebsiella pneumoniae  may be induced by environmental pressure during infection. 
         [0008]    Recently, capsular serotype K1 was found to be the major virulence strain for  Klebsiella pneumoniae  causing PLA. And magA is one of the major components in capsular genetic determinants. Mutations in magA lost the exopolysaccharides and became avirulent. Therefore, the magA deletion mutant is not considered to be an ideal vaccine strain because of the lack of exopolysaccharides and immunogenicity. 
         [0009]      Klebsiella pneumoniae  vaccines using purified capsules of  Klebsiella pneumoniae  and other non-capsular compositions such as lipopolysaccharides, type 3 pilus, or even capsular gene deletion mutants were developed but with little success. Generally patients with PLA have been treated with catheter drainage and antibiotic therapy, but still showed a mortality of 10-40%. There is room for improvement since inactivated dead bacteria used in vaccine preparation limited the efficiency of antibody induction. 
       SUMMARY OF THE INVENTION 
       [0010]    The present invention provides a novel  Klebsiella pneumoniae  tonB deletion mutant and a producing method based on the research of previous studies to improve the efficiency of antibody induction. 
         [0011]    The objective of the present invention is to provide a  Klebsiella pneumoniae  tonB deletion mutant used as an immunogenically effective amount of a live attenuated vaccine. The tonB deletion mutant was stored in Bioresource Collection and Research Center (Food Industry Research and Development Institute, Hsinchu, Taiwan) with an accession number of BCRC 910405. The live attenuated vaccine used for immunization against  Klebsiella pneumoniae  caused PLA has no pathogenicity to BALB/c mouse after intraperitoneal injection. 
         [0012]    Another object of the present invention is to provide a method for producing the  Klebsiella pneumoniae  tonB deletion mutant. The tonB deletion mutant contains exopolysaccharides K1 and serum resistance, and grows in a condition containing iron ions. And the live attenuated vaccine further comprises a pharmaceutical carrier, diluent, or excipient. 
         [0013]    The abovementioned method for producing a  Klebsiella pneumoniae  tonB deletion mutant comprises the steps of: (a) constructing a plasmid containing a tonB gene of  Klebsiella pneumoniae  and flanking regions of the tonB gene using a first primer set and a second primer set; (b) digesting the plasmid with a restriction enzyme to form a restriction fragment containing the flanking regions of tonB gene, and ligating the restriction fragment with a vector containing a temperature-sensitive region, an antibiotic selection marker and a negative selection marker; (c) transforming the vector into a  Klebsiella pneumoniae  strain; and (d) screening the tonB deletion mutant by an antibiotic and a negative selection drug, and obtaining the tonB deletion mutant containing the directly ligated flanking regions without tonB gene, and no selection markers. 
         [0014]    The first primer set of step (a) is SEQ ID NO: 43 and SEQ ID NO: 44, and the second primer set is SEQ ID NO: 45 and SEQ ID NO: 46, the plasmid is a TA plasmid. The restriction enzyme of step (b) is NotI, and the vector is a pKO3 plasmid containing a temperature-sensitive origin, an antibiotic selection marker and a negative selection marker. The screened tonB deletion mutant was proved to be not pathogenicity to BALB/c mouse after activity assay but could be used for immunization against PLA—causing  Klebsiella pneumoniae  to induce anti-EPS IgG. 
         [0015]    The present invention is further explained in the following embodiment illustration and examples. Those examples below should not, however, be considered to limit the scope of the invention, it is contemplated that modifications will readily occur to those skilled in the art, which modifications will be within the spirit of the invention and the scope of the appended claims. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]      FIG. 1 . Diagram of microarray gene expression profile of cloned fragments in the present invention. 
           [0017]      FIG. 2   a . The genes of  Yersinia  HPI and iucABCDiutA distributed in  Klebsiella pneumoniae  genome. 
           [0018]      FIG. 2   b . The genes of iroA and hmuRSTUV distributed in  Klebsiella pneumoniae  genome. 
           [0019]      FIG. 3   a . Construction of the deletion mutants of          irp2 and          iuc. 
           [0020]      FIG. 3   b . Construction of the deletion mutants of          iroA and          kfu. 
           [0021]      FIG. 4 . The flow chart of the unmarked deletion method using a pKO3 plasmid. 
           [0022]      FIG. 5 . The growth curves of the NTUH-K2044 strain and other deletion mutants in iron-deficient conditions. 
           [0023]      FIG. 6 . The survival curves of BALB/c mice after infection of each deletion mutants. 
           [0024]      FIG. 7 . Results of injection with the NTUH-K2044 strain to the immunized BALB/c mice. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0025]    The present invention used polysaccharide-protein conjugate vaccine which is the most effective vaccine. 
         [0026]    The known virulence factors of PLA-causing  Klebsiella pneumoniae  include capsular associated antigens, metabolic gene, iron uptake gene, and a regulatory gene for capsular exopolysaccharide synthesis. Among them, the gene involved in iron transport system, such as tonB, is important since iron can be used in anti-oxidation of the hosts. Therefore, several  Klebsiella pneumoniae  deletion mutants are constructed in the present invention to against PLA causing  Klebsiella pneumoniae . The non-pathogenic infection tonB deletion mutant was screened as the major active ingredient of vaccine candidates because it can induce the immunity to the same pathogens. 
         [0027]    Single dose of tonB deletion mutant was found to be enough for the BALB/c mice to generate immunity from the screening studies on deletion mutants of PLA causing  Kiebsiella pneumonia  in the present invention. This showed that the tonB deletion mutant is able to stimulate BALB/c mice to generate protective memory immune responses. The tonB deletion mutant would not be cleaned immediately by the immune responses of the hosts since it contains complete K1 capsular exopolysaccharides, serum resistance and also induces anti-EPS IgG production in comparison to magA mutants. 
         [0028]    The original  Klebsiella pneumoniae  strain of the tonB mutant was screened and isolated from the blood of the PLA patient in National Taiwan University Hospital (NTUH) with an accession number of NTUH-K2044. The whole genome size is 5.2×10 6  bases, including about 5093 open reading frames (ORFs), a GC ratio of 57.68% and a plasmid pK2044 (large plasmid) of 2.24×10 5  bases, 299 ORFs, a GC ratio of 50.17%. 
         [0029]    All the containers for NTUH-K2044 RNA extraction were washed by a solution of 0.1% DEPC to prevent the degradation of RNase in the air. 
         [0030]    Overnight culture of NTUH-K2044  Klebsiella pneumoniae  were cultured in 1 ml of LB media and 1 ml of diluted healthy human blood at 4% to 8%, or in 2 ml of LB for the control group. The cells were grown at 37° C. for 3-4 h to log phase, then centrifuged at 14000 g, 4° C. for 2 h. The supernatant was removed and the cell pellet was washed twice with 0.1% DEPC ddH 2 O. The cells were centrifuged again for 2 h then the supernatant was removed. Each ml of the cell pellet solution was resuspended into 0.25 ml of lysis buffer and then boiled at 100° C. for 5 min till solution was clear. The RNA of the strain was isolated with RNeasy Mini Kit (Qiagen, cat. No. 74106) and residual DNA was removed with RNase-Free DNase Set (cat. No. 79254) after cool down. RNA concentration and purity was determined using agarose electrophoresis and spectrophotometrically at OD 260/280 . 
         [0031]    Microarray hybridization was used to analyze the expression level of extracted RNA from NTUH-K2044. To 5 μl of the RNA, 7.5 μl of random hexamer primer and 14.75 μl of DEPC-dH 2 O were added and heated at 70° C. for 3 min, followed by addition of 12.5 μl of 5× First RT buffer, 0.1 M DTT, and 2.5 μl each of dATP, dCTP, dGTP, and dTTP (in the concentration of 25 μl), 6 μl of biotin-16-dUTP (25 mM), 1.5 μl of RNaseOut™ Ribonuclease and 5 μl of Superscript II reverse transcriptase. The reverse transcription-PCR (RT-PCR) was carried out at 25° C. for 10 min, 42° C. for 90 min and 94° C. for 5 min. The biotin-16-dUTP could then be incorporated into the first strand of cDNA. This biotin-16-dUTP cDNA can be used as labeled probe after incubated with 6.9 μl NaOH (3M) at 50° C. for 30 min to remove the residual RNA and neutralized with 6.9 μl acetic acid (3M) at 50° C. for 30 min. The labeled probe can be precipitated with ethanol. 
         [0032]    The microarray filter was incubated with 2 ml of hybridization buffer at 63° C. for 1.5-2 h. One ml of the hybridization buffer containing the labeled probe was added onto the surface of filter and incubated at 63° C. for 16-18 h. The filter was washed with 2×SSC and 0.1% SDS twice at room temperature (evenly shaking) for 5 min, with a final wash of 0.1×SSC and 0.1% SDS three times at 63° C. for 15 min. 
         [0033]    Color development was performed after the microarray filter was treated in the following order: incubating with 2 ml of blocking buffer with shaking at room temperature for 1 h, incubating with 2 ml of reaction buffer at room temperature for 1 h, washing with PBST buffer for 4 times at room temperature for 5 min each, reacting with 1.5 ml of NBT/BCIP buffer at room temperature for 20-40 min, and stopping the reaction with water. The color developed in the filter was scanned in a scanner and stored in the computer followed by quantification of the images with NIH ImageJ software. The 23SrRNA was served as the internal control. 
         [0034]    Referring to  FIG. 1 , the microarray gene expression profile of 14 cloned sequences, including  Yersinia  HPI iucABCDiutA, iroA and hmuRSTUV, showed significant increase of RNA expression levels in human serum-induction group but not in control group.  Yersinia  HPI, iucABCDiutA and iroA belong to the siderophore-dependent iron acquisition systems, while hmuRSTUV is an ATP-binding cassette (ABC) hemoprotein transport system. In contrast, 5 clones revealed a decrease in RNA expression level, including yjdL, cadABC and tdcABCDE gene loci. 
       Example 1 
     Method for Establishing  Klebsiella Pneumoniae  Gene Deletion Mutants 
       [0035]    All the cloned fragments with higher gene expression levels than the control group were selected as candidates of deletion mutants. The method for producing a  Klebsiella pneumoniae  tonB deletion mutant comprises the steps of: (a) constructing a plasmid containing a tonB gene of Klebsiella pneumoniae and flanking regions of the tonB gene using a first primer set and a second primer set; (b) digesting the plasmid with a restriction enzyme to form a restriction fragment containing the flanking regions of tonB gene, and ligating the restriction fragment with a vector containing a temperature-sensitive region, an antibiotic selection marker and a negative selection marker; (c) transforming the vector from step (b) into a  Klebsiella pneumoniae  strain; and (d) screening the tonB deletion mutant by an antibiotic and a negative selection drug, and obtaining the tonB deletion mutant containing the directly ligated flanking regions without tonB gene, and no selection markers. The abovementioned primer sets were listed in Table 1: 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 primers 
                 sequence 
                 usage 
                 SEQ ID NO 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1040-F 
                 GGTGCTCTTTACATCATTGC 
                 prevalence analysis 
                 SEQ ID NO:1 
                   
               
               
                 936-R 
                 GCAATGGCCATTTGCGTTAG 
                 magA 
                 SEQ ID NO:2 
               
               
                   
               
               
                 10E4-2-5F 
                 AGTCGGCCTGGGGTTTAAGG 
                 prevalence analysis allS 
                 SEQ ID NO:3 
               
               
                 10E4-2- 
                 CAGTCAACGTGGCGATTCGC 
               
               
                 475R 
               
               
                   
               
               
                 kfu-F1179 
                 GAAGTGACGCTGTTTCTGGC 
                 prevalence analysis 
                 SEQ ID NO:5 
               
               
                 kfuC-R649 
                 TTTCGTGTGTGGCCAGTGACTC 
                 kfu/PTS 
                 SEQ ID NO:6 
               
               
                   
               
               
                 ybtU-F 
                 TTGTGCGCAACACATTACGC 
                 prevalence analysis 
                 SEQ ID NO:7 
               
               
                 ubtU-R 
                 TCACAGCGCCTCCTTATCAT 
                 Yersinia HPI 
                 SEQ ID NO:8 
               
               
                 ybtA-F 
                 ATGACGGAGTCACCGCAAAC 
                   
                 SEQ ID NO:9 
               
               
                 ybtA-R 
                 TTACATCACGCGTTTAAAGG 
                   
                 SEQ ID NO:10 
               
               
                   
               
               
                 iucB-F 
                 ATGTCTAAGGCAAACATCGT 
                 iucABCDiutA prevalence 
                 SEQ ID NO:11 
               
               
                 iucB-R 
                 TTACAGACCGACCTCCGTGA 
                 analysis &amp; iucB qPCR 
                 SEQ ID NO;12 
               
               
                   
               
               
                 iroN-F 
                 GTCCGGCGGTAACTTCAGCC 
                 iroA(iroNDCB) 
                 SEQ ID NO:13 
               
               
                 iroN-P-R 
                 TCAGAATGAAACTACCGCCC 
                 prevalence analysis &amp; 
                 SEQ ID NO:14 
               
               
                   
                   
                 iroN qPCR 
               
               
                   
               
               
                 iroNB-F 
                 GGCTACTGATACTTGACTATTC 
                 iroA(iroBCDN) 
                 SEQ ID NO:15 
               
               
                 iroNB-R 
                 CAGGATACAATAGCCCATAG 
                 prevalence analysis 
                 SEQ ID NO:16 
               
               
                   
               
               
                 hmuR-F 
                 GTGGCGACTATGTACAAATC 
                 hmuRSTUV prevalence 
                 SEQ ID NO:17 
               
               
                 hmuR-R 
                 GCTGTTGTTTTCAGTTTCCT 
                 analysis &amp; human qPCR 
                 SEQ ID NO:18 
               
               
                   
               
               
                 KP-23-F 
                 GGTTAAGCGACTAAGCGTACACGGT 
                 23S rRNA qPCR 
                 SEQ ID NO:19 
               
               
                 KP-23-R 
                 ACGAGGCGCTACCTAAATAGCTTTC 
                   
                 SEQ ID NO:20 
               
               
                   
               
               
                 irp2-2200F 
                 GCATGACAGGGTGCTGGCCC 
                 irp2 qPCR 
                 SEQ ID NO:21 
               
               
                 irp2-2899R 
                 CTCCGACTTTGACCTGCTTGTC 
                   
                 SEQ ID NO:22 
               
               
                   
               
               
                 ybtA-F 
                 ATGACGGAGTCACCGCAAAC 
                 irp2 deletion construct 
                 SEQ ID NO:23 
               
               
                 irp1-R 
                 CGGTATAGCCGACCTTTCTG 
                   
                 SEQ ID NO:24 
               
               
                 ybtA-R 
                 TTACATCACGCGTTTAAAGG 
                   
                 SEQ ID NO:25 
               
               
                 irp1-F 
                 ATGGATAACTTGCGCTTCTC 
                   
                 SEQ ID NO:26 
               
               
                   
               
               
                 Iuc-FR 
                 GAGCCGCCCCAAACGACAGC 
                 iucABCDiutA deletion 
                 SEQ ID NO:27 
               
               
                 Iuc-RR 
                 GGCTTTTCTGATACCAATCT 
                 construct 
                 SEQ ID NO:28 
               
               
                 iuc-Fout 
                 AAGCACAATCAATATATGG 
                   
                 SEQ ID NO:29 
               
               
                 iuc-Rout 
                 CCGGTATTCCTTTACAACAA 
                   
                 SEQ ID NO:30 
               
               
                   
               
               
                 iro-PF 
                 TCCTGTTGGCCAGCGTCTAT 
                 iroA(iroNDCB) deletion 
                 SEQ ID NO:31 
               
               
                 iro-PR 
                 CTCCTTCAGCCCGAACAAAC 
                 construct 
                 SEQ ID NO:32 
               
               
                 iro-P-Fout 
                 TCGTAATTATTAGGACTAAG 
                   
                 SEQ ID NO:33 
               
               
                 iro-P-Rout 
                 GCTCCTGTATACTATGGCAG 
                   
                 SEQ ID NO:34 
               
               
                   
               
               
                 iroN-R 
                 TCAGAATGATGCGGTGACAC 
                 iroA(iroBCDN) deletion 
                 SEQ ID NO:35 
               
               
                 iroD-R 
                 TCAACCTTTTAGTAAACC 
                 construct 
                 SEQ ID NO:36 
               
               
                 iroN-F 
                 GTCCGGCGGTAACTTCAGCC 
                   
                 SEQ ID NO:37 
               
               
                 iroD-F 
                 ATGCTGAACATGCAACAAC 
                   
                 SEQ ID NO:38 
               
               
                   
               
               
                 kfu-FR 
                 GCAGCAGATGAATATTCTGG 
                 kfuABC deletion 
                 SEQ ID NO:39 
               
               
                 kfu-RR 
                 TTCCGACGCCAATGCTGATC 
                 construct 
                 SEQ ID NO:40 
               
               
                 kfu-Fout 
                 TCTGGGTGCAGAACCAAATG 
                   
                 SEQ ID NO:41 
               
               
                   
               
               
                 kfu-Rout 
                 ATGGAGTGGTAGTACGTTGG 
                   
                 SEQ ID NO:42 
               
               
                   
               
               
                 tonB-FR 
                 AGCAACTTAACGCTGGCAGC 
                 tonB deletion construct 
                 SEQ ID NO:43 
               
               
                 tonB-RR 
                 TGAGCTGGGTACCAACACC 
                   
                 SEQ ID NO:44 
               
               
                 tonB-Fout 
                 GCAATCATATTCAATAAGG 
                   
                 SEQ ID NO:45 
               
               
                 tonB-Rout 
                 TAAAACGCTGCGGCGGACCG 
                   
                 SEQ ID NO:46 
               
               
                   
               
               
                 iro-pP 
                 GAAAATCCCTCTTTTAACGC 
                 salmochelin receptor 
                 SEQ ID NO:47 
               
               
                 iroN-P-R 
                 TCAGAATGAAACTACCGCCC 
                 IroN from 
                 SEQ ID NO:48 
               
               
                   
                   
                 iroA(iroNDCB) 
               
               
                   
               
               
                 iro-pC 
                 TCGATCCGGTTGTTTGCAGG 
                 salmochelin receptor 
                 SEQ ID NO:49 
               
               
                 iroN-R 
                 TCAGAATGATGCGGTGACAC 
                 IroN from 
                 SEQ ID NO:50 
               
               
                   
                   
                 iroA(iroBCDN) 
               
               
                   
               
               
                 irp2-p 
                 CCCCTTCGACCTTTAAACGC 
                 irp2 complementation 
                 SEQ ID NO:51 
               
               
                 irp2-R 
                 CTATATCCGCCGCTGACGAC 
                   
                 SEQ ID NO:52 
               
               
                   
               
               
                 pIuc-F 
                 CCAGTACAGGGATCGCGACC 
                 iucABCDiutA 
                 SEQ ID NO:53 
               
               
                 iutA-R 
                 TCAGAACAGCACAGAGTAGTTCAG 
                 complementation 
                 SEQ ID NO:54 
               
               
                   
               
               
                 tonB-F 
                 CGTAAAGCACGGCAAAGCTC 
                 tonB complementation 
                 SEQ ID NO:55 
               
               
                 tonB-R 
                 TCAGTTAATCTCGACGCCG 
                   
                 SEQ ID NO:56 
               
               
                   
               
             
          
         
       
     
         [0036]    Primer sets listed in the above table and PCR performed in step (a) were used as reference for gene deletion to detect the distribution of gene fragments such as  Yersinia  HPI, iucABCDiutA, iroA and hmuRSTUV in community acquired PLA or non-community acquired PLA. The cycling program consisted of one denaturation step of 3 min at 96° C. and 30 cycles of 30 s at 96° C., 15 s at 52° C., and 1 min at 74° C., followed by 10 min at 72° C. Referring to  FIGS. 2   a  and  2   b , respectively the gene distribution diagram of  Yersinia  HPI and iucABCDiutA, iroA and hmuRSTUV in  Klebsiella pneumoniae  genome was shown. The distribution of Yersinia HPI iucABCDiutA, iroA and hmuRSTUV was shown after PCR. These 4 genes were shown to be more common (associated with) in community acquired PLA than in non-community acquired PLA. Therefore, the deletion mutants of Yersinia HPI, iucABCDiutA, iroA and hmuRSTUV are candidates of the live attenuated vaccine against community acquired PLA. 
         [0037]    Referring to  FIGS. 3   a  and  3   b , respectively the deletion mutants of          irp2 and          iuc,          iroA and          kfu were constructed. Unmarked deletion method is one of the several methods used to construct the deletion mutants in the invention. The targeted deletion genes such as          irp2,          iuc,          iroA and          kfu in dotted lines were the few examples shown in  FIG. 3 . Other single or multiple gene deletion such as          tonB,          irp2 iuc iroA and          irp2 iuc iroA kfu can further be included.  FIG. 4  shows the unmarked deletion method. The flanking regions of the target gene were ligated into the plasmid pKO3, a gene replacement vector that contains a temperature-sensitive origin of replication and markers for positive and negative selection for chromosomal integration and excision. The recombinant pKO3 plasmid was transformed into  Klebsiella pneumoniae  using electroporation and cultured at 43° C. in LB plates containing kanamycin. This plasmid would integrate into the chromosome of NTUH-K2044 through homologous recombination. PCR was carried out to screen the pKO3 integrated clones. These clones were cultured at 30° C. in LB plates containing 5% sucrose. The plasmid pKO3 can be generated again through homologous recombination to form gene deletion mutants of NTUH-K2044. PCR was carried out to screen the mutants. 
         [0038]    The          tonB deletion mutant was taken as an example. Primers tonB-FR (SEQ ID NO: 43) and tonB-RR (SEQ ID NO: 44) were used to synthesize a complete tonB gene through PCR, including 1 kb of the upstream and downstream flanking regions in step (a). This gene fragment was ligated into a plasmid such as TA. A reverse PCR was performed using the resulting plasmid as a template, plus the primers tonB-Fout (SEQ ID NO: 45) and tonB-Rout (SEQ ID NO: 46, LA-Taq polymerase to yield products. The A-tail of the 3′-end was removed from the products using T4 DNA polymerase (NEB) and phosphate was attached to the 5′-end of the products using polynucleotide kinase (NEB) for self-ligation. 
         [0039]    The restriction enzymes used to digest the tonB gene and the flanking regions from the TA cloning vector in Step (b) including but not limited to NotI, the vector containing temperature-sensitive region, an antibiotic selection marker and a negative selection marker including but not limited to pKO3, the antibiotic selection marker including but not limited to kanamycin, the negative selection marker including but not limited to sacB (encoding levansucrase). 
         [0040]    Electroporation can be used in step (c) to transform the flanking regions of tonB gene into the NTUH-K2044 strain. 
         [0041]    A tonB deletion mutant containing the directly ligated flanking regions without tonB gene was screened by the antibiotic and the negative selection marker in step (d), which contained no selection marker in the genome. The sequence of mutant was confirmed with PCR and sequence analysis. The negative selection marker was sucrose in step (d). 
         [0042]    The functional analysis was carried out with trans-complementation, cross-feeding assay, growth study, String test, Serum resistance assays, serotyping, animal study, Enzyme-Linked Immunosorbent Assay (ELISA) and immunoblotting. 
         [0043]    The growth curves of the NTUH-K2044 strain and each deletion mutants (irp2, iuc, or iroA single mutants, irp2 iuc iroA triple mutant and irp2 iuc iroA kfu quadruple mutant) were shown in  FIG. 5 . All mutant strains as well as the wild type parental strain showed no difference in both iron-replete and iron-deficient conditions in vitro. Wild-type NTUH-K2044 secreted yersiniabactin and aerobactin into the culture supernatants but the deletion mutants irp2 or iuc single mutants, triple mutant and quadruple mutant produced neither yersiniabactin nor aerobactin as determined by cross-feeding assays. However, plasmid CopyControl pCC1 containing corresponding genes used in trans-complementation restored the ability to produce yersiniabactin or aerobactin of these mutants as shown in Table 2. 
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                 Genotype or 
                 Synthesis 
                 Synthesis of 
                   
                 Serum 
               
               
                 Strain 
                 phenotype* 
                 of Ybt †   
                 Aerobactin †   
                 K1 CPS Ag 
                 resistance ‡   
               
               
                   
               
             
             
               
                 NTUH-K2044 
                 Wild-type/m +   
                 + 
                 + 
                 + 
                 R 
               
               
                 Δirp2 
                 irp2/m +   
                 − 
                 ND 
                 ND 
                 ND 
               
               
                 Δiuc 
                 iuc/m +   
                 ND 
                 − 
                 ND 
                 ND 
               
               
                 ΔiroA 
                 iroA/m +   
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 Δkfu 
                 kfu/m +   
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 Δirp2 iuc iroA 
                 irp2 iuc iroA/m +   
                 − 
                 − 
                 + 
                 ND 
               
               
                 Δirp2 iuc iroA 
                 irp2 iuc iroA kfu/ 
                 − 
                 − 
                 + 
                 R 
               
               
                 kfu 
                 m +   
               
               
                 Δirp2::pCC1-irp2 
                 Irp2/m +   
                 + 
                 ND 
                 ND 
                 ND 
               
               
                 Δiuc::pCC1-iuc 
                 Iuc/m +   
                 ND 
                 + 
                 ND 
                 ND 
               
               
                 ΔtonB 
                 tonB/m +   
                 ND 
                 ND 
                 + 
                 R 
               
               
                 ΔtonB::pCC1- 
                 TonB/m +   
                 ND 
                 ND 
                 + 
                 ND 
               
               
                 tonB 
               
               
                   
               
               
                 *m + , mucoviscosity; 
               
               
                 +, synthesizing ability ≧5 mm; 
               
               
                 ND, not detected; 
               
               
                   ‡ R, resistance. 
               
             
          
         
       
     
         [0044]    The ΔtonB deletion mutant was shown to remain hyperviscous, K1 capsular exopolysaccharides, and serum resistance according to Table 2. Animal studies were carried out to test the possibility for mutants as vaccine candidates. Five-week-old female BALB/c mice were obtained from National Laboratory Animal Center. The BALB/c mice were infected intraperitoneally (IP) or intragastrically (IG) with  Klebsiella pneumoniae  NTIH-K2044 mutants including          irp2 iuc iroA triple mutant,          irp2 iuc iroA kfu quadruple mutant,          tonB mutant,          magA mutant (not able to synthesize K1 capsular exopolysaccharide) at the dose of 1×10 3  CFU to          irp2 iuc iroA triple mutant,          irp2 iuc iroA kfu quadruple mutant, and 1×10 4  CFU or 1×10 5  CFU to          tonB mutant, and 1×10 6  CFU to          magA mutant; saline was used in the control group. After 4 weeks, immunized and non-immunized control BALB/c mice were challenged with 1×10 3  CFU of NTUH-K2044 (greater than the wild-type LD 50  value). The challenged mice were monitored for another 4 weeks. The pathogenicity was analyzed with Kaplan-Meier analysis (SPSS 12 th  ed.) and a log-rank test; P&lt;0.05 was considered to be statistically significant. 
         [0045]    Single mutants including          irp2,          iuc,          iroA, and          kfu were shown to have the same pathogenicity with the wild type NTIH-K2044 (LD 50 &lt;1×10 2  CFU) after IP infection. However, the mice survived after injection of the dose of 1×10 3  CFU to          irp2 iuc iroA triple mutant or          irp2 iuc iroA kfu quadruple mutant (LD 50  of 1.3×10 4  CFU for irp2 iuc iroA triple mutant and 5.5×10 4  CFU for irp2 iuc iroA kfu quadruple mutant). In addition, there was no significant difference for the pathogenicity between the          irp2 iuc iroA triple mutant and the wild type strain after IG feeding (LD 50  of 5.6×10 5  CFU for irp2 iuc iroA triple mutant and 1×10 5  CFU for the wild-type strain). Referring to  FIG. 6 , the pathogenicity of the          irp2 iuc iroA kfu quadruple mutant was even lower than the wild type strain or the          kfu mutant after IG feeding (LD 50  of &gt;1×10 7  CFU for irp2 iuc iroA kfu quadruple mutant and LD 50  of 6.3×10 6  CFU for          kfu mutant).          tonB mutant was found to have no pathogenicity in mice after 28 days observation and the survival rate was 100%. 
         [0046]    Analysis of the genomic sequence of  Klebsiella pneumoniae  NTUH-K2044 identified ten putative iron transport systems, and 7 of them were TonB dependent, such as  Yersinia  HPI, iucABCDiutA, and iroA. The tonB deletion mutant was unable to grow in iron-deficient media, whereas complementation of the tonB deletion mutants restored growth in iron-restricted media. The tonB deletion mutant was therefore confirmed to be defective in iron uptake. In comparison with the parental NTUH-K2044 strain, the tonB deletion mutant formed smaller colonies on either blood agar or LB media. IP inoculation of BALB/c mice with the tonB deletion mutant resulted in a less efficient spread in the body than IP injection of wild type strain at the same dose. A string test was examined in these deletion mutants and revealed that the tonB deletion mutant remained hyperviscous. There was no significant difference in CPS antigen serotyping using double immunodiffusion and serum sensitivity assays between the tonB mutant and the wild type NTUH-K2044 strain. Therefore, the immunogenicity was regarded the same for both strains. 
         [0047]    Referring to  FIG. 7 , the results of injection of NTUH-K2044 strain to the immunized BALB/c mice were shown. The protective efficacy of the live vaccines using mutants of magA,          irp2 iuc iroA,          irp2 iuc iroA kfu, and          tonB were compared with non-immunized BALB/c mice. These attenuated strains of each mutant were injected into BALB/c mice respectively to get immunity, followed by challenging with the lethal dose of NTUH-K2044. Four weeks later, all tonB deletion mutant-immunized BALB/c mice survived without any symptoms of disease, whereas 75% of the non-immunized control BALD/c mice or magA mutant-immunized BALB/c mice died within 5 days of infection. The survival rates of          irp2 iuc iroA and          irp2 iuc iroA kfu-mutant immunized BALB/c mice were lower than that of tonB mutant-immunized BALB/c mice. ELISA analysis showed that BALB/c mice immunized with the tonB mutant had EPS serum immunoglobulin G (anti-EPS IgG) responses against NTUH-K2044 strain, while control mice or magA mutant-immunized mice did not. 
         [0048]    From the description and results of the abovementioned animal studies, the tonB deletion mutant was shown to be a potential live vaccine since the strain could survive in human blood and has the ability to synthesize K1 capsular exopolysaccharides. Therefore the tonB deletion strain can be prepared as live vaccine in large scale, resulting in induction of anti-EPS antibody in human and immediate protect against the  Klebsiella pneumoniae —causing community acquired PLA. The tonB deletion mutant was stored in Bioresource Collection and Research Center (Food industry Research and Development Institute, Hsinchu, Taiwan) on Oct. 30, 2008 with an accession number of BCRC910405.