Differential proteomic analysis of Aeromonas salmonicida outer membrane proteins in response to low iron and in vivo growth conditions

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis, a serious infectious disease of salmonids. Bacterial phenotypes are known to change in vivo compared to the in vitro state. Proteomic analysis of in vivo phenotypes is usually not possible due to insufficient biomass. Using an in vivo growth chamber model, the pathogenic fish bacterium A. salmonicida was cultured in pure culture in vivo in its host, the Atlantic salmon, to obtain sufficient biomass to allow proteomic analysis. Growth of A. salmonicida under in vitro iron-restricted conditions resulted in the expression of outer membrane proteins of 73, 76 and 85 kDa, which were not present when grown under in vitro iron-replete conditions. Mass spectrometry analysis identified the 73 kDa protein as a colicin receptor, the 76 kDa protein as an outer membrane heme receptor, and the 85 kDa protein as a ferric siderophore receptor. When cultured in vivo, A. salmonicida up-regulated the identical 73, 76 and 85 kDa proteins. The results of this study also suggest, at least with respect to the outer membrane proteins, that the in vitro iron-restricted growth model largely reproduces the results obtained from growth of A. salmonicida within the peritoneal cavity of salmon.     

Improved protection against lung colonization by Actinobacillus pleuropneumoniae ghosts: characterization of a genetically inactivated vaccine

Pigs immunized with Actinobacillus pleuropneumoniae ghosts or a formalin-inactivated bacterin were found to be protected against clinical disease in both vaccination groups, whereas colonization of the lungs with A. pleuropneumoniae was only prevented in ghost-vaccinated pigs. Bacterial ghosts are empty cell envelopes created by the expression of a cloned bacteriophage lysis gene and, unlike formalin-inactivated bacteria, suffer no denaturing steps during their production. This quality may lead to a superior presentation of surface antigens to the immune system. Analysis by SDS-PAGE and immunoblotting of the two vaccine preparations revealed different contents of antigenic proteins. In order to better understand the immunogenic properties of A. pleuropneumoniae ghosts and formalin-inactivated bacteria, we compared the serum antibody response induced in both treatment groups. Immune sera were tested on whole cell antigen or purified virulence factors including outer membrane protein preparations (OMPs), outer membrane lipoprotein OmlA1, transferrin binding proteins (TfbA1, TfbA7 and TfbB) and Apx toxins (ApxI, II and III). SDS-PAGE and immunoblots revealed no specific antibody response against the single virulence factors tested in any vaccinated animal. The two vaccination groups showed different recognition patterns of whole cell antigen and OMP-enriched preparations. A 100 kDa protein was recognized significantly stronger by ghost-vaccinated pigs than convalescent pigs. This unique antibody population induced by ghosts could play a determining role in the prevention of lung colonization. The same 100 kDa antigen was recognized by ghost-sera in homologous as well as heterologous serotype A. pleuropneumoniae protein preparations. Indications for a crossprotective potential in the ghost vaccine were supported by studies on rabbit hyperimmune sera.     

Structural and immunochemical homogeneity of Aeromonas salmonicida lipopolysaccharide.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the lipopolysaccharides of typical and atypical strains of the fish pathogen Aeromonas salmonicida. 32P intrinsically radiolabeled lipopolysaccharide in sarcosinate-extracted outer membrane preparations, lipopolysaccharide stained by silver in proteinase K-digested outer membrane preparations and whole cell lysates, as well as purified lipopolysaccharide, displayed O-polysaccharide chains which were unusually homogeneous with respect to chain length. Chemical analysis further revealed that the sugar composition of the smooth lipopolysaccharide purified from three typical strains was very similar. Immunoblotting and immunofluorescent staining with both polyclonal and monoclonal antibody showed that the O-polysaccharide chains were strongly immunogenic and were antigenically cross-reactive on typical and atypical strains from diverse origins. Immunofluorescence analysis and phage binding studies demonstrated that a number of these O-polysaccharide chains traversed the surface protein array of virulent strains of A. salmonicida and were exposed on the cell surface.     

The outer membrane proteins of the fish pathogens Aeromonas hydrophila, Aeromonas salmonicida and Edwardsiella tarda

Abstract The profiles of the outer membrane proteins contained many major proteins and were markedly different in a number of Aeromonas hydrophila strains isolated from various origins. In contrast, the profiles of outer membrane proteins in Aeromonas salmonicida from different sources were identical and quite different from those of A. hydrophila .
In the strains of Edwardsiella tarda tested, protein components in the outer membrane were quite similar. 2 Major proteins, of 36 and approx. 46 kDa, were found in the outer membrane. Major proteins in A. hydrophila and A. salmonicida were detected in the 32–36 kDa range, as observed in many Enterobacteriaceae. In all strains tested, several 68–90 kDa polypeptides appeared in the outer membrane during iron starvation.     

Intranasal Immunization with Nontypeable Haemophilus influenzae Outer Membrane Vesicles Induces Cross-Protective Immunity in Mice

Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.     

Immunogenicity of a Haemophilus influenzae polysaccharide-Neisseria meningitidis outer membrane protein complex conjugate vaccine

Polysaccharide-protein conjugate vaccines made with different carriers vary in their ability to elicit antipolysaccharide IgG antibody responses in young infants and an adult mouse model, suggesting that the carrier proteins used in the conjugate vaccines differ in their ability to act as carriers, or that additional mechanisms of immunogenicity play a role. A conjugate vaccine of Haemophilus influenzae PRP coupled to the outer membrane protein complex (OMPC) of Neisseria meningitidis serogroup B is immunogenic in children as young as 2 mo of age and is immunogenic in infant rhesus monkeys, an animal model for infant humans. In the present study, PRP-OMPC was found to induce efficient IgM to IgG switching of anti-PRP serum antibody in adult mice, whereas PRP conjugated to two other protein carriers did not. Thus the PRP-OMPC conjugate was examined in order to determine why PRP coupled to OMPC was so immunogenic, even more immunogenic than conjugates made with other carrier proteins. The OMPC carrier differs from the other protein carriers in that the proteins are present in a liposomal form containing lipids (including LPS) derived from the outer membrane of N. meningitidis. We studied the OMPC to see whether the different components or the nature of the OMPC carrier could contribute to its enhanced immunogenicity. Specifically we evaluated the OMPC for both classic Th cell carrier activity and adjuvanticity, and the LPS component of OMPC for systemic polyclonal B cell activation. Carrier recognition of the OMPC moiety of PRP-OMPC was demonstrated. In addition the PRP-OMPC conjugate vaccine was observed to have adjuvant properties for both T cell-dependent and T cell-independent Ag in the absence of LPS-induced systemic polyclonal B cell activation. These observations suggest that in addition to functioning as a classic protein carrier whereby the proteins in OMPC provide Th cell epitopes, the OMPC also has adjuvant activity that distinguishes it from other protein carriers and may contribute to the increased immunogenicity of PRP-OMPC conjugates in animal models.     

A gel-free proteomic-based method for the characterization of Bordetella pertussis clinical isolates

Bordetella pertussis (Bp) is the etiologic agent of pertussis or whooping cough, a highly contagious respiratory disease occurring primarily in infants and young children. Although vaccine preventable, pertussis cases have increased over the years leading researchers to re-evaluate vaccine control strategies. Since bacterial outer membrane proteins, comprising the surfaceome, often play roles in pathogenesis and antibody-mediated immunity, three recent Bp circulating isolates were examined using proteomics to identify any potential changes in surface protein expression. Fractions enriched for outer membrane proteins were digested with trypsin and the peptides analyzed by nano liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS), followed by database analysis to elucidate the surfaceomes of our three Bp isolates. Furthermore, a less labor intensive non-gel based antibody affinity capture technology in conjunction with MS was employed to assess each Bp strains' immunogenic outer membrane proteins. This novel technique is generally applicable allowing for the identification of immunogenic surface expressed proteins on pertussis and other pathogenic bacteria.     

Purification and disposition of a surface protein associated with virulence of Aeromonas salmonicida.

Virulent strains of Aeromonas salmonicida observed by electron microscopy were characterized by an outer layer exhibiting a tetragonal repeat pattern. Attenuated strains had a 2.5 X 10(3)- to 5 X 10(3)-fold reduction in virulence and lost the outer layer, autoaggregating properties, and a 49-kilodalton protein (A protein) simultaneously. The A protein is the major protein component of outer membrane fractions of virulent strains. A variety of radiolabeling studies showed that this protein was surface localized and that it provided an effective barrier against iodination of other outer membrane proteins with either lactoperoxidase or diazoiodosulfanilic acid; A protein was not labeled with lactoperoxidase but was specifically labeled with diazoidosulfanilic acid. The A protein was purified by selective extraction with detergent and guanidine hydrochloride, and its amino acid composition was determined. The properties of A protein are compared with those of other bacterial surface layer proteins.     

Identification of the major outer membrane proteins of Aeromonas salmonicida

Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the etiological agent of furunculosis, a serious infectious disease of salmonids. Aeromonas spp. are ubiquitous waterborne bacteria responsible for a wide spectrum of diseases among aquatic organisms and humans. Bacterial outer membrane proteins (OMPs) play a significant role in virulence as they comprise the outermost surface in contact with host cells and immune defense factors. To identify the major OMPs of A. salmonicida a proteomic analysis was undertaken using a carbonate OMP-enrichment protocol. The enriched OMP-extracts were separated by 2-dimensional electrophoresis (2-DE) and the spots identified using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) via an electrospray ionization source. In total, 76 unique proteins were identified from the 125 spots observed on the 2-D gel. The surface layer (S-layer) VapA protein dominated the A. salmonicida OMP 2-D profile, accounting for 60% of the protein on the 2-D gels. Among the other outer membrane proteins identified were at least 10 porins and various receptors involved in nutrient acquisition. Also identified in the carbonate insoluble fraction were phosphoglycerate kinase, enolase and others that lacked classical export sorting signals. The putative association of these proteins with the cell surface might provide new insights concerning the biological and pathogenic roles of these molecules in A. salmonicida infection. This work represents the first systematic attempt to characterize the cell surface of A. salmonicida.     

Antigenic and cross-protection studies of biotype 1 and biotype 2 isolates of Yersinia ruckeri in rainbow trout, Oncorhynchus mykiss (Walbaum)

Aims: The study investigated antigen characteristics of biotype (bt) 1 and bt 2 isolates of Yersinia ruckeri. Methods and Results: The cell surface characteristics of Y. ruckeri were compared for their antigenic characteristics using polyclonal antibodies that revealed that both biotypes had a homogenous whole‐cell protein antigenic profile. Notable differences in the antigenic properties were observed in the lipopolysaccharide profile of both biotypes. Two iron‐regulated outer membrane proteins (IROMP) of c. 90 and 100 kDa were shown to be major specific antigens. The results demonstrate for the first time differences in antigens between bt 1 and bt 2 isolates of serotype O1 isolates of Y. ruckeri. The protection induced in rainbow trout by a commercial monovalent, and bivalent inactivated vaccine was tested with the outcome that the ability of isolates to cause mortality in vaccinated fish varied with geographical location. In this context, vaccination studies suggested that the O antigen was the dominant immunogenic molecule involved in protection against the disease. Conclusions: The O antigen of Y. ruckeri was the dominant immunogenic molecule involved in the protection of rainbow trout against enteric redmouth disease. Significance and Impact of the Study: There are distinct phenotypic and antigenic differences in Y. ruckeri bt 1 and bt 2 with O antigen recognized as the dominant immunogenic molecule. The data have significance in explaining the lack of success of the earlier monovalent vaccine and demonstrate the effectiveness of the newer bivalent vaccine.     

Comparative proteome analysis of Brucella melitensis vaccine strain Rev 1 and a virulent strain, 16M

The genus Brucella consists of bacterial pathogens that cause brucellosis, a major zoonotic disease characterized by undulant fever and neurological disorders in humans. Among the different Brucella species, Brucella melitensis is considered the most virulent. Despite successful use in animals, the vaccine strains remain infectious for humans. To understand the mechanism of virulence in B. melitensis, the proteome of vaccine strain Rev 1 was analyzed by two-dimensional gel electrophoresis and compared to that of virulent strain 16M. The two strains were grown under identical laboratory conditions. Computer-assisted analysis of the two B. melitensis proteomes revealed proteins expressed in either 16M or Rev 1, as well as up- or down-regulation of proteins specific for each of these strains. These proteins were identified by peptide mass fingerprinting. It was found that certain metabolic pathways may be deregulated in Rev 1. Expression of an immunogenic 31-kDa outer membrane protein, proteins utilized for iron acquisition, and those that play a role in sugar binding, lipid degradation, and amino acid binding was altered in Rev 1.     

Differences in immunogenicity and protection in mice and guinea pigs following intranasal immunization with Helicobacter pylori outer membrane antigens

Mice and guinea pigs were intranasally immunized with either recombinant lipoprotein 20 or Helicobacter pylori outer membrane vesicles (OMV). Cholera toxin was used as mucosal adjuvant. In mice, both vaccines elicited systemic and local IgG responses, which correlated with significantly lower levels of H. pylori colonization. In contrast, only OMV proved immunogenic in guinea pigs, with the development of both systemic and local immune responses. These antibodies did not, however, correlate with protection in these animals, which suggests that vaccine formulation is as important as choice of antigen in the development of an H. pylori vaccine.     

Identification of Omp38 by immunoproteomic analysis and evaluation as a potential vaccine antigen against Aeromonas hydrophila in Chinese breams

Aeromonas hydrophila is a fish pathogen causing systemic infections in aquatic environments, and determining its antigenic proteins is important for vaccine development to reduce economic losses in aquaculture worldwide. Here, an immunoproteomic approach was used to identify immunogenic outer membrane proteins (OMPs) of the Chinese vaccine strain J-1 using convalescent sera from Chinese breams. Seven unique immunogenic proteins were identified by two-dimensional (2-D) electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-TOF-MS). One protein of interest, Omp38, was expressed, and its immunogenicity and protective efficacy were evaluated in Chinese breams. The two groups of fish immunized with the inactivated vaccine and recombinant Omp38 protein showed significant serum IgM antibody levels after vaccination, compared with the fish injected with PBS buffer. In addition, the superoxide dismutase (SOD) activity, lysozyme (LSZ) activity and phagocytosis activity of head kidney lymphocytes of immunized groups were significantly higher than those of the control. The fish receiving inactivated vaccine and recombinant Omp38 protein developed a protective response to a live A. hydrophila challenge 45 days post-immunization, as demonstrated by increased survival of vaccinated fish over the control and by decreased histological alterations in vaccinated fish. Furthermore, protective effect was better in Omp38 group than in the inactivated vaccine group. These results suggest that the recombinant Omp38 protein could effectively stimulate both specific and non-specific immune responses and protect against A. hydrophila infection. Therefore, Omp38 may be developed as a potential vaccine candidate against A. hydrophila infection.     

Eukaryotic cell signaling and transcriptional activation induced by bacterial porins

The protein composition of the outer membrane of Gram-negative bacteria consists of about 20 immunochemically distinct proteins, termed outer membrane proteins (OMPs). Apart from their structural role, OMPs have been shown to have other functions, particularly with regard to transport, and have been classified as permeases and porins. Porins, during their interaction with the host, are immunogenic and also directly stimulate several cellular functions. Porins work both as molecules present on the bacterial surface and as molecules released by bacteria. Lipopolysaccharide and OMPs, the major structural macromolecular constituents of the outer membrane, carry out a fundamental role in the pathogenesis of Gram-negative infections. This brief review describes the multiple facets of the biological activities of porins both in vitro and in vivo, particularly focusing on their ability to induce the expression of cytokines and other factors that modulate cellular activities with either pathological or adaptive consequences. This brief discussion will focus on the signal transmission mechanisms induced by bacterial porins.     

Cloning and expression of an outer membrane protein ompTS of Aeromonas hydrophila and study of immunogenicity in fish

The outer membrane proteins of the warm water fish pathogen, Aeromonas hydrophila have a role in the virulence of the organism and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein designated ompTS was amplified by PCR excluding the region coding for signal peptide, cloned in pQE 30-UA Vector and expressed using induction with isopropyl thiogalactoside (IPTG). The size of the expressed protein was 37 kDa as estimated by migration in 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal antibodies were raised in mice and rabbit against the purified protein and the reaction of the antibody was confirmed by Western blotting using the purified protein and A. hydrophila cultures. The Indian major carp, Labeo rohita Hamilton was immunized using the purified protein and developed antibodies with mean titers of 1:4000 on day 14 and 1:12,000 on day 28 showing promise that the protein is highly immunogenic in fish.     

Vaccine efficacy in spotted wolffish Anarhichas minor: relationship to molecular variation in A-layer protein of atypical Aeromonas salmonicida

Atypical Aeromonas salmonicida strains comprise a heterogeneous group in terms of molecular and phenotypic characteristics. They cause various conditions of ulcer diseases or atypical furunculosis and are being isolated in increasing number from various fish species and geographical areas. Several marine fish species susceptible to atypical A. salmonicida, including spotted wolffish Anarhichas minor O., are now being farmed and new vaccines may be needed. A commercial furunculosis vaccine for salmon is reported to protect wolffish poorly against experimental challenge with atypical A. salmonicida. The protective antigen(s) in furunculosis vaccines is still unclear, but in oil-adjuvanted vaccine for Atlantic salmon Salmo salar L., the surface A-layer was shown to be important for protection. In spotted wolffish, the efficacy of atypical furunculosis vaccines seems to vary with the atypical A. salmonicida strains used as bacterin in the vaccine. In the present study we investigated whether differences in the A-layer protein among atypical strains might be responsible for the observed variation in vaccine efficacy. Atypical A. salmonicida strains from 16 fish species in 11 countries were compared by genome polymorphism analysis using amplified fragment length polymorphism (AFLP) fingerprinting and by comparative sequencing of the vapA genes encoding the A-protein. The A-protein sequences appeared to be highly conserved except for a variable region between Residues 90 to 170. Surprisingly, the grouping of strains based on AFLP- or A-protein sequence similarities was consistent. In addition, serological differences in the A-protein among the strains were demonstrated by an A-protein-specific monoclonal antibody. Vaccines based on atypical A. salmonicida strains possessing genetically and serologically different A-layer proteins were shown to result in significantly different protection in spotted wolffish.     

Biochemical and immunological characterization of the cell surface of the fish pathogenic bacterium Aeromonas salmonicida

The identification of lipopolysaccharide as periodic acid-Schiff positive material, present in the membrane fraction of the fish pathogenic Gram-negative bacterium Aeromonas salmonicida, analyzed by SDS-polyacrylamide gel electrophoresis, is shown. Such analysis has revealed several periodic acid-Schiff positive bands and many membrane proteins among which a pathogenicity-related Mr 54000 protein as a constituent of an additional surface layer outside the outer membrane (Evenberg et al., (1982) Biochim. Biophys. Acta 684, 241-248). The latter protein, designated as additional cell envelope protein or ACE protein, has been purified and characterized in our laboratory (Evenberg and Lugtenberg, (1982) Biochim. Biophys. Acta 684, 249-254). Most strains produce both high and low molecular weight lipopolysaccharide species, presumably corresponding with the presence and (virtual) absence, respectively, of an O-antigenic chain. The property to produce high molecular weight lipopolysaccharide can be lost upon subculturing in laboratory growth media and such is greatly enhanced by the prior loss of the ability to produce ACE protein. Lipopolysaccharide and ACE protein were identified as the major antigens. A new polysaccharide-like antigen, designated as PS-antigen, was detected. Moreover, immunological indications for the presence of a lipoprotein in A. salmonicida are described. The surface localization of the antigens was determined by testing whether preadsorption of antisera by intact cells decreased the binding of IgG to these antigens, or decreased the ability of the sera to agglutinate cells. According to these criteria lipopolysaccharide, ACE protein and PS-antigen are the major surface-located antigens. Material cross-reactive with lipopolysaccharide, ACE protein and PS-antigen has been found in a large number of strains. Several lines of evidence indicate the presence of interactions between ACE protein and lipopolysaccharide. Based on these results a molecular model of the cell envelope of virulent A. salmonicida is presented.     

Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimensional gel electrophoresis and mass spectrometry

Helicobacter pylori is one of the most common bacterial pathogens and causes a variety of diseases, such as peptic ulcer or gastric cancer. Despite intensive study of this human pathogen in the last decades, knowledge about its membrane proteins and, in particular, those which are putative components of the type IV secretion system encoded by the cag pathogenicity island (PAI) remains limited. Our aim is to establish a dynamic two-dimensional electrophoresis-polyacrylamide gel electrophoresis (2-DE-PAGE) database with multiple subproteomes of H. pylori (http://www.mpiib-berlin.mpg.de/2D-PAGE) which facilitates identification of bacterial proteins important in pathogen-host interactions. Using a proteomic approach, we investigated the protein composition of two H. pylori fractions: soluble proteins and structure-bound proteins (including membrane proteins). Both fractions differed markedly in the overall protein composition as determined by 2-DE. The 50 most abundant protein spots in each fraction were identified by peptide mass fingerprinting. We detected four cag PAI proteins, numerous outer membrane proteins (OMPs), the vacuolating cytotoxin VacA, other potential virulence factors, and few ribosomal proteins in the structure-bound fraction. In contrast, catalase (KatA), gamma-glutamyltranspeptidase (Ggt), and the neutrophil-activating protein NapA were found almost exclusively in the soluble protein fraction. The results presented here are an important complement to genome sequence data, and the established 2-D PAGE maps provide a basis for comparative studies of the H. pylori proteome. Such subproteomes in the public domain will be effective instruments for identifying new virulence factors and antigens of potential diagnostic and/or curative value against infections with this important pathogen.     

Molecular cloning and nucleotide sequence analysis of the maltose-inducible porin gene of Aeromonas salmonicida

Abstract The gene for the Aeromonas salmonicida maltose-inducible porin (maltoporin) was cloned into phagemid pTZ18R in two restriction fragments, 0.6-kb Pst I/ Kpn I and 1.7-kb Sph I, of genomic DNA and their nucleotide sequences were determined. Open reading frames of 1329 and 1335 bp translated into sequences of 443 and 445 amino acids, with a 23 or 25 amino acid signal sequence and a 420 amino acid mature protein of molecular mass 46424 Da. Putative ribosome binding sites, AGGA and GGGAA, occurred 9 bp upstream of two possible ATG initiation codons. The A. salmonicida gene product showed a high degree of similarity with Escherichia coli LamB, and codon usage was very similar to that of another A. salmonicida outer membrane protein but markedly different from those of extracellular proteins.     

Identification of vaccine candidate antigens of an ESBL producing Klebsiella pneumoniae clinical strain by immunoproteome analysis

Klebsiella pneumoniae is an opportunistic pathogen which causes pneumoniae, urinary tract infections and septicemia in immunocompromised patients. Hospital outbreaks of multidrug-resistant K. pneumoniae, especially those in neonatal wards, are often caused by strains producing the extended-spectrum-beta-lactamases (ESBLs). An immunoproteome based approach was developed to identify candidate antigens of K. pneumoniae for vaccine development. Sera from patients with acute K. pneumoniae infections (n = 55) and a control group of sera from healthy individuals (n = 15) were analyzed for reactivity by Western blot against ESBL K. pneumoniae outer membrane proteins separated by 2-DE. Twenty highly immunogenic protein spots were identified by immunoproteomic analysis. The immunogenic proteins that are most frequently recognized by positive K. pneumoniae sera were OmpA, OmpK36, FepA, OmpK17, OmpW, Colicin I receptor protein and three novel proteins. Two of the vaccine candidate genes, OmpA (Struve et al. Microbiology 2003, 149, 167-176) and FepA (Lai, Y. C. et al.. Infect Immun 2001, 69, 7140-7145), have recently been shown to be essential in colonization and infection in an in vivo mouse model. Hence, these two immunogenic proteins could serve as potential vaccine candidates.