Decoration of Burkholderia Hcp1 protein to virus-like particles as a vaccine delivery platform.

Virus-like particles (VLPs) are protein-based nanoparticles frequently used as carriers in conjugate vaccine platforms. VLPs have been used to display foreign antigens for vaccination and to deliver immunotherapy against diseases. Hemolysin-coregulated proteins 1 (Hcp1) is a protein component of the Burkholderia type 6 secretion system, which participates in intracellular invasion and dissemination. This protein has been reported as a protective antigen and is used in multiple vaccine candidates with various platforms against melioidosis, a severe infectious disease caused by the intracellular pathogen Burkholderia pseudomallei. In this study, we used P22 VLPs as a surface platform for decoration with Hcp1 using chemical conjugation. C57BL/6 mice were intranasally immunized with three doses of either PBS, VLPs, or conjugated Hcp1-VLPs. Immunization with Hcp1-VLPs formulation induced Hcp1-specific IgG, IgG1, IgG2c, and IgA antibody responses. Furthermore, the serum from Hcp1-VLPs immunized mice enhanced the bacterial uptake and opsonophagocytosis by macrophages in the presence of complement. This study demonstrated an alternative strategy to develop a VLPs-based vaccine platform against Burkholderia species.

Biofilm-producing Escherichia coli O104:H4 overcomes bile salts toxicity by expressing virulence and resistance proteins.

We investigated bile salts' ability to induce phenotypic changes in biofilm production and protein expression of pathogenic Escherichia coli strains. For this purpose, 82 pathogenic E. coli strains isolated from humans (n = 70), and animals (n = 12), were examined for their ability to form biofilms in the presence or absence of bile salts. We also identified bacterial proteins expressed in response to bile salts using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-electrophoresis) and liquid chromatography-mass spectrometry (LC-MS/MS). Lastly, we evaluated the ability of these strains to adhere to Caco-2 epithelial cells in the presence of bile salts. Regarding biofilm formation, two strains isolated from an outbreak in Republic of Georgia in 2009 were the only ones that showed a high and moderate capacity to form biofilm in the presence of bile salts. Further, we observed that those isolates, when in the presence of bile salts, expressed different proteins identified as outer membrane proteins (i.e. OmpC), and resistance to adverse growth conditions (i.e. F0F1, HN-S, and L7/L12). We also found that these isolates exhibited high adhesion to epithelial cells in the presence of bile salts. Together, these results contribute to the phenotypic characterization of E. coli O104: H4 strains.

Evaluating the Contribution of the Predicted Toxin-Antitoxin System HigBA to Persistence, Biofilm Formation, and Virulence in Burkholderia pseudomallei.

Melioidosis is an underreported human disease caused by the Gram-negative intracellular pathogen Burkholderia pseudomallei (Bpm). Both the treatment and the clearance of the pathogen are challenging, with high relapse rates leading to latent infections. This has been linked to the bacterial persistence phenomenon, a growth arrest strategy that allows bacteria to survive under stressful conditions, as in the case of antibiotic treatment, within a susceptible clonal population. At a molecular level, this phenomenon has been associated with the presence of toxin-antitoxin (TA) systems. We annotated the Bpm K96243 genome and selected 11 pairs of genes encoding for these TA systems, and their expression was evaluated under different conditions (supralethal antibiotic conditions; intracellular survival bacteria). The predicted HigB toxin (BPSL3343) and its predicted antitoxin HigA (BPS_RS18025) were further studied using mutant construction. The phenotypes of two mutants (ΔhigB and ΔhigB ΔhigA) were evaluated under different conditions compared to the wild-type (WT) strain. The ΔhigB toxin mutant showed a defect in intracellular survival on macrophages, a phenotype that was eliminated after levofloxacin treatment. We found that the absence of the toxin provides an advantage over the WT strain, in both in vitro and in vivo models, during persister conditions induced by levofloxacin. The lack of the antitoxin also resulted in differential responses to the conditions evaluated, and under some conditions, it restored the WT phenotype, overall suggesting that both toxin and antitoxin components play a role in the persister-induced phenotype in Bpm.

Development of Melioidosis Subunit Vaccines Using an Enzymatically Inactive Burkholderia pseudomallei AhpC.

Burkholderia pseudomallei, the causative agent of melioidosis, is a facultative intracellular, Gram-negative pathogen that is highly infectious via the respiratory route and can cause severe, debilitating, and often fatal diseases in humans and animals. At present, no licensed vaccines for immunization against this CDC Tier 1 select agent exist. Studies in our lab have previously demonstrated that subunit vaccine formulations consisting of a B. pseudomallei capsular polysaccharide (CPS)-based glycoconjugate (CPS-CRM197) combined with hemolysin-coregulated protein (Hcp1) provided C57BL/6 mice with high-level protection against an acute inhalational challenge of B. pseudomallei. In this study, we evaluated the immunogenicity and protective capacity of B. pseudomallei alkyl hydroperoxide reductase subunit C (AhpC) in combination with CPS-CRM197. AhpC is a peroxiredoxin involved in oxidative stress reduction and is a potential protective antigen. To facilitate our studies and maximize safety in animals, recombinant B. pseudomallei AhpC harboring an active site mutation (AhpCC57G) was expressed in Escherichia coli and purified using tandem nickel-cobalt affinity chromatography. Immunization of C57BL/6 mice with CPS-CRM197 combined with AhpCC57G stimulated high-titer IgG responses against the CPS component of the glycoconjugate as well as stimulated high-titer IgG and robust interferon gamma (IFN-γ)-, interleukin-5 (IL-5)-, and IL-17-secreting T cell responses against AhpCC57G. When challenged via an inhalational route with a high dose (~27 50% lethal doses [LD50s]) of B. pseudomallei, 70% of the immunized mice survived 35 days postchallenge. Collectively, our findings demonstrate that AhpCC57G is a potent activator of cellular and humoral immune responses and may be a promising candidate to include in future melioidosis subunit vaccines.

Unexplored Challenges of Minoritized Microbiologists in Academia.

The scientific community is making significant efforts to be inclusive and to promote diversity and equity. The microbial sciences are not the exception, and organizations, such as the American Society for Microbiology (ASM), are implementing strategic plans to advance these initiatives. However, one unexplored topic is whether the recruitment of minoritized microbiologists should use tailored programs for the success of trainees and faculty. Some challenges and opportunities are presented for consideration while developing recruitment, retention, and advancement programs in the microbial sciences.

Genomic Diversity of Burkholderia pseudomallei Isolates, Colombia.

We report an analysis of the genomic diversity of isolates of Burkholderia pseudomallei, the cause of melioidosis, recovered in Colombia from routine surveillance during 2016-2017. B. pseudomallei appears genetically diverse, suggesting it is well established and has spread across the region.

Description of two fatal cases of melioidosis in Mexican children with acute pneumonia: case report.

BACKGROUND: Melioidosis is an infectious disease caused by Burkholderia pseudomallei. In Mexico, the disease is rarely diagnosed in humans and there is no evidence of simultaneous environmental isolation of the pathogen. Here, we describe clinical profiles of fatal cases of melioidosis in two children, in a region without history of that disease. CASE PRESENTATION: About 48 h before onset of symptoms, patients swam in a natural body of water, and thereafter they rapidly developed fatal septicemic illness. Upon necropsy, samples from liver, spleen, lung, cerebrospinal fluid, and bronchial aspirate tissues contained Burkholderia pseudomallei. Environmental samples collected from the locations where the children swam also contained B. pseudomallei. All the clinical and environmental strains showed the same BOX-PCR pattern, suggesting that infection originated from the area where the patients were swimming. CONCLUSIONS: The identification of B. pseudomallei confirmed that melioidosis disease exists in Sonora, Mexico. The presence of B. pseudomallei in the environment may suggest endemicity of the pathogen in the region. This study highlights the importance of strengthening laboratory capacity to prevent and control future melioidosis cases.

Comparative genomics of a subset of Adherent/Invasive Escherichia coli strains isolated from individuals without inflammatory bowel disease.

There is increased evidence demonstrating the association between Crohn's Disease (CD), a type of Inflammatory Bowel Disease (IBD), and non-diarrheagenic Adherent/Invasive Escherichia coli (AIEC) isolates. AIEC strains are phenotypically characterized by their adhesion, invasion and intra-macrophage survival capabilities. In the present study, the genomes of five AIEC strains isolated from individuals without IBD (four from healthy donors and one from peritoneal liquid) were sequenced and compared with AIEC prototype strains (LF82 and NRG857c), and with extra-intestinal uropathogenic strain (UPEC CFT073). Non-IBD-AIEC strains showed an Average Nucleotide Identity up to 98% compared with control strains. Blast identities of the five non-IBD-AIEC strains were higher when compared to AIEC and UPEC reference strains than with another E. coli pathotypes, suggesting a relationship between them. The SNPs phylogeny grouped the five non-IBD-AIEC strains in one separated cluster, which indicates the emergence of these strains apart from the AIEC group. Additionally, four genomic islands not previously reported in AIEC strains were identified. An incomplete Type VI secretion system was found in non-IBD-AIEC strains; however, the Type II secretion system was complete. Several groups of genes reported in AIEC strains were searched in the five non-IBD-AIEC strains, and the presence of fimA, fliC, fuhD, chuA, irp2 and cvaC were confirmed. Other virulence factors were detected in non-IBD-AIEC strains, which were absent in AIEC reference strains, including EhaG, non-fimbrial adhesin 1, PapG, F17D-G, YehA/D, FeuC, IucD, CbtA, VgrG-1, Cnf1 and HlyE. Based on the differences in virulence determinants and SNPs, it is plausible to suggest that non-IBD AIEC strains belong to a different pathotype.

Burkholderia pseudomallei as an Enteric Pathogen: Identification of Virulence Factors Mediating Gastrointestinal Infection.

Burkholderia pseudomallei is a Gram-negative bacterium and the causative agent of melioidosis. Despite advances in our understanding of the disease, B. pseudomallei poses a significant health risk, especially in regions of endemicity, where treatment requires prolonged antibiotic therapy. Even though the respiratory and percutaneous routes are well documented and considered the main ways to acquire the pathogen, the gastrointestinal tract is believed to be an underreported and underrecognized route of infection. In the present study, we describe the development of in vitro and in vivo models to study B. pseudomallei gastrointestinal infection. Further, we report that the type 6 secretion system (T6SS) and type 1 fimbriae are important virulence factors required for gastrointestinal infection. Using a human intestinal epithelial cell line and mouse primary intestinal epithelial cells (IECs), we demonstrated that B. pseudomallei adheres, invades, and forms multinucleated giant cells, ultimately leading to cell toxicity. We demonstrated that mannose-sensitive type 1 fimbria is involved in the initial adherence of B. pseudomallei to IECs, although the impact on full virulence was limited. Finally, we also showed that B. pseudomallei requires a functional T6SS for full virulence, bacterial dissemination, and lethality in mice infected by the intragastric route. Overall, we showed that B. pseudomallei is an enteric pathogen and that type 1 fimbria is important for B. pseudomallei intestinal adherence, and we identify a new role for T6SS as a key virulence factor in gastrointestinal infection. These studies highlight the importance of gastrointestinal melioidosis as an understudied route of infection and open a new avenue for the pathogenesis of B. pseudomallei.

Evaluating the role of Burkholderia pseudomallei K96243 toxins BPSS0390, BPSS0395, and BPSS1584 in persistent infection.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with a mortality rate of up to 40% even with treatment. Despite the ability of certain antibiotics to control initial infection, relapse occurs in treated patients. The inability of antibiotics to clear this bacterial infection is in part due to persistence, an evasion mechanism against antibiotics and the effect of host defenses. Evaluation of antibiotic efficacy against B. pseudomallei revealed that up to 48% of in vitro grown populations can survive in a persister state. Toxin-antitoxin (TA) systems have been previously implicated in modulating bacterial persistence. We generated three isogenic TA mutants and found that loss of each toxin gene did not alter antibiotic persistence or macrophage survival. In response to macrophage-induced persistence, all three toxin mutants demonstrated increased intracellular susceptibility to levofloxacin which in part was due to the inability of the mutants to induce persistence after nitric oxide or nutrient starvation. In an inhalational model of murine melioidosis, both ΔBPSS0395 and ΔBPSS1584 strains were attenuated, and treatment with levofloxacin led to significant reduction in lung colonisation and reduced splenic colonisation by ΔBPSS0395. Based on our findings, these toxins deserve additional evaluation as putative therapeutic targets.

Increased Mortality in Mice following Immunoprophylaxis Therapy with High Dosage of Nicotinamide in Burkholderia Persistent Infections.

Bacterial persistence, known as noninherited antibacterial resistance, is a factor contributing to the establishment of long-lasting chronic bacterial infections. In this study, we examined the ability of nicotinamide (NA) to potentiate the activity of different classes of antibiotics against Burkholderia thailandensis persister cells. Here we demonstrate that addition of NA in in vitro models of B. thailandensis infection resulted in a significant depletion of the persister population in response to various classes of antibiotics. We applied microfluidic bioreactors with a continuous medium flow to study the effect of supplementation with an NA gradient on the recovery of B. thailandensis persister populations. A coculture of human neutrophils preactivated with 50 µM NA and B. thailandensis resulted in the most efficient reduction in the persister population. Applying single-cell RNA fluorescence in situ hybridization analysis and quantitative PCR, we found that NA inhibited gene expression of the stringent response regulator relA, implicated in the regulation of the persister metabolic state. We also demonstrate that a therapeutic dose of NA (250 mg/kg of body weight), previously applied as immunoprophylaxis against antibiotic-resistant bacterial species, produced adverse effects in an in vivo murine model of infection with the highly pathogenic bacterium Burkholderia pseudomallei, indicating that therapeutic dose and metabolite effects have to be carefully evaluated and tailored for every case of potential clinical application.

Development of Subunit Vaccines That Provide High-Level Protection and Sterilizing Immunity against Acute Inhalational Melioidosis.

Burkholderia pseudomallei, the etiologic agent of melioidosis, causes severe disease in humans and animals. Diagnosis and treatment of melioidosis can be challenging, and no licensed vaccines currently exist. Several studies have shown that this pathogen expresses a variety of structurally conserved protective antigens that include cell surface polysaccharides and cell-associated and cell-secreted proteins. Based on those findings, such antigens have become important components of the subunit vaccine candidates that we are currently developing. In the present study, the 6-deoxyheptan capsular polysaccharide (CPS) from B. pseudomallei was purified, chemically activated, and covalently linked to recombinant CRM197 diphtheria toxin mutant (CRM197) to produce CPS-CRM197. Additionally, tandem nickel-cobalt affinity chromatography was used to prepare highly purified recombinant B. pseudomallei Hcp1 and TssM proteins. Immunization of C57BL/6 mice with CPS-CRM197 produced high-titer IgG and opsonizing antibody responses against the CPS component of the glycoconjugate, while immunization with Hcp1 and TssM produced high-titer IgG and robust gamma interferon-secreting T cell responses against the proteins. Extending upon these studies, we found that when mice were vaccinated with a combination of CPS-CRM197 and Hcp1, 100% of the mice survived a lethal inhalational challenge with B. pseudomallei Remarkably, 70% of the survivors had no culturable bacteria in their lungs, livers, or spleens, indicating that the vaccine formulation had generated sterilizing immune responses. Collectively, these studies help to better establish surrogates of antigen-induced immunity against B. pseudomallei as well as provide valuable insights toward the development of a safe, affordable, and effective melioidosis vaccine.

Burkholderia mallei CLH001 Attenuated Vaccine Strain Is Immunogenic and Protects against Acute Respiratory Glanders.

Burkholderia mallei is the causative agent of glanders, an incapacitating disease with high mortality rates in respiratory cases. Its endemicity and ineffective treatment options emphasize its public health threat and highlight the need for a vaccine. Live attenuated vaccines are considered the most viable vaccine strategy for Burkholderia, but single-gene-deletion mutants have not provided complete protection. In this study, we constructed the select-agent-excluded B. mallei ΔtonB Δhcp1 (CLH001) vaccine strain and investigated its ability to protect against acute respiratory glanders. Here we show that CLH001 is attenuated, safe, and effective at protecting against lethal B. mallei challenge. Intranasal administration of CLH001 to BALB/c and NOD SCID gamma (NSG) mice resulted in complete survival without detectable colonization or abnormal organ histopathology. Additionally, BALB/c mice intranasally immunized with CLH001 in a prime/boost regimen were fully protected against lethal challenge with the B. mallei lux (CSM001) wild-type strain.

Polysorbates prevent biofilm formation and pathogenesis of Escherichia coli O104:H4.

Escherichia coli biotype O104:H4 recently caused the deadliest E. coli outbreak ever reported. Based on prior results, it was hypothesized that compounds inhibiting biofilm formation by O104:H4 would reduce its pathogenesis. The nonionic surfactants polysorbate 80 (PS80) and polysorbate 20 (PS20) were found to reduce biofilms by ≥ 90% at submicromolar concentrations and elicited nearly complete dispersal of preformed biofilms. PS80 did not significantly impact in vivo colonization in a mouse infection model; however, mice treated with PS80 exhibited almost no intestinal inflammation or tissue damage while untreated mice exhibited robust pathology. As PS20 and PS80 are classified as 'Generally Recognized as Safe' (GRAS) compounds by the Food and Drug Administration (FDA), these compounds have clinical potential to treat future O104:H4 outbreaks.

Finding Regulators Associated with the Expression of the Long Polar Fimbriae in Enteropathogenic Escherichia coli.

UNLABELLED: Enteropathogenic Escherichia coli (EPEC) is a human pathogen that requires initial adhesion to the intestine in order to cause disease. Multiple adhesion factors have been identified in E. coli strains, among them the long polar fimbriae (Lpf), a colonization factor associated with intestinal adhesion. The conditions of Lpf expression are well understood in enterohemorrhagic E. coli (EHEC); however, the expression of EPEC lpf has been found to be repressed under any in vitro condition tested. Therefore, we decided to identify those factors silencing expression of EPEC lpf. Because histone-like nucleoid structuring protein (H-NS) is a known repressor of EHEC lpf, we tested it and found that H-NS is a repressor of EPEC lpf. We also found that the adhesion of the EPEC Δhns strain was significantly enhanced compared to the wild-type strain. Because lpf expression was modestly increased in the hns mutant, transposon mutagenesis was performed to find a strain displaying higher lpf expression than EPEC Δhns. One Tn5 insertion was identified within the yhjX gene, and further in vitro characterization revealed increased lpf expression and adhesion to Caco-2 cells compared with EPEC Δhns. However, in a murine model of intestinal infection, the EPEC Δhns and EPEC Δhns Tn5 mutants had only a slight change in colonization pattern compared to the wild-type strain. Our data showed that EPEC Lpf is transcribed, but its role in EPEC intestinal colonization requires further analysis. IMPORTANCE: Data are presented demonstrating that the long polar fimbriae (lpf) operon in enteropathogenic E. coli (EPEC) is highly regulated; however, derepression occurs by mutagenizing two proteins associated with its control. The study demonstrates that the EPEC lpf operon can be expressed and, therefore, participates in the EPEC adherence phenotype.

The Escherichia coli O157:H7 cattle immunoproteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine rectoanal junction squamous epithelial (RSE) cells.

Building on previous studies, we defined the repertoire of proteins comprising the immunoproteome (IP) of Escherichia coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (O157 IP), a ß-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called "proteomics-based expression library screening" (PELS; Kudva et al., 2006). The E. coli O157 IP (O157-IP) comprised 91 proteins, and included those identified previously using proteomics-based expression library screening, and also proteins comprising DMEM and bovine rumen fluid proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured HEp-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine rectoanal junction squamous epithelial cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to rectoanal junction squamous epithelial cells, and additionally implicate a possible role for the outer membrane protein A regulator, TdcA, in the expression of such adhesins. Our observations have implications for the development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract.

The IbeA invasin of adherent-invasive Escherichia coli mediates interaction with intestinal epithelia and macrophages.

Adherent-invasive Escherichia coli (AIEC) pathogroup isolates are a group of isolates from the intestinal mucosa of Crohn's disease patients that can invade intestinal epithelial cells (IECs) or macrophages and survive and/or replicate within. We have identified the ibeA gene in the genome of AIEC strain NRG857c and report the contribution of IbeA to the interaction of AIEC with IECs and macrophages and colonization of the mouse intestine. An ibeA deletion mutant strain (NRG857cΔibeA) was constructed, and the in vitro effect on AIEC adhesion and invasion of nonpolarized and polarized Caco-2 cells, the adhesion and transcytosis of M-like cells, the intracellular survival in THP-1 macrophages, and the contribution to intestinal colonization of the CD-1 murine model of infection were evaluated. A significant reduction in invasion was observed with the ibeA mutant in Caco-2 and M-like cells, whereas adhesion was not affected. Complementation of the mutant reestablished Caco-2 invasive phenotype to wild-type levels. Reduction in invasion did not significantly affect transcytosis through M-like cells at early time points. The absence of ibeA significantly affected AIEC intramacrophage survival up to 24 h postinfection. No significant changes associated with IbeA were found in AIEC colonization across the murine gastrointestinal tract, but a slight reduction of gamma interferon was observed in the ceca of mice infected with the ibeA mutant. In addition, a decrease in the pathology scores was observed in the ilea and ceca of mice infected with the ibeA mutant. Our data support the function of IbeA in the AIEC invasion process, macrophage survival, and inflammatory response in the murine intestine.

Consensus on the development of vaccines against naturally acquired melioidosis.

Several candidates for a vaccine against Burkholderia pseudomallei, the causal bacterium of melioidosis, have been developed, and a rational approach is now needed to select and advance candidates for testing in relevant nonhuman primate models and in human clinical trials. Development of such a vaccine was the topic of a meeting in the United Kingdom in March 2014 attended by international candidate vaccine developers, researchers, and government health officials. The focus of the meeting was advancement of vaccines for prevention of natural infection, rather than for protection from the organism's known potential for use as a biological weapon. A direct comparison of candidate vaccines in well-characterized mouse models was proposed. Knowledge gaps requiring further research were identified. Recommendations were made to accelerate the development of an effective vaccine against melioidosis.

A gold nanoparticle-linked glycoconjugate vaccine against Burkholderia mallei.

Burkholderia mallei are Gram-negative bacteria, responsible for the disease glanders. B. mallei has recently been classified as a Tier 1 agent owing to the fact that this bacterial species can be weaponised for aerosol release, has a high mortality rate and demonstrates multi-drug resistance. Furthermore, there is no licensed vaccine available against this pathogen. Lipopolysaccharide (LPS) has previously been identified as playing an important role in generating host protection against Burkholderia infection. In this study, we present gold nanoparticles (AuNPs) functionalised with a glycoconjugate vaccine against glanders. AuNPs were covalently coupled with one of three different protein carriers (TetHc, Hcp1 and FliC) followed by conjugation to LPS purified from a non-virulent clonal relative, B. thailandensis. Glycoconjugated LPS generated significantly higher antibody titres compared with LPS alone. Further, they improved protection against a lethal inhalation challenge of B. mallei in the murine model of infection. FROM THE CLINICAL EDITOR: Burkholderia mallei is associated with multi-drug resistance, high mortality and potentials for weaponization through aerosol inhalation. The authors of this study present gold nanoparticles (AuNPs) functionalized with a glycoconjugate vaccine against this Gram negative bacterium demonstrating promising results in a murine model even with the aerosolized form of B. Mallei.

Comparative genomics and immunoinformatics approach for the identification of vaccine candidates for enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains are major human food-borne pathogens, responsible for bloody diarrhea and hemolytic-uremic syndrome worldwide. Thus far, there is no vaccine for humans against EHEC infections. In this study, a comparative genomics analysis was performed to identify EHEC-specific antigens useful as potential vaccines. The genes present in both EHEC EDL933 and Sakai strains but absent in nonpathogenic E. coli K-12 and HS strains were subjected to an in silico analysis to identify secreted or surface-expressed proteins. We obtained a total of 65 gene-encoding protein candidates, which were subjected to immunoinformatics analysis. Our criteria of selection aided in categorizing the candidates as high, medium, and low priority. Three members of each group were randomly selected and cloned into pVAX-1. Candidates were pooled accordingly to their priority group and tested for immunogenicity against EHEC O157:H7 using a murine model of gastrointestinal infection. The high-priority (HP) pool, containing genes encoding a Lom-like protein (pVAX-31), a putative pilin subunit (pVAX-12), and a fragment of the type III secretion structural protein EscC (pVAX-56.2), was able to induce the production of EHEC IgG and sIgA in sera and feces. HP candidate-immunized mice displayed elevated levels of Th2 cytokines and diminished cecum colonization after wild-type challenge. Individually tested HP vaccine candidates showed that pVAX-12 and pVAX-56.2 significantly induced Th2 cytokines and production of fecal EHEC sIgA, with pVAX-56.2 reducing EHEC cecum colonization. We describe here a bioinformatics approach able to identify novel vaccine candidates potentially useful for preventing EHEC O157:H7 infections.