Most pathogens enter the body through mucosal surfaces. Mucosal immunization, a non-invasive needle-free route, often stimulates a mucosal immune response that is both effective against mucosal and systemic pathogens. The development of mucosally administered heat-stable vaccines with long shelf life would therefore significantly enhance immunization programs in developing countries by avoiding the need for a cold chain or systemic injections. Currently, recombinant vaccine carriers are being used for antigen delivery. Engineering Bacillus subtilis for use as a non-invasive and heat stable antigen delivery system has proven successful. Bacterial spores protected by multiple layers of protein are known to be robust and resistant to desiccation. Stable constructs have been created by integration into the bacterial chromosome of immunogens. The spore coat has been used as a vehicle for heterologous antigen presentation and protective immunization. Sublingual (SL) and intranasal (IN) routes have recently received attention as delivery routes for therapeutic drugs and vaccines and recent attempts by several investigators, including our group, to develop vaccines that can be delivered intranasally and sublingually have met with a lot of success.
As discussed in this Review, the use of Bacillus subtilis to express antigens that can be administered either intranasally or sublingually is providing new insights in the area of mucosal vaccines. In our work, we evaluated the efficacy of SL and IN immunizations with B. subtilis engineered to express tetanus toxin fragment C (TTFC) in mice and piglets. These bacteria engineered to express heterologous antigen either on the spore surface or within the vegetative cell have been used for oral, IN and SL delivery of antigens. A Bacillus subtilis spore coat protein, CotC was used as a fusion partner to express the tetanus fragment C. B. subtilis spores known to be highly stable and safe are also easy to purify making this spore-based display system a potentially powerful approach for surface expression of antigens. These advances will help to accelerate the development and testing of new mucosal vaccines against many human and animal diseases.
Bacillus subtilis; tetanus; vaccine
Shiga toxin (Stx)-producing Escherichia coli cause severe intestinal infections involving colonization of epithelial Peyer’s patches and formation of attachment/effacement (A/E) lesions. These lesions trigger leukocyte infiltration followed by inflammation and intestinal hemorrhage. Systems biology, which explores the crosstalk of Stx-producing Escherichia coli with the in vivo host environment, may elucidate novel molecular pathogenesis aspects.
Enterohemorrhagic E. coli strain 86–24 produces Shiga toxin-2 and belongs to the serotype O157:H7. Bacterial cells were scrapped from stationary phase cultures (the in vitro condition) and used to infect gnotobiotic piglets via intestinal lavage. Bacterial cells isolated from the piglets’ guts constituted the in vivo condition. Cell lysates were subjected to quantitative 2D gel and shotgun proteomic analyses, revealing metabolic shifts towards anaerobic energy generation, changes in carbon utilization, phosphate and ammonia starvation, and high activity of a glutamate decarboxylase acid resistance system in vivo. Increased abundance of pyridine nucleotide transhydrogenase (PntA and PntB) suggested in vivo shortage of intracellular NADPH. Abundance changes of proteins implicated in lipopolysaccharide biosynthesis (LpxC, ArnA, the predicted acyltransferase L7029) and outer membrane (OM) assembly (LptD, MlaA, MlaC) suggested bacterial cell surface modulation in response to activated host defenses. Indeed, there was evidence for interactions of innate immunity-associated proteins secreted into the intestines (GP340, REG3-γ, resistin, lithostathine, and trefoil factor 3) with the bacterial cell envelope.
Proteomic analysis afforded insights into system-wide adaptations of strain 86–24 to a hostile intestinal milieu, including responses to limited nutrients and cofactor supplies, intracellular acidification, and reactive nitrogen and oxygen species-mediated stress. Protein and lipopolysaccharide compositions of the OM were altered. Enhanced expression of type III secretion system effectors correlated with a metabolic shift back to a more aerobic milieu in vivo. Apparent pathogen pattern recognition molecules from piglet intestinal secretions adhered strongly to the bacterial cell surface.
Easy access to next generation sequencing has enabled the rapid analysis of complex microbial populations. To take full advantage of these technologies, animal models enabling the manipulation of human microbiomes and the study of the impact of such perturbations on the host are needed. To this aim we are developing experimentally tractable and clinically relevant pig models of the human adult and infant gastro-intestinal tract. The intestine of germ-free piglets was populated with human adult or infant fecal microbial populations, and the piglets were maintained on solid or milk diet, respectively. Amplicons of 16S rRNA V6 region were deep-sequenced to monitor to what extent the transplanted human microbiomes changed in the pig. Within 24 h of transfer of human fecal microbiome to pigs, bacterial microbiomes rich in Proteobacteria emerged. These populations evolved toward a more diverse composition rich in Bacteroidetes and Firmicutes. In the experiment where infant microbiome was used, the phylogenetic composition of the transplanted bacterial population converged toward that of the human inoculum. A majority of sequences belonged to a relatively small number of operational taxonomic units, whereas at the other end of the abundance spectrum, a large number of rare and transient OTUs were detected. Analysis of fecal and colonic microbiomes originating from the same animal indicate that feces closely replicate the colonic microbiome. We conclude that the pig intestine can be colonized with human fecal microbiomes to generate a realistic model of the human GI tract.
pig model; intestinal microbiome; 16S rRNA amplicon sequencing; principal coordinate analysis; microbial diversity
Cryptosporidium hominis and Cryptosporidium
parvum, which infect humans equally, are genetically/antigenically almost identical. It remains unclear, however, whether infection with C. hominis protects against C. parvum. Gnotobiotic piglets were used to investigate cross-protection. After ≥3 days of recovery from C. hominis infection, the piglets were completely protected against subsequent challenge with C. hominis but only partially against challenge with C. parvum, as compared with age-matched control animals challenged with either species. In conclusion, C. hominis–specific immunity was sufficient to completely protect against challenge with the same species but insufficient to provide the same level of protection against C. parvum.
Clostridium difficile infection (CDI) causes moderate to severe disease, resulting in diarrhea and pseudomembranous colitis. CDI is difficult to treat due to production of inflammation-inducing toxins, resistance development, and high probability of recurrence. Only two antibiotics are approved for the treatment of CDI, and the pipeline for therapeutic agents contains few new drugs. MBX-500 is a hybrid antibacterial, composed of an anilinouracil DNA polymerase inhibitor linked to a fluoroquinolone DNA gyrase/topoisomerase inhibitor, with potential as a new therapeutic for CDI treatment. Since MBX-500 inhibits three bacterial targets, it has been previously shown to be minimally susceptible to resistance development. In the present study, the in vitro and in vivo efficacies of MBX-500 were explored against the Gram-positive anaerobe, C. difficile. MBX-500 displayed potency across nearly 50 isolates, including those of the fluoroquinolone-resistant, toxin-overproducing NAP1/027 ribotype, performing as well as comparator antibiotics vancomycin and metronidazole. Furthermore, MBX-500 was a narrow-spectrum agent, displaying poor activity against many other gut anaerobes. MBX-500 was active in acute and recurrent infections in a toxigenic hamster model of CDI, exhibiting full protection against acute infections and prevention of recurrence in 70% of the animals. Hamsters treated with MBX-500 displayed significantly greater weight gain than did those treated with vancomycin. Finally, MBX-500 was efficacious in a murine model of CDI, again demonstrating a fully protective effect and permitting near-normal weight gain in the treated animals. These selective anti-CDI features support the further development of MBX 500 for the treatment of CDI.
(See the editorial commentary by Johnson, on pages 353–4.)
Background. Clostridium difficile infection (CDI) can cause a wide range of disease, from mild diarrhea to fulminant systemic disease. The incidence of systemic CDI with fatal consequence has increased rapidly in recent years.
Methods. Using an ultrasensitive cytotoxicity assay, we measured C. difficile toxin A (TcdA) and C. difficile toxin B (TcdB) in sera and body fluids of piglets and mice exposed to C. difficile to investigate the relationship between the presence of toxins in body fluids and systemic manifestations of CDI.
Results. We found that both TcdA and TcdB disseminate systemically, with toxins present in the sera and body fluids of infected animals, and toxemia is significantly correlated with the development of systemic CDI. The systemic administration of neutralizing antibodies against both toxins blocked the development of systemic disease in mice. We measured cytokine concentrations in the sera of mice and piglets with systemic and nonsystemic CDI and found that proinflammatory mediators were considerably elevated in animals with systemic CDI.
Conclusion. Our study demonstrates the existence of a strong correlation between toxemia and the occurrence of systemic disease, supporting the hypothesis that systemic CDI is most likely due to the toxicity of TcdA and TcdB and the induction of proinflammatory cytokines by the toxins.
The global emergence of Clostridium difficile infection (CDI) has contributed to the recent surge in severe antibiotic-associated diarrhea and colonic inflammation. C. difficile produces two homologous glucosylating exotoxins, TcdA and TcdB, both of which are pathogenic and require neutralization to prevent disease occurrence. However, because of their large size and complex multifunctional domain structures, it has been a challenge to produce native recombinant toxins that may serve as vaccine candidates. Here, we describe a novel chimeric toxin vaccine that retains major neutralizing epitopes from both toxins and confers complete protection against primary and recurrent CDI in mice. Using a nonpathogenic Bacillus megaterium expression system, we generated glucosyltransferase-deficient holotoxins and demonstrated their loss of toxicity. The atoxic holotoxins induced potent antitoxin neutralizing antibodies showing little cross-immunogenicity or protection between TcdA and TcdB. To facilitate simultaneous protection against both toxins, we generated an active clostridial toxin chimera by switching the receptor binding domain of TcdB with that of TcdA. The toxin chimera was fully cytotoxic and showed potent proinflammatory activities. This toxicity was essentially abolished in a glucosyltransferase-deficient toxin chimera, cTxAB. Parenteral immunization of mice or hamsters with cTxAB induced rapid and potent neutralizing antibodies against both toxins. Complete and long-lasting disease protection was conferred by cTxAB vaccinations against both laboratory and hypervirulent C. difficile strains. Finally, prophylactic cTxAB vaccination prevented spore-induced disease relapse, which constitutes one of the most significant clinical issues in CDI. Thus, the rational design of recombinant chimeric toxins provides a novel approach for protecting individuals at high risk of developing CDI.
Botulinum neurotoxin (BoNT) is responsible for causing botulism, a potentially fatal disease characterized by paralysis of skeletal muscle. Existing specific treatments include polyclonal antisera derived from immunized humans or horses. Both preparations have similar drawbacks, including limited supply, risk of adverse effects and batch to batch variation. Here, we describe a panel of six highly protective sheep monoclonal antibodies (SMAbs) derived from sheep immunized with BoNT/A1 toxoid (SMAbs 2G11, 4F7) or BoNT/A1 heavy chain C-terminus (HcC) (SMAbs 1G4, 5E2, 5F7, 16F9) with or without subsequent challenge immunization with BoNT/A1 toxin. Although each SMAb bound BoNT/A1 toxin, differences in specificity for native and recombinant constituents of BoNT/A1 were observed. Structural differences were suggested by pI (5E2 = 8.2; 2G11 = 7.1; 4F7 = 8.8; 1G4 = 7.4; 5F7 = 8.0; 16F9 = 5.1). SMAb protective efficacy vs. 10,000 LD50 BoNT/A1 was evaluated using the mouse lethality assay. Although not protective alone, divalent and trivalent combinations of SMabs, IG4, 5F7 and/or 16F9 were highly protective. Divalent combinations containing 0.5–4 μg/SMAb (1–8 μg total SMAb) were 100% protective against death with only mild signs of botulism observed; relative efficacy of each combination was 1G4 + 5F7 > 1G4 + 16F9 >> 5F7 + 16F9. The trivalent combination of 1G4 + 5F7 + 16F9 at 0.25 μg/SMAb (0.75 μg total SMAb) was 100% protective against clinical signs and death. These results reflect levels of protective potency not reported previously.
botulinum neurotoxin; botulinum toxin; BoNT; BoNT/A1; BoNT/A2; monoclonal antibodies; sheep monoclonal antibodies; immunotherapy; passive immunization; botulism
The intestinal immune dysfunction due to loss of mucosal and peripheral CD4+ T cells in individuals with HIV/AIDS is presumably responsible for the establishment of persistent cryptosporidiosis. Simian immunodeficiency virus (SIV)-infected macaques were used to investigate the phase/timing in SIV infection, which permits a self-limiting Cryptosporidium parvum infection to become persistent in immunodeficient hosts because of significant mucosal immune defects. Two groups of SIV-infected macaques were challenged with C. parvum; one was challenged during the acute SIV infection phase (2 weeks post-SIV infection) and the second was challenged during the chronic SIV phase (CD4 counts 200–500 cells/μl of blood). Samples (fecal, blood, biopsy, and necropsy) were collected at different time points after infection to correlate the progression of disease with the immune status of the animals. All seven SIV-infected macaques challenged during the acute phase of SIV infection became persistently infected and excreted oocysts for 1–4 months. However, four of the six in the chronic SIV phase became infected with cryptosporidiosis, of which one survived 2 weeks and one became naturally infected. Sequential analysis of CD4+ in blood and intestines of coinfected macaques exhibited pronounced losses of CD4 T cells during the first 2 weeks after SIV infection, followed by transient rebound of CD4 T cells in the gut after C. parvum infection, and then a gradual loss over subsequent months. Persistent cryptosporidiosis was more consistently induced during the acute SIV phase indicating that profound viral damage to gut lymphoid tissue during the acute phase was more conducive, compared with the chronic phase, to establishing persistent cryptosporidiosis than low circulating CD4 T cells.
Most Cryptosporidium infections in humans are caused by C. parvum or C. hominis. However, genotyping techniques have identified infections caused by unusual Cryptosporidium species. Cryptosporidium meleagridis has been identified in ≤ 1% of persons with diarrhea, although prevalence is higher in developing nations. We examined the infectivity of C. meleagridis in healthy adults. Five volunteers were challenged with 105 C. meleagridis oocysts and monitored six weeks for fecal oocysts and clinical manifestations. Four volunteers had diarrhea; three had detectable fecal oocysts; and one infected volunteer remained asymptomatic. Fecal DNA from two volunteers was amplified by using a polymerase chain reaction specific for the Cryptosporidium small subunit ribosomal RNA gene. Nucleotide sequence of these amplicons was diagnostic for C. meleagridis. All infections were self-limited; oocysts were cleared within ≤ 12 days of challenge. These studies establish that healthy adults can be infected and become ill from ingestion of C. meleagridis oocysts.
Hemolytic uremic syndrome (HUS) leading to acute kidney failure, is a condition linked to the production of primarily Shiga toxin 2 (Stx2) by some E. coli serotypes. We have previously shown that Stx2 A subunit-specific human monoclonal antibody (HuMAb) 5C12, and B subunit-specific HuMAb 5H8 inhibit cultured cell death, and protect mice and piglets from fatal Stx2-intoxication. We have also shown that 5H8 blocks binding of Stx2 to its cell-surface receptor globotriaosyl ceramide (Gb3), whereas Stx2 when complexed with 5C12 binds Gb3 with higher affinity than Stx2. The mechanism by which 5C12 neutralizes Stx2 in vitro involves trapping of Stx2 in the recycling endosomes and releasing it into the extracellular environment. Because of the clinical implications associated with the formation of Stx2/antibody complexes and the potential for trapping and clearance through a severely damaged kidney associated with HUS, we investigated the likely site(s) of Stx2/antibody localization and clearance in intoxicated mice treated with antibody or placebo.
Mice were injected with radiolabeled Stx2 (125I-Stx2) 4 hours after administration of 5C12, 5H8, or phosphate buffered saline (PBS) and the sites of localization of labeled Stx2, were investigated 3, 24 and 48 hours later. The liver recorded statistically much higher concentrations of labeled Stx2 for groups receiving 5C12 and 5H8 antibodies after 3, 24 and 48 hours, as compared with the PBS group. In contrast, highest levels of labeled Stx2 were detected in the kidneys of the PBS group at all 3 sampling times. Mice receiving either of the two HuMAbs were fully protected against the lethal effect of Stx2, as compared with the fatal outcome of the control group.
The results suggest that HuMAbs 5C12 and 5H8 promoted hepatic accumulation and presumably clearance of toxin/antibody complexes, significantly diverting Stx2 localization in the kidneys, the target of Stx2 and the cause of HUS. This is in contrast to the fatal outcome of the control group receiving PBS. The results also confirm earlier observations that both HuMAbs are highly and equally protective against Stx2 intoxication in mice.
Shiga toxin; Radiolabel; Antibody; Toxin elimination; Toxin concentration; Pharmacokinetic; Human monoclonal antibody
Enterocytozoon bieneusi is clinically the most significant microsporidian parasite associated with persistent diarrhea, wasting and cholangitis in 30-50% of individuals with HIV/AIDS, as well as in malnutritional children and in recipients of immunosuppressive therapy. However, the host immune responses to E. bieneusi have not been investigated until recently due to lack of sources of spores, cell culture system, and animal models. In this study, we purified spores from heavily infected human or monkey feces by serial salt-Percoll-sucrose-iodixanol centrifugation and the purity of spores was confirmed by FACS and scanning electron microscopy. Exposure of dendritic cells to E. bieneusi spores induced up-regulation of the surface markers and production of pro-inflammatory cytokines. The cytokine production was independent of Toll-like receptor 4, but MyD88-dependent, since dendritic cells from MyD88 knockout mice failed to secrete these pro-inflammatory cytokines, whereas dendritic cells from C3H/HeJ (a Toll-like receptor 4 mutant) were activated by E. bieneusi and secreted these cytokines. Furthermore, MyD88 deficient mice were susceptible to E. bieneusi infection, in contrast to wild type mice which resisted the infection. Collectively the data demonstrate innate recognition of E. bieneusi by dendritic cells and the importance of MyD88-dependent signaling in resisting infection in a murine challenge model.
Dendritic cells; Enterocytozoon bieneusi; innate immunity; MyD88; TLR
The intravascular trematode Schistosoma mansoni is a causative agent of schistosomiasis, a disease that constitutes a major health problem globally. In this study we cloned and characterized the schistosome tegumental phosphodiesterase SmNPP-5 and evaluated its role in parasite virulence. SmNPP-5 is a 52.5-kDa protein whose gene is rapidly turned on in the intravascular parasitic life stages, following invasion of the definitive host. Highest expression is found in mated adult males. As revealed by immunofluorescence analysis, SmNPP-5 protein is found prominently in the dorsal surface of the tegument of males. Localization by immuno-electron microscopy illustrates a unique pattern of immunogold-labeled SmNPP-5 within the tegument; some immunogold particles are scattered throughout the tissue, but many are clustered in tight arrays. To determine the importance of the protein for the parasites, RNA interference (RNAi) was employed to knock down expression of the SmNPP-5-encoding gene in schistosomula and adult worms. Both quantitative real-time PCR (qRT-PCR) and Western blotting confirmed successful and robust gene suppression. In addition, the suppression and the ectolocalization of this enzyme in live parasites were evident because of a significantly impaired ability of the suppressed parasites to hydrolyze exogenously added phosphodiesterase substrate p-nitrophenyl 5′-dTMP (p-Nph-5′-TMP). The effects of suppressing expression of the SmNPP-5 gene in vivo were tested by injecting parasites into mice. It was found that, unlike controls, parasites whose SmNPP-5 gene was demonstrably suppressed at the time of host infection were greatly impaired in their ability to establish infection. These results demonstrate that SmNPP-5 is a virulence factor for schistosomes.
Upon binding to intestinal epithelial cells, enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium trigger formation of actin pedestals beneath bound bacteria. Pedestal formation has been associated with enhanced colonization, and requires intimin, an adhesin that binds to the bacterial effector translocated intimin receptor (Tir), which is translocated to the host cell membrane and promotes bacterial adherence and pedestal formation. Intimin has been suggested to also promote cell adhesion by binding one or more host receptors, and allelic differences in intimin have been associated with differences in tissue and host specificity. We assessed the function of EHEC, EPEC, or C. rodentium intimin, or a set of intimin derivatives with varying Tir-binding abilities in animal models of infection. We found that EPEC and EHEC intimin were functionally indistinguishable during infection of gnotobiotic piglets by EHEC, and that EPEC, EHEC, and C. rodentium intimin were functionally indistinguishable during infection of C57BL/6 mice by C. rodentium. A derivative of EHEC intimin that bound Tir but did not promote robust pedestal formation on cultured cells was unable to promote C. rodentium colonization of conventional mice, indicating that the ability to trigger actin assembly, not simply to bind Tir, is required for intimin-mediated intestinal colonization. Interestingly, streptomycin pre-treatment of mice eliminated the requirement for Tir but not intimin during colonization, and intimin derivatives that were defective in Tir-binding still promoted colonization of these mice. These results indicate that EPEC, EHEC, and C. rodentium intimin are functionally interchangeable during infection of gnotobiotic piglets or conventional C57BL/6 mice, and that whereas the ability to trigger Tir-mediated pedestal formation is essential for colonization of conventional mice, intimin provides a Tir-independent activity during colonization of streptomycin pre-treated mice.
Citrobacter rodentium; intimin; enterohemorrhagic Escherichia coli; invasin; enteropathogenic Escherichia coli
Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant ‘targeting agent’ that binds a toxin at two unique sites and a ‘clearing Ab’ that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab VH (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit.
Cryptosporidium parvum induces the formation of an actin-dense plaque which is essential for the successful invasion of epithelial cells. Host molecules that are involved in the regulation of this cytoskeleton reorganization are unknown. Here we identified that calcium-dependent thiol protease calpain is critical for regulating parasite-induced actin polymerization. C. parvum invasion induced activation of calpain. Inhibition of calpain activity by overexpression of the endogenous inhibitor calpastatin diminished the formation of the actin-dense plaque and decreased the initial invasion of parasites. Our data indicates a key role of calpain activity of host cell in C. parvum infection via regulating cytoskeleton reorganization.
CRYPTOSPORIDIUM PARVUM; CALPAIN; INVASION
Upon intestinal colonization, enterohemorrhagic Escherichia coli (EHEC) induces epithelial cells to generate actin “pedestals” beneath bound bacteria, lesions that promote colonization. To induce pedestals, EHEC utilizes a type III secretion system to translocate into the mammalian cell bacterial effectors such as translocated intimin receptor (Tir), which localizes in the mammalian cell membrane and functions as a receptor for the bacterial outer membrane protein intimin. Whereas EHEC triggers efficient pedestal formation during mammalian infection, EHEC cultured in vitro induces pedestals on cell monolayers with relatively low efficiency. To determine whether growth within the mammalian host enhances EHEC pedestal formation, we compared in vitro-cultivated bacteria with EHEC directly isolated from infected piglets. Mammalian adaptation by EHEC was associated with a dramatic increase in the efficiency of cell attachment and pedestal formation. The amounts of intimin and Tir were significantly higher in host-adapted than in in vitro-cultivated bacteria, but increasing intimin or Tir expression, or artificially increasing the level of bacterial attachment to mammalian cells, did not enhance pedestal formation by in vitro-cultivated EHEC. Instead, a functional assay suggested that host-adapted EHEC translocate Tir much more efficiently than does in vitro-cultivated bacteria. These data suggest that adaptation of EHEC to the mammalian intestine enhances bacterial cell attachment, expression of intimin and Tir, and translocation of effectors that promote actin signaling.
host adaptation; actin assembly; translocation; EHEC; intimin; Tir
The recent outbreak of severe infections with Shiga toxin (Stx) producing Escherichia coli (STEC) serotype O104:H4 highlights the need to understand horizontal gene transfer among E. coli strains, identify novel virulence factors and elucidate their pathogenesis. Quantitative shotgun proteomics can contribute to such objectives, allowing insights into the part of the genome translated into proteins and the connectivity of biochemical pathways and higher order assemblies of proteins at the subcellular level.
We examined protein profiles in cell lysate fractions of STEC strain 86-24 (serotype O157:H7), following growth in cell culture or bacterial isolation from intestines of infected piglets, in the context of functionally and structurally characterized biochemical pathways of E. coli. Protein solubilization in the presence of Triton X-100, EDTA and high salt was followed by size exclusion chromatography into the approximate Mr ranges greater than 280 kDa, 280-80 kDa and 80-10 kDa. Peptide mixtures resulting from these and the insoluble fraction were analyzed by quantitative 2D-LC-nESI-MS/MS. Of the 2521 proteins identified at a 1% false discovery rate, representing 47% of all predicted E. coli O157:H7 gene products, the majority of integral membrane proteins were enriched in the high Mr fraction. Hundreds of proteins were enriched in a Mr range higher than that predicted for a monomer supporting their participation in protein complexes. The insoluble STEC fraction revealed enrichment of aggregation-prone proteins, including many that are part of large structure/function entities such as the ribosome, cytoskeleton and O-antigen biosynthesis cluster.
Nearly all E. coli O157:H7 proteins encoded by prophage regions were expressed at low abundance levels or not detected. Comparative quantitative analyses of proteins from distinct cell lysate fractions allowed us to associate uncharacterized proteins with membrane attachment, potential participation in stable protein complexes, and susceptibility to aggregation as part of larger structural assemblies.
Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1) in vitro (derived from LB cell cultures) and in vivo (derived from gnotobiotic piglets) was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology.
Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate), including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM) proteins (38% of in silico predicted SD1 membrane proteome) contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA) and mixed acid fermentation (PflA/PflB) indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB) implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB) were increased, while β-barrel OM proteins (OmpA), OM lipoproteins (NlpD), chaperones involved in OM protein folding pathways (YraP, NlpB) and lipopolysaccharide biosynthesis (Imp) were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins) required for invasion of colonic epithelial cells, and release of bacteria into the host cell cytosol were increased in vivo.
Global proteomic profiling of SD1 comparing in vivo vs. in vitro proteomes revealed differential expression of proteins geared towards survival of the pathogen in the host gut environment, including increased abundance of proteins involved in anaerobic energy respiration, acid resistance and virulence. The immunogenic OspC2, OspC3 and IpgA virulence proteins were detected solely under in vivo conditions, lending credence to their candidacy as potential vaccine targets.
Enterocytozoon bieneusi is the most common and clinically significant microsporidium associated with chronic diarrhea and wasting in immunocompromised humans. Albendazole, which is effective against several helminths, protozoa, and microsporidia, is relatively ineffective against infections due to E. bieneusi. A likely explanation for the observed clinical resistance to albendazole was discovered from sequence analysis of the E. bieneusi β-tubulin from isolates from an infected human and a naturally infected rhesus macaque. The β-tubulin of E. bieneusi has a substitution at Glu198, which is one of six amino acids reported to be associated with benzimidazole sensitivity.
Albendazole resistance; beta-tubulin gene; microsporidia
Respiratory cryptosporidiosis is recognized as a late-stage complication in persons with HIV/AIDS. However, respiratory signs and symptoms are common in otherwise healthy children with intestinal cryptosporidiosis, suggesting that respiratory infection may occur in immunocompetent hosts.
We recruited children aged 9–36 months who presented with diarrhea to Mulago Hospital in Kampala, Uganda. Children with Cryptosporidium-positive and -negative stools were selected for further evaluation, including sputum induction in those with cough or unexplained respiratory signs, and collection of saliva and blood. Sputum samples were subjected to comprehensive bacteriological testing, and both sputum and saliva were tested for Cryptosporidium by nested-PCR.
Of 926 fecal samples screened, 116 (12.5%) were Cryptosporidium positive. Seventeen of 48 (35.4%) sputum samples tested from stool-positive children were positive for Cryptosporidium. Sixteen of the 17 children with confirmed respiratory cryptosporidiosis were HIV-seronegative and 10/17 (58.8%) children were normally nourished. None of the 12 sputum specimens tested from stool-negative children were Cryptosporidium positive (p=0.013 compared to stool-positive children). Parasite DNA was only detected in 2/103 (1.9%) saliva samples (p<0.0001 compared to sputum).
Respiratory cryptosporidiosis was documented in one third of HIV-seronegative children who were tested. These novel findings suggest the potential for respiratory transmission. This study is registered with ClinicalTrials.gov, number NCT00507871.
cryptosporidiosis; respiratory; HIV; transmission
Bacillus subtilis vaccine strains engineered to express either group A bovine or murine rotavirus VP6 were tested in adult mice for their ability to induce immune responses and provide protection against rotavirus challenge. Mice were inoculated intranasally with spores or vegetative cells of the recombinant strains of B. subtilis. To enhance mucosal immunity, whole cholera toxin (CT) or a mutant form (R192G) of Escherichia coli heat-labile toxin (mLT) were included as adjuvants. To evaluate vaccine efficacy, the immunized mice were challenged orally with EDIM EW murine rotavirus and monitored daily for 7 days for virus shedding in feces. Mice immunized with either VP6 spore or VP6 vegetative cell vaccines raised serum anti-VP6 IgG enzyme-linked immunosorbent assay (ELISA) titers, whereas only the VP6 spore vaccines generated fecal anti-VP6 IgA ELISA titers. Mice in groups that were immunized with VP6 spore vaccines plus CT or mLT showed significant reductions in virus shedding, whereas the groups of mice immunized with VP6 vegetative cell vaccines showed no difference in virus shedding compared with mice immunized with control spores or cells. These results demonstrate that intranasal inoculation with B. subtilis spore-based rotavirus vaccines is effective in generating protective immunity against rotavirus challenge in mice.
Shiga toxin 2 (Stx2) producing Escherichia coli (STEC) strains are more frequently isolated from hemolytic-uremic syndrome cases than strains that produce Stx1 and Stx2, and rarely the strains that produce only Stx1. Studies have implicated Stx2 as the sole contributor to acute kidney failure and other systemic complications in humans, and our study adds further support to this assumption since Stx2- and Stx1-specific antibodies protected 100% and 0% of piglets, respectively, against an oral challenge with a Stx1- and Stx2-producing STEC strain. We conclude that Stx2-specific antibody is sufficient to protect piglets, and possibly humans, against STEC producing both toxins.
Shiga toxin; Stx; antibody; enterohemorrhagic; piglet; hemolytic uremic syndrome; kidney failure; E. coli
The lack of a standardized laboratory animal model that mimics key aspects of human shigellosis remains major obstacle to addressing questions on pathogenesis, screening therapeutics and evaluating vaccines.
We characterize a piglet model for Shigella dysenteriae type 1.
Piglets developed acute diarrhea, anorexia, dehydration and often fatal, with severity depending on age and dose. Bacteria were apparent in the lumen and on surface epithelium throughout the gut initially, but severe mucosal damage and bacterial cellular invasion were most profound in the colon. Detached necrotic colonocytes were present in the lumen, with inflammatory cells outpouring from damaged mucosa. High levels of IL-8 and IL-12, were followed by other proinflammatory cytokines. Elevated TNF-α, IL-1β, IL-6, and IL-10 were detected in feces and in gut segments of infected animals. Bacteria were present inside epithelial cells and within colonic lamina propria. In contrast, an isogenic strain lacking Shiga toxin, induced similar but milder symptoms, with moderate mucosal damage and lower cytokine levels.
We conclude that piglets are highly susceptible to shigellosis providing a useful tool to compare vaccine candidates for immunogenicity, reactogenicity and response to challenge, investigate the role of virulence factors and test the efficacy of microbial agents.
Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. We present the first comprehensive proteome analysis of this pathogen, profiling proteins from bacteria cultured in vitro and bacterial isolates from the large bowel of infected gnotobiotic piglets (in vivo). Overall, 1061 distinct gene products were identified. Differential display analysis revealed that SD1 cells switched to an anaerobic energy metabolism in vivo. High in vivo abundances of amino acid decarboxylases (GadB and AdiA) which enhance pH homeostasis in the cytoplasm and protein disaggregation chaperones (HdeA, HdeB and ClpB) were indicative of a coordinated bacterial survival response to acid stress. Several type III secretion system (T3SS) effectors were increased in abundance in vivo, including OspF, IpaC and IpaD. These proteins are implicated in invasion of colonocytes and subversion of the host immune response in S. flexneri. These observations likely reflect an adaptive response of SD1 to the hostile host environment. Seven proteins, among them the T3SS effectors OspC2 and IpaB, were detected as antigens in western blots using piglet antisera. The outer membrane protein OmpA, the heat shock protein HtpG and OspC2 represent novel SD1 subunit vaccine candidates and drug targets.
acid stress; bacillary dysentery; proteome analysis; Shigella dysenteriae