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Although clinical and laboratory evidence support roles for both staphylococcal infection and environmental allergens in the pathogenesis of atopic dermatitis, human studies have largely considered these variables independently. We sought to test the hypothesis that staphylococcal superantigen influences the allergen-specific T cell response. We first mapped a Der p 1 epitope and used HLA DRB1*1501 class II tetramer-based cell sorted populations to show that specific CD4(+) T cells were able to recognize the peptide presented by HLA DR-matched keratinocytes. We observed that staphylococcal enterotoxin B (SEB) enhanced the IL-4 Der p 1-specific T cell response. This response was mediated by two synergistic mechanisms: first, SEB-induced IFN-gamma promoted class II and intercellular adhesion molecule-1 expression by presenting keratinocytes; and second, SEB-induced IL-4 directly amplified allergen-specific CD4(+) T cell production of many cytokines. We propose that handling of staphylococcal infection is a critical step in the amplification of the allergen-specific T cell response, linking two common disease associations and with implications for the prevention and treatment of atopic disease.

CagA is a bacterial effector protein of Helicobacter pylori that is translocated via a type IV secretion system into gastric epithelial cells. We previously described that H. pylori require CagA to disrupt the organization and assembly of apical junctions in polarized epithelial cells. In this study, we provide evidence that CagA expression is not only sufficient to disrupt the apical junctions but also perturbs epithelial differentiation. CagA-expressing cells lose apicobasal polarity and cell-cell adhesion, extend migratory pseudopodia, and degrade basement membranes, acquiring an invasive phenotype. Expression of the CagA C-terminal domain, which contains the tyrosine phosphorylated EPIYA motifs, induces pseudopodial activity but is not sufficient to induce cell migration. Conversely, the N terminus targets CagA to the cell-cell junctions. Neither domain is sufficient to disrupt cell adhesion or cell polarity, but coexpressed in trans, the N terminus determines the localization of both polypeptides. We show that CagA induces a morphogenetic program in polarized Madin-Darby canine kidney cells resembling an epithelial-to-mesenchymal transition. We propose that altered cell-cell and cell matrix interactions may serve as an early event in H. pylori-induced carcinogenesis.

The three proteins that constitute anthrax toxin self-assemble into toxic complexes after one of these proteins, protective antigen (PA), binds to tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2) cellular receptors. The toxin receptor complexes are internalized, and acidic endosomal pH triggers pore formation by PA and translocation of the catalytic subunits into the cytosol. In this study we show that the pH threshold for conversion of the PA prepore to the pore and for translocation differs by approximately a pH unit, depending on whether the TEM8 or CMG2 receptor is used. For TEM8-associated toxin, these events can occur at close to neutral pH values, and they show relatively low sensitivity to ammonium chloride treatment in cells. In contrast, with CMG2-associated toxin, these events require more acidic conditions and are highly sensitive to ammonium chloride. We show, furthermore, that PA dissociates from TEM8 and CMG2 upon pore formation. Our results are consistent with a model in which translocation depends on pore formation and pore formation, in turn, depends on release of PA from its receptor. We propose that because PA binds to CMG2 with much higher affinity than it does to TEM8, a lower pH is needed to attenuate CMG2 binding to allow pore formation. Our results suggest that toxin can form pores at different points in the endocytic pathway, depending on which receptor is used for entry.

Persistent gastritis induced by Helicobacter pylori is the strongest known risk factor for adenocarcinoma of the distal stomach, yet only a fraction of colonized persons ever develop gastric cancer. The H. pylori cytotoxin-associated gene (cag) pathogenicity island encodes a type IV secretion system that delivers the bacterial effector CagA into host cells after bacterial attachment, and cag+ strains augment gastric cancer risk. A host effector that is aberrantly activated in gastric cancer precursor lesions is beta-catenin, and activation of beta-catenin leads to targeted transcriptional up-regulation of genes implicated in carcinogenesis. We report that in vivo adaptation endowed an H. pylori strain with the ability to rapidly and reproducibly induce gastric dysplasia and adenocarcinoma in a rodent model of gastritis. Compared with its parental noncarcinogenic isolate, the oncogenic H. pylori strain selectively activates beta-catenin in model gastric epithelia, which is dependent on translocation of CagA into host epithelial cells. Beta-catenin nuclear accumulation is increased in gastric epithelium harvested from gerbils infected with the H. pylori carcinogenic strain as well as from persons carrying cag+ vs. cag- strains or uninfected persons. These results indicate that H. pylori-induced dysregulation of beta-catenin-dependent pathways may explain in part the augmentation in the risk of gastric cancer conferred by this pathogen.

Mycobacterium tuberculosis secretes ESAT-6, a virulence factor that triggers cell-mediated immune responses and IFN-gamma production during tuberculosis. ESAT-6 is transported across the bacterial envelope by a specialized secretion system with a FSD (FtsK-SpoIIIE domain) membrane protein. Although the presence of ESAT-6-like genes has been identified in the genomes of other microbes, the possibility that they may encode general virulence functions has hitherto not been addressed. Herein we show that the human pathogen Staphylococcus aureus secretes EsxA and EsxB, ESAT-6-like proteins, across the bacterial envelope. Staphylococcal esxA and esxB are clustered with six other genes and some of these are required for synthesis or secretion of EsxA and EsxB. Mutants that failed to secrete EsxA and EsxB displayed defects in the pathogenesis of S. aureus murine abscesses, suggesting that this specialized secretion system may be a general strategy of human bacterial pathogenesis.

The lethality of infection by Bacillus anthracis is largely due to its plasmid-encoded toxins, which consist of a carrier protein, the protective antigen (PA), in combination with either the lethal-factor or edema-factor moiety. During B. anthracis infections, PA secreted by bacteria binds to membrane receptors of susceptible cells, is cleaved proteolytically, attaches to lethal factor or edema factor, undergoes oligomerization and internalization, and transports its toxin partners to acidic endosomes where they are released into the cytosol. To identify specific host functions that mediate these events, we used RNA encoded by a lentivirus-based library of approximately 40,000 human ESTs to inactivate chromosomal genes in a human cell population, and we isolated clones that survived PA-dependent toxin-induced death. This phenotypic screen and subsequent analysis identified ARAP3, which is a phosphoinositide-binding protein implicated previously in membrane vesicle trafficking and cytoskeletal organization, as a mammalian host-cell gene that is essential for normal anthrax toxicity. ARAP3 deficiency produced by antisense expression of an ARAP3 EST impaired entry of PA and its bound toxigenic moieties into both human and mouse cells, resulting in reduced toxin sensitivity. Our results demonstrate the usefulness of antisense EST libraries for global chromosomal gene inactivation, establish the practicality of loss-of-function phenotypic screens for the identification of genomic loci required for pathogen effects in mammalian cells, and reveal an important role for ARAP3 in cellular internalization of anthrax toxin.

Molecular factors that contribute to the emergence of new virulent bacterial subclones and epidemics are poorly understood. We hypothesized that analysis of a population-based strain sample of serotype M3 group A Streptococcus (GAS) recovered from patients with invasive infection by using genome-wide investigative methods would provide new insight into this fundamental infectious disease problem. Serotype M3 GAS strains (n = 255) cultured from patients in Ontario, Canada, over 11 years and representing two distinct infection peaks were studied. Genetic diversity was indexed by pulsed-field gel electrophoresis, DNA-DNA microarray, whole-genome PCR scanning, prophage genotyping, targeted gene sequencing, and single-nucleotide polymorphism genotyping. All variation in gene content was attributable to acquisition or loss of prophages, a molecular process that generated unique combinations of proven or putative virulence genes. Distinct serotype M3 genotypes experienced rapid population expansion and caused infections that differed significantly in character and severity. Molecular genetic analysis, combined with immunologic studies, implicated a 4-aa duplication in the extreme N terminus of M protein as a factor contributing to an epidemic wave of serotype M3 invasive infections. This finding has implications for GAS vaccine research. Genome-wide analysis of population-based strain samples cultured from clinically well defined patients is crucial for understanding the molecular events underlying bacterial epidemics.

Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette-Guérin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting DeltaRD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis DeltaRD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the DeltaRD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette-Guérin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness.

We have investigated the effect of sequence-specific antisense phosphorothioate-modified oligodeoxyribonucleotides (PS-ODNs) targeting different regions of each of the 3032-kDa protein complex (antigen 85 complex) encoding genes on the multiplication of Mycobacterium tuberculosis. Single PS-ODNs to one of the three mycolyl transferase transcripts, added either once or weekly over the 6-wk observation period, inhibited bacterial growth by up to 1 log unit. A combination of three PS-ODNs specifically targeting all three transcripts inhibited bacterial growth by approximately 2 logs; the addition of these PS-ODNs weekly for 6 wk was somewhat more effective than a one-time addition. Targeting the 5' end of the transcripts was more inhibitory than targeting internal sites; the most effective PS-ODNs and target sites had minimal or no secondary structure. The effect of the PS-ODNs was specific, as mismatched PS-ODNs had little or no inhibitory activity. The antisense PS-ODNs, which were highly stable in M. tuberculosis cultures, specifically blocked protein expression by their gene target. PS-ODNs targeting the transcript of a related 24-kDa protein (mpt51) had little inhibitory effect by themselves and did not increase the effect of PS-ODNs against the three members of the 3032-kDa protein complex. The addition of PS-ODNs against the transcripts of glutamine synthetase I (glnA1) and alanine racemase (alr) modestly increased the inhibitory efficacy of the 3032-kDa protein complex-specific PS-ODNs to approximately 2.5 logs. This study shows that the three mycolyl transferases are highly promising targets for antituberculous therapy by using antisense or other antimicrobial technologies.

The bacterial pathogen Shigella flexneri causes bacillary dysentery in humans by invading coloncytes. Upon contact with epithelial cells, S. flexneri elicits localized plasma membrane projections sustained by long actin filaments which engulf the microorganism. The products necessary for Shigella entry include three secretory proteins: IpaB, IpaC, and IpaD. Extracellular IpaB and IpaC associate in a soluble complex, the Ipa complex. We have immunopurified this Ipa complex on latex beads and found that they were efficiently internalized into HeLa cells. Like S. flexneri entry, uptake of the beads bearing the Ipa complex was associated with membrane projections and polymerization of actin at the site of cell-bead interaction and was dependent on small Rho GTPases. These results indicate that a secreted factor can promote S. flexneri entry into epithelial cells.

Monoclonal antibody (mAb) 1604 is directed against a cell surface antigen of Myxococcus xanthus. Purified antibody 1604 inhibited development of M. xanthus under conditions of submerged culture procedure otherwise leading to fruiting body formation. Intact molecules of mAb 1604, as well as its Fab fragments, inhibited developmental aggregation, autolysis, fruiting body formation, and sporulation. The addition of relatively small amounts of antibody every 4 hr was much more effective than a single large dose given at the onset of development. The inhibitory action of mAb 1604 on development was reversible after prolonged incubation of the antibody with cells; this was probably due to proteolytic degradation of the antibody. The effect of mAb 1604 on submerged bacterial development was neutralized by affinity-purified 1604 cell surface antigen. Another antibody, mAb 2788, directed against an M. xanthus cell surface antigen, did not block development. These data suggest that 1604 cell surface antigens is involved in contact-mediated cell interactions in M. xanthus.