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Bacterial pathogens coordinate virulence using two-component regulatory systems (TCS). The Bordetella virulence gene (BvgAS) phosphorelay-type TCS controls expression of all known protein virulence factor-encoding genes and is considered the "master virulence regulator" in Bordetella pertussis, the causal agent of pertussis, and related organisms, including the broad host range pathogen Bordetella bronchiseptica We recently discovered an additional sensor kinase, PlrS [for persistence in the lower respiratory tract (LRT) sensor], which is required for B. bronchiseptica persistence in the LRT. Here, we show that PlrS is required for BvgAS to become and remain fully active in mouse lungs but not the nasal cavity, demonstrating that PlrS coordinates virulence specifically in the LRT. PlrS is required for LRT persistence even when BvgAS is rendered constitutively active, suggesting the presence of BvgAS-independent, PlrS-dependent virulence factors that are critical for bacterial survival in the LRT. We show that PlrS is also required for persistence of the human pathogen B. pertussis in the murine LRT and we provide evidence that PlrS most likely functions via the putative cognate response regulator PlrR. These data support a model in which PlrS senses conditions present in the LRT and activates PlrR, which controls expression of genes required for the maintenance of BvgAS activity and for essential BvgAS-independent functions. In addition to providing a major advance in our understanding of virulence regulation in Bordetella, which has served as a paradigm for several decades, these results indicate the existence of previously unknown virulence factors that may serve as new vaccine components and therapeutic or diagnostic targets.

Bacteria of the Bordetella genus cause respiratory tract infections. Both broad host range (e.g. Bordetella bronchiseptica) and human-adapted (e.g. Bordetella pertussis) strains produce a surface-exposed and secreted protein called filamentous haemagglutinin (FHA) that functions in adherence and immunomodulation. Previous studies using B. pertussis and cultured mammalian cells identified several FHA domains with potential roles in host cell interactions, including an Arg-Gly-Asp (RGD) triplet that was reported to bind integrins on epithelial cells and monocytes to activate host signalling pathways. We show here that, in contrast to our previous report, the fhaB genes of B. pertussis and B. bronchiseptica are functionally interchangeable, at least with regard to the various in vitro and in vivo assays investigated. This result is significant because it indicates that information obtained studying FHA using B. bronchiseptica and natural-host animal models should apply to B. pertussis FHA as well. We also show that the C-terminus of mature FHA, which we name the MCD, mediates adherence to epithelial and macrophage-like cells and is required for colonization of the rat respiratory tract and modulation of the inflammatory response in mouse lungs. We could not, however, detect a role for the RGD in any of these processes.

Salmonella enterica serovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety of dam mutant Salmonella vaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Since dam mutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-AP r ) isolates in systemic tissues of mice infected with dam mutant Salmonella . Such 2-AP r derivatives were isolated following intraperitoneal but not oral administration and were shown to be competent for infectivity via intraperitoneal but not oral infection of naïve mice. These 2-AP r derivatives were deficient in methyl-directed mismatch repair and were resistant to nitric oxide, yet they retained the bile-sensitive phenotype of the parental dam mutant strain. Additionally, introduction of a mutH null mutation into dam mutant cells suppressed the inherent defects in intraperitoneal infectivity and nitric oxide resistance, as well as overexpression of SpvB, an actin cytotoxin required for Salmonella systemic survival. These data suggest that restoration of intraperitoneal virulence of dam mutant strains is associated with deficiencies in methyl-directed mismatch repair that correlate with the production of systemically related virulence functions.

Filamentous hemagglutinin (FHA) is a large (>200 kDa), rod-shaped protein expressed by bordetellae that is both surface-associated and secreted. FHA mediates bacterial adherence to epithelial cells and macrophages in vitro and is absolutely required for tracheal colonization in vivo. The recently sequenced Bordetella bronchiseptica genome revealed the presence of a gene, fhaS , that is nearly identical to fhaB , the FHA structural gene. We show that although fhaS expression requires the BvgAS virulence control system, it is maximal only under a subset of conditions in which BvgAS is active, suggesting an additional level of regulation. We also show that, like FHA, FhaS undergoes a C-terminal proteolytic processing event and is both surface-associated and secreted and that export across the outer membrane requires the channel-forming protein FhaC. Unlike FHA, however, FhaS was unable to mediate adherence of B. bronchiseptica to epithelial cell lines in vitro and was not required for respiratory tract colonization in vivo. In a coinfection experiment, a Δ fhaS strain was out-competed by wild-type B. bronchiseptica , indicating that fhaS is expressed in vivo and that FhaS contributes to bacterial fitness in a manner revealed when the mutant must compete with wild-type bacteria. These data suggest that FHA and FhaS perform distinct functions during the Bordetella infectious cycle. A survey of various Bordetella strains revealed two distinct fhaS alleles that segregate according to pathogen host range and that B. parapertussis hu most likely acquired its fhaS allele from B. pertussis horizontally, suggesting fhaS may contribute to host-species specificity.

Yersinia pseudotuberculosis mutants that overproduce the DNA adenine methylase (Dam) are highly attenuated, confer fully protective immune responses, and secrete several Yersinia virulence proteins ( Yersinia outer proteins [Yops]) under conditions that are nonpermissive for secretion in wild-type strains. We examined here the effects of Dam overproduction on Yersinia virulence determinant expression and secretion, as well as the host immune response to Yersinia antigens. Western blot analysis with convalescent antisera identified several low-calcium-responsive antigens whose synthesis was affected by Dam overproduction. One of these antigens was shown to be the type III secretion effector protein, YopE, a cytotoxin involved in antiphagocytosis. Dam overproduction disrupted both the thermal and calcium regulation of YopE synthesis and relaxed the thermal but not the calcium dependence of YopE secretion. Altered expression and/or secretion of Yersinia proteins in Dam-overproducing strains may contribute to the decreased virulence and heightened immunity observed in vaccinated hosts and may provide a means by which to deliver heterologous antigens and/or immune modulators of the inflammatory response.

In vivo expression technology (IVET) has resulted in the isolation of more than 100 Salmonella typhimurium genes that are induced during infection. Many of these in vivo induced (ivi) genes, as well as other virulence genes, are clustered in regions of the chromosome that are specific for Salmonella and are not present in Escherichia coli (e.g., pathogenicity islands). It would be desirable to be able to delete such putative virulence regions of the chromosome, and if the deletion removes genes that play a role in pathogenesis subsequent efforts can then be focused on individual genes that reside within that region. We therefore have developed a strategy for constructing chromosomal deletions which are not limited in size, have defined endpoints with a selectable marker at the joint point, and are not dependent on prior knowledge of sequences contained within the deleted region. Such deletion strategies can be applied to almost any bacterium with homologous recombination and to plasmid-based mutational systems where homologous recombination is not desired or feasible.

In vivo e xpression t echnology (IVET) has been used to identify >100 Salmonella typhimurium genes that are specifically expressed during infection of BALB/c mice and/or murine cultured macrophages. Induction of these genes is shown to be required for survival in the animal under conditions of the IVET selection. One class of in vivo i nduced ( ivi ) genes, iviVI-A and iviVI-B , constitute an operon that resides in a region of the Salmonella genome with low G+C content and presumably has been acquired by horizontal transfer. These ivi genes encode predicted proteins that are similar to adhesins and invasins from prokaryotic and eukaryotic pathogens ( Escherichia coli [ tia ], Plasmodium falciparum [PfEMP1]) and have coopted the PhoPQ regulatory circuitry of Salmonella virulence genes. Examination of the in vivo induction profile indicates ( i ) many ivi genes encode regulatory functions (e.g., phoPQ and pmrAB ) that serve to enhance the sensitivity and amplitude of virulence gene expression (e.g., spvB ); ( ii ) the biochemical function of many metabolic genes may not represent their sole contribution to virulence; ( iii ) the host ecology can be inferred from the biochemical functions of ivi genes; and ( iv ) nutrient limitation plays a dual signaling role in pathogenesis: to induce metabolic functions that complement host nutritional deficiencies and to induce virulence functions required for immediate survival and spread to subsequent host sites.

Bordetella pertussis, the causative agent of the acute childhood respiratory disease whooping cough, is a human-adapted variant ofBordetella bronchiseptica, which displays a broad host range and typically causes chronic, asymptomatic infections. These pathogens express a similar but not identical surface-exposed and secreted protein called filamentous hemagglutinin (FHA) that has been proposed to function as both a primary adhesin and an immunomodulator. To test the hypothesis that FHA plays an important role in determining host specificity and/or the propensity to cause acute versus chronic disease, we constructed aB. bronchisepticastrain expressing FHA fromB. pertussis(FHABp) and compared it with wild-typeB. bronchisepticain several natural-host infection models. FHABpwas able to substitute for FHA fromB. bronchiseptica(FHABb) with regard to its ability to mediate adherence to several epithelial and macrophage-like cell linesin vitro, but it was unable to substitute for FHABbin vivo. Specifically, FHABb, but not FHABp, allowedB. bronchisepticato colonize the lower respiratory tracts of rats, to modulate the inflammatory response in the lungs of immunocompetent mice, resulting in decreased lung damage and increased bacterial persistence, to induce a robust anti-Bordetellaantibody response in these immunocompetent mice, and to overcome innate immunity and cause a lethal infection in immunodeficient mice. These results indicate a critical role for FHA inB. bronchiseptica-mediated immunomodulation, and they suggest a role for FHA in host specificity.

Salmonella strains that lack or overproduce DNA adenine methylase (Dam) elicit a protective immune response to different Salmonella species. To generate vaccines against other bacterial pathogens, the dam genes of Yersinia pseudotuberculosis and Vibrio cholerae were disrupted but found to be essential for viability. Overproduction of Dam significantly attenuated the virulence of these two pathogens, leading to, in Yersinia , the ectopic secretion of virulence proteins ( Yersinia outer proteins) and a fully protective immune response in vaccinated hosts. Dysregulation of Dam activity may provide a means for the development of vaccines against varied bacterial pathogens.

Escherichia coli ssrA encodes a small stable RNA molecule, tmRNA, that has many diverse functions, including tagging abnormal proteins for degradation, supporting phage growth, and modulating the activity of DNA binding proteins. Here we show that ssrA plays a role in Salmonella enterica serovar Typhimurium pathogenesis and in the expression of several genes known to be induced during infection. Moreover, the phage-like attachment site, attL , encoded within ssrA , serves as the site of integration of a region of Salmonella -specific sequence; adjacent to the 5′ end of ssrA is another region of Salmonella -specific sequence with extensive homology to predicted proteins encoded within the unlinked Salmonella pathogenicity island SPI4. S. enterica serovar Typhimurium ssrA mutants fail to support the growth of phage P22 and are delayed in their ability to form viable phage particles following induction of a phage P22 lysogen. These data indicate that ssrA plays a role in the pathogenesis of Salmonella , serves as an attachment site for Salmonella -specific sequences, and is required for the growth of phage P22.

Analysis of several Salmonella typhimurium in vivo -induced genes located in regions of atypical base composition has uncovered acquired genetic elements that cumulatively engender pathogenicity. Many of these regions are associated with mobile elements, encode predicted adhesin and invasin-like functions, and are required for full virulence. Some of these regions distinguish broad host range from host-adapted Salmonella serovars and may contribute to inherent differences in host specificity, tissue tropism, and disease manifestation. Maintenance of this archipelago of acquired sequence by selection in specific hosts reveals a fossil record of the evolution of pathogenic species.