In vivo expression technology (IVET) has been used to identify >100 Salmonella typhimurium genes that are specifically expressed during infection of BALB͞c mice and͞or 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 induced (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.Microbial pathogenicity may be defined by the ability to propagate and persist at sites in the host that are inaccessible to commensal species (1). Many virulence determinants that contribute to this ability share a unique phenotype: induction in the host. Previously, we have established a genetic approach, termed in vivo expression technology (IVET), which uses the animal as a selective medium to identify bacterial genes specifically induced during infection (2, 3). These in vivo induced (ivi) genes were shown to be poorly expressed on laboratory medium but exhibit relatively elevated levels of expression in host tissues or in cultured macrophages. It is not anticipated that all ivi genes will have an essential role in virulence. However, their in vivo induction suggests that they contribute to growth in restricted host tissues and thus enhance pathogenicity.The IVET selection is a promoter trap, whereby bacterial promoters are selected that drive the expression of a gene that is required for virulence (Fig. 1). A promoterless lacZ gene is fused downstream of the promoterless purA gene to monitor the expression of the entire synthetic operon when cells are grown on laboratory medium or in animal tissues. Two variations of the IVET selection strategy have been employed, using purA and cat (chloramphenicol acetyltransferase) as the in vivo-selected markers (2, 3). A positive selection for ivi genes is provided by the need to complement a purA nutritional deficiency or to express cat in response to a host drug regimen. Here we show that induction of ivi genes is required for survi...
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
Bordetella pertussis, the causative agent of the acute childhood respiratory disease whooping cough, is a human-adapted variant of Bordetella 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 and͞or the propensity to cause acute versus chronic disease, we constructed a B. bronchiseptica strain expressing FHA from B. pertussis (FHA Bp) and compared it with wild-type B. bronchiseptica in several naturalhost infection models. FHA Bp was able to substitute for FHA from B. bronchiseptica (FHA Bb) with regard to its ability to mediate adherence to several epithelial and macrophage-like cell lines in vitro, but it was unable to substitute for FHA Bb in vivo. Specifically, FHA Bb, but not FHABp, allowed B. bronchiseptica to 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-Bordetella antibody 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 in B. bronchiseptica-mediated immunomodulation, and they suggest a role for FHA in host specificity.inflammation ͉ adhesin ͉ respiratory infection ͉ filamentous hemagglutinin D espite widespread vaccine coverage, whooping cough, or pertussis, remains a serious threat to human health, and its incidence has been increasing in recent years (1, 2). The causative agents, Bordetella pertussis and Bordetella parapertussis hu , are human-adapted pathogens that belong to a clade of very closely related Gram-negative bacteria that cause respiratory infections in mammals. Phylogenetic analyses indicate that Bordetella bronchiseptica, which displays a broad host range and typically colonizes its hosts chronically and asymptomatically, was the progenitor of this clade, with B. pertussis diverging relatively early and B. parapertussis hu diverging independently and much more recently than B. pertussis (3-6). Adaptation to humans and the propensity to cause acute disease (in which the infection is eventually cleared) rather than chronic disease (characterized by persistence of the bacteria, often for the lifetime of the host) has, therefore, evolved twice within this group of bacteria. Although a variety of Bordetella virulence factors have been characterized (4, 7-10), the mechanisms that determine host specificity and disease characteristics are not understood.Filamentous hemagglutinin (FHA), a primary component of acellular pertussis vaccines, is a large, -helical, highly immunogenic protein that is both surface-associated and secreted (11)(12)(13). In vitro...
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