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Regulation and Mechanisms of Bacterial Virulence
Summary: Andrew Camilli is interested in understanding how bacterial pathogens cause disease and spread to new hosts.
Bacterial pathogens that infect mucosal surfaces in the respiratory and intestinal tracts are the leading causes of disease and death worldwide. Our laboratory investigates bacterial determinants that are important in the transmission and virulence of mucosal pathogens and how expression of these factors is regulated. For facultative pathogens, i.e., those that normally live outside the human body, a major shift in gene expression occurs upon entry into (and exit from) the human host. We are using Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, as a model facultative pathogen. We are investigating the spatial and temporal patterns of gene expression upon entry into and exit from the intestinal tract, and the factors important in transmission during cholera outbreaks.
Human commensal bacteria are also a frequent source of disease in both immunocompromised and healthy people. When commensal bacteria invade protected mucosal surfaces such as in the lung, they too undergo a shift in gene expression that contributes to disease. We are studying Streptococcus pneumoniae as a model commensal pathogen. S. pneumoniae normally resides in the nasopharynx of healthy people but occasionally invades the lung or bloodstream, leading to pneumonia, bacteremia, or meningitis. Understanding these shifts in gene expression and how they are orchestrated, as well as deciphering the roles of the induced virulence factors, will lead to a better understanding of transmission and disease by V. cholerae and S. pneumoniae and will aid in the design of improved diagnostics, therapeutics, and vaccines.
The Shift in Gene Expression when V. cholerae Enters the Host Intestine
Bacteria are extremely efficient organisms and generally do not express genes unnecessarily. This makes the discovery and study of virulence factors challenging, since many such factors are only expressed during infection of the host. As a graduate student and then as a postdoctoral fellow, I developed genetic tools to identify and study bacterial virulence factors in the context of the host. One such tool, which our laboratory has continued to develop, is recombination-based in vivo expression technology (RIVET). This method uses a site-specific DNA recombinase as a reporter gene. When expressed, the recombinase mediates the excision and loss of a selectable marker from the genome. In this way, gene induction is coupled to a heritable phenotypic change in the bacteria, allowing us to know if, when, and where a gene is turned on during infection.
Using RIVET, we have so far identified 53 genes that are turned on when V. cholerae enters the host intestine, and these genes have provided insight into important host-pathogen interactions. Through a recent collaboration with investigators at the University of Maryland, we used RIVET to identify V. cholerae genes turned on during experimental infection of human volunteers, and found good overlap with the set of genes known to be turned on during experimental infection of mice. The products encoded by these genes play diverse roles in nutrient acquisition, motility, gene regulation, and resistance to chemical stresses. We have also used RIVET to investigate the expression of known virulence genes, including those for cholera toxin (CT) and the toxin-coregulated pilus (TCP). TCP and CT are critical virulence factors required for colonization of the intestinal wall and for eliciting profuse watery diarrhea. Although CT and TCP are coexpressed during in vitro growth under conditions that mimic the host, we found that during infection TCP is expressed several hours earlier than CT. In addition, we discovered that TCP-mediated colonization is required for the later expression of CT. These and other data show that V. cholerae expresses virulence factors in a highly orchestrated manner, with specific times of induction and functional interdependencies that could never be observed in vitro (see Figure 1).
We investigated the requirements for the timing of CT induction during infection and identified a signal transduction phosphorelay system composed of three proteins—VieS, VieA, and VieB—that is necessary for correct expression. VieS is a membrane-localized sensor kinase that detects certain amino acids in the small intestine. Upon binding these amino acids, VieS activates VieA by phosphorylation. VieA is a response regulator that uses its DNA-binding domain to amplify its own expression. Importantly, VieA harbors a phosphodiesterase domain that cleaves a cyclic dinucleotide called cyclic di-guanylate (c-di-GMP). By lowering the cellular concentration of c-di-GMP, VieS and VieA turn off environmental survival genes (specifically biofilm formation genes) while simultaneously turning on virulence genes. Because amino acid stimulation of VieS results in a reduced c-di-GMP concentration, amino acids are the first messengers and c-di-GMP is the second messenger in this signal transduction pathway. Among the many unanswered questions associated with this model are these: What factors in V. cholerae sense c-di-GMP levels and impart changes in gene expression? Why are certain free amino acids a specific host signal for the bacteria?
The Shift in Gene Expression when V. cholerae Exits the Host
Our observation that genes involved in resistance to chemical stress are turned on during infection led us to hypothesize that a preinduction of such genes might increase the infectivity of V. cholerae. Indeed, we found that such a preinduction increases infectivity by an order of magnitude; i.e., 10-fold fewer bacteria need be ingested to cause a full-blown infection. In an independent line of investigation, we found that V. cholerae shed by cholera patients in their watery stools are also more infectious, having a similar 10-fold reduction in infectious dose. To test the hypothesis that this heightened state of infectivity may have evolved to aid in transmission of cholera during outbreaks, we are investigating the properties of stool V. cholerae that lead to increased infectivity. One clue, which came from our determining the transcriptome of stool V. cholerae using DNA microarrays, is that chemotaxis in the shed bacteria is temporarily altered in a way that increases infectivity. We have confirmed this model, and we are investigating other factors that might contribute to transmission of cholera. (The above work has been supported by grants from the National Institutes of Health and a grant from the Pew Charitable Trusts.)
A Novel Surface Structure Mediates Adherence of S. pneumoniae to Host Surfaces
In a separate project, we are addressing basic questions concerning the virulence properties of S. pneumoniae. Using a genetic screen, we identified more than 200 genes that are required for infection of the lung. Seven of these genes are contiguous in the genome and form a "pathogenicity islet," a mobile cluster of genes endowing bacteria with a particular virulence trait. In this case, the trait appears to be adherence to host mucosal surfaces. We dissected the regulation of the genes in this islet and identified a cis-encoded transcriptional regulator (RlrA), as well as an unlinked regulator (MgrA). MgrA controls the expression of rlrA in response to unknown signals. Recent findings suggest that uptake and metabolism of host sugars by S. pneumoniae may serve as a signal to regulate the activity of MgrA. This has prompted us to study the mechanisms and regulation of sugar metabolism in S. pneumoniae, and this ongoing work has established a connection between the physiology and virulence of this pathogen, which we are investigating further.
RlrA induces expression of six genes in the pathogenicity islet that code for three surface-anchored proteins (RrgA, RrgB, and RrgC) and three sortase enzymes (SrtB, SrtC, and SrtD). Sortases are membrane-localized sorting proteins that covalently attach particular secreted proteins to the cell wall so they can perform cell surface functions such as cell wall turnover or interaction with the host. We found that RrgA, RrgB, and RrgC are the subunits of a multiprotein, cell wall–anchored pilus. The subunits are covalently linked together to build up the pilus fiber. We are investigating the function and mechanism of assembly of this pilus. (This work has been supported by a grant from the National Institutes of Health and a grant from the Pew Charitable Trusts.)
Last updated March 05, 2009
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