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Mobile Genetic Elements and Bacterial Pathogenicity
Summary: Matthew Waldor studies the evolution and pathogenicity of microorganisms that cause human disease.
Horizontal exchange of genetic information is pervasive in prokaryotes. Lateral gene transfer is a major factor in the evolution of pathogenic bacteria because most virulence factors are encoded on mobile genetic elements. Our work is focused on two virulence-linked mobile elements: (1) CTXφ, an integrating filamentous phage that encodes cholera toxin, the principal virulence factor of Vibrio cholerae, the cause of cholera, and (2) SXT, a V. cholerae–derived integrating conjugative element that encodes multiple antibiotic resistance genes. By studying the interactions of these and other mobile elements with their respective hosts, we have identified environmental and genetic factors that control dissemination of virulence factors and discovered that mobile elements can directly influence pathogenicity.
Bacterial genomics has revealed that many prokaryotes, including V. cholerae, have more than one chromosome. A new focus of our lab is study of the mechanisms that control and coordinate the replication and segregation of the two V. cholerae chromosomes.
CTXφ Biology
CTXφ infection of nontoxigenic V. cholerae strains can render them fully pathogenic. We have dissected many of the events in the CTXφ life cycle (Figure 1) and demonstrated the profound dependence of CTXφ on its host. Cellular factors directly mediate integration of the CTXφ genome into the V. cholerae chromosome, secretion of viral particles, and regulation of phage gene transcription.
In all characterized isolates of V. cholerae from the ongoing seventh pandemic of cholera, CTXφ DNA is maintained as part of a chromosomal array that contains one or more prophages plus one or more copies of a related genetic element known as RS1. We have discovered that these arrays routinely yield hybrid virions, composed of DNA from two adjacent prophages or from a prophage and a downstream RS1. The presence of tandem elements is required for production of virions. Generation of the replicative (plasmid) form of CTXφ, pCTX, does not depend on reversal of the process for site-specific integration of CTXφ DNA into the chromosome; instead, our work suggests that the CTXφ-specific proteins required for replication of pCTX can also function on a chromosomal substrate, and that, unlike the processes used by other integrating phages, production of CTXφ does not require excision of the prophage from the chromosome. Use of this replication strategy maximizes vertical transmission of prophage DNA, while still enabling dissemination of CTXφ to new hosts. (Grants from the National Institutes of Health partially supported our work on CTXφ.)
SXT Biology
Many determinants of antibiotic resistance are borne by mobile genetic elements. To learn about the environmental and genetic factors that control dissemination of antibiotic resistance genes, we study SXT, a V. cholerae-derived integrative conjugative element (ICE) that encodes resistance to multiple antibiotics. Like conjugative plasmids, ICEs are transferred between cells in a cell-contact-dependent fashion; unlike plasmids, ICEs do not autonomously replicate but integrate into the chromosome of the new host. We have carried out genomic and functional analyses of the ~100-kb SXT and identified many of the genes that mediate its integration, excision, and conjugation. We discovered that SXT is part of a family of closely related ICEs, and we have defined some key components of a regulatory circuit that controls SXT transfer (Figure 2). The bacterial response to DNA damage (SOS) promotes SXT transfer by diminishing repression by the SXT repressor of the SXT transcriptional activators. The discovery of this novel stimulus of conjugative transfer suggests that the use of antimicrobial agents that induce SOS may promote the dissemination of resistance genes.
We found that ICEs, like conjugative plasmids, encode exclusion factors that inhibit entry of identical ICEs into host cells. Our data suggest that direct interactions between the cytoplasmic domains of inner membrane proteins located in donor and recipient cells dictate exclusion specificity. These observations suggest that complex topological rearrangements of conjugative proteins must occur during mating to enable these domains to interact.
Shiga Toxin–Encoding Phages and Pathogenicity of Enterohemorrhagic Escherichia coli (EHEC)
EHEC (E. coli O157) are emerging foodborne pathogens that cause diseases ranging from mild diarrhea to the potentially fatal hemolytic uremic syndrome. Shiga toxin (Stx), the principal EHEC virulence factor, is encoded in the genome of a λ-like prophage. In collaboration with David Friedman (University of Michigan), we discovered that transcription of genes encoding Stx is largely dependent on a phage promoter and that toxin release depends on phage-mediated cell lysis. Thus, Stx prophages directly contribute to pathogenesis. We found that factors that promote prophage induction, including certain antibiotics, enhance Stx production. Such induction may underlie the epidemiological observation that antibiotics worsen the course of EHEC. Our current goals are to explore Stx prophage induction within the intestine and to identify previously uncharacterized horizontally transmitted genes that influence EHEC intestinal colonization. (Grants from the National Institutes of Health partially supported our work on EHEC).
Vibrio cholerae Chromosome Replication and Segregation
Studies of prokaryotic chromosome replication and segregation have focused almost exclusively on organisms with one chromosome. We defined and characterized the origins of replication of the two V. cholerae chromosomes, oriCIvc and oriCIIvc. The two differ, and oriCIIvc is unrelated to any previously characterized chromosome origin of replication. OriCIIvc-based replication requires an origin-binding protein that is conserved among diverse genera of the family Vibrionaceae. We are defining the activity of this protein and exploring the mechanisms that coordinate the replication of the two chromosomes. Our studies of replication in V. cholerae indicate that microorganisms having multiple chromosomes may utilize unique mechanisms for the control of replication.
We used fluorescence microscopy to visualize the localization and segregation of oriCIvc and oriCIIvc. In all stages of the cell cycle, the two origins localized to distinct subcellular locations (Figure 3). The differences in localization and timing suggest that distinct mechanisms govern the segregation of the two V. cholerae chromosomes. Both V. cholerae chromosomes encode homologs of plasmid partitioning (Par) proteins, and we established that these proteins mediate the localization of the respective oris. The biologic necessity for these two sets of Par proteins differs. The chromosome I Par proteins appear to act as components of an apparatus that pulls the oriCIvc region to the cell pole and anchors it there. These Par proteins do not, however, appear to be essential for this chromosome's segregation to daughter cells. In contrast, deletion of the genes encoding the chromosome II Par proteins results in frequent loss of this chromosome. The resulting cells, containing only chromosome I, undergo a consistent set of cytologic changes prior to their death, suggesting that prokaryotes, like eukaryotes, possess characteristic death pathways. We are attempting to reconstitute DNA movement by the chromosome I and II Par systems in vitro.
Vibrio cholerae sRNAs
Recently it has become clear from studies in E. coli that small untranslated RNAs (sRNAs) regulate many cellular processes. Hfq is an RNA-binding protein that is important for the activity of many sRNAs. We found that V. cholerae lacking Hfq fail to grow in the intestine, despite the production of the principal V. cholerae intestinal colonization factor by our hfq mutant. These findings suggest that sRNAs, in conjunction with Hfq, control previously unrecognized processes that are critical for V. cholerae pathogenicity. We developed sRNAPredict, a computer program that enables the rapid identification of putative sRNAs in intergenic regions of bacterial genomes. Using this software, we have annotated hundreds of putative sRNA-encoding genes in 12 pathogens, and we are investigating the targets and mechanisms of action of several of the sRNAs. (Grants from the National Institutes of Health partially supported our work on sRNAs.)
Last updated: July 23, 2007
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