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Summary: Matthew Waldor studies the evolution, cell biology, and pathogenicity of enteric bacteria that cause human disease.

We are exploring the evolution, pathogenicity, and cell biology of Vibrio cholerae and enterohemorrhagic Escherichia coli (EHEC, two clinically important enteric pathogens. For both pathogens we are exploring (1) how mobile genetic elements, like bacteriophages, have influenced their evolution as pathogens, (2) new animal models of the diarrheal diseases these pathogens cause, and (3) the small noncoding RNAs (sRNAs) that control their virulence. A new focus of our lab is the study of several aspects of V. cholerae cell biology, including the mechanisms that control and coordinate the replication and segregation of the two V. cholerae chromosomes and the mechanisms that determine V. cholerae cell shape.

Mobile Genetic Elements
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) the integrating filamentous phage CTXφ, which encodes cholera toxin, the principal virulence factor of the cholera-inducing bacterium V. cholerae, 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.

	Figure 1: Steps in the CTXf life cycle... Figure 2: SOS control of SXT transfer... Figure 3: Segregation of the two V. cholerae chromosomes... Figure 4: Model of ParA segregation of the chromosome...

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. We are studying the molecular mechanisms that govern CTXφ production by V. cholerae.

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 are exploring (1) whether the SXT-encoded conjugative machinery serves as a conduit to translocate proteins from donor to recipient cell, (2) the diversity of SXT-related ICEs, and (3) the mechanisms that promote the maintenance of SXT in the host. (Grants from the National Institutes of Health partially support our work on CTXφ and SXT.)

Animal Models of EHEC and V. cholerae Virulence
EHEC (e.g., E. coli O157) are emerging foodborne pathogens that cause diseases ranging from mild diarrhea to the potentially fatal hemolytic uremic syndrome. We have found that infant rabbits develop severe diarrhea following intragastric inoculation with EHEC. We are using this animal model to determine which horizontally transmitted EHEC genes contribute to the organism's pathogenicity and to explore EHEC gene expression in the intestine using confocal microscopy.

We have recently found that suckling rabbits also develop profound diarrhea after intragastric inoculation with toxin-producing V. cholerae. The diarrheal fluid from infected rabbits is similar in appearance and chemical composition to "rice-water" stool from cholera patients. Using this model, we routinely recover virtually pure cultures of 10⁸ V. cholerae cfu/ml from the cecal fluid of infected rabbits. We are using this model host to explore (1) V. cholerae intraintestinal physiology, (2) transmission of V. cholerae (uninfected littermates develop cholera), and (3) new therapeutics and vaccines for cholera.

V. cholerae and EHEC Virulence-Associated sRNAs
Recently it has become clear from studies in E. coli that 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. We also observed that EHEC hfq mutants exhibit marked overexpression of the components of the type III secretion system as well as the numerous "effectors" that this secretion apparatus injects into host cells. These findings suggest that sRNAs, in conjunction with Hfq, control previously unrecognized processes that are critical for V. cholerae and EHEC pathogenicity. Our group recently developed SIPHT (sRNA identification protocol using high-throughput technologies), a Web-accessible computational tool for identifying and annotating intergenic sRNA-encoding genes in bacterial genomes. We are carrying out screens to identify the sRNAs that influence V. cholerae and EHEC pathogenicity and, in collaboration with Andrew Camilli (HHMI, Tufts University School of Medicine), developing new techniques employing deep sequencing technologies to enable high-throughput identification of sRNAs in bacteria.

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, oriCI_vc and oriCII_vc. The two differ, and oriCII_vc is unrelated to any previously characterized chromosome origin of replication. OriCII_vc-based replication requires an origin-binding protein (RctB) that is conserved among diverse genera of the family Vibrionaceae. RctB binds to and unwinds oriCII_vc DNA, thereby initiating chromosome II replication. We are carrying out high-throughput screens to identify small molecules that inhibit RctB activity, since such compounds could serve as a new class of antibiotics that exclusively target vibrios, 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 oriCI_vc and oriCII_vc. 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 oriCI_vc region to the cell pole and anchors it there (Figure 4). 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.

Last updated October 31, 2008