We are exploring the evolution, cell biology, and pathogenicity of Vibrio cholerae, Vibrio parahaemolyticus, and enterohemorrhagic Escherichia coli (EHEC), which are important causes of diarrheal disease in many parts of the world. We are addressing five questions and goals related to these human enteric pathogens:
- How have mobile genetic elements influenced their evolution as pathogens?
- What are the mechanisms that control and coordinate the replication and segregation of the two chromosomes found in all Vibrio species?
- What small non-translated RNAs (sRNAs) regulate virulence?
- How do D-amino acids regulate bacterial cell wall remodeling?
- Can we develop small-animal models of enteric diseases that enable comprehensive assessment of in vivo bacterial physiology and host-pathogen interactions, as well as testing of novel therapeutics?
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. We have also discovered that mobile elements can directly influence pathogenicity (Figures 1 and 2).
Vibrio 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, oriCIIvc is unrelated to any previously characterized chromosome origin of replication, and oriCIIvc-based replication requires an origin-binding protein (RctB) that is restricted to and conserved within the diverse genera of the family Vibrionaceae. RctB binds to and unwinds oriCIIvc 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 establish a new class of antibiotics that exclusively target vibrios. We are also 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.
In addition to relying upon distinct mechanisms for replication of each of its two chromosomes, V. cholerae uses distinct processes for segregation of these genetic elements. We used fluorescence microscopy to visualize oriCIvc and oriCIIvc: the two origins were distributed differently at all stages of the cell cycle (Figure 3). Additionally, we established that homologs of plasmid partitioning (par) genes, which are found near the origin of each chromosome, mediate the localization of their respective oris. The biologic necessity for the two sets of Par proteins differs. The chromosome I Par proteins, which appear to be components of an apparatus that pulls the oriCIvc region to the cell pole and anchors it there (Figure 4), do not 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.
V. cholerae and EHEC Virulence-Associated sRNAs
Studies of E. coli have revealed 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 production of the principal V. cholerae intestinal colonization factor. We also observed that EHEC hfq mutants exhibit marked overexpression of the bacterium's type III secretion apparatus, as well as the numerous "effectors" that it injects into host cells. These findings suggest that sRNAs, in conjunction with Hfq, control processes that are critical for V. cholerae and EHEC pathogenicity. Our group developed 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 developing new techniques employing deep sequencing technologies to enable high-throughput identification of sRNAs in bacteria.
Regulation of Cell Wall Remodeling by D-Amino Acids
Peptidoglycan (PG) is a strong but elastic polymer that constitutes the bacterial cell wall. We discovered that V. cholerae modulates the amount and structure of PG in stationary phase by releasing D-amino acids. These amino acids (predominantly D-Met and D-Leu) are generated from their L-enantiomers by an amino acid racemase, BsrV, whose expression is induced as cells enter stationary phase. In most bacteria, the only D-amino acids previously known to be produced in significant quantities were D-Ala and D-Glu, which are components of PG; however, we detected numerous additional D-amino acids released by bacteria from diverse phyla, at up to millimolar concentrations.
In collaboration with Miguel de Pedro (Universidad Autonoma de Madrid), we compared PG isolated from wild-type and bsrV mutant V. cholerae at stationary phase, and observed differences in quantity, chemical composition, and structure. The PG in wild-type cells appeared to be stronger, despite being less abundant than in bsrV cells. These data suggest that D-amino acids prompt V. cholerae to remodel its PG, presumably to adapt to stationary-phase conditions. In aggregate, our findings suggest that D-amino acids are potent, yet previously unrecognized, regulators of cell wall remodeling. Notably, because D-amino acids are released extracellularly, they can be encountered by cells that have not produced them. Thus, D-amino acids may also act in trans within bacterial communities.
The precise mechanisms by which D-amino acids alter PG amount and architecture remain to be defined. Currently, we are identifying the PG-modifying enzymes, such as penicillin-binding proteins (PBPs), that bind D-amino acids and investigating how D-amino acids influence their activities. Since PBPs are targets of numerous antibiotics, these studies may facilitate development of new antimicrobial agents that exploit D-amino acid regulation of PBP activity. We are also identifying the enzymes that incorporate D-amino acids into PG. Characterization of this process will also further understanding of PG remodeling.
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 within the host intestine.
We 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. We are using this model, coupled with emerging high-throughput technologies for studying transcriptomes, proteomes, and metabolomes, to explore V. cholerae intraintestinal physiology and transmission (uninfected littermates develop cholera). Our animal models are also facilitating evaluation of new therapeutics and vaccines for cholera and EHEC.
Grants from the National Institutes of Health provided partial support for these projects.
As of May 05, 2010