Research

We are exploring the evolution, cell biology, and pathogenicity of Vibrio cholerae, Vibrio parahaemolyticus, and Shiga toxin-producing Escherichia coli (including E. coli O157 and O104:H4), important causes of diarrheal disease in many parts of the world. Our work spans a large terrain that ranges from questions concerning basic mechanisms underlying bacterial cell processes (e.g. the genesis of cell polarity) to development of new therapeutics and vaccines. We often apply and develop emerging technologies (e.g. PacBio single molecule DNA sequencing) to issues we are exploring. We are addressing five questions and goals related to the human enteric pathogens we study:

1. Determining the mechanisms that mediate the replication and segregation of the two chromosomes found in all vibrio species.

2. Determing the factors and mechanisms that contribute to the establishment of cell polarity in vibrios.

3. Deciphering the functions regulated by D-amino acids and the mechanisms by which they control processes in single cells and bacterial communities.

4. Applying single molecule real time DNA sequencing to assess the extent, diversity and functional consequences of DNA modifications (e.g. methylation) in enteric pathogens.

5. Developing small animal models of enteric diseases that will enable comprehensive assessment of in vivo bacterial physiology and host-pathogen interactions, as well as testing of novel therapeutics.

VopZ

The Vibrio parahaemolyticus T3SS Effector VopZ Mediates Pathogenesis by Independently Enabling Intestinal Colonization and Inhibiting TAK1 Activation

Vibrio parahaemolyticus type III secretion system 2 (T3SS2) is essential for the organism’s virulence, but the effectors required for intestinal colonization and induction of diarrhea by this pathogen have not been identified. The type III secretion system (T3SS2)-secreted effector, VopZ, is essential for V. parahaemolyticus pathogenicity. VopZ plays distinct, genetically separable roles in enabling intestinal colonization and diarrheagenesis. Truncation of VopZ prevents V. parahaemolyticus colonization, whereas deletion of VopZ amino acids 38-62 abrogates V. parahaemolyticus-induced diarrhea and intestinal pathology but does not impair colonization. VopZ inhibits activation of the kinase TAK1 and thereby prevents the activation of MAPK and NF-κB signaling pathways, which lie downstream. In contrast, the VopZ internal deletion mutant cannot counter the activation of pathways regulated by TAK1.

Zhou X, Gewurz BE et al. Cell Rep 2013 (http://www NULL.sciencedirect NULL.com/science/article/pii/S2211124713001630)

cell pole maturation

Cell pole maturation and the specific spatiotemporal recruitment of proteins to the new pole during the V. cholerae cell cycle

The intracellular localization of chemotaxis proteins in V. cholerae is regulated and coordinated with the cell cycle. CheW1, CheY3, and ParC, which are all encoded in the main V. cholerae chemotaxis operon, all localize to the old flagellated pole in newborn cells. Then, as cells elongate, ParC is recruited to the new pole, where it facilitates the recruitment of CheW1 and CheY3. Thus, ParC promotes the maturation of V. cholerae’s new pole, readying this site for its development into a functional old pole. Importantly, ATP hydrolysis is required for ParC function, and thus, in contrast to E. coli, it appears that active rather than stochastic processes mediate the subcellular distribution of chemotactic proteins in V. cholerae.

Ringgaard S et al. Genes Dev. 2011 (http://genesdev NULL.cshlp NULL.org/content/25/14/1544 NULL.long)

D-amino acids

Peptidoglycan remodeling governed by D-amino acid release in stationary phase

Peptidoglycan in V. cholerae is composed of linear glycan strands made up of repeating disaccharide units of N-acetyl glucosamine and N-acetylmuramic acid cross-linked by short peptides that consist of L-Ala, D-Glu, meso-diaminopimelic acid (m-DAP), and D-Ala. In stationary phase, D-Met (blue circles) and D-Leu (red circles) are produced by BsrV, a periplasmic racemase. These D-amino acids (1) are incorporated at the 4th position of the PG-peptide bridge where D-Ala is usually found, (2) regulate the activity of periplasmic enzymes including penicillin-binding proteins (PBPs), which synthesize and modify PG, and (3) are released into the extracellular milieu where D-amino acids regulate the PG of other bacteria. OM Outer membrane, IM inner membrane

Cava F et al. Cell Mol Life Sci. 2011 (http://www NULL.springerlink NULL.com/content/7mtw912282635186/fulltext NULL.pdf)

D-amino acids

RNA-Seq-based monitoring of infection-linked changes in Vibrio cholerae gene expression

We used RNA-seq to generate comprehensive transcriptome profiles of V. cholerae during growth in the intestines of infant rabbits as well as in laboratory cultures. Genes induced in vivo included all the known V. cholerae virulence factors including genes for CT and TCP biosynthesis as well as many genes encoding proteins and small RNAs not previously linked to infection. A) Profile of V. cholerae gene expression in culture and during infection (Illumina). B) Strand-specific coverage per nucleotide across the genes within the TCP island.

Mandlik A et al. Cell Host Microbe. 2011 (http://www NULL.ncbi NULL.nlm NULL.nih NULL.gov/pmc/articles/PMC3166260/?tool=pubmed)

rabbit sections

Studying cholera pathogenesis using infant rabbits

Histological findings in the distal small intestines of rabbits inoculated with wild-type (WT) or mutant V. cholerae. (A and B) Representative H&E-stained sections taken from rabbits at 22 h postinfection showing edema (arrows) and capillary congestion (arrowheads) in V. cholerae-infected rabbits (A), which is not evident in mock-infected rabbits (B). (C) The arrows point to heterophils present in the lamina propria of V. cholerae-infected rabbits. (D to F) PAS-stained sections show that mucin (magenta stain; arrows) is absent from goblet cells in rabbits infected with wild-type V. cholerae (D) but not in mock-infected rabbits (E) or rabbits infected with the ctxAB mutant (F). Bars, 500 µm (A, B, D, E, and F) and 100 µm (C).

Ritchie JM et al. MBio. 2010 (http://www NULL.ncbi NULL.nlm NULL.nih NULL.gov/pmc/articles/PMC2912669/pdf/mBio NULL.00047-10 NULL.pdf)

V parahemolyticus schematic

Kinetics of V. parahaemolyticus-induced damage to the intestinal epithelial surface

Following initial attachment, V. parahaemolyticus induces erosion of microvilli and depletion of cytoplasmic contents resulting in the formation of bacterial clusters located just below the level of the surrounding epithelium. Continued depletion of epithelial cell contents either by cytoplasmic ‘blebbing’, whole cell extrusion and microvilli elongation around the edge of the cluster, leaves V. parahaemolyticus clusters situated within deeper cavities in the epithelium. Eventually, this leads to disintegration of normal villus structure and the generation of large amounts of luminal debris. These pathological changes appear to be attributed to T3SS2 as a similar pathology was observed in rabbits infected with mutants lacking TDH or T3SS1. The purple rods represent V. parahaemolyticus.

Ritchie JM et al. PLoS Pathog. 2012 (http://www NULL.ncbi NULL.nlm NULL.nih NULL.gov/pmc/articles/PMC3305451/pdf/ppat NULL.1002593 NULL.pdf)