Coordinate Regulation of Virulence in Vibrio Cholerae

Harvard Medical School, Boston MA

Investigators

Linked publications & trials

Abstract

PROJECT SUMMARY: 1. Continued efforts to understand the signals that trigger the CDD anti-phage system in V. cholerae and defining how it works biochemically. We've shown that palindromic sequences trigger the DdmABC+PirA anti-phage system which we call the Cell Density-dependent Death or CDD system. We plan to define the precise mechanism of CDD by address several key questions: Is this DdmA nuclease activated in the cytosol of cells that are exposed to DNA palindromic hairpins or the whole VIBO4 phage genome at high MOI? Is the host chromosome degraded DdmA nuclease in the presence of palindromic DNA? Answers to these questions will guide in vitro biochemistry that will seek to reconstitute the signalling complex (e.g., DdnABC +/- PirA +/- palindromic DNA hairpin). Such a complex might trigger DdnA nuclease activity. We already have active Hexa-His versions of these proteins for purification or doing pull down experiments to define stable complexes. We plan to work with our colleagee Dr. Philip Kranzusch to obtain a cryo- EM structure of the complexes in active and inactive biochemical states. We successfully did this with Dr. Kranzusch with two other CBASS nucleases as well as the DNA bound version of eukaryotic cGAS. 2. Understanding the host range of V. cholerae phage and antiphage systems. While the genes that trigger CDD can block some phage infections, many phages are immune to the system. We will determine if these are devoid of palindromic sequences by phage genome sequencing and analysis. If they do have palindromes, we will clone them and confirm they are actively recognized by the CDD system. A positive result would suggest that these phages likely encode anti-CDD proteins. Thus, we will attempt to clone these anti-CDD genes as trans-suppressors for palindromic hairpin-induced CDD. 3. Using CRISPRi to understand more about phage resistance phenotypes in V. cholerae. Our computational pipeline called PCOVA has identified many new anti-phage systems based solely on their covariance with other anti-phage systems. We will use our new genome-wide CRISPRi system to ask whether these are functional. For example, PCOVA has identfied a new anti-phage gene called VC0812 which is not associated with chromosomal islands, and which likely encodes a nuclease. Knockout of VC0812 sensitize V. cholerae to unique phages. We will further characterize VC0812 because this gene correlates highly with pandemic lineages of V. cholerae that include both Classical and El Tor biotypes. We also plan to use CRISPRi to identify essential genes involved in CDD as these are impossible to inactivate. For example, if DNA synthesis is required for a step in the CDD pathway, then gwCRISPi might uncover essential genes like polA and gyrAB that are involved in DNA replication as being needed for efficient CDD. Knocking down their transcription with CRISPRi would be picked up as enrichment of sgRNA against these targets in cells that survive brief exposure to CDD conditions. Of course genes like DdmABC would serve as positive controls since we know the level of transcription of these genes is controled by quorum sensing and thus their knockdown should block CDD. Pair-wise pwCRISPRi willl be imployed to provide information about the CDD components that become most critical when a particular CDD component such as PriA is knocked down. 4. Exploring Biological Signal Amplifiers (BSA) in vivo during V. cholerae infection. We've shown that T6SS killing of commensal E. coli greatly enhances mucosal colonization of V. cholerae. As a way to explore the effects of bacterial cell death on mucosal biology, we've developed two potential BSA systems that cause V. cholerae to lyse during infection -- 1) by using CRISPRi to knockdown essential genes (e.g., cell wall biosynthesis gene murG), and 2) by expressing various cell wall hydrolytic enzymes. We plan to monitor the transcription of bacteria and host animals that are infected with BSA strains plus or minus a coninfecting WT. In parallel, we will prepare cell-free supernatant fluids from V. cholerae that have undergone lysis due to BSA expression. These will be tested for their ability to signal host cells and trigger cytokine responses consistent with activation of innate immune pathways. We will define which PAMPs are released by different BSAs. Preliminary results suggest that muramyl di-peptide (MDP) is indeed one PAMP that can enhance V. cholerae colonization. Phage infection in vitro and in vivo will also be used to generate phage-induced PAMPs that will be studied the same way. Given that phages do grow on V. cholerae during human infection, this could be another way phages influence V. cholerae virulence and pathogenesis. Thus, phage infection of a fraction of the in vivo V. cholerae might act as a BSA as well. 5. Exploring the mechanism of how Aeromonas A603 specifically targets V. cholerae for killing with its T6SS and phenazine natural product. We plan to wrap up our work on A603 with experiments focused on the activity of two T6SS effectors (TseBC) that may permeabilize Vibrio cells to phenazine uptake. The structure of the A603 phenazine and the identity and mechanism of resistance proteins will be determined. We also plan to study whether A603 can interupt transmission of V. cholerae from one linfected animal to another. This would suggest A603 could be used to control cholera outbreaks.