Type VI secretion systems (T6SS) are organelles that are encoded by gene clusters present in many Gram-negative bacterial species in including Pseudomonas aeruginosa, an opportunistic pathogen of Cystic Fibrosis (CF) patients. In many bacterial organisms, T6SS are known virulence factors but recently it has been further recognized that these systems can also kill bacterial as well as eukaryotic cells. Our laboratory has shown that functionally these nanomachines transport proteins into target host cells by a novel mechanism that is analogous to bacteriophage tail contraction. This process can be visualized in real time using appropriate fluorescent reporter protein fusions. The T6SS dynamic organelle assembles and attacks prey cells by initially penetrating them with a large protein ensemble called the VgrG/PAAR/Hcp spike/ tube complex which we hypothesize is decorated with toxic effector proteins by several mechanisms. Furthermore, we have shown that P. aeruginosa can use its T6SS to counterattack and kill other T6SS+ aggressive prey organisms (such as Vibrio cholerae and Acinetobacter baylyi) that attack its cell envelope. A P. aeruginosa sensory system called TagQRST-PpkA-Fha1 regulatory cascade drives T6SS organelle assembly at the site of prey attack and also responds to the outer membrane active, cationic antibiotic polymyxin B (PxB). Therefore, we propose to investigate the following questions in the context of P. aeruginosa: 1) Will PxB activate T6SS assembly in PxB resistant P. aeruginosa mutants and will these mutants still counterattack other aggressive T6SS+ prey cells? 2) Will other membrane stress signals activate organelle assembly including exposure to host defense molecules that attack the bacterial cell envelope (such as human defensins and other toxic cationic and antimicrobial peptides)? 3) Can we use PxB activation to facilitate the large-scale purification of T6SS organelles from P. aeruginosa for structural analysis? 4) Are different PAAR and VgrG proteins required for secretion of different VgrG proteins and T6SS effectors? 5) Will a new saturation mutagenesis technique developed in our lab called Mut-seq allow us to perform a comprehensive analysis of the protein sequences required for T6SS function and allow us further to isolate dominant-negative versions of some of its essential components? 6) Can we define the genes that encode the T6SS armor which allows P. aeruginosa to resist killing by other antibacterial T6SS+ species and their effectors? In pursuing the answers to these questions we will also determine if a broad panel of P. aeruginosa strains express T6SS organelles and characterize these using state-of-the-art imaging methodologies including fluorescence microscopy and electron cryo tomography. Our studies offer novel opportunities for development of therapeutics that attack the cell envelope of P. aeruginosa, a notoriously antibiotic-resistant organism. Thus, these studies may ultimately benefit CF and other patients suffering from P. aeruginosa infections and potentially also contribute to the development of new strategies to inhibit Gram-negative bacteria including those that use the T6SS as a virulence factor.
The bacterium Pseudomonas aeruginosa is the cause of severe infections in cystic fibrosis patients, burn victims and the immune-compromised. This bacterium expresses a dynamic structure called the Type VI secretion system (T6SS) and this nanomachine has been implicated in the pathogenic properties of numerous bacterial species. P. aeruginosa also responds to other aggressive bacterial cells by using its T6SS to attack and kill them. The proposed study seeks to understand how this organism senses exogenous cells and other host signals, and describes studies that will likely define features of P. aeruginosa and its T6SS that could advance anti-bacterial drug discovery, thus impacting medical sciences well beyond P. aeruginosa.
|Skurnik, David; Clermont, Olivier; Guillard, Thomas et al. (2016) Emergence of Antimicrobial-Resistant Escherichia coli of Animal Origin Spreading in Humans. Mol Biol Evol 33:898-914|
|Roux, Damien; Danilchanka, Olga; Guillard, Thomas et al. (2015) Fitness cost of antibiotic susceptibility during bacterial infection. Sci Transl Med 7:297ra114|
|Fu, Yang; Mekalanos, John J (2014) Infant Rabbit Colonization Competition Assays. Bio Protoc 4:|
|Ho, Brian T; Dong, Tao G; Mekalanos, John J (2014) A view to a kill: the bacterial type VI secretion system. Cell Host Microbe 15:9-21|
|Basler, Marek; Ho, Brian T; Mekalanos, John J (2013) Tit-for-tat: type VI secretion system counterattack during bacterial cell-cell interactions. Cell 152:884-94|
|Ho, Brian T; Basler, Marek; Mekalanos, John J (2013) Type 6 secretion system-mediated immunity to type 4 secretion system-mediated gene transfer. Science 342:250-3|
|Fu, Yang; Waldor, Matthew K; Mekalanos, John J (2013) Tn-Seq analysis of Vibrio cholerae intestinal colonization reveals a role for T6SS-mediated antibacterial activity in the host. Cell Host Microbe 14:652-63|
|Skurnik, David; Roux, Damien; Cattoir, Vincent et al. (2013) Enhanced in vivo fitness of carbapenem-resistant oprD mutants of Pseudomonas aeruginosa revealed through high-throughput sequencing. Proc Natl Acad Sci U S A 110:20747-52|
|Danilchanka, Olga; Mekalanos, John J (2013) Cyclic dinucleotides and the innate immune response. Cell 154:962-970|
|Shneider, Mikhail M; Buth, Sergey A; Ho, Brian T et al. (2013) PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 500:350-353|
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