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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI026289-26A1
Application #
8756136
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Taylor, Christopher E,
Project Start
1988-05-01
Project End
2019-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
26
Fiscal Year
2014
Total Cost
$538,137
Indirect Cost
$220,652
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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
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 500:350-3
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
Danilchanka, Olga; Mekalanos, John J (2013) Cyclic dinucleotides and the innate immune response. Cell 154:962-70
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
Basler, M; Pilhofer, M; Henderson, G P et al. (2012) Type VI secretion requires a dynamic contractile phage tail-like structure. Nature 483:182-6
Davies, Bryan W; Bogard, Ryan W; Dupes, Nicole M et al. (2011) DNA damage and reactive nitrogen species are barriers to Vibrio cholerae colonization of the infant mouse intestine. PLoS Pathog 7:e1001295
Chin, Chen-Shan; Sorenson, Jon; Harris, Jason B et al. (2011) The origin of the Haitian cholera outbreak strain. N Engl J Med 364:33-42
Ma, Amy T; Mekalanos, John J (2010) In vivo actin cross-linking induced by Vibrio cholerae type VI secretion system is associated with intestinal inflammation. Proc Natl Acad Sci U S A 107:4365-70

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