Bacteria have evolved sophisticated mechanisms to inhibit the growth of competitors. One such mechanism involves type VI secretion systems, which bacteria can use to inject antibacterial toxins directly into neighbouring cells. Many of these toxins target the integrity of the cell envelope, but the full range of growth inhibitory mechanisms remains unknown. Here we identify a type VI secretion effector, Tas1, in the opportunistic pathogen Pseudomonas aeruginosa. The crystal structure of Tas1 shows that it is similar to enzymes that synthesize (p)ppGpp, a broadly conserved signalling molecule in bacteria that modulates cell growth rate, particularly in response to nutritional stress. However, Tas1 does not synthesize (p)ppGpp; instead, it pyrophosphorylates adenosine nucleotides to produce (p)ppApp at rates of nearly 180,000 molecules per minute. Consequently, the delivery of Tas1 into competitor cells drives rapid accumulation of (p)ppApp, depletion of ATP, and widespread dysregulation of essential metabolic pathways, thereby resulting in target cell death. Our findings reveal a previously undescribed mechanism for interbacterial antagonism and demonstrate a physiological role for the metabolite (p)ppApp in bacteria.
See the Commentary at Nature.
Type III Secretion Systems (T3SSs) are structurally conserved nanomachines that span the inner and outer bacterial membranes, and via a protruding needle complex contact host cell membranes and deliver type III effector proteins. T3SS are phylogenetically divided into several families based on structural basal body components. Here we have studied the evolutionary and functional conservation of four T3SS proteins from the Inv/Mxi-Spa family: a cytosolic chaperone, two hydrophobic translocators that form a plasma membrane-integral pore, and the hydrophilic ‘tip complex’ translocator that connects the T3SS needle to the translocon pore. Salmonella enterica serovar Typhimurium (S. Typhimurium), a common cause of food-borne gastroenteritis, possesses two T3SSs, one belonging to the Inv/Mxi-Spa family. We used invasion-deficient S. Typhimurium mutants as surrogates for expression of translocator orthologs identified from an extensive phylogenetic analysis, and type III effector translocation and host cell invasion as a readout for complementation efficiency, and identified several Inv/Mxi-Spa orthologs that can functionally substitute for the S. Typhimurium chaperone and translocator proteins. Functional complementation correlates with amino acid sequence identity between orthologs, but varies considerably between the four proteins. This is the first in-depth survey of the functional interchangeability of Inv/Mxi-Spa T3SS proteins acting directly at the host-pathogen interface.
Three of our undergraduate students gave poster presentations in the last month. Arjun Sharma (Biochemistry & Biomedical Sciences 3rd year) outlined his work on developing the Comprehensive Antibiotic Resistance Database’s (arpcard.mcmaster.ca) CARD*Shark text mining algorithms at the Michael G. DeGroote Institute for Infectious Disease Research (IIDR) Trainee Day while Kirill Pankov (Biomedical Discovery & Commercialization 4th year) presented the results of his summer NSERC Undergraduate Student Research Award (USRA) research in the Laboratory of Dr. Joanna Wilson into the origin of Cnidarian P450 enzymes, work he is continuing in our lab as part of his thesis research. Mohammad Khan (Biomedical Discovery & Commercialization 4th year), a thesis student in the Laboratory of Dr. Eric Brown that collaborates with our group, also presented a poster at IIDR Trainee Day on his work on chemical-genetic interaction database design.
Sharma, A.N., S. Doshi, A.R. Raphenya, B. Alcock, B.M. Dave, B.A. Lago, K.K. Tsang, & A.G. McArthur. 2016. CARDShark: Computer-assisted biocuration of the Comprehensive Antibiotic Resistance Database. Poster presentation at the 2016 Michael G. DeGroote Institute for Infectious Disease Research (IIDR) Trainee Day, Hamilton, Ontario, Canada.
Pankov, K., A.G. McArthur & J.Y. Wilson. 2016. The Cytochrome P450 (CYP) superfamily in the Cnidarian phylum. Poster presentation at the 2016 Undergraduate Student Research Awards (USRA) Poster Session, Hamilton, Ontario, Canada.
Khan, M.A., S. French, B. Aubie, A.G McArthur & E.D. Brown. 2016. Challenging common screening filters through analysis of a chemical-genetic screening database. Poster presentation at the 2016 Michael G. DeGroote Institute for Infectious Disease Research (IIDR) Trainee Day, Hamilton, Ontario, Canada.
Cytochrome P450 (CYP) proteins compose a highly diverse superfamily found in all domains of life. These proteins are enzymes involved in metabolism of endogenous and exogenous compounds. In vertebrates, the CYP2 family is one of the largest, most diverse and plays an important role in mammalian drug metabolism. However, there are more than 20 vertebrate CYP2 subfamilies with uncertain evolution and fairly discrete subfamily composition within vertebrate classes, hindering extrapolation of knowledge across subfamilies. To better understand CYP2 diversity, a phylogenetic analysis of 196 CYP2 protein sequences from 16 species was performed using a maximum likelihood approach and Bayesian inference. The analyses included the CYP2 compliment from human, fugu, zebrafish, stickleback, medaka, cow, and dog genomes. Additional sequences were included from rabbit, marsupial, platypus, chicken, frog, and salmonid species. Three CYP2 sequences from the tunicate Ciona intestinalis were utilized as the outgroup. Results indicate a single ancestral vertebrate CYP2 gene and monophyly of all CYP2 subfamilies. Two subfamilies (CYP2R and CYP2U) pre-date vertebrate diversification, allowing direct comparison across vertebrate classes, while all other subfamilies originated during vertebrate diversification, often within specific vertebrate lineages. Analysis of site-specific evolution indicates that some substrate recognition sites (SRS) previously proposed for CYP genes do not have elevated rates of evolution, suggesting that these regions of the protein are not necessarily important in recognition of CYP2 substrates. Type II functional divergence analysis identified multiple residues in the active site of CYP2F, CYP2A, and CYP2B proteins that have undergone radical biochemical changes and may be functionally important.
Following up on his doctoral research, Dr. McArthur joined the NMNH as a Postdoctoral Fellow to use the tools of molecular systematics to test the hypothesis of ancient origins of deep-sea gastropods. This included his first serious foray into bioinformatics as part of the Laboratory of Molecular Systematics, with use of advanced algorithms for phylogenetic analysis.