Stenotrophomonas maltophilia TfcA and TfcB: Antibacterial T4SS effectors from an emerging human pathogen

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PROJECT SUMMARY Gram- Stenotrophomonas maltophilia (Sm) is an emergent, multi-drug resistant pathogen. Sm infection occurs throughout the body but is most often manifest as pneumonia. Sm is notable in cystic fibrosis (CF) patients, often heightening risk of lung exacerbations, and its significance is increased further by recent reports of Sm infecting COVID-19 patients. Despite this clinical importance, knowledge of Sm is still relatively limited. Recently, we found that Sm encodes a type IV secretion system (T4SS) that promotes, in a contact-dependent manner, apoptosis of macrophages, while blunting apoptosis in lung epithelial cells. Yet, perhaps even more significant was our finding that the Sm T4SS mediates, in contact-dependent fashion, killing of E. coli (Ec) and multiple clinical isolates of Pseudomonas aeruginosa (Pa), including those from CF patients. Thus, we posited that the antibacterial effect of Sm T4SS impacts human infection, as Sm and Pa are often together in water systems in hospitals and in various types of infections, especially in the (CF) lungs. Based on the analysis of mutants and their complements, we identified two putative effectors (TfcA and TfcB) as being required for the bactericidal activity of the Sm T4SS. A mutant lacking both these proteins was as impaired as a mutant lacking the T4SS apparatus, indicating that TfcA and TfcB account for (nearly all) the killing effects seen. Using both a 2-hybrid assay that measures binding to the T4SS coupling protein and an interbacterial protein translocation assay, we determined that TfcA and TfcB are bona fide substrates of the T4SS, a result confirmed by testing mutants lacking both the T4SS apparatus and the individual effectors. Delivery of cloned TfcA (alone) into the periplasm resulted in the killing of target bacteria, indicating that this effector is both necessary and sufficient for bactericidal activity. Bioinformatics suggested that TfcA is representative of a large but uncharacterized branch of microbial lipases, whereas TfcB appears to signify a large but ill-defined group of lysozyme-like enzymes. In contrast to the vast data re T4SSs impacting mammalian hosts and the many studies on the antibacterial role of type VI secretion systems, knowledge of the antibacterial role of T4SSs is very minimal. Indeed, Sm T4SS is thus far the only antibacterial T4SS reported for a human pathogen, and TfcA and TfcB its only documented bactericidal effectors. Thus, we propose to i) define the enzyme activities encoded by TfcA and TfcB, ii) discern if those activities promote killing of Pa and Ec, and iii) explore if Sm T4SS, TfcA, and TfcB also kill other co-inhabitants of the CF lung, ranging from other emerging Gram- bacteria to classic Gram+ pathogens to fungi. Besides improving our knowledge of Sm, the data obtained will have broad implications for the roles of other T4SSs and possible new links between T4SS effectors and anti-microbial therapies.