ABSTRACT
Previously, we documented that Stenotrophomonas maltophilia encodes a type IV secretion system (T4SS) that allows the organism to kill, in contact-dependent fashion, heterologous bacteria, including wild-type Pseudomonas aeruginosa. Bioinformatic screens based largely on the presence of both a C-terminal consensus sequence and an adjacent gene encoding a cognate immunity protein identified 13 potential antibacterial effectors, most of which were highly conserved among sequenced strains of S. maltophilia. The immunity proteins of two of these proved especially capable of protecting P. aeruginosa and Escherichia coli against attack from the Stenotrophomonas T4SS. In turn, S. maltophilia mutants lacking the putative effectors RS14245 and RS14255 were impaired for killing not only laboratory E. coli but clinical isolates of P. aeruginosa, including ones isolated from the lungs of cystic fibrosis patients. That complemented mutants behaved as wild type did confirmed that RS14245 and RS14255 are required for the bactericidal activity of the S. maltophilia T4SS. Moreover, a mutant lacking both of these proteins was as impaired as a mutant lacking the T4SS apparatus, indicating that RS14245 and RS14255 account for (nearly) all of the bactericidal effects seen. Utilizing an interbacterial protein translocation assay, we determined that RS14245 and RS14255 are bona fide substrates of the T4SS, a result confirmed by examination of mutants lacking both the T4SS and the individual effectors. Delivery of the cloned 14245 protein (alone) into the periplasm resulted in the killing of target bacteria, indicating that this effector, a putative lipase, is both necessary and sufficient for bactericidal activity. IMPORTANCE S. maltophilia is an increasingly important opportunistic pathogen. Inherently resistant to many antibiotics, S. maltophilia is often associated with lung infection, being, among other things, a complicating factor in cystic fibrosis patients. Moreover, it is a common form of coinfection in COVID-19 patients. In these various clinical settings and in natural habitats, S. maltophilia coexists with other pathogens, including P. aeruginosa. Previously, we documented that S. maltophilia possesses a T4SS that kills other bacteria, a notable observation given that most prior work on interbacterial competition has highlighted bactericidal effects of type VI secretion systems. By utilizing approaches ranging from bioinformatics to mutant analysis to protein translocation assays, we have now identified two substrates of the Stenotrophomonas T4SS that largely mediate the killing of pathogenic P. aeruginosa. These results represent a major advance in understanding S. maltophilia, the roles of T4SSs, concepts regarding clinically relevant, interbacterial competition, and activities of bactericidal effectors.