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Stenotrophomonas maltophilia encodes a type II protein secretion system that promotes detrimental effects on lung epithelial cells.

ABSTRACT: The Gram-negative bacterium Stenotrophomonas maltophilia is increasingly identified as a multidrug-resistant pathogen, being associated with pneumonia, among other infections. Despite this increasing clinical problem, the genetic and molecular basis of S. maltophilia virulence is quite minimally defined. We now report that strain K279a, the first clinical isolate of S. maltophilia to be sequenced, encodes a functional type II protein secretion (T2S) system. Indeed, mutants of K279a that contain a mutation in the xps locus exhibit a loss of at least seven secreted proteins and three proteolytic activities. Unlike culture supernatants from the parental K279a, supernatants from multiple xps mutants also failed to induce the rounding, detachment, and death of A549 cells, a human lung epithelial cell line. Supernatants of the xps mutants were also unable to trigger a massive rearrangement in the host cell's actin cytoskeleton that was associated with K279a secretion. In all assays, a complemented xpsF mutant behaved as the wild type did, demonstrating that Xps T2S is required for optimal protein secretion and the detrimental effects on host cells. The activities that were defined as being Xps dependent in K279a were evident among other respiratory isolates of S. maltophilia. Utilizing a similar type of genetic analysis, we found that a second T2S system (Gsp) encoded by the K279a genome is cryptic under all of the conditions tested. Overall, this study represents the first examination of T2S in S. maltophilia, and the data obtained indicate that Xps T2S likely plays an important role in S. maltophilia pathogenesis.

SUBMITTER: Karaba SM

PROVIDER: S-EPMC3754218 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Stenotrophomonas maltophilia encodes a type II protein secretion system that promotes detrimental effects on lung epithelial cells.

Karaba Sara M SM White Richard C RC Cianciotto Nicholas P NP

Infection and immunity 20130617 9

The Gram-negative bacterium Stenotrophomonas maltophilia is increasingly identified as a multidrug-resistant pathogen, being associated with pneumonia, among other infections. Despite this increasing clinical problem, the genetic and molecular basis of S. maltophilia virulence is quite minimally defined. We now report that strain K279a, the first clinical isolate of S. maltophilia to be sequenced, encodes a functional type II protein secretion (T2S) system. Indeed, mutants of K279a that contain ...[more]

PMID: 23774603

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Project description:Bacterial type IV secretion systems (T4SS) are a highly diversified but evolutionarily related family of macromolecule transporters that can secrete proteins and DNA into the extracellular medium or into target cells. It was recently shown that a subtype of T4SS harboured by the plant pathogen Xanthomonas citri transfers toxins into target cells. Here, we show that a similar T4SS from the multi-drug-resistant opportunistic pathogen Stenotrophomonas maltophilia is proficient in killing competitor bacterial species. T4SS-dependent duelling between S. maltophilia and X. citri was observed by time-lapse fluorescence microscopy. A bioinformatic search of the S. maltophilia K279a genome for proteins containing a C-terminal domain conserved in X. citri T4SS effectors (XVIPCD) identified twelve putative effectors and their cognate immunity proteins. We selected a putative S. maltophilia effector with unknown function (Smlt3024) for further characterization and confirmed that it is indeed secreted in a T4SS-dependent manner. Expression of Smlt3024 in the periplasm of E. coli or its contact-dependent delivery via T4SS into E. coli by X. citri resulted in reduced growth rates, which could be counteracted by expression of its cognate inhibitor Smlt3025 in the target cell. Furthermore, expression of the VirD4 coupling protein of X. citri can restore the function of S. maltophilia ΔvirD4, demonstrating that effectors from one species can be recognized for transfer by T4SSs from another species. Interestingly, Smlt3024 is homologous to the N-terminal domain of large Ca2+-binding RTX proteins and the crystal structure of Smlt3025 revealed a topology similar to the iron-regulated protein FrpD from Neisseria meningitidis which has been shown to interact with the RTX protein FrpC. This work expands our current knowledge about the function of bacteria-killing T4SSs and increases the panel of effectors known to be involved in T4SS-mediated interbacterial competition, which possibly contribute to the establishment of S. maltophilia in clinical and environmental settings.

Project description:Clinical strains of Stenotrophomonas maltophilia are often highly resistant to multiple antibiotics, although the mechanisms of resistance are generally poorly understood. Multidrug resistant (MDR) strains were readily selected by plating a sensitive reference strain of the organism individually onto a variety of antibiotics, including tetracycline, chloramphenicol, ciprofloxacin, and norfloxacin. Tetracycline-selected MDR strains typically showed cross-resistance to erythromycin and fluoroquinolones and, in some instances, aminoglycosides. MDR mutants selected with the other agents generally displayed resistance to chloramphenicol and fluoroquinolones only, although two MDR strains (e.g., K1385) were also resistant to erythromycin and hypersusceptible to aminoglycosides. Many of the MDR strains expressed either moderate or high levels of a novel outer membrane protein (OMP) of ca. 50 kDa molecular mass, a phenotype typical of MDR strains of Pseudomonas aeruginosa hyperexpressing drug efflux systems. Indeed, the 50-kDa OMP of these S. maltophilia MDR strains reacted with antibody to OprM, the outer membrane component of the MexAB-OprM MDR efflux system of P. aeruginosa. Similarly, a ca. 110-kDa cytoplasmic membrane protein of these MDR strains also reacted with antibody to the MexB component of the P. aeruginosa pump. The outer and cytoplasmic membranes of several clinical S. maltophilia strains also reacted with the anti-OprM and anti-MexB antibodies. N-terminal amino acid sequencing of a cyanogen bromide-generated peptide of the 50-kDa OMP of MDR strain K1385, dubbed SmeM (Stenotrophomonas multidrug efflux), revealed it to be very similar to a number of outer membrane multidrug efflux components of P. aeruginosa and Pseudomonas putida. Deletion of the L1 and L2 beta-lactamase genes confirmed that these enzymes were responsible for the bulk of the beta-lactam resistance of K1385 and its parent. Still, overexpression of the MDR efflux mechanism in an L1- and L2-deficient derivative of K1385 did yield a modest increase in resistance to a few beta-lactams. These data are consistent with the MDR efflux mechanism(s) playing a role in the multidrug resistance of S. maltophilia.

Dataset Information

Stenotrophomonas maltophilia encodes a type II protein secretion system that promotes detrimental effects on lung epithelial cells.

Publications

Stenotrophomonas maltophilia encodes a type II protein secretion system that promotes detrimental effects on lung epithelial cells.

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