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.

Type II Secretion-Dependent Degradative and Cytotoxic Activities Mediated by Stenotrophomonas maltophilia Serine Proteases StmPr1 and StmPr2

“…Mutants used in this study are also listed in Table 1. S. maltophilia strains were routinely cultured at 37°C on Luria-Bertani (LB) agar (Becton, Dickinson, Franklin Lakes, NJ) or LB broth (21). In order to obtain supernatants for enzymatic assays and other analyses, S. maltophilia strains were cultured in a version of buffered yeast extract (BYE) broth consisting of 10 g per liter of yeast extract, 54.9 mM N-(2-acetamido)-2-aminoethanesulfonic acid buffer, and 39.2 mM potassium hydroxide at pH 6.85 to 6.95.…”

“…By constructing and characterizing gsp and xps mutants of the clinical isolate K279a, we concluded that Xps T2S promotes detrimental effects on the human lung epithelial cell line A549. Specifically, S. maltophilia supernatants caused cell rounding, detachment, and actin rearrangement and exhibited cytotoxicity of A549 cells in an Xps T2S-dependent manner (21). It remains to be determined whether Gsp T2S is functional, as gsp mutants do not lack any of the observed activities exhibited by strain K279a.…”

“…T2S occurs through a multistep process, where T2S substrates are first translocated across the in-ner membrane into the periplasm, predominantly through the Sec pathway. The T2S apparatus then promotes translocation of the substrates through the outer membrane secretin into the extracellular milieu via the piston-like motion of the pseudopilus (21). Our laboratory has recently investigated the functionality of two T2S systems in S. maltophilia; i.e., Gsp T2S and Xps T2S (21).…”

“…The T2S apparatus then promotes translocation of the substrates through the outer membrane secretin into the extracellular milieu via the piston-like motion of the pseudopilus (21). Our laboratory has recently investigated the functionality of two T2S systems in S. maltophilia; i.e., Gsp T2S and Xps T2S (21). By constructing and characterizing gsp and xps mutants of the clinical isolate K279a, we concluded that Xps T2S promotes detrimental effects on the human lung epithelial cell line A549.…”

“…It remains to be determined whether Gsp T2S is functional, as gsp mutants do not lack any of the observed activities exhibited by strain K279a. Analysis of culture supernatants from strain K279a by SDS-PAGE revealed that at least seven proteins are secreted in an Xps T2S-dependent manner (21). T2S substrates, such as degradative enzymes, are commonly associated with the virulence of Gram-negative pathogens (22,23), and the genome of strain K279a encodes such factors as proteases, lipases, esterases, DNases, RNases, and fibrolysins (19).…”