Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM

Abstract

bBurkholderia pseudomallei, the etiologic agent of melioidosis, is an opportunistic pathogen that harbors a wide array of secretion systems, including a type II secretion system (T2SS), three type III secretion systems (T3SS), and six type VI secretion systems (T6SS). The proteins exported by these systems provide B. pseudomallei with a growth advantage in vitro and in vivo, but relatively little is known about the full repertoire of exoproducts associated with each system. In this study, we constructed deletion mutations in gspD and gspE, T2SS genes encoding an outer membrane secretin and a cytoplasmic ATPase, respectively. The secretion profiles of B. pseudomallei MSHR668 and its T2SS mutants were noticeably different when analyzed by SDS-PAGE. We utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins present in the supernatants of B. pseudomallei MSHR668 and B. pseudomallei ⌬gspD grown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent or substantially reduced in the supernatants of ⌬gspD strains. Many of these proteins were putative hydrolytic enzymes, including 12 proteases, two phospholipases, and a chitinase. Biochemical assays validated the LC-MS/MS results and demonstrated that the export of protease, phospholipase C, and chitinase activities is T2SS dependent. Previous studies had failed to identify the mechanism of secretion of TssM, a deubiquitinase that plays an integral role in regulating the innate immune response. Here we present evidence that TssM harbors an atypical signal sequence and that its secretion is mediated by the T2SS. This study provides the first in-depth characterization of the B. pseudomallei T2SS secretome.

Type VI secretion and anti-host effectors

“…Interference with host immunity pathways is achieved in Bpmt infection by the deubiquitinase TssM (unrelated to the T6SS structural component of the same name), which inhibits NF-kB and type I interferon signaling [51,52]. TssM is a T2SS-dependent protein but is in fact encoded within the T6SS-5 cluster [53].…”

“…The evolved VgrG5, with unknown catalytic activity, is responsible for the fusion of host cell membranes leading to multinucleated giant cell formation[57,58]. The T6SS-5 cluster also encodes an autotransporter, BimA, involved in actin-mediated propulsion; and TssM, a T2SS-dependent effector that dampens NF-kB and type I interferon signaling[51,53,55]. II.…”

Melioidosis: molecular aspects of pathogenesis

“…The tssM gene is physically linked to both the T3SS-3 and T6SS-1 gene clusters and is co-regulated with T6SS-1, but neither system is involved in TssM export [71]. It has recently been shown that secretion of TssM is instead dependent on a functional type II secretion system (T2SS) [72]. When secreted inside macrophages, TssM interferes with the toll-like receptor (TLR) mediated NF-κB signaling pathways associated with innate immune responses [71].…”

Acinetobacter baumannii Is Dependent on the Type II Secretion System and Its Substrate LipA for Lipid Utilization and In Vivo Fitness

“…Second, the T2S system is commonly required for the secretion of several proteins in other species. For example, proteomic analyses of culture supernatants of L. pneumophila, V. cholerae, and Burkholderia pseudomallei indicate that more than 20 different proteins may be dependent on the T2S system for extracellular release (30,39,76). Future work will focus on identifying additional T2S substrates and determining their role in A. baumannii pathogenesis.…”