Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung

Abstract

Type II protein secretion is critical for Legionella pneumophila infection of amoebae, macrophages, and mice. Previously, we found several enzymes to be secreted by this (Lsp) secretory pathway. To better define the L. pneumophila type II secretome, a 2D electrophoresis proteomic approach was used to compare proteins in wild-type and type II mutant supernatants. We identified 20 proteins that are type II-dependent, including aminopeptidases, an RNase, and chitinase, as well as proteins with no homology to known proteins. Because a chitinase had not been previously reported in Legionella, we determined that wild type secretes activity against both p-nitrophenyl triacetyl chitotriose and glycol chitin. An lsp mutant had a 70 -75% reduction in activity, confirming the type II dependency of the secreted chitinase. Newly constructed chitinase (chiA) mutants also had Ϸ75% less activity, and reintroduction of chiA restored the mutants to normal levels of activity. Although chiA mutants were not impaired for in vitro intracellular infection, they were defective upon intratracheal inoculation into the lungs of A/J mice, and antibodies against ChiA were detectable in infected animals. In contrast, mutants lacking a secreted phosphatase, protease, or one of several lipolytic enzymes were not defective in vivo. In sum, this study shows that the output of type II secretion is greater in magnitude than previously appreciated and includes previously undescribed proteins. Our data also indicate that an enzyme with chitinase activity can promote infection of a mammalian host.bacterial protein secretion ͉ bacterial virulence ͉ Legionnaires' disease

Bacterial chitinases and chitin-binding proteins as virulence factors

“…However, the ability of the Legionella pneumophila mutants to survive in macrophage cell cultures is similar to that of the wild-type. This is also the case for Listeria monocytogenes mutants infecting epithelial and macrophage cell cultures (Chaudhuri et al, 2010;DebRoy et al, 2006;Larsen et al, 2010), despite the notion of enhanced intracellular chitinase transcription in macrophages (Mraheil et al, 2011). The in vivo virulence effects of chitinases might not be limited to bacteria, as the trypanosomatid protozoan pathogen, Leishmania mexicana, produces a chitinase that contributes to formation of lesions in a mouse model as well as promoting survival in host macrophage cells (Joshi et al, 2005).…”

“…In some instances chitinase-encoding genes are important for virulence in in vivo animal models but not in in vitro cellular assays. Examples include the intracellular pathogens Legionella pneumophila and Listeria monocytogenes in which chitinase deficiency causes defective growth in mice lungs and spleens, respectively (Chaudhuri et al, 2010;DebRoy et al, 2006). However, the ability of the Legionella pneumophila mutants to survive in macrophage cell cultures is similar to that of the wild-type.…”

“…Such analyses have predicted that the efChi18A chitinase (Table 1) from the Gram-positive Enterococcus faecalis is secreted by the Sec pathway. The Gram-negative Escherichia coli, Legionella pneumophila and V. cholerae chitinases also carry signal sequences and are secreted by means of the type II secretion system (Table 1) (DebRoy et al, 2006;Francetic et al, 2000b;Sikora et al, 2011), whereas this does not seem to be the case for P. aeruginosa (Folders et al, 2001). A number of chitinases (Fig.…”

“…There is an increasing amount of direct or indirect evidence suggesting that some chitinases and CBPs additionally serve as virulence factors for bacterial pathogens during infection. Examples include chitinases from Legionella pneumophila (DebRoy et al, 2006), Listeria monocytogenes (Chaudhuri et al, 2010;Larsen et al, 2010) and Salmonella enterica serovar Typhimurium (S. Typhimurium) (Larsen et al, 2011) as well as a CBP from Vibrio cholerae (Kirn et al, 2005). A summary of cell culture/in vivo indications of this role is presented in Tables 1 and 2.…”

Two-dimensional gel electrophoresis in bacterial proteomics

“…In Legionella pneumophila (the bacterial pathogen responsible for legionnaire's disease), type II protein secretion is known to be involved in virulence. DebRoy et al used a comparative 2-DE analysis of the culture supernatant of wild type L. pneumophila versus a type-II secretion mutant, to identify 20 proteins via the type-II secretion pathway (DebRoy et al, 2006). This included a previously undescribed chitinase A protein, which was subsequently implicated in infection using an A/J mouse model, promoting persistence in the lung.…”