Legionella Pneumophila Type II Secretome Reveals Unique Exoproteins and a Chitinase That Promotes Bacterial Persistence in the Lung
Total Page：16
File Type：pdf, Size：1020Kb
Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung Sruti DebRoy, Jenny Dao, Maria So¨ derberg, Ombeline Rossier, and Nicholas P. Cianciotto* Department of Microbiology–Immunology, Northwestern University Medical School, Chicago, IL 60611 Edited by Thomas J. Silhavy, Princeton University, Princeton, NJ, and approved October 27, 2006 (received for review September 20, 2006) Type II protein secretion is critical for Legionella pneumophila acid phosphatases, lipases, phospholipase A, phospholipase C, infection of amoebae, macrophages, and mice. Previously, we lysophospholipase A, cholesterol acyltransferase, and ribonucle- found several enzymes to be secreted by this (Lsp) secretory ase (4–6, 10–15). Both the Sec and Tat pathways are implicated pathway. To better define the L. pneumophila type II secretome, a in the processing of L. pneumophila type II substrates (16). 2D electrophoresis proteomic approach was used to compare Because mutants lacking individual type II effectors infect proteins in wild-type and type II mutant supernatants. We identi- normally (10, 12–14, 17, 18), we hypothesize that more proteins fied 20 proteins that are type II-dependent, including aminopep- are secreted by Lsp, and that one or more of them are virulence tidases, an RNase, and chitinase, as well as proteins with no determinants. Although L. pneumophila has been sequenced (19, homology to known proteins. Because a chitinase had not been 20), it is not possible to readily identify effectors by bioinfor- previously reported in Legionella, we determined that wild type matics, because there is no motif that specifically defines a secretes activity against both p-nitrophenyl triacetyl chitotriose protein as a type II substrate (3). Thus, we embarked on a 2D and glycol chitin. An lsp mutant had a 70–75% reduction in activity, electrophoresis (2DE)-based proteomic approach to more fully confirming the type II dependency of the secreted chitinase. Newly define the scope of L. pneumophila type II secretion. This effort constructed chitinase (chiA) mutants also had Ϸ75% less activity, has revealed a large set of effectors, including previously unde- and reintroduction of chiA restored the mutants to normal levels scribed exoproteins and a chitinase, which remarkably promotes of activity. Although chiA mutants were not impaired for in vitro bacterial persistence in the lungs. intracellular infection, they were defective upon intratracheal inoculation into the lungs of A/J mice, and antibodies against ChiA Results were detectable in infected animals. In contrast, mutants lacking a Analysis of the L. pneumophila Type II Secretome. To further define secreted phosphatase, protease, or one of several lipolytic en- the proteins secreted by Lsp, we analyzed proteins in wild-type zymes were not defective in vivo. In sum, this study shows that the and lsp mutant supernatants by 2DE and then used mass output of type II secretion is greater in magnitude than previously spectrometry to obtain the identity of the secreted proteins. appreciated and includes previously undescribed proteins. Our Twenty-seven proteins present in wild-type strain 130b super- data also indicate that an enzyme with chitinase activity can natants but absent or greatly reduced in supernatants of the promote infection of a mammalian host. NU275 lspF mutant were identified [Table 1; 2D gel images ͉ ͉ appear as supporting information (SI) Fig. 4]. Three of these, the bacterial protein secretion bacterial virulence Legionnaires’ disease ProA metalloprotease, PlaA lysophospholipase A, and Map acid phosphatase, were defined as type II exoproteins before (Table egionella pneumophila is the etiological agent of Legion- 1; refs. 4–6, 9, 12, and 21). A fourth protein was identified as a Lnaires’ disease, a potentially fatal pneumonia that especially ribonuclease, undoubtedly representing the type II-dependent affects immunocompromised individuals (1). As an environmen- ribonuclease activity that we had reported before, but for which tal pathogen, this Gram-negative bacterium exists in fresh the gene had been unknown (Table 1; refs. 6 and 11). Of the waters, in protozoan hosts, and as a part of biofilms. It is also remaining 23 proteins, 13 were also predicted to contain a signal present in man-made systems, such as potable water systems and sequence and likely represent new type II-dependent exopro- cooling towers. Transmission occurs when aerosolized bacteria teins (Table 1). Two of these were annotated in the database as are inhaled. L. pneumophila colonizes the respiratory tract, a leucine aminopeptidase and a chitinase. Four others were where it invades alveolar macrophages. Bacterial degradative annotated as hypothetical proteins having similarities to bacte- enzymes, host cell lysis, and excessive inflammation combine to rial enzymes and included an amidase, aminopeptidase, cysteine promote damage to lung tissue. We have shown that a type II protease, and endoglucanase. Two others had similarities to protein secretion system is important in the ecology and patho- eukaryotic proteins; i.e., spot 27 had a protein with collagen-like genesis of L. pneumophila (2). Present in many but not all gram repeats and spot 21 a protein with greatest homology to a negatives, including animal and plant pathogens (2), type II eukaryotic zinc proteinase. Five other proteins were annotated secretion is a two-step process in which nascent proteins are first translocated across the inner membrane by the Sec or Tat pathway and then exit the cell through an outer membrane Author contributions: S.D. and N.P.C. designed research; S.D., J.D., M.S., and O.R. per- secretin (3). L. pneumophila type II secretion pathway (Lsp) formed research; S.D., O.R., and N.P.C. analyzed data; and S.D., O.R., and N.P.C. wrote the mutants display a growth defect in amoebae, macrophages, and paper. the lungs of mice (4–6). L. pneumophila is the only intracellular The authors declare no conflict of interest. pathogen shown to possess a functional type II system. Other This article is a PNAS direct submission. phenotypes ascribed to L. pneumophila type II mutants include Abbreviations: Lsp, Legionella secretion pathway; pNP-[GlcNAc]3, p-nitrophenyl -D- reduced growth at low temperatures, altered colony morphol- N,NЈ,NЉ triacetyl chitotriose; 2DE, 2D electrophoresis. ogy, and diminished invasion of host cells (6–9). Thus far, *To whom correspondence should be addressed. E-mail: [email protected]. proteins secreted through the L. pneumophila type II system This article contains supporting information online at www.pnas.org/cgi/content/full/ have been first identified based on the detection of enzymatic 0608279103/DC1. activities in culture supernatants, including a metalloprotease, © 2006 by The National Academy of Sciences of the USA 19146–19151 ͉ PNAS ͉ December 12, 2006 ͉ vol. 103 ͉ no. 50 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0608279103 Downloaded by guest on September 25, 2021 Table 1. Type II-dependent exoproteins of L. pneumophila identified by 2DE analysis Gene designation† PubMed Sequence Identity (as defined in the Spot no.* accession no. Score covered, % annotated genome) Philadelphia Paris Lens SignalP 1, 42 2110146 108 16 Zinc metalloprotease lpg0467 lpp0532 lpl0508 Y 2 11119504 209 18 Major acid phosphatase lpg1119 lpp1120 lpl1124 Y 3 15448271 146 11 Ribonuclease, T2 family lpg2848 lpp2906 lpl2760 Y 4 12379686 229 15 Lysophospholipase A lpg2343 lpp2291 lpl2264 Y 5, 11, 12, 37 15448271 50 29 Leucine aminopeptidase lpg2814 lpp2866 lpl2729 Y 6–10, 34 11923356 261 32 LvrE lpg1244 lpp0181 lpl0163 Y 13 15448271 174 7 Chitinase lpg1116 lpp1117 lpl1121 Y 14, 15, 17 15003709 448 36 IcmX lpg2689 lpp2743 lpl2616 Y 16 15448271 383 26 Hypothetical protein lpg0189 lpp0250 lpl0249 Y 18, 35, 41 15448271 92 9 Hypothetical protein lpg1809 lpp1772 lpl1773 Y 19, 41 15467720 345 46 Hypothetical protein - has VirK domain lpg1832 lpp1795 lpl1796 Y 20 15467720 319 46 Hypothetical protein - weak similarity lpg0264 lpp0335 lpl0316 Y to amidase 21 15448271 415 39 Similar to eukaryotic zinc lpg2999 lpp3071 lpl2927 Y metalloproteinase 22, 25, 26 15467720 583 41 Hypothetical protein - weakly similar lpg1918 lpp1893 lpl1882 Y to endoglucanase 23, 24, 38 15448271 357 23 Hypothetical protein lpg0956 lpp1018 lpl0985 Y 27, 36, 39 15448271 363 27 Tail fiber protein (collagen-like protein lpg2644 lpp2697 lpl2569 Y SclB) 28, 40, 42 15448271 227 33 Hypothetical protein lpg0873 lpp0936 lpl0906 Y 29 15467720 431 12 Aconitate hydratase lpg1690 lpp1659 lpl1653 N 30 9332363 506 12 Chaperone protein dnaK lpg2025 lpp2007 lpl2002 N 31 7790062 590 46 Flagellin lpg1340 lpp1294 lpl1293 N 32, 33 15467720 92 25 Hypothetical protein - lpp1177 lpl1183 N 34 15448271 102 11 Hypothetical protein - weakly similar lpg2622 lpp2675 lpl2547 Y to cysteine protease 35 15448271 105 25 Hypothetical protein - lpg0406 lpp0472 lpl0048 N carboxymuconolactone decarboxylase 36 15467720 415 24 Hypothetical protein - some similarity lpg2526 lpp2591 lpl2446 N to nucleotidase 37 15467720 489 25 Hypothetical protein - similar to lpg0032 lpp0031 lpl0032 Y aminopeptidase 38 15467720 146 19 Hypothetical protein lpg1954 lpp1936 lpl1923 N 40 15448271 153 16 Hypothetical protein lpg1385 lpp1340 lpl1336 Y *As is commonly seen, some proteins were represented in more than one spot, and some spots contained multiple proteins. For those proteins found in multiple spots, comparable scores and sequence coverage were obtained from the