Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung - PubMed (original) (raw)

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

Sruti DebRoy et al. Proc Natl Acad Sci U S A. 2006.

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 approximately 75% 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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Secreted chitinase activity of L. pneumophila wild type and lspF and chiA mutants. (A) Culture supernatants of wild-type 130b (pMMB2002; black bar), lspF mutant NU275 (pMMB2002; dark gray bar), NU275 (p_lspF_; light gray bar), chiA mutant NU318 (pMMB2002; white bar), and NU318 (p_chiA_; crosshatched bar) were assayed for chitinase activity against p_-NP-[GlcNAc]3. Data represent the mean and standard deviation for triplicate cultures for each strain. The reductions in enzymatic activity for NU275 (pMMB2002) and NU318 (pMMB2002) were significant (Student's t test; P < 0.05). 130b, NU275, and NU318 behaved identically to their corresponding derivatives that contained only the cloning vector pMMB2002 (data not shown). (B) Utilization of glycol chitin by the secreted chitinase in the supernatants of strains 130b (1), 130b (pMMB2002; 2), 130b (p_chiA; 3), NU275 (4), NU275 (pMM2002; 5), NU275 (p_lspF_; 6), NU318 (7), NU318 (pMMB2002; 8), and NU318 (p_chiA_; 9). Twenty microunits of S. griseus chitinase (a) and BYE medium (b) were used as the positive and negative controls, respectively. Similar results for A and B were obtained on at least two other occasions. The independently derived chiA mutant NU319 had a phenotype identical to that of mutant NU318 (data not shown).

Fig. 2.

Fig. 2.

In vivo competition of L. pneumophila wild type and a chiA mutant in the lung. An equal mixture of wild-type strain 130b and chiA mutant NU318 was inoculated intratracheally into the lungs of A/J mice. The ratio of the wild type to the mutant was determined at days 1 and 3 after inoculation. Data are representative of actual values obtained per mouse (n = 5), and the solid bar indicates the mean value. The differences in the ratios of 130b and NU318 on day 1 vs. day 3 were significant (Student's t test; P < 0.05).

Fig. 3.

Fig. 3.

Growth and persistence of wild type and mutant L. pneumophila in the lung. A/J mice were intratracheally inoculated with equal numbers of 130b (♦; A) and the chiA mutant NU318 (□) and 130b (♦; B), NU318 (□) and the type II mutant NU275 (■), and the cfus in the infected lungs were determined at various time points. The data represent the mean and standard deviations of four to six mice for each strain. The differences between the cfus recovered for 130b and NU318 at 48 and 72 h after inoculation (A) and 130b and NU275 and 130b and NU318 at 48 h after inoculation (B) were significant (Student's t test, P < 0.01).

Similar articles

Cited by

References

    1. Fields BS, Benson RF, Besser RE. Clin Microbiol Rev. 2002;15:506–526. - PMC - PubMed
    1. Cianciotto NP. Trends Microbiol. 2005;13:581–588. - PubMed
    1. Johnson TL, Abendroth J, Hol WG, Sandkvist M. FEMS Microbiol Lett. 2006;255:175–186. - PubMed
    1. Liles MR, Edelstein PH, Cianciotto NP. Mol Microbiol. 1999;31:959–970. - PubMed
    1. Hales LM, Shuman HA. Infect Immun. 1999;67:3662–3666. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources