Legionella Pneumophila Type II Secretome Reveals Unique Exoproteins and a Chitinase That Promotes Bacterial Persistence in the Lung

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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 28, 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 different spots. †Gene designation is given for the three sequenced strains of L. pneumophila, i.e., Philadelphia-1, Lens, and Paris (19, 20).

as hypothetical with no similarities to any known protein or tumefaciens protein encoded within the region of the VirB/D4 domain in the database and therefore may represent novel type IV secretion system but not required for Ti-plasmid-DNA activities. As noted in Table 1, several of the type II exoproteins, transfer (27, 28). These data raise the possibility of a mech- including the leucine aminopeptidase, some of the hypothetical anistic connection between types II and IV secretion. Alter- proteins, and others (see IcmX and LvrE, below) were in nately, these data indicate that IcmX, LvrE, and the VirK-like multiple spots. This is common and can be due to protein protein are merely genetically linked to type IV secretion, and modifications such as phosphorylation, glycosylation, or limited that their secretion is in fact by the type II system. proteolysis. The seven remaining proteins that were differentially ob- Three more proteins identified in wild-type but not mutant served in supernatants were not predicted to have a signal supernatants had signal sequences (Table 1), but other infor- peptide and thus are unlikely to be Lsp substrates (Table 1). mation makes a simple connection to type II secretion less The presence of flagellin (29) in supernatants may not be immediate. Two of these, IcmX and LvrE, are linked to type surprising, because the release of flagellar proteins into the IV secretion systems of L. pneumophila. IcmX is linked to extracellular medium is commonly seen. The detection of MICROBIOLOGY Dot/Icm and is required for the biogenesis of the intracellular aconitase and DnaK, cytosolic proteins of L. pneumophila (30, phagosome, although its precise function is unknown (22, 23). 31), might be the result of some cell lysis. The remaining four A previous study also found IcmX in supernatants, in addition proteins were not predicted to be cytoplasmic by PSORTb. to being in the periplasm, but that secretion depended on Beyond lytic or flagellar release, it is conceivable that one or Dot/Icm (23). The gene for LvrE is linked to those encoding more of these proteins exit the cell by type IV or I systems (32). the Lvh system, but it is still not clear whether LvrE has a role It is not apparent why type II mutant supernatants should lack in type IV secretion (24, 25). LvrE has also been observed in these proteins, but perhaps there is a feedback loop triggered supernatants (26). The third protein, spot 19, was annotated as in the absence of type II secretion that decreases expression of hypothetical but with homology to VirK, an Agrobacterium these factors.

DebRoy et al. PNAS ͉ December 12, 2006 ͉ vol. 103 ͉ no. 50 ͉ 19147 Downloaded by guest on September 28, 2021 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 (plspF; light gray bar), chiA mutant NU318 (pMMB2002; white bar), and NU318 (pchiA; cross- 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 hatched bar) were assayed for chitinase activity against p-NP-[GlcNAc]3. Data represent the mean and standard deviation for triplicate cultures for each was inoculated intratracheally into the lungs of A/J mice. The ratio of the wild strain. The reductions in enzymatic activity for NU275 (pMMB2002) and NU318 type to the mutant was determined at days 1 and 3 after inoculation. Data are ϭ (pMMB2002) were signiﬁcant (Student’s t test; P Ͻ 0.05). 130b, NU275, and representative of actual values obtained per mouse (n 5), and the solid bar NU318 behaved identically to their corresponding derivatives that contained indicates the mean value. The differences in the ratios of 130b and NU318 on Ͻ only the cloning vector pMMB2002 (data not shown). (B) Utilization of glycol day 1 vs. day 3 were signiﬁcant (Student’s t test; P 0.05). chitin by the secreted chitinase in the supernatants of strains 130b (1), 130b (pMMB2002; 2), 130b (pchiA; 3), NU275 (4), NU275 (pMM2002; 5), NU275 (plspF; 6), NU318 (7), NU318 (pMMB2002; 8), and NU318 (pchiA; 9). Twenty growth (data not shown). Mutant supernatants had a Ϸ75% microunits of S. griseus chitinase (a) and BYE medium (b) were used as the reduction in activity against pNP-[GlcNAc]3 and no clearance on positive and negative controls, respectively. Similar results for A and B were chitin plates (Fig. 1). That NU318 and NU319 gave similar obtained on at least two other occasions. The independently derived chiA results, coupled with the fact that chiA is monocistronic, indi- mutant NU319 had a phenotype identical to that of mutant NU318 (data not cated that the observed secretion defect was due to the loss of shown). chiA and not a polar effect or spontaneous second-site mutation. As expected, a plasmid-borne copy of chiA (pchiA) restored the ChiA Encodes the Type II-Secreted Chitinase of L. pneumophila. One mutant to wild-type levels of activity (Fig. 1). That chiA encodes of the newly identified type II dependent proteins was annotated a chitinase was further confirmed when lysates of chiA- as a chitinase. In all three sequenced L. pneumophila strains (19, expressing Escherichia coli had activity against pNP-[GlcNAc]3 20), the gene encoding the protein is monocistronic, with the and chitin (SI Fig. 5). Because the type II and chiA mutants had gene on either side of it being transcribed in the opposite similar reductions in activity, we believe that ChiA is the only L. direction. The C terminus of the protein has a glycosyl hydrolase pneumophila type II chitinase. domain and a conserved active site characteristic of the family 18 of glycosyl hydrolases, the group that contains most of the ChiA Promotes Persistence in the Lungs. To begin to investigate the known bacterial chitinases (33, 34). The protein did not contain role of ChiA in L. pneumophila ecology and infection, we the chitin-binding or fibronectin type III-like domains that are analyzed the capacity of chiA mutants to grow in Hartmannella sometimes present in chitinases (35–38). For reasons below, we vermiformis amoebae and macrophage-like U937 cells. The designated this protein and its gene as ChiA and chiA, respec- mutants grew similarly to wild type in both cells (data not tively. Because Legionella had not been previously investigated shown), indicating chiA is not required for intracellular growth. for chitinases, we next assayed supernatants from wild-type 130b We next analyzed the chiA mutants in the A/J mouse model of for activity against known substrates of chitinases. The super- Legionnaires’ disease, initially doing a competition assay. After natants had activity against p-nitrophenyl ␤-D-N,NЈ,NЉ triacetyl introduction into the mouse lung, mutant NU318 exhibited a chitotriose (pNP-[GlcNAc]3), indicating that L. pneumophila 3.5-fold deficiency compared with wild type (Fig. 2). That secretes an endochitinase (Fig. 1A; ref. 39). No activity was seen independently derived NU319 had a 4-fold deficiency (SI Fig. 6) against exochitinases substrates, p-NP-N-acetyl-␤-D-glu- indicated that the defects were due to inactivation of monocis- cosamine and p-NP ␤-D-N,NЈ-diacetylchitobiose (data not tronic chiA and not spontaneous second-site mutation. Further shown; refs. 39 and 40). 130b supernatants also showed clearance confirmation was not possible, because plasmids containing on chitin-containing plates (Fig. 1B). When assayed with pNP- cloned chiA reduced the growth of wild type in the host (data not [GlcNAc]3, the type II mutant NU275 showed a 70–75% reduc- shown), precluding complementation analysis. At the least, these tion in secreted chitinase activity and minimal clearance on data indicate that ChiA is indirectly involved in persistence in the chitin plates (Fig. 1). Both activities were restored to wild-type lung. To further explore the in vivo defect, we compared the levels in NU275 containing a plasmid copy of lspF (ref. 6; Fig. ability of wild type and a chiA mutant to exist in the lungs when 1). Together, these data confirm that L. pneumophila secretes a injected into separate groups of animals. Once again, NU318 had chitinase secreted by the type II system. a 4-fold deficiency (Fig. 3A). In another trial, the reduced To determine whether ChiA is the secreted chitinase activity, recoverability of the chiA mutant was found to be about one-half two independent chiA mutants of strain 130b were constructed. that of a type II mutant (Fig. 3B). Taken together, these data Mutants NU318 and NU319 showed normal behavior when indicate that ChiA, directly or indirectly, is required for optimal cultured in buffered yeast extract broth and on buffered charcoal survival of L. pneumophila in the lung. That ChiA is expressed yeast extract agar, indicating that chiA is not required for general in vivo was confirmed by Western blot by using antisera from

19148 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0608279103 DebRoy et al. Downloaded by guest on September 28, 2021 MS analysis. Third, our samples were from bacteria grown under a single growth condition. Therefore, we suspect that L. pneumophila type II effectors number Ͼ25. Indeed, an in silico screen of the L. pneumophila genome revealed 254 proteins that both contain a signal peptide and are predicted by a combination of programs not to be in the cytoplasm, periplasm, or outer membrane or have a function clearly linked to the cell interior (SI Tables 2 and 3). Among these proteins, 60 were predicted to be extracellular by at least one program and 13 by two programs. Our proteomic analysis identified proteins belonging to this group as well as some proteins whose predicted location is unknown. We do not imagine that the output of the type II system is 254, but it is reasonable to think that the system can process 60 substrates. Even at 25, the experimentally defined catalog of L. pneumophila type II effectors (i.e., proteins/ activities that are in wild-type but not mutant supernatants and whose ORF had a signal sequence) is the largest known in bacteria. Our data increase our appreciation not only for the magnitude of the type II output but also for the types of proteins exported. First, we confirmed that a number of types of enzymes secreted by other bacterial type II systems are elaborated by L. pneumophila, including peptidases, lipolytic enzymes, cellulases, chitinases, and phosphatases (2). Second, we reaffirmed that L. pneumophila is unique in its secretion of a ribonuclease and gained the first indication of a type II-dependent amidase. Third, we documented that the secretome of L. pneumophila includes proteins that show their greatest similarity to eukaryotic proteins. We had previously reported that a secreted acid phosphatase of L. pneumophila has eukaryotic-like properties (12), and now we add a protein with collagen-like repeats and a protein related to a eukaryotic zinc metalloproteinase. Fourth, based on the type II dependency of IcmX and LvrE, we obtained an indication of a possible connection between types II and IV secretion. Our in silico analysis also revealed secreted proteins that are predicted to have known and novel types of activities, homology to eukaryotic factors, or a connection to type IV Fig. 3. Growth and persistence of wild type and mutant L. pneumophila in secretion (SI Tables 2 and 4). Fifth, and perhaps most signifi- the lung. A/J mice were intratracheally inoculated with equal numbers of 130b cantly, we uncovered a number of secreted proteins that do not ࡗ ᮀ ࡗ ᮀ ( ; A) and the chiA mutant NU318 ( ) and 130b ( ; B), NU318 ( ) and the bear any similarity to known proteins, raising the possibility of 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 there being novel effectors secreted by the type II system. From of four to six mice for each strain. The differences between the cfus recovered proteomic and in silico analysis, the type of proteins most for 130b and NU318 at 48 and 72 h after inoculation (A) and 130b and NU275 represented are proteases/peptidases (Tables 1, 2, and 4), a and 130b and NU318 at 48 h after inoculation (B) were signiﬁcant (Student’s finding consistent with the fact that amino acids are the main t test, P Ͻ 0.01). source of carbon and energy for broth-grown L. pneumophila (41). The legionellae may also be able to catabolize carbohydrate derivatives (42), and our list of putative Lsp exoproteins also animals intratracheally inoculated with wild-type L. pneumo- includes proteins with predicted glycosidase activity (SI Tables phila (SI Fig. 7). ChiA is first type II effector to be implicated in 2 and 4), such as a eukaryotic-like glucoamylase. Paralogs of L. pneumophila virulence, because no other effector mutant genes from the enhABC locus, including EnhC and LpnE, which tested, including strains lacking acid phosphatase (map), lipases promote invasion of macrophages (43–45), were found in our AandB(lipAB), lysophospholipase A (plaA), metalloprotease screen (SI Tables 2 and 4). Such a finding is compatible with the (proA), or phospholipase C (plcA), displayed a defect in the mice fact that, under some growth conditions, Lsp mutants exhibit a (SI Fig. 8). In sum, these data represent an indication that a defect in amoebae and macrophage invasion (8) (O.R. and protein having chitinase activity can promote survival of a N.P.C., unpublished results). Based on both the number of pathogen in a mammalian host. proteins uncovered and the types of proteins detected, our analysis highlights more than ever the impact that type II Discussion secretion has on bacterial function. Our proteomic analysis identified 20 proteins that are substrates for One of the type II exoproteins was a chitinase. Chitin is an type II secretion; i.e., they were in wild-type but not mutant insoluble polymer consisting of alternating ␤-1,4-linked N-

supernatants, and their corresponding ORF had a signal sequence. acetylglucosamine residues and is the second most-abundant MICROBIOLOGY These 20 are not, however, the complete set of type II substrates, organic compound in nature after cellulose (46). It is promi- for several reasons. First, five enzymes, lipases, phospholipases, and nently found in the cell walls of mold, fungi, and algae and is the lysophospholipases that were previously defined as type II- major constituent of cuticles and exoskeletons of worms, mol- dependent were not identified in this study. The reasons that these lusks, and arthropods (46). Chitinases, in turn, are present in proteins and perhaps other type II effectors were not seen include plants, insects, crustaceans, bacteria (especially marine bacte- low-level expression that precluded detection by Coomassie stain ria), protozoa, fish, animals, and humans (46–49). Bacterial and degradation due to the processing of the sample or to the chitinases are often involved in nutrient acquisition (50, 51). specific action of a secreted protease. Second, although we exam- However, chitinases may also provide protection/advantage ined 42 spots, there are additional spots that we did not submit for against invading or competing microorganisms that have chitin

DebRoy et al. PNAS ͉ December 12, 2006 ͉ vol. 103 ͉ no. 50 ͉ 19149 Downloaded by guest on September 28, 2021 in their composition (52). Thus, it is quite plausible that ChiA is buffered charcoal yeast extract agar (6). E. coli DH5␣ (Invitro- important for L. pneumophila to succeed in the environment. gen, Carlsbad, CA) was grown as before (64). Chemicals, unless Although mutating chiA did not reduce replication in H. vermi- otherwise noted, were from Sigma (St. Louis, MO.). formis trophozoites, ChiA may play a role in the usage or degradation of chitin in amoebal cysts (53). Beyond obviously 2DE Analysis and Protein Identification. L. pneumophila were grown acting upon other chitins in the environment, ChiA might act on in 50 ml of broth to late-log phase, and supernatants were concen- peptidoglycan fragments that have similarity to chitin (54). trated 100-fold as described (15). Samples were treated with the Our most striking observation is the reduced ability of the Ready Prep 2-D Cleanup Kit (BioRad, Hercules, CA) and sub- chitinase mutant to persist in the lung. As such, L. pneumophila jected to 2DE (details are published as SI Supporting Text). Protein ChiA is the first protein with chitinase activity to be implicated spots were excised and submitted for identification to Stanford in infection of a mammalian host and a type II-dependent Mass Spectrometry Services (Stanford University, Stanford, CA). virulence factor of L. pneumophila. Because the chiA mutant The MS/MS spectra were searched by MASCOT (Matrix Science, grew normally in U937 cells, we believe that the role of ChiA is Boston, MA) by using the bacterial database NCBI-nr. Peptide not simply associated with macrophage infection. Because the mass tolerance was set at 2 Da, and fragment mass tolerance was reduced recoverability of the mutant was not manifest in the first set at 0.8 Da. Unambiguous matches were established by consid- 24 h but rather at 72 h, we further suspect that ChiA most ering the number of peptides matched, the percentage of the ORF significantly promotes persistence vs. replication. At a molecular covered, and the agreement between the experimental and pre- level, the infection defect could have several explanations. In one dicted masses and isoelectric points for the protein. Proteins scenario, the effect of ChiA could be due to the protein’s direct containing signal peptides were identified by using SignalP (65). action on a host target. Because mammals do not have chitin, this Further predictions concerning the cellular location of proteins leads to the hypothesis that there is a chitin-like substance in the were determined by using PSORTb (66, 67). lung whose degradation by ChiA aids bacterial persistence. Compatible with this hypothesis is the recent suggestion that Genetic Techniques. To construct and complement chitinase mu- O-GlcNAcylated proteins in eukaryotic cells can be acted on by tants, we used our standard cloning, allelic exchange, and chitinases/chitinase-like enzymes (55). Alternatively, ChiA might be a novel bifunctional enzyme that has another uniden- transformation procedures (details are published as SI Support- tified substrate, whose loss enhances bacterial survival. In a ing Text). NU318 and NU319 are independent mutants, because second scenario, the effect of ChiA is not directly due to a they were derived from separate transformations. For comple- bacterial enzymatic activity but involves the host response to the mentation, chiA was cloned into pMMB2002 (6), yielding pchiA. protein; when ChiA is present, the host response is compromised sufficiently to allow progression to pneumonia, but when ChiA Detection of Chitinase Activity. L. pneumophila supernatants and is absent, a more effective response is mounted. That chitinases E. coli lysates were assayed for chitinase by monitoring the can modulate host response is recently evident from studies of release of p-nitrophenol from the chromogenic substrates, p- ␤ ␤ Ј the asthmatic lung in which overexpression of mammalian nitrophenyl-N-acetyl- -D-glucosamine, p-nitrophenyl -D-N,N - chitinases contribute to pathogenesis by the recruitment of diacetylchitobiose, and pNP-[GlcNAc]3, as described previously inflammatory cells (56). Incidentally, this type of observation (39, 68). Briefly, filtered supernatants from late-log broth cul- may help explain the detrimental effect that overexpression of tures of L. pneumophila and lysates from overnight cultures of E. cloned chiA had on legionellae in the lung. Our observations coli were prepared as described previously (11). Then, 200 ␮lof have implications for the pathogenesis of other bacteria that supernatant or lysate was incubated in 600 ␮l of sodium phos- elaborate chitinases, including E. coli, Listeria monocytogenes, phate buffer (pH 6.5) containing 10 ␮g of substrate for4hat Vibrio cholerae, and Vibrio vulnificus (57–60). The V. cholerae, V. 37°C. The reaction was ended by the addition of 200 ␮l of sodium vulnificus, and E. coli chitinases are type II-dependent (59–61), carbonate, and absorbance was measured at 410 nm. The ability but they have not been assessed for a role in infection. of 50-␮l samples of supernatants or lysates to degrade glycol Another major implication of this study is that factors such as chitin was measured on agarose plates containing 0.1% glycol chitinases that are traditionally viewed as being relevant only in chitin, as described (69, 70). Immunoblot detection of chitinase the environment may in fact have great importance in human in recombinant E. coli was done as before, by using mouse sera and animal infection. In support of this view, a chitin-binding from animals intratracheally inoculated with wild-type 130b (6). protein of V. cholerae was recently shown to promote attachment to intestinal epithelia (62). Thus, future investigations into Infection Assays. H. vermiformis and U937 cells were infected with pathogenic processes may well benefit from the inclusion of L. pneumophila, as described (4, 6). To assess in vivo growth, 6- ‘‘environmental’’ enzymes such as chitinases and cellulases. to 8-week-old A/J mice (The Jackson Laboratory, Bar Harbor, ME) were inoculated intratracheally, and then the numbers of Materials and Methods legionellae in lung homogenates were determined by plating (6). Strains, Growth Media, and Chemicals. L. pneumophila 130b (ATCC Animal experiments were approved by the Animal Care and Use BAA-74) was used as the wild type in this study. Its derivatives Committee of Northwestern University. NU275, NU255, NU267, NU270, NU268, and AA200, which contain mutations in lspF, map, lipAB, plaA, plcA, and proA, We thank Cianciotto laboratory members as well as Mark Strom for respectively, were described before (6, 12–14, 17, 63). Legionel- helpful discussions. This work was supported by National Institutes of lae were cultured at 37°C in buffered yeast extract broth or on Health Grant AI43987 (to N.P.C.).

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