Multiple Legionella pneumophila Type II Secretion Substrates,Including a Novel Protein, Contribute to Differential Infection of theAmoebae Acanthamoeba castellanii, Hartmannella vermiformis, andNaegleria lovaniensis

Jessica Y. Tyson, Meghan M. Pearce, Paloma Vargas, Sreya Bagchi, Brendan J. Mulhern, Nicholas P. Cianciotto

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, Illinois, USA

Type II protein secretion (T2S) by Legionella pneumophila is required for intracellular infection of host cells, including macro-phages and the amoebae Acanthamoeba castellanii and Hartmannella vermiformis. Previous proteomic analysis revealed thatT2S by L. pneumophila 130b mediates the export of >25 proteins, including several that appeared to be novel. Following confir-mation that they are unlike known proteins, T2S substrates NttA, NttB, and LegP were targeted for mutation. nttA mutants wereimpaired for intracellular multiplication in A. castellanii but not H. vermiformis or macrophages, suggesting that novel exopro-teins which are specific to Legionella are especially important for infection. Because the importance of NttA was host cell depen-dent, we examined a panel of T2S substrate mutants that had not been tested before in more than one amoeba. As a result, RNaseSrnA, acyltransferase PlaC, and metalloprotease ProA all proved to be required for optimal intracellular multiplication in H.vermiformis but not A. castellanii. Further examination of an lspF mutant lacking the T2S apparatus documented that T2S is alsocritical for infection of the amoeba Naegleria lovaniensis. Mutants lacking SrnA, PlaC, or ProA, but not those deficient for NttA,were defective in N. lovaniensis. Based upon analysis of a double mutant lacking PlaC and ProA, the role of ProA in H. vermifor-mis was connected to its ability to activate PlaC, whereas in N. lovaniensis, ProA appeared to have multiple functions. Together,these data document that the T2S system exports multiple effectors, including a novel one, which contribute in different ways tothe broad host range of L. pneumophila.

The aquatic bacterium Legionella pneumophila is the agent ofLegionnaires’ disease (1). The environmental persistence of L.

pneumophila is largely dependent upon its ability to infect andgrow in protozoa, notably amoebae belonging to the Acantham-oeba and Hartmannella genera (2–4). Human infection occursafter the inhalation of L. pneumophila-containing droplets that aregenerated from a variety of devices (5). In the lung, L. pneumo-phila multiplies in alveolar macrophages although persistencelikely also involves growth in lung epithelial cells and extracellularsurvival (6, 7). Much of the ecology and pathogenesis of L. pneu-mophila is mediated by secreted factors (8–11). For secreting pro-teins into the extracellular milieu and/or target host cells, the or-ganism uses both type II secretion (T2S) and the type IV secretionsystems, large membrane-spanning machines that are composedof more than 10 component proteins (6, 9).

T2S promotes the growth, ecology, and virulence of manyGram-negative bacteria (12). T2S substrates are translocatedacross the inner membrane by the Sec or Tat pathway, and then apseudopilus may act to push the proteins through a dedicatedouter membrane pore (13, 14). On many occasions, we along withothers have observed that L. pneumophila mutants lacking the T2Sapparatus are severely impaired for infection of Acanthamoebacastellanii and Hartmannella vermiformis, indicating that proteinssecreted via T2S are required for infection of protozoan hosts(15–21). The magnitude of the defects is similar in infections ofthe two types of amoebae. The T2S mutants are not impaired forentry into the amoebae, indicating that T2S is promoting bacterialresistance to intracellular killing and/or facilitating replication it-self (22). Others of our studies have shown that T2S also promotesthe persistence of L. pneumophila in the environment by facilitat-

ing extracellular survival in low-temperature water samples (18,22–25). Furthermore, L. pneumophila T2S helps to mediate thesecretion of a surfactant that confers both sliding motility andantibacterial activity (26, 27). In the mammalian host, L. pneumo-phila T2S also has a multifactorial role by fostering intracellularmultiplication in both macrophages and lung epithelial cells,dampening the chemokine and cytokine output from infectedhost cells, and elaborating tissue-destructive enzymes (7, 16, 28).Based primarily upon studies examining strain 130b, L. pneumo-phila T2S promotes the export of at least 25 proteins and 18 enzy-matic activities (9, 28–32). From the analysis of a secreted acidphosphatase, we observed, early on, that some T2S-dependentexoproteins have striking similarity to eukaryotic proteins (33). Ametalloprotease (ProA) and RNase (SrnA) have been shown to berequired for infection of H. vermiformis, and a secreted chitinase(ChiA) has been linked to bacterial persistence in the lungs (19, 28,34). However, T2S-dependent exoproteins that are required for

Received 11 January 2013 Returned for modification 7 February 2013Accepted 11 February 2013

Published ahead of print 19 February 2013

Editor: J. B. Bliska

Address correspondence to Nicholas P. Cianciotto,n-cianciotto@northwestern.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00045-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00045-13

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infection of A. castellanii or macrophages or epithelial cells haveyet to be identified. In our previous proteomic analysis of L. pneu-mophila T2S, a number of the proteins that were in wild-typestrain 130b but not in lspF T2S mutant supernatants appeared tohave very little if any similarity to known proteins (28). Therefore,to begin the present study, we sought to determine the importanceof these potentially novel T2S substrates in infection. We demon-strate, among other things, that one of these Legionella-specificexoproteins significantly promotes infection of A. castellanii. In-terestingly, the importance of this protein as well as several otherT2S substrates proved to be dependent upon the amoebal hostthat is being infected.

MATERIALS AND METHODSBacterial strains and media. L. pneumophila strain 130b (American TypeCulture Collection [ATCC] strain BAA-74) served as our wild-type strain(see Table S1 in the supplemental material). Mutants of strain 130b thatwere used in this study are listed in Table S1. Strains representing otherLegionella species that were also examined are listed in Table S2 in thesupplemental material. Legionellae were routinely grown at 37°C on buff-ered charcoal-yeast extract (BCYE) agar, which, when appropriate, wassupplemented with chloramphenicol at 3 �g/ml, kanamycin at 25 �g/ml,or gentamicin at 2.5 �g/ml (27). Escherichia coli strain DH5� was the hostfor recombinant plasmids (Life Technologies, Carlsbad, CA). E. coli cellswere grown in Luria-Bertani medium with kanamycin (50 �g/ml), chlor-amphenicol (30 �g/ml), or ampicillin (100 �g/ml). Unless otherwisenoted, chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

Assessments of bacterial extracellular growth and secreted factors.In order to monitor the extracellular growth of L. pneumophila strains aswell as to isolate RNA, legionellae grown on BCYE agar were inoculatedinto buffered yeast extract (BYE) broth and then incubated at 37°C withshaking (35). The optical densities of the cultures were determined at 660nm using a DU520 or DU720 spectrophotometer (Beckman Coulter, In-dianapolis, IN). The secretion of pyomelanin was ascertained by the pres-ence or absence of browning following bacterial growth in BYE broth tolate stationary phase (36). Cell-free supernatants collected from late-log-phase BYE cultures were assayed for protease activity, as measured byazocasein hydrolysis, and for phosphatase activity, as measured by therelease of p-nitrophenol from p-nitrophenol phosphate (33, 37). Starch-degrading activity was monitored as previously described (31). To lookfor the presence of astacin-like activity, culture supernatants were assayedfor their ability to trigger the release of Congo red from elastin-Congo red,as previously described (38, 39). Swimming motility was determined bywet mount, and sliding motility was assessed by examining bacteria spot-ted onto BCYE medium containing only 0.5% agar (27).

DNA and protein sequence analysis. DNA was isolated from L. pneu-mophila as described previously (27). Primers used for sequencing or PCRwere obtained from Integrated DNA Technologies (Coralville, IA).Primer names and their sequences are listed in Table S3 in the supplemen-tal material. DNA sequences were analyzed using Lasergene (DNASTAR,Madison, WI), and protein alignments were done using the Clustalmethod. Phyre (www.sbg.bio.ic.ac.uk/�phyre/) and BLAST homologysearches were done using the GenBank at the NCBI and the other L.pneumophila databases at http://genolist.pasteur.fr/LegioList/.

Southern hybridizations. Southern blotting was carried out usingEcoRI-restricted DNA. A digoxigenin nonradioactive labeling, detection,and stripping system was used (Roche Molecular Biochemicals, Indianap-olis, IN). Probes were produced by PCR incorporation using 130b DNAas a template and primers LPW13951-F3 South and LPW13951-R3 Southfor detection of lpw13951 (nttA), LPW28721-F2 South andLPW28721-R2 South for detection of lpw28721 (nttB), LEGP-F2 Southand LEGP-R2 South for detection of lpw32851 (legP), and LSPD-F1 Southand LSPD-R1 South for detection of lspD. Low-stringency hybridization

conditions (i.e., approximately 30% base pair mismatch allowed) wereused as described previously (40).

RT-PCR analysis. Reverse transcription-PCR (RT-PCR) was done es-sentially as described previously (35). Bacterial RNA was isolated fromBYE cultures by using RNA STAT-60 reagent (Tel-Test, Friendswood,TX) and following the manufacturer’s instructions, with the exceptionthat 80 �g/�l glycogen (Roche, Mannheim, Germany) and 300 mM so-dium acetate were added during RNA precipitation (41). In order to iso-late bacterial RNA from infected host cells, A. castellanii amoebae wereinfected with L. pneumophila (below). After removal of the culture me-dium, the monolayer was lysed with 50% RNA Protect (Qiagen, Valencia,CA)–1% saponin, and RNA was extracted using RNA STAT-60. Sampleswere treated with DNase I (Life Technologies, Carlsbad, CA) for 45 min at37°C, extracted using acid-phenol-chloroform (Life Technologies), andprecipitated with sodium acetate-ethanol (42). The purity and concentra-tion of the RNA were confirmed by spectrophotometry (Synergy H1 Hy-brid Reader; BioTek, Winooski, VT). cDNA was synthesized in a 20-�lreaction mixture containing 1 �g of RNA and the following items ob-tained from Life Technologies: 0.12 �g of random primers, 1� FirstStrand buffer, 2 mM each deoxynucleoside triphosphate (dNTP), 10 mMdithiothreitol (DTT), 40 U of RNaseOut, and 200 U of SuperScript IIIreverse transcriptase. Primers LPW13951-F4 and LPW13951-R4 wereused to examine transcription of lpw13951, LPW28721-F3 andLPW28721-R3 were used for lpw28721, and LEGP-F3 and LEGP-R3 wereused for legP. As a control, 16S rRNA gene transcription was assessedusing primers 16S-F1 and 16S-F2. Control experiments in which the re-verse transcriptase was omitted from the reaction mixture were done torule out contributions from contaminating DNA. Relative, endpointPCRs were separated by agarose-gel electrophoresis and detected withethidium bromide staining.

Mutant constructions. To make mutants of L. pneumophila 130blacking individual T2S-dependent exoproteins, we performed variationson allelic exchange as previously employed (19, 27, 30, 35). To obtain amutant lacking lpw13951 (nttA), a 1,783-bp fragment containinglpw13951 was amplified using LPW13951-F1 and LPW13951-R1 andthen ligated into pGEM-T Easy (Promega, Madison, WI) to yieldpG13951. Next, pG13951 was digested with BglII, and blunt ends weregenerated with T4 DNA polymerase (Life Technologies). A kanamycinresistance gene (Kmr) from pMB2190 (35) was inserted approximatelyone-third of the way through lpw13951, yielding pG13951::Km. Follow-ing transformation of strain 130b (27) with pG13951::Km, mutant colo-nies were obtained by plating on BCYE medium containing kanamycin.Verification of the mutant genotype was carried out by PCR, using prim-ers LPW13951-F1 and LPW13951-R1. The two independently derivedlpw13951 mutants were designated NU415 and NU416. To obtain a mu-tant that lacked lpw28721 (nttB), a 1,862-bp fragment containing the genewas amplified using primers LPW28721-F1 and LPW28721-R1 and thenligated into pGEM-T Easy to yield pG28721. Next, pG28721 was digestedwith BsmI, and blunt ends were generated. A gentamicin resistance gene(Gmr) from pX1918-GT (35) was then inserted to yield pG28721::Gt,which has its Gmr cassette approximately one-fourth of the way throughlpw28721. pG28721::Gt was introduced into strain 130b by transforma-tion, and mutant colonies were selected for on BCYE agar containinggentamicin. Verification of the mutant (NU417) genotype was deter-mined by PCR, using primers LPW28721-F1 and LPW28721-R1. To ob-tain a mutant lacking lpw32851 (legP), a 1,268-bp fragment containinglegP as the only intact open reading frame (ORF) was amplified usingprimers LEGP-F1 and LEGP-R1 and ligated into pGEM-T Easy to yieldpGLegP. A 665-bp fragment was removed from legP in pGLegP by diges-tion with Bst98I and BamHI. Blunt ends were generated, and a Kmr cas-sette was inserted to yield pGLegP::Km. Next, the SacI-SphI fragment ofpGLegP::Km containing the disrupted gene was cloned into pRE112 (40),yielding pRELegP::Km. After pRELegP::Km was electroporated intostrain 130b, colonies that were Kmr, chloramphenicol (Cm) sensitive, andsucrose resistant were obtained. The mutant (NU418) genotype was con-

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firmed by PCR using primers LEGP-F1 and LEGP-R1. To obtain anlpw05041 (gamA) mutant, a 1,866-bp fragment containing the gene wasamplified using primers GAMA-F1 and GAMA-R1 and ligated intopGEM-T Easy. The resulting pGgamA was digested with BamHI, and afterblunt ends were generated, a Kmr cassette was inserted to yield pGgamA::Km. Following transformation of strain 130b with pGgamA::Km, mu-tants were obtained by plating on BCYE agar containing kanamycin. Ver-ification of the mutant (NU419) genotype was done by PCR, usingprimers GAMA-F1 and GAMA-R1. The loss of GamA activity was con-firmed by the absence of starch hydrolysis on indicator plates (data notshown) (31). In order to isolate an lpw30971 (plaC) mutant, plasmidpGplaC (7) was digested with BamHI, and blunt ends were generated. Achloramphenicol resistance gene obtained from pRE112 was then insertedat bp 165 in the gene to yield pGplaC::Cm. Next, pGplaC::Cm was intro-duced into strain 130b by transformation, and mutant colonies were ob-tained on chloramphenicol-containing BCYE agar. Verification of themutant (NU420) genotype was performed by PCR, using primersPLAC-F1 and PLAC-R1. A double mutant lacking both plaC and proA(NU421) was obtained by introducing pGplaC::Cm into the proA mutantAA200 (19) and then selecting for acquisition of chloramphenicol resis-tance.

Genetic complementation. Complementation analysis of the plaCmutants was done by making a derivative of NU420 that had the plaC gene(alone) inserted into a neutral site in the chromosome, analogously towhat we have previously done for other mutants (35). To that end, a1,512-bp fragment that included 172 bp of the plaC promoter region wasamplified using primers PLAC-F2 and PLAC-R2 and then digested usingKpnI and XbaI. The resulting fragment was then ligated into a KpnI/XbaI-digested pMMB2002 (16), yielding pMplaC. Next, the XbaI/SacI frag-ment of pMplaC was cloned into XbaI/SacI-digested pZL1153 (35), re-sulting in pZLplaC. Finally, pZLplaC was transformed into NU420 andplated on kanamycin-containing BCYE agar. A Kmr, sucrose-resistantclone (NU423) was obtained and verified as having the plaC gene insertedbetween lpw27541 and lpw27551. Insertions at this site do not affect theability of L. pneumophila to grow in standard medium and infection assays(35). Complementation of the lpw13951 mutants was achieved by a sim-ilar approach, inserting lpw13951 into the site between lpw27541 andlpw27551 in NU415. To that end, a 922-bp fragment containing thelpw13951 ORF plus 377 bp of upstream sequences was amplified usingprimers LPW13951-F2 and LPW13951-R2 and then ligated into pGEM-TEasy to yield pG13951c. The lpw13951-containing KpnI/XbaI fragment ofpG13951c was ligated into pMMB2002, yielding pM13951c. Then, theSalI/SacI fragment of pM13951c containing lpw13951 was cloned intoSalI/SacI-digested pZL1153Gt, resulting in pZL13951c. The pZL1153Gt

vector had been generated by removing the Kmr cassette from pZL1153and replacing it with the Gmr cassette obtained from pX1918-GT. Aftertransformation of NU415 with pZL13951c, the Gmr, sucrose-resistantclone NU422 was obtained. Introduction of the two integrating plasmids,pZLplaC and pZL13951c, into the chromosome of wild-type strain 130bdid not alter intracellular multiplication (data not shown).

Intracellular infection assays. To assess L. pneumophila growthwithin mammalian cells, we infected human U937 macrophages (ATCCCRL-1593.2) as previously done (7). To examine the ability of L. pneumo-phila strains to grow in protozoa, A. castellanii (ATCC 30234), H. vermi-formis (ATCC 50237), and Naegleria lovaniensis (ATCC 30569) were in-fected as described previously (19, 27, 30, 43, 44). H. vermiformis and N.lovaniensis were infected in ATCC medium number 1034 that lacked itsserum component, whereas infection of A. castellanii utilized proteasepeptone-yeast extract (PY) medium.

RESULTSIdentification of novel proteins that are secreted via T2S. Thisstudy began with a further examination of three T2S-dependentsubstrates of L. pneumophila strain 130b that had appeared to benovel based upon preliminary sequence analysis (28). The firstsubstrate was a 53-kDa protein that, based upon the now-com-plete database of strain 130b (45), is encoded by ORF lpw13951(Fig. 1A). Based upon current searches, the Lpw13951 protein hadno similarity to anything outside the Legionella database. Thus,lpw13951 was designated nttA, for novel type two secreted proteinA. A gene that is 98 to 99% identical to nttA was detected in theother currently sequenced strains of L. pneumophila. An NttA-likeprotein was identified in databases that are available for strainsrepresenting three non-pneumophila species of Legionella (see Ta-ble S2 in the supplemental material). Southern blot analysis fur-ther revealed the presence of an nttA-like gene in two of sevenother non-pneumophila species examined (see Table S2). To-gether, these data indicated that NttA is conserved among L. pneu-mophila strains, present in a subset of the other Legionella species,and absent outside the Legionella genus.

The second T2S-dependent substrate that we sought to furtherinvestigate was a 39-kDa protein that was encoded by lpw28721(Fig. 1B). Lpw28721 displayed only very slight similarity (i.e., Evalue of �2 � 10�5) to hypothetical proteins in non-Legionellabacteria. In some cases, the proteins were annotated as putative

FIG 1 L. pneumophila loci encoding novel secreted proteins. Depiction of the region of the strain 130b chromosome containing nttA (A), nttB (B), and legP (C).The horizontal arrows denote the locations, orientations, and relative sizes of the genes. The “lpw” numbers indicated within the arrows are ORF designationsused in the database. Gene names and annotations are below the arrows. Sizes of the genes (bp) are given above the arrows, and the sizes of intergenic regions arenoted between the arrows.

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cysteine proteases. Thus, we named lpw28721 nttB, for novel typetwo secreted protein B. A gene that is 97 to 99% identical to nttBwas detected in the other sequenced L. pneumophila strains. L.pneumophila also had an NttB paralog; i.e., NttB shared 44% iden-tity and 61% similarity across approximately 300 amino acids (aa)(E value of 2 � 10�75) with Lpw28341. No homolog or paralog ofNttB was found in databases of Legionella longbeachae, Legionelladrancourtii, and Legionella dumoffii. However, when we didSouthern hybridizations, an nttB-like gene was detected in three ofthe seven species tested (see Table S2 in the supplemental mate-rial). These data indicated that NttB is another T2S-dependentsubstrate that appears to be unique to members of the Legionellagenus.

The final T2S substrate that was reexamined was a 29-kDaprotein encoded by ORF lpw32851. Previously, we had noted thatLpw32851 contained a short sequence that is similar to a metallo-protease domain that was first characterized in the crayfish en-zyme astacin (28, 46). Other bioinformatic analyses noted thegene in L. pneumophila strains Philadelphia-1, Paris, and Lens asbeing “eukaryotic-like,” and one of the studies dubbed the genelegP for Legionella eukaryotic-like gene protease (47–49). Currentresults confirmed that LegP has similarity to hypothetical proteinsin invertebrates, such as Drosophila sp. (XP_001999174, with an Evalue of 9 � 10�41), and vertebrates, such as Mus musculus(NP_766127, with an E value of 9 � 10�37). However, an exami-nation of 130b culture supernatants failed to detect astacin-likeactivity (data not shown). Furthermore, the new BLASTP analysisrevealed that LegP, despite its appellation, has its greatest similar-ity to hypothetical proteins of other bacteria, such as one fromRoseobacter sp. (YP_004692382; E value of 1 � 10�68), and Cellu-lomonas fimi (YP_004453501; E value of 2 � 10�69). Althoughthere was no homolog or paralog of LegP in databases of L. long-beachae, L. drancourtii, or L. dumoffii, Southern blotting detecteda legP-like gene in Legionella cardiaca, Legionella moravica, andLegionella worsleiensis (see Table S2 in the supplemental material).In sum, even though LegP did not prove to have the level ofuniqueness that the other two T2S substrates had, it is represen-tative of an uncharacterized family of proteins that exists in bothprokaryotes and eukaryotes.

Compatible with our previous proteomic analysis of strain130b culture supernatants, RT-PCR analysis determined thatnttA, nttB, and legP are all expressed when strain 130b is grown tostationary phase in BYE broth at 37°C (see Fig. S1A in the supple-mental material).

L. pneumophila mutants lacking novel T2S substrates. In or-der to discern if the newly defined T2S substrates are needed for L.pneumophila growth, we generated 130b mutants specifically lack-ing nttA, nttB, or legP. All of the mutants grew normally in BYEbroth (see Fig. S2 in the supplemental material), indicating thatthe mutants do not have a generalized growth defect and that nttA,nttB, and legP are not required for extracellular growth. These datawere compatible with the fact that T2S (lsp) mutants of L. pneu-mophila grow normally in BYE broth (16). The new mutants dis-played typical L. pneumophila colony morphology when grown onBCYE agar as well as normal cell shape and swimming motility(data not shown). They also behaved as parental 130b cells did interms of surface translocation (i.e., sliding motility) and surfac-tant production (see Fig. S3 in the supplemental material). Finally,the mutants had normal levels of pyomelanin pigment, acid phos-phatase, and protease activity within their BYE culture superna-

tants (data not shown), indicating that they do not have general-ized defects in T2S and other forms of secretion.

Novel T2S substrate NttA is required for intracellular infec-tion of A. castellanii but not infection of H. vermiformis. Tobegin to determine the importance of novel T2S substrates in in-fection, we assessed the ability of the new mutants to grow in U937cell macrophages. The nttB and legP mutants grew as well as thewild type did (Fig. 2A), indicating that NttB and LegP are notneeded for optimal infection of macrophages. Turning to proto-zoan models of intracellular infection, we observed that the sametwo mutants grew normally within H. vermiformis and A. castel-lanii, indicating that NttB and LegP are also not required for in-fection of multiple types of amoebae (Fig. 2B and C).

NttA mutant NU415, although growing normally in macro-phages and in hartmannellae (Fig. 3A and B), exhibited reducedrecovery upon infection of acanthamoebae (Fig. 3C). Indeed, at 48

FIG 2 Intracellular infection of macrophages, H. vermiformis, and A. castella-nii by L. pneumophila wild-type, nttB mutant, and legP mutant strains. U937cells, H. vermiformis, and A. castellanii, as indicated on the figure, were infectedwith either the wild-type (WT) 130b, nttB mutant NU417, or legP mutantNU418 strain, and at the indicated times, CFU counts from the infected mono-layers were determined. Data are the means and standard deviations for threeto four infected wells. Data in panel A are representative of two independentexperiments, whereas data in panels B and C represent three trials.

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h and 72 h postinoculation, the nttA mutant-infected A. castellaniicultures contained about 30-fold fewer bacteria. The nttA mutantdid not exhibit reduced survivability when incubated in the PYassay medium (see Fig. S4A in the supplemental material) or inconditioned PY medium obtained from infected monolayers (seeFig. S4B), affirming that the strain’s reduced recovery from in-fected monolayers was due to impaired intracellular infection.That NU415 was not defective when infecting H. vermiformis in

PY medium (see Fig. S4C) further indicated that the reduced re-coverability of the nttA mutant from A. castellanii was not simplydue to the particular medium used in infection of those amoebae.Because a second, independently derived nttA mutant (i.e.,NU416) had the same defect (Fig. 3D) and because the mutantphenotype was complemented by reintroduction of nttA into thechromosome (Fig. 3E), we concluded that NttA is required foroptimal infection of A. castellanii. Compatible with this conclu-

FIG 3 Intracellular infection of macrophages, H. vermiformis, and A. castellanii by L. pneumophila wild type, nttA mutants, and a complemented nttA mutant.U937 cells (A), H. vermiformis (B) and A. castellanii (C) were infected with WT and nttA mutant NU415 strains. In addition, A. castellanii was infected with thelspF mutant NU275 (C) nttA mutant NU416 (D), and complemented nttA mutant NU422 (E), and at the indicated times, CFU counts from the infectedmonolayers were determined. Data are the means and standard deviations for three to four infected wells. Data in panels A to D are representative of threeindependent experiments, and data in panel E are representative of two trials. As shown in panel C, the recovery of the nttA mutant NU415 was significantly lessthan that of the WT at both 48 h (P � 0.024; Student’s t test) and 72 h (P � 0.010). The recovery of the nttA mutant NU416 was significantly less than that of theWT at 48 h (P � 0.017) and 72 h (P � 0.009) (D). The recovery of the nttA mutant was again different from that of WT and also significantly less than that of thecomplemented nttA mutant at 48 h (P � 0.006) and 72 h (P � 0.005) (E). For the data shown in panel E, P � 0.012 and P � 0.024 at 48 and 72 h, respectively,for recovery of the WT compared to that of the complement.

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sion, RT-PCR analysis determined that nttA is expressed during L.pneumophila infection of A. castellanii (see Fig. S1B). Since thenttA mutants were not as defective in A. castellanii as an lspF mu-tant was (Fig. 3C), we posit that more T2S effectors facilitate in-tracellular infection of these amoebae.

T2S substrate SrnA is required for intracellular infection ofH. vermiformis but not infection of A. castellanii. The fact thatthe nttA mutant was impaired for infection of A. castellanii but notfor infection of H. vermiformis indicated that the importance of aT2S substrate can be dependent upon the amoebal host that isbeing infected. We had reached a similar conclusion before whentesting a 130b mutant lacking secreted ProA although, in thatinstance, the mutant was impaired in H. vermiformis not in A.castellanii (19). However, the only other T2S substrate mutantsthat were tested in two amoebae were strains lacking either theLapA and LapB aminopeptidases or the endoglucanase CelA, andin these cases, no infection defects were seen (19, 30). In light ofthe results involving nttA and past data concerning proA, we con-sidered the possibility that another T2S substrate(s) that had beenpreviously assessed in only one amoebal model might prove tohave a different importance if a second amoebal model were tried.

In one past study, an L. pneumophila mutant defective forgamA was tested only in A. castellanii and found to grow normally(31). Therefore, we made a 130b mutant lacking gamA and testedit in H. vermiformis. The gamA mutant was not impaired for in-fection of H. vermiformis (see Fig. S5A in the supplemental mate-rial), nor was it defective in A. castellanii as was expected (data notshown). Since a gamA mutant had not been previously tested inmacrophages, we took the opportunity to test the new mutant inU937 cells, but once again it grew as well as the wild type did (seeFig. S5B). These data indicated that GamA is another T2S-depen-dent exoprotein that is not required for infection of H. vermiformisor A. castellanii.

In our own past studies, mutants of 130b lacking either chiA,lipA, lipB, map, plaA, plcA, or srnA were tested only for their abilityinfect H. vermiformis, and with the exception of the srnA mutant,all grew normally. Thus, we tested mutants lacking these genes inA. castellanii. In testing the importance of plcA, we utilized a re-cently made double mutant that lacks both PlcA and PlcB, a pu-tative T2S-dependent phospholipase C that is closely related toPlcA (7). All of the mutants tested infected A. castellanii as well asthe wild type did (Fig. 4). We also found that the plcA plcB mutantgrew normally in H. vermiformis and U937 cells (data not shown).These data indicated that ChiA, LipA, LipB, Map, PlaA, PlcA, andPlcB, like CelA, LapA, and LapB, are not required for infection ofH. vermiformis or A. castellanii. But the data did document thatSrnA, like ProA, is required for infection of H. vermiformis but notinfection of A. castellanii.

T2S substrate PlaC is required for intracellular infection ofH. vermiformis but not infection of A. castellanii. In anotherprevious study (50), an L. pneumophila mutant lacking plaC wastested in A. castellanii (and macrophages) and found to grow aswell as the wild type did. Thus, we generated a new 130b mutant(NU420) lacking plaC and tested it in H. vermiformis. The plaCmutant, although still not impaired in A. castellanii, was defectivein H. vermiformis (Fig. 5A and B). At 48 and 72 h postinoculation,the plaC mutant-infected cultures contained 4- to 10-fold fewerbacteria. The plaC mutant did not show reduced survivabilitywhen incubated in the 1034 assay medium alone with and withoutconditioning (data not shown). We also observed that NU420 was

not defective when infecting A. castellanii in 1034 assay medium(data not shown), indicating that the reduced recovery of the mu-tant was not due to the medium used. Because a second, indepen-dent plaC mutant (NU367) had the same defect (Fig. 5C) andbecause complementation of the mutant phenotype was obtained(Fig. 5D), we concluded that PlaC is required for optimal intra-cellular infection of H. vermiformis. These data showed that PlaCis required for infection of H. vermiformis but not infection of A.castellanii, reinforcing our conclusion that T2S substrates can beimportant in a host cell-specific manner.

Role of ProA in H. vermiformis infection is related to its sub-strate PlaC. One of our earlier reports on T2S indicated that someT2S substrates might be cleaved by ProA (51), and subsequentwork found that the lipolytic activity of PlaC is, in fact, dependentupon cleavage of PlaC by ProA (50, 52). Therefore, we made aproA plaC double mutant (NU421) and assessed its infectivityrelative to that of the proA mutant and plaC mutant. In the H.vermiformis infection model, the double mutant was no more de-fective than either of the single mutants (Fig. 5E), suggesting thatthe importance of ProA in H. vermiformis infection is related to itsrole in activating PlaC.

T2S and substrates PlaC, ProA, and SrnA promote infectionof N. lovaniensis. By fully utilizing two amoebal models, we wereable to learn more about the importance of individual T2S-depen-dent substrates. Thus, we next sought to further improve our ap-preciation of T2S by incorporating a third amoebal host into ouranalysis. Although hartmannellae and acanthamoebae are theamoebae most frequently implicated as natural hosts for L. pneu-mophila, studies have implicated Naegleria species as being an-other natural reservoir (2, 53, 54). Therefore, we developed amodel using N. lovaniensis. To begin, we determined that strain130b can infect the naegleriae quite well, achieving a level ofgrowth that was comparable to that observed with acanthamoebaeand hartmannellae (Fig. 6A). An lspF mutant of 130b, but not itscomplement, was severely impaired in N. lovaniensis (Fig. 6A),demonstrating that L. pneumophila T2S is critical in at least threedifferent amoebal hosts. Turning to an analysis of T2S substrates,we found that the nttA, nttB, and legP mutants were not impairedin N. lovaniensis (Fig. 6B to D), nor were most of our other mu-tants (see Fig. S6 in the supplemental material). However, mu-tants lacking plaC, proA, or srnA were impaired in N. lovaniensis(Fig. 6E). Unlike the result obtained in infection of H. vermiformis,the proA mutant and the plaC proA double mutant were moredefective than the plaC mutant (Fig. 6F), suggesting that in somehost cells ProA can have key activities beyond that of activatingPlaC.

DISCUSSION

The results presented here advance our appreciation of T2S by L.pneumophila in several ways. With the demonstration of a role forNttA in A. castellanii infection, we have our first example of asubstrate promoting growth in an Acanthamoeba host. The docu-mentation of a role for PlaC in growth within H. vermiformisbrings to three (PlaC, ProA, and SrnA) the number of substratesthat are required for infection of a Hartmannella host. Together,these data have confirmed the hypothesis that the inability of T2S(lsp) mutants to infect A. castellanii and H. vermiformis is due, atleast in part, to the loss of secreted substrates as opposed to beingdue to potential cell-associated defects. The relatively modest ef-fect of each of the individual substrate mutations is compatible

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with a scenario in which the role of T2S is an additive effect ofmultiple, secreted proteins. That the importance of NttA was re-vealed after testing only three of the novel substrates (NttA, NttB,and LegP)—whereas a greater effort that had focused on proteinswith similarity to known enzymes yielded only three required pro-moters of intracellular infection (PlaC, ProA, and SrnA)—sug-gests that substrates that are more unique in sequence and specificto Legionella may be critical for L. pneumophila persistence. Withthe demonstration of a role for lspF, plaC, proA, and srnA in N.lovaniensis infection, we conclude that T2S is required for L. pneu-mophila infection of at least three genera of amoebae. The fact thatan nttA mutant was impaired in A. castellanii but not H. vermifor-

mis or N. lovaniensis and that a plaC mutant, proA mutant, andsrnA mutant were impaired in H. vermiformis and N. lovaniensisbut not in A. castellanii documents that the importance of a par-ticular T2S substrate can be dependent on the amoeba being in-fected and implies that the exoprotein repertoire has a role inshaping host range. Presumably, different amoebae present differ-ent intracellular environments for infecting legionellae (to sur-mount); e.g., NttA may have evolved to interact with a factor thatis present or highly expressed in A. castellanii but absent from orpoorly expressed in H. vermiformis. Alternatively, the expressionlevels of T2S substrates are different in different amoebae. Basedon the behavior of our mutants, infection of H. vermiformis and

FIG 4 Intracellular infection of A. castellanii by L. pneumophila wild type and various other T2S substrate mutants. Acanthamoebae were infected, as indicatedon the figure, with either the WT 130b, chiA mutant NU318, lipA lipB mutant NU373, map mutant NU254, plaA mutant NU270, plcA plcB mutant NU371, orsrnA mutant NU328 strain, and then at the indicated times, CFU counts from the infected monolayers were determined. Data are the means and standarddeviations for four infected wells, and each panel is representative of three experiments.

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infection of N. lovaniensis appear to be more similar to each otherthan they are to infection of A. castellanii.

Given the novelty of NttA at this moment, it is difficult topredict what the protein might be doing within the infected amoe-bae. One possible direction to pursue is determining the protein’sthree-dimensional (3D) structure as it may reveal a conserved foldthat can then give a clue to function. Unlike NttA, PlaC is an

known enzyme, a glycerophospholipid:cholesterol acyltransferasethat has phospholipase A and lysophospholipase activities (50).Thus, we posit that PlaC is altering ergosterol-containing mem-branes (52) of H. vermiformis and N. lovaniensis (but not A. cas-tellanii) and thereby might influence processes such as the traf-ficking of the phagosome or the flux of factors into or out of it.Another consideration is that the fatty acid profile and position of

FIG 5 Intracellular infection of H. vermiformis and A. castellanii by L. pneumophila wild type, plaC mutants, a complemented plaC mutant, and a plaC proAmutant. A. castellanii and H. vermiformis were infected, as indicated on the figure, with either the WT 130b, plaC mutant NU420, plaC mutant NU367,complemented plaC mutant NU423, proA mutant AA200, or plaC proA mutant NU421 strain, and then at the indicated times, CFU counts from the infectedmonolayers were ascertained by plating. Data are the means and standard deviations for four infected wells. Data in all panels represent three independentexperiments, except for the data in panel D, which represent two trials. As shown in panel B, the recovery of the plaC mutant NU420 was significantly less thanthat of the WT at both 48 h (P � 0.046; Student’s t test) and 72 h (P � 0.029). The recovery of the plaC mutant NU367 was significantly less than that of the WTat 48 h (P � 0.005) and 72 h (P � 0.0005) (C). Recovery of the plaC mutant was again different from that of WT and also significantly less than that ofcomplemented plaC mutant at 48 h (P � 0.027) and 72 h (P � 0.015) (D). For panel D, P � 0.501 and P � 0.668 at 48 and 72 h, respectively, for a comparisonof the recovery of the WT to that of the complement. As shown in panel E, the recovery of the plaC mutant was again less than that of WT at 48 h (P � 0.025) and72 h (P � 0.007), as were the recoveries of the proA mutant and proA plaC double mutant at 72 h (P � 0.008).

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fatty acids in glycerophospholipids may be different in the differ-ent amoebae. Based on our analysis of the plaC proA mutant, thekey role of ProA in H. vermiformis infection appears to be to acti-vate PlaC. However, this does not appear to be the case duringinfection of N. lovaniensis. Hence, ProA may, in certain hosts, behelping L. pneumophila obtain amino acids or cleave other se-creted proteins (besides PlaC) that promote intracellular growth.As the last of the known T2S-dependent potentiators of intracel-lular infection, SrnA might be degrading H. vermiformis and N.lovaniensis RNA in order to obtain nucleotides and phosphate.

Although NttB and LegP proved not to be required for infec-tion of macrophages, A. castellanii, H. vermiformis, or N. lovani-ensis, this does not necessarily mean that they are irrelevant forinfection. Indeed, another factor may compensate for their loss, orthey may be more important in another host or other niches suchas biofilms (2, 55). LegP joins the phosphatase Map (33) as beinga T2S-dependent substrate that is eukaryotic-like, reflecting therelationship that L. pneumophila has with eukaryotic hosts. Al-though we along with others have shown that LegP is secreted intoculture supernatants in a T2S-dependent manner (28, 32), other

FIG 6 Intracellular infection of N. lovaniensis by L. pneumophila wild type and various T2S substrate mutants. The naegleriae were infected with either the WT130b, lspF mutant NU275 and its complemented derivative ([dagger]), legP mutant NU418, nttA mutant NU415, nttB mutant NU417, srnA mutant NU328, plaCmutant NU420 and proA mutant AA200, or plaC proA double mutant strain, as indicated on the figure, and then at the indicated times, the numbers of CFU fromthe infected monolayers were determined by plating. Data are the means and standard deviations for four infected wells. Data in panels A to D are representativeof two independent trials, and data in panels E and F represent three independent experiments. As shown in panel A, the recovery of the lspF mutants wassignificantly less than that of the WT and the complemented lspF mutant at 24, 48, and 72 h postinoculation (P � 0.05; Student’s t test). The recoveries of the srnAand plaC mutants (E and F) were less than the recovery of the WT at 48 and 72 h, whereas the recoveries of the proA and plaC proA mutants were less than theWT recovery at 24, 48, and 72 h (P � 0.05; Student’s t test).

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work indicates that during infection of J774 mouse macrophages,the protein is translocated out of the bacterial phagosome and intothe host cell cytoplasm in a type IV secretion (Dot/Icm)-depen-dent manner (56). This raises the intriguing scenario wherebyLegP and perhaps other proteins are directly secreted or indirectlyinfluenced by multiple secretion pathways, with environmentalconditions (e.g., extracellular versus intracellular) dictating whichsecretion pathway is most critical.

Table 1 is a summary of the T2S-dependent exoproteins of L.pneumophila that have now been examined for their broad role ininfection. Based upon these data, it may be instructive to incorpo-rate even more hosts into the analysis; e.g., L. pneumophila cangrow in at least 18 more amoebae, including seven other species ofAcanthamoeba, five other species of Naegleria, another species ofHartmannella, and species of Balamuthia, Dictyostelium, Echi-namoeba, Vahlkampfia, and Willaertia (58–77), as well as threespecies of Tetrahymena (64, 69, 78–80). It has often been arguedthat when an effector mutant lacks an infection defect, it is theresult of functional redundancy; i.e., the loss of a single, secretedprotein is compensated for by the (up-) expression of anotherprotein. In light of current data, which follow from our earlierassessment of ProA (19), mutants are best tested in multiple pro-tozoa before the argument of functional redundancy is invoked.This strategy may be even more important for investigation of L.pneumophila type IV secretion which mediates the secretion of�270 effectors (6, 81).

The Legionella genus consists of 57 species (82). Previously, wefound that all seven non-pneumophila species examined con-tained lsp genes encoding T2S (16). Based on the data in Table S2in the supplemental material, we can now say that nine morenon-pneumophila species have lsp genes. Thus, we suspect that lspgenes exist throughout the Legionella genus, as one would predict,

given the prevalence of T2S among Proteobacteria (12). However,we have been gaining evidence that the T2S output varies betweenLegionella species (22, 26, 82). Hybridization analysis of nttA, nttB,and legP, coupled with BLAST analysis of recently sequenced non-pneumophila genomes, indicates that the presence of genes encod-ing substrates varies among species. Thus, the variations in T2Soutput are most likely due to differences in substrate gene contentversus the presence or absence of the T2S apparatus. It will beinteresting to more systematically assess substrate genotypesamong different species as this may give clues to evolutionaryrelationships. A molecular evolution analysis of (just) L. pneumo-phila strains has already concluded that ProA and SrnA have beenselected due to their role in virulence mechanisms (83).

ACKNOWLEDGMENTS

We thank members of the Cianciotto lab for their helpful comments. Wealso thank Felizza Gunderson for providing RNA, Kessler McCoy-Si-mandle for assisting with the plcA plcB and chiA mutants, and CatherineStewart for help with analyzing plaC.

J.Y.T. was partly supported by NIH training grant T32 AI0007476.This study was funded by NIH grant AI043987 awarded to N.P.C.

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TABLE 1 Role of T2S and individual T2S substrates in intracellular infection

Strain 130bprotein

Strain 130bORF

Protein activity or sequencenovelty

Role in intracellular infection of:aReference(s) orsourcebU937 A. castellanii H. vermiformis N. lovaniensis

LspFc lpw13701 T2S apparatus 16, 19, 22CelA lpw19571 Endoglucanase � � � � 30ChiA lpw11641 Chitinase � � � � 28GamA lpw05041 Glucoamylase � � � � 31LapA lpw30701 Leu/Tyr aminopeptidase � � � � 19LapB lpw00321 Lys/Arg aminopeptidase � � � � 19LegP lpw32851 Eukaryotic-like � � � � This studyLipA lpw08251 Monoacylglyercol lipase � � � � 57LipB lpw12111 Triacylglycerol lipase � � � � 57Map lpw11671 Acid phosphatase � � � � 33NttA lpw13951 Novel at this time � � � This studyNttB lpw28721 Novel at this time � � � � This studyPlaA lpw25361 Lysophospholipase A � � � � 51PlaC lpw30971 GCATd � � 50PlcA lpw05821 Phospholipase C � � � � 57PlcB lpw14741 Phospholipase Ce � � � � 7ProA lpw05471 Metalloprotease � � 18, 20SrnA lpw31111 T2 RNase � � 34a Based upon comparing the infectivity of parental strain 130b to a mutant lacking the indicated protein. �, no difference; , mutant impaired 2- to 10-fold; , mutant impaired11- to 100-fold; , mutant impaired �100-fold. U937, U937 cell macrophages.b Where appropriate, relevant previous studies are cited.c Similar results were obtained when mutants lacking LspD, LspE, or LspG were examined (15).d GCAT, glycerophospholipid:cholesterol acyltransferase.e Putative, based upon very high similarity to PlcA.

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