Stenotrophomonas maltophilia Encodes a Type II Protein SecretionSystem That Promotes Detrimental Effects on Lung Epithelial Cells

Sara M. Karaba, Richard C. White, Nicholas P. Cianciotto

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

The Gram-negative bacterium Stenotrophomonas maltophilia is increasingly identified as a multidrug-resistant pathogen, beingassociated with pneumonia, among other infections. Despite this increasing clinical problem, the genetic and molecular basis ofS. maltophilia virulence is quite minimally defined. We now report that strain K279a, the first clinical isolate of S. maltophilia tobe sequenced, encodes a functional type II protein secretion (T2S) system. Indeed, mutants of K279a that contain a mutation inthe xps locus exhibit a loss of at least seven secreted proteins and three proteolytic activities. Unlike culture supernatants fromthe parental K279a, supernatants from multiple xps mutants also failed to induce the rounding, detachment, and death of A549cells, a human lung epithelial cell line. Supernatants of the xps mutants were also unable to trigger a massive rearrangement inthe host cell’s actin cytoskeleton that was associated with K279a secretion. In all assays, a complemented xpsF mutant behavedas the wild type did, demonstrating that Xps T2S is required for optimal protein secretion and the detrimental effects on hostcells. The activities that were defined as being Xps dependent in K279a were evident among other respiratory isolates of S. malto-philia. Utilizing a similar type of genetic analysis, we found that a second T2S system (Gsp) encoded by the K279a genome iscryptic under all of the conditions tested. Overall, this study represents the first examination of T2S in S. maltophilia, and thedata obtained indicate that Xps T2S likely plays an important role in S. maltophilia pathogenesis.

Stenotrophomonas maltophilia is a Gram-negative bacteriumfound ubiquitously in soil, water, and plants and is increas-

ingly being identified as an opportunistic and nosocomial patho-gen (1–3). The most common type of infection is pneumoniafollowed by bloodstream infections although the bacterium hasbeen associated with many other types of infection as well. S.maltophilia accounts for 4.5% of nosocomial pneumonia and 6%of ventilator-associated pneumonia and is reported to be amongthe 11 most isolated organisms in intensive care units (ICUs) inthe United States (1, 3). Mortality rates for patients with S. malto-philia pneumonia are between 23 to 77%, while a separate studyfound that the overall attributable mortality rate for S. maltophiliainfections is 37.5% (1, 4). Some of the risk factors for S. malto-philia infection are prolonged mechanical ventilation, presence ofindwelling devices, compromised health status, malignancy, ex-posure to broad-spectrum antibiotics, and long-term hospitaliza-tion or ICU stays (1, 3). The incidence and prevalence of S. malto-philia are also increasing in cystic fibrosis (CF) patients in NorthAmerica and Europe, with the prevalence of S. maltophilia being ashigh as 25% (1, 3, 5). Additionally, chronic S. maltophilia infectionin CF patients is an independent risk factor for lung exacerbations(3, 6). Another reason for clinical concern is the intrinsic antibi-otic resistance that S. maltophilia possesses, making infections dif-ficult to treat (1, 3, 7, 8).

Despite the increasing clinical importance of S. maltophilia,our understanding of this bacterium’s pathogenicity and viru-lence is very minimal. Phenotyping of S. maltophilia strains sug-gests that the organism has traits that are linked to the virulence ofother bacteria (3, 5). Inoculation of S. maltophilia into the lungs ofmice results in bacterial replication and a marked inflammatoryresponse (9–11). However, documentation of the genetic basis ofS. maltophilia pathogenicity is in its infancy. From the sequencingof the clinical isolate K279a, S. maltophilia is predicted to encodefour types of protein secretions systems; i.e., types I, II, IV, and V(2, 12). Based upon myriad studies in other Gram-negative patho-

gens, one or more of these secretion systems is likely encodingvirulence determinants.

Type II protein secretion (T2S) systems are common, althoughnot universal, among Gram-negative bacteria (13). T2S is a mul-tistep process (14–16). Proteins that are to be secreted are trans-located across the inner membrane. In most cases, unfolded sub-strates cross that membrane via the Sec pathway; however, insome cases, folded substrates cross via the twin-arginine translo-con. Once in the periplasm, unfolded substrates take on their ter-tiary conformation and may oligomerize. Finally, substrates aretransported across the outer membrane by a complex of proteinsthat is dedicated to T2S. The T2S apparatus consists of 12 coreproteins: a cytosolic ATPase (T2S E), inner membrane proteinsthat form a platform for T2S E (T2S F, L, and M), major and minorpseudopilins that form a pilus-like structure which spans theperiplasm (T2S G, H, I, J, and K), an inner membrane peptidasethat processes pseudopilins (T2S O), an outer membrane “secre-tin” that oligomerizes to form the secretion pore (T2S D), and aprotein that appears to bridge inner and outer membrane factors(T2S C). The overall model is that substrates are recognized by theT2S apparatus, and then, using energy generated at the innermembrane, the pseudopilus acts like a piston to push the proteins

Received 1 May 2013 Returned for modification 8 June 2013Accepted 11 June 2013

Published ahead of print 17 June 2013

Editor: S. M. Payne

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.00546-13.

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

doi:10.1128/IAI.00546-13

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through the secretin pore. T2S promotes the growth of environ-mental bacteria as well as the virulence of many human, animal,and plant pathogens (13–15, 17). Therefore, we initiated studiesaimed at assessing the functionality of T2S in S. maltophilia andnow report that the Xps T2S system of strain K279a mediates,among other things, detrimental effects on lung epithelial cells.

MATERIALS AND METHODSBacterial strains, media, and growth assays. S. maltophilia strain K279a(American Type Culture Collection [ATCC] strain BAA-2423) served asour wild-type strain (Table 1). K279a is a multidrug-resistant strain thatwas isolated from the blood of a cancer patient (18). Mutants of K279athat were used in this study are listed in Table 1. Clinical isolates of S.maltophilia that had been previously obtained from patients were alsoincluded in this study (Table 1). Because of variations in the secretedactivities produced by these isolates (see below), we used 16S rRNA se-quencing to confirm the identity of the strains as S. maltophilia (data notshown). In performing this analysis, a fifth isolate (i.e., UPSm4) that hadbeen previously reported to us as being S. maltophilia (9) proved to be astrain of Achromobacter xylosoxidans and therefore was not studied fur-ther here. S. maltophilia was routinely cultured at 37°C on Luria-Bertani(LB) agar (Becton, Dickinson, Franklin Lakes, NJ). When appropriate,medium was supplemented with chloramphenicol at 10 �g/ml, gentami-cin (Corning, Tewksbury, MA) at 20 �g/ml, tetracycline at 20 �g/ml,norfloxacin at 5 �g/ml, 1 mM isopropyl �-1-thiogalactopyranoside(IPTG), or 10% sucrose. Growth of S. maltophilia was assessed by incu-bating strains in 25 ml (in a 125-ml flask) of buffered yeast extract (BYE)broth (19) at 37°C with agitation and monitoring optical densities of thecultures at 600 nm (OD600) using a DU 720 spectrophotometer (BeckmanCoulter, Indianapolis, IN). Escherichia coli DH5� (Life Technologies,Carlsbad, CA) was used as a host for recombinant plasmids. E. coli wasgrown in LB medium at 37°C. When appropriate, the medium was sup-plemented with ampicillin (Research Products International, Mt. Pros-pect, IL) at 100 �g/ml, chloramphenicol at 30 �g/ml, gentamicin at 5�g/ml, tetracycline at 10 �g/ml, or 10% sucrose. Chemicals were fromSigma-Aldrich (St. Louis, MO), unless otherwise noted.

Detection of secreted enzymatic activities. Initially, casein hydrolysiswas determined by spotting a 5-�l aliquot of a bacterial suspension onMueller-Hinton agar (Becton, Dickinson) containing 3% (wt/vol) skimmilk (Nestle Carnation, Solon, OH) while gelatinase activity was tested byspotting bacteria on LB agar containing 4% (wt/vol) gelatin (20). Prior tobeing spotted on the indicator plates, bacteria were grown overnight onLB agar, resuspended in phosphate-buffered saline (PBS; Corning) to anOD600 equal to 0.1, and diluted 1:10 in PBS. Plates were incubated at 37°Cfor 2 to 3 days. Whereas clearing due to caseinolytic activity was visible tothe eye, the visualization of clearing due to gelatinase was aided by flood-ing the plate with ammonium sulfate for 10 min (21). In order to quan-titate the levels of secreted enzymes, bacteria were grown in 25 ml of BYEbroth for various periods of time, and then following centrifugation of the

cultures, cell-free supernatants were obtained by filtering the culture su-pernatants through 0.22-�m-pore-size syringe filters (EMD Millipore,Billerica, MA). To measure caseinolytic activity, 100 �l of supernatant wasincubated with 100 �l of 25 mg/ml azocasein powder in 0.1 M potassiumphosphate buffer (pH 7.6). After 30 min, 800 �l of 5% (vol/vol) trichlo-roacetic acid was added, and then after centrifugation at 2,000 � g for 10min, 100 �l of the supernatant was combined with 100 �l of 0.5N NaOH;absorbance was read at 440 nm using a Synergy H1 plate reader (BioTek,Winooski, VT) (22). Serine protease activity was determined as describedpreviously (23). Briefly, 25 �l of supernatant was mixed with 100 �l of 0.5mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroaniline (pNA) in 20 mM so-dium phosphate (pH 9.0) containing 400 mM NaCl. After 2 h of incuba-tion at 37°C, the absorbance of the sample was read at 405 nm. The back-ground absorbance from samples containing no substrate was subtractedfrom each sample determination.

Detection of secreted proteins. S. maltophilia strains were grown in300 ml of BYE broth (in 1-liter flasks) to early stationary phase, and cul-ture supernatants were obtained as noted above. A total of 100 ml ofsupernatant was precipitated by the addition of 2 volumes of isopropanolat �20°C. After centrifugation of the sample at 10,000 � g, pellets weresuspended in 10 ml of water and then concentrated another 20- to 40-foldby passage through a 30-kDa Amicon filter (EMD Millipore). Samples(100 �l) were then treated with a ReadyPrep 2D Cleanup Kit (Bio-Rad,Hercules, CA), according to the manufacturer’s specifications. Finally,proteins were suspended in 2� Laemmli sample buffer containing 5%�-mercaptoethanol. The amount of protein in each sample was quantifiedusing the RC DC (reducing agent and detergent compatible) Protein As-say (Bio-Rad). Samples containing equivalent amounts of protein wereboiled for 5 min and subjected to electrophoresis through a 10% SDS-polyacrylamide gel. Protein bands were stained with a SilverQuest stain-ing kit (Life Technologies) and compared with molecular weight stan-dards. Images were converted to grayscale, and brightness and contrastwere adjusted using Adobe Photoshop.

Examination of lung epithelial cells. The human A549 cell line(ATCC CCL-185) was passaged in RPMI medium (Corning) containing10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Law-renceville, GA) at 37°C in 5% CO2 (19). In order to obtain bacterial su-pernatants to be used on A549 cells, S. maltophilia strains were brought toan OD600 of 0.215 in PBS and then diluted in RPMI medium to approxi-mately 5 �105 CFU/ml. After 24 h of static incubation at 37°C in 5% CO2,cell-free supernatants were obtained as above. Supernatants were usedimmediately or were stored at �20°C for periods of up to 1 month. Toexamine the effect of the supernatants on host cell rounding, monolayerscontaining 5 �105 A549 cells were established in the wells of a 24-welltissue culture plate (BD Falcon, Franklin Lakes, NJ), washed three timeswith fresh RPMI medium, and treated with 1 ml of supernatant for vari-ous periods of time; images were then captured using the 40� objective ofan EVOS system (AMG, Life Technologies, Carlsbad, CA). Brightness andcontrast of images were adjusted using ImageJ (NIH). To examine theeffect of live bacteria on host cell rounding, monolayers containing 5 �105

A549 cells were established as above, washed three times with fresh RPMImedium, and treated with 1 ml of bacterial strains or medium alone. S.maltophilia strains were brought to an OD600 of 0.215 in PBS and thendiluted in RPMI medium to approximately 5 �105 CFU/ml. After 24 h ofcoculture, images were captured using a 40� objective.

To monitor effects on the actin cytoskeleton, 5 � 105 A549 cells werefirst seeded onto a glass coverslip (Fisher Scientific, Pittsburgh, PA) placedwithin a 24-well tissue culture plate and incubated overnight. After themonolayers were washed three times with RPMI medium, 1 ml of super-natant was added for 1 h. The treated cells were fixed with 4% parafor-maldehyde (vol/vol) (Electron Microscopy Sciences, Hatfield, PA) in PBSand then incubated for 20 min with a 1:300 dilution (in PBS) of AlexaFluor 488-phalloidin (Life Technologies) and a 1:1,000 dilution (in PBS)of 5 mg/ml 4=,6-diamidino-2-phenylindole (DAPI) (Life Technologies).Coverslips were then mounted on slides with ProLong Gold (Life Tech-

TABLE 1 S. maltophilia strains used in this study

Strain DescriptionSource orreference

K279a Clinical isolate from blood 9, 18NUS1 gspF mutant of K279a This studyNUS2 xpsD mutant of K279a This studyNUS3 gspF xpsD mutant of K279a This studyNUS4 xpsF mutant of K279a This studyNUS4(pBxpsF) Complemented xpsF mutant This studyUPSm1 Clinical isolate, tracheal aspirate 9UPSm2 Clinical isolate, sputum 9UPSm3 Clinical isolate, respiratory sinus 9UPSm5 Clinical isolate, respiratory sinus 9

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nologies), and images were captured using the 60� objective of a NikonC2� confocal microscope (Nikon, Melville, NY). To monitor the effect ofsupernatants on the ability of A549 cells to remain attached, monolayerscontaining 2.5 � 105 cells/well were maintained for 24 h, washed withRPMI medium, and then treated with supernatants. After 3 h, the mono-layers were washed three times to remove the nonadherent cells. As de-scribed previously (24), the cells that had remained attached were treatedwith a 1:10 dilution of 1� trypsin (Corning) in PBS and then counted ona hemocytometer. To assess effects on host cell viability, A549 cells wereseeded into wells within a 96-well tissue culture plate (BD Falcon) at adensity of 5 �104 cells/well. After a series of washes, 90 �l of supernatantwas added for an incubation period of 23 h. Ten microliters of PrestoBlue(Life Technologies) was added in a 1:10 dilution and incubated with cellsat 37°C for 1 h. Fluorescence was read with an excitation of 560 nm andemission of 590 nm, and values obtained were normalized to those ofuntreated cells. A549 cell viability was also monitored using AlamarBlue(Life Technologies). Following the addition of 180 �l of supernatant andincubation for 20 h, 20 �l of AlamarBlue (1:10 dilution) was added, theplates were incubated for 4 h at 37°C, and then samples were read in theSynergy H1, as noted above.

DNA, RNA, and protein sequence analysis. S. maltophilia DNA andRNA were isolated from early-stationary-phase BYE broth cultures usingmethods and reagents previously described (17). Primers used for se-quencing, PCR, and reverse transcription-PCR (RT-PCR) were obtainedfrom Integrated DNA Technologies (Coralville, IA). Primer names andsequences are listed in Table S1 in the supplemental material. For RT-PCR, the primer pair SK106 and SK107 was used to examine transcriptionof gspF, and the pair SK179 and SK180 and the pair SK181A and SK181Bwere used for xpsF. Control experiments in which the reverse transcrip-tase was omitted from the reaction mixture were done to rule out contri-butions from contaminating DNA. For PCR amplification of 16S rRNAgene sequences, the primers SK148 and SK149 were used (25, 26). Thereaction mixtures for sequencing of the rRNA genes included the ampli-fication primer pair, as well as internal sequencing primers SK150, SK151,SK152, SK153, SK154, SK155, SK156, SK157, SK158, and SK159. DNAsequences were analyzed using Lasergene (DNASTAR, Madison, WI).BLASTP homology searches were done using GenBank at the NCBI andthe K279a database on the GenoList server (genodb.pasteur.fr/cgi-bin/WebObjects/GenoList).

Mutant construction and complementation. Mutants of S. malto-philia K279a were constructed using the gene replacement vectorpEX18Tc as described previously (27, 28). To obtain a mutant (NUS1)specifically lacking the gspF gene (i.e., Smlt2740 in the K279a genomedatabase), the 5= and 3= ends of the gene and flanking DNA were separatelyPCR amplified from K279a DNA by using the primer pair SK87 and SK89and the pair SK88 and SK90, respectively. Each of the generated fragmentswas ligated into pGEM-T Easy (Promega, Madison, WI), and then the tworesulting plasmids were digested with SmaI and SacI. A trimolecular liga-tion was performed by placing a gentamicin resistance (Gmr)-containingcassette, obtained from pX1918 (29) digested with PvuII and HincII, be-tween the beginning and end of gspF. The plasmid thus obtained (i.e.,pG�gspF) carried a 920-bp deletion in the central gspF coding region. The�gspF fragment was ligated into pEX18Tc by digestion with EcoRI, yield-ing pEX�gspF. pEX�gspF was moved into E. coli S17-1 (30) and mobilizedinto S. maltophilia K279a via conjugation. Transconjugants were selectedon LB agar supplemented with tetracycline, gentamicin, and norfloxacin.Emerging resistant colonies were streaked on LB agar supplemented with10% sucrose and gentamicin. Mutation of gspF was confirmed by PCRusing mutant strain DNA as a template with primers SK87 and SK88.

A similar strategy was employed for constructing an xpsD (Smlt0697)mutant (NUS2) and a gspF xpsD double mutant (NUS3). The 5= and 3=ends of the gene and flanking DNA were separately PCR amplified fromK279a DNA using the primer pair SK91 and SK93 and the pair SK92 andSK94, respectively. Each of the generated fragments was ligated intopGEM-T Easy, and then the two resulting plasmids were digested with

SacI and StuI. A trimolecular ligation was performed by placing a chlor-amphenicol resistance (Cmr)-containing cassette, obtained from pRE112(17) digested with ApaI, between the beginning and end of xpsD. Thisplasmid thus obtained (i.e., pG�xpsD) carried a 1,972-bp deletion in thecentral xpsD coding region. The �xpsD fragment was PCR amplified outof pGEM-T Easy with primers SK112 and SK113. The resultant productwas digested with KpnI and HindIII and ligated into pEX18Tc digestedwith the same enzymes, yielding pEX�xpsD. pEX�xpsD was moved intoE. coli S17-1 and mobilized into K279a (for xpsD mutant) and gspF mu-tant NUS1 (for gspF xpsD double mutant) by conjugation. Transconju-gants were selected on LB agar supplemented with tetracycline, chloram-phenicol, and norfloxacin. Resistant colonies were streaked on LB agarsupplemented with 10% sucrose and chloramphenicol. Mutation of xpsDwas confirmed by PCR using primers SK91 and SK92.

A deletion mutant (NUS4) of xpsF (Smlt0688) was constructed usingFlp-mediated excision as previously described (31). Briefly, xpsF with ap-proximately 500 bp flanking on either side was PCR amplified with prim-ers SK209 and SK210 and ligated into pGEM-T Easy, resulting in pGxpsF.The entire coding sequence for xpsF was replaced by a Flp recognitiontarget (FRT)-flanked chloramphenicol cassette from pKD3 (31) amplifiedusing primers SK205 and SK206 using recombineering and E. coli DY330(31), resulting in the plasmid pG�xpsF. The �xpsF construction was PCRamplified using primers SK211 and SK212. The resultant product wasreligated into pGEM-T Easy, and then �xpsF was digested out withBamHI and HindIII and ligated into pEX18Tc digested with the sameenzymes, yielding pEX�xpsF. pEX�xpsF was moved into E. coli S17-1 andmobilized into K279a via conjugation. Transconjugants were selected onLB agar supplemented with tetracycline, chloramphenicol, and norfloxa-cin. Resistant colonies were streaked onto LB agar supplemented with10% sucrose and chloramphenicol. Replacement of xpsF with chloram-phenicol-flanked FRT sites (xpsF::frt-cat-frt strain SK3.2) was confirmedby PCR using primers SK213 and SK214. To perform Flp-mediated exci-sion of the Cmr cassette, pBSFlp (31) was electroporated into SK3.2, andtransformants were selected on LB agar supplemented with gentamicinand IPTG. Individual colonies were patched onto LB agar containingeither chloramphenicol or gentamicin or no selection. Colonies whichwere either chloramphenicol or gentamicin sensitive were streaked ontoLB agar with 10% sucrose. Deletion of xpsF was confirmed by PCR usingprimers SK213 and SK214.

For trans-complementation of the xpsF mutant NUS4, a 1.3-kb PCR frag-ment containing the xpsF coding region plus 24 bp upstream was amplifiedfrom K279a DNA using primers SK213 and SK214. The resulting fragmentwas A-tailed using T4 polymerase (Life Technologies) and ligated intopGEM-T Easy, resulting in pGxpsFC=. pGxpsFC= was digested with ApaI andSacI, and the resulting fragment was cloned into pBBR1MCS-5 (32) cut withthe same enzymes, yielding pBxpsFC=. pBxpsFC= was electroporated into thexpsF mutant NUS4 and transformants were selected on LB agar supple-mented with gentamicin. Gmr clones carrying pBxpsFC= were confirmed byPCR utilizing SK214 and the vector-specific primer SK74.

RESULTSS. maltophilia strains have two T2S loci. Examination of thegenome of the clinical isolate K279a (12) revealed the presence oftwo unlinked loci (i.e., gsp and xps) predicted to encode a T2Sapparatus (Fig. 1A and B). Each locus had 11 T2S genes, corre-sponding to the core components T2S C through T2S M. In the gsplocus, gspCHIJ and gspFEDMLKG were separated by three unre-lated genes (Fig. 1A). In the xps locus, the T2S genes occurredwithout interruption (Fig. 1B). Elsewhere in the K279a chromo-some the Smlt3760 open reading frame encodes a predicted prepi-lin peptidase (T2S O) (Fig. 1C). All of the T2S genes were full-length, indicating that K279a has the potential to express two T2Sapparatuses. RT-PCR analysis determined that both gsp and xpsgenes are expressed when K279a is grown on bacteriological me-

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dium (data not shown). We identified a similar set of T2S genes inthe genomes of four other sequenced S. maltophilia strains, i.e.,R551-3, D457, JV3, and SKA14 (see Table S2 in the supplementalmaterial) (33, 34). Whereas SKA14 had its T2S genes arranged in aunique pattern, R551-3, D457, and JV3 exhibited gene syntenywith K279a. Thus, two T2S loci were evident in both clinical(K279a and D457) and environmental (R551-3, JV3, and SKA14)isolates of S. maltophilia. When K279a protein sequences wereused as the query in BLASTP analysis, the T2S proteins of S. malto-philia were most similar to those from species of Pseudomonas andXanthomonas. This result is compatible with the genetic relation-ship that exists between Pseudomonas, Stenotrophomonas, andXanthomonas (5). In summary, our bioinformatic analysis indi-cated that T2S is conserved in the S. maltophilia species. Further-more, S. maltophilia strains appear to uniformly encode two T2Ssystems, as is the case for Pseudomonas aeruginosa and Xanthomo-nas campestris (35, 36).

Xps T2S mediates the secretion of multiple proteolytic activ-ities. When K279a was inoculated onto plates containing skimmilk or gelatin, a zone of clearing developed around the areas ofbacterial growth (Fig. 2), indicating that K279a secretes caseino-lytic and gelatinase activities. To determine if one or both of theT2S loci contribute to these secreted activities, we made mutantsof K279a that contained a mutation in the gsp and/or xps locus andthen compared them to the parental wild type on the indicatorplates (Fig. 2). A gspF mutant (i.e., NUS1) behaved as the wild-type K279a did, indicating that the Gsp T2S system is not requiredfor the protease activities. In contrast, an xpsD mutant (NUS2)and an xpsF mutant (NUS4) displayed no or very small zones ofclearing on the skim milk and gelatin plates. This reduction insecreted activities was also exhibited by NUS3, a mutant lackingboth xpsD and gspF. These data indicated that, at least under thesetest conditions, Xps T2S mediates the secretion of caseinolytic andgelatinase activity. When an intact copy of xpsF was introducedinto the NUS4 mutant, there was a restoration of secreted activity(Fig. 2), confirming that T2S is functional in S. maltophilia andthat Xps T2S is required for secretion.

To quantitate the degree to which Xps T2S is responsible forsecreted protease activities, we grew K279a and its various mu-tants in liquid medium, collected cell-free culture supernatants,and measured the levels of activity using two standard proteaseassays. In the course of this experiment, the xps and gsp mutantsgrew comparably to the parental K279a (see Fig. S1 in the supple-mental material), indicating that mutations in the T2S loci do not

C

C H I J 2735

2739F

2737

EDMLKG2747

0686 E F G H I J K L M D 0698

2729

A

B

C

3759 O 3761~1 kb

gsp locus

xps locus

pre-pilin peptidase locus

FIG 1 T2S loci in S. maltophilia strain K279a. Horizontal arrows denote the relative size and orientation of genes within the gsp locus (A), the xps locus (B), anda third locus encoding the prepilin peptidase (T2S O) gene (C). Genes shown in gray are those that encode T2S-related proteins, with the name of the gene beingdesignated by its single-letter abbreviation; e.g., F for gspF or xpsF. Genes shown in white are those that encode a protein that does not contribute to the T2Sapparatus and are identified by their locus numbers (e.g., Smlt2729). In panel A, the absence of open reading frames Smlt2736 and Smlt2739 reflects thedesignations given in the current genome database. In panel B, we indicated the presence of xpsC (rather than xpsN) in order to follow the precedent set in X.campestris whereby the gene originally annotated as xpsN was renamed xpsC (14).

A

B

Skim milk

WTK279a

gspFNUS1

xpsDNUS2

gspFxpsDNUS3

xpsFNUS4

xpsF/xpsF+NUS4 (pBxpsF)

GelatinWT

K279agspFNUS1

xpsDNUS2

gspFxpsDNUS3

xpsFNUS4

xpsF/xpsF+NUS4 (pBxpsF)

FIG 2 Caseinolytic and gelatinase activities associated with wild-type and T2Smutant S. maltophilia strains. Strains as indicated were spotted onto an agarmedium containing 3% skim milk (A) or 4% gelatin (B). Areas of growth andthe associated presence or absence of zones of clearing were photographedafter 2 (A) or 3 (B) days of incubation at 37°C. The data presented are repre-sentative of at least three independent experiments.

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result in a generalized growth defect. Wild-type supernatants ef-fectively hydrolyzed azocasein, and gspF mutant supernatants dis-played no loss of that activity (Fig. 3A). In contrast, the levels ofactivity in supernatants of the xpsD mutant, xpsF mutant, and gspFxpsD mutant were significantly lower than those of the wild typeand equivalent to levels seen with medium alone. The comple-mented xpsF mutant had a level of activity that was comparable tothat of the parental K279a (Fig. 3A). A similar result was obtainedwhen we examined supernatants for serine protease activity; i.e.,the ability to cleave Suc-Ala-Ala-Pro-Phe-pNA (Fig. 3B). Takentogether, these data indicate that Xps T2S is responsible for all ofthe secreted caseinolytic activity and serine protease activity inK279a supernatants.

Xps T2S mediates the secretion of multiple proteins. As analternate means of judging the contribution of Xps and Gsp to

secretion by S. maltophilia, supernatants from broth cultures ofK279a and its mutants were examined by SDS-PAGE (Fig. 4).Wild-type K279a exhibited the presence of seven or more proteinbands, ranging in size from approximately 25 to 66 kDa. All ofthese protein species were absent from supernatants of the xpsDmutant and xpsF mutant (Fig. 4A). Complementation of the xpsFmutant resulted in a pattern of secreted proteins that was akin tothat of the wild type (Fig. 4A). When the gspF mutant’s superna-tant was examined, there was no loss of protein bands (Fig. 4B). Asexpected, the gspF xpsD double mutant produced a supernatantthat was devoid of protein species (data not shown). These dataindicate that S. maltophilia secretes at least seven different proteinsand that Xps T2S is responsible for the secretion of all of thoseprotein species.

Xps T2S causes structural and viability changes in lung epi-thelial cells. As a first step toward assessing the role of T2S in S.maltophilia pathogenesis, we compared the wild type and mutantstrains for their effects on a human cell line. Given the rise of S.maltophilia as a pathogen involved in pneumonia, we used as thecell target A549 cells, a human cell line of type II lung epithelialcells that is widely used to investigate respiratory pathogens (19,24, 37–39). Initially, we examined the effect of coculturing bacte-ria with the A549 cells. Whereas monolayers incubated with wild-type K279a or the gsp mutant were rounded after 24 h, monolayersincubated with an xps mutant appeared analogous to monolayerstreated with medium alone (see Fig. S2 in the supplemental ma-terial). When supernatants obtained from K279a cultures wereadded to a monolayer of A549 cells, the epithelial cells began toround up after 1 h of incubation, and after 3 h of incubation allcells appeared rounded (Fig. 5A). Whereas supernatants fromgspF mutant cultures produced a pattern of rounding similar tothat of the wild type, supernatants obtained from the xpsD mu-tant, the xpsF mutant, and the gspF xpsD double mutant did not

Pro

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gspF NUS1

xpsD NUS2

gspFxpsD NUS3

xpsF NUS4

xpsF/xpsF+ NUS4 (pBxpsF)BYE

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B

0

0.2

0.4

0.6

0.8

1.0

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WT K279a

gspF NUS1

xpsD NUS2

gspFxpsD NUS3

xpsF NUS4

xpsF/xpsF+ NUS4 (pBxpsF)BYE

Abs

orba

nce

OD

405

0

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0.1

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FIG 3 Caseinolytic activity and serine protease activity in culture superna-tants of wild-type and T2S mutant S. maltophilia. (A) Strains as indicated weregrown in BYE broth at 37°C to late stationary phase, and then cell-free super-natants were examined for their ability to hydrolyze azocasein. (B) Strains asindicated were grown in BYE broth at 37°C to early stationary phase, and thencell-free supernatants were examined for activity against the substrate Suc-Ala-Ala-Pro-Phe-pNA. Data are the means and standard deviations from du-plicate culture supernatants, and the results presented are representative of atleast three independent experiments. In both panels, the levels of activity ex-hibited by the xps mutants were significantly less than those of the wild typeand the other strains examined (P � 0.02; Student’s t test).

31

36.5

55.4

66.397.4116.3

WT K279a

gpsF NUS1B

31

36.5

55.4

66.397.4116.3

WT K279a

xpsD NUS2

xpsF NUS4

xpsF/xpsF+ NUS4 (pBxpsF)

A

*

*

*

***

*

*

*

***

*

FIG 4 Proteins present in culture supernatants of wild-type and T2S mutantS. maltophilia. Strains as indicated were grown in BYE broth at 37°C to earlystationary phase, and then concentrated supernatants were electrophoresedthrough a 10% SDS-polyacrylamide gel and silver stained. The gels wereloaded with 8 �g (A) or 5 �g (B) of total protein. The migration of molecularmass standards (in kDa) is indicated to the left of the gel images. Some of theprotein bands present in the wild-type samples but not the xps mutant samplesare denoted by asterisks. The very faint bands that appear in the xpsD mutantlane are likely the result of spillover from the adjacent wild-type lane sincethese bands were not observed in other gels that had the xpsD mutant laneplaced farther apart from the wild-type lane. Overall, the data presented arerepresentative of at least three independent experiments.

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cause the A549 cells to round (Fig. 5A) even after 24 h of incuba-tion (data not shown). The complemented xpsF mutant gave re-sults similar to those with the wild type (Fig. 5A). Together, thesedata indicate that Xps T2S is required for the ability of S. malto-philia to trigger rounding of A549 cells.

Cell rounding is often associated with changes in the actin cy-toskeleton (24). To determine if K279a and its T2S-dependentproteins induce changes in host actin cytoskeleton, A549 cellswere treated with supernatants and then stained for F-actin.Whereas untreated A549 cells exhibited typical actin filaments and

FIG 5 A549 cell morphology after treatment with supernatants from wild-type and T2S mutant S. maltophilia strains. Strains as indicated were grown in RPMImedium at 37°C in the presence of 5% CO2 for 24 h, and then cell-free culture supernatants (as well as medium controls) were obtained and added to A549 cellmonolayers (n 3). (A) Following 1 and 3 h of incubation, the morphology of the treated A549 cells was determined by phase-contrast light microscopy. Scalebar, 100 �m. The data presented are representative of at least three independent experiments. (B) After 1 h of incubation with supernatants or medium control,A549 cells were fixed and treated with Alexa Fluor 488-phalloidin to label actin (left column) and DAPI to label nuclei (right column) and then visualized byconfocal fluorescence microscopy. Scale bar, 20 �m. Data are representative of at least two independent experiments.

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stress fibers, cells treated with K279a supernatants displayed anabsence of stress fibers, and the actin was marginalized to theperiphery of the cell (Fig. 5B). These data indicate, for the firsttime, that S. maltophilia secretes a factor(s) that results in majorchanges in the host actin cytoskeleton. Supernatants obtainedfrom the gspF mutant gave a pattern similar to that of the wild type(Fig. 5B), indicating that Gsp T2S is not required for cell roundingor cytoskeletal rearrangements. In contrast, supernatants from thexpsD mutant, the xpsF mutant, and the gspF xpsD double mutantdid not lead to changes in the actin cytoskeleton (Fig. 5B). Theinability of the xpsF mutant to trigger changes was reversed whenan intact copy of xpsF was introduced into the mutant (Fig. 5B).Thus, Xps T2S mediates the secretion of the factor(s) responsiblefor rounding and actin rearrangement.

Given the effect of Xps T2S on cell morphology and actin, weposited that secreted factors would also cause A549 cells to detachfrom their substrata. After 3 h of incubation with supernatantsfrom K279a, only about 10% of A549 cells remained attached (Fig.6). Whereas supernatants obtained from the gspF mutant caused alevel of detachment similar to that of the wild type, supernatantsfrom the xpsD mutant, xpsF mutant, and gspF xpsD double mutantfailed to cause any detachment (Fig. 6). The complemented xpsFmutant gave results similar to those of the wild type (Fig. 6). To-gether, these data indicate that the Xps T2S system of S. malto-philia can cause host cells to detach from surfaces.

Next, to determine whether T2S-dependent activities pro-moted a cytotoxic effect, we assessed the viability of the A549 cellsfollowing their treatment with supernatants (Fig. 7). At 24 h, 30 to40% of cells exposed to K279a products lost reactivity with vitalstains, indicating that the wild-type strain secretes a factor(s) that,directly or indirectly, promotes loss of viability. Supernatantsfrom the gspF mutant elicited a similar level of cell death, implying

that Gsp T2S does not encode the cytotoxic factor(s). However,supernatants from the xpsD mutant, xpsF mutant, and gspF xpsDmutant failed to induce a loss of cell viability. The complementedxpsF mutant triggered cell death to a degree that was comparableto levels with the wild type and the gspF mutant. Thus, Xps T2S ofS. maltophilia mediates the secretion of a factor(s) that leads to thedeath of lung epithelial cells.

Xps-dependent phenotypes are expressed to different de-grees by other clinical isolates. Four isolates of S. maltophiliaobtained from the respiratory tract (Table 1) were examined forcaseinolytic activity, serine protease activity, and the ability tocause A549 cells to round (Table 2). In terms of caseinolytic activ-ity, strains UPSm1 and UPSm2 displayed zones of clearing onskim milk plates that were similar in size to those of K279a. Incontrast, UPSm3 exhibited no clearing, and UPSm5 gave moreclearing. For serine protease activity, UPSm1, UPSm2, andUPSm5 had levels of activity similar to those of strain K279a.However, UPSm3 displayed a reduced level of serine protease.Lastly, when the ability of bacterial supernatants to cause round-ing was examined, UPSm1, UPSm3, and UPSm5 behaved simi-larly to K279a, whereas supernatants from UPSm2 produced

0

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WT K279a

gspF NUS1

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xpsF NUS4

xpsF/xpsF+ NUS4 (pBxpsF)Medium

FIG 6 Attachment of A549 cells after treatment with supernatants from wild-type and T2S mutant S. maltophilia strains. Strains as indicated were grown inRPMI medium at 37°C in the presence of 5% CO2 for 24 h, and then cell-freeculture supernatants (as well as medium controls) were obtained and added toA549 cell monolayers. Following 3 h of incubation, the numbers of A549 cellsthat still remained attached to the wells of the microtiter plate were deter-mined. Values were normalized to the monolayers that had been treated withmedium alone. Data are the means and standard deviations from three treatedmonolayers, and the results presented are representative of at least three inde-pendent experiments. Levels of attachment after treatment with supernatantsfrom the wild type, the gspF mutant, or the complemented xpsF mutant weresignificantly reduced compared to those of the untreated controls (P � 0.001;Student’s t test).

0

20

40

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iabl

e ce

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gspF NUS1

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gspFxpsD NUS3

xpsF NUS4

xpsF/xpsF+ NUS4 (pBxpsF)Medium

FIG 7 Viability of A549 cells after treatment with supernatants from wild-typeand T2S mutant S. maltophilia. Strains as indicated were grown in RPMI me-dium for 24 h, and then cell-free culture supernatants were obtained andadded to A549 cell monolayers. After 24 h of incubation, the numbers of viableA549 cells that still remained in the well were determined by staining withPrestoBlue. Values were normalized to cells treated with medium alone. Thelevels of cell viability after treatment with supernatants of the wild type, thegspF mutant, or the complemented xpsF mutant were significantly reducedcompared to those of the untreated controls (P � 0. 008; Student’s t test). Dataare the means and standard deviations from three treated monolayers and arerepresentative of two experiments using PrestoBlue. A similar result was ob-tained in a third trial that utilized the vital stain AlamarBlue (data not shown).

TABLE 2 T2S-dependent activities expressed by strains of S. maltophilia

Strain

Relative level of the indicated Xps-dependent activitya

Casein hydrolysis Serine protease A549 rounding

K279a �� �� ��UPSm1 �� �� ��UPSm2 �� �� �UPSm3 � � ��UPSm5 ��� �� ��a ���, greatest activity; ��, intermediate activity; �, lower activity; �, no activity.

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rounding only after 3 h or more of incubation. Together, thesedata indicate that multiple activities ascribed to Xps T2S in K279aare expressed by the other clinical isolates and that the T2S systemis likely to be functional in many strains of S. maltophilia. That asingle activity was absent or poorly expressed in certain strains(e.g., the lack of casein hydrolysis in UPSm3) suggests that thegene encoding the Xps-dependent exoprotein is not always pres-ent or well expressed.

DISCUSSION

The data presented here represent the first experimental definitionof a protein secretion system in S. maltophilia. That Xps T2S wasshown to promote damage of lung epithelial cells also marks animportant development in our understanding of the genetic basisof S. maltophilia pathogenicity. Although many advances havebeen made in our understanding of the genetic basis of antibioticresistance in S. maltophilia (40), only a few previous studies haveused mutants to define (potential) virulence factors of S. malto-philia (3). Key earlier studies focused on the role of flagella inbacterial adherence to bronchial epithelial cells in vitro, the impactof a diffusible, signal factor (cis-�2-11-metyl-dodecenoic acid)and a secreted cell-signaling protein (Ax21) in nematode andmoth models of toxicity and virulence, the connection betweenphosphoglucomutase and lipopolysaccharide (LPS) and virulencein a rat lung model of infection, and the need for the RNA chap-erone Hfq in adherence to bronchial cells (27, 41–44). Thus, em-barking upon a systematic assessment of protein secretion systemsin S. maltophilia is significant when one considers the current stateof the S. maltophilia pathogenesis field.

Based upon our analysis of culture supernatants from strainK279a, Xps T2S mediates the secretion of at least seven proteinsand three types of proteolytic activity. Given the conservation ofxps genes among four other S. maltophilia genomes, we infer thatXps T2S is active in other strains of S. maltophilia. In support ofthis hypothesis, we along with other investigators have found pro-tease activities in supernatants from a variety of other strains of S.maltophilia (3, 20, 23, 45, 46). The 47-kDa protein that we ob-served in the K279a supernatant but not in the xps mutants’ su-pernatants is likely to be StmPr1, a 47-kDa serine protease whosegene sequence encodes a signal sequence (23). That Xps T2S ofK279a mediates the secretion of this number of proteins and thesetypes of degradative enzymes as well as having substrates in thesize range of 25 to 66 kDa is entirely compatible with what isknown about T2S in other Gram-negatives (13). From work donein other bacteria, where the number of T2S-dependent substratescan be �25 (47–50), we hypothesize that the output of S. malto-philia Xps T2S should prove to be greater than what we found inthis initial report. Future studies using different growth condi-tions (e.g., other media or temperatures) and incorporating addi-tional enzymatic assays (e.g., degradation of lipid, nucleic acids,and carbohydrates) are likely to uncover more Xps substrates.

We have documented that strain K279a secretes an Xps-depen-dent factor(s) that damages A549 cells. The detrimental effectswere cell rounding, actin rearrangement, and cell detachmentwithin a 3-h time period as well as cell death after 24 h. Compatiblewith these data are two past studies that reported that superna-tants from other S. maltophilia strains elicit rounding and death ofHEp-2 (human larynx), HeLa (human cervix), and Vero (Africangreen monkey) cells as well as rounding and detachment of hu-man fibroblasts (23, 45). Though it is tempting to speculate that

the protease activities that we identified in K279a supernatants areresponsible for the effects on A549 cells, one of the previous stud-ies found that the toxic activity, though sensitive to heat (56°C)treatment, was resistant to protease inhibitors, including thosethat impede serine proteases (45). On the one hand, it is possiblethat the different effects on A549 cells that we observed are inter-connected and due to a single secreted protein. For example, anXps-dependent substrate might enter into the host cell and alterthe actin cytoskeleton, and then this triggers rounding and detach-ment and ultimately death. Alternatively, a single exoprotein actsexternally and affects matrix material and/or a surface receptor,and this leads to changes in internal signaling and ultimately mor-phological and viability changes. On the other hand, it is alsopossible that the effects seen derive from the independent actionof multiple Xps substrates; e.g., one exoprotein triggers relativelyrapid morphological changes, and another more slowly inducesthe cell death. Thus, future work will be aimed at identifying newXps substrates that are responsible for damaging lung epithelialcells and determining how they achieve their effect on the host cell.

We have found that all S. maltophilia strains examined encodetwo T2S loci. However, all of the secreted proteins/activities ofK279a that were identified in this paper were dependent upon XpsT2S; nothing was ascribed to Gsp T2S. This finding is akin to thecurrent situation in X. campestris pv. vesicatoria (36). In contrast,in P. aeruginosa, both the Xcp and Hxc systems are known to beactive, with Xcp being more broadly expressed and Hxc showingactivity under phosphate-limiting growth conditions (35). Thus,it is quite possible that the Gsp T2S system of S. maltophilia isfunctional but under different growth conditions.

T2S contributes to the virulence of several human pathogens,including E. coli, Legionella pneumophila, P. aeruginosa, Yersiniaenterocolitica, and Vibrio cholerae (14). Notable T2S-dependentsubstrates include cholera toxin of V. cholerae, exotoxin A of P.aeruginosa, and heat-labile toxin of enterotoxigenic E. coli (15).Other pathogenic processes linked to T2S include adherence tohost cells, resistance to complement, and biofilm formation byvarious pathogenic E. coli, as well as lung infection, intracellularinfection of macrophages, and dampening of the host innate im-mune response by L. pneumophila (13, 19, 51). Given this prece-dent as well as the connection between Xps T2S and damage tolung epithelial cells, the T2S system(s) of S. maltophilia likely pro-motes human infection and bacterial virulence. The Xps sub-strate(s) that damages A549 cells in vitro likely also damages epi-thelial cells in the infected respiratory tract, perhaps leading toincreased bacterial spread and loss of lung function. Other plau-sible targets for Xps T2S are polymorphonuclear leukocytes(PMNs) and other phagocytes that attempt to contain S. malto-philia in the lung, bloodstream, or other body sites. Examining thebehavior of the S. maltophilia T2S mutants in mammalian (mu-rine) models of infection (9–11) will be an important next step intesting this hypothesis.

ACKNOWLEDGMENTS

We thank members of the Cianciotto lab, past and present, for helpfulcomments. We also thank Peter Sporn and Marina Matsuda for their helpwith actin staining, Alan Hauser for providing us with strain S17-1, andMichelle Swanson for providing pKD3 and strain DY330.

Imaging work was done at the Northwestern University Cell ImagingFacility supported by NCI CCSG P30 CA060553 awarded to the Lurie

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Comprehensive Cancer Center. This study was supported in part by NIHgrants AI082541 and AI043987 awarded to N.P.C.

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