Stenotrophomonas maltophilia Encodes a VirB/VirD4 Type IV Secretion System That Modulates Apoptosis in Human Cells and Promotes Competition against Heterologous Bacteria, Including Pseudomonas aeruginosa

Stenotrophomonas maltophilia is an emerging opportunistic and nosocomial pathogen. S. maltophilia is also a risk factor for lung exacerbations in cystic fibrosis patients. S. maltophilia attaches to various mammalian cells, and we recently documented that the bacterium encodes a type II secretion system which triggers detachment-induced apoptosis in lung epithelial cells.

ABSTRACT

Stenotrophomonas maltophilia is an emerging opportunistic and nosocomial pathogen. S. maltophilia is also a risk factor for lung exacerbations in cystic fibrosis patients. S. maltophilia attaches to various mammalian cells, and we recently documented that the bacterium encodes a type II secretion system which triggers detachment-induced apoptosis in lung epithelial cells. We have now confirmed that S. maltophilia also encodes a type IVA secretion system (VirB/VirD4 [VirB/D4] T4SS) that is highly conserved among S. maltophilia strains and, looking beyond the Stenotrophomonas genus, is most similar to the T4SS of Xanthomonas. To define the role(s) of this T4SS, we constructed a mutant of strain K279a that is devoid of secretion activity due to loss of the VirB10 component. The mutant induced a higher level of apoptosis upon infection of human lung epithelial cells, indicating that a T4SS effector(s) has antiapoptotic activity. However, when we infected human macrophages, the mutant triggered a lower level of apoptosis, implying that the T4SS also elaborates a proapoptotic factor(s). Moreover, when we cocultured K279a with strains of Pseudomonas aeruginosa, the T4SS promoted the growth of S. maltophilia and reduced the numbers of heterologous bacteria, signaling that another effector(s) has antibacterial activity. In all cases, the effect of the T4SS required S. maltophilia contact with its target. Thus, S. maltophilia VirB/D4 T4SS appears to secrete multiple effectors capable of modulating death pathways. That a T4SS can have anti- and prokilling effects on different targets, including both human and bacterial cells, has, to our knowledge, not been seen before.

INTRODUCTION

Existing naturally in water, soil, and plants, Stenotrophomonas maltophilia is an opportunistic pathogen within the hospital setting (1–3). Recent reports also document community-acquired S. maltophilia infections, including those in immunocompetent individuals (3, 4). Thus, the Gram-negative S. maltophilia is the best-studied member of the Stenotrophomonas genus, which currently has 17 species (5). Pneumonia and bloodstream infections are the most frequent form of S. maltophilia infection, with some of the risk factors for infection being mechanical ventilation, indwelling devices, exposure to broad-range antibiotics, and stays in the intensive-care unit (1, 3, 6, 7). The incidence of S. maltophilia is also rising in cystic fibrosis (CF) patients (1, 8, 9). Moreover, S. maltophilia infection is a documented risk factor for CF lung exacerbations, and S. maltophilia can be dominant in patients with severe disease (1, 2, 8, 10–12). A key reason for the S. maltophilia problem is the inherent resistance of the bacterium to β-lactams, aminoglycosides, tetracycline, and fosfomycin and acquired resistance to fluoroquinolones, carbapenem, and colistin (3, 13–16). We and others have shown that delivery of S. maltophilia into the lungs of mice results in bacterial outgrowth, tissue damage, and inflammation (17–19). S. maltophilia is thought to be an extracellular pathogen binding to host cells, including lung and bronchial epithelia (20–22). The organism also has other traits that are linked to virulence in a variety of bacteria, including biofilm formation, quorum sensing, and siderophore production (23–27). We have shown that S. maltophilia encodes a type II protein secretion system (T2SS) which secretes, among other things, a protease that cleaves extracellular matrix and triggers apoptosis in epithelial cells (28–30). Based on genome sequencing, S. maltophilia has type I, IV, V, and VI secretion systems in addition to T2SS (24, 31–34).

Type IV secretion systems (T4SS) deliver DNA and/or proteins (effectors) into eukaryotic or bacterial targets (35–37). The T4SS apparatus typically consists of 12 proteins (VirB1 to VirB11 and VirD4) that exist in four subcomplexes (36, 38, 39). The first subcomplex is the VirD4 ATPase that is a “coupling protein” (40) for the recruitment of substrates to an inner membrane complex made of VirB3, VirB6, VirB8, VirB4, and VirB11. After transfer across the inner membrane, substrates are translocated out via a periplasm-outer membrane-spanning subcomplex made of VirB7, VirB9, and VirB10. Finally, VirB2 and VirB5 form a “pilus” for contacting target membranes, with VirB1 promoting peptidoglycan degradation during apparatus assembly. The T4SS is important in a range of environmental bacteria, including species of Agrobacterium, Piscirickettsia, Sinorhizobium, Wolbachia, and Xanthomonas, promoting symbiotic or pathogenic relationships with plants, insects, and fish (35, 41–43). In human and other mammal hosts, T4SS effectors are important for both extracellular pathogens such as Bordetella pertussis and Helicobacter pylori and intracellular pathogens, including species of Anaplasma, Bartonella, Brucella, Coxiella, Ehrlichia, Legionella, and Rickettsia (35, 36, 44–58), and DNA release by T4SS is important for Neisseria gonorrhoeae (36, 59). A host process that is often targeted by T4SS is apoptosis. Indeed, the T4SS of Anaplasma, Bartonella, Brucella, Coxiella, Ehrlichia, Helicobacter, and Legionella all blunt apoptosis (60–71), since maintaining host cell viability can be beneficial to pathogen persistence in intra- and/or extracellular spaces. T4SS also secrete proapoptotic effectors, as occurs for extracellular Actinobacillus actinomycetemcomitans; the induction of apoptosis helps a pathogen when directed toward cells that mediate host defense (72–74). Nothing is known about the S. maltophilia T4SS, other than it being encoded by the genome (24, 31–34, 75). Here, we document that the S. maltophilia T4SS promotes an antiapoptotic effect on lung epithelial cells but a proapoptotic effect on macrophages. Moreover, T4SS allows S. maltophilia to more effectively grow amid other bacteria, including Pseudomonas species that can coinfect the CF lung.

RESULTS

Strain K279a encodes a T4SS that is highly conserved among S. maltophilia strains.

Inspection of the genome of the S. maltophilia clinical isolate K279a (31) confirmed the presence of two T4SS loci in the bacterial chromosome (24, 32). The first set of genes (trb) was encoded by open reading frames (ORFs) RS06205 to RS06255 and the second set (virB-virD4) by RS14275 to RS14330. BLAST analysis of all available complete genomes of S. maltophilia strains revealed that the VirB/VirD4 (VirB/D4) T4SS genes are fully intact in 19/22 strains, being located in the same position within the chromosome (see Table S1A in the supplemental material). This indicates that the VirB/D4 T4SS is highly conserved within the S. maltophilia species, being prevalent in both clinical and environmental isolates. Upon further analysis of the three genomes lacking the VirB/D4 T4SS, we determined that strains ISMM3, AA1, and SJTL3 were likely misclassified as S. maltophilia, as their average nucleotide identities (ANI) to the completely sequenced S. maltophilia type strain (NCTC10257) were 90.97%, 87.94%, and 91.87%, respectively, which are well below the ANI cutoff of 94% for delineating Stenotrophomonas species (76). Thus, we report the intact VirB/D4 in all 19 completed S. maltophilia genomes. This finding updates an earlier analysis which had found these T4SS genes in 3/5 strains (24). The levels of amino acid identity shared among the different VirB/D4 T4SS proteins ranged from 100 to 92% for VirD4, VirB7, VirB9, VirB11, VirB3, and VirB4 to 28% for some of the VirB5 proteins (Table S1A). BLAST analysis revealed that the Trb T4SS is present in only 3/19 sequenced strains (Table S1B), indicating that this T4SS is not common within the S. maltophilia species. These data are compatible with earlier reports, which indicated the Trb genes are on a genomic island that is in <50% of S. maltophilia strains (24, 32). Given the high conservation of the VirB/D4 system, especially relative to the Trb system, we focused our efforts for the remainder of this study on the VirB/D4 system. The VirB1 to VirB11 proteins are encoded within a single operon, with the genes occurring in the order virB7, virB8, virB9, virB10, virB11, virB1, virB2, virB3, virB4, virB5, and virB6. The remaining T4SS gene, virD4, was monocistronic and mapped directly upstream of virB7 in the same transcriptional orientation. Based upon its components and the sequences of those constituents, this S. maltophilia T4SS was classified as a type IVA secretion system, which is exemplified by the VirB/D4 system of Agrobacterium tumefaciens (38, 77, 78). This stands in contrast to the type IVB secretion systems, which are typified by the Dot/Icm system of L. pneumophila (39). Further analysis of the database examined the presence of VirB/D4 T4SS in other species of Stenotrophomonas. We analyzed all currently sequenced type strain genomes for type strains representing 15/17 validated species and found that 9/15 had a complete or nearly complete T4SS (Fig. S1, blue clade). Six of the 9 clearly had a complete VirB/D4 T4SS, which was encoded by a single locus, as in strain K279a. Although two others, Stenotrophomonas ginsengisoli and Stenotrophomonas koreensis, appeared to lack a VirB5 homolog, they did possess a hypothetical protein encoded by a gene between virB4 and virB6 that had 21% and 23% identity to VirB5 of K279a. Due to the low sequence similarity, the E values for S. ginsengisoli and S. koreensis, 0.003E−5 and 4.31E−5, respectively, were below the cutoff for orthologs and were not included in the alignment for tree building (Fig. S1). For the 9th species, Stenotrophomonas pictorum, virD4, virB4, and virB6 were pseudogenized due to frameshift mutation, suggesting that its T4SS is not functional. Stenotrophomonas acidaminiphila, Stenotrophomonas nitritireducens, Stenotrophomonas panacihumi, Stenotrophomonas rhizophila, and Stenotrophomonas terrae lacked all of the VirB/D4 T4SS proteins, whereas S. humi only had a homolog of VirB11, which had 34% identity to the K279a counterpart (E value of 2.6E−44). An earlier study comparing S. maltophilia and S. rhizophila had also reported a lack of T4SS in S. rhizophila (33). Looking beyond the genus, we determined that this S. maltophilia T4SS is most related in terms of amino acid similarity to one encoded by the chromosome of Xanthomonas spp. (41, 79–81) (Fig. S1, blue and purple clades). We also confirmed that the arrangement of the VirB/D4 T4SS genes in S. maltophilia is identical to that in Xanthomonas (32). The Xanthomonas floridensis (82) T4SS was the one most related to that of S. maltophilia K279a, with its components having amino acid identities ranging from 89% for VirB11 to 31% for VirB5. After Xanthomonas, the genera in the order Xanthomonadales that had the T4SS next most related to that of S. maltophilia were Pseudoxanthomonas and Frateuria (Fig. S1). Bacteria that are in Xanthomonadales but did not encode a related T4SS are listed in Table S2.

Mutation of the VirB/D4 T4SS of S. maltophilia.

To begin to determine the roles of T4SS in S. maltophilia physiology and pathogenesis, we introduced an unmarked deletion into the virB10 gene (i.e., RS14310), using methods that we previously employed to construct a variety of strain K279a mutants (27–30). We continued to use K279a as the parental strain for our mutational analyses, because it is representative of contemporary virulent strains of S. maltophilia (83, 84). Since VirB10 is part of the periplasm-outer membrane-spanning subcomplex that mediates ultimate secretion of substrates (36, 38, 39, 85), inactivation of virB10 abolishes T4SS function. The newly generated virB10 mutant (strain NUS15) survived normally in bacteriological media (Fig. S2A), indicating that the loss of the VirB/D4 T4SS does not result in a generalized growth defect in S. maltophilia. The T4SS mutant also had a typical colony morphology on Luria-Bertani (LB) agar (Fig. S2B). Reverse transcription-PCR (RT-PCR) analysis determined that the virB-virD4 locus, though not required, is expressed when strain K279a is grown on or in LB media (data not shown).

S. maltophilia VirB/D4 T4SS inhibits apoptosis of human lung epithelial cells in a contact-dependent manner.

S. maltophilia strain K279a induces cell rounding and detachment and then death when it is added to monolayers of human A549 lung epithelial cells and other human and murine cell lines (28, 29). The A549 cell line is widely used for the study of lung pathogens, including S. maltophilia (86–90). We previously linked the death of the A549 cells to an induction of apoptosis (i.e., caspase-3/7 activation) that is largely due to a protease released by the T2SS (28–30). Such detachment-induced apoptosis is referred to as anoikis (30). Given this, coupled with the fact that T4SS in other bacteria often modulate apoptosis (60–73, 91, 92), we infected A549 monolayers with the new virB10 mutant and monitored cell detachment and death. Infection with the VirB/D4 T4SS mutant first resulted in an increase in detachment (i.e., fewer remaining adherent A549 cells) relative to what occurred upon infection with parental, wild-type (WT) K279a (Fig. 1A). Subsequently, the mutant resulted in fewer viable A549 cells within the wells compared to the level in the wild type (Fig. 1B). Normal levels of detachment and death were restored when the mutant was complemented with plasmid-borne virB10 (Fig. 1A and B), confirming that the heightened death was due to the loss of VirB10 and, by extension, the VirB/D4 T4SS, as opposed to a second-site mutation(s). Compatible with this, RT-PCR analysis demonstrated that virB-virD4 locus genes are expressed when S. maltophilia is incubated with A549 cells (data not shown). Since the virB10 mutant did not exhibit altered growth/survival when incubated in the tissue culture medium (Fig. S2C), we surmised that the mutant phenotype was directly due to an altered interaction with the host cells. More specifically, we posited that the mutant had lost one or more secreted effectors that normally act to impede apoptosis. To test this hypothesis, we compared the wild type and the virB10 mutant for the ability to inhibit cell death caused by staurosporine, a well-known inducer of apoptosis (30). While exhibiting wild-type levels of association with the A549 cells (Fig. S3A), the virB10 mutant was still less able to inhibit staurosporine-induced cell death than its parent and the complemented mutant (Fig. 2A). Wild-type levels of induced host cell death were seen when the mutant infected the staurosporine-treated cells in the presence of a pan-caspase inhibitor (30) (Fig. 2A), further supporting the idea that the S. maltophilia T4SS inhibits apoptosis. Incidentally, the ability of S. maltophilia to inhibit staurosporine-induced cell death was similarly observed when we infected the A549 cells with other clinical isolates (Fig. 2B), indicating that we were not studying a peculiarity of K279a. Compared to infections with the wild type or the complemented mutant, infections with the virB10 mutant resulted in increased levels of caspase-3/7 activation, as measured by the CellEvent caspase-3/7 green detection reagent (Fig. 3A), further suggesting that the VirB/D4 T4SS normally inhibits S. maltophilia-induced caspase-dependent death. Confirmation of this result was obtained by Western blot analysis, which detected more of the two caspase-7 cleavage products that trigger cell death in the mutant-infected cells than in the wild-type-infected cells (Fig. 3B) (93). When we inactivated the T2SS in the virB10 mutant by mutating its xpsF gene and then infected the A549 cells, the virB10 xpsF mutant (i.e., strain NUS16) induced a lower level of death than the virB10 mutant (Fig. 1C), indicating that the antiapoptotic effect of the T4SS effector(s) acts in opposition to the proapoptotic effect caused by T2SS-dependent proteases (30). This is reminiscent of some other pathogens that deliver both pro- and antiapoptotic factors to a host cell target, presumably creating a situation that is most conducive to their survival (66, 67, 91, 92). Importantly, the ability of the S. maltophilia T4SS to inhibit the apoptotic pathway was also seen when we infected primary human bronchial/tracheal epithelial cells (Fig. 3C), showing that the effect was not an oddity of using A549 cells. Except for the secretion of pertussis toxin, the delivery of T4SS effectors typically requires contact between the T4SS apparatus and the target cell membrane (35, 85). Thus, we inoculated the S. maltophilia strains and A549 cells on opposite sides of a transwell apparatus, such that the bacteria were prevented by a membrane filter from contacting the host cells. As a result, the elevated level of cell death activation that had been seen for the virB10 mutant was lost (Fig. 4A). A similar result was obtained when we blocked contact between the bacteria and primary epithelial cells (Fig. 4B). These results documented that the antiapoptosis effect of T4SS is not due to a soluble factor that is secreted into the culture medium but requires contact between S. maltophilia and the host cell. We posit that, following S. maltophilia attachment to the epithelial surface by a mechanism that is yet to be determined, the T4SS apparatus penetrates the plasma membrane and delivers effectors into the epithelial cell cytoplasm, with one or more of those effectors inhibiting apoptosis.

FIG 1.

Adherence and viability of A549 lung epithelial cells following infection with S. maltophilia wild-type, virB10 mutant, and virB10 xpsF mutant strains. (A and B) A549 cell monolayers were either uninfected (UI) or infected with parental strain K279a (WT), virB10 mutant NUS15 (virB10), or complemented mutant NUS15(pvirB10) (virB10/virB10+). At 8 h postinoculation, the amounts of adherent host cells were enumerated (A), and at 24 h postinoculation, the total numbers of viable A549 cells (both attached and detached) were determined (B). (C) A549 cells were uninfected or infected with either the virB10 xpsF double mutant NUS16 (virB10 xpsF) or one of the three other strains indicated in panels A and B, and at 24 h postinoculation, the numbers of viable host cells (both attached and detached) were determined. For panels A to C, the raw data appear in the left panels, and data normalized to the value for uninfected cells are in the right panels. Asterisks indicate significant differences in adherence or viability (*, P < 0.05; **, P < 0.01). Data are presented as the means and standard deviations of results from three independent experiments (n = 3 each).

FIG 2.

Effect of S. maltophilia wild-type and virB10 mutant strains on A549 cell monolayers that had been treated with apoptosis-inducing staurosporine. (A) A549 cells were either uninfected (UI) or infected with parental strain K279a (WT), virB10 mutant NUS15 (virB10), or complemented mutant NUS15(pvirB10) (virB10/virB10+), and at 6 h postinoculation, the amounts of viable host cells were enumerated. As indicated, one set of cells that had been infected with the virB10 mutant was also treated with the pan-caspase inhibitor Z-VAD-FMK. UI cells and UI cells treated with the lysing agent Triton X-100 provided controls for cell death induced by staurosporine alone. (B) Staurosporine-treated A549 cells were infected with WT strains K279a, UPSm1, UPSm2, UPSm3, and UPSm5, and then the numbers of viable host cells were determined, as indicated for panel A. In panels A and B, raw data appear in the left panels, and data are normalized to the value for UI cells in the right panels. Asterisks indicate significant differences in death following infection with the strains or treatments (*, P < 0.05; **, P < 0.01). Data are presented as the means and standard deviations of results from three independent experiments (n = 3 each).

FIG 3.

Caspase-3/7 activation in A549 cells and primary lung epithelia following infection with S. maltophilia wild-type and virB10 mutant strains. Host cell monolayers were uninfected (UI) or infected with either parental strain K279a (WT), virB10 mutant NUS15 (virB10), or complemented mutant NUS15(pvirB10) (virB10/virB10+) for 24 h. (A and C) The levels of caspase-3/7 cleavage in A549 cells (A) and human bronchial/tracheal epithelial cells (ATCC PCS-300-010) (C) were determined by CellEvent caspase-3/7 green detection reagent that fluoresces upon binding the active caspase cleavage products. The raw data appear in the left panels, and data are normalized to the value for WT cells in the right panels. Asterisks indicate significant differences in the activation induced by the strains (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Data are presented as the means and standard deviations of results from three independent experiments (n = 3 each). (B) Cell lysates were collected and analyzed by SDS-PAGE and then immunoblotted using an anti-caspase-7 antibody. (Left) Immunoblot from a representative experiment, with the migration of molecular mass standards (in kDa) indicated to the left of the gel image and the migration of caspase-7 and its active 20- and 10-kDa cleaved products to the right of the gel image. Immunoblot analysis using anti-GAPDH antibodies was performed to confirm equal loading. (Right) Graph showing the levels of the active cleaved products relative to GAPDH pooled from three independent experiments. Asterisks indicate significant differences between strains (**, P < 0.01). Whereas the 20-kDa species was seen in all trials, the 10-kDa cleaved product was only visible in two of three immunoblots.

FIG 4.

Effect of bacterial and host cell contact on the ability of VirB/D4 T4SS to inhibit caspase-3/7 activation in S. maltophilia-infected epithelial cells. Following the addition of A549 cells (A) or primary lung epithelial cells (B) into the lower chamber of a transwell apparatus, parental strain K279a (WT), virB10 mutant NUS15 (virB10), or complemented mutant NUS15(pvirB10) (virB10/virB10+) was added to the upper chamber of the transwell apparatus. A filter within the apparatus prevented contact between the bacterial inoculum and the host cells. At 24 h postinoculation, the levels of caspase-3/7 cleavage were determined by CellEvent caspase-3/7 green detection reagent. Uninfected (UI) A549 cells added to the lower chamber but not exposed to any bacteria served as a negative control. Asterisks indicate significant differences in the activation induced by the strains compared to that in UI cells (**, P < 0.01). As in Fig. 3, the data are normalized to the value for cells infected with the WT, which was set at 100% activation. Data are presented as the means and standard deviations of results from three independent experiments (n = 3 each).

S. maltophilia VirB/D4 T4SS triggers apoptosis of human macrophages in a contact-dependent manner.

When we extended our analysis to include infection of U937 cells, a human macrophage line that is widely used for infection studies (74, 94–96), the virB10 mutant attached to the host cells at a wild-type level (Fig. S3B) but resulted in less cell death (i.e., more viable host cells) than did the wild-type strain (Fig. 5A). The mutant phenotype was also evident when we assayed for caspase-3/7 activation (Fig. 5B and C). The complemented mutant showed wild-type levels of U937 cell death and caspase activation (Fig. 5A to C), confirming that the reductions in macrophage death and caspase activation were due to the loss of VirB10 and its T4SS. Incidentally, other isolates of S. maltophilia showed a level of macrophage killing that was comparable to that of strain K279a (Fig. S4). When contact with the U937 cells was blocked, the parental K279a, virB10 mutant, and complemented mutant behaved alike (Fig. 5D). The ability of T4SS to promote host cell death in a contact-dependent manner was also apparent when the infections used primary macrophages derived from the bone marrow (BMDM) of A/J mice (Fig. S5). Not surprisingly, the death induced by wild-type strain K279a was more apparent with the primary cells than the cell line. Together, these data indicate that the S. maltophilia VirB/D4 T4SS imparts a contact-dependent, proapoptotic effect on macrophages. Presumably, upon S. maltophilia contact, the T4SS apparatus pierces the macrophage membrane and dispenses an effector(s) that then promotes death of the phagocyte.

FIG 5.

Viability and caspase-3/7 activation in U937 cells following infection with S. maltophilia wild-type and virB10 mutant strains. (A) Macrophage monolayers were left uninfected (UI) or infected with either parental strain K279a (WT), virB10 mutant NUS15 (virB10), or complemented mutant NUS15(pvirB10) (virB10/virB10+), and at 24 h postinoculation, the amount of viable host cells was enumerated. The raw data appear in the left panel, and data normalized to the value for uninfected cells are in the right panel. (B) Cell lysates were collected and analyzed by SDS-PAGE and then immunoblotted using an anti-caspase-7 antibody. The left panel depicts the immunoblot from a representative experiment, with the migration of molecular mass standards (in kDa) indicated to the left of the gel image and the migration of caspase-7 and its active 20- and 10-kDa cleaved products to the right of the gel image. Immunoblot analysis using anti-GAPDH antibodies was performed to confirm equal loading. The graph in the right panel shows the levels of the active cleaved products relative to levels for GAPDH pooled from three independent experiments. Asterisks indicate significant differences between strains (*, P < 0.05). (C) U937 macrophages were infected with the strains indicated in panel A, and at 24 h postinoculation, levels of caspase-3/7 cleavage were determined by CellEvent caspase-3/7 green detection reagent. UI cells were included as a negative control. The data are normalized to the value for cells infected with the WT, which was set at 100% activation. (D) After the addition of U937 cells into the lower chamber of a transwell apparatus, the bacterial strains indicated in panel C were added to the upper chamber of the transwell apparatus with a filter preventing contact between the bacteria and the host cells, and at 24 h postinoculation, the levels of caspase-3/7 cleavage were determined and presented as described for panel C. In panels A, C, and D, asterisks indicate significant differences between the strains and/or relative to the UI control (*, P < 0.05; **, P < 0.01; ***, P < 0.001), and data are presented as the means and standard deviations of results from three independent experiments (n = 3 each).

VirB/D4 T4SS enhances S. maltophilia growth in the presence of other bacteria.

As noted above, the S. maltophilia T4SS apparatus is most akin to that of Xanthomonas species, bacteria that infect plants (97). In Xanthomonas axonopodis pv. citri , T4SS does not target plants but kills other bacteria in a contact-dependent manner (81, 98). Compared to the attention given to the bactericidal effects of type VI secretion (99, 100), our appreciation for the role of T4SS and related systems in bacterial competition is limited to that X. axonopodis pv. citri paper and a recent Bartonella report (101). Thus, to judge the role of the S. maltophilia T4SS in interbacterial relationships, we performed the same assay used in the X. axonopodis pv. citri study as well as in many studies on type VI-mediated killing (98, 102, 103). More specifically, we spotted onto LB agar an aliquot of Escherichia coli DH5α together with wild-type S. maltophilia K279a, its virB10 mutant, or the complemented mutant using a starting S. maltophilia/E. coli ratio of ∼200, 150, 100, or 50 and then monitored changes in the S. maltophilia/E. coli ratio at 24 h. Whereas parental K279a either outcompeted E. coli or maintained its standing against E. coli with all starting ratios except 50, the virB10 mutant consistently showed a clear reduction in relative CFU (Fig. 6A), suggesting that the VirB/D4 T4SS helps S. maltophilia grow in the presence of other bacteria. This type of result was not seen when we cultured the S. maltophilia strains with Klebsiella pneumoniae strain KPPR1 (104) (Fig. 6B), signifying that there is some specificity to the effect of the VirB/D4 T4SS. Since a role for the T4SS in interbacterial competition may be important in infected lungs, especially the CF lung, where multiple genera usually coexist (105–107), we coincubated our S. maltophilia strains with a P. aeruginosa strain. Wild-type K279a outcompeted P. aeruginosa strain 7700 (108, 109) at all starting ratios, including 50, and even more importantly, the virB10 mutant defect was much more dramatic than it had been with the E. coli cocultures (Fig. 6C), confirming that it was not an artifact of using E. coli as the competing organism. In this experiment as well as in the one using E. coli, the complemented virB10 mutant behaved like the wild type (Fig. 6A and C), indicating that the mutant defect was due to the loss of VirB10 and the T4SS. In considering changes in the CFU of each strain, it was clear that the S. maltophilia strains increased in number and to similar extents during the 24 h of incubation with P. aeruginosa (Fig. S6). The P. aeruginosa strain also increased its numbers during the cocultures; however, it increased significantly less in the presence of wild-type K279a than in the presence of the virB10 mutant (Fig. S6). This suggested that the VirB/D4 T4SS was promoting S. maltophilia growth in the coculture by impeding the growth and/or survival of the competing P. aeruginosa strain. A similar conclusion was made when considering the changes in the CFU of each bacterial strain included in the S. maltophilia-E. coli coculture experiment (data not shown). Overall, these data indicated that the VirB/D4 T4SS secretes factors that are antagonistic to heterologous bacteria, including P. aeruginosa strains that share ecological niches with S. maltophilia.

FIG 6.

Survival of S. maltophilia wild-type and virB10 mutant strains when cocultured with heterologous bacteria. S. maltophilia (Sm) K279a (WT), virB10 mutant NUS15 (virB10), or complemented mutant NUS15(pvirB10) (virB10/virB10+) was mixed with E. coli strain DH5α (Ec) (A), K. pneumoniae strain KPPR1 (Kp) (B), or P. aeruginosa strain 7700 (Pa) (C) at ratios of ∼200:1, 150:1, 100:1, and 50:1, spotted onto LB agar. After 24 h of incubation, the numbers of each strain were determined by plating serial dilutions of the bacterial growth area on selective media. Results are presented as the ratios of S. maltophilia CFU to the CFU of other species at t = 0 and t =24 h. The dagger symbols indicate when the ending ratio was <5. Asterisks denote significant differences in the ratio at t = 0 h compared to the ratio at t = 24 h (*, P < 0.05; **, P < 0.01). Data are presented as the means and standard deviations of results from three independent experiments (n = 3 each).

S. maltophilia VirB/D4 T4SS promotes the death of multiple P. aeruginosa strains in a contact-dependent manner.

In order to pursue the basis for the antibacterial role of the VirB/D4 system, we used P. aeruginosa as the competing organism rather than E. coli DH5α because of the greater clinical and ecological implications associated with it. We next performed a coculture using a transwell apparatus such that the S. maltophilia strains and P. aeruginosa strain 7700 could be physically separated from each other depending upon the pore size of the membrane filter within the transwell apparatus. When bacterial mixing and contact were not blocked, the wild type and the complemented virB10 mutant were, once again, more adept at competing against P. aeruginosa than was the virB10 mutant, indicating that the antibacterial effect of the VirB/D4 T4SS can occur when the bacteria are in a liquid phase as well as on a solid surface, as shown in Fig. 6. This finding was evident whether we monitored the changes in the ratio of S. maltophilia to P. aeruginosa (Fig. S7A, left) or the changes in the numbers of P. aeruginosa (Fig. S7B, left). In contrast, when the pore size of the membrane filter within the transwell apparatus precluded bacterial mixing and contact, wild-type K279a, the virB10 mutant, and the complemented mutant limited P. aeruginosa growth/survival to similar extents (Fig. S7A and B, right). Overall, these data demonstrated that the antibacterial effect of the VirB/D4 T4SS is contact dependent. Upon mixing wild-type K279a or the complemented virB10 mutant with strain 7700 for only 2 h, rather than the 24 h used in the earlier cocultures, we observed, for the first time, a reduction in the CFU of the P. aeruginosa strain (Fig. 7A, left). In contrast, when the virB10 mutant was combined with strain 7700, this reduction did not occur. These data indicated that the VirB/D4 system of strain K279a is capable of promoting the death of a P. aeruginosa strain. Indeed, many studies examining the bactericidal effects of type VI secretion systems utilized reductions in CFU during a 2-h incubation as evidence of interbacterial killing (110). Wild-type K279a and the complemented virB10 mutant, but not the virB10 mutant, were also able to reduce the numbers of P. aeruginosa strains PAO1 (Fig. 7B, left) and PAK (Fig. 7C, left), indicating that the anti-P. aeruginosa effect was not a peculiarity of using P. aeruginosa 7700. Moreover, these results documented that the VirB/D4 T4SS of strain K279a could lead to the death of both clinical (PAO1 and PAK) and environmental (7700) isolates of P. aeruginosa. During these short cocultures, the three S. maltophilia strains did not show an increase in CFU, except for a slight increase when incubated with strain 7700 (Fig. 7, right). Incidentally, four other isolates of S. maltophilia, but not a strain of Achromobacter xylosoxidans, could also reduce the CFU of P. aeruginosa (Fig. S8). Strain K279a and the complemented virB10 mutant, but not the virB10 mutant, were also able to reduce the P. mendocina CFU (Fig. 8A) but not the CFU of P. fluorescens, P. putida, or P. stutzeri (Fig. 8B and D). In the cocultures with other Pseudomonas species, the S. maltophilia strains did not increase in number of CFU or differ in survival (data not shown). These data showed that the bactericidal effect of the VirB/D4 T4SS extended to multiple but not all species of Pseudomonas. Based on this data set, we posit that the S. maltophilia VirB/D4 T4SS secretes an effector(s) that leads to the death of other bacteria, such as P. aeruginosa and other Pseudomonas species.

FIG 7.

Bactericidal effect of S. maltophilia wild-type and virB10 mutant strains on various strains of P. aeruginosa. S. maltophilia K279a (WT), virB10 mutant NUS15 (virB10), or complemented mutant NUS15(pvirB10) (virB10/virB10+) was mixed with P. aeruginosa strain 7700 (A), PAO1 (B), and PAK (C) at a ratio of ∼100:1 and spotted onto LB agar, and after 2 h of incubation, the numbers of each strain were determined by plating dilutions of the entire bacterial growth area on selective media. The results presented are the CFU at t = 0 h and t = 2 h for each time that the P. aeruginosa strain had been coincubated with the S. maltophilia WT, mutant, or complemented mutant strain (A to C, left) as well as the CFU at t = 0 h and t = 24 h for the three S. maltophilia strains under each growth condition (A to C, right). Asterisks indicate significant differences in CFU obtained at t = 0 versus t = 2 h (*, P < 0.05; **, P < 0.01). Data are presented as the means and standard deviations of results from three independent experiments (n = 3 each).

FIG 8.

Bactericidal effect of S. maltophilia wild-type and virB10 mutant strains on various species of Pseudomonas. S. maltophilia K279a (WT), virB10 mutant NUS15 (virB10), or complemented mutant NUS15(pvirB10) (virB10/virB10+) was mixed with P. mendocina (A), P. fluorescens (B), P. putida (C), and P. stutzeri (D) at a ratio of ∼100:1 and spotted onto LB agar, and after 2 h of incubation, the numbers of each strain were determined by plating on selective media. The results presented are the CFU at t = 0 h and t = 2 h for each of the P. aeruginosa strains that had been coincubated with the WT, mutant, and complemented mutant S. maltophilia (left) as well as the CFU for the S. maltophilia strains (right). Asterisks indicate significant differences in CFU obtained at t = 0 versus t = 2 h (*, P < 0.05). Data are presented as the means and standard deviations of results from three independent experiments (n = 3 each).

DISCUSSION

The current study represents the first genetic and functional analysis of S. maltophilia T4SS. After documenting that the VirB/D4 T4SS exemplified in strain K279a is well conserved among both clinical and environmental isolates of S. maltophilia, we determined that the T4SS (i) inhibits apoptosis in infected human epithelial cells, (ii) promotes apoptosis in infected mammalian macrophages, (iii) enhances the growth of S. maltophilia when it is cocultured with various genera of heterologous bacteria, and (iv) promotes the death of multiple strains of P. aeruginosa and at least one strain of P. mendocina. In all cases, the impact of the T4SS required S. maltophilia contact with its target, suggesting that the VirB/D4 apparatus is able to interact with a range of different membranes/receptors. We infer that the S. maltophilia VirB/D4 T4SS secretes multiple protein effectors that are injected into target cells. The finding that a single T4SS can have anti- and prokilling effects on disparate targets, including both human and bacterial cells, has, to our knowledge, not been reported before. We suspect that different secreted proteins are responsible for the different effects seen; e.g., in one possible scenario, one effector is antiapoptotic in epithelial cells, another is proapoptotic in macrophages, and yet another effector(s) is antagonistic toward heterologous bacteria. We intuit that the antiapoptotic effect of the T4SS on epithelial cells promotes S. maltophilia growth, perhaps by allowing prolonged bacterial attachment to an epithelial cell layer and/or nutrient assimilation from viable host cells. Given that macrophages are a key phagocytic cell of the innate immune system, we surmise that the proapoptotic effect we have observed helps S. maltophilia to resist being engulfed and killed and/or recognized by the immune system. Finally, it is probable that the anti-Pseudomonas effect of the VirB/D4 T4SS helps S. maltophilia to survive in a variety of niches, including perhaps the human host and the CF lung, which can be infected with P. mendocina in addition to the well-known P. aeruginosa (111, 112). Based upon the range of target cells utilized in this study, we strongly suspect that the S. maltophilia T4SS can modulate the death program of a wide range of cellular targets, including killing other mammalian cells that are part of the immune system (e.g., neutrophils) or other bacteria that inhabit its niches, whether planktonic in nature or coinfecting plant, animal, or human hosts. In regard to the proapoptotic effect that the K279a VirB/D4 T4SS has on macrophages, it is tempting to speculate that this function evolved as a result of S. maltophilia interacting with predatory protozoans (amoebae) in nature (113–117). Despite demonstrating the importance of the S. maltophilia T4SS in influencing the death of other cells, we do not believe that this is the only function of this VirB/D4 T4SS, given what is known about T4SS in general (35–37).

It is highly likely that the virB10 mutant phenotypes that we observed are due to the loss of T4SS effectors. A number of software programs have been designed for predicting the output of a T4SS (118–124). These bioinformatic tools tag candidates based on a number of traits, including C-terminal features and the presence of eukaryotic-like domains. Thus, a recent bioinformatic study encompassing a very wide variety of organisms and their secretion systems suggested that S. maltophilia K279a encodes more than 25 putative T4SS effectors (125). Such a sized output is entirely compatible with the output of known T4SSs, which ranges from 1 (B. pertussis and H. pylori) to ∼7 to 25 (Bartonella, Brucella, Anaplasma, and Ehrlichia) to >140 (Coxiella and Legionella) (35, 45, 46, 49, 126). Moreover, it could account for the multiple effects of the VirB/D4 system that we have defined in our study as well as other potential functions. In contemplating the types of S. maltophilia effectors that might modulate death in mammalian cells and how they operate, it is helpful to consider the range of mechanisms that have already been defined in other T4SS-encoding pathogens. For example, in C. burnettii, the effector CaeB inhibits apoptosis at the level of mitochondria, whereas effector AnkG modulates p32 (63, 127). In L. pneumophila, some Dot/Icm effectors target proapoptotic members of the Bcl2 family within macrophages (69). In thinking about the bactericidal effects of the newly described S. maltophilia T4SS, it is instructive to reflect on the recently discovered antibacterial effect of the X. axonopodis pv. citri T4SS (98). In this case, the effector X-Tfe^XAC2609 is a peptidoglycan hydrolase that can promote the killing of both E. coli DH5α and a strain of Chromobacterium violaceum. Importantly, we used the same assay as that in this earlier study in order to measure the antibacterial effect of the S. maltophilia VirB/D4 T4SS on E. coli DH5α and P. aeruginosa 7700 (Fig. 6), and the magnitude of the effect that we observed was comparable to that found in the X. axonopodis pv. citri study. Interestingly, proteins with domains similar to those in protein X-Tfe^XAC2609 are encoded by the genomes of some S. maltophilia strains and other Xanthomonadaceae species (98). Finally, it is possible that the antibacterial effector(s) of the S. maltophilia VirB/D4 T4SS is (also) functionally similar to antibacterial proteins that are secreted by various type VI secretion systems, e.g., lipases, peptidases, and nucleases, in addition to muramidases (128).

Based upon our analysis of completed genomes, the VirB/D4 T4SS that exists in K279a and most, if not all, strains of S. maltophilia is also present in eight other species of Stenotrophomonas, i.e., S. chelatiphaga, S. daejeonensis, S. ginsengisoli, S. indicatrix, S. koreensis, S. lactitubi, S. pavanii, and S. pictorum (5, 129–134). Thus, the impact of this VirB/D4 system is undoubtedly quite broad. That this Stenotrophomonas T4SS was most related to a T4SS in Xanthomonas is not surprising given that S. maltophilia was, until 1993, classified as a Xanthomonas species (135). Given the positions of the various genera encoding a T4SS in the phylogenetic tree that we generated using orthologous Vir T4SS proteins and the complete lack of T4SS in other closely related species, we posit that the VirB/D4 system typified in S. maltophilia strain K279a has been acquired horizontally after the divergence of the families Xanthomonadaceae and Rhodanobacteraceae. The fact that the %GC for the VirB/D4 locus is 61.5 whereas the overall %GC for S. maltophilia is 66.1 further suggests that the VirB/D4 system of S. maltophilia was acquired horizontally. Our genomic analysis also confirmed that some strains of S. maltophilia, including strain K279a, have two T4SS, i.e., Trb T4SS in addition to the VirB/D4 system (see Table S1 in the supplemental material) (24, 32). The function of the Trb T4SS in S. maltophilia is unknown, although it has been suggested that this T4SS has some similarities to one in a strain of P. aeruginosa (32). Based upon its genetic makeup (24, 32), the Trb T4SS, more so than the VirB/D4 system, may mediate conjugal transfer of plasmids between S. maltophilia strains.

In summary, our finding that the T4SS has a role in the interplay between S. maltophilia and mammalian hosts as well as against other bacteria that inhabit the human host is a novel development in our understanding of S. maltophilia. Although many advances have been made concerning antibiotic resistance in S. maltophilia, relatively few studies have defined aspects of S. maltophilia pathogenesis. Thus, the further study of S. maltophilia T4SS is significant when one considers the present state of the field. This effort will also have implications for the hundreds of uncharacterized T4SS that have been detected in the genomes of other bacteria (36, 136).

MATERIALS AND METHODS

Bacterial strains, media, and extracellular growth.

S. maltophilia K279a (American Type Culture Collection [ATCC] strain BAA-2423) served as both our main wild-type strain and the parent for mutants (17, 28, 29, 31). S. maltophilia isolates UPSm1, UPSm2, UPSm3, and UPSm5 were also examined (28). Other strains used in the bacterial competition assays were E. coli DH5α (Life Technologies); K. pneumoniae KPPR1, P. aeruginosa ATCC 7700, PAO1, and PAK, P. fluorescens ATCC 1759, P. putida ATCC 49128, P. mendocina, and P. stutzeri (obtained from Alan Hauser, Northwestern University); and A. xylosoxidans strain UPAx4 (28). E. coli DH5α was also used as the host for recombinant plasmids. All strains were cultured routinely at 37°C on Luria-Bertani (LB) agar or broth (Becton, Dickinson) (29). Bacterial growth in LB broth cultures was monitored by measuring the optical density at 600 nm (OD₆₀₀) of the cultures (30). Bacterial survival in RPMI media (Corning) containing 10% fetal bovine serum (FBS; Atlanta Biologicals) was assessed by plating samples onto LB agar to enumerate CFU.

DNA, RNA, and protein analysis.

S. maltophilia K279a DNA and RNA were isolated as before (28, 29). DNA sequences were analyzed and primers were designed using Lasergene software (DNAStar). Primers (Integrated DNA Technology) are listed in Table S3 in the supplemental material. RT-PCR analysis was done as before (28), with the primers targeting virD4 (MN30 and MN31) and virB6 (MN32 and MN33) designed using the Primer-BLAST tool at NCBI. BLAST was done using the genome sequences in the GenBank database at NCBI. Pairwise ANI was calculated for selected genomes using OrthoANIu (137). A reciprocal best BLAST strategy was used to identify orthologs of the S. maltophilia K279a VirB/D4 T4SS within each of the analyzed genomes (138, 139). An E value cutoff of 1E−5 was used to filter highly divergent sequences out of subsequent analyses. The amino acid sequences of the orthologous proteins were concatenated, and an alignment was generated using MUSCLE (140). A maximum-likelihood tree was constructed with RaxML using the LG + gamma evolutionary model (141).

Mutant constructions and complementation analysis.

We introduced an unmarked deletion into virB10 of K279a, using previous methods (27–30). The entire virB10 (SMLT_RS14310) coding region was deleted using a recombineering approach and FLP excision. The virB10 gene, including 600 bp of flanking DNA on both sides, was PCR amplified from K279a genomic DNA using primers AD116 and AD117 and then ligated into pGEM-T Easy (Promega), creating the plasmid pGvirB10. Primers AD118 and AD119 were used to PCR amplify an FLP recombination target (FRT)-flanked chloramphenicol cassette. Two micrograms of pGvirB10 DNA and 700 ng of the FRT-flanked cassette next were transformed into E. coli DY330 (142), and bacteria containing pGEMΔvirB10::frt-cat-frt were selected on chloramphenicol-containing agar. After PCR confirmation of the recombination event using M13 universal primers, mutated virB10 was PCR amplified using pGEMΔvirB10::frt-cat-frt as the template and primers AD120 and AD121. This PCR product was digested using BamHI and HindIII and ligated into pEX18Tc (28), yielding pEXΔvirB10::frt-cat-frt. This plasmid was transferred into E. coli S17-1 and mobilized from there into S. maltophilia K279a by conjugation (28). Tetracycline-, chloramphenicol-, and norfloxacin-resistant conjugants were streaked onto LB agar supplemented with 10% (wt/vol) sucrose and chloramphenicol to induce and select for loss of the pEX18Tc vector. FLP-mediated excision of the chloramphenicol cassette was achieved by electroporating pBSFlp (143) into the virB10 mutant and selecting transformants on LB agar containing gentamicin and 1 mM isopropyl-β-d-thiogalactopyranoside, incubated overnight at 37°C, and then incubated for 24 h at room temperature. Smaller colonies appeared after this second incubation, and these were patched onto LB containing either chloramphenicol, gentamicin, or no selection drug. Those that were either gentamicin or chloramphenicol sensitive were streaked onto sucrose-containing LB agar at 37°C overnight, and desired gene deletions were confirmed by PCR using the primers AD122 and AD123. The newly made virB10 mutant was designated NUS15. For trans-complementation of the virB10 mutant, a 2.2-kb PCR fragment containing the virB10 coding region and its promoter was PCR amplified from K279a DNA using primers MN13 and MN14. These fragments were digested by KpnI and XbaI and cloned into pBBR1MCS (30, 144), yielding pBvirB10, which was electroporated into the mutant. Transformants were selected on LB agar containing chloramphenicol, and clones were confirmed as carrying pBvirB10 by PCR using M13 primers. A mutant lacking both virB10 and xpsF was constructed by mobilizing pEXΔvirB10::frt-xpsF-frt carried by the E. coli S17-1 strain into mutant NUS15 containing the virB10 deletion. Transconjugants were selected as described above, and mutation of xpsF was confirmed by PCR using the primers SK213 and SK214. The virB10 xpsF mutant was designated NUS16.

Assays for host cell detachment, viability, and apoptosis.

The human A549 cell line (ATCC CCL-185) and U937 cell line (ATCC CRL-1593.2) were maintained by passage in RPMI medium with 10% fetal bovine serum (FBS) (29, 145). Primary human bronchial/tracheal epithelial cells were obtained from the ATCC (PCS-300-010) and maintained according to their guidelines. BMDM from A/J mice (Jackson Laboratory) were cultured as before (145). Epithelial and macrophage cells were infected with S. maltophilia essentially as before (28). Monolayers of 5 × 10⁵ A549 or PCS-300-010 cells or 1 × 10⁶ U937 cells or BMDM were established in the wells of a 24-well tissue culture plate, and bacteria that had been grown overnight on LB agar and resuspended in phosphate-buffered saline (PBS) were added at a multiplicity of infection of 50. The numbers of viable cells in infected wells, whether only those that are adherent or the combination of adherent and detached cells, were determined as before (28), except that cells were quantitated using a Scepter 2.0 cell counter (Millipore-Sigma) (146, 147). The ability of S. maltophilia to inhibit staurosporine-induced apoptosis was measured by first pretreating monolayers with 5 μM staurosporine (Santa Cruz Biotech) in the absence or presence of 200 μM caspase inhibitor Z-VAD-FMK (R&D Systems). Cells were then infected with S. maltophilia, and viability was measured using the Scepter cell counter. The ability of S. maltophilia to activate caspase-3/7 was measured as previously described (30). Mammalian cells were preloaded with 5 μM CellEvent caspase-3/7 green detection regent (Thermo Fisher) and infected with S. maltophilia or not infected but given 2 μM staurosporine. Caspase activation was revealed by measuring fluorescence at 503 nm and 530 nm. Western blot analysis was done to probe for the loss of intact caspase-7 and the appearance of its two cleavage products, as has been done by others (148, 149). Membranes were blocked for 1 h in Tris-buffered saline with 0.1% Tween (TBST) and 5% milk. Proteins were exposed to anti-caspase-7 (1:1,000; Cell Signaling) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000; Santa Cruz) diluted in TBST with 5% milk and incubated at 4°C overnight. The secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit (Santa Cruz), was diluted 1:10,000 in TBST plus milk and incubated for 1 h at room temperature. Blots were developed by adding the enhanced chemiluminescence reagent (GE Healthcare). To judge the effect of bacterium-to-host cell contact on these phenomena, epithelial cells and macrophages were added to the bottom of a 24-well plate, and bacteria were added to a transwell insert (Corning) that was placed into the well. One set of transwells contained an 8-μm filter such that bacterial mixing and contact could occur, and the other set had a 0.4-μm filter such that mixing and contact was impeded (150, 151).

Assays for bacterial competition and killing.

Interbacterial competition was assessed using previously described methods (98, 102, 103, 110). Single colonies for each strain to be tested were resuspended in LB broth, and the bacteria were grown at 37°C to mid-log phase. Using fresh LB medium, each bacterial suspension was then diluted to an OD₆₀₀ of 0.3, providing approximately equivalent CFU/ml. Aliquots of the cell suspensions were then combined so as to yield ratios of S. maltophilia to the heterologous bacterium approximating 200:1, 150:1, 100:1, and 50:1. Fifty microliters of the mixture was spotted on LB agar plates and incubated at 37°C either for 2 h in order to measure bactericidal effects (110) or for 24 h in order to gauge effects on bacterial growth (98, 102, 103). After incubation, the area of bacterial growth was removed and resuspended into 1 ml of PBS. The resultant cell suspensions were serially diluted and plated onto both standard LB agar and LB agar containing norfloxacin. The colonies formed on the antibiotic-containing plates represented the numbers of S. maltophilia in the cell mixture (29), whereas the colonies on the standard plates reflected the total cell population. Thus, the numbers of heterologous bacteria surviving in the coculture were determined by calculating the difference in CFU on the two plates. To measure the effect of cell-to-cell contact on interbacterial competition, P. aeruginosa was suspended in LB broth to 10⁷ CFU/ml and 1 ml added to the bottom of a 24-well plate. S. maltophilia strains also resuspended in LB broth were added to the transwell insert such that an S. maltophilia/P. aeruginosa ratio of 100:1 was achieved. One set of inoculated transwells contained an 8-μm filter such that bacterial mixing and contact could occur, and the other contained a 0.4-μm filter such that mixing and contact were impeded. After 24 h of incubation, the liquids in the two chambers were combined, serially diluted, and plated on both LB plates and LB plates containing norfloxacin in order to calculate the numbers of each type of bacterium as described above.

Statistical procedures.

In all experiments, each sample or condition was assessed using at least three technical replicates. All experiments were repeated at least three times. The resultant values obtained were presented as the means and standard deviations from the three independent experiments, and statistical analysis was applied using the Student's t test as appropriate. P values are presented in the figure legends.