Pseudomonas aeruginosa surface motility and invasion into competing communities enhance interspecies antagonism

ABSTRACT

Chronic polymicrobial infections involving Pseudomonas aeruginosa and Staphylococcus aureus are prevalent, difficult to eradicate, and associated with poor health outcomes. Therefore, understanding interactions between these pathogens is important to inform improved treatment development. We previously demonstrated that P. aeruginosa is attracted to S. aureus using type IV pili (TFP)-mediated chemotaxis, but the impact of attraction on S. aureus growth and physiology remained unknown. Using live single-cell confocal imaging to visualize microcolony structure, spatial organization, and survival of S. aureus during coculture, we found that interspecies chemotaxis provides P. aeruginosa a competitive advantage by promoting invasion into and disruption of S. aureus microcolonies. This behavior renders S. aureus susceptible to P. aeruginosa antimicrobials. Conversely, in the absence of TFP motility, P. aeruginosa cells exhibit reduced invasion of S. aureus colonies. Instead, P. aeruginosa builds a cellular barrier adjacent to S. aureus and secretes diffusible, bacteriostatic antimicrobials like 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) into the S. aureus colonies. Reduced invasion leads to the formation of denser and thicker S. aureus colonies with increased HQNO-mediated lactic acid fermentation, a physiological change that could complicate treatment strategies. Finally, we show that P. aeruginosa motility modifications of spatial structure enhance competition against S. aureus. Overall, these studies expand our understanding of how P. aeruginosa TFP-mediated interspecies chemotaxis facilitates polymicrobial interactions, highlighting the importance of spatial positioning in mixed-species communities.

IMPORTANCE

The polymicrobial nature of many chronic infections makes their eradication challenging. Particularly, coisolation of Pseudomonas aeruginosa and Staphylococcus aureus from airways of people with cystic fibrosis and chronic wound infections is common and associated with severe clinical outcomes. The complex interplay between these pathogens is not fully understood, highlighting the need for continued research to improve management of chronic infections. Our study unveils that P. aeruginosa is attracted to S. aureus, invades into neighboring colonies, and secretes anti-staphylococcal factors into the interior of the colony. Upon inhibition of P. aeruginosa motility and thus invasion, S. aureus colony architecture changes dramatically, whereby S. aureus is protected from P. aeruginosa antagonism and responds through physiological alterations that may further hamper treatment. These studies reinforce accumulating evidence that spatial structuring can dictate community resilience and reveal that motility and chemotaxis are critical drivers of interspecies competition.

INTRODUCTION

Microorganisms exist in complex polymicrobial environments, such as the soil and the human body, where they interact with each other and respond to changes in their surroundings (1–3). These interactions can lead to the emergence of community-level properties not observed in monoculture (4–10). The resulting collective behavior can have significant implications for the microbial physiology, evolution, and interactions with the host. For example, bacterial pathogens can enhance both virulence and antibiotic tolerance in mixed communities (2, 4, 11–16), potentially undermining current chronic infection treatments.

Pseudomonas aeruginosa and Staphylococcus aureus are the most prevalent and abundant pathogens in individuals with cystic fibrosis (CF) (5, 17) and persist in significant quantities in the lungs for decades (18). Critically, their coinfection is linked with more severe lung disease, increased rates of hospitalization, and reduced lung function in patients (19–23). Additionally, coinfection in chronic burn wounds can delay healing time (24). Thus, there is a need to further understand how interactions between these two organisms exacerbate the outcomes of polymicrobial infections.

Several in vitro studies support clinical observations that P. aeruginosa and S. aureus increase each other’s virulence during coculture (12, 25). P. aeruginosa secretes numerous anti-staphylococcal factors including the siderophores pyoverdine and pyochelin, phenazines, rhamnolipids, staphylolytic proteases like LasA, and quinolones (26–32). Many of these secreted products alter S. aureus physiology, enhancing its antibiotic tolerance (13, 15, 16, 33–35). One example is the small molecule, 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), which inhibits S. aureus cellular respiration, shifting its metabolism to fermentation (11, 30, 36, 37). HQNO has been detected in CF sputum (30) and can increase S. aureus tolerance to several antibiotics used clinically (13, 15, 16). However, the effect of these antimicrobials (AM) on S. aureus has been mainly studied in the presence of P. aeruginosa cell-free spent medium in a well-mixed environment (13, 15, 16, 35, 38). Interestingly, recent studies found that HQNO modifies the spatial organization of P. aeruginosa and S. aureus in a synthetic CF sputum medium (SCFM2) (39) and chronic murine wounds (40), highlighting the importance of visualizing communities in a structured environment. Therefore, to elucidate how interspecies interactions negatively impact clinical outcomes, experimental models are needed that better reflect how microbes naturally encounter each other, namely, under spatial constraint.

Previously, we demonstrated that P. aeruginosa responds to S. aureus from a distance by increasing type IV pili (TFP) motility, mediated by retractile appendages that allow P. aeruginosa to move across surfaces through twitching motility (41). Specifically, when P. aeruginosa and S. aureus start as spatially separated single cells, we observed that P. aeruginosa uses the Pil-Chp chemoreceptor, PilJ, to respond from a distance by directionally moving toward S. aureus (42, 43). This attraction requires secretion of S. aureus peptides called phenol-soluble modulins (42, 44). However, it remains unknown how P. aeruginosa chemotaxis toward S. aureus influences S. aureus physiology and survival.

Here, we sought to understand the consequences of P. aeruginosa TFP-mediated attraction on S. aureus. We visualized P. aeruginosa and S. aureus interactions at the single-cell level over time using resonant scanning confocal microscopy and discovered that P. aeruginosa utilizes a combination of TFP-mediated motility and secreted antimicrobials to effectively outcompete S. aureus under these conditions. Particularly, we found that wild-type (WT) P. aeruginosa is attracted to, invades, and disrupts S. aureus colonies. Moreover, P. aeruginosa-secreted antimicrobials HQNO, pyoverdine, pyochelin, and LasA were necessary for negatively influencing S. aureus growth, but not for invasion and disruption of S. aureus colonies. Conversely, in the absence of TFP motility, P. aeruginosa cannot invade S. aureus colonies but rather grows around them, leading to an altered S. aureus colony architecture resulting in compact, thicker colonies with increased biomass compared with coculture with WT P. aeruginosa. In addition to these effects on S. aureus colony architecture, we also found that coculture with a TFP-deficient P. aeruginosa leads to altered physiology through the induction of an HQNO-mediated increase in S. aureus fermentation. Moreover, TFP motility was crucial for modulating the spatial arrangement and competitive dynamics between P. aeruginosa and S. aureus in conditions that capture essential features of the CF airway environment. Overall, our findings highlight the importance of spatial organization in community-based behaviors and the need for a more thorough understanding of the interplay between polymicrobial communities in the context of infection.

RESULTS

TFP are necessary for P. aeruginosa invasion into S. aureus colonies

We previously reported that P. aeruginosa responds to S. aureus from a distance by using TFP to chemotax toward and surround S. aureus colonies (42), but how this behavior affects S. aureus physiology remained unclear. To test the consequences of P. aeruginosa chemotaxis on S. aureus, we first visualized P. aeruginosa interactions with S. aureus colonies in three dimensions by performing live resonant scanning confocal microscopy of S. aureus in mono- or coculture with P. aeruginosa WT or a TFP-deficient mutant (∆pilA). Here, S. aureus and P. aeruginosa constitutively expressed sgfp (pseudocolored orange) and mCherry (pseudocolored cyan), respectively. Bacteria were inoculated between a cover slip and an agarose pad for visualization in the same visual field over time. Imaging was initiated with S. aureus and P. aeruginosa as single cells, positioned approximately 30 to 50 µm apart to provide sufficient time and distance for P. aeruginosa to respond to the presence of S. aureus. As previously demonstrated (42), at approximately 5 hours, we observed that WT P. aeruginosa responds to S. aureus by breaking into single cells and moving toward it with TFP motility, which eventually leads to P. aeruginosa surrounding, invading, and disrupting S. aureus cells from the colony (Fig. 1). This invasion is dependent on TFP motility, as P. aeruginosa ∆pilA exhibited significantly decreased invasion compared with WT (Fig. 1B). While the ∆pilA mutant is amotile, it eventually grows against the S. aureus colony at later time points (Fig. 1A and C). These data suggest that TFP motility is not only necessary for P. aeruginosa chemotaxis toward S. aureus but also enables effective invasion of P. aeruginosa into S. aureus colonies.

Fig 1.

Type IV pili are necessary for P. aeruginosa invasion into S. aureus colonies. Resonant scanning confocal live imaging of S. aureus and P. aeruginosa. (A) Representative micrographs of WT S. aureus (pseudocolored orange) in monoculture or in coculture with P. aeruginosa (pseudocolored cyan; WT or TFP-deficient mutant ΔpilA). (B) Quantification of P. aeruginosa single-cell invasion into S. aureus colonies t ~ 7 hours in mono- or coculture with P. aeruginosa (WT or ΔpilA). At least four biological replicates with two technical replicates each were analyzed. Each data point represents one technical replicate. Statistical significance was determined by a Mann-Whitney U-test. ****P < 0.0001. (C) Zoomed micrograph of S. aureus colony edge in coculture with P. aeruginosa (WT or ΔpilA). White arrows indicate dispersed S. aureus cells. S. aureus: pCM29 P_sarAP1-sgfp; P. aeruginosa: chromosomal P_{A1/04/03-mCherry}.

P. aeruginosa TFP motility-mediated invasion influences S. aureus growth and architecture

To investigate how P. aeruginosa invasion affects S. aureus colony physiology, we imaged S. aureus in mono- or coculture with WT or ∆pilA P. aeruginosa following ~24 hours of incubation. At later time points, visualizing P. aeruginosa becomes challenging due to reduced fluorescence from decreased mCherry production and photobleaching. Nevertheless, phase contrast microscopy confirmed that P. aeruginosa cells surround S. aureus colonies after ~24 hours (Fig. S1).

We found that in coculture with WT P. aeruginosa, S. aureus forms smaller colonies than in monoculture by measuring the area at the base of the S. aureus colony (Fig. 2A and B). Moreover, P. aeruginosa TFP motility-mediated invasion resulted in S. aureus colony edges exhibiting reduced fluorescence, likely caused by dispersed, lysed cells, or a combination thereof (Fig. 2A). In the presence of ∆pilA, the area of S. aureus colonies was comparable to that in the presence of WT P. aeruginosa (Fig. 2B). However, despite similar growth area, S. aureus colonies exhibited less dispersal at the colony edges in coculture with ∆pilA, possibly due to loss of P. aeruginosa invasion (Fig. 2A; top and middle rows). Additionally, S. aureus colonies appeared thicker and denser than in coculture with WT P. aeruginosa, likely due to reduced cell dispersal.

Fig 2.

P. aeruginosa type IV pili motility-mediated invasion influences the architecture of S. aureus colonies independently of P. aeruginosa-secreted antimicrobials. Analysis of S. aureus colony edge disruption and thickness. (A) Representative resonant scanning confocal micrographs of the whole colony (top row) or Galvano scanner colony edge micrographs of WT S. aureus (orange) in monoculture or coculture with P. aeruginosa (not shown; WT or ΔpilA) at t ~ 24 hours shown from the top (middle row) of the colony or the side (bottom row). The micrographs in A (bottom row) show the colonies on the Z-plane and demonstrate how the height was quantified. Quantification of S. aureus whole colony area at t ~ 24 hours (B) or height at the edge of S. aureus (Sa) colony (µm) at t ~ 24 hours (C) in monoculture or coculture with P. aeruginosa (WT, ΔpilA, ΔlasA, ΔAM^B [bacteriostatic antimicrobials; HQNO, pyoverdine, and pyochelin], ΔAM^C [complete antimicrobials; HQNO, pyoverdine, pyochelin, and LasA], or ΔpilA ΔAM^C). (D–G) Representative BiofilmQ heatmaps (D and E) and quantification (F and G) of local surface roughness and cell packing analysis at S. aureus colony edge in mono- or coculture with P. aeruginosa (WT or ΔpilA). Each data point represents the average of two technical replicates within one biological replicate. Statistical significance was determined by a Mann-Whitney U-test with an ad hoc Bonferroni correction for multiple comparisons. (H) Cell packing distribution within S. aureus colony edge in the abovementioned conditions. A Kolmogorov-Smirnov cumulative distribution test was performed, and all three conditions were significantly different (****P < 0.0001) from one another. A total of 15 biological replicates with two technical replicates each were analyzed in panels F–H. (I) The number of invading P. aeruginosa (Pa) single cells inside S. aureus was quantified at t ~ 7 hours in mono- or coculture with the P. aeruginosa strains described above. At least four biological replicates with two technical replicates each were analyzed per condition in panels B, C, and I. Each data point represents one technical replicate. Statistical significance in panels B, C, and I was determined by Kruskal-Wallis followed by Dunn’s multiple comparisons test. n.s., not significant; *P < 0.05, ***P < 0.001, and ****P < 0.0001.

To further investigate this, we visualized and quantified S. aureus colony architecture in more detail. Since thickness and density were more distinct on the colony edges, images were acquired with higher magnification and spatial resolution using galvanometric point-scanning confocal microscopy at the end time point (Fig. 2A; middle row). We then measured the height at the edge of S. aureus colonies at 15 µm from the edge using the Z-plane (Fig. 2A, bottom row, and Fig. 2C). As expected, the height at the colony edge was significantly higher in coculture with ∆pilA than in mono- or coculture with WT P. aeruginosa (Fig. 2C). To quantitatively analyze colony density, we measured both cell packing and colony surface roughness using the microscopy image analysis software BiofilmQ (45). These parameters quantify density by measuring the amount of surface or volume within a specified area. In BiofilmQ, S. aureus colony edges were separated from the background by segmentation onto a 3D grid, with each cubic grid unit measuring 0.72 µm per side. Neighborhood surface roughness and cell packing were then calculated by determining the biovolume fraction and surface height variance of S. aureus for each grid cube within 4 and 6 µm, respectively. Representative heatmaps in Fig. 2D and E provide a two-dimensional visualization of the quantified data in Fig. 2F through H, using color coding to represent local surface roughness and cell packing. S. aureus colonies in monoculture show low surface roughness and uniform cell packing (Fig. 2D and E [first column] and Fig. 2F and G). Conversely, when WT P. aeruginosa is present, S. aureus edges exhibit significantly increased surface roughness and slightly decreased cell packing (Fig. 2D and E [middle column] and Fig. 2F and G), which suggests reduced colony density is caused by WT P. aeruginosa. When cocultured with ∆pilA (i.e., lacking invasion and colony disruption), S. aureus colonies portrayed significantly reduced colony surface roughness and increased cell packing compared with WT P. aeruginosa coculture (Fig. 2D and E [last column] and Fig. 2F and G). While the colony cell roughness was not different between S. aureus coculture with ∆pilA and monoculture, the mean colony cell packing was significantly increased (Fig. 2F and G). Additionally, we analyzed the cell packing distribution within S. aureus colony edges and found all three conditions to be statistically different (Fig. 2H). The majority of the monoculture colony edge population was distributed between 0.5 and 1.0, with almost no low-density areas. In S. aureus coculture with ∆pilA, the large peak at 1.0 indicates that the majority of cells within these edges are highly packed. On the other hand, in the presence of WT P. aeruginosa, the cell packing is more evenly distributed with an increased proportion of the population at low-density values compared with monoculture or coculture with ∆pilA. However, there is also an increased proportion of the population at high-density values portrayed as a small peak at 1.0. This peak may be attributed to the colony edge height being slightly higher than monoculture S. aureus as reported in Fig. 2C, leading to higher cell packing as this calculation considers the three-dimensional space.

Thus, while the base area of the colony is similar in the presence of WT or ∆pilA, the colonies are more densely packed when P. aeruginosa lacks TFP. Altogether, these observations reveal the crucial role of P. aeruginosa TFP motility in altering S. aureus architecture. Without TFP motility, P. aeruginosa does not invade or disrupt S. aureus colonies; instead, it grows alongside them, resulting in increased compaction and altered S. aureus colony structure.

P. aeruginosa secretes antimicrobials that affect S. aureus growth but do not influence S. aureus colony architecture

Next, we wondered how invasion changes S. aureus colony architecture and enhances competition. One hypothesis is that invasion increases the local concentration of P. aeruginosa antimicrobials within S. aureus colonies. Additionally, these anti-staphylococcal factors could aid P. aeruginosa invasion. If the former is correct, S. aureus colonies grown in the presence of the P. aeruginosa ∆pilA would be protected from P. aeruginosa antimicrobials. P. aeruginosa secretes many factors known to inhibit or lyse S. aureus, including HQNO, a respiratory toxin that inhibits the S. aureus electron transport chain (30, 36), the siderophores pyoverdine and pyochelin, which aid in iron scavenging (29, 32), and an anti-staphylococcal protease, staphylolysin or LasA, which lyses S. aureus by cleaving the peptidoglycan pentaglycine cross-links (31).

Since ∆pilA had reduced invasion and disruption of S. aureus exterior structure compared with WT, we first determined if this difference is due to variations in levels of secreted antimicrobials between the P. aeruginosa strains and tested whether ∆pilA produces similar levels of exoproducts as WT. The cell-free supernatant from ∆pilA or WT P. aeruginosa was added to S. aureus to examine its growth and lysis over time. No differences were observed in either S. aureus lysis or growth rate when exposed to supernatant from WT or ∆pilA P. aeruginosa, confirming that each produces similar levels of antimicrobials (Fig. S2). Here, supernatant from P. aeruginosa ∆lasA served as a control to confirm that staphylolysin is the main driver of S. aureus lysis.

To test the hypothesis that P. aeruginosa invasion enhances competition by increasing diffusion and local antimicrobial concentration within S. aureus colonies, we next examined S. aureus colony growth dynamics in the presence of P. aeruginosa strains lacking genes encoding antimicrobials. These include a staphylolysin mutant (∆lasA), a strain without both HQNO and siderophores (∆pqsL ∆pvdA ∆pchE), referred to as “∆AM^B” (antimicrobials^{Bacteriostatic}), and a mutant with all four antimicrobials deleted (∆pqsL ∆pvdA ∆pchE ∆lasA) which we call “∆AM^C” (AM^Complete) in Fig. 2B, C and I. The interactions between S. aureus and these antimicrobial-deficient strains were assessed by live imaging as described for Fig. 1, and P. aeruginosa invasion and S. aureus colony height (as a proxy for biomass) were quantified. No detectable differences were observed between coculture with WT P. aeruginosa and the antimicrobial mutants for either the S. aureus colony edge height (Fig. 2C) or the number of invading cells (Fig. 2I), which suggests that these antimicrobials do not play a role in P. aeruginosa invasion or increased S. aureus colony height observed in coculture with ∆pilA. Yet, it is known that these antimicrobials can impact S. aureus growth in vitro (11, 13, 15, 16, 35, 37, 38, 46). Therefore, we measured S. aureus colony base area to examine antimicrobial influence on growth under these conditions. The S. aureus colony area did not significantly increase when cocultured with ∆AM^B or ∆lasA, compared with the WT (Fig. 2B). However, colony area did increase upon deletion of all four antimicrobials (∆AM^C), which suggests that while these antimicrobials do not influence S. aureus colony architecture, their combinatorial effect alters S. aureus growth and colony area.

To determine if S. aureus colony edge height and P. aeruginosa invasion are driven by motility alone or a combined effect of motility and antimicrobials, we deleted pilA in the ∆AM^C mutant, generating ∆pqsL ∆pvdA ∆pchE ∆lasA ∆pilA (∆pilA ∆AM^C). S. aureus colony height and invasion of P. aeruginosa ∆pilA ∆AM^C phenocopied ∆pilA (Fig. 2C and I), suggesting that TFP motility plays a prominent role in driving these phenotypes. Furthermore, when comparing the effect of ∆pilA on the WT background to the ∆AM^C background, significant antimicrobial influence on the colony area is only observed when TFP are functional, supporting that motility may enhance antimicrobial action against S. aureus under these conditions (Fig. 2B).

Overall, these data suggest that thicker and denser S. aureus colony architecture is exclusively mediated by the absence of P. aeruginosa TFP-mediated colony invasion and that the main P. aeruginosa anti-staphylococcal factors do not substantially influence this observation. Furthermore, P. aeruginosa TFP motility may enhance antimicrobial access into the colony to fully affect S. aureus growth, revealing the important role P. aeruginosa motility plays in antagonistic interactions against S. aureus.

Increased cell packing enhances HQNO-mediated S. aureus fermentation

Although P. aeruginosa antimicrobials did not influence S. aureus structure, we next explored how colony morphology differences affect S. aureus physiological response to HQNO by utilizing a fluorescent reporter system. HQNO poisons the S. aureus respiratory chain, forcing a shift to fermentative metabolism (11); therefore, S. aureus fermentation can be used as a proxy for HQNO activity. A fluorescent transcriptional fusion to the promoter of the lactate dehydrogenase gene (P_ldh1-sgfp) was used to measure fermentation (47). If HQNO penetrates densely packed colonies, we expect to see increased fluorescence compared with coculture with P. aeruginosa lacking HQNO production. To test this prediction, we live imaged S. aureus in mono- or coculture with P. aeruginosa and quantified the mean fluorescence intensity (MFI) per S. aureus colony over 18 hours (Fig. 3). S. aureus P_ldh1-sgfp fluorescence began to increase at approximately 12 hours in coculture with WT P. aeruginosa but did not increase in ∆pqsL coculture, confirming prior reports that HQNO increases ldh expression (11). To test if fermentation increases in the absence of invasion, P_ldh1-sgfp expression was quantified in coculture with P. aeruginosa ∆pilA. Notably, we observed a sharp increase in fermentation of densely packed colonies produced by coculture with ∆pilA (shown in Fig. 3A; quantified in Fig. 3B and C). One interpretation of these data is that HQNO concentrates within densely packed colonies, inducing a more dramatic change in S. aureus physiology. Additionally, since ∆pilA cells grow around and against S. aureus, it is possible that the striking increase in S. aureus fermentation is due to more HQNO-producing cells surrounding S. aureus colonies, although precisely quantifying the cell number under these conditions is technically challenging. Alternatively, the increased P_ldh1-sgfp signal may result from increased cell density, independent of HQNO, potentially due to oxygen restriction within the colony. To differentiate these possibilities, fermentation was measured in the presence of a ∆pqsL ∆pilA mutant. As seen with motile ∆pqsL, the double mutant does not induce S. aureus fermentation over time (Fig. 3B), suggesting HQNO mediates this increased fermentation. Importantly, the phenotypes of both ∆pilA and ∆pqsL mutants were genetically complemented by expressing their respective genes in cis (∆pilA) or trans (∆pqsL) under control of an inducible promoter (Fig. S3). These findings show that HQNO likely diffuses into S. aureus colonies independently of P. aeruginosa invasion and plays a crucial role in mediating interspecies interactions by pushing S. aureus toward fermentation.

Fig 3.

Increased S. aureus cell packing enhances HQNO-mediated S. aureus fermentation. S. aureus lactic acid fermentation (P_ldh1-sgfp) was measured in the presence of the indicated P. aeruginosa strains. (A) Representative resonant scanning confocal micrographs of S. aureus fermentation in coculture with P. aeruginosa (WT, ΔpilA, ΔpqsL, or ΔpqsL ΔpilA) t = 18 hours, S. aureus channel only. (B) MFI (fluorescence/colony volume) was quantified over time. (C) MFI at 18 hours. Data represent the mean and standard deviation from three biological replicates with two technical replicates per condition. Each data point represents one technical replicate. Statistical analyses were performed at 18 hours using one-way ANOVA followed by Dunnett’s multiple comparisons test comparing each condition to +WT P. aeruginosa. **P < 0.01 and ****P < 0.0001.

P. aeruginosa TFP motility is necessary for competition against S. aureus in conditions that mimic CF lung secretions

So far, we see a role for TFP motility in competition against S. aureus under conditions that constrain cells to the surface. While useful for high spatial and temporal resolution, this approach does not accurately reflect other attributes of the CF airway infection environment. Thus, we sought to determine whether TFP motility drives interactions when cocultured under conditions that mimic CF lung secretions by using artificial sputum media (ASM) (5), a modified version of SCFM2 (48). ASM captures some of the essential features of the CF environment, like constraints on movement and diffusion, that shaken liquid culture methods do not, and similar recipes have been shown to recapitulate approximately 86% of P. aeruginosa gene expression in human-expectorated CF sputum, outperforming both laboratory media and the acute mouse pneumonia model of infection (49, 50).

S. aureus and P. aeruginosa (WT or ∆pilA) at a 1:1 ratio were grown statically for 22 hours and imaged with resonant scanning confocal microscopy to visualize their spatial organization. The end time point was plated for colony-forming units to assess bacterial viability. In the presence of WT motile P. aeruginosa, S. aureus was suppressed relative to its monoculture condition: very few S. aureus cells could be observed or counted (Fig. 4A and B), compared with approximately 10⁸ CFUs/well recovered in S. aureus monoculture. However, when S. aureus was grown with P. aeruginosa ∆pilA, a 100–10,000-fold increase in S. aureus cells was recovered in comparison to coculture with WT P. aeruginosa (Fig. 4B). Overall, this suggests that TFP motility is necessary for effective competition with S. aureus in ASM. TFP are also necessary for P. aeruginosa biofilm formation and attachment to surfaces (51–53). However, we observed that P. aeruginosa biofilm formation and spatial organization were similar in appearance between WT and ∆pilA in coculture with S. aureus in ASM (Fig. 4A). While the CFUs/well recovered for ∆pilA were significantly lower than WT P. aeruginosa in mono- or coculture with S. aureus (Fig. 4C), the difference is modest (~95% of WT) and thus not expected to account for the increase in S. aureus survival. Next, we tested if the mere presence of TFP has a role in competition or if TFP motility is required. To differentiate between these two outcomes, a hyperpiliated, non-twitching P. aeruginosa mutant (∆pilT) was cocultured with S. aureus. This mutant lacks the main retraction ATPase of the TFP machinery, PilT, and is well documented to ineffectively retract extended pili (54). S. aureus survival in the presence of ∆pilT phenocopied ∆pilA (Fig. 4A and B), suggesting that functional TFP are necessary for competitive interactions with S. aureus in ASM. Collectively, these data demonstrate that under CF-relevant conditions, P. aeruginosa TFP motility aids in interspecies competition.

Fig 4.

P. aeruginosa type IV pili motility is necessary for competition against S. aureus in artificial sputum media. Resonant scanning confocal imaging of S. aureus and P. aeruginosa under static conditions in artificial sputum media, with CFU quantification. (A) Representative images of resonant scanning confocal micrographs of WT S. aureus (pseudocolored orange) in monoculture or in coculture with P. aeruginosa (pseudocolored cyan; WT, ΔpilA, or ΔpilT) t ~ 22 hours. White indicates areas of overlap between S. aureus and P. aeruginosa suggesting colocalization. S. aureus (B) or P. aeruginosa (C) CFU quantification in monoculture or in coculture with P. aeruginosa (WT, ΔpilA, or ΔpilT) (B) or in mono- or coculture with S. aureus (C) at t = 24 hours. The CFUs/well in Y-axes are portrayed as log₁₀ transformed. Three biological replicates with one technical replicate each were analyzed, and the mean and standard deviation are shown. Each data point represents one biological replicate. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. n.s., not significant; *P < 0.05 and ****P < 0.0001.

P. aeruginosa TFP motility is necessary for disruption and competition against pre-formed S. aureus biofilms

Next, we asked if P. aeruginosa TFP motility is necessary for competition against pre-formed (5 hours) S. aureus biofilms in ASM. P. aeruginosa WT, ∆pilA, or ∆pilT were added to S. aureus and allowed to grow for an additional 24 hours before imaging and plating for viability. Remarkably, we found that WT P. aeruginosa invades and disrupts pre-formed S. aureus biofilms, as depicted in Fig. 5A (Z plane) and Movie S1, where a layer of the S. aureus biofilm detaches from the surface and is blanketed by P. aeruginosa cells. Disruption was dependent on P. aeruginosa TFP motility, as WT P. aeruginosa disrupted S. aureus pre-formed biofilms significantly more than ∆pilA or ∆pilT (Fig. S4). Notably, S. aureus and P. aeruginosa ∆pilA remained segregated into monoculture aggregates, while cells were well mixed during coculture with motile P. aeruginosa (Fig. 5A, inset). These observations were consistent with results under agarose pads. We also observed that a higher number of WT P. aeruginosa colonized the surface of the coverslip compared with the ∆pilA or ∆pilT mutants in the presence of S. aureus (Fig. 5A; Fig. S4). These data suggest that TFP motility is necessary for P. aeruginosa cells to invade from the top of S. aureus biofilms and traverse through to access the coverslip, potentially disrupting and detaching the biofilms in the process. This results in a significant reduction in S. aureus viability (10–15-fold) when comparing S. aureus coculture with WT versus ∆pilA or ∆pilT (Fig. 5B). Importantly, no viability differences were observed between P. aeruginosa WT and ∆pilT (Fig. 5C). While ∆pilA shows a significant decrease in viability compared with WT, it is unlikely to have a biological influence on S. aureus growth (Fig. 5C). Altogether, these observations suggest that TFP motility enhances P. aeruginosa competitive fitness, allowing it to disrupt and potentially render S. aureus cells more vulnerable to P. aeruginosa antimicrobials.

Fig 5.

P. aeruginosa type IV pili motility is necessary for disruption and competition against pre-formed S. aureus biofilms. Resonant scanning confocal imaging of S. aureus and P. aeruginosa under static conditions in artificial sputum media, with CFU quantification at late time points. P. aeruginosa was added to S. aureus pre-formed biofilms at 5 hours. (A) Representative images of resonant scanning confocal micrographs of WT S. aureus (pseudocolored orange) in monoculture or in coculture with P. aeruginosa (pseudocolored cyan; WT, ΔpilA, or ΔpilT) t ~ 26 hours. The insets show ~3× zoomed images at 30 µm from the base of the coverslip. White indicates areas of overlap between S. aureus and P. aeruginosa suggesting colocalization. S. aureus (B) or P. aeruginosa (C) CFU quantification in mono- or in coculture with P. aeruginosa (WT, ΔpilA, or ΔpilT) (B) or in mono- or coculture with S. aureus (C) at t ~ 29 hours. The CFUs/well in Y-axes are portrayed as log₁₀ transformed. Five biological replicates with one technical replicate each were analyzed, and the mean and standard deviation are shown. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. n.s., not significant; *P < 0.05, ***P < 0.001, and ****P < 0.0001.

DISCUSSION

Growing data support the hypothesis that spatial organization is crucial in shaping microbial communities and influencing community-based behaviors (4, 5, 39, 55–61). In this study, we found that P. aeruginosa motility plays a vital role in shaping the biogeography in S. aureus cocultures. By influencing spatial aggregation, P. aeruginosa TFP motility ultimately dictates S. aureus physiology and survival.

While P. aeruginosa antimicrobials have been well documented to influence S. aureus growth and survival (11–16), P. aeruginosa motility in interspecies competition has only begun to be explored. We recently reported that P. aeruginosa senses S. aureus-secreted PSM peptides from a distance by the PilJ chemoreceptor (43). Consequently, it employs TFP motility to chemotax toward S. aureus colonies or PSMs alone (42, 44). In addition to chemotaxis, S. aureus PSMs also trigger a “competition sensing” response whereby P. aeruginosa upregulates type VI secretion system and pyoverdine biosynthesis pathways (44). Similarly, P. aeruginosa has been reported to utilize TFP-mediated motility to perform “suicidal chemotaxis” toward antibiotics (62). The upregulation of these common interbacterial competition pathways supports a model where P. aeruginosa senses potential danger in the environment and responds with directional twitching, while simultaneously activating defense mechanisms to combat the “enemy”. Additionally, it has been reported that P. aeruginosa upregulates antimicrobial production upon sensing N-acetylglucosamine alone or shed from Gram-positive bacteria (63).

Our single-cell level temporal analysis also revealed that P. aeruginosa TFP motility is necessary for invading and disrupting S. aureus colonies (Fig. 1). Interestingly, loss of invasion leads P. aeruginosa to grow adjacent to S. aureus colonies, potentially acting as a “wall” to prevent expansion of the S. aureus colonies, which become thicker and denser (Fig. 2). While P. aeruginosa anti-staphylococcal factors did not mediate invasion into S. aureus colonies, they did influence growth as S. aureus formed larger colonies in the absence of P. aeruginosa antimicrobials HQNO, pyoverdine, pyochelin, and staphylolysin (Fig. 2B), as expected based on prior reports (11, 13, 16). However, most studies have been performed with P. aeruginosa cell-free supernatant and not with live P. aeruginosa present. Imaging P. aeruginosa and S. aureus in coculture at the single-cell level has allowed us to visualize the importance of P. aeruginosa motility in their interactions and, therefore, start to build a model whereby TFP motility aids in competition by disrupting S. aureus single cells away from the colony, leaving them exposed and more vulnerable to P. aeruginosa-secreted factors (Fig. 6). Therefore, when P. aeruginosa cannot move, we hypothesize that S. aureus cells remain protected within the colony and resist infiltration of P. aeruginosa antimicrobials. Altogether, these findings provide additional support of how TFP motility can either enhance competition or foster coexistence with S. aureus.

Fig 6.

Model for motility-driven interspecies competition. We propose that P. aeruginosa (Pa) TFP motility-mediated attraction toward, invasion, and disruption of S. aureus (Sa) colonies promote the diffusion of antimicrobials (AM) to maximize interspecies competition. Lack of motility affects S. aureus spatial organization and physiology in a manner that promotes coexistence.

Different S. aureus colony morphology is a consequence of limited invasion by P. aeruginosa ∆pilA, compared with WT, and shows a striking change in physiology by increasing fermentation (P_ldh1-sgfp) (Fig. 3). While we initially hypothesized that S. aureus cells remained protected from P. aeruginosa antimicrobials in the absence of invasion, these data suggest that HQNO can diffuse into S. aureus colonies and alter growth and physiology without P. aeruginosa TFP-mediated invasion. Nevertheless, deletion of antimicrobial production in the ∆pilA mutant background does not significantly improve S. aureus survival, as it does in the WT background. This observation supports the role of TFP-mediated invasion and disruption in allowing these antimicrobials to access S. aureus cells within the colonies for greater impact on its growth (Fig. 2B). Therefore, we hypothesize that without invasion and disruption, antimicrobials with higher molecular weight, such as staphylolysin (20 kDa), are precluded from freely diffusing into S. aureus colonies, while smaller compounds like HQNO (0.259 kDa) can diffuse and concentrate within S. aureus, eliciting physiological changes that could pose greater challenges for the effective treatment of infections.

Importantly, P. aeruginosa TFP motility’s role in mediating interspecies competition and spatial aggregation was highlighted under conditions that mimic the nutritional and viscoelastic properties of CF airways. Of note, P. aeruginosa was capable of detaching preformed S. aureus biofilms and significantly reducing S. aureus viability in a motility-dependent manner when grown in ASM. These results emphasize the importance of motility in this CF-like polymicrobial environment.

The mucoid P. aeruginosa phenotype, a common adaptation that P. aeruginosa exhibits during CF infections, is associated with decreased competition against S. aureus due to the reduced production of anti-staphylococcal factors (46). Additional studies have demonstrated that another common adaptation of P. aeruginosa linked to chronic CF infections is reduced motility (64). Interestingly, P. aeruginosa mucoid and reduced twitching phenotypes have been identified as the best phenotypic predictors of future pulmonary exacerbations in children with CF (64). Our studies revealed that impairing twitching motility hinders P. aeruginosa competitiveness and promotes coexistence with S. aureus under CF-relevant conditions. This may be a contributing explanation for why P. aeruginosa and S. aureus are still found in high numbers in people with CF (18).

Extensive discussion surrounds whether P. aeruginosa and S. aureus encounter each other in CF lungs and if they compete or coexist (4, 39, 46, 55). CF airways are indeed a complex environment with multiple, distinct niches within; therefore, we predict that P. aeruginosa and S. aureus can coexist in some areas, while P. aeruginosa might outcompete S. aureus in others. They may be well mixed in some spaces and spatially segregated in others but still influence each other through the diffusion of secreted factors. As evidenced by both this study and others, it is clear that interspecies competition or coexistence can greatly depend on bacterial and host genotype and phenotype. Our in vitro data show how spatial organization can determine the outcome of microbe-microbe interactions and inform the potential interactions of these bacteria during infection. However, the CF airways are complex and involve other microorganisms and host factors that must be considered. Therefore, future experiments should explore these interactions in vivo and ex vivo to map the community biogeography and further elucidate interspecies dynamics.

Overall, our study reveals how P. aeruginosa TFP motility aids in the disruption of S. aureus colonies and biofilms, which potentiates the effect of P. aeruginosa-secreted anti-staphylococcal factors on S. aureus cells. Ultimately, P. aeruginosa motility plays a crucial and previously unexplored role in determining S. aureus outcome.

MATERIALS AND METHODS

For additional details on all the methods, see the supplemental materials and methods in Text S1.

Bacterial strains and growth conditions

See Text S1 for details on bacterial growth conditions. A list of strains used in this study can be found in Table S1.

Generation of P. aeruginosa deletion mutants

Markerless deletion mutants of genes in PA14 were constructed through homologous recombination as previously described (65).

Time-lapse microscopy

P. aeruginosa and S. aureus were cocultured under agarose pads as previously described (42) and live imaged with resonant scanning confocal microscopy.

S. aureus colony edge height and density measurements

P. aeruginosa and S. aureus were cocultured under agarose pads for ~24 hours and imaged with galvanometer scanning confocal microscopy. S. aureus colony edge height and density were analyzed in Nikon Elements and BiofilmQ (45), respectively.

S. aureus lactic acid fermentation (P_ldh1-sgfp) quantification

P. aeruginosa and S. aureus were cocultured under agarose pads and live imaged with resonant scanning confocal microscopy. S. aureus lactic acid fermentation was quantified by measuring the mean fluorescence intensity (P_ldh1-sgfp) of whole colonies over time.

Artificial sputum media assay

P. aeruginosa and S. aureus were cocultured in ASM for ~24 or 29 hours and imaged with resonant scanning confocal microscopy. ASM was prepared as previously described (5).

S. aureus lysis assay and growth curve with P. aeruginosa supernatant

S. aureus lysis in the presence of P. aeruginosa cell-free supernatant was assessed by modifying a previously published method (66). S. aureus growth with P. aeruginosa supernatant was assessed in a plate reader for 18 hours.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/mbio.00956-24.

Text S1. mbio.00956-24-s0001.pdf.

Supplemental materials and methods.

Figure S1. mbio.00956-24-s0002.tif.

P. aeruginosa cells surround S. aureus colonies at the end time point.

Figure S2. mbio.00956-24-s0003.tif.

Exoproducts from ΔpilA lyse and inhibit S. aureus to the same levels as WT P. aeruginosa factors.

Figure S3. mbio.00956-24-s0004.tif.

Genetic complementation of pqsL or pilA.

Figure S4. mbio.00956-24-s0005.tif.

P. aeruginosa type IV pilus motility is necessary for disrupting pre-formed S. aureus biofilms.

Supplemental Figures. mbio.00956-24-s0006.pdf.

Supplemental figures.

Supplemental Tables. mbio.00956-24-s0007.pdf.

Tables S1 to S3.

Movie S1. mbio.00956-24-s0008.mov.

WT P. aeruginosa (cyan) disrupts S. aureus (orange) biofilms in ASM. Movie is at 29 hours.

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