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.

Keywords: Pseudomonas aeruginosa; Staphylococcus aureus; biofilms; polymicrobial; spatial organization; type IV pili motility.

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}.

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.

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.

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.

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.

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.