RESUMO
The genome of Pseudomonas aeruginosa encodes three type VI secretion systems, each comprising a dozen distinct proteins, which deliver toxins upon T6SS sheath contraction. The least conserved T6SS component, TssA, has variations in size which influence domain organisation and structure. Here we show that the TssA Nt1 domain interacts directly with the sheath in a specific manner, while the C-terminus is essential for oligomerisation. We built chimeric TssA proteins by swapping C-termini and showed that these can be functional even when made of domains from different TssA sub-groups. Functional specificity requires the Nt1 domain, while the origin of the C-terminal domain is more permissive for T6SS function. We identify two regions in short TssA proteins, loop and hairpin, that contribute to sheath binding. We propose a docking mechanism of TssA proteins with the sheath, and a model for how sheath assembly is coordinated by TssA proteins from this position.
Assuntos
Proteínas de Bactérias , Domínios Proteicos , Pseudomonas aeruginosa , Sistemas de Secreção Tipo VI , Sistemas de Secreção Tipo VI/metabolismo , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/química , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
IMPORTANCE: Although E. faecalis is a common wound pathogen, its pathogenic mechanisms during wound infection are unexplored. Here, combining a mouse wound infection model with in vivo transposon and RNA sequencing approaches, we identified the E. faecalis purine biosynthetic pathway and galactose/mannose MptABCD phosphotransferase system as essential for E. faecalis acute replication and persistence during wound infection, respectively. The essentiality of purine biosynthesis and the MptABCD PTS is driven by the consumption of purine metabolites by E. faecalis during acute replication and changing carbohydrate availability during the course of wound infection. Overall, our findings reveal the importance of the wound microenvironment in E. faecalis wound pathogenesis and how these metabolic pathways can be targeted to better control wound infections.
Assuntos
Infecções Urinárias , Infecção dos Ferimentos , Animais , Camundongos , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Carboidratos , PurinasRESUMO
Pseudomonas aeruginosa is widely attributed as the leading cause of hospital-acquired infections. Due to intrinsic antibiotic resistance mechanisms and the ability to form biofilms, P. aeruginosa infections are challenging to treat. P. aeruginosa employs multiple virulence mechanisms to establish infections, many of which are controlled by the global virulence regulator Vfr. An attractive strategy to combat P. aeruginosa infections is thus the use of anti-virulence compounds. Here, we report the discovery that FDA-approved drug auranofin attenuates virulence pathways in P. aeruginosa, including quorum sensing (QS) and Type IV pili (TFP). We show that auranofin acts via multiple targets, one of which being Vfr. Consistent with inhibition of QS and TFP expression, we show that auranofin attenuates biofilm maturation, and when used in combination with colistin, displays strong synergy in eradicating P. aeruginosa biofilms. Auranofin may have immediate applications as an anti-virulence drug against P. aeruginosa infections.
Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/metabolismo , Auranofina/farmacologia , Auranofina/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Fatores de Virulência/metabolismo , Fatores de Virulência/farmacologia , Fatores de Virulência/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Biofilmes , Percepção de Quorum , Proteínas de Bactérias/farmacologiaRESUMO
Enterococcus faecalis is often coisolated with Pseudomonas aeruginosa in polymicrobial biofilm-associated infections of wounds and the urinary tract. As a defense strategy, the host innately restricts iron availability at infection sites. Despite their coprevalence, the polymicrobial interactions of these two species in biofilms and under iron-restricted conditions remain unexplored. Here, we show that E. faecalis inhibits P. aeruginosa growth within biofilms when iron is restricted. E. faecalis lactate dehydrogenase (ldh1) gives rise to l-lactate production during fermentative growth. We find that an E. faecalis ldh1 mutant fails to inhibit P. aeruginosa growth. Additionally, we demonstrate that ldh1 expression is induced under iron-restricted conditions, resulting in increased lactic acid exported and, consequently, a reduction in local environmental pH. Together, our results suggest that E. faecalis synergistically inhibits P. aeruginosa growth by decreasing environmental pH and l-lactate-mediated iron chelation. Overall, this study emphasizes the importance of the microenvironment in polymicrobial interactions and how manipulating the microenvironment can impact the growth trajectory of bacterial communities. IMPORTANCE Many infections are polymicrobial and biofilm-associated in nature. Iron is essential for many metabolic processes and plays an important role in controlling infections, where the host restricts iron as a defense mechanism against invading pathogens. However, polymicrobial interactions between pathogens are underexplored under iron-restricted conditions. Here, we explore the polymicrobial interactions between commonly coisolated E. faecalis and P. aeruginosa within biofilms. We find that E. faecalis modulates the microenvironment by exporting lactic acid which further chelates already limited iron and also lowers the environmental pH to antagonize P. aeruginosa growth under iron-restricted conditions. Our findings provide insights into polymicrobial interactions between bacteria and how manipulating the microenvironment can be taken advantage of to better control infections.
Assuntos
Enterococcus faecalis , Pseudomonas aeruginosa , Biofilmes , Enterococcus faecalis/metabolismo , Ferro/metabolismo , Ácido Láctico/metabolismo , Pseudomonas aeruginosa/metabolismoRESUMO
Enterococcus faecalis forms single and mixed-species biofilms on both tissue and medical devices in the host, often under exposure to fluid flow, giving rise to infections that are recalcitrant to treatment. The factors that drive enterococcal biofilm formation in the host, however, remain unclear. Recent reports in other pathogens show how surface sensing by bacteria can trigger the transition from planktonic to sessile lifestyle. Fluid flow can enhance initial adhesion, but also influence quorum sensing. Biofilm-specific factors, as well as biofilm size and extracellular polymeric substances, can compromise opsonization and phagocytosis. Bacterial interspecies synergy can create favorable conditions in the host for biofilm formation. Through these concepts, we define the knowledge gaps in understanding host-associated E. faecalis biofilm formation and propose a roadmap for future investigations.