Biology subjects

Jean-Pierre, F.

Publications and source records attributed to Jean-Pierre, F..

10 recordsLinked to original sources

bioRxiv2026

Highly effective modulator therapy hinders the emergence of hyperbiofilm variants of Pseudomonas aeruginosa PA14 grown in a cystic fibrosis lung model

Pseudomonas aeruginosa is an opportunistic pathogen that often adopts persistent phenotypes -- such as biofilm formation-- that are associated with chronic infections including those observed in the cystic fibrosis (CF) lung Recently, highly effective modulator therapy (HEMT) such as elexacaftor/tezacaftor/ivacaftor (ETI) has significantly improved the quality of life of people with CF (pwCF). Yet a potential direct impact of ETI on the physiology of P. aeruginosa during growth to a remodeled CF lung environment has remained unexplored. To address this, we conducted an experimental evolution using P. aeruginosa PA14 grown in CF-like conditions in the presence or absence of ETI. We observed a marked reduction in biofilm formation and in the number of small colony variants (SCVs) for P. aeruginosa populations evolved under ETI treatment. Also, sequencing of specific evolved clones exhibiting distinct morphotypes revealed two major observations: (i) P. aeruginosa-evolved communities exposed to ETI retained a wild type-like morphotype and, (ii) P. aeruginosa populations evolved in the absence of ETI adopted a SCV-like phenotype with mutations acquired in the Wsp chemosensory pathway. Furthermore, analysis of evolved populations revealed that ETI treatment likely modulates c-di-GMP pools by driving mutations in an enzyme catalyzing the degradation of this second messenger. Overall, our work suggests that ETI has the potential to hinder the acute to chronic biofilm transition of P. aeruginosa thereby limiting the emergence of variants typically associated with long-term CF lung colonization.

microbiology↗

bioRxiv2026

Polymicrobial extracellular vesicles reduce the innate immune response of human cystic fibrosis bronchial epithelial cells

Chronic antibiotic-resistant cystic fibrosis (CF) lung infections are the leading cause of death in adults with CF. Despite advances in highly effective modulator therapies, microbial communities persist in the CF lung. The pathogenesis of CF airway infections can be exacerbated by pathogens such as Pseudomonas aeruginosa, which communicates with primary human bronchial epithelial cells (pHBEC) by secreting bacterial extracellular vesicles (bEVs) that diffuse through mucus and deliver virulence factors, DNA, and RNA to pHBEC. However, most CF lung infections are polymicrobial in nature, and therefore, the contribution of polymicrobial bEVs remains to be determined. By using a polymicrobial culture model representing a pulmotype detected in [~]34% of lung infections in people with CF (pwCF), comprised of P. aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in synthetic sputum medium under anoxia, we report that each bacterial genus in the polymicrobial community secretes bEVs containing proteins and RNAs predicted to promote the establishment of chronic infection by enhancing virulence, biofilm formation, and upregulating the stress response and pro-inflammatory pathways in pHBEC. This response is most pronounced in CF pHBEC. Elexacaftor/Tezacaftor/Ivacaftor (ETI), a highly effective modulator therapy, does not ameliorate the response or return it to WT levels. Bacterial EVs also inhibited ETI CFTR Cl- currents by CF pHBEC. These studies provide insight into why ETI does not eliminate polymicrobial lung infections and a hyperinflammatory lung environment in pwCF. IMPORTANCECystic fibrosis (CF) is a genetic disease characterized by chronic polymicrobial lung infections that, if untreated, are one of the primary causes of death in CF. Elexacaftor/Tezacaftor/Ivacaftor (ETI) has many positive clinical outcomes, but it does not eliminate chronic polymicrobial lung infections or inflammation. Using a new biologically relevant co-culture model, we have demonstrated that bacteria secrete vesicles (bEVs) that contain proteins and RNAs. We observed that these RNA-loaded bEVs are predicted to promote the pathogenesis of chronic CF lung infections by enhancing bacterial virulence and biofilm formation, as well as upregulating the pro-inflammatory response in lung cells. ETI does not ameliorate the response of lung cells to bEVs. Our research will facilitate the development of more effective approaches to eliminate infection and inflammation in CF and other lung diseases characterized by chronic polymicrobial infections and excessive inflammation.

microbiology↗

bioRxiv2025

Role of Pseudomonas aeruginosa Dnr-regulated denitrification in oxic conditions

Pseudomonas aeruginosa causes acute and chronic infections such as those that occur in the lungs of people with cystic fibrosis (CF). In infection environments, oxygen (O2) concentrations are often low. The transcription factor Anr responds to low O2 by upregulating genes necessary for P. aeruginosa fitness in microoxic and anoxic conditions. Anr regulates dnr, a gene encoding a transcriptional regulator that promotes the expression of genes required for using nitrate as an alternative electron acceptor during denitrification. In CF sputum, transcripts involved in denitrification are highly expressed. While Dnr is necessary for the anoxic growth of P. aeruginosa in CF sputum and artificial sputum media (ASMi), the contribution of denitrification to P. aeruginosa fitness in oxic conditions has not been well described. Here we show that P. aeruginosa requires dnr for fitness in ASMi and the requirement for dnr is abolished when nitrate is excluded from the media. Additionally, we show that P. aeruginosa consumes nitrate in lysogeny broth (LB) under microoxic conditions. Furthermore, strains without a functioning quorum sensing regulator LasR, which leads to elevated Anr activity, consume nitrate in LB even in normoxia. There was no growth advantage for P. aeruginosa when nitrate was present at concentrations from 100 {micro}M to 1600 {micro}M. However, P. aeruginosa consumption of nitrate in oxic conditions created a requirement for Dnr and Dnr-regulated NorCB likely due to the need to detoxify nitric oxide. These studies suggest that Anr- and Dnr-regulated processes may impact P. aeruginosa physiology in many common culture conditions. ImportancePseudomonas aeruginosa is an opportunistic pathogen commonly isolated from low-oxygen environments such as the lungs of people with cystic fibrosis. While the importance of P. aeruginosa energy generation by denitrification is clear in anoxic environments, the effects of denitrification in oxic cultures is not clear. Here, we show that nitrate is consumed even in oxic environments and while it does not appear to stimulate growth, it does impact fitness. Further, we report that two regulators that are best known for their roles in anoxic conditions also contribute to P. aeruginosa fitness in commonly- used laboratory media in presence of oxygen.

microbiology↗

bioRxiv2024

Pseudomonas aeruginosa supports the survival of Prevotella melaninogenica in a cystic fibrosis lung polymicrobial community through metabolic cross-feeding

Cystic fibrosis (CF) is a multi-organ genetic disorder that affects more than 100,000 individuals worldwide. Chronic respiratory infections are among the hallmark complications associated with CF lung disease, and these infections are often due to polymicrobial communities that colonize the airways of persons with CF (pwCF). Such infections are a significant cause of morbidity and mortality, with studies indicating that pwCF who are co-infected with more than one organism experience more frequent pulmonary exacerbations, leading to a faster decline in lung function. Previous work established an in vitro CF-relevant polymicrobial community model composed of P. aeruginosa, S. aureus, S. sanguinis, and P. melaninogenica. P. melaninogenica cannot survive in monoculture in this model. In this study, we leverage this model to investigate the interactions between P. aeruginosa and P. melaninogenica, allowing us to understand the mechanisms by which the two microbes interact to support the growth of P. melaninogenica specifically in the context of the polymicrobial community. We demonstrate a cross-feeding mechanism whereby P. melaninogenica metabolizes mucin into short-chain fatty acids that are in turn utilized by P. aeruginosa and converted into metabolites (succinate, acetate) that are cross-fed to P. melaninogenica, supporting the survival of this anaerobe in the CF lung-relevant model. ImportancePolymicrobial interactions impact disease outcomes in pwCF who suffer from chronic respiratory infections. Previous work established a CF-relevant polymicrobial community model that allows experimental probing of these microbial interactions to achieve a better understanding of the factors that govern the mechanisms by which CF lung microbes influence each other. In this study, we investigate the interaction between P. aeruginosa and P. melaninogenica, which are two highly prevalent and abundant CF lung microbes. We uncover a cross-feeding mechanism that requires the metabolism of mucin by P. melaninogenica to generate short-chain fatty acids that are cross-fed to P. aeruginosa, and into metabolized into metabolites which are then cross-fed back to P. melaninogenica to support the growth of this anaerobe.

microbiology↗

bioRxiv2024

DPR-MEDIATED H2O2 RESISTANCE CONTRIBUTES TO STREPTOCOCCI SURVIVAL IN A CYSTIC FIBROSIS AIRWAY MODEL SYSTEM

The cystic fibrosis (CF) lung environment is conducive to the colonization of bacteria as polymicrobial biofilms, which are associated with poor clinical outcomes for persons with CF (pwCF). Streptococcus spp. is highly prevalent in the CF airway, but its role in the CF lung microbiome is poorly understood. Some studies have shown Streptococcus spp. to be associated with better clinical outcomes for pwCF, while others show that high abundance of Streptococcus spp. is correlated with exacerbations. Our lab previously reported a polymicrobial culture system consisting of four CF-relevant pathogens that can be used to study microbial behavior in a more clinically relevant setting. Here, we use this model system to identify genetic pathways that are important for Streptococcus sanguinis survival in the context of the polymicrobial community. We identified genes related to reactive oxygen species (ROS) as differentially expressed in S. sanguinis monoculture versus growth of this microbe in the mixed community. Genetic studies identified Dpr as important for S. sanguinis survival in the community. We show that Dpr, a DNA binding ferritin-like protein, and PerR, a peroxide-responsive transcriptional regulator of Dpr, are important for protecting S. sanguinis from phenazine-mediated toxicity in co-culture with P. aeruginosa and when exposed to ROS, both of which mimic the CF lung environment. Characterizing such interactions in a clinically relevant model system contributes to our understanding of microbial behavior in the context of polymicrobial biofilm infections. IMPORTANCEStreptococcus spp. is recognized as a highly prevalent pathogen in CF airway infections. However, its role in clinical outcomes for pwCF is poorly understood. Here, we leverage a polymicrobial community system previously developed by our group to model CF airway infections as a tool to investigate a Pseudomonas-Streptococcus interaction involving ROS. We show that protection against ROS is required for S. sanguinis survival in a clinically relevant polymicrobial system. Using this model system to study interspecies interactions contributes to our broader understanding of the complex role of Streptococcus spp. in the CF lung.

microbiology↗

bioRxiv2024

Growing a cystic fibrosis-relevant polymicrobial biofilm to probe community phenotypes.

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat chronic and polymicrobial biofilm-based infections detected in the airways of persons with cystic fibrosis (pwCF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of pwCF, no in vitro models thus far had fully integrated critical cystic fibrosis (CF)-relevant lung features. Therefore, a significant knowledge-gap in our capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections remained. To tackle this challenge, we have built a four-species microbial community model including Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobial (Abx) recalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of this in vitro model resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections. SUMMARYIn this protocol, we describe a cystic fibrosis (CF)-lung relevant four-species polymicrobial biofilm model that can be used to explore the impact of bacterial interspecies interactions.

microbiology↗

bioRxiv2023

Transcriptional Profiling and Genetic Analysis of a Cystic Fibrosis Airway-Relevant Model Shows Asymmetric Responses to Growth in a Polymicrobial Community

Bacterial infections in the lungs of persons with cystic fibrosis are typically composed of multispecies biofilm-like communities, which modulate clinically relevant phenotypes that cannot be explained in the context of a single species culture. Most analyses to-date provide a picture of the transcriptional responses of individual pathogens, however, there is relatively little data describing the transcriptional landscape of clinically-relevant multispecies communities. Harnessing a previously described cystic fibrosis-relevant, polymicrobial community model consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis and Prevotella melaninogenica, we performed an RNA-Seq analysis to elucidate the transcriptional profiles of the community grown in artificial sputum medium (ASM) as compared to growth in monoculture, without mucin, and in fresh medium supplemented with tobramycin. We provide evidence that, although the transcriptional profile of P. aeruginosa is community agnostic, the transcriptomes of S. aureus and S. sanguinis are community aware. Furthermore, P. aeruginosa and P. melaninogenica are transcriptionally sensitive to the presence of mucin in ASM, whereas S. aureus and S. sanguinis largely do not alter their transcriptional profiles in the presence of mucin when grown in a community. Only P. aeruginosa shows a robust response to tobramycin. Genetic studies of mutants altered in community-specific growth provide complementary data regarding how these microbes adapt to a community context. ImportancePolymicrobial infections constitute the majority of infections in the cystic fibrosis (CF) airway, but their study has largely been neglected in a laboratory setting. Our lab previously reported a polymicrobial community that can explain clinical outcomes in the lungs of persons with CF. Here we obtain transcriptional profiles of the community versus monocultures to provide transcriptional information about how this model community responds to CF-related growth conditions and perturbations. Genetic studies provide complementary functional outputs to assess how the microbes adapt to life in a community.

microbiology↗

bioRxiv2022

Community composition shapes microbial-specific phenotypes in a cystic fibrosis polymicrobial model system

Interspecies interactions can drive the emergence of unexpected microbial phenotypes that are not observed when studying monocultures. The cystic fibrosis (CF) lung consists of a complex environment where particular microbes, living as polymicrobial biofilm-like communities, are associated with negative clinical outcomes for persons with CF (pwCF). However, the current lack of in vitro models integrating the microbial diversity observed in the CF airway hampers our understanding of why polymicrobial communities are recalcitrant to therapy in this disease. Here, integrating computational approaches informed by clinical data, we built a mixed community of clinical relevance to the CF lung composed of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis and Prevotella melaninogenica. We developed and validated this model biofilm community with multiple isolates of these four genera. When challenged with tobramycin, a front-line antimicrobial used to treat pwCF, the microorganisms in the polymicrobial community show altered sensitivity to this antibiotic compared to monospecies biofilms. We observed that wild-type P. aeruginosa is sensitized to tobramycin in a mixed community versus monoculture, and this observation holds across a range of community relative abundances. We also report that LasR loss-of-function, a variant frequently detected in the CF airway, induces tolerance of P. aeruginosa to tobramycin specifically in the mixed community. The molecular basis of this community-specific recalcitrance to tobramycin for the LasR mutant variant is the increased production of redox-active phenazines. Our data support the importance of studying clinically-relevant model polymicrobial biofilms to understand community-specific traits relevant to infections. SIGNIFICANCE STATEMENTThe CF lung is colonized by biofilm-like microbial communities that exhibit both resistance and tolerance (collectively called "recalcitrance") to antimicrobials used in the clinic. Here, we leveraged clinical data from pwCF to inform our understanding of communities exhibiting recalcitrance. We developed and validated an in vitro model that revealed novel, community-specific phenotypes relevant to the clinic. We used this model to explore the underlying mechanism associated with a community-specific emergent behavior. We posit that in vitro models of polymicrobial communities may help in developing new antimicrobial strategies to improve patient outcomes, and that the approach used here can be applied to other polymicrobial models.

microbiology↗

bioRxiv2020

An experimentally evolved variant of RsmA confirms its central role in the control of Pseudomonas aeruginosa social motility

Bacteria can colonize a variety of different environments by modulating their gene regulation using two-component systems. The versatile opportunistic pathogen Pseudomonas aeruginosa has been studied for its capacity to adapt to a broad range of environmental conditions. The Gac/Rsm pathway is composed of the sensor kinase GacS, that detects environmental cues, and the response regulator GacA, that modulates the expression of a specific genes. This system, through the sRNA repressors RsmY and RsmZ, negatively controls the activity of the protein RsmA, which is centrally involved in the transition from chronic to acute infections by post-transcriptionally regulating several virulence functions. RsmA positively regulates swarming motility, a social surface behaviour. Through a poorly defined mechanism, RsmA is also indirectly regulated by HptB, and a {Delta}hptB mutant exhibits a severe swarming defect. Since a {Delta}hptB mutant retains all the known functions required for that type of motility, we used an experimental evolution approach to identify elements responsible for its swarming defect. After a few passages under swarming conditions, the defect of the {Delta}hptB mutant was rescued by the emergence of spontaneous single nucleotide substitutions in the gacA and rsmA genes. Since GacA indirectly represses RsmA activity, it was coherent that an inactivating mutation in gacA would compensate the {Delta}hptB swarming defect. However, the effect of the mutation in rsmA was unexpected since RsmA promotes swarming; indeed, using expression reporters, we found that the mutation that does not abolish its activity. Instead, using electrophoretic mobility shift assays and molecular simulations, we show that this variant of RsmA is actually less amenable to titration by its cognate repressor RsmY, supporting the other phenotypes observed for this mutant. These results confirm the central role of RsmA as a regulator of swarming motility in P. aeruginosa and identify residues crucial for RsmA function in social motility. Author summaryBacteria need to readily adapt to their environment. Two-component systems (TCS) allow such adaption by triggering bacterial regulation changes through the detection of environmental cues. The opportunistic pathogen Pseudomonas aeruginosa possesses more than 60 TCS in its genome. The Gac/Rsm is a TCS extensively studied for its implication in virulence regulation. This system regulates the transition between chronic and acute bacterial infection behaviours. To acquire a better understanding of this regulation, we performed a directed experimental evolution on a swarming-deficient mutant in a poorly understood regulatory component of the Gac/Rsm pathway. We observed single nucleotide substitutions that allowed restoration of a swarming phenotype similar to the wild-type behaviour. More specifically, mutations were found in the gacA and rsmA genes. Interestingly, the observed mutation in rsmA does not result in loss of function of the protein but rather alters its susceptibility to repression by its cognate interfering sRNA. Since modification in the RNA sequence of RsmA results in the rescue of swarming motility, we confirm the central role of this posttranscriptional repressor in this social lifestyle.

microbiology↗

bioRxiv2020

Pseudomonas aeruginosa enhances the efficacy of norfloxacin against Staphylococcus aureus biofilms

The thick mucus within the airways of individuals with cystic fibrosis (CF) promotes frequent respiratory infections that are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent pathogens that cause CF pulmonary infections, and both have been associated with worse lung function. Furthermore, the ability of P. aeruginosa and S. aureus to form biofilms promotes the establishment of chronic infections that are often difficult to eradicate using antimicrobial agents. In this study, we found that multiple LasR-regulated exoproducts of P. aeruginosa, including HQNO, siderophores, phenazines, and rhamnolipids, likely contribute to the ability of P. aeruginosa to shift S. aureus norfloxacin susceptibility profiles. Here, we observe that exposure to P. aeruginosa exoproducts leads to an increase in intracellular norfloxacin accumulation by S. aureus. We previously showed that P. aeruginosa supernatant dissipates S. aureus membrane potential, and furthermore, depletion of the S. aureus proton-motive force recapitulates the effect of P. aeruginosa supernatant on shifting norfloxacin sensitivity profiles of biofilm-grown S. aureus. From these results, we hypothesize that exposure to P. aeruginosa exoproducts leads to increased uptake of the drug and/or an impaired ability of S. aureus to efflux norfloxacin. Our results illustrate that microbially-derived products can greatly alter the ability of antimicrobial agents to kill bacterial biofilms. ImportancePseudomonas aeruginosa and Staphylococcus aureus are frequently co-isolated from multiple infection sites, including the lungs of individuals with cystic fibrosis (CF) and non-healing diabetic foot ulcers. Co-infection with P. aeruginosa and S. aureus has been shown to produce worse outcomes compared to infection with one organism alone. Furthermore, the ability of these pathogens to form biofilms enables them to cause persistent infection and withstand antimicrobial therapy. In this study, we found that P. aeruginosa-secreted products dramatically increase the ability of the antibiotic norfloxacin to kill S. aureus biofilms. Understanding how interspecies interactions alter the antibiotic susceptibility of bacterial biofilms may inform treatment decisions and inspire the development of new therapeutic strategies.

microbiology↗