Biology subjects

O'Toole, G.

Publications and source records attributed to O'Toole, G..

At least 19 recordsLinked to original sources

bioRxiv2025

In Situ Photoactivated Hydrogel Adhesive Dressings for Post Colon Polypectomies (PolypCures)

Colon polypectomy is a widely performed endoscopic procedure that reduces the incidence of colorectal cancer but leaves exposed colonic wounds susceptible to bleeding, perforation, and infection. The current standard of wound care, mechanical clips, is limited by technical complexity, high cost, and poor efficacy for large (> 2 cm in diameter) or difficult-to-access lesions. Here, we present PolypCure, an in situ photoactivated hydrogel adhesive dressing delivered via a single catheter, through either oozing or spraying methods, to achieve rapid, spatiotemporally controlled sealing and hemostasis (within 2 min) of colon lesions using the white light source integrated into standard endoscopic instruments. The hydrogel, composed of norbornene- and thiol-functionalized polyethylene glycol, carboxymethyl cellulose, and Eosin Y, exhibits mechanical properties comparable to colon tissue (G [~] 6 kPa), strong shear strength (> 15 kPa), low swelling (< 200%), and small pore size ([~] 20 {micro}m), ensuring long-term stability (up to 30 days) and protection against bacterial infiltration. The formulation also demonstrates biocompatibility and hemocompatibility. In an in vivo pig colon endoscopic mucosal resection (EMR) model, PolypCure fully adheres to large lesions for at least 3 days and promotes early-stage wound healing. Overall, PolypCure is a promising solution to wound management post-polypectomy.

bioengineering↗

bioRxiv2025

The Regional Landscape of the Human Colon Culturome in Health and Cystic Fibrosis

Cystic fibrosis (CF) alters gut physiology, yet its impact on microbial communities across colonic regions (ascending, transverse, descending colon) and microhabitats (lumen, mucosa) remains incompletely understood. Here, we applied culturomics to characterize gut microbiota in 32 individuals (22 nonCF, 10 CF). Persons with CF (pwCF) exhibited significantly higher viable bacterial loads than nonCF individuals, particularly in mucosal samples. Anaerobes predominated overall, with relative enrichment of aerobes in the mucosa of pwCF. Alpha diversity was reduced in mucosal samples and aerobic cultures for pwCF, whereas beta diversity was influenced by all the tested variables except the colonic region. Phylum-level analyses revealed enrichment of Proteobacteria and depletion of Actinobacteria, Bacteroidota, and Firmicutes in samples from pwCF, consistent with stool analysis. Random forest models identified selected oral-associated microbes as key predictive taxa and accurately classified polyp status with very high accuracy. Whole-genome sequencing of Bacteroides fragilis (n=21) and Escherichia coli (n=15) isolates, representing a subset of 109 gut bacterial genomes sequenced from this cohort, revealed minimal genomic variation across colonic regions and sample types, indicating intra-individual strain stability. The understandings from this pilot culturome study may help in developing targeted microbial therapeutic approaches to address the gut dysbiosis of CF. ImportanceThis pilot study represents the first culturome analysis of the cystic fibrosis colon. Our preliminary findings demonstrate that CF-associated gut dysbiosis is spatially specific, with mucosal bacterial communities showing pronounced alterations while luminal communities remain unchanged. This spatial specificity suggests the mucosal microenvironment as a potential therapeutic target and indicates that interventions focused solely on luminal bacteria may be insufficient. The promising predictive accuracy of culturome-based machine learning models in this small cohort suggests these viable bacterial signatures could serve as biomarkers for CF management pending larger validation studies. Additionally, our initial observations of complex CFTR modulator effects on gut microbial communities provide insights for future studies optimizing combination therapies.

microbiology↗

bioRxiv2025

A genotoxin associated with colorectal cancer linked to gut dysbiosis in children with cystic fibrosis

Cystic fibrosis (CF) substantially alters the gastrointestinal microbiome from an early age, leading to significant changes in microbial composition and functionality. This study explores the physiological and microbiological factors contributing to dysbiosis in children with cystic fibrosis (cwCF), characterized by an increase in potentially pathogenic Escherichia coli and a decrease in beneficial anaerobes such as Bacteroides. In this study, we employed an in vitro medium representative of the nutritional environment of the CF colon to test the role of factors including mucin, fat, bile, pH, antibiotics and features associated with inflammation (e.g., nitrate, sulfate, formate, reactive oxygen species) on growth of clinical isolates of E. coli and Bacteroides spp. We further examined interactions between these two microbes under CF-like conditions to understand modulators of microbial competition, and identified glycerol, a surrogate of increased fat, as a significant driver of altered microbial competition. Finally, we investigated genetic determinants influencing these microbial interactions, with the focus on glycerol metabolism, by performing a transposon mutagenesis screen in E. coli. Results of this screen pointed to the role of colibactin production in mediating this microbial competition; colibactin is a DNA-damaging genotoxin associated with the increased risk of colorectal cancer (CRC) in CF populations. This work enhances our understanding of mechanisms of microbial competition in the CF gut, while potentially enhancing our understanding of colorectal cancer risk in persons with CF through the identification of early-life microbial biomarkers. Significance StatementThe risk of CRC development in CF populations is significantly increased. This study examines the interplay of altered intestinal physiology in the microbial dysbiosis common in the CF gut, implicating the high fat environment in a competition-mediated depletion of immune-modulating Bacteroides. This work identifies candidate features of the young CF intestine and gut microbiome that may contribute to advanced development of CRC in these populations, informing potential therapeutic approaches.

microbiology↗

bioRxiv2025

Sex and regional effects of Bacteroides in the gut

Bacteroides spp. is a key immune-programming microbe in healthy individuals - these bacteria have been shown to be reduced in abundance across a variety of disease states. Our study investigated the systemic and region-specific responses to Bacteroides colonization in the gut, including sex-related differences, in mice. Utilizing C57BL/6 mice, we administered Bacteroides to conventional, antibiotic-treated mice, then assessed this microbes influence on the gut microbiota composition and inflammatory responses following an airway lipopolysaccharide challenge to assess effects on the gut-lung axis. We found that Bacteroides successfully colonizes the intestinal tract of antibiotic-treated mice, particularly the colon lumen of the large intestine as evidenced by 16S rRNA amplicon gene sequencing and culturing. Differential gene expression analysis using NanoString technology revealed significant immune response variations across the gut regions, with notable differences in adaptive immune response genes. A striking sex-dependent outcome was noted in the regulation of atg12 in the cecum, potentially enhancing autophagic function, particularly in female mice. Additionally, Bacteroides intestinal colonization was associated with altered expression of macrophage markers such as cd163, cd84, and ms4a4a, which may reflect shifts in the macrophage profile within the cecum. These findings pave the way for novel therapeutic approaches that leverage microbial impacts on gut and systemic health, offering a deeper understanding of Bacteroides role in human health and disease. Our study highlights the necessity for further research to elucidate the intricate relationships between gut microbiota, host immunity, biological sex and their interplay. ImportanceThis research marks an investigation into how specific microbiota, like Bacteroides, regulate host responses across different gut regions to influence systemic health. By dissecting the impact of Bacteroides across multiple regions of the intestinal tract, this study offers new insights into the localized and whole-body effects of this important immune-programming microbe. Such an understanding is crucial as it helps in unraveling the complex interplay between gut microbes and the hosts immune system. This research helps bridge the gap between local intestinal ecology and overall systemic health, addresses important questions relevant to the gut-lung axis, and helps pave the way for innovative therapies.

microbiology↗

bioRxiv2025

Profiling Bile Acids in the Stools of Humans and Animal Models of Cystic Fibrosis

Cystic fibrosis (CF) is associated with dysbiosis of the gut microbiome, alterations in intestinal mucus production, aberrant bile acid (BA) metabolism, fat malabsorption, and chronic inflammation. As little is known about BAs in CF, we performed both comprehensive and targeted BA profiling in stool of children with or without CF. Our results reveal that select BA species and metabolites are significantly different between children with CF (cwCF) and healthy controls. There is also a trend towards higher primary cBA and total BA levels for cwCF. Matched bacterial metagenomic analyses showed no change in alpha-diversity between groups in our small cohort, at odds with previous studies, whereas changes in relative abundance of Bacteroides (lower) and E. coli (increased) species is consistent with prior reports. A robust trend was noted toward reduced abundance of bsh gene families (Wilcox test, p = 0.052), a key rate-limiting enzyme required for bacterial synthesis of secondary BAs, in cwCF. Modest changes in both BAs and microbial BA metabolism-related gene abundances may be attributable to small sample sizes, but also suggest likely combination defects in both host and microbial BA metabolic pathways in cwCF. Importantly, although fecal BA profiles from both ferret and mouse CF models showed significant differences from human BA profiles, only the ferret model reproduced significant differences between CF and nonCF animals, highlighting ferrets as a potentially more appropriate model for studying BA in stool in the context of CF. Together, these results provide new insights into CF-related BA dysmetabolism in cwCF, and highlight limitations of CF animal models for BA functional studies. IMPORTANCEChanges in the abundance and/or composition of intestinal bile acids (BAs) may contribute to dysbiosis and altered gastrointestinal physiology in CF. Here, we report shifts in select fecal BA classes and species for children with CF (cwCF). Matched metagenomic analysis suggest possible defects in both host intestinal BA absorption and gut microbial BA metabolism. Additional analyses of mouse and ferret CF stool for BA composition suggest great care must be taken when interpreting BA functional studies using these animal models. Together, this work lays technical and conceptual foundations for interrogating BA-microbe interactions in cwCF.

microbiology↗

bioRxiv2024

In vitro biofilm formation only partially predicts beneficial Pseudomonas fluorescens protection against rhizosphere pathogens

Plant roots form associations with both beneficial and pathogenic soil microorganisms. While members of the rhizosphere microbiome can protect against pathogens, the mechanisms are poorly understood. We hypothesized that the ability to form a robust biofilm on the root surface is necessary for the exclusion of pathogens; however, it is not known if the same biofilm formation components required in vitro are necessary in vivo. Pseudomonas fluorescens WCS365 is a beneficial strain that is phylogenetically closely related to an opportunistic pathogen P. fluorescens N2C3 and confers robust protection against P. fluorescens N2C3 in the rhizosphere. We used this plant-mutualist-pathogen model to screen collections of P. fluorescens WCS365 increased attachment mutants (iam) and surface attachment defective (sad) transposon insertion mutants that form increased or decreased levels of biofilm on an abiotic surface, respectively. We found that while the P. fluorescens WCS365 mutants had altered biofilm formation in vitro, only a subset of these mutants, including those involved in large adhesion protein (Lap) biosynthesis, flagellin biosynthesis and O-antigen biosynthesis, lost protection against P. fluorescens N2C3. We found that the inability of P. fluorescens WCS365 mutants to grow in planta, and the inability to suppress pathogen growth, both partially contributed to loss of plant protection. We did not find a correlation between the extent of biofilm formed in vitro and pathogen protection in planta indicating that biofilm formation on abiotic surfaces may not fully predict pathogen exclusion in planta. Collectively, our work provides insights into mechanisms of biofilm formation and host colonization that shape the outcomes of host-microbe-pathogen interactions.

microbiology↗

bioRxiv2024

Genetic Analysis of Flagellar-Mediated Surface Sensing by Pseudomonas aeruginosa PA14

Surface sensing is a key aspect of the early stage of biofilm formation. For P. aeruginosa, the type IV pili (TFP), the TFP alignment complex and PilY1 were shown to play a key role in c-di-GMP signaling upon surface contact. The role of the flagellar machinery in surface sensing is less well understood in P. aeruginosa. Here we show, consistent with findings from other groups, that a mutation in the gene encoding the flagellar hook protein ({Delta}flgK) or flagellin ({Delta}fliC) results in a strain that overproduces the Pel exopolysaccharide (EPS) with a concomitant increase in c-di-GMP levels. We use a candidate gene approach and genetic screens, combined with phenotypic assays, to identify key roles for the MotAB and MotCD stators and the FliG protein, a component of the flagellar switch complex, in stimulating the surface-dependent, increased c-di-GMP level noted for these flagellar mutants. These findings are consistent with previous studies showing a role for the stators in surface sensing. We also show that mutations in the genes coding for the diguanylate cyclases SadC and RoeA as well as SadB, a protein involved in early surface colonization, abrogate the increased c-d-GMP-related phenotypes of the {Delta}flgK mutant. Together, these data indicate that bacteria monitor the status of flagellar synthesis and/or function during surface sensing as a means to trigger the biofilm program. ImportanceUnderstanding how the flagellum contributes to surface sensing by P. aeruginosa is key to elucidating the mechanisms of biofilm initiation by this important opportunistic pathogen. Here we take advantage of the observation that mutations in the flagellar hook protein or flagellin enhance surface sensing. We exploit this phenotype to identify key players in this signaling pathway, a critical first step in understanding the mechanistic basis of flagellar-mediated surface sensing. Our findings establish a framework for the future study of flagellar-based surface sensing.

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

An energy coupling factor transporter of Streptococcus sanguinis impacts antibiotic susceptibility as well as metal and membrane homeostasis

Streptococcus sanguinis is a prevalent member of human microbiome capable of acting as a causative agent of oral and respiratory infections. S. sanguinis competitive success within the infection niche is dependent on acquisition of metal ions and vitamins. Among the systems that bacteria use for micronutrient uptake is the energy coupling factor (ECF) transporter system EcfAAT. Here we describe physiological changes arising from EcfAAT transporter disruption. We found that EcfAAT contributes to S. sanguinis antibiotic sensitivity as well as metal and membrane homeostasis. Specifically, our work found that disruption of EcfAAT results in increased polymyxin susceptibility. We performed assessment of cell-associated metal content and found depletion of iron, magnesium, and manganese. Furthermore, membrane composition analysis revealed significant enrichment in unsaturated fatty acid species resulting in increased membrane fluidity. Our results demonstrate how disruption of a single EcfAAT transporter can have broad consequences on bacterial cell homeostasis. ECF transporters are of interest within the context of infection biology in bacterial species other than streptococci, hence work described here will further the understanding of how micronutrient uptake systems contribute to bacterial pathogenesis. ImportanceProficiency in micronutrient uptake is key for pathogen success in bacteria-bacteria and bacteria-host interactions within the infection context. Micronutrient uptake mechanisms are of interest in furthering the understanding of bacterial physiology within infection niche and as targets for design of antimicrobials. Here we describe how a deletion of a nutrient uptake transporter in S. sanguinis alters bacterial sensitivity to antibiotics. We also show that a defect in this candidate nutrient uptake system has consequences on the intracellular metal content, and also results in changes in membrane fatty acid composition and fluidity. This study demonstrates how disruption of a single nutrient uptake system disrupts bacterial physiology resulting in increased antibiotic sensitivity.

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↗

bioRxiv2023

Reconstitution of a Biofilm Adhesin System from a Sulfate-Reducing Bacterium in Pseudomonas fluorescens

Biofilms of the sulfate reducing bacterium (SRB) Desulfovibrio vulgaris Hildenborough (DvH) can facilitate metal corrosion in various industrial and environmental settings leading to substantial economic losses; however, the mechanisms of biofilm formation by DvH are not yet well-understood. Evidence suggests that a large adhesin, DvhA, may be contributing to biofilm formation in DvH. The dvhA gene and its neighbors encode proteins that resemble the Lap system, which regulates biofilm formation by Pseudomonas fluorescens, including a LapG-like protease DvhG and effector protein DvhD, which has key differences from the previously described LapD. By expressing the Lap-like adhesion components of DvH in P. fluorescens, our data support the model that the N-terminal fragment of the large adhesin DvhA serves as an adhesin "retention module" and is the target of the DvhG/DvhD regulatory module, thereby controlling cell-surface location of the adhesin. By heterologously expressing the DvhG/DvhD-like proteins in a P. fluorescens background lacking native regulation ({Delta}lapG{Delta}lapD) we also show that cell surface regulation of the adhesin is dependent upon the intracellular levels of c-di-GMP. This study provides insight into the key players responsible for biofilm formation by DvH, thereby expanding our understanding of Lap-like systems. SignificanceCorrosion leads to [~]2.5 trillion US dollars in economic losses, 20% of which are estimated to be microbially induced. Biofilms of sulfate reducing bacteria (SRB), especially the genus Desulfovibrio, are important members of the corrosion consortium and accelerate deterioration of metals. Understanding how biofilms are formed by SRB can provide important clues to mitigate this challenge. In this study, we used genetic and biochemical tools to investigate the mechanism of biofilm formation by Desulfovibrio vulgaris Hildenborough. Our study reveals critical genes responsible for regulating the secretion of a large adhesin known to be required for biofilm formation.

microbiology↗

bioRxiv2023

An In Vitro Medium for Modeling Gut Dysbiosis Associated with Cystic Fibrosis

The gut physiology of pediatric and adult persons with cystic fibrosis (pwCF) is altered relative to healthy persons. The CF gut is characterized, in part, as having excess mucus, increased fat content, acidic pH, increased inflammation, increased antibiotic perturbation and the potential for increased oxygen availability. These physiological differences shift nutritional availability and the local environment for intestinal microbes, thus likely driving significant changes in microbial metabolism, colonization and competition with other microbes. The impact of any specific change in this physiological landscape is difficult to parse using human or animal studies. Thus, we have developed a novel culture medium representative of the CF gut environment, inclusive of all the aforementioned features. This medium, called CF-MiPro, maintains CF gut microbiome communities, while significantly shifting non-CF gut microbiome communities toward a CF-like microbial profile, characterized by low Bacteroidetes and high Proteobacteria abundance. This medium is able to maintain this culture composition for up to 5 days of passage. Additionally, microbial communities passaged in CF-MiPro produce significantly less immunomodulatory short chain fatty acids (SCFA), including propionate and butyrate, than communities passaged in MiPro, a culture medium representative of healthy gut physiology, confirming not only a shift in microbial composition but altered community function. Our results support the potential for this in vitro culture medium as a new tool for the study of gut dysbiosis in CF. ImportanceCystic fibrosis is an autosomal recessive disease that disrupts ion transport at mucosal surfaces, leading to mucus accumulation and altered physiology of both the lungs and the intestines, among other organs, with the resulting altered environment contributing to an imbalance of microbial communities. Culture media representative of the CF airway have been developed and validated; however, no such medium exists for modeling the CF intestine. Here, we develop and validate a first-generation culture medium inclusive of features that are altered in the CF colon. Our findings suggest this novel medium, called CF-MiPro, as a maintenance medium for CF gut microbiome samples and a flexible tool for studying key drivers of CF-associated gut dysbiosis.

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↗

bioRxiv2023

The Regulator FleQ Post-Transcriptionally Regulates the Production of RTX Adhesins by Pseudomonas fluorescens

Biofilm formation by the Gram-negative gammaproteobacterium Pseudomonas fluorescens relies on the production of the repeat-in-toxin (RTX) adhesins LapA and MapA in the cytoplasm, secretion of these adhesins through their respective type 1 secretion systems, and retention at the cell surface. Published work has shown that retention of the adhesins occurs via a post-translational mechanism involving the cyclic-di-GMP receptor LapD and the protease LapG. However, little is known about the underlying mechanisms that regulate the production of these adhesins. Here, we demonstrate that the master regulator FleQ modulates biofilm formation by post-transcriptionally regulating the production of LapA and MapA. We find that a {Delta}fleQ mutant has a biofilm formation defect compared to the WT strain, which is attributed in part to a decrease in LapA and MapA production, despite the {Delta}fleQ mutant having increased levels of lapA and mapA transcripts compared to the WT strain. Through transposon mutagenesis and subsequent genetic analysis, we found that over-stimulation of the Gac/Rsm pathway partially rescues biofilm formation in the {Delta}fleQ mutant background. Collectively, these findings provide evidence that FleQ regulates biofilm formation by post-transcriptionally regulating the production of LapA and MapA, and that activation of the Gac/Rsm pathway can enhance biofilm formation by P. fluorescens. ImportanceBiofilm formation is a highly coordinated process that bacteria undergo to colonize a variety of surfaces. For Pseudomonas fluorescens, biofilm formation requires the production and localization of RTX adhesins to the cell surface. To date, little is known about the underlying mechanisms that regulate biofilm formation by P. fluorescens. Here, we identify FleQ as a key regulator of biofilm formation that modulates the production of LapA and MapA through a post-transcriptional mechanism. We provide further evidence implicating activation of the Gac/Rsm system in FleQ-dependent regulation of biofilm formation. Together, our findings uncover evidence for a mechanism of post-transcriptional regulation of the LapA/MapA adhesins.

microbiology↗

bioRxiv2023

Evidence for the Type IV Pili Retraction Motor PilT as a Component of the Surface Sensing System in Pseudomonas aeruginosa

Biofilm formation begins when bacteria contacting a surface induce cellular changes to become better adapted for surface growth. One of the first changes to occur for Pseudomonas aeruginosa after surface contact is an increase in the nucleotide second messenger 3,5-cyclic adenosine monophosphate (cAMP). It has been demonstrated that this increase in intracellular cAMP is dependent on functional Type IV pili (T4P) relaying a signal to the Pil-Chp system, but the mechanism by which this signal is transduced remains poorly understood. Here, we investigate the role of the Type IV pili retraction motor PilT in sensing a surface and relaying that signal to cAMP production. We show that mutations affecting the structure of PilT and in particular ATPase activity of this motor protein, reduce surface-dependent cAMP production. We identify a novel interaction between PilT and PilJ, a member of the Pil-Chp system, and propose a new model whereby P. aeruginosa uses its retraction motor to sense a surface and to relay that signal via PilJ to increased production of cAMP. We discuss these findings in light of current TFP-dependent surface sensing models for P. aeruginosa. ImportanceT4P are cellular appendages that allow P. aeruginosa to sense a surface leading to the production of cAMP. This second messenger not only activates virulence pathways but leads to further surface adaptation and irreversible attachment of cells. Here, we demonstrate the importance of the retraction motor PilT in surface sensing. We also present a new surface sensing model in P. aeruginosa whereby the T4P retraction motor PilT senses and transmits the surface signal, likely via its ATPase domain and interaction with PilJ, to mediate production of the second messenger cAMP.

microbiology↗

bioRxiv2023

Assessment of the Glycan-Binding Profile of Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is an opportunistic pathogen that can establish acute and chronic infections in individuals that lack fully functional innate immunity. In particular, phagocytosis by neutrophils and macrophages is a key mechanism that modulates host control and clearance of P. aeruginosa. Individuals with neutropenia or cystic fibrosis are highly susceptible to P. aeruginosa infection thus underscoring the importance of the host innate immune response. Cell-to-cell contact between host innate immune cells and the pathogen, a first step in phagocytic uptake, is facilitated by simple and complex glycan structures present at the host cell surface. We have previously shown that endogenous polyanionic N-linked glycans localized to the cell surface of phagocytes mediate binding and subsequent phagocytosis of P. aeruginosa. However, the suite of glycans that P. aeruginosa binds to on host phagocytic cells remains poorly characterized. Here we demonstrate, with the use of exogenous N-linked glycans and a glycan array, that P. aeruginosa PAO1 preferentially attaches to a subset of glycans, including a bias towards monosaccharide versus more complex glycan structures. Consistent with these findings, we were able to competitively inhibit bacterial adherence and uptake by the addition of exogenous N-linked mono- and di-saccharide glycans. We discuss of findings in the context of previous reports of P. aeruginosa glycan binding. IMPORTANCEP. aeruginosa binds to a variety of glycans as part of its interaction with host cells, and a number of P. aeruginosa-encoded receptors and target ligands have been described that allow this microbe to bind to such glycans. Here we extend this work by studying the glycans used by P. aeruginosa PAO1 to bind to phagocytic cells and by using a glycan array to characterize the suite of such molecules that could facilitate host cell-binding by this microbe. This study provides an increased understanding of the glycans bound by P. aeruginosa, and furthermore, provides a useful dataset for future studies of P. aeruginosa-glycan interactions.

microbiology↗

bioRxiv2022

Predicting Clinical Outcomes in Infants With Cystic Fibrosis From Stool Microbiota using Random Forest Algorithms

The gut-lung axis describes the interaction between microbiota in the gut and health status of the airway, whereby there is a bidirectional relationship facilitated by systemic transport of microbially-derived metabolites and immune factors. Cystic fibrosis (CF) is a genetic disease that is associated with dysbiosis of the gut microbiota. Recent literature has shown that the microbial dysbiosis in the CF gut can alter the hosts inflammatory status and that there are distinct microbial compositions in children with CF who have low versus high intestinal inflammation. These distinct microbial profiles can be used as signatures in children with CF (cwCF) to predict health outcomes. Here, we use supervised machine learning to train a random forest model on the distinct microbial composition of cwCF to predict: age (as a validation of the method), frequency of upper respiratory infection (URIfreq), and neutrophil to lymphocyte ratio (NLR), a clinical marker for systemic inflammation that negatively correlates with lung function. We find that the out of bag error, a measure of model accuracy, is lower when predicting age for cwCF compared to children without CF, consistent with previous data. We are able to predict high URIfreq with only 16% error and high NLR with 27% error. This machine learning pipeline may allow physicians and microbiome researchers to use the stool microbiota of cwCF as a tool for identifying individuals with the more negative airway clinical outcomes from this population, and potentially allow for early intervention. ImportanceChildren with CF (cwCF) often experience chronic respiratory infections, leading to progressive, irreversible lung function decline and significant morbidity and premature mortality. Modulator therapy has revolutionized the treatment of eligible adults with CF. cwCF as young as age 6 are now eligible to receive modulator therapy, although by early childhood many children have already experienced pulmonary exacerbations. Here we show that for cwCF, stool microbiota composition is associated with higher upper respiratory infection frequency and increased systemic inflammation. Our findings may aid in developing diagnostic tools that can allow physicians further understanding of which intestinal microbiota profiles are associated with health outcomes and to identify targets for preventative treatment for cwCF.

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↗