Tissue remodeling by an opportunistic pathogen triggers allergic inflammation.

Different effector arms of the immune system are optimized to protect from different classes of pathogens. In some cases, pathogens manipulate the host immune system to promote the wrong type of effector response-a phenomenon known as immune deviation. Typically, immune deviation helps pathogens to avoid destructive immune responses. Here, we report on a type of immune deviation whereby an opportunistic pathogen, Pseudomonas aeruginosa (P. aeruginosa), induces the type 2 immune response resulting in mucin production that is used as an energy source by the pathogen. Specifically, P. aeruginosa-secreted toxin, LasB, processed and activated epithelial amphiregulin to induce type 2 inflammation and mucin production. This "niche remodeling" by P. aeruginosa promoted colonization and, as a by-product, allergic sensitization. Our study thus reveals a type of bacterial immune deviation by increasing nutrient supply. It also uncovers a mechanism of allergic sensitization by a bacterial virulence factor.

Evaluation of expanded 2-aminobenzothiazole library as inhibitors of a model histidine kinase and virulence suppressors in Pseudomonas aeruginosa.

Bacterial resistance to antibiotics is a rapidly increasing threat to human health. New strategies to combat resistant organisms are desperately needed. One potential avenue is targeting two-component systems, which are the main bacterial signal transduction pathways used to regulate development, metabolism, virulence, and antibiotic resistance. These systems consist of a homodimeric membrane-bound sensor histidine kinase, and a cognate effector, the response regulator. Histidine kinases play an essential role in the regulation of multiple virulence mechanisms including toxin production, immune evasion, and antibiotic resistance. Targeting virulence, as opposed to development of bactericidal compounds, could reduce evolutionary pressure for acquired resistance. Additionally, compounds targeting the highly conserved catalytic and adenosine triphosphate-binding (CA) domain have the potential to impair multiple two-component systems that regulate virulence in one or more pathogens. We conducted in vitro structure-activity relationship studies of 2-aminobenzothiazole-based inhibitors designed to target the CA domain. We found that these compounds, which inhibit the model histidine kinase, HK853 from Thermotoga maritima, have anti-virulence activities inPseudomonas aeruginosa, reducing motility phenotypes and toxin production associated with the pathogenic functions of this bacterium.

Respiratory Influenza Virus Infection Causes Dynamic Tuft Cell and Innate Lymphoid Cell Changes in the Small Intestine.

Influenza A viruses (IAV) can cause severe disease and death in humans. IAV infection and the accompanying immune response can result in systemic inflammation, leading to intestinal damage and disruption of the intestinal microbiome. Here, we demonstrate that a specific subset of epithelial cells, tuft cells, increase across the small intestine during active respiratory IAV infection. Upon viral clearance, tuft cell numbers return to baseline levels. Intestinal tuft cell increases were not protective against disease, as animals with either increased tuft cells or a lack of tuft cells did not have any change in disease morbidity after infection. Respiratory IAV infection also caused transient increases in type 1 and 2 innate lymphoid cells (ILC1 and ILC2, respectively) in the small intestine. ILC2 increases were significantly blunted in the absence of tuft cells, whereas ILC1s were unaffected. Unlike the intestines, ILCs in the lungs were not altered in the absence of tuft cells. This work establishes that respiratory IAV infection causes dynamic changes to tuft cells and ILCs in the small intestines and that tuft cells are necessary for the infection-induced increase in small intestine ILC2s. These intestinal changes in tuft cell and ILC populations may represent unexplored mechanisms preventing systemic infection and/or contributing to severe disease in humans with preexisting conditions. IMPORTANCE Influenza A virus (IAV) is a respiratory infection in humans that can lead to a wide range of symptoms and disease severity. Respiratory infection can cause systemic inflammation and damage in the intestines. Few studies have explored how inflammation alters the intestinal environment. We found that active infection caused an increase in the epithelial population called tuft cells as well as type 1 and 2 innate lymphoid cells (ILCs) in the small intestine. In the absence of tuft cells, this increase in type 2 ILCs was seriously blunted, whereas type 1 ILCs still increased. These findings indicate that tuft cells are necessary for infection-induced changes in small intestine type 2 ILCs and implicate tuft cells as regulators of the intestinal environment in response to systemic inflammation.

Single cell resolution of SARS-CoV-2 tropism, antiviral responses, and susceptibility to therapies in primary human airway epithelium.

The human airway epithelium is the initial site of SARS-CoV-2 infection. We used flow cytometry and single cell RNA-sequencing to understand how the heterogeneity of this diverse cell population contributes to elements of viral tropism and pathogenesis, antiviral immunity, and treatment response to remdesivir. We found that, while a variety of epithelial cell types are susceptible to infection, ciliated cells are the predominant cell target of SARS-CoV-2. The host protease TMPRSS2 was required for infection of these cells. Importantly, remdesivir treatment effectively inhibited viral replication across cell types, and blunted hyperinflammatory responses. Induction of interferon responses within infected cells was rare and there was significant heterogeneity in the antiviral gene signatures, varying with the burden of infection in each cell. We also found that heavily infected secretory cells expressed abundant IL-6, a potential mediator of COVID-19 pathogenesis.

Staphylococcus aureus Overcomes Anaerobe-Derived Short-Chain Fatty Acid Stress via FadX and the CodY Regulon.

Chronic rhinosinusitis (CRS) is characterized by immune dysfunction, mucus hypersecretion, and persistent infection of the paranasal sinuses. While Staphylococcus aureus is a primary CRS pathogen, recent sequence-based surveys have found increased relative abundances of anaerobic bacteria, suggesting that S. aureus may experience altered metabolic landscapes in CRS relative to healthy airways. To test this possibility, we characterized the growth kinetics and transcriptome of S. aureus in supernatants of the abundant CRS anaerobe Fusobacterium nucleatum. While growth was initially delayed, S. aureus ultimately grew to similar levels as in the control medium. The transcriptome was significantly affected by F. nucleatum metabolites, with the agr quorum sensing system notably repressed. Conversely, expression of fadX, encoding a putative propionate coenzyme A (CoA)-transferase, was significantly increased, leading to our hypothesis that short-chain fatty acids (SCFAs) produced by F. nucleatum could mediate S. aureus growth behavior and gene expression. Supplementation with propionate and butyrate, but not acetate, recapitulated delayed growth phenotypes observed in F. nucleatum supernatants. A fadX mutant was found to be more sensitive than wild type to propionate, suggesting a role for FadX in the S. aureus SCFA stress response. Interestingly, spontaneous resistance to butyrate, but not propionate, was observed frequently. Whole-genome sequencing and targeted mutagenesis identified codY mutants as resistant to butyrate inhibition. Together, these data show that S. aureus physiology is dependent on its cocolonizing microbiota and metabolites they exchange and indicate that propionate and butyrate may act on different targets in S. aureus to suppress its growth. IMPORTANCE Staphylococcus aureus is an important CRS pathogen, and yet it is found in the upper airways of 30% to 50% of people without complications. The presence of strict and facultative anaerobic bacteria in CRS sinuses has recently spurred research into bacterial interactions and how they influence S. aureus physiology and pathogenesis. We show here that propionate and butyrate produced by one such CRS anaerobe, namely, Fusobacterium nucleatum, alter the growth and gene expression of S. aureus. We show that fadX is important for S. aureus to resist propionate stress and that the CodY regulon mediates growth in inhibitory concentrations of butyrate. This work highlights the possible complexity of S. aureus-anaerobe interactions and implicates membrane stress as a possible mechanism influencing S. aureus behavior in CRS sinuses.

Anaerobic Microbiota Derived from the Upper Airways Impact Staphylococcus aureus Physiology.

Staphylococcus aureus is associated with the development of persistent and severe inflammatory diseases of the upper airways. Yet, S. aureus is also carried asymptomatically in the sinonasal cavity of ∼50% of healthy adults. The causes of this duality and host and microbial factors that tip the balance between S. aureus pathogenesis and commensalism are poorly understood. We have shown that by degrading mucins, anaerobic microbiota support the growth of airway pathogens by liberating metabolites that are otherwise unavailable. Given the widely reported culture-based detection of anaerobes from individuals with chronic rhinosinusitis (CRS), here we tested our hypothesis that CRS microbiota is characterized by a mucin-degrading phenotype that alters S. aureus physiology. Using 16S rRNA gene sequencing, we indeed observed an increased prevalence and abundance of anaerobes in CRS relative to non-CRS controls. PICRUSt2-based functional predictions suggested increased mucin degradation potential among CRS microbiota that was confirmed by direct enrichment culture. Prevotella, Fusobacterium, and Streptococcus comprised a core mucin-degrading community across CRS subjects that generated a nutrient pool that augmented S. aureus growth on mucin as a carbon source. Finally, using transcriptome sequencing (RNA-seq), we observed that S. aureus transcription is profoundly altered in the presence of mucin-derived metabolites, though expression of several key metabolism- and virulence-associated pathways varied between CRS-derived bacterial communities. Together, these data support a model in which S. aureus metabolism and virulence in the upper airways are dependent upon the composition of cocolonizing microbiota and the metabolites they exchange.

Stochasticity in the enterococcal sex pheromone response revealed by quantitative analysis of transcription in single cells.

In Enterococcus faecalis, sex pheromone-mediated transfer of antibiotic resistance plasmids can occur under unfavorable conditions, for example, when inducing pheromone concentrations are low and inhibiting pheromone concentrations are high. To better understand this paradox, we adapted fluorescence in situ hybridization chain reaction (HCR) methodology for simultaneous quantification of multiple E. faecalis transcripts at the single cell level. We present direct evidence for variability in the minimum period, maximum response level, and duration of response of individual cells to a specific inducing condition. Tracking of induction patterns of single cells temporally using a fluorescent reporter supported HCR findings. It also revealed subpopulations of rapid responders, even under low inducing pheromone concentrations where the overall response of the entire population was slow. The strong, rapid induction of small numbers of cells in cultures exposed to low pheromone concentrations is in agreement with predictions of a stochastic model of the enterococcal pheromone response. The previously documented complex regulatory circuitry controlling the pheromone response likely contributes to stochastic variation in this system. In addition to increasing our basic understanding of the biology of a horizontal gene transfer system regulated by cell-cell signaling, demonstration of the stochastic nature of the pheromone response also impacts any future efforts to develop therapeutic agents targeting the system. Quantitative single cell analysis using HCR also has great potential to elucidate important bacterial regulatory mechanisms not previously amenable to study at the single cell level, and to accelerate the pace of functional genomic studies.

Mapping a multiplexed zoo of mRNA expression.

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.

Evidence and Role for Bacterial Mucin Degradation in Cystic Fibrosis Airway Disease.

Chronic lung infections in cystic fibrosis (CF) patients are composed of complex microbial communities that incite persistent inflammation and airway damage. Despite the high density of bacteria that colonize the lower airways, nutrient sources that sustain bacterial growth in vivo, and how those nutrients are derived, are not well characterized. In this study, we examined the possibility that mucins serve as an important carbon reservoir for the CF lung microbiota. While Pseudomonas aeruginosa was unable to efficiently utilize mucins in isolation, we found that anaerobic, mucin-fermenting bacteria could stimulate the robust growth of CF pathogens when provided intact mucins as a sole carbon source. 16S rRNA sequencing and enrichment culturing of sputum also identified that mucin-degrading anaerobes are ubiquitous in the airways of CF patients. The collective fermentative metabolism of these mucin-degrading communities in vitro generated amino acids and short chain fatty acids (propionate and acetate) during growth on mucin, and the same metabolites were also found in abundance within expectorated sputum. The significance of these findings was supported by in vivo P. aeruginosa gene expression, which revealed a heightened expression of genes required for the catabolism of propionate. Given that propionate is exclusively derived from bacterial fermentation, these data provide evidence for an important role of mucin fermenting bacteria in the carbon flux of the lower airways. More specifically, microorganisms typically defined as commensals may contribute to airway disease by degrading mucins, in turn providing nutrients for pathogens otherwise unable to efficiently obtain carbon in the lung.

Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle.

The candidate phylum TM7 is globally distributed and often associated with human inflammatory mucosal diseases. Despite its prevalence, the TM7 phylum remains recalcitrant to cultivation, making it one of the most enigmatic phyla known. In this study, we cultivated a TM7 phylotype (TM7x) from the human oral cavity. This extremely small coccus (200-300 nm) has a distinctive lifestyle not previously observed in human-associated microbes. It is an obligate epibiont of an Actinomyces odontolyticus strain (XH001) yet also has a parasitic phase, thereby killing its host. This first completed genome (705 kb) for a human-associated TM7 phylotype revealed a complete lack of amino acid biosynthetic capacity. Comparative genomics analyses with uncultivated environmental TM7 assemblies show remarkable conserved gene synteny and only minimal gene loss/gain that may have occurred as TM7x adapted to conditions within the human host. Transcriptomic and metabolomic profiles provided the first indications, to our knowledge, that there is signaling interaction between TM7x and XH001. Furthermore, the induction of TNF-α production in macrophages by XH001 was repressed in the presence of TM7x, suggesting its potential immune suppression ability. Overall, our data provide intriguing insights into the uncultivability, pathogenicity, and unique lifestyle of this previously uncharacterized oral TM7 phylotype.

Genome-Wide Survey of Pseudomonas aeruginosa PA14 Reveals a Role for the Glyoxylate Pathway and Extracellular Proteases in the Utilization of Mucin.

Chronic airway infections by the opportunistic pathogen Pseudomonas aeruginosa are a major cause of mortality in cystic fibrosis (CF) patients. Although this bacterium has been extensively studied for its virulence determinants, biofilm growth, and immune evasion mechanisms, comparatively little is known about the nutrient sources that sustain its growth in vivo Respiratory mucins represent a potentially abundant bioavailable nutrient source, although we have recently shown that canonical pathogens inefficiently use these host glycoproteins as a growth substrate. However, given that P. aeruginosa, particularly in its biofilm mode of growth, is thought to grow slowly in vivo, the inefficient use of mucin glycoproteins may be relevant to its persistence within the CF airways. To this end, we used whole-genome fitness analysis, combining transposon mutagenesis with high-throughput sequencing, to identify genetic determinants required for P. aeruginosa growth using intact purified mucins as a sole carbon source. Our analysis reveals a biphasic growth phenotype, during which the glyoxylate pathway and amino acid biosynthetic machinery are required for mucin utilization. Secondary analyses confirmed the simultaneous liberation and consumption of acetate during mucin degradation and revealed a central role for the extracellular proteases LasB and AprA. Together, these studies describe a molecular basis for mucin-based nutrient acquisition by P. aeruginosa and reveal a host-pathogen dynamic that may contribute to its persistence within the CF airways.

Caenorhabditis elegans NPR-1-mediated behaviors are suppressed in the presence of mucoid bacteria.

Caenorhabditis elegans exhibits a diverse range of behaviors in response to bacteria. The presence of bacterial food influences C. elegans aerotaxis, aggregation, locomotion, and pathogen avoidance behaviors through the activity of the NPR-1 neuropeptide receptor. Here, we show that mucoid strains of bacteria that produce an exopolysaccharide matrix do not induce NPR-1-dependent behaviors. In the presence of mucoid strains of bacteria, the C. elegans laboratory wild-type (WT) strain N2 exhibits behaviors characteristic of wild isolates and mutants with reduced NPR-1 activity. Specifically, N2 exhibits lawn bordering and roaming behavior on mucoid nonpathogenic bacteria and loss of pathogen avoidance on mucoid Pseudomonas aeruginosa. Alginate biosynthesis by laboratory and clinical isolates of mucoid P. aeruginosa is necessary and sufficient to attenuate NPR-1-mediated behavior and it suppresses C. elegans pathogen avoidance behavior. Our data suggest that the specific interaction with nonmucoid bacteria induces NPR-1-dependent behaviors of C. elegans. These observations provide an example of how exopolysaccharide matrix biosynthesis by a community of bacteria may inhibit specific host responses to microbes.

The RND-family transporter, HpnN, is required for hopanoid localization to the outer membrane of Rhodopseudomonas palustris TIE-1.

Rhodopseudomonas palustris TIE-1 is a gram-negative bacterium that produces structurally diverse hopanoid lipids that are similar to eukaryotic steroids. Its genome encodes several homologues to proteins involved in eukaryotic steroid trafficking. In this study, we explored the possibility that two of these proteins are involved in intracellular hopanoid transport. R. palustris has a sophisticated membrane system comprising outer, cytoplasmic, and inner cytoplasmic membranes. It also divides asymmetrically, producing a mother and swarmer cell. We deleted genes encoding two putative hopanoid transporters that belong to the resistance-nodulation-cell division superfamily. Phenotypic analyses revealed that one of these putative transporters (HpnN) is essential for the movement of hopanoids from the cytoplasmic to the outer membrane, whereas the other (Rpal_4267) plays a minor role. C(30) hopanoids, such as diploptene, are evenly distributed between mother and swarmer cells, whereas hpnN is required for the C(35) hopanoid, bacteriohopanetetrol, to remain localized to the mother cell type. Mutant cells lacking HpnN grow like the WT at 30 °C but slower at 38 °C. Following cell division at 38 °C, the ΔhpnN cells remain connected by their cell wall, forming long filaments. This phenotype may be attributed to hopanoid mislocalization because a double mutant deficient in both hopanoid biosynthesis and transport does not form filaments. However, the lack of hopanoids severely compromises cell growth at higher temperatures more generally. Because hopanoid mutants only manifest a strong phenotype under certain conditions, R. palustris is an attractive model organism in which to study their transport and function.

Microbiome and metabolome patterns after lung transplantation reflect underlying disease and chronic lung allograft dysfunction.

BACKGROUND: Progression of chronic lung disease may lead to the requirement for lung transplant (LTx). Despite improvements in short-term survival after LTx, chronic lung allograft dysfunction (CLAD) remains a critical challenge for long-term survival. This study investigates the molecular and microbial relationships between underlying lung disease and the development of CLAD in bronchoalveolar lavage fluid (BALF) from subjects post-LTx, which is crucial for tailoring treatment strategies specific to allograft dysfunctions. METHODS: Paired 16S rRNA gene amplicon sequencing and untargeted LC-MS/MS metabolomics were performed on 856 BALF samples collected over 10 years from LTx recipients (n = 195) with alpha-1-antitrypsin disease (AATD, n = 23), cystic fibrosis (CF, n = 47), chronic obstructive pulmonary disease (COPD, n = 78), or pulmonary fibrosis (PF, n = 47). Data were analyzed using random forest (RF) machine learning and multivariate statistics for associations with underlying disease and CLAD development. RESULTS: The BALF microbiome and metabolome after LTx differed significantly according to the underlying disease state (PERMANOVA, p = 0.001), with CF and AATD demonstrating distinct microbiome and metabolome profiles, respectively. Uniqueness in CF was mainly driven by Pseudomonas abundance and its metabolites, whereas AATD had elevated levels of phenylalanine and a lack of shared metabolites with the other underlying diseases. BALF microbiome and metabolome composition were also distinct between those who did or did not develop CLAD during the sample collection period (PERMANOVA, p = 0.001). An increase in the average abundance of Veillonella (AATD, COPD) and Streptococcus (CF, PF) was associated with CLAD development, and decreases in the abundance of phenylalanine-derivative alkaloids (CF, COPD) and glycerophosphorylcholines (CF, COPD, PF) were signatures of the CLAD metabolome. Although the relative abundance of Pseudomonas was not associated with CLAD, the abundance of its virulence metabolites, including siderophores, quorum-sensing quinolones, and phenazines, were elevated in those with CF who developed CLAD. There was a positive correlation between the abundance of these molecules and the abundance of Pseudomonas in the microbiome, but there was no correlation between their abundance and the time in which BALF samples were collected post-LTx. CONCLUSIONS: The BALF microbiome and metabolome after LTx are particularly distinct in those with underlying CF and AATD. These data reflect those who developed CLAD, with increased virulence metabolite production from Pseudomonas, an aspect of CF CLAD cases. These findings shed light on disease-specific microbial and metabolic signatures in LTx recipients, offering valuable insights into the underlying causes of allograft rejection. Video Abstract.

To Compare the Effects of a Standard Versus Hydrophilic Polymer Coated Airway Stent in a Porcine Model: A Randomized, Single-Blinded Study.

BACKGROUND: Stent encrustation with debris and mucostasis is a significant cause of airway injury and comorbidity, leading to ~25% of stent exchanges (1-3). Previous work from our group has shown that the experimental coating can reduce mucous adhesion in bench testing and demonstrated a signal for reducing airway injury and mucostasis in a feasibility study. OBJECTIVES: The aim of this study is to continue our inquiry in a randomized, single-blinded multi-animal trial to investigate the degree of airway injury and mucostasis using silicone stents with and without this specialized coating. METHODS: We modified commercially available silicone stents with a hydrophilic polymer from Toray Industries. We conducted an in vivo survival study in 6 mainstem airways (3 coated and 3 uncoated) of 3 pigs to compare the degree of airway injury and mucostasis between coated versus noncoated stented airways. Both stents were randomized to either left or right mainstem bronchus. The pathologist was blinded to the stent type. RESULTS: We implanted a total of six 14×15 mm silicone stents (1 per mainstem bronchi) into 3 pigs. All animals survived to termination at 4 weeks. All stents were intact; however, 1 uncoated stent migrated out. On average, all the coated stents demonstrated reduced pathology and tissue injury scores (75 vs. 68.3, respectively). The average total dried mucous weight was slightly higher in the coated stents (0.07 g vs. 0.05 g; respectively). CONCLUSION: Coated stents had lower airway injury compared with uncoated stents in this study. Of all the stents, 1 uncoated stent migrated out and was not included in the dried mucous weight totals. This could explain the slightly higher mucous weight in the coated stents. Nevertheless, this current study demonstrates promising results in lowering airway injury in stents incorporated with the hydrophilic coating, and future studies, including a larger number of subjects, would be needed to corroborate our findings.

Commensal-derived short-chain fatty acids disrupt lipid membrane homeostasis in Staphylococcus aureus.

The role of commensal anaerobic bacteria in chronic respiratory infections is unclear, yet they can exist in abundances comparable to canonical pathogens in vivo. Their contributions to the metabolic landscape of the host environment may influence pathogen behavior by competing for nutrients and creating inhospitable conditions via toxic metabolites. Here, we reveal a mechanism by which the anaerobe-derived short chain fatty acids (SCFAs) propionate and butyrate negatively affect Staphylococcus aureus physiology by disrupting branched chain fatty acid (BCFA) metabolism. In turn, BCFA impairment results in impaired growth, diminished expression of the agr quorum sensing system, as well as increased sensitivity to membrane-targeting antimicrobials. Altered BCFA metabolism also reduces S. aureus fitness in competition with Pseudomonas aeruginosa, suggesting that airway microbiome composition and the metabolites they produce and exchange directly impact pathogen succession over time. The pleiotropic effects of these SCFAs on S. aureus fitness and their ubiquity as metabolites in animals also suggests that they may be effective as sensitizers to traditional antimicrobial agents when used in combination.

Evaluation of Expanded 2-Aminobenzothiazole Library for Inhibition of Pseudomonas aeruginosa Virulence Phenotypes.

Bacterial resistance to antibiotics is a rapidly increasing threat to human health. New strategies to combat resistant organisms are desperately needed. One potential avenue is targeting two-component systems, which are the main bacterial signal transduction pathways used to regulate development, metabolism, virulence, and antibiotic resistance. These systems consist of a homodimeric membrane-bound sensor histidine kinase, and a cognate effector, the response regulator. The high sequence conservation in the catalytic and adenosine triphosphate-binding (CA) domain of histidine kinases and their essential role in bacterial signal transduction could enable broad-spectrum antibacterial activity. Through this signal transduction, histidine kinases regulate multiple virulence mechanisms including toxin production, immune evasion, and antibiotic resistance. Targeting virulence, as opposed to development of bactericidal compounds, could reduce evolutionary pressure for acquired resistance. Additionally, compounds targeting the CA domain have the potential to impair multiple two-component systems that regulate virulence in one or more pathogens. We conducted structure-activity relationship studies of 2-aminobenzothiazole-based inhibitors designed to target the CA domain of histidine kinases. We found these compounds have anti-virulence activities in Pseudomonas aeruginosa, reducing motility phenotypes and toxin production associated with the pathogenic functions of this bacterium.

Bacterial community morphogenesis is intimately linked to the intracellular redox state.

Many microbial species form multicellular structures comprising elaborate wrinkles and concentric rings, yet the rules governing their architecture are poorly understood. The opportunistic pathogen Pseudomonas aeruginosa produces phenazines, small molecules that act as alternate electron acceptors to oxygen and nitrate to oxidize the intracellular redox state and that influence biofilm morphogenesis. Here, we show that the depth occupied by cells within colony biofilms correlates well with electron acceptor availability. Perturbations in the environmental provision, endogenous production, and utilization of electron acceptors affect colony development in a manner consistent with redox control. Intracellular NADH levels peak before the induction of colony wrinkling. These results suggest that redox imbalance is a major factor driving the morphogenesis of P. aeruginosa biofilms and that wrinkling itself is an adaptation that maximizes oxygen accessibility and thereby supports metabolic homeostasis. This type of redox-driven morphological change is reminiscent of developmental processes that occur in metazoans.

High-throughput quantification of microbial-derived organic acids in mucin-rich samples via reverse phase high performance liquid chromatography.

Organic acids (short chain fatty acids, amino acids, etc.) are common metabolic byproducts of commensal bacteria of the gut and oral cavity in addition to microbiota associated with chronic infections of the airways, skin, and soft tissues. A ubiquitous characteristic of these body sites in which mucus-rich secretions often accumulate in excess, is the presence of mucins; high molecular weight (HMW), glycosylated proteins that decorate the surfaces of non-keratinized epithelia. Owing to their size, mucins complicate quantification of microbial-derived metabolites as these large glycoproteins preclude use of 1D and 2D gel approaches and can obstruct analytical chromatography columns. Standard approaches for quantification of organic acids in mucin-rich samples typically rely on laborious extractions or outsourcing to laboratories specializing in targeted metabolomics. Here we report a high-throughput sample preparation process that reduces mucin abundance and an accompanying isocratic reverse phase high performance liquid chromatography (HPLC) method that enables quantification of microbial-derived organic acids. This approach allows for accurate quantification of compounds of interest (0.01 mM - 100 mM) with minimal sample preparation, a moderate HPLC method run time, and preservation of both guard and analytical column integrity. This approach paves the way for further analyses of microbial-derived metabolites in complex clinical samples.