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Previous urinary tract infections (UTIs) can predispose one to future infections; however, the underlying mechanisms affecting recurrence are poorly understood. We previously found that UTIs in mice cause differential bladder epithelial (urothelial) remodelling, depending on disease outcome, that impacts susceptibility to recurrent UTI. Here we compared urothelial stem cell (USC) lines isolated from mice with a history of either resolved or chronic uropathogenic Escherichia coli (UPEC) infection, elucidating evidence of molecular imprinting that involved epigenetic changes, including differences in chromatin accessibility, DNA methylation and histone modification. Epigenetic marks in USCs from chronically infected mice enhanced caspase-1-mediated cell death upon UPEC infection, promoting bacterial clearance. Increased Ptgs2os2 expression also occurred, potentially contributing to sustained cyclooxygenase-2 expression, bladder inflammation and mucosal wounding—responses associated with severe recurrent cystitis. Thus, UPEC infection acts as an epi-mutagen reprogramming the urothelial epigenome, leading to urothelial-intrinsic remodelling and training of the innate response to subsequent infection.

Background Unbiased assessment of the risks associated with acquisition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to informing mitigation efforts during pandemics. The objective of our study was to understand the risk factors for acquiring coronavirus disease 2019 (COVID-19) in a large prospective cohort of adult residents in a large US metropolitan area. Methods We designed a fully remote longitudinal cohort study involving monthly at-home SARS-CoV-2 polymerase chain reaction (PCR) and serology self-testing and monthly surveys. Results Between October 2020 and January 2021, we enrolled 10 289 adults reflective of the Boston metropolitan area census data. At study entry, 567 (5.5%) participants had evidence of current or prior SARS-CoV-2 infection. This increased to 13.4% by June 15, 2021. Compared with Whites, Black non-Hispanic participants had a 2.2-fold greater risk of acquiring COVID-19 (hazard ratio [HR], 2.19; 95% CI, 1.91–2.50; P < .001), and Hispanics had a 1.5-fold greater risk (HR, 1.52; 95% CI, 1.32–1.71; P < .016). Individuals aged 18–29, those who worked outside the home, and those living with other adults and children were at an increased risk. Individuals in the second and third lowest disadvantaged neighborhood communities were associated with an increased risk of acquiring COVID-19. Individuals with medical risk factors for severe disease were at a decreased risk of SARS-CoV-2 acquisition. Conclusions These results demonstrate that race/ethnicity and socioeconomic status are the biggest determinants of acquisition of infection. This disparity is significantly underestimated if based on PCR data alone, as noted by the discrepancy in serology vs PCR detection for non-White participants, and points to persistent disparity in access to testing. Medical conditions and advanced age, which increase the risk for severity of SARS-CoV-2 disease, were associated with a lower risk of COVID-19 acquisition, suggesting the importance of behavior modifications. These findings highlight the need for mitigation programs that overcome challenges of structural racism in current and future pandemics.

The bacterial pathogen Salmonella enterica is able to establish persistent infections, evading antibiotics and the host immune system and providing a reservoir for recrudescence and transmission to new hosts. Non-typhoidal serovars (NTS) of S. enterica can establish and maintain symptomatic and asymptomatic long term human infections that may predispose carriers to inflammatory bowel diseases and cancer. Defining the adaptations and host-pathogen interactions enabling these persistent infections is key to devising more effective strategies to combat and prevent persistent bacterial infections. Using comparative genomics of 639 Salmonella NTS isolates belonging to 49 serovars that were longitudinally obtained from 256 salmonelosis patients during different stages of infection, we identified numerous genetic variations accruing over time in strains isolated from the same patient. Many of these changes were found in the same gene across multiple patients and serovars. Among these variant loci, genes encoding global transcriptional regulators were found to be the most commonly mutated between early and late same-patient isolates. Genetic changes in the SirA/BarA two-component regulatory system were particularly frequent, with mutations identified in 24 independent patients. Comparative RNA-Seq analysis revealed that distinct mutations in sirA/barA that arose independently in late isolates of multiple patients lead to significantly diminished expression of virulence-associated genes encoded in the Salmonella Pathogenicity Islands (SPIs) 1 and 4, many of which are known to be critical for host cell invasion and the production of enteritis. Using the salmonellosis mouse model we showed that these mutations in sirA/barA genes confer attenuated virulence in-vivo. Taken together, these data suggest that selection of mutations in the SirA/BarA pathway facilitates persistent Salmonella infection in humans, possibly by attenuating Salmonella virulence and its ability to cause inflammation.

SARS-CoV-2 infection leads to a broad range of outcomes and immune responses, with the development of neutralizing antibodies generally correlated with protection against reinfection. Here, we have characterized both neutralizing activity and T cell responses in a cluster of subjects with mild disease linked to a single spreading event. Surprisingly, we observed sex-specific associations between spike- and particularly nucleoprotein-specific T cell responses and neutralization, with pro-inflammatory cytokines being linked to higher titers only in males. Using single cell immunoprofiling, which provided matched transcriptome and T-cell receptor (TCR) profiles in restimulated CD4 + and CD8 + cells from these subjects, we identified differences in type I IFN signaling that may underlie this difference in antibody generation. Finally, we also identified several TCRs associated with cytokine producing T cells. Altogether, our work maps the breadth of immunological outcomes of SARS-CoV2 infections and highlight the potential role of sex-specific feedback loops during the generation of neutralizing antibodies.

ImportanceUnbiased assessment of risks associated with acquisition of SARS-CoV-2 is critical to informing mitigation efforts during pandemics.ObjectiveUnderstand risk factors for acquiring COVID-19 in a large, prospective cohort of adult residents recruited to be representative of a large US metropolitan area.DesignFully remote longitudinal cohort study launched in October 2020 and ongoing; Study data reported through June 15, 2021.SettingBrigham and Women’s Hospital, Boston MA.ParticipantsAdults within 45 miles of Boston, MA.InterventionMonthly at-home SARS-CoV-2 viral and antibody testing.Main OutcomesBetween October 2020 and January 2021, we enrolled 10,289 adults reflective of Massachusetts census data. At study entry, 567 (5.5%) participants had evidence of current or prior SARS-CoV-2 infection. This increased to 13.4% by June 15, 2021. Compared to whites, Black non-Hispanic participants had a 2.2 fold greater risk of acquiring COVID-19 (HR 2.19, 95% CI 1.91-2.50; p=ConclusionsThese results demonstrate that race/ethnicity and socioeconomic status are not only risk factors for severity of disease but are also the biggest determinants of acquisition of infection. Importantly, this disparity is significantly underestimated if based on PCR data alone as noted by the discrepancy in serology vs. PCR detection for non-white participants, and points to persistent disparity in access to testing. Meanwhile, medical conditions and advanced age that increase the risk for severity of SARS-CoV-2 disease were associated with a lower risk of acquisition of COVID-19 suggesting the importance of behavior modifications. These findings highlight the need for mitigation programs that overcome challenges of structural racism in current and future pandemics.Trial RegistrationN/AKEY POINTSQuestionWhat population and occupational groups in the United States are at increased risk for acquiring COVID-19?FindingsIn this remote, longitudinal cohort study involving monthly PCR and serology self-testing of 10,289 adult residents of the Boston metropolitan area, 9257 (90.0%) of TestBoston participants acquired evidence of immunity to SARS-CoV-2 through vaccination, infection, or both as of June 15, 2021. Residents identifying as Black, Hispanic/Latinx had an increased risk of acquisition of COVID-19. Healthcare workers were not at increased risk of SARS-CoV-2 acquisition. Individuals with medical risk factors for severe COVID-19 disease were at a decreased risk of SARS-CoV-2 acquisition.MeaningThese results demonstrate that race/ethnicity and socioeconomic status are not only risk factors for severity of disease but also are the biggest determinants of acquisition of infection. These findings highlight the need to address the consequences of structural racism during the development of mitigation programs for current and future pandemics.

Multidrug resistant organisms are a serious threat to human health1,2. Fast, accurate antibiotic susceptibility testing (AST) is a critical need in addressing escalating antibiotic resistance, since delays in identifying multidrug resistant organisms increase mortality3,4 and use of broad-spectrum antibiotics, further selecting for resistant organisms. Yet current growth-based AST assays, such as broth microdilution5, require several days before informing key clinical decisions. Rapid AST would transform the care of patients with infection while ensuring that our antibiotic arsenal is deployed as efficiently as possible. Growth-based assays are fundamentally constrained in speed by doubling time of the pathogen, and genotypic assays are limited by the ever-growing diversity and complexity of bacterial antibiotic resistance mechanisms. Here we describe a rapid assay for combined genotypic and phenotypic AST through RNA detection, GoPhAST-R, that classifies strains with 94–99% accuracy by coupling machine learning analysis of early antibiotic-induced transcriptional changes with simultaneous detection of key genetic resistance determinants to increase accuracy of resistance detection, facilitate molecular epidemiology and enable early detection of emerging resistance mechanisms. This two-pronged approach provides phenotypic AST 24–36 h faster than standard workflows, with

Rapid and accurate diagnosis of infections is fundamental to individual patient care and public health management. Nucleic acid detection methods are critical to this effort, but are limited either in the breadth of pathogens targeted or by the expertise and infrastructure required. We present here a high-throughput system that enables rapid identification of bacterial pathogens, bCARMEN, which utilizes: (1) modular CRISPR-Cas13-based nucleic acid detection with enhanced sensitivity and specificity; and (2) a droplet microfluidic system that enables thousands of simultaneous, spatially multiplexed detection reactions at nanoliter volumes; and (3) a novel pre-amplification strategy that further enhances sensitivity and specificity. We demonstrate bCARMEN is capable of detecting and discriminating 52 clinically relevant bacterial species and several key antibiotic resistance genes. We further develop a proof of principle system for use with stabilized reagents and a simple workflow with optical readout using a cell phone camera, opening up the possibility of a rapid point-of-care multiplexed bacterial pathogen identification and antibiotic susceptibility testing.Significance StatementIn this paper, we use a novel primer design method combined with droplet-based CRISPR Cas13 detection to distinguish 52 clinically relevant bacterial pathogens in a single assay. We also apply the method to detect and distinguish a panel of major antibiotic resistance genes, which is of critical importance in this era of rising antibiotic resistance. Finally, we make key advances towards making our diagnostic assay deployable at the point-of-care, with a simplified emulsion-free assay process that uses mobile phone camera for detection and reduces infrastructure/skilled labor requirements.

Interspecies interactions are known to activate specialized metabolism in diverse actinomycetes. However, how interspecies cues are sensed and ultimately lead to induction of specialized metabolite biosynthetic gene clusters remains largely unexplored. Using transcriptome sequencing (RNA-seq), we analyzed genes that were transcriptionally induced in the model actinomycete Streptomyces coelicolor during interactions with four different actinomycetes, including genes that encode unusual regulatory systems known as conservons. Deletions in one such system, encoded by the cvn8 genes, led to altered patterns of pigmented antibiotic production by S. coelicolor during interactions. Further transcriptomic analysis of mutants lacking each of the five genes in the cvn8 locus demonstrated that this system is a global regulator of at least four different specialized metabolite biosynthetic pathways. How conservon systems work at the mechanistic level to regulate gene expression is not well understood, although it has been hypothesized that they may function in a way similar to eukaryotic G-protein-coupled receptors. The data presented here indicate that the gene products of the cvnA8 and cvnF8 (SCO6939) genes likely function together in one part of the Cvn8 signaling cascade, while the cvnC8 and cvnD8 gene products likely function together in another part. Importantly, because cvnD8 likely encodes a Ras-like GTPase, these results connect G-protein-mediated signaling to gene regulation in a bacterium. Additionally, deletion of any of the cvn8 genes led to abnormally high expression of an adjacent cryptic lanthipeptide biosynthetic gene cluster, indicating that conservon systems may be fruitful targets for manipulation to activate silent specialized metabolite biosynthetic pathways. IMPORTANCE Interactions between different species of actinomycete bacteria often trigger one of the strains to produce specialized metabolites, such as antibiotics. However, how this induction occurs at the genetic level is poorly understood. Using transcriptomic methods, we show that an unusual regulatory system, known as a conservon system, is responsible for regulating expression of multiple specialized metabolite biosynthetic gene clusters in the organism Streptomyces coelicolor during interactions. Conservon systems are unusual because they appear to employ small GTPases as an important component of their signaling cascades. Small GTPases are common in eukaryotic signaling pathways, but the results presented here are notable since they implicate a system that includes a small GTPase in global gene regulation in a bacterium. Mutants lacking this conservon system also showed abnormally high expression of a gene cluster involved in making an unknown specialized metabolite, suggesting that conservon mutants might be useful for driving natural product discovery.

Interspecies interactions are known to activate specialized metabolism in diverse actinomycetes. However, how interspecies cues are sensed and ultimately lead to induction of specialized metabolite biosynthetic gene clusters remains largely unexplored. Using transcriptome sequencing (RNA-seq), we analyzed genes that were transcriptionally induced in the model actinomycete Streptomyces coelicolor during interactions with four different actinomycetes, including genes that encode unusual regulatory systems known as conservons. Deletions in one such system, encoded by the cvn8 genes, led to altered patterns of pigmented antibiotic production by S. coelicolor during interactions. Further transcriptomic analysis of mutants lacking each of the five genes in the cvn8 locus demonstrated that this system is a global regulator of at least four different specialized metabolite biosynthetic pathways. How conservon systems work at the mechanistic level to regulate gene expression is not well understood, although it has been hypothesized that they may function in a way similar to eukaryotic G-protein-coupled receptors. The data presented here indicate that the gene products of the cvnA8 and cvnF8 (SCO6939) genes likely function together in one part of the Cvn8 signaling cascade, while the cvnC8 and cvnD8 gene products likely function together in another part. Importantly, because cvnD8 likely encodes a Ras-like GTPase, these results connect G-protein-mediated signaling to gene regulation in a bacterium. Additionally, deletion of any of the cvn8 genes led to abnormally high expression of an adjacent cryptic lanthipeptide biosynthetic gene cluster, indicating that conservon systems may be fruitful targets for manipulation to activate silent specialized metabolite biosynthetic pathways. IMPORTANCE Interactions between different species of actinomycete bacteria often trigger one of the strains to produce specialized metabolites, such as antibiotics. However, how this induction occurs at the genetic level is poorly understood. Using transcriptomic methods, we show that an unusual regulatory system, known as a conservon system, is responsible for regulating expression of multiple specialized metabolite biosynthetic gene clusters in the organism Streptomyces coelicolor during interactions. Conservon systems are unusual because they appear to employ small GTPases as an important component of their signaling cascades. Small GTPases are common in eukaryotic signaling pathways, but the results presented here are notable since they implicate a system that includes a small GTPase in global gene regulation in a bacterium. Mutants lacking this conservon system also showed abnormally high expression of a gene cluster involved in making an unknown specialized metabolite, suggesting that conservon mutants might be useful for driving natural product discovery.

Recurrent bacterial infections are a major health burden worldwide, yet the mechanisms dictating host susceptibility to recurrence are poorly understood. Here we demonstrate that an initial bacterial infection of the urinary bladder with uropathogenic E. coli (UPEC) can induce sustained epigenetic changes in the bladder epithelial (urothelial) stem cells that reprogram the differentiated urothelium. We established urothelial stem cell (USC) lines from isogenic mice with different urinary tract infection histories (naïve, chronic or self-resolving). Differentiation of the USC lines in Transwell culture resulted in polarized urothelial cultures that recapitulated distinct remodeling morphologies seen in vivo. In addition, we discovered differences in chromatin accessibility that segregated by disease history, resulting in differences in gene expression upon differentiation of the USC lines in vitro, based on ATAC-seq analysis of the USC lines. Differential basal expression of Caspase-1 led to divergent susceptibilities to inflammatory cell death upon UPEC infection. In mice with a history of chronic infection, enhanced caspase 1-mediated inflammatory cell death was found to be a protective response that enhanced bacterial clearance upon challenge infection. Thus, UPEC infection reshapes the epigenome leading to epithelial-intrinsic remodeling that trains the mucosal immune response to subsequent infection. These findings may have broad implications for the prevention of chronic/recurrent bacterial infections.

Background Antibody response duration following severe acute respiratory syndrome coronavirus 2 infection tends to be variable and depends on severity of disease and method of detection. Study design and methods COVID‐19 convalescent plasma from 18 donors was collected longitudinally for a maximum of 63–129 days following resolution of symptoms. All the samples were initially screened by the Ortho total Ig test to confirm positivity and subsequently tested with seven additional direct sandwich or indirect binding assays (Ortho, Roche, Abbott, Broad Institute) directed against a variety of antigen targets (S1, receptor binding domain, and nucleocapsid [NC]), along with two neutralization assays (Broad Institute live virus PRNT and Vitalant Research Institute [VRI] Pseudovirus reporter viral particle neutralization [RVPN]). Results The direct detection assays (Ortho total Ig total and Roche total Ig) showed increasing levels of antibodies over the time period, in contrast to the indirect IgG assays that showed a decline. Neutralization assays also demonstrated declining responses; the VRI RVPN pseudovirus had a greater rate of decline than the Broad PRNT live virus assay. Discussion These data show that in addition to variable individual responses and associations with disease severity, the detection assay chosen contributes to the heterogeneous results in antibody stability over time. Depending on the scope of the research, one assay may be preferable over another. For serosurveillance studies, direct, double Ag‐sandwich assays appear to be the best choice due to their stability; in particular, algorithms that include both S1‐ and NC‐based assays can help reduce the rate of false‐positivity and discriminate between natural infection and vaccine‐derived seroreactivity.

Our understanding of how the gut microbiome interacts with its human host has been restrained by limited access to longitudinal datasets to examine stability and dynamics, and by having only a few isolates to test mechanistic hypotheses. Here, we present the Broad Institute-OpenBiome Microbiome Library (BIO-ML), a comprehensive collection of 7,758 gut bacterial isolates paired with 3,632 genome sequences and longitudinal multi-omics data. We show that microbial species maintain stable population sizes within and across humans and that commonly used ‘omics’ survey methods are more reliable when using averages over multiple days of sampling. Variation of gut metabolites within people over time is associated with amino acid levels, and differences across people are associated with differences in bile acids. Finally, we show that genomic diversification can be used to infer eco-evolutionary dynamics and in vivo selection pressures for strains within individuals. The BIO-ML is a unique resource designed to enable hypothesis-driven microbiome research. A comprehensive biobank of bacterial isolates with longitudinal and multi-omics characterization will advance understanding of the diversity and functions of human gut bacteria.

Mycobacterium tuberculosis ( Mtb ) killed more people in 2017 than any other single infectious agent. This dangerous pathogen is able to withstand stresses imposed by the immune system and tolerate exposure to antibiotics, resulting in persistent infection. The global tuberculosis (TB) epidemic has been exacerbated by the emergence of mutant strains of Mtb that are resistant to frontline antibiotics. Thus, both phenotypic drug tolerance and genetic drug resistance are major obstacles to successful TB therapy. Using a chemical approach to identify compounds that block stress and drug tolerance, as opposed to traditional screens for compounds that kill Mtb , we identified a small molecule, C10, that blocks tolerance to oxidative stress, acid stress, and the frontline antibiotic isoniazid (INH). In addition, we found that C10 prevents the selection for INH-resistant mutants and restores INH sensitivity in otherwise INH-resistant Mtb strains harboring mutations in the katG gene, which encodes the enzyme that converts the prodrug INH to its active form. Through mechanistic studies, we discovered that C10 inhibits Mtb respiration, revealing a link between respiration homeostasis and INH sensitivity. Therefore, by using C10 to dissect Mtb persistence, we discovered that INH resistance is not absolute and can be reversed.

Rapid bacterial identification remains a critical challenge in infectious disease diagnostics. We developed a novel molecular approach to detect and identify a wide diversity of bacterial pathogens in a single, simple assay, exploiting the conservation, abundance, and rich phylogenetic content of ribosomal RNA in a rapid fluorescent hybridization assay that requires no amplification or enzymology. Of 117 isolates from 64 species across 4 phyla, this assay identified bacteria with >89% accuracy at the species level and 100% accuracy at the family level, enabling all critical clinical distinctions. In pilot studies on primary clinical specimens, including sputum, blood cultures, and pus, bacteria from 5 different phyla were identified.

BackgroundAntibody response duration following SARS-CoV-2 infection tends to be variable and depends on severity of disease and method of detection.Study design and methodsCOVID-19 convalescent plasma (CCP) from 18 donors was collected longitudinally for a maximum of 63 - 129 days following resolution of symptoms. All the samples were initially screened by the Ortho Total Ig test to confirm positivity and subsequently tested with 7 additional direct sandwich or indirect binding assays (Ortho, Roche, Abbott, Broad Institute) directed against a variety of antigen targets (S1, RBD, and NC), along with 2 neutralization assays (Broad Institute live virus PRNT and Vitalant Research Institute Pseudovirus RVPN).ResultsThe direct detection assays (Ortho Total Ig total and Roche Total Ig) showed increasing levels of antibodies over the time period, in contrast to the indirect IgG assays that showed a decline.Neutralization assays also demonstrated declining responses; the VRI RVPN pseudovirus had a greater rate of decline than the Broad PRNT live virus assay.DiscussionThese data show that in addition to variable individual responses and associations with disease severity, the detection assay chosen contributes to the heterogeneous results in antibody stability over time. Depending on the scope of the research, one assay may be preferable over another. For serosurveillance studies, direct, double Ag-sandwich assays appear to be the best choice due to their stability; in particular, algorithms that include both S1 and NC based assays can help reduce the rate of false-positivity and discriminate between natural infection and vaccine-derived seroreactivity.

In this era of rising antibiotic resistance, in contrast to our increasing understanding of mechanisms that cause resistance, our understanding of mechanisms that influence the propensity to evolve resistance remains limited. Here, we identified genetic factors that facilitate the evolution of resistance to carbapenems, the antibiotic of “last resort,” inKlebsiella pneumoniae, the major carbapenem resistant species. In clinical isolates, we found that high-level transposon insertional mutagenesis plays an important role in contributing to high-level resistance frequencies in several major and emerging carbapenem-resistant lineages. A broader spectrum of resistance-conferring mutations for select carbapenems such as ertapenem also enables higher resistance frequencies and importantly, creates stepping-stones to achieve high-level resistance to all carbapenems. These mutational mechanisms can contribute to the evolution of resistance, in conjunction with the loss of systems that restrict horizontal resistance gene uptake, such as the CRISPR-Cas system. Given the need for greater antibiotic stewardship, these findings argue that in addition to considering the current efficacy of an antibiotic for a clinical isolate in antibiotic selection, considerations of future efficacy are also important. The genetic background of a clinical isolate and the exact antibiotic identity can and should also be considered as it is a determinant of a strain’s propensity to become resistant. Together, these findings thus provide a molecular framework for understanding acquisition of carbapenem resistance inK. pneumoniaewith important implications for diagnosing and treating this important class of pathogens.

The genomes of gut Bacteroidales contain numerous invertible regions, many of which contain promoters that dictate phase-variable synthesis of surface molecules such as polysaccharides, fimbriae, and outer surface proteins. Here, we characterize a different type of phase-variable system of Bacteroides fragilis, a Type I restriction modification system (R-M). We show that reversible DNA inversions within this R-M locus leads to the generation of eight specificity proteins with distinct recognition sites. In vitro grown bacteria have a different proportion of specificity gene combinations at the expression locus than bacteria isolated from the mammalian gut. By creating mutants, each able to produce only one specificity protein from this region, we identified the R-M recognition sites of four of these S-proteins using SMRT sequencing. Transcriptome analysis revealed that the locked specificity mutants, whether grown in vitro or isolated from the mammalian gut, have distinct transcriptional profiles, likely creating different phenotypes, one of which was confirmed. Genomic analyses of diverse strains of Bacteroidetes from both host-associated and environmental sources reveal the ubiquity of phase-variable R-M systems in this phylum.

Significance This work describes the most extensive discovery and functional characterization of small regulatory RNAs (sRNAs) in Mycobacterium tuberculosis to date. We comprehensively define the sRNAs expressed in M. tuberculosis under five host-like stress conditions. This reference dataset comprehensively defines the expression patterns and boundaries of mycobacterial sRNAs. We perform in-depth characterization of one sRNA, mycobacterial regulatory sRNA in iron (MrsI), which is induced in M. tuberculosis in multiple stress conditions. MrsI is critical for the iron-sparing response in mycobacteria by binding directly to mRNAs encoding nonessential iron-containing proteins to repress their expression. Interestingly, MrsI acts in an anticipatory manner, in which its induction by a variety of stresses primes M. tuberculosis to enter an iron-sparing state more rapidly upon iron deprivation., One key to the success of Mycobacterium tuberculosis as a pathogen is its ability to reside in the hostile environment of the human macrophage. Bacteria adapt to stress through a variety of mechanisms, including the use of small regulatory RNAs (sRNAs), which posttranscriptionally regulate bacterial gene expression. However, very little is currently known about mycobacterial sRNA-mediated riboregulation. To date, mycobacterial sRNA discovery has been performed primarily in log-phase growth, and no direct interaction between any mycobacterial sRNA and its targets has been validated. Here, we performed large-scale sRNA discovery and expression profiling in M. tuberculosis during exposure to five pathogenically relevant stresses. From these data, we identified a subset of sRNAs that are highly induced in multiple stress conditions. We focused on one of these sRNAs, ncRv11846, here renamed mycobacterial regulatory sRNA in iron (MrsI). We characterized the regulon of MrsI and showed in mycobacteria that it regulates one of its targets, bfrA, through a direct binding interaction. MrsI mediates an iron-sparing response that is required for optimal survival of M. tuberculosis under iron-limiting conditions. However, MrsI is induced by multiple host-like stressors, which appear to trigger MrsI as part of an anticipatory response to impending iron deprivation in the macrophage environment.

Summary The omnigenic/polygenic theory, which states that complex traits are not shaped by single/few genes, but by situation-specific large networks, offers an explanation for a major enigma in microbiology: deletion of specific small RNAs (sRNAs) playing key roles in various aspects of bacterial physiology, including virulence and antibiotic resistance, results in surprisingly subtle phenotypes. A recent study uncovered polar accumulation of most sRNAs upon osmotic stress, the majority not known to be involved in the applied stress. Here we show that cells deleted for a handful of pole-enriched sRNAs exhibit fitness defect in several stress conditions, as opposed to single, double, or triple sRNA-knockouts, implying that regulation by sRNA relies on sets of genes. Moreover, analysis of RNA-seq data of Escherichia coli and Salmonella typhimurium exposed to antibiotics and/or infection-relevant conditions reveals the involvement of multiple sRNAs in all cases, in line with the existence of a polygenic plan for sRNA-mediated regulation., Graphical abstract, Highlights • The level of numerous sRNAs increases upon various environmental conditions • Cells deleted for five pole-enriched sRNAs show a fitness defect in various stresses • No fitness defect is detected with single, double, or triple sRNA knockouts • Our data support the existence of a polygenic plan for sRNA-mediated regulation, Complex system biology; Microbiology; Molecular biology

Growing evidence suggests that microbes can influence the efficacy of cancer therapies. By studying colon cancer models, we found that bacteria can metabolize the chemotherapeutic drug gemcitabine (2′,2′-difluorodeoxycytidine) into its inactive form, 2′,2′-difluorodeoxyuridine. Metabolism was dependent on the expression of a long isoform of the bacterial enzyme cytidine deaminase (CDDL), seen primarily in Gammaproteobacteria. In a colon cancer mouse model, gemcitabine resistance was induced by intra-tumor Gammaproteobacteria, dependent on bacterial CDDL expression, and abrogated by co-treatment with the antibiotic ciprofloxacin. Gemcitabine is commonly used to treat pancreatic ductal adenocarcinoma (PDAC), and we hypothesized that intra-tumor bacteria might contribute to drug resistance of these tumors. Consistent with this possibility, we found that of the 113 human PDACs that were tested, 86 (76%) were positive for bacteria, mainly Gammaproteobacteria.

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications., United States. Air Force Office of Scientific Research (Grant FA9550-14-1-0060), Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-14-1-0006), National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706), National Institutes of Health (U.S.) (Grant 1R01-MH110049)

A mucosal infectious disease episode can render the host either more or less susceptible to recurrent infection, but the specific mechanisms that tip the balance remain unclear. We investigated this question in a mouse model of recurrent urinary tract infection and found that a prior bladder infection resulted in an earlier onset of tumor necrosis factor-alpha (TNFɑ)-mediated bladder inflammation upon subsequent bacterial challenge, relative to age-matched naive mice. However, the duration of TNFɑ signaling activation differed according to whether the first infection was chronic (Sensitized) or self-limiting (Resolved). TNFɑ depletion studies revealed that transient early-phase TNFɑ signaling in Resolved mice promoted clearance of bladder-colonizing bacteria via rapid recruitment of neutrophils and subsequent exfoliation of infected bladder cells. In contrast, sustained TNFɑ signaling in Sensitized mice prolonged damaging inflammation, worsening infection. This work reveals how TNFɑ signaling dynamics can be rewired by a prior infection to shape diverse susceptibilities to future mucosal infections.

Dual transcriptional profiling of host and bacteria during infection is challenging due to the low abundance of bacterial mRNA. We report Pathogen Hybrid Capture (PatH-Cap), a method to enrich for bacterial mRNA and deplete bacterial rRNA simultaneously from dual RNA-seq libraries using transcriptome-specific probes. By addressing both the differential RNA content of the host relative to the infecting bacterium and the overwhelming abundance of uninformative structural RNAs (rRNA, tRNA) of both species in a single step, this approach enables analysis of very low-input RNA samples. By sequencing libraries before (pre-PatH-Cap) and after (post-PatH-Cap) enrichment, we achieve dual transcriptional profiling of host and bacteria, respectively, from the same sample. Importantly, enrichment preserves relative transcript abundance and increases the number of unique bacterial transcripts per gene in post-PatH-Cap libraries compared to pre-PatH-Cap libraries at the same sequencing depth, thereby decreasing the sequencing depth required to fully capture the transcriptional profile of the infecting bacteria. We demonstrate that PatH-Cap enables the study of low-input samples including single eukaryotic cells infected by 1–3 Pseudomonas aeruginosa bacteria and paired host-pathogen temporal gene expression analysis of Mycobacterium tuberculosis infecting macrophages. PatH-Cap can be applied to the study of a range of pathogens and microbial species, and more generally, to lowly-abundant species in mixed populations.

Summary RNA localization in eukaryotes is a mechanism to regulate transcripts fate. Conversely, bacterial transcripts were not assumed to be specifically localized. We previously demonstrated that E. coli mRNAs may localize to where their products localize in a translation-independent manner, thus challenging the transcription-translation coupling extent. However, the scope of RNA localization in bacteria remained unknown. Here, we report the distribution of the E. coli transcriptome between the membrane, cytoplasm, and poles by combining cell fractionation with deep-sequencing (Rloc-seq). Our results reveal asymmetric RNA distribution on a transcriptome-wide scale, significantly correlating with proteome localization and prevalence of translation-independent RNA localization. The poles are enriched with stress-related mRNAs and small RNAs, the latter becoming further enriched upon stress in an Hfq-dependent manner. Genome organization may play a role in localizing membrane protein-encoding transcripts. Our results show an unexpected level of intricacy in bacterial transcriptome organization and highlight the poles as hubs for regulation.

Toxin synthesis and endospore formation are two of the most critical factors that determine the outcome of infection by Clostridioides difficile. The two major toxins, TcdA and TcdB, are the principal factors causing damage to the host. Spores are the infectious form of C. difficile, permit survival of the bacterium during antibiotic treatment and are the predominant cell form that leads to recurrent infection. Toxin production and sporulation have their own specific mechanisms of regulation, but they share negative regulation by the global regulatory protein CodY. Determining the extent of such regulation and its detailed mechanism is important for understanding the linkage between two apparently independent biological phenomena and raises the possibility of creating new ways of limiting infection. The work described here shows that a codY null mutant of a hypervirulent (ribotype 027) strain is even more virulent than its parent in a mouse model of infection and that the mutant expresses most sporulation genes prematurely during exponential growth phase. Moreover, examining the expression patterns of mutants producing CodY proteins with different levels of residual activity revealed that expression of the toxin genes is dependent on total CodY inactivation, whereas most sporulation genes are turned on when CodY activity is only partially diminished. These results suggest that, in wild-type cells undergoing nutrient limitation, sporulation genes can be turned on before the toxin genes.

The ability to simultaneously characterize the bacterial and host expression programs during infection would facilitate a comprehensive understanding of pathogen-host interactions. Although RNA sequencing (RNA-seq) has greatly advanced our ability to study the transcriptomes of prokaryotes and eukaryotes separately, limitations in existing protocols for the generation and analysis of RNA-seq data have hindered simultaneous profiling of host and bacterial pathogen transcripts from the same sample. Here we provide a detailed protocol for simultaneous analysis of host and bacterial transcripts by RNA-seq. Importantly, this protocol details the steps required for efficient host and bacteria lysis, barcoding of samples, technical advances in sample preparation for low-yield sample inputs and a computational pipeline for analysis of both mammalian and microbial reads from mixed host-pathogen RNA-seq data. Sample preparation takes 3 d from cultured cells to pooled libraries. Data analysis takes an additional day. Compared with previous methods, the protocol detailed here provides a sensitive, facile and generalizable approach that is suitable for large-scale studies and will enable the field to obtain in-depth analysis of host-pathogen interactions in infection models.

The global regulator CodY controls the expression of dozens of metabolism and virulence genes in the opportunistic pathogen Staphylococcus aureus in response to the availability of isoleucine, leucine and valine (ILV), and GTP. Using RNA-Seq transcriptional profiling and partial activity variants, we reveal that S. aureus CodY activity grades metabolic and virulence gene expression as a function of ILV availability, mediating metabolic reorganization and controlling virulence factor production in vitro. Strains lacking CodY regulatory activity produce a PIA-dependent biofilm, but development is restricted under conditions that confer partial CodY activity. CodY regulates the expression of thermonuclease (nuc) via the Sae two-component system, revealing cascading virulence regulation and factor production as CodY activity is reduced. Proteins that mediate the host-pathogen interaction and subvert the immune response are shut off at intermediate levels of CodY activity, while genes coding for enzymes and proteins that extract nutrients from tissue, that kill host cells, and that synthesize amino acids are among the last genes to be derepressed. We conclude that S. aureus uses CodY to limit host damage to only the most severe starvation conditions, providing insight into one potential mechanism by which S. aureus transitions from a commensal bacterium to an invasive pathogen.

This study was conducted to investigate the role of iron deprivation in the persistence of Mycobacterium tuberculosis. We present evidence of iron restriction in human necrotic granulomas and demonstrate that under iron starvation M. tuberculosis persists, refractive to antibiotics and capable of restarting replication when iron is made available. Transcriptomics and metabolomic analyses indicated that the persistence of M. tuberculosis under iron starvation is dependent on strict control of endogenous Fe utilization and is associated with upregulation of pathogenicity and intrinsic antibiotic resistance determinants. M. tuberculosis mutants compromised in their ability to survive Fe starvation were identified. The findings of this study advance the understanding of the physiological settings that may underpin the chronicity of human tuberculosis (TB) and are relevant to the design of effective antitubercular therapies., IMPORTANCE One-third of the world population may harbor persistent M. tuberculosis, causing an asymptomatic infection that is refractory to treatment and can reactivate to become potentially lethal tuberculosis disease. However, little is known about the factors that trigger and maintain M. tuberculosis persistence in infected individuals. Iron is an essential nutrient for M. tuberculosis growth. In this study, we show, first, that in human granulomas the immune defense creates microenvironments in which M. tuberculosis likely experiences drastic Fe deprivation and, second, that Fe-starved M. tuberculosis is capable of long-term persistence without growth. Together, these observations suggest that Fe deprivation in the lung might trigger a state of persistence in M. tuberculosis and promote chronic TB. We also identified vulnerabilities of iron-restricted persistent M. tuberculosis, which can be exploited for the design of new antitubercular therapies.

Urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC) strains. In contrast to many enteric E. coli pathogroups, no genetic signature has been identified for UPEC strains. We conducted a high-resolution comparative genomic study using E. coli isolates collected from the urine of women suffering from frequent recurrent UTIs. These isolates were genetically diverse and varied in their urovirulence, that is, their ability to infect the bladder in a mouse model of cystitis. We found no set of genes, including previously defined putative urovirulence factors (PUFs), that were predictive of urovirulence. In addition, in some patients, the E. coli strain causing a recurrent UTI had fewer PUFs than the supplanted strain. In competitive experimental infections in mice, the supplanting strain was more efficient at colonizing the mouse bladder than the supplanted strain. Despite the lack of a clear genomic signature for urovirulence, comparative transcriptomic and phenotypic analyses revealed that the expression of key conserved functions during culture, such as motility and metabolism, could be used to predict subsequent colonization of the mouse bladder. Together, our findings suggest that UTI risk and outcome may be determined by complex interactions between host susceptibility and the urovirulence potential of diverse bacterial strains.