Transcriptional Response of Burkholderia cenocepacia H111 to Severe Zinc Starvation. - Related Documents




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829601.0000Transcriptional Response of Burkholderia cenocepacia H111 to Severe Zinc Starvation. Burkholderia cenocepacia is an opportunistic pathogen that is primarily associated with severe respiratory infections in people with cystic fibrosis. These bacteria have significant intrinsic resistance to antimicrobial therapy, and there is a need for more effective treatments. Bacterial zinc uptake and homeostasis systems are attractive targets for new drugs, yet our understanding of how bacteria acquire and utilise zinc remains incomplete. Here we have used RNA-sequencing and differential gene expression analysis to investigate how B. cenocepacia H111 is able to survive in zinc poor environments, such as those expected to be encountered within the host. The data shows that 201 genes are significantly differentially expressed when zinc supply is severely limited. Included in the 85 upregulated genes, are genes encoding a putative ZnuABC high affinity zinc importer, two TonB-dependent outer membrane receptors that may facilitate zinc uptake across the outer cell membrane, and a COG0523 family zinc metallochaperone. Amongst the 116 downregulated genes, are several zinc-dependent enzymes suggesting a mechanism of zinc sparring to reduce the cells demand for zinc when bioavailability is low.202337822354
892210.9997Transitioning from Soil to Host: Comparative Transcriptome Analysis Reveals the Burkholderia pseudomallei Response to Different Niches. Burkholderia pseudomallei, a soil and water saprophyte, is responsible for the tropical human disease melioidosis. A hundred years since its discovery, there is still much to learn about B. pseudomallei proteins that are essential for the bacterium's survival in and interaction with the infected host, as well as their roles within the bacterium's natural soil habitat. To address this gap, bacteria grown under conditions mimicking the soil environment were subjected to transcriptome sequencing (RNA-seq) analysis. A dual RNA-seq approach was used on total RNA from spleens isolated from a B. pseudomallei mouse infection model at 5 days postinfection. Under these conditions, a total of 1,434 bacterial genes were induced, with 959 induced in the soil environment and 475 induced in bacteria residing within the host. Genes encoding metabolism and transporter proteins were induced when the bacteria were present in soil, while virulence factors, metabolism, and bacterial defense mechanisms were upregulated during active infection of mice. On the other hand, capsular polysaccharide and quorum-sensing pathways were inhibited during infection. In addition to virulence factors, reactive oxygen species, heat shock proteins, siderophores, and secondary metabolites were also induced to assist bacterial adaptation and survival in the host. Overall, this study provides crucial insights into the transcriptome-level adaptations which facilitate infection by soil-dwelling B. pseudomallei. Targeting novel therapeutics toward B. pseudomallei proteins required for adaptation provides an alternative treatment strategy given its intrinsic antimicrobial resistance and the absence of a vaccine. IMPORTANCE Burkholderia pseudomallei, a soil-dwelling bacterium, is the causative agent of melioidosis, a fatal infectious disease of humans and animals. The bacterium has a large genome consisting of two chromosomes carrying genes that encode proteins with important roles for survival in diverse environments as well as in the infected host. While a general mechanism of pathogenesis has been proposed, it is not clear which proteins have major roles when the bacteria are in the soil and whether the same proteins are key to successful infection and spread. To address this question, we grew the bacteria in soil medium and then in infected mice. At 5 days postinfection, bacteria were recovered from infected mouse organs and their gene expression was compared against that of bacteria grown in soil medium. The analysis revealed a list of genes expressed under soil growth conditions and a different set of genes encoding proteins which may be important for survival, replication, and dissemination in an infected host. These proteins are a potential resource for understanding the full adaptation mechanism of this pathogen. In the absence of a vaccine for melioidosis and with treatment being reliant on combinatorial antibiotic therapy, these proteins may be ideal targets for designing antimicrobials to treat melioidosis.202336856434
830820.9996PhoPQ Regulates Quinolone and Cephalosporin Resistance Formation in Salmonella Enteritidis at the Transcriptional Level. The two-component system (TCS) PhoPQ has been demonstrated to be crucial for the formation of resistance to quinolones and cephalosporins in Salmonella Enteritidis (S. Enteritidis). However, the mechanism underlying PhoPQ-mediated antibiotic resistance formation remains poorly understood. Here, it was shown that PhoP transcriptionally regulated an assortment of genes associated with envelope homeostasis, the osmotic stress response, and the redox balance to confer resistance to quinolones and cephalosporins in S. Enteritidis. Specifically, cells lacking the PhoP regulator, under nalidixic acid and ceftazidime stress, bore a severely compromised membrane on the aspects of integrity, fluidity, and permeability, with deficiency to withstand osmolarity stress, an increased accumulation of intracellular reactive oxygen species, and dysregulated redox homeostasis, which are unfavorable for bacterial survival. The phosphorylated PhoP elicited transcriptional alterations of resistance-associated genes, including the outer membrane porin ompF and the aconitate hydratase acnA, by directly binding to their promoters, leading to a limited influx of antibiotics and a well-maintained intracellular metabolism. Importantly, it was demonstrated that the cavity of the PhoQ sensor domain bound to and sensed quinolones/cephalosporins via the crucial surrounding residues, as their mutations abrogated the binding and PhoQ autophosphorylation. This recognition mode promoted signal transduction that activated PhoP, thereby modulating the transcription of downstream genes to accommodate cells to antibiotic stress. These findings have revealed how bacteria employ a specific TCS to sense antibiotics and combat them, suggesting PhoPQ as a potential drug target with which to surmount S. Enteritidis. IMPORTANCE The prevalence of quinolone and cephalosporin-resistant S. Enteritidis is of increasing clinical concern. Thus, it is imperative to identify novel therapeutic targets with which to treat S. Enteritidis-associated infections. The PhoPQ two-component system is conserved across a variety of Gram-negative pathogens, by which bacteria adapt to a range of environmental stimuli. Our earlier work has demonstrated the importance of PhoPQ in the resistance formation in S. Enteritidis to quinolones and cephalosporins. In the current work, we identified a global profile of genes that are regulated by PhoP under antibiotic stresses, with a focus on how PhoP regulated downstream genes, either positively or negatively. Additionally, we established that PhoQ sensed quinolones and cephalosporins in a manner of directly binding to them. These identified genes and pathways that are mediated by PhoPQ represent promising targets for the development of a drug potentiator with which to neutralize antibiotic resistance in S. Enteritidis.202337184399
830430.9996A Shift to Human Body Temperature (37°C) Rapidly Reprograms Multiple Adaptive Responses in Escherichia coli That Would Facilitate Niche Survival and Colonization. One of the first environmental cues sensed by a microbe as it enters a human host is an upshift in temperature to 37°C. In this dynamic time point analysis, we demonstrate that this environmental transition rapidly signals a multitude of gene expression changes in Escherichia coli. Bacteria grown at 23°C under aerobic conditions were shifted to 37°C, and mRNA expression was measured at time points after the shift to 37°C (t = 0.5, 1, and 4 h). The first hour is characterized by a transient shift to anaerobic respiration strategies and stress responses, particularly acid resistance, indicating that temperature serves as a sentinel cue to predict and prepare for various niches within the host. The temperature effects on a subset of stress response genes were shown to be mediated by RpoS and directly correlated with RpoS, DsrA, and RprA levels, and increased acid resistance was observed that was dependent on 23°C growth and RpoS. By 4 h, gene expression shifted to aerobic respiration pathways and decreased stress responses, coupled with increases in genes associated with biosynthesis (amino acid and nucleotides), iron uptake, and host defense. ompT, a gene that confers resistance to antimicrobial peptides, was highly thermoregulated, with a pattern conserved in enteropathogenic and uropathogenic E. coli strains. An immediate decrease in curli gene expression concomitant with an increase in flagellar gene expression implicates temperature in this developmental decision. Together, our studies demonstrate that temperature signals a reprogramming of gene expression immediately upon an upshift that may predict, prepare, and benefit the survival of the bacterium within the host. IMPORTANCE As one of the first cues sensed by the microbe upon entry into a human host, understanding how bacteria like E. coli modulate gene expression in response to temperature improves our understanding of how bacteria immediately initiate responses beneficial for survival and colonization. For pathogens, understanding the various pathways of thermal regulation could yield valuable targets for anti-infective chemotherapeutic drugs or disinfection measures. In addition, our data provide a dynamic examination of the RpoS stress response, providing genome-wide support for how temperature impacts RpoS through changes in RpoS stability and modulation by small regulatory RNAs.202134516284
77240.9996A Transcriptomic Approach to Identify Novel Drug Efflux Pumps in Bacteria. The core genomes of most bacterial species include a large number of genes encoding putative efflux pumps. The functional roles of most of these pumps are unknown, however, they are often under tight regulatory control and expressed in response to their substrates. Therefore, one way to identify pumps that function in antimicrobial resistance is to examine the transcriptional responses of efflux pump genes to antimicrobial shock. By conducting complete transcriptomic experiments following antimicrobial shock treatments, it may be possible to identify novel drug efflux pumps encoded in bacterial genomes. In this chapter we describe a complete workflow for conducting transcriptomic analyses by RNA sequencing, to determine transcriptional changes in bacteria responding to antimicrobials.201829177833
829850.9996Cellular Management of Zinc in Group B Streptococcus Supports Bacterial Resistance against Metal Intoxication and Promotes Disseminated Infection. Zinc is an essential trace element for normal bacterial physiology but, divergently, can intoxicate bacteria at high concentrations. Here, we define the molecular systems for Zn detoxification in Streptococcus agalactiae, also known as group B streptococcus, and examine the effects of resistance to Zn stress on virulence. We compared the growth of wild-type bacteria and mutants deleted for the Zn exporter, czcD, and the response regulator, sczA, using Zn-stress conditions in vitro Macrophage antibiotic protection assays and a mouse model of disseminated infection were used to assess virulence. Global bacterial transcriptional responses to Zn stress were defined by RNA sequencing and quantitative reverse transcription-PCR. czcD and sczA enabled S. agalactiae to survive Zn stress, with the putative CzcD efflux system activated by SczA. Additional genes activated in response to Zn stress encompassed divalent cation transporters that contribute to regulation of Mn and Fe homeostasis. In vivo, the czcD-sczA Zn management axis supported virulence in the blood, heart, liver, and bladder. Additionally, several genes not previously linked to Zn stress in any bacterium, including, most notably, arcA for arginine deamination, also mediated resistance to Zn stress, representing a novel molecular mechanism of bacterial resistance to metal intoxication. Taken together, these findings show that S. agalactiae responds to Zn stress by sczA regulation of czcD, with additional novel mechanisms of resistance supported by arcA, encoding arginine deaminase. Cellular management of Zn stress in S. agalactiae supports virulence by facilitating bacterial survival in the host during systemic infection.IMPORTANCEStreptococcus agalactiae, also known as group B streptococcus, is an opportunistic pathogen that causes various diseases in humans and animals. This bacterium has genetic systems that enable zinc detoxification in environments of metal stress, but these systems remain largely undefined. Using a combination of genomic, genetic, and cellular assays, we show that this pathogen controls Zn export through CzcD to manage Zn stress and utilizes a system of arginine deamination never previously linked to metal stress responses in bacteria to survive metal intoxication. We show that these systems are crucial for survival of S. agalactiaein vitro during Zn stress and also enhance virulence during systemic infection in mice. These discoveries establish new molecular mechanisms of resistance to metal intoxication in bacteria; we suggest these mechanisms operate in other bacteria as a way to sustain microbial survival under conditions of metal stress, including in host environments.202134011683
829960.9996Regulatory cross-talk supports resistance to Zn intoxication in Streptococcus. Metals such as copper (Cu) and zinc (Zn) are important trace elements that can affect bacterial cell physiology but can also intoxicate bacteria at high concentrations. Discrete genetic systems for management of Cu and Zn efflux have been described in several bacterial pathogens, including streptococci. However, insight into molecular cross-talk between systems for Cu and Zn management in bacteria that drive metal detoxification, is limited. Here, we describe a biologically consequential cross-system effect of metal management in group B Streptococcus (GBS) governed by the Cu-responsive copY regulator in response to Zn. RNAseq analysis of wild-type (WT) and copY-deficient GBS subjected to metal stress revealed unique transcriptional links between the systems for Cu and Zn detoxification. We show that the Cu-sensing role of CopY extends beyond Cu and enables CopY to regulate Cu and Zn stress responses that effect changes in gene function for central cellular processes, including riboflavin synthesis. CopY also supported GBS intracellular survival in human macrophages and virulence during disseminated infection in mice. In addition, we show a novel role for CovR in modulating GBS resistance to Zn intoxication. Identification of the Zn resistome of GBS using TraDIS revealed a suite of genes essential for GBS growth in metal stress. Several of the genes identified are novel to systems that support bacterial survival in metal stress and represent a diverse set of mechanisms that underpin microbial metal homeostasis during cell stress. Overall, this study reveals a new and important mechanism of cross-system complexity driven by CopY in bacteria to regulate cellular management of metal stress and survival.202235862444
830170.9996Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance. Aminoglycoside antibiotics target ribosomes and are effective against a wide range of bacteria. Here, we demonstrated that knockout strains related to energy metabolism in Escherichia coli showed increased tolerance to aminoglycosides during the mid-exponential growth phase. Contrary to expectations, these mutations did not reduce the proton motive force or aminoglycoside uptake, as there were no significant changes in metabolic indicators or intracellular gentamicin levels between wild-type and mutant strains. Our comprehensive proteomics analysis unveiled a noteworthy upregulation of proteins linked to the tricarboxylic acid (TCA) cycle in the mutant strains during the mid-exponential growth phase, suggesting that these strains compensate for the perturbation in their energy metabolism by increasing TCA cycle activity to maintain their membrane potential and ATP levels. Furthermore, our pathway enrichment analysis shed light on local network clusters displaying downregulation across all mutant strains, which were associated with both large and small ribosomal binding proteins, ribosome biogenesis, translation factor activity, and the biosynthesis of ribonucleoside monophosphates. These findings offer a plausible explanation for the observed tolerance of aminoglycosides in the mutant strains. Altogether, this research provides valuable insights into the mechanisms of aminoglycoside tolerance, paving the way for novel strategies to combat such cells.202439093940
834080.9996Iron-Induced Respiration Promotes Antibiotic Resistance in Actinomycete Bacteria. The bacterial response to antibiotics eliciting resistance is one of the key challenges in global health. Despite many attempts to understand intrinsic antibiotic resistance, many of the underlying mechanisms still remain elusive. In this study, we found that iron supplementation promoted antibiotic resistance in Streptomyces coelicolor. Iron-promoted resistance occurred specifically against bactericidal antibiotics, irrespective of the primary target of antibiotics. Transcriptome profiling revealed that some genes in the central metabolism and respiration were upregulated under iron-replete conditions. Iron supported the growth of S. coelicolor even under anaerobic conditions. In the presence of potassium cyanide, which reduces aerobic respiration of cells, iron still promoted respiration and antibiotic resistance. This suggests the involvement of a KCN-insensitive type of respiration in the iron effect. This phenomenon was also observed in another actinobacterium, Mycobacterium smegmatis. Taken together, these findings provide insight into a bacterial resistance strategy that mitigates the activity of bactericidal antibiotics whose efficacy accompanies oxidative damage by switching the respiration mode. IMPORTANCE A widely investigated mode of antibiotic resistance occurs via mutations and/or by horizontal acquisition of resistance genes. In addition to this acquired resistance, most bacteria exhibit intrinsic resistance as an inducible and adaptive response to different classes of antibiotics. Increasing attention has been paid recently to intrinsic resistance mechanisms because this may provide novel therapeutic targets that help rejuvenate the efficacy of the current antibiotic regimen. In this study, we demonstrate that iron promotes the intrinsic resistance of aerobic actinomycetes Streptomyces coelicolor and Mycobacterium smegmatis against bactericidal antibiotics. A surprising role of iron to increase respiration, especially in a mode of using less oxygen, appears a fitting strategy to cope with bactericidal antibiotics known to kill bacteria through oxidative damage. This provides new insights into developing antimicrobial treatments based on the availability of iron and oxygen.202235357210
896790.9996Distinct transcriptomic response of S. coelicolor to ciprofloxacin in a nutrient-rich environment. With the rising threat of anti-microbial resistance (AMR), there is an urgent need to enhance efficacy of existing antibiotics. Understanding the myriad mechanisms through which bacteria evade these drugs would be of immense value to designing novel strategies against them. Streptomyces coelicolor A3(2) M145 belongs to the actinomyctes species that are responsible for more than two-thirds of antibiotics. This group of bacteria therefore encodes for various mechanisms that can resist both endogenous and non-endogenous antibiotics. In an earlier study, we had studied the transcriptomic response of these bacteria to ciprofloxacin, when cultured in a minimal media. In this work, we investigate why the minimum inhibitory concentration of the drug increases by fourfold when the bacteria are grown in a nutrient-rich media. Through transcriptomic, biochemical, and microscopic studies, we show that S. coelicolor responds to ciprofloxacin in a concentration-dependent manner. While, sub-inhibitory concentration of the drug primarily causes oxidative stress, the inhibitory concentration of ciprofloxacin evokes a more severe genome-wide response in the cell, which ranges from the familiar upregulation of the SOS response and DNA repair pathways to the widespread alterations in the central metabolism pathway to accommodate the increased needs of nucleotides and other precursors. Further, the upregulation of peptidoglycan synthesis genes, along with microscopy images, suggest alterations in the cell morphology to increase fitness of the bacteria during the antibiotic stress. The data also points to the enhanced efflux activity in cells cultured in rich media that contributes significantly towards reducing intracellular drug concentration and thus promotes survival.201830327831
8305100.9996Light Modulates Metabolic Pathways and Other Novel Physiological Traits in the Human Pathogen Acinetobacter baumannii. Light sensing in chemotrophic bacteria has been relatively recently ascertained. In the human pathogen Acinetobacter baumannii, light modulates motility, biofilm formation, and virulence through the blue-light-sensing-using flavin (BLUF) photoreceptor BlsA. In addition, light can induce a reduction in susceptibility to certain antibiotics, such as minocycline and tigecycline, in a photoreceptor-independent manner. In this work, we identified new traits whose expression levels are modulated by light in this pathogen, which comprise not only important determinants related to pathogenicity and antibiotic resistance but also metabolic pathways, which represents a novel concept for chemotrophic bacteria. Indeed, the phenylacetic acid catabolic pathway and trehalose biosynthesis were modulated by light, responses that completely depend on BlsA. We further show that tolerance to some antibiotics and modulation of antioxidant enzyme levels are also influenced by light, likely contributing to bacterial persistence in adverse environments. Also, we present evidence indicating that surfactant production is modulated by light. Finally, the expression of whole pathways and gene clusters, such as genes involved in lipid metabolism and genes encoding components of the type VI secretion system, as well as efflux pumps related to antibiotic resistance, was differentially induced by light. Overall, our results indicate that light modulates global features of the A. baumannii lifestyle.IMPORTANCE The discovery that nonphototrophic bacteria respond to light constituted a novel concept in microbiology. In this context, we demonstrated that light could modulate aspects related to bacterial virulence, persistence, and resistance to antibiotics in the human pathogen Acinetobacter baumannii In this work, we present the novel finding that light directly regulates metabolism in this chemotrophic bacterium. Insights into the mechanism show the involvement of the photoreceptor BlsA. In addition, tolerance to antibiotics and catalase levels are also influenced by light, likely contributing to bacterial persistence in adverse environments, as is the expression of the type VI secretion system and efflux pumps. Overall, a profound influence of light on the lifestyle of A. baumannii is suggested to occur.201728289081
8310110.9996Dynamic heterogeneity in an E. coli stress response regulon mediates gene activation and antimicrobial peptide tolerance. The bacterial stress response is an intricately regulated system that plays a critical role in cellular resistance to drug treatment. The complexity of this response is further complicated by cell-to-cell heterogeneity in the expression of bacterial stress response genes. These genes are often organized into networks comprising one or more transcriptional regulators that control expression of a suite of downstream genes. While the expression heterogeneity of many of these upstream regulators has been characterized, the way in which this variability affects the larger downstream stress response remains hard to predict, prompting two key questions. First, how does heterogeneity and expression noise in stress response regulators propagate to the diverse downstream genes in their regulons. Second, when expression levels vary, how do multiple downstream genes act together to protect cells from stress. To address these questions, we focus on the transcription factor PhoP, a critical virulence regulator which coordinates pathogenicity in several gram-negative species. We use optogenetic stimulation to precisely control PhoP expression levels and examine how variations in PhoP affect the downstream activation of genes in the PhoP regulon. We find that these downstream genes exhibit differences both in mean expression level and sensitivity to increasing levels of PhoP. These response functions can also vary between individual cells, increasing heterogeneity in the population. We tie these variations to cell survival when bacteria are exposed to a clinically-relevant antimicrobial peptide, showing that high expression of the PhoP-regulon gene pmrD provides a protective effect against Polymyxin B. Overall, we demonstrate that even subtle heterogeneity in expression of a stress response regulator can have clear consequences for enabling bacteria to survive stress.202439677761
8302120.9996Auxin-mediated regulation of susceptibility to toxic metabolites, c-di-GMP levels, and phage infection in the rhizobacterium Serratia plymuthica. The communication between plants and their microbiota is highly dynamic and involves a complex network of signal molecules. Among them, the auxin indole-3-acetic acid (IAA) is a critical phytohormone that not only regulates plant growth and development, but is emerging as an important inter- and intra-kingdom signal that modulates many bacterial processes that are important during interaction with their plant hosts. However, the corresponding signaling cascades remain largely unknown. Here, we advance our understanding of the largely unknown mechanisms by which IAA carries out its regulatory functions in plant-associated bacteria. We showed that IAA caused important changes in the global transcriptome of the rhizobacterium Serratia plymuthica and multidisciplinary approaches revealed that IAA sensing interferes with the signaling mediated by other pivotal plant-derived signals such as amino acids and 4-hydroxybenzoic acid. Exposure to IAA caused large alterations in the transcript levels of genes involved in amino acid metabolism, resulting in significant metabolic alterations. IAA treatment also increased resistance to toxic aromatic compounds through the induction of the AaeXAB pump, which also confers resistance to IAA. Furthermore, IAA promoted motility and severely inhibited biofilm formation; phenotypes that were associated with decreased c-di-GMP levels and capsule production. IAA increased capsule gene expression and enhanced bacterial sensitivity to a capsule-dependent phage. Additionally, IAA induced the expression of several genes involved in antibiotic resistance and led to changes in the susceptibility and responses to antibiotics with different mechanisms of action. Collectively, our study illustrates the complexity of IAA-mediated signaling in plant-associated bacteria. IMPORTANCE: Signal sensing plays an important role in bacterial adaptation to ecological niches and hosts. This communication appears to be particularly important in plant-associated bacteria since they possess a large number of signal transduction systems that respond to a wide diversity of chemical, physical, and biological stimuli. IAA is emerging as a key inter- and intra-kingdom signal molecule that regulates a variety of bacterial processes. However, despite the extensive knowledge of the IAA-mediated regulatory mechanisms in plants, IAA signaling in bacteria remains largely unknown. Here, we provide insight into the diversity of mechanisms by which IAA regulates primary and secondary metabolism, biofilm formation, motility, antibiotic susceptibility, and phage sensitivity in a biocontrol rhizobacterium. This work has important implications for our understanding of bacterial ecology in plant environments and for the biotechnological and clinical applications of IAA, as well as related molecules.202438837409
8309130.9996The expression of virulence genes increases membrane permeability and sensitivity to envelope stress in Salmonella Typhimurium. Virulence gene expression can represent a substantial fitness cost to pathogenic bacteria. In the model entero-pathogen Salmonella Typhimurium (S.Tm), such cost favors emergence of attenuated variants during infections that harbor mutations in transcriptional activators of virulence genes (e.g., hilD and hilC). Therefore, understanding the cost of virulence and how it relates to virulence regulation could allow the identification and modulation of ecological factors to drive the evolution of S.Tm toward attenuation. In this study, investigations of membrane status and stress resistance demonstrate that the wild-type (WT) expression level of virulence factors embedded in the envelope increases membrane permeability and sensitizes S.Tm to membrane stress. This is independent from a previously described growth defect associated with virulence gene expression in S.Tm. Pretreating the bacteria with sublethal stress inhibited virulence expression and increased stress resistance. This trade-off between virulence and stress resistance could explain the repression of virulence expression in response to harsh environments in S.Tm. Moreover, we show that virulence-associated stress sensitivity is a burden during infection in mice, contributing to the inherent instability of S.Tm virulence. As most bacterial pathogens critically rely on deploying virulence factors in their membrane, our findings could have a broad impact toward the development of antivirulence strategies.202235389980
8964140.9996Analysis of the Oxidative Stress Regulon Identifies soxS as a Genetic Target for Resistance Reversal in Multidrug-Resistant Klebsiella pneumoniae. In bacteria, the defense system deployed to counter oxidative stress is orchestrated by three transcriptional factors, SoxS, SoxR, and OxyR. Although the regulon that these factors control is known in many bacteria, similar data are not available for Klebsiella pneumoniae. To address this data gap, oxidative stress was artificially induced in K. pneumoniae MGH78578 using paraquat and the corresponding oxidative stress regulon recorded using transcriptome sequencing (RNA-seq). The soxS gene was significantly induced during oxidative stress, and a knockout mutant was constructed to explore its functionality. The wild type and mutant were grown in the presence of paraquat and subjected to RNA-seq to elucidate the soxS regulon in K. pneumoniae MGH78578. Genes that are commonly regulated both in the oxidative stress and soxS regulons were identified and denoted as the oxidative SoxS regulon; these included a group of genes specifically regulated by SoxS. Efflux pump-encoding genes and global regulators were identified as part of this regulon. Consequently, the isogenic soxS mutant was found to exhibit a reduction in the minimum bactericidal concentration against tetracycline compared to that of the wild type. Impaired efflux activity, allowing tetracycline to be accumulated in the cytoplasm to bactericidal levels, was further evaluated using a tetraphenylphosphonium (TPP(+)) accumulation assay. The soxS mutant was also susceptible to tetracycline in vivo in a zebrafish embryo model. We conclude that the soxS gene could be considered a genetic target against which an inhibitor could be developed and used in combinatorial therapy to combat infections associated with multidrug-resistant K. pneumoniae. IMPORTANCE Antimicrobial resistance is a global health challenge. Few new antibiotics have been developed for use over the years, and preserving the efficacy of existing compounds is an important step to protect public health. This paper describes a study that examines the effects of exogenously induced oxidative stress on K. pneumoniae and uncovers a target that could be useful to harness as a strategy to mitigate resistance.202134098732
6342150.9995Determinants of Extreme β-Lactam Tolerance in the Burkholderia pseudomallei Complex. Slow-growing bacteria are insensitive to killing by antibiotics, a trait known as antibiotic tolerance. In this study, we characterized the genetic basis of an unusually robust β-lactam (meropenem) tolerance seen in Burkholderia species. We identified tolerance genes under three different slow-growth conditions by extensive transposon mutant sequencing (Tn-seq), followed by single mutant validation. There were three principal findings. First, mutations in a small number of genes reduced tolerance under multiple conditions. Most of the functions appeared to be specific to peptidoglycan synthesis and the response to its disruption by meropenem action rather than being associated with more general physiological processes. The top tolerance genes are involved in immunity toward a type VI toxin targeting peptidoglycan (BTH_I0069), peptidoglycan recycling (ldcA), periplasmic regulation by proteolysis (prc), and an envelope stress response (rpoE and degS). Second, most of the tolerance functions did not contribute to growth in the presence of meropenem (intrinsic resistance), indicating that the two traits are largely distinct. Third, orthologues of many of the top Burkholderia thailandensis tolerance genes were also important in Burkholderia pseudomallei Overall, these studies show that the determinants of meropenem tolerance differ considerably depending on cultivation conditions, but that there are a few shared functions with strong mutant phenotypes that are important in multiple Burkholderia species.201829439964
9607160.9995Transcriptome-Level Signatures in Gene Expression and Gene Expression Variability during Bacterial Adaptive Evolution. Antibiotic-resistant bacteria are an increasingly serious public health concern, as strains emerge that demonstrate resistance to almost all available treatments. One factor that contributes to the crisis is the adaptive ability of bacteria, which exhibit remarkable phenotypic and gene expression heterogeneity in order to gain a survival advantage in damaging environments. This high degree of variability in gene expression across biological populations makes it a challenging task to identify key regulators of bacterial adaptation. Here, we research the regulation of adaptive resistance by investigating transcriptome profiles of Escherichia coli upon adaptation to disparate toxins, including antibiotics and biofuels. We locate potential target genes via conventional gene expression analysis as well as using a new analysis technique examining differential gene expression variability. By investigating trends across the diverse adaptation conditions, we identify a focused set of genes with conserved behavior, including those involved in cell motility, metabolism, membrane structure, and transport, and several genes of unknown function. To validate the biological relevance of the observed changes, we synthetically perturb gene expression using clustered regularly interspaced short palindromic repeat (CRISPR)-dCas9. Manipulation of select genes in combination with antibiotic treatment promotes adaptive resistance as demonstrated by an increased degree of antibiotic tolerance and heterogeneity in MICs. We study the mechanisms by which identified genes influence adaptation and find that select differentially variable genes have the potential to impact metabolic rates, mutation rates, and motility. Overall, this work provides evidence for a complex nongenetic response, encompassing shifts in gene expression and gene expression variability, which underlies adaptive resistance. IMPORTANCE Even initially sensitive bacteria can rapidly thwart antibiotic treatment through stress response processes known as adaptive resistance. Adaptive resistance fosters transient tolerance increases and the emergence of mutations conferring heritable drug resistance. In order to extend the applicable lifetime of new antibiotics, we must seek to hinder the occurrence of bacterial adaptive resistance; however, the regulation of adaptation is difficult to identify due to immense heterogeneity emerging during evolution. This study specifically seeks to generate heterogeneity by adapting bacteria to different stresses and then examines gene expression trends across the disparate populations in order to pinpoint key genes and pathways associated with adaptive resistance. The targets identified here may eventually inform strategies for impeding adaptive resistance and prolonging the effectiveness of antibiotic treatment.201728217741
8297170.9995Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase. Gram-negative bacteria have effective methods of excluding toxic compounds, including a largely impermeable outer membrane (OM) and a range of efflux pumps. Furthermore, when cells become nutrient limited, RpoS enacts a global expression change providing cross-protection against many stresses. Here, we utilized sensitivity to an anionic detergent (sodium dodecyl sulfate [SDS]) to probe changes occurring to the cell's permeability barrier during nutrient limitation. Escherichia coli is resistant to SDS whether cells are actively growing, carbon limited, or nitrogen limited. In actively growing cells, this resistance depends on the AcrAB-TolC efflux pump; however, this pump is not necessary for protection under either carbon-limiting or nitrogen-limiting conditions, suggesting an alternative mechanism(s) of SDS resistance. In carbon-limited cells, RpoS-dependent pathways lessen the permeability of the OM, preventing the necessity for efflux. In nitrogen-limited but not carbon-limited cells, the loss of rpoS can be completely compensated for by the AcrAB-TolC efflux pump. We suggest that this difference simply reflects the fact that nitrogen-limited cells have access to a metabolizable energy (carbon) source that can efficiently power the efflux pump. Using a transposon mutant pool sequencing (Tn-Seq) approach, we identified three genes, sanA, dacA, and yhdP, that are necessary for RpoS-dependent SDS resistance in carbon-limited stationary phase. Using genetic analysis, we determined that these genes are involved in two different envelope-strengthening pathways. These genes have not previously been implicated in stationary-phase stress responses. A third novel RpoS-dependent pathway appears to strengthen the cell's permeability barrier in nitrogen-limited cells. Thus, though cells remain phenotypically SDS resistant, SDS resistance mechanisms differ significantly between growth states. IMPORTANCE: Gram-negative bacteria are intrinsically resistant to detergents and many antibiotics due to synergistic activities of a strong outer membrane (OM) permeability barrier and efflux pumps that capture and expel toxic molecules eluding the barrier. When the bacteria are depleted of an essential nutrient, a program of gene expression providing cross-protection against many stresses is induced. Whether this program alters the OM to further strengthen the barrier is unknown. Here, we identify novel pathways dependent on the master regulator of stationary phase that further strengthen the OM permeability barrier during nutrient limitation, circumventing the need for efflux pumps. Decreased permeability of nutrient-limited cells to toxic compounds has important implications for designing new antibiotics capable of targeting Gram-negative bacteria that may be in a growth-limited state.201727821607
8311180.9995Perturbation of Quorum Sensing after the Acquisition of Bacteriophage Resistance Could Contribute to Novel Traits in Vibrio alginolyticus. Bacteria employ a wide range of molecular mechanisms to confer resistance to bacteriophages, and these mechanisms are continuously being discovered and characterized. However, there are instances where certain bacterial species, despite lacking these known mechanisms, can still develop bacteriophage resistance through intricate metabolic adaptation strategies, potentially involving mutations in transcriptional regulators or phage receptors. Vibrio species have been particularly useful for studying the orchestrated metabolic responses of Gram-negative marine bacteria in various challenges. In a previous study, we demonstrated that Vibrio alginolyticus downregulates the expression of specific receptors and transporters in its membrane, which may enable the bacterium to evade infection by lytic bacteriophages. In our current study, our objective was to explore how the development of bacteriophage resistance in Vibrio species disrupts the quorum-sensing cascade, subsequently affecting bacterial physiology and metabolic capacity. Using a real-time quantitative PCR (rt-QPCR) platform, we examined the expression pattern of quorum-sensing genes, auto-inducer biosynthesis genes, and cell density regulatory proteins in phage-resistant strains. Our results revealed that bacteriophage-resistant bacteria downregulate the expression of quorum-sensing regulatory proteins, such as LuxM, LuxN, and LuxP. This downregulation attenuates the normal perception of quorum-sensing peptides and subsequently diminishes the expression of cell density regulatory proteins, including LuxU, aphA, and LuxR. These findings align with the diverse phenotypic traits observed in the phage-resistant strains, such as altered biofilm formation, reduced planktonic growth, and reduced virulence. Moreover, the transcriptional depletion of aphA, the master regulator associated with low cell density, was linked to the downregulation of genes related to virulence. This phenomenon appears to be phage-specific, suggesting a finely tuned metabolic adaptation driven by phage-host interaction. These findings contribute to our understanding of the role of Vibrio species in microbial marine ecology and highlight the complex interplay between phage resistance, quorum sensing, and bacterial physiology.202337764117
8322190.9995Pathogen-induced damage in Drosophila: Uncoupling disease tolerance from resistance. Immune response against infections can be divided into mechanisms of resistance that ensure active pathogen elimination, and mechanisms of disease tolerance, which include processes that return the host to physiological homeostasis without direct control of pathogen load. Studies on host immune response to infection have targeted mechanisms of resistance, and consequently, these are now well-described in both vertebrates and invertebrates. By comparison, the mechanistic basis of disease tolerance is poorly understood. This is in part because both processes interact and can be difficult to disentangle under an infection scenario. Using the insect model Drosophila melanogaster exposed to its natural entomopathogen, Pseudomonas entomophila, we aimed to tease apart mechanisms of disease tolerance from those of resistance. To this end, we reasoned that the response to oral exposure to heat-killed entomopathogenic bacteria, whilst initially triggering both resistance and disease tolerance mechanisms, would be resolved mainly by disease tolerance alone. Using this method, we observe that oral exposure to heat-killed P. entomophila causes mortality and reduced fecundity in D. melanogaster. We confirm that this reduction in fitness-related traits depends on the duration of the exposure, is sexually dimorphic, and is dependent on the virulence of the bacterium. We also found the microbiota to play a role, with its presence exacerbating the deleterious effect on host survival. In addition, we show that the Imd pathway, but not effector genes, is involved in the process of surviving exposure to HK bacteria. This experimental framework, which may be extended to other systems, can be instrumental towards an understanding of the molecular, genetic, and physiological basis of disease tolerance and its interactions with resistance mechanisms.202540971962