CpxAR of Actinobacillus pleuropneumoniae Contributes to Heat Stress Response by Repressing Expression of Type IV Pilus Gene apfA. - Related Documents




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67801.0000CpxAR of Actinobacillus pleuropneumoniae Contributes to Heat Stress Response by Repressing Expression of Type IV Pilus Gene apfA. Acute pleuropneumonia in swine, caused by Actinobacillus pleuropneumoniae, is characterized by a high and sustained fever. Fever creates an adverse environment for many bacteria, leading to reduced bacterial proliferation; however, most pathogenic bacteria can tolerate higher temperatures. CpxAR is a two-component regulation system, ubiquitous among Gram-negative bacteria, which senses and responds to envelope alterations that are mostly associated with protein misfolding in the periplasm. Our previous study showed that CpxAR is necessary for the optimal growth of Actinobacillus pleuropneumoniae under heat stress. Here, we showed that mutation of the type IV pilin gene apfA rescued the growth defect of the cpxAR deletion strain under heat stress. RNA sequencing (RNA-seq) analyses revealed that 265 genes were differentially expressed in the ΔcpxAR strains grown at 42°C, including genes involved in type IV pilus biosynthesis. We also demonstrated direct binding of the CpxR protein to the promoter of the apf operon by an electrophoretic mobility shift assay and identified the binding site by a DNase I footprinting assay. In conclusion, our results revealed the important role of CpxAR in A. pleuropneumoniae resistance to heat stress by directly suppressing the expression of ApfA. IMPORTANCE Heat acts as a danger signal for pathogens, especially those infecting mammalian hosts in whom fever indicates infection. However, some bacteria have evolved exquisite mechanisms to survive under heat stress. Studying the mechanism of resistance to heat stress is crucial to understanding the pathogenesis of A. pleuropneumoniae during the acute stage of infection. Our study revealed that CpxAR plays an important role in A. pleuropneumoniae resistance to heat stress by directly suppressing expression of the type IV pilin protein ApfA.202236259970
634210.9995Determinants 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
68320.9995Integrative Multiomics Analysis of the Heat Stress Response of Enterococcus faecium. A continuous heat-adaptation test was conducted for one Enterococcus faecium (E. faecium) strain wild-type (WT) RS047 to obtain a high-temperature-resistant strain. After domestication, the strain was screened with a significantly higher ability of heat resistance. which is named RS047-wl. Then a multi-omics analysis of transcriptomics and metabolomics was used to analyze the mechanism of the heat resistance of the mutant. A total of 98 differentially expressed genes (DEGs) and 115 differential metabolites covering multiple metabolic processes were detected in the mutant, which indicated that the tolerance of heat resistance was regulated by multiple mechanisms. The changes in AgrB, AgrC, and AgrA gene expressions were involved in quorum-sensing (QS) system pathways, which regulate biofilm formation. Second, highly soluble osmotic substances such as putrescine, spermidine, glycine betaine (GB), and trehalose-6P were accumulated for the membrane transport system. Third, organic acids metabolism and purine metabolism were down-regulated. The findings can provide target genes for subsequent genetic modification of E. faecium, and provide indications for screening heat-resistant bacteria, so as to improve the heat-resistant ability of E. faecium for production.202336979372
68230.9995Comparative transcriptome analysis of Brucella melitensis in an acidic environment: Identification of the two-component response regulator involved in the acid resistance and virulence of Brucella. Brucella melitensis, encounters a very stressful environment in phagosomes, especially low pH levels. So identifying the genes that contribute to the replication and survival within an acidic environment is critical in understanding the pathogenesis of the Brucella bacteria. In our research, comparative transcriptome with RNA-seq were used to analyze the changes of genes in normal-medium culture and in pH4.4-medium culture. The results reveal that 113 genes expressed with significant differences (|log2Ratio| ≥ 3); about 44% genes expressed as up-regulated. With GO term analysis, structural constituent of the ribosome, rRNA binding, structural molecule activity, and cation-transporting ATPase activity were significantly enriched (p-value ≤ 0.05). These genes distributed in 51 pathways, in which ribosome and photosynthesis pathways were significantly enriched. Six pathways (oxidative phosphorylation, iron-transporting, bacterial secretion system, transcriptional regulation, two-component system, and ABC transporters pathways) tightly related to the intracellular survival and virulence of Brucella were analyzed. A two-component response regulator gene in the transcriptional regulation pathway, identified through gene deletion and complementary technologies, played an important role in the resistance to the acid-resistance and virulence of Brucella.201626691825
68540.9995Implication of a Key Region of Six Bacillus cereus Genes Involved in Siroheme Synthesis, Nitrite Reductase Production and Iron Cluster Repair in the Bacterial Response to Nitric Oxide Stress. Bacterial response to nitric oxide (NO) is of major importance for bacterial survival. NO stress is a main actor of the eukaryotic immune response and several pathogenic bacteria have developed means for detoxification and repair of the damages caused by NO. However, bacterial mechanisms of NO resistance by Gram-positive bacteria are poorly described. In the opportunistic foodborne pathogen Bacillus cereus, genome sequence analyses did not identify homologs to known NO reductases and transcriptional regulators, such as NsrR, which orchestrate the response to NO of other pathogenic or non-pathogenic bacteria. Using a transcriptomic approach, we investigated the adaptation of B. cereus to NO stress. A cluster of 6 genes was identified to be strongly up-regulated in the early phase of the response. This cluster contains an iron-sulfur cluster repair enzyme, a nitrite reductase and three enzymes involved in siroheme biosynthesis. The expression pattern and close genetic localization suggest a functional link between these genes, which may play a pivotal role in the resistance of B. cereus to NO stress during infection.202134064887
68450.9994Transcriptome analysis reveals mechanisms by which Lactococcus lactis acquires nisin resistance. Nisin, a posttranslationally modified antimicrobial peptide produced by Lactococcus lactis, is widely used as a food preservative. Yet, the mechanisms leading to the development of nisin resistance in bacteria are poorly understood. We used whole-genome DNA microarrays of L. lactis IL1403 to identify the factors underlying acquired nisin resistance mechanisms. The transcriptomes of L. lactis IL1403 and L. lactis IL1403 Nis(r), which reached a 75-fold higher nisin resistance level, were compared. Differential expression was observed in genes encoding proteins that are involved in cell wall biosynthesis, energy metabolism, fatty acid and phospholipid metabolism, regulatory functions, and metal and/or peptide transport and binding. These results were further substantiated by showing that several knockout and overexpression mutants of these genes had strongly altered nisin resistance levels and that some knockout strains could no longer become resistant to the same level of nisin as that of the wild-type strain. The acquired nisin resistance mechanism in L. lactis is complex, involving various different mechanisms. The four major mechanisms are (i) preventing nisin from reaching the cytoplasmic membrane, (ii) reducing the acidity of the extracellular medium, thereby stimulating the binding of nisin to the cell wall, (iii) preventing the insertion of nisin into the membrane, and (iv) possibly transporting nisin across the membrane or extruding nisin out of the membrane.200616641446
63260.9994The Role of the Two-Component System PhoP/PhoQ in Intrinsic Resistance of Yersinia enterocolitica to Polymyxin. Polymyxin is the "last resort" of antibiotics. The self-induced resistance to polymyxin in Gram-negative bacteria could be mediated by lipopolysaccharide (LPS) modification, which is regulated by the two-component system, PhoP/PhoQ. Yersinia enterocolitica is a common foodborne pathogen. However, PhoP/PhoQ has not been thoroughly studied in Y. enterocolitica. In this study, the functions of PhoP/PhoQ in Y. enterocolitica intrinsic resistance were investigated. The resistance of Y. enterocolitica was found to decrease with the deletion of PhoP/PhoQ. Further, PhoP/PhoQ was found to play an important role in maintaining membrane permeability, intercellular metabolism, and reducing membrane depolarization. Based on subsequent studies, the binding ability of polymyxin to Y. enterocolitica was decreased by the modification of LPS with structures, such as L-Ara4N and palmitate. Analysis of the gene transcription levels revealed that the LPS modification genes, pagP and arn operon, were downregulated with the deletion of PhoP/PhoQ in Y. enterocolitica during exposure to polymyxin. In addition, pmrA, pmrB, and eptA were downregulated in the mutants compared with the wild-type strain. Such findings demonstrate that PhoP/PhoQ contributes to the intrinsic resistance of Y. enterocolitica toward polymyxins. LPS modification with L-Ara4N or palmitate is mainly responsible for the resistance of Y. enterocolitica to polymyxins. The transcription of genes related to LPS modification and PmrA/PmrB can be both affected by PhoP/PhoQ in Y. enterocolitica. This study adds to current knowledge regarding the role of PhoP/PhoQ in intrinsic resistance of Y. enterocolitica to polymyxin.202235222323
621870.9994An allele of an ancestral transcription factor dependent on a horizontally acquired gene product. Changes in gene regulatory circuits often give rise to phenotypic differences among closely related organisms. In bacteria, these changes can result from alterations in the ancestral genome and/or be brought about by genes acquired by horizontal transfer. Here, we identify an allele of the ancestral transcription factor PmrA that requires the horizontally acquired pmrD gene product to promote gene expression. We determined that a single amino acid difference between the PmrA proteins from the human adapted Salmonella enterica serovar Paratyphi B and the broad host range S. enterica serovar Typhimurium rendered transcription of PmrA-activated genes dependent on the PmrD protein in the former but not the latter serovar. Bacteria harboring the serovar Typhimurium allele exhibited polymyxin B resistance under PmrA- or under PmrA- and PmrD-inducing conditions. By contrast, isogenic strains with the serovar Paratyphi B allele displayed PmrA-regulated polymyxin B resistance only when experiencing activating conditions for both PmrA and PmrD. We establish that the two PmrA orthologs display quantitative differences in several biochemical properties. Strains harboring the serovar Paratyphi B allele showed enhanced biofilm formation, a property that might promote serovar Paratyphi B's chronic infection of the gallbladder. Our findings illustrate how subtle differences in ancestral genes can impact the ability of horizontally acquired genes to confer new properties.201223300460
67980.9994RNA-Seq Analysis Discovers the Critical Role of Rel in ppGpp Synthesis, Pathogenicity, and the VBNC State of Clavibacter michiganensis. The viable but nonculturable (VBNC) state is a unique survival strategy of bacteria in response to stress conditions. It was confirmed that Clavibacter michiganensis, the causal agent of bacterial canker in tomato, could be induced into the VBNC state by exposure to CuSO(4) in an oligotrophic solution. RNA-sequencing analysis was used to monitor the mechanisms of the VBNC state during CuSO(4) induction in C. michiganensis. The results identified that numerous genes involved in stringent response, copper resistance, and stress resistance were upregulated, and some involved in cell division were downregulated significantly. The study investigated the importance of Rel, which is an essential enzyme in the synthesis of the molecular alarmone ppGpp, via the generation of a Δrel mutant and its complementation strain. Biological characterization revealed that deficiency of rel reduced the bacterial growth, production of exopolysaccharides, and pathogenicity as well as ppGpp production. The Δrel mutant increased the sensitivity to environmental stress, exhibiting reduced growth on minimal media and a propensity to enter the VBNC state in response to CuSO(4). These findings have important implications for the understanding of survival mechanism and management of C. michiganensis and other phytopathogenic bacteria.202235341314
68790.9994RpoS-Regulated Genes and Phenotypes in the Phytopathogenic Bacterium Pectobacterium atrosepticum. The alternative sigma factor RpoS is considered to be one of the major regulators providing stress resistance and cross-protection in bacteria. In phytopathogenic bacteria, the effects of RpoS have not been analyzed with regard to cross-protection, and genes whose expression is directly or indirectly controlled by RpoS have not been determined at the whole-transcriptome level. Our study aimed to determine RpoS-regulated genes and phenotypes in the phytopathogenic bacterium Pectobacterium atrosepticum. Knockout of the rpoS gene in P. atrosepticum affected the long-term starvation response, cross-protection, and virulence toward plants with enhanced immune status. The whole-transcriptome profiles of the wild-type P. atrosepticum strain and its ΔrpoS mutant were compared under different experimental conditions, and functional gene groups whose expression was affected by RpoS were determined. The RpoS promoter motif was inferred within the promoter regions of the genes affected by rpoS deletion, and the P. atrosepticum RpoS regulon was predicted. Based on RpoS-controlled phenotypes, transcriptome profiles, and RpoS regulon composition, the regulatory role of RpoS in P. atrosepticum is discussed.202338139177
6338100.9994Transcriptome Analysis of the Intracellular Facultative Pathogen Piscirickettsia salmonis: Expression of Putative Groups of Genes Associated with Virulence and Iron Metabolism. The intracellular facultative bacteria Piscirickettsia salmonis is one of the most important pathogens of the Chilean aquaculture. However, there is a lack of information regarding the whole genomic transcriptional response according to different extracellular environments. We used next generation sequencing (NGS) of RNA (RNA-seq) to study the whole transcriptome of an isolate of P. salmonis (FAVET-INBIOGEN) using a cell line culture and a modified cell-free liquid medium, with or without iron supplementation. This was done in order to obtain information about the factors there are involved in virulence and iron acquisition. First, the isolate was grown in the Sf21 cell line; then, the bacteria were cultured into a cell-free liquid medium supplemented or not with iron. We identified in the transcriptome, genes associated with type IV secretion systems, genes related to flagellar structure assembly, several proteases and sigma factors, and genes related to the development of drug resistance. Additionally, we identified for the first time several iron-metabolism associated genes including at least two iron uptake pathways (ferrous iron and ferric iron uptake) that are actually expressed in the different conditions analyzed. We further describe putative genes that are related with the use and storage of iron in the bacteria, which have not been previously described. Several sets of genes related to virulence were expressed in both the cell line and cell-free culture media (for example those related to flagellar structure; such as basal body, MS-ring, C-ring, proximal and distal rod, and filament), which may play roles in other basic processes rather than been restricted to virulence.201628033422
8946110.9994Role of the CpxAR two-component signal transduction system in control of fosfomycin resistance and carbon substrate uptake. Although fosfomycin is an old antibiotic, it has resurfaced with particular interest. The antibiotic is still effective against many pathogens that are resistant to other commonly used antibiotics. We have found that fosfomycin resistance of enterohemorrhagic Escherichia coli (EHEC) O157:H7 is controlled by the bacterial two-component signal transduction system CpxAR. A cpxA mutant lacking its phosphatase activity results in constitutive activation of its cognate response regulator, CpxR, and fosfomycin resistance. We have shown that fosfomycin resistance requires CpxR because deletion of the cpxR gene in the cpxA mutant restores fosfomycin sensitivity. We have also shown that CpxR directly represses the expression of two genes, glpT and uhpT, which encode transporters that cotransport fosfomycin with their native substrates glycerol-3-phosphate and glucose-6-phosphate, and repression of these genes leads to a decrease in fosfomycin transport into the cpxA mutant. However, the cpxA mutant had an impaired growth phenotype when cultured with glycerol-3-phosphate or glucose-6-phosphate as a sole carbon substrate and was outcompeted by the parent strain, even in nutrient-rich medium. This suggests a trade-off between fosfomycin resistance and the biological fitness associated with carbon substrate uptake. We propose a role for the CpxAR system in the reversible control of fosfomycin resistance. This may be a beneficial strategy for bacteria to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin.201424163343
8308120.9994PhoPQ 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
8891130.9994Analysis of Shigella flexneri Resistance, Biofilm Formation, and Transcriptional Profile in Response to Bile Salts. The Shigella species cause millions of cases of watery or bloody diarrhea each year, mostly in children in developing countries. While many aspects of Shigella colonic cell invasion are known, crucial gaps in knowledge regarding how the bacteria survive, transit, and regulate gene expression prior to infection remain. In this study, we define mechanisms of resistance to bile salts and build on previous research highlighting induced virulence in Shigella flexneri strain 2457T following exposure to bile salts. Typical growth patterns were observed within the physiological range of bile salts; however, growth was inhibited at higher concentrations. Interestingly, extended periods of exposure to bile salts led to biofilm formation, a conserved phenotype that we observed among members of the Enterobacteriaceae Characterization of S. flexneri 2457T biofilms determined that both bile salts and glucose were required for formation, dispersion was dependent upon bile salts depletion, and recovered bacteria displayed induced adherence to HT-29 cells. RNA-sequencing analysis verified an important bile salt transcriptional profile in S. flexneri 2457T, including induced drug resistance and virulence gene expression. Finally, functional mutagenesis identified the importance of the AcrAB efflux pump and lipopolysaccharide O-antigen synthesis for bile salt resistance. Our data demonstrate that S. flexneri 2457T employs multiple mechanisms to survive exposure to bile salts, which may have important implications for multidrug resistance. Furthermore, our work confirms that bile salts are important physiological signals to activate S. flexneri 2457T virulence. This work provides insights into how exposure to bile likely regulates Shigella survival and virulence during host transit and subsequent colonic infection.201728348056
686140.9994SigB-dependent general stress response in Bacillus subtilis and related gram-positive bacteria. One of the strongest and most noticeable responses of Bacillus subtilis cells to a range of stress and starvation stimuli is the dramatic induction of about 150 SigB-dependent general stress genes. The activity of SigB itself is tightly regulated by a complex signal transduction cascade with at least three main signaling pathways that respond to environmental stress, energy depletion, or low temperature. The SigB-dependent response is conserved in related gram-positive bacteria but is missing in strictly anaerobic or in some facultatively anaerobic gram-positive bacteria. It covers functions from nonspecific and multiple stress resistance to the control of virulence in pathogenic bacteria. A comprehensive understanding of this crucial stress response is essential not only for bacterial physiology but also for applied microbiology, including pathogenicity and pathogen control.200718035607
696150.9994Identification of a two-component regulatory system involved in antimicrobial peptide resistance in Streptococcus pneumoniae. Two-component regulatory systems (TCS) are among the most widespread mechanisms that bacteria use to sense and respond to environmental changes. In the human pathogen Streptococcus pneumoniae, a total of 13 TCS have been identified and many of them have been linked to pathogenicity. Notably, TCS01 strongly contributes to pneumococcal virulence in several infection models. However, it remains one of the least studied TCS in pneumococci and its functional role is still unclear. In this study, we demonstrate that TCS01 cooperates with a BceAB-type ABC transporter to sense and induce resistance to structurally-unrelated antimicrobial peptides of bacterial origin that all target undecaprenyl-pyrophosphate or lipid II, which are essential precursors of cell wall biosynthesis. Even though tcs01 and bceAB genes do not locate in the same gene cluster, disruption of either of them equally sensitized the bacterium to the same set of antimicrobial peptides. We show that the key function of TCS01 is to upregulate the expression of the transporter, while the latter appears the main actor in resistance. Electrophoretic mobility shift assays further demonstrated that the response regulator of TCS01 binds to the promoter region of the bceAB genes, implying a direct control of these genes. The BceAB transporter was overexpressed and purified from E. coli. After reconstitution in liposomes, it displayed substantial ATPase and GTPase activities that were stimulated by antimicrobial peptides to which it confers resistance to, revealing new functional features of a BceAB-type transporter. Altogether, this inducible defense mechanism likely contributes to the survival of the opportunistic microorganism in the human host, in which competition among commensal microorganisms is a key determinant for effective host colonization and invasive path.202235395062
688160.9994The cop operon is required for copper homeostasis and contributes to virulence in Streptococcus pneumoniae. High levels of copper are toxic and therefore bacteria must limit free intracellular levels to prevent cellular damage. In this study, we show that a number of pneumococcal genes are differentially regulated by copper, including an operon encoding a CopY regulator, a protein of unknown function (CupA) and a P1-type ATPase, CopA, which is conserved in all sequenced Streptococcus pneumoniae strains. Transcriptional analysis demonstrated that the cop operon is induced by copper in vitro, repressed by the addition of zinc and is autoregulated by the copper-responsive CopY repressor protein. We also demonstrate that the CopA ATPase is a major pneumococcal copper resistance mechanism and provide the first evidence that the CupA protein plays a role in copper resistance. Our results also show that copper homeostasis is important for pneumococcal virulence as the expression of the cop operon is induced in the lungs and nasopharynx of intranasally infected mice, and a copA(-) mutant strain, which had decreased growth in high levels of copper in vitro, showed reduced virulence in a mouse model of pneumococcal pneumonia. Furthermore, using the copA(-) mutant we observed for the first time in any bacteria that copper homeostasis also appears to be required for survival in the nasopharynx.201121736642
698170.9994Genome-wide transcriptional changes induced by phagocytosis or growth on bacteria in Dictyostelium. BACKGROUND: Phagocytosis plays a major role in the defense of higher organisms against microbial infection and provides also the basis for antigen processing in the immune response. Cells of the model organism Dictyostelium are professional phagocytes that exploit phagocytosis of bacteria as the preferred way to ingest food, besides killing pathogens. We have investigated Dictyostelium differential gene expression during phagocytosis of non-pathogenic bacteria, using DNA microarrays, in order to identify molecular functions and novel genes involved in phagocytosis. RESULTS: The gene expression profiles of cells incubated for a brief time with bacteria were compared with cells either incubated in axenic medium or growing on bacteria. Transcriptional changes during exponential growth in axenic medium or on bacteria were also compared. We recognized 443 and 59 genes that are differentially regulated by phagocytosis or by the different growth conditions (growth on bacteria vs. axenic medium), respectively, and 102 genes regulated by both processes. Roughly one third of the genes are up-regulated compared to macropinocytosis and axenic growth. Functional annotation of differentially regulated genes with different tools revealed that phagocytosis induces profound changes in carbohydrate, amino acid and lipid metabolism, and in cytoskeletal components. Genes regulating translation and mitochondrial biogenesis are mostly up-regulated. Genes involved in sterol biosynthesis are selectively up-regulated, suggesting a shift in membrane lipid composition linked to phagocytosis. Very few changes were detected in genes required for vesicle fission/fusion, indicating that the intracellular traffic machinery is mostly in common between phagocytosis and macropinocytosis. A few putative receptors, including GPCR family 3 proteins, scaffolding and adhesion proteins, components of signal transduction and transcription factors have been identified, which could be part of a signalling complex regulating phagocytosis and adaptational downstream responses. CONCLUSION: The results highlight differences between phagocytosis and macropinocytosis, and provide the basis for targeted functional analysis of new candidate genes and for comparison studies with transcriptomes during infection with pathogenic bacteria.200818559084
713180.9994OxyR-activated expression of Dps is important for Vibrio cholerae oxidative stress resistance and pathogenesis. Vibrio cholerae is the causative agent of cholera, a dehydrating diarrheal disease. This Gram-negative pathogen is able to modulate its gene expression in order to combat stresses encountered in both aquatic and host environments, including stress posed by reactive oxygen species (ROS). In order to further the understanding of V. cholerae's transcriptional response to ROS, we performed an RNA sequencing analysis to determine the transcriptional profile of V. cholerae when exposed to hydrogen hydroperoxide. Of 135 differentially expressed genes, VC0139 was amongst the genes with the largest induction. VC0139 encodes a protein homologous to the DPS (DNA-binding protein from starved cells) protein family, which are widely conserved and are implicated in ROS resistance in other bacteria. Using a promoter reporter assay, we show that during exponential growth, dps is induced by H2O2 in a manner dependent on the ROS-sensing transcriptional regulator, OxyR. Upon entry into stationary phase, the major stationary phase regulator RpoS is required to transcribe dps. Deletion of dps impaired V. cholerae resistance to both inorganic and organic hydroperoxides. Furthermore, we show that Dps is involved in resistance to multiple environmental stresses. Finally, we found that Dps is important for V. cholerae adult mouse colonization, but becomes dispensable in the presence of antioxidants. Taken together, our results suggest that Dps plays vital roles in both V. cholerae stress resistance and pathogenesis.201728151956
681190.9994Global transcriptional response to vancomycin in Mycobacterium tuberculosis. In order to gain additional understanding of the physiological mechanisms used by bacteria to maintain surface homeostasis and to identify potential targets for new antibacterial drugs, we analysed the variation of the Mycobacterium tuberculosis transcriptional profile in response to inhibitory and subinhibitory concentrations of vancomycin. Our analysis identified 153 genes differentially regulated after exposing bacteria to a concentration of the drug ten times higher than the MIC, and 141 genes differentially expressed when bacteria were growing in a concentration of the drug eightfold lower than the MIC. Hierarchical clustering analysis indicated that the response to these different conditions is different, although with some overlap. This approach allowed us to identify several genes whose products could be involved in the protection from antibiotic stress targeting the envelope and help to confer the basal level of M. tuberculosis resistance to antibacterial drugs, such as Rv2623 (UspA-like), Rv0116c, PE20-PPE31, PspA and proteins related to toxin-antitoxin systems. Moreover, we also demonstrated that the alternative sigma factor sigma(E) confers basal resistance to vancomycin, once again underlining its importance in the physiology of the mycobacterial surface stress response.200919332811