RESUMO
Intracellular pathogens like Brucella face challenges during the intraphagocytic adaptation phase, where the modulation of gene expression plays an essential role in taking advantage of stressors to persist inside the host cell. This study aims to explore the expression of antisense virB2 RNA strand and related genes under intracellular simulation media. Sense and antisense virB2 RNA strands increased expression when nutrient deprivation and acidification were higher, being starvation more determinative. Meanwhile, bspB, one of the T4SS effector genes, exhibited the highest expression during the exposition to pH 4.5 and nutrient abundance. Based on RNA-seq analysis and RACE data, we constructed a regional map depicting the 5' and 3' ends of virB2 and the cis-encoded asRNA_0067. Without affecting the CDS or a possible autonomous RBS, we generate the deletion mutant ΔasRNA_0067, significantly reducing virB2 mRNA expression and survival rate. These results suggest that the antisense asRNA_0067 expression is promoted under exposure to the intraphagocytic adaptation phase stressors, and its deletion is associated with a lower transcription of the virB2 gene. Our findings illuminate the significance of these RNA strands in modulating the survival strategy of Brucella within the host and emphasize the role of nutrient deprivation in gene expression.
Assuntos
Brucella abortus , Regulação Bacteriana da Expressão Gênica , Brucella abortus/genética , Brucella abortus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Transcrição Gênica , RNA Antissenso/genética , RNA Antissenso/metabolismo , Estresse Fisiológico , Animais , Macrófagos/microbiologiaRESUMO
BACKGROUND: In fast-growing bacteria, the genomic location of ribosomal protein (RP) genes is biased towards the replication origin (oriC). This trait allows optimizing their expression during exponential phase since oriC neighboring regions are in higher dose due to multifork replication. Relocation of s10-spc-α locus (S10), which codes for most of the RP, to ectopic genomic positions shows that its relative distance to the oriC correlates to a reduction on its dosage, its expression, and bacterial growth rate. However, a mechanism linking S10 dosage to cell physiology has still not been determined. RESULTS: We hypothesized that S10 dosage perturbations impact protein synthesis capacity. Strikingly, we observed that in Vibrio cholerae, protein production capacity was independent of S10 position. Deep sequencing revealed that S10 relocation altered chromosomal replication dynamics and genome-wide transcription. Such changes increased as a function of oriC-S10 distance. Since RP constitutes a large proportion of cell mass, lower S10 dosage could lead to changes in macromolecular crowding, impacting cell physiology. Accordingly, cytoplasm fluidity was higher in mutants where S10 is most distant from oriC. In hyperosmotic conditions, when crowding differences are minimized, the growth rate and replication dynamics were highly alleviated in these strains. CONCLUSIONS: The genomic location of RP genes ensures its optimal dosage. However, besides of its essential function in translation, their genomic position sustains an optimal macromolecular crowding essential for maximizing growth. Hence, this could be another mechanism coordinating DNA replication to bacterial growth.
Assuntos
Proteínas de Bactérias/metabolismo , Dosagem de Genes , Genes Bacterianos , Origem de Replicação , Proteínas Ribossômicas/metabolismo , Vibrio cholerae/genética , Replicação do DNA , DNA Bacteriano/fisiologia , Vibrio cholerae/crescimento & desenvolvimentoRESUMO
LuxR-type transcription factors control diverse physiological functions necessary for bacterial adaptation to environmental changes. In the intracellular pathogen Brucella, the LuxR homolog VjbR has been shown to regulate the expression of virulence factors acting at early stages of the intracellular infection and, directly or indirectly, hundreds of additional genes. However, the precise determination of VjbR direct targets has so far proved elusive. Here, we performed chromatin immunoprecipitation of VjbR followed by next-generation sequencing (ChIP-seq). We detected a large amount of VjbR-binding sites distributed across the Brucella genome and determined a markedly asymmetric binding consensus motif, an unusual feature among LuxR-type regulators. RNA-seq analysis performed under conditions mimicking the eukaryotic intracellular environment revealed that, among all loci associated to VjbR-binding, this regulator directly modulated the expression of only a subset of genes encoding functions consistent with an intracellular adaptation strategy for survival during the initial stages of the host cell infection. Other VjbR-binding events, however, showed to be dissociated from transcription and may require different environmental signals to produce a transcriptional output. Taken together, our results bring new insights into the extent and functionality of LuxR-type-related transcriptional networks.
Assuntos
Proteínas de Bactérias/genética , Brucella abortus/genética , Brucella abortus/patogenicidade , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Proteínas Repressoras/genética , Transativadores/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Brucella abortus/metabolismo , Imunoprecipitação da Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Motivos de Nucleotídeos , Ligação Proteica , Percepção de Quorum/genética , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Transcrição Gênica , VirulênciaRESUMO
Regulatory network plasticity is a key attribute underlying changes in bacterial gene expression and a source of phenotypic diversity to interact with the surrounding environment. Here, we sought to study the transcriptional circuit of HutC, a regulator of both metabolic and virulence genes of the facultative intracellular pathogen Brucella. Using in silico and biochemical approaches, we identified a novel functional HutC-binding site upstream of btaE, a trimeric-autotransporter adhesin involved in the attachment of Brucella to host extracellular matrix components. Moreover, we identified two additional regulators, one of which, MdrA, acts in concert with HutC to exert a combinatorial control of both btaE promoter activity and attachment of Brucella to HeLa cells. Analysis of btaE promoter sequences of different species indicated that this HutC-binding site was generated de novo by a single point mutation in a virulent Brucella strain, indicative of a transcriptional rewiring event. In addition to major domain organization differences existing between BtaE proteins within the genus Brucella, our analyses revealed that sequences upstream of btaE display high variability probably associated to intrinsic promoter structural features, which may serve as a substrate for reciprocal selection during co-evolution between this pathogen and its mammalian host.
Assuntos
Brucella abortus/genética , Brucella abortus/metabolismo , Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases/genética , Sítios de Ligação/genética , Brucella abortus/fisiologia , Biologia Computacional/métodos , Matriz Extracelular/microbiologia , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Sistemas de Secreção Tipo V/metabolismo , Virulência/fisiologiaRESUMO
Phytochromes constitute a major photoreceptor family found in plants, algae, fungi, and prokaryotes, including pathogens. Here, we report that Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot disease which affects cruciferous crops worldwide, codes for a functional bacteriophytochrome (XccBphP). XccBphP possesses an N-terminal PAS2-GAF-PHY photosensory domain triad and a C-terminal PAS9 domain as its output module. Our results show that illumination of Xcc, prior to plant infection, attenuates its virulence in an XccBphP-dependent manner. Moreover, in response to light, XccBphP downregulates xanthan exopolysaccharide production and biofilm formation, two known Xcc virulence factors. Furthermore, the XccbphP null mutant shows enhanced virulence, similar to that of dark-adapted Xcc cultures. Stomatal aperture regulation and callose deposition, both well-established plant defense mechanisms against bacterial pathogens, are overridden by the XccbphP strain. Additionally, an RNA-Seq analysis reveals that far-red light or XccBphP overexpression produces genomewide transcriptional changes, including the inhibition of several Xcc virulence systems. Our findings indicate that Xcc senses light through XccBphP, eliciting bacterial virulence attenuation via downregulation of bacterial virulence factors. The capacity of XccBphP to respond to light both in vitro and in vivo was abolished by a mutation on the conserved Cys13 residue. These results provide evidence for a novel bacteriophytochrome function affecting an infectious process.
Assuntos
Proteínas de Bactérias/genética , Fitocromo/metabolismo , Doenças das Plantas/microbiologia , Xanthomonas campestris/metabolismo , Xanthomonas campestris/patogenicidade , Biofilmes/crescimento & desenvolvimento , Produtos Agrícolas , Regulação Bacteriana da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Luz , Mutação , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/metabolismo , Fatores de Virulência/genética , Xanthomonas campestris/genéticaRESUMO
Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world. The success of Brucella as pathogen relies in its ability to adapt to the harsh environmental conditions found in mammalian hosts. One of its main adaptations is the induction of the expression of different genes involved in respiration at low oxygen tension. In this report we describe a regulatory network involved in this adaptation. We show that Brucella abortusâ PrrBA is a functional two-component signal transduction system that responds to the redox status and acts as a global regulator controlling the expression of the regulatory proteins NtrY, FnrN and NnrA, which are involved in the adaptation to survive at low oxygen tension. We also show that the two-component systems PrrBA and NtrYX co-ordinately regulate the expression of denitrification and high-affinity cytochrome oxidase genes. Strikingly, a double mutant strain in the prrB and ntrY genes is severely impaired in growth and virulence, while the ntrY and prrB single mutant strains are similar to wild-type B. abortus. The proposed regulatory network may contribute to understand the mechanisms used by Brucella for a successful adaptation to its replicative niche inside mammalian cells.
Assuntos
Adaptação Fisiológica/genética , Proteínas de Bactérias/metabolismo , Brucella abortus/fisiologia , Regulação Bacteriana da Expressão Gênica , Oxigênio/farmacologia , Proteínas Quinases/metabolismo , Animais , Proteínas de Bactérias/genética , Brucella abortus/efeitos dos fármacos , Brucella abortus/genética , Brucella abortus/metabolismo , Brucelose/microbiologia , Desnitrificação , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Histidina Quinase , Camundongos , Oxirredução , Consumo de Oxigênio/fisiologia , Proteínas Quinases/genética , Transdução de SinaisRESUMO
Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major human pathogen and a research priority for developing new antimicrobial agents. CRAB is a causative agent of a variety of infections in different body sites. One of the manifestations is catheter-associated urinary tract infection, which exposes the bacteria to the host's urine, creating a particular environment. Exposure of two CRAB clinical isolates, AB5075 and AMA40, to human urine (HU) resulted in the differential expression levels of 264 and 455 genes, respectively, of which 112 were common to both strains. Genes within this group play roles in metabolic pathways such as phenylacetic acid (PAA) catabolism, the Hut system, the tricarboxylic acid (TCA) cycle, and other processes like quorum sensing and biofilm formation. These results indicate that the presence of HU induces numerous adaptive changes in gene expression of the infecting bacteria. These modifications presumably help bacteria establish and thrive in the hostile conditions in the urinary tract. These analyses advance our understanding of CRAB's metabolic adaptations to human fluids, as well as expanding knowledge on bacterial responses to distinct human fluids containing different concentrations of human serum albumin (HSA).
RESUMO
Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major human pathogen and a research priority for developing new antimicrobial agents. CRAB is a causative agent of a variety of infections in different body sites. One of the manifestations is catheter-associated urinary tract infection, which exposes the bacteria to the host's urine, creating a particular environment. Exposure of two CRAB clinical isolates, AB5075 and AMA40, to human urine (HU) resulted in the differential expression levels of 264 and 455 genes, respectively, of which 112 were common to both strains. Genes within this group play roles in metabolic pathways such as phenylacetic acid (PAA) catabolism, the Hut system, the tricarboxylic acid (TCA) cycle, and other processes like quorum sensing and biofilm formation. These results indicate that the presence of HU induces numerous adaptive changes in gene expression of the infecting bacteria. These changes presumably help bacteria establish and thrive in the hostile conditions in the urinary tract. These analyses advance our understanding of CRAB's metabolic adaptations to human fluids, as well as expand knowledge on bacterial responses to distinct human fluids containing different concentrations of human serum albumin (HSA).
Assuntos
Acinetobacter baumannii , Carbapenêmicos , Urina , Acinetobacter baumannii/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Humanos , Carbapenêmicos/farmacologia , Urina/microbiologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Antibacterianos/farmacologia , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/urina , Adaptação Fisiológica/genética , Infecções Urinárias/microbiologia , Biofilmes/crescimento & desenvolvimento , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana/genéticaRESUMO
Brucella is responsible for brucellosis, one of the most common zoonoses worldwide that causes important economic losses in several countries. Increasing evidence indicates that adhesion of Brucella spp. to host cells is an important step to establish infection. We have previously shown that the BmaC unipolar monomeric autotransporter mediates the binding of Brucella suis to host cells through cell-associated fibronectin. Our genome analysis shows that the B. suis genome encodes several additional potential adhesins. In this work, we characterized a predicted trimeric autotransporter that we named BtaE. By expressing btaE in a nonadherent Escherichia coli strain and by phenotypic characterization of a B. suis ΔbtaE mutant, we showed that BtaE is involved in the binding of B. suis to hyaluronic acid. The B. suis ΔbtaE mutant exhibited a reduction in the adhesion to HeLa and A549 epithelial cells compared with the wild-type strain, and it was outcompeted by the wild-type strain in the binding to HeLa cells. The knockout btaE mutant showed an attenuated phenotype in the mouse model, indicating that BtaE is required for full virulence. BtaE was immunodetected on the bacterial surface at one cell pole. Using old and new pole markers, we observed that both the BmaC and BtaE adhesins are consistently associated with the new cell pole, suggesting that, in Brucella, the new pole is functionally differentiated for adhesion. This is consistent with the inherent polarization of this bacterium, and its role in the invasion process.
Assuntos
Adesinas Bacterianas/metabolismo , Brucella suis/metabolismo , Brucella suis/patogenicidade , Brucelose/microbiologia , Proteínas de Transporte/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Adesinas Bacterianas/genética , Animais , Anticorpos Antibacterianos , Aderência Bacteriana/fisiologia , Brucella suis/genética , Proteínas de Transporte/genética , Polaridade Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Família Multigênica , VirulênciaRESUMO
Carbapenem-resistant Acinetobacter baumannii (CRAB) is a recognized nosocomial pathogen with limited antibiotic treatment options. Lactic acid bacteria (LAB) constitute a promising therapeutic alternative. Here we studied the antibacterial properties of a collection of LAB strains using phenotypic and transcriptomic analysis against A. baumannii clinical strains. One strain, Lacticaseibacillus rhamnosus CRL 2244, demonstrated a potent inhibitory capacity on A. baumannii with a significant killing activity. Scanning electron microscopy images showed changes in the morphology of A. baumannii with an increased formation of outer membrane vesicles. Significant changes in the expression levels of a wide variety of genes were also observed. Interestingly, most of the modified genes were involved in a metabolic pathway known to be associated with the survival of A. baumannii . The paa operon, Hut system, and fatty acid degradation were some of the pathways that were induced. The analysis reveals the impact of Lcb. rhamnosus CRL 2244 on A. baumannii response, resulting in bacterial stress and subsequent cell death. These findings highlight the antibacterial properties of Lcb. rhamnosus CRL 2244 and its potential as an alternative or complementary strategy for treating infections. Further exploration and development of LAB as a treatment option could provide valuable alternatives for combating CRAB infections.
RESUMO
Carbapenem-resistant Acinetobacter baumannii (CRAB) is a recognized nosocomial pathogen with limited antibiotic treatment options. Lactic acid bacteria (LAB) constitute a promising therapeutic alternative. Here we studied the antibacterial properties of a collection of LAB strains using phenotypic and transcriptomic analysis against A. baumannii clinical strains. One strain, Lacticaseibacillus rhamnosus CRL 2244, demonstrated a potent inhibitory capacity on A. baumannii with a significant killing activity. Scanning electron microscopy images showed changes in the morphology of A. baumannii with an increased formation of outer membrane vesicles. Significant changes in the expression levels of a wide variety of genes were also observed. Interestingly, most of the modified genes were involved in a metabolic pathway known to be associated with the survival of A. baumannii. The paa operon, Hut system, and fatty acid degradation were some of the pathways that were induced. The analysis reveals the impact of Lcb. rhamnosus CRL 2244 on A. baumannii response, resulting in bacterial stress and subsequent cell death. These findings highlight the antibacterial properties of Lcb. rhamnosus CRL 2244 and its potential as an alternative or complementary strategy for treating infections. Further exploration and development of LAB as a treatment option could provide valuable alternatives for combating CRAB infections.
Assuntos
Acinetobacter baumannii , Lacticaseibacillus rhamnosus , Lactobacillales , Acinetobacter baumannii/genética , Lacticaseibacillus , Antibacterianos/farmacologia , Morte Celular , Carbapenêmicos/farmacologiaRESUMO
Type IV secretion systems (T4SS) are multiprotein structures that direct the translocation of specific molecules across the bacterial cell envelope. As in other bacteria, pathogenicity of the genus Brucella essentially depends on the integrity of the T4SS-encoding virB operon, whose expression is regulated by multiple transcription factors belonging to different families. Previously, we identified IHF and HutC, two direct regulators of the virB genes that were isolated from total protein extracts of Brucella. Here, we report the identification of MdrA, a third regulatory element that was isolated using the same screening procedure. This transcription factor, which belongs to the MarR-family of transcriptional regulators, binds at two different sites of the virB promoter and regulates expression in a growth phase-dependent manner. Like other members of the MarR family, specific ligands were able to dissociate MdrA from DNA in vitro. Determination of the MdrA-binding sites by DNase I footprinting and analyses of protein-DNA complexes by electrophoresis mobility shift assays (EMSAs) showed that MdrA competes with IHF and HutC for the binding to the promoter because their target DNA sequences overlap. Unlike IHF, both MdrA and HutC bound to the promoter without inducing bending of DNA. Moreover, the two latter transcription factors activated virB expression to similar extents, and in doing so, they are functionally redundant. Taken together, our results show that MdrA is a regulatory element that directly modulates the activity of the virB promoter and is probably involved in coordinating gene expression in response to specific environmental signals.
Assuntos
Brucella abortus/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Virulência/biossíntese , Sítios de Ligação , Brucella abortus/patogenicidade , Pegada de DNA , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
The MerR family is a group of transcriptional activators with conserved N-terminal helix-turn-helix DNA binding domains and variable C-terminal effector binding regions. In most MerR proteins the effector binding domain (EBD) contains a cysteine center suited for metal binding and mediates the response to environmental stimuli, such as oxidative stress, heavy metals or antibiotics. We here present a novel transcriptional regulator classified in the MerR superfamily that lacks an EBD domain and has neither conserved metal binding sites nor cysteine residues. This regulator from the psychrotolerant bacteria Bizionia argentinensis JUB59 is involved in iron homeostasis and was named MliR (MerR-like iron responsive Regulator). In silico analysis revealed that homologs of the MliR protein are widely distributed among different bacterial species. Deletion of the mliR gene led to decreased cell growth, increased cell adhesion and filamentation. Genome-wide transcriptomic analysis showed that genes associated with iron homeostasis were downregulated in mliR-deletion mutant. Through nuclear magnetic resonance-based metabolomics, ICP-MS, fluorescence microscopy and biochemical analysis we evaluated metabolic and phenotypic changes associated with mliR deletion. This work provides the first evidence of a MerR-family regulator involved in iron homeostasis and contributes to expanding our current knowledge on relevant metabolic pathways and cell remodeling mechanisms underlying in the adaptive response to iron availability in bacteria.
RESUMO
According to the Centers for Disease Control and Prevention, Acinetobacter baumannii is listed among the most threatening pathogens. A. baumannii is mainly a nosocomial pathogen with a distinctive ability to survive in multiple environments. These characteristics together with this bacterium's ability to acquire antibiotic resistance determinants make it a notorious pathogen. The presence of human serum albumin (HSA) is associated with modification of expression levels in numerous genes. The presence of HSA in the culture medium is also correlated with a reduction in levels of the global suppressor histone-like nucleoid structure protein, H-NS. Comparative transcriptome analysis of the wild type and isogenic Δhns strains cultured in lysogeny broth (LB) in the presence or absence of HSA revealed that the expression of a subset of eleven genes are modified in the Δhns cultured in LB and the wild-type strain in the presence of HSA, pointing out these genes as candidates to be regulated by the presence of HSA through H-NS. Six and five of these genes were up- or down-regulated, respectively. Three of these genes have functions in quorum sensing (acdA, kar and fadD), one in quorum quenching (aidA), two in stress response (katE, ywrO), three in metabolism (phaC, yedL1, and yedL2), one in biofilm formation (csuAB), and one in ß-oxidation of fatty acids (fadA). The regulation of these genes was assessed by: (i) transcriptional analysis and qPCR at the transcriptional level; and (ii) by determining the phenotypic characteristics of each function. The results of these studies support the hypothesis that HSA-mediated reduction of H-NS levels may be one very important regulatory circuit utilized by A. baumannii to adapt to selected environments, such as those where HSA-containing human fluids are abundant.
Assuntos
Acinetobacter baumannii , Antibacterianos , Proteínas de Bactérias , Biofilmes , Regulação Bacteriana da Expressão Gênica , Histonas , Humanos , Percepção de Quorum , Albumina Sérica HumanaRESUMO
Cefiderocol (CFDC) is a novel chlorocatechol-substituted siderophore antibiotic approved to treat complicated urinary tract infections (cUTI) and hospital-acquired and ventilator-acquired pneumonia (HAP/VAP). Previous work determined that albumin-rich human fluids increase the minimum inhibitory concentration (MICs) of Acinetobacter baumannii against CFDC and reduce the expression of genes related to iron uptake systems. This latter effect may contribute to the need for higher concentrations of CFDC to inhibit growth. The presence of human urine (HU), which contains low albumin concentrations, did not modify MIC values of two carbapenem-resistant A. baumannii. Levels of resistance to CFDC were not modified by HU in strain AMA40 but were reduced in strain AB5075. Expanding the studies to other carbapenem-resistant A. baumannii isolates showed that the presence of HU resulted in unmodified or reduced MIC of CDFC values. The expression of piuA, pirA, bauA, and bfnH determined by qRT-PCR was enhanced in A. baumannii AMA40 and AB5075 by the presence of HU in the culture medium. All four tested genes code for functions related to recognition and transport of ferric-siderophore complexes. The effect of HU on expression of pbp1, pbp3, blaOXA-51-like, blaADC, and blaNDM-1, genes associated with resistance to ß-lactams, as well as genes coding for efflux pumps and porins was variable, showing dependence with the strain analyzed. We conclude that the lack of significant concentrations of albumin and free iron in HU makes this fluid behave differently from others we tested. Unlike other albumin rich fluids, the presence of HU does not impact the antibacterial activity of CFDC when tested against A. baumannii.
Assuntos
Acinetobacter baumannii , Albuminas/farmacologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Carbapenêmicos/farmacologia , Cefalosporinas , Humanos , Ferro/farmacologia , Testes de Sensibilidade Microbiana , Sideróforos , beta-Lactamases/genética , CefiderocolRESUMO
Acinetobacter baumannii is a nosocomial pathogen capable of causing serious infections associated with high rates of morbidity and mortality. Due to its antimicrobial drug resistance profile, A. baumannii is categorized as an urgent priority pathogen by the Centers for Disease Control and Prevention in the United States and a priority group 1 critical microorganism by the World Health Organization. Understanding how A. baumannii adapts to different host environments may provide critical insights into strategically targeting this pathogen with novel antimicrobial and biological therapeutics. Exposure to human fluids was previously shown to alter the gene expression profile of a highly drug-susceptible A. baumannii strain A118 leading to persistence and survival of this pathogen. Herein, we explore the impact of human pleural fluid (HPF) and human serum albumin (HSA) on the gene expression profile of a highly multi-drug-resistant strain of A. baumannii AB5075. Differential expression was observed for ~30 genes, whose products are involved in quorum sensing, quorum quenching, iron acquisition, fatty acid metabolism, biofilm formation, secretion systems, and type IV pilus formation. Phenotypic and further transcriptomic analysis using quantitative RT-PCR confirmed RNA-seq data and demonstrated a distinctive role of HSA as the molecule involved in A. baumannii's response.
RESUMO
In the multidrug resistant (MDR) pathogen Acinetobacter baumannii the global repressor H-NS was shown to modulate the expression of genes involved in pathogenesis and stress response. In addition, H-NS inactivation results in an increased resistance to colistin, and in a hypermotile phenotype an altered stress response. To further contribute to the knowledge of this key transcriptional regulator in A. baumannii behavior, we studied the role of H-NS in antimicrobial resistance. Using two well characterized A. baumannii model strains with distinctive resistance profile and pathogenicity traits (AB5075 and A118), complementary transcriptomic and phenotypic approaches were used to study the role of H-NS in antimicrobial resistance, biofilm and quorum sensing gene expression. An increased expression of genes associated with ß-lactam resistance, aminoglycosides, quinolones, chloramphenicol, trimethoprim and sulfonamides resistance in the Δhns mutant background was observed. Genes codifying for efflux pumps were also up-regulated, with the exception of adeFGH. The wild-type transcriptional level was restored in the complemented strain. In addition, the expression of biofilm related genes and biofilm production was lowered when the transcriptional repressor was absent. The quorum network genes aidA, abaI, kar and fadD were up-regulated in Δhns mutant strains. Overall, our results showed the complexity and scope of the regulatory network control by H-NS (genes involved in antibiotic resistance and persistence). These observations brings us one step closer to understanding the regulatory role of hns to combat A. baumannii infections.
Assuntos
Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla , Histonas/metabolismo , Acinetobacter baumannii/genética , Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana Múltipla/genética , Regulação Bacteriana da Expressão Gênica , Genótipo , Testes de Sensibilidade Microbiana , Fenótipo , Percepção de Quorum/genéticaRESUMO
Most Acinetobacter baumannii strains are naturally competent. Although some information is available about factors that enhance or reduce the frequency of the transformation of this bacterium, the regulatory elements and mechanisms are barely understood. In this article, we describe studies on the role of the histone-like nucleoid structuring protein, H-NS, in the regulation of the expression of genes related to natural competency and the ability to uptake foreign DNA. The expression levels of the natural transformation-related genes pilA, pilT, pilQ, comEA, comEC, comF, and drpA significantly increased in a Δhns derivative of A. baumannii A118. The complementation of the mutant with a recombinant plasmid harboring hns restored the expression levels of six of these genes (pilT remained expressed at high levels) to those of the wild-type strain. The transformation frequency of the A. baumannii A118 Δhns strain was significantly higher than that of the wild-type. Similar, albeit not identical, there were consequences when hns was deleted from the hypervirulent A. baumannii AB5075 strain. In the AB5075 complemented strain, the reduction in gene expression in a few cases was not so pronounced that it reached wild-type levels, and the expression of comEA was enhanced further. In conclusion, the expression of all seven transformation-related genes was enhanced after deleting hns in A. baumannii A118 and AB5075, and these modifications were accompanied by an increase in the cells' transformability. The results highlight a role of H-NS in A. baumannii's natural competence.
RESUMO
In a recent report by the Centers for Disease Control and Prevention (CDC), multidrug resistant (MDR) Acinetobacter baumannii is a pathogen described as an "urgent threat." Infection with this bacterium manifests as different diseases such as community and nosocomial pneumonia, bloodstream infections, endocarditis, infections of the urinary tract, wound infections, burn infections, skin and soft tissue infections, and meningitis. In particular, nosocomial meningitis, an unwelcome complication of neurosurgery caused by extensively-drug resistant (XDR) A. baumannii, is extremely challenging to manage. Therefore, understanding how A. baumannii adapts to different host environments, such as cerebrospinal fluid (CSF) that may trigger changes in expression of virulence factors that are associated with the successful establishment and progress of this infection is necessary. The present in-vitro work describes, the genetic changes that occur during A. baumannii infiltration into CSF and displays A. baumannii's expansive versatility to persist in a nutrient limited environment while enhancing several virulence factors to survive and persist. While a hypervirulent A. baumannii strain did not show changes in its transcriptome when incubated in the presence of CSF, a low-virulence isolate showed significant differences in gene expression and phenotypic traits. Exposure to 4% CSF caused increased expression of virulence factors such as fimbriae, pilins, and iron chelators, and other virulence determinants that was confirmed in various model systems. Furthermore, although CSF's presence did not enhance bacterial growth, an increase of expression of genes encoding transcription, translation, and the ATP synthesis machinery was observed. This work also explores A. baumannii's response to an essential component, human serum albumin (HSA), within CSF to trigger the differential expression of genes associated with its pathoadaptibility in this environment.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Líquido Cefalorraquidiano , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/patogenicidade , Animais , Farmacorresistência Bacteriana Múltipla , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Larva/microbiologia , Mariposas/crescimento & desenvolvimento , Mariposas/microbiologia , Albumina Sérica/farmacologia , Transcriptoma/efeitos dos fármacos , Fatores de Virulência/genéticaRESUMO
VjbR is a LuxR-type quorum-sensing (QS) regulator that plays an essential role in the virulence of the intracellular facultative pathogen Brucella, the causative agent of brucellosis. It was previously described that VjbR regulates a diverse group of genes, including the virB operon. The latter codes for a type IV secretion system (T4SS) that is central for the pathogenesis of Brucella. Although the regulatory role of VjbR on the virB promoter (P(virB)) was extensively studied by different groups, the VjbR-binding site had not been identified so far. Here, we identified the target DNA sequence of VjbR in P(virB) by DNase I footprinting analyses. Surprisingly, we observed that VjbR specifically recognizes a sequence that is identical to a half-binding site of the QS-related regulator MrtR of Mesorhizobium tianshanense. As shown by DNase I footprinting and electrophoretic mobility shift assays, generation of a palindromic MrtR-like-binding site in P(virB) increased both the affinity and the stability of the VjbR-DNA complex, which confirmed that the QS regulator of Brucella is highly related to that of M. tianshanense. The addition of N-dodecanoyl homoserine lactone dissociated VjbR from the promoter, which confirmed previous reports that indicated a negative effect of this signal on the VjbR-mediated activation of P(virB). Our results provide new molecular evidence for the structure of the virB promoter and reveal unusual features of the QS target DNA sequence of the main regulator of virulence in Brucella.