RESUMO
Intracellular bacterial pathogens have developed a variety of strategies to avoid degradation by the host innate immune defense mechanisms triggered upon phagocytocis. Upon infection of mammalian host cells, the intracellular pathogen Francisella replicates exclusively in the cytosolic compartment. Hence, its ability to escape rapidly from the phagosomal compartment is critical for its pathogenicity. Here, we show for the first time that a glutamate transporter of Francisella (here designated GadC) is critical for oxidative stress defense in the phagosome, thus impairing intra-macrophage multiplication and virulence in the mouse model. The gadC mutant failed to efficiently neutralize the production of reactive oxygen species. Remarkably, virulence of the gadC mutant was partially restored in mice defective in NADPH oxidase activity. The data presented highlight links between glutamate uptake, oxidative stress defense, the tricarboxylic acid cycle and phagosomal escape. This is the first report establishing the role of an amino acid transporter in the early stage of the Francisella intracellular lifecycle.
Assuntos
Ciclo do Ácido Cítrico , Francisella tularensis/metabolismo , Ácido Glutâmico/metabolismo , Macrófagos/microbiologia , Fagossomos/metabolismo , Tularemia/metabolismo , Sistema X-AG de Transporte de Aminoácidos/genética , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Feminino , Francisella tularensis/genética , Francisella tularensis/patogenicidade , Ácido Glutâmico/genética , Macrófagos/metabolismo , Macrófagos/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mutação , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Fagossomos/genética , Fagossomos/microbiologia , Fagossomos/patologia , Tularemia/genéticaRESUMO
In order to develop a successful infectious cycle, intracellular bacterial pathogens must be able to adapt their metabolism to optimally utilize the nutrients available in the cellular compartments and tissues where they reside. Francisella tularensis, the agent of the zoonotic disease tularaemia, is a highly infectious bacterium for a large number of animal species. This bacterium replicates in its mammalian hosts mainly in the cytosol of infected macrophages. We report here the identification of a novel amino acid transporter of the major facilitator superfamily of secondary transporters that is required for bacterial intracellular multiplication and systemic dissemination. We show that inactivation of this transporter does not affect phagosomal escape but prevents multiplication in the cytosol of all cell types tested. Remarkably, the intracellular growth defect of the mutant was fully and specifically reversed by addition of asparagine or asparagine-containing dipeptides as well as by simultaneous addition of aspartic acid and ammonium. Importantly, bacterial virulence was also restored in vivo, in the mouse model, by asparagine supplementation. This work unravels thus, for the first time, the importance of asparagine for cytosolicmultiplication of Francisella. Amino acid transporters are likely to constitute underappreciated players in bacterial intracellular parasitism.
Assuntos
Sistemas de Transporte de Aminoácidos/genética , Asparagina/metabolismo , Proteínas de Bactérias/genética , Francisella tularensis/crescimento & desenvolvimento , Compostos de Amônio/farmacologia , Animais , Asparagina/farmacologia , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacologia , Proteínas de Bactérias/farmacocinética , Linhagem Celular Tumoral , Francisella tularensis/metabolismo , Francisella tularensis/patogenicidade , Células Hep G2 , Humanos , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Fagossomos/microbiologia , Tularemia/microbiologiaRESUMO
Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. In vivo, this facultative intracellular bacterium survives and replicates mainly in the cytoplasm of infected cells. We have recently identified a genetic locus, designated moxR that is important for stress resistance and intramacrophage survival of F. tularensis. In the present work, we used tandem affinity purification coupled to mass spectrometry to identify in vivo interacting partners of three proteins encoded by this locus: the MoxR-like ATPase (FTL_0200), and two proteins containing motifs predicted to be involved in protein-protein interactions, bearing von Willebrand A (FTL_0201) and tetratricopeptide (FTL_0205) motifs. The three proteins were designated here for simplification, MoxR, VWA1, and TPR1, respectively. MoxR interacted with 31 proteins, including various enzymes. VWA1 interacted with fewer proteins, but these included the E2 component of 2-oxoglutarate dehydrogenase and TPR1. The protein TPR1 interacted with one hundred proteins, including the E1 and E2 subunits of both oxoglutarate and pyruvate dehydrogenase enzyme complexes, and their common E3 subunit. Remarkably, chromosomal deletion of either moxR or tpr1 impaired pyruvate dehydrogenase and oxoglutarate dehydrogenase activities, supporting the hypothesis of a functional role for the interaction of MoxR and TPR1 with these complexes. Altogether, this work highlights possible links between stress resistance and metabolism in F. tularensis virulence.
Assuntos
Proteínas de Bactérias/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Francisella tularensis/metabolismo , Francisella tularensis/patogenicidade , Estresse Fisiológico , Sequência de Aminoácidos , Animais , DNA Bacteriano/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Análise de Sequência de DNA , TularemiaRESUMO
Francisella tularensis, a Gram-negative bacterium that causes the disease tularemia in a large number of animal species, is thought to reside preferentially within macrophages in vivo. F. tularensis has developed mechanisms to rapidly escape from the phagosome into the cytoplasm of infected cells, a habitat with a rich supply of nutrients, ideal for multiplication. SLC1A5 is a neutral amino acid transporter expressed by human cells, which serves, along with SLC7A5 to equilibrate cytoplasmic amino acid pools. We herein analysed whether SLC1A5 was involved in F. tularensis intracellular multiplication. We demonstrate that expression of SLC1A5 is specifically upregulated by F. tularensis in infected THP-1 human monocytes. Furthermore, we show that SLC1A5 downregulation decreases intracellular bacterial multiplication, supporting the involvement of SLC1A5 in F. tularensis infection. Notably, after entry of F. tularensis into cells and during the whole infection, the highly glycosylated form of SLC1A5 was deglycosylated only by bacteria capable of cytosolic multiplication. These data suggest that intracellular replication of F. tularensis depends on the function of host cell SLC1A5. Our results are the first, which show that Francisella intracellular multiplication in human monocyte cytoplasm is associated with a post-translational modification of a eukaryotic amino acid transporter.
Assuntos
Sistema ASC de Transporte de Aminoácidos/biossíntese , Francisella tularensis/patogenicidade , Interações Hospedeiro-Patógeno , Monócitos/microbiologia , Sistemas de Transporte de Aminoácidos , Bactérias , Linhagem Celular , Francisella , Francisella tularensis/crescimento & desenvolvimento , Humanos , Antígenos de Histocompatibilidade Menor , Regulação para CimaRESUMO
Staphylococcus aureus is a predominant cause of chronic lung infections. While the airway environment is rich in highly sialylated mucins, the interaction of S. aureus with sialic acid is poorly characterized. Using S. aureus USA300 as well as clinical isolates, we demonstrate that quorum-sensing dysfunction, a hallmark of S. aureus adaptation, correlates with a greater ability to consume free sialic acid, providing a growth advantage in an air-liquid interface model and in vivo. Furthermore, RNA-seq experiment reveals that free sialic acid triggers transcriptional reprogramming promoting S. aureus chronic lifestyle. To support the clinical relevance of our results, we show the co-occurrence of S. aureus, sialidase-producing microbiota and free sialic acid in the airway of patients with cystic fibrosis. Our findings suggest a dual role for sialic acid in S. aureus airway infection, triggering virulence reprogramming and driving S. aureus adaptive strategies through the selection of quorum-sensing dysfunctional strains.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Percepção de Quorum/genética , Ácido N-Acetilneuramínico , Sistema Respiratório , Proteínas de BactériasRESUMO
We studied three Fur-regulated systems of Listeria monocytogenes: the srtB region, that encodes sortase-anchored proteins and a putative ABC transporter, and the fhu and hup operons, that produce putative ABC transporters for ferric hydroxamates and haemin (Hn)/haemoglobin (Hb) respectively. Deletion of lmo2185 in the srtB region reduced listerial [(59) Fe]-Hn transport, and purified Lmo2185 bound [(59) Fe]-Hn (K(D) = 12 nM), leading to its designation as a Hn/Hb binding protein (hbp2). Purified Hbp2 also acted as a haemophore, capturing and supplying Hn from the environment. Nevertheless, Hbp2 only functioned in [(59) Fe]-Hn transport at external concentrations less than 50 nM: at higher Hn levels its uptake occurred with equivalent affinity and rate without Hbp2. Similarly, deletion of sortase A had no effect on ferric siderophore or Hn/Hb transport at any concentration, and the srtA-independence of listerial Hn/Hb uptake distinguished it from comparable systems of Staphylococcus aureus. In the cytoplasmic membrane, the Hup transporter was specific for Hn: its lipoprotein (HupD) only showed high affinity for the iron porphyrin (K(D) = 26 nM). Conversely, the FhuD lipoprotein encoded by the fhu operon had broad specificity: it bound both ferric siderophores and Hn, with the highest affinity for ferrioxamine B (K(D) = 123 nM). Deletions of Hup permease components hupD, hupG or hupDGC reduced Hn/Hb uptake, and complementation of ΔhupC and ΔhupG by chromosomal integration of hupC(+) and hupG(+) alleles on pPL2 restored growth promotion by Hn/Hb. However, ΔhupDGC did not completely eliminate [(59) Fe]-Hn transport, implying the existence of another cytoplasmic membrane Hn transporter. The overall K(M) of Hn uptake by wild-type strain EGD-e was 1 nM, and it occurred at similar rates (V(max) = 23 pmol 10(9) cells(-1) min(-1)) to those of ferric siderophore transporters. In the ΔhupDGC strain uptake occurred at a threefold lower rate (V(max) = 7 pmol 10(9) cells(-1) min(-1)). The results show that at low (< 50 nM) levels of Hn, SrtB-dependent peptidoglycan-anchored proteins (e.g. Hbp2) bind the porphyrin, and HupDGC or another transporter completes its uptake into the cytoplasm. However, at higher concentrations Hn uptake is SrtB-independent: peptidoglycan-anchored binding proteins are dispensable because HupDGC directly absorbs and internalizes Hn. Finally, ΔhupDGC increased the LD(50) of L. monocytogenes 100-fold in the mouse infection model, reiterating the importance of this system in listerial virulence.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Heme/metabolismo , Hemoglobinas/metabolismo , Listeria monocytogenes/metabolismo , Aminoaciltransferases/genética , Animais , Proteínas de Bactérias/genética , Cisteína Endopeptidases/genética , Feminino , Humanos , Listeria monocytogenes/enzimologia , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Camundongos , Óperon , VirulênciaRESUMO
Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. This facultative intracellular bacterium replicates in vivo mainly inside macrophages and therefore has developed strategies to resist this stressful environment. Here, we identified a novel genetic locus that is important for stress resistance and intracellular survival of F. tularensis. In silico and transcriptional analyses suggest that this locus (genes FTL_0200 to FTL_0209 in the live vaccine strain [LVS]) constitutes an operon controlled by the alternative sigma factor σ³². The first gene, FTL_0200, encodes a putative AAA+ ATPase of the MoxR subfamily. Insertion mutagenesis into genes FTL_0200, FTL_0205, and FTL_0206 revealed a role for the locus in both intracellular multiplication and in vivo survival of F. tularensis. Deletion of gene FTL_0200 led to a mutant bacterium with increased vulnerability to various stress conditions, including oxidative and pH stresses. Proteomic analyses revealed a pleiotropic impact of the ΔFTL_0200 deletion, supporting a role as a chaperone for FTL_0200. This is the first report of a role for a MoxR family member in bacterial pathogenesis. This class of proteins is remarkably conserved among pathogenic species and may thus constitute a novel player in bacterial virulence.
Assuntos
Francisella tularensis/genética , Francisella tularensis/patogenicidade , Genes Bacterianos/genética , Chaperonas Moleculares/genética , Estresse Fisiológico/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Southern Blotting , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tularemia/genética , Tularemia/metabolismo , Virulência/genéticaRESUMO
Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. Its ability to multiply and survive in macrophages is critical for its virulence. By screening a bank of HimarFT transposon mutants of the F. tularensis live vaccine strain (LVS) to isolate intracellular growth-deficient mutants, we selected one mutant in a gene encoding a putative gamma-glutamyl transpeptidase (GGT). This gene (FTL_0766) was hence designated ggt. The mutant strain showed impaired intracellular multiplication and was strongly attenuated for virulence in mice. Here we present evidence that the GGT activity of F. tularensis allows utilization of glutathione (GSH, gamma-glutamyl-cysteinyl-glycine) and gamma-glutamyl-cysteine dipeptide as cysteine sources to ensure intracellular growth. This is the first demonstration of the essential role of a nutrient acquisition system in the intracellular multiplication of F. tularensis. GSH is the most abundant source of cysteine in the host cytosol. Thus, the capacity this intracellular bacterial pathogen has evolved to utilize the available GSH, as a source of cysteine in the host cytosol, constitutes a paradigm of bacteria-host adaptation.
Assuntos
Cisteína/metabolismo , Dipeptídeos/metabolismo , Francisella tularensis/metabolismo , Francisella tularensis/patogenicidade , Glutationa/metabolismo , Viabilidade Microbiana , Animais , Linhagem Celular , Citosol/metabolismo , Escherichia coli/genética , Feminino , Francisella tularensis/genética , Genes Bacterianos , Teste de Complementação Genética , Macrófagos/citologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Plasmídeos , Virulência , gama-Glutamiltransferase/genética , gama-Glutamiltransferase/metabolismoRESUMO
BACKGROUND: Regulation of bacterial gene expression by small RNAs (sRNAs) have proved to be important for many biological processes. Francisella tularensis is a highly pathogenic Gram-negative bacterium that causes the disease tularaemia in humans and animals. Relatively little is known about the regulatory networks existing in this organism that allows it to survive in a wide array of environments and no sRNA regulators have been identified so far. RESULTS: We have used a combination of experimental assays and in silico prediction to identify sRNAs in F. tularensis strain LVS. Using a cDNA cloning and sequencing approach we have shown that F. tularensis expresses homologues of several sRNAs that are well-conserved among diverse bacteria. We have also discovered two abundant putative sRNAs that share no sequence similarity or conserved genomic context with any previously annotated regulatory transcripts. Deletion of either of these two loci led to significant changes in the expression of several mRNAs that likely include the cognate target(s) of these sRNAs. Deletion of these sRNAs did not, however, significantly alter F. tularensis growth under various stress conditions in vitro, its replication in murine cells, or its ability to induce disease in a mouse model of F. tularensis infection. We also conducted a genome-wide in silico search for intergenic loci that suggests F. tularensis encodes several other sRNAs in addition to the sRNAs found in our experimental screen. CONCLUSION: Our findings suggest that F. tularensis encodes a significant number of non-coding regulatory RNAs, including members of well conserved families of structural and housekeeping RNAs and other poorly conserved transcripts that may have evolved more recently to help F. tularensis deal with the unique and diverse set of environments with which it must contend.
Assuntos
Francisella tularensis/genética , RNA Bacteriano/análise , RNA Bacteriano/genética , Animais , Vacinas Bacterianas/imunologia , Sequência de Bases , Northern Blotting , Clonagem Molecular , Biologia Computacional , DNA Complementar/genética , Francisella tularensis/imunologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Espaço Intracelular/microbiologia , Macrófagos/microbiologia , Camundongos , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Transporte de RNA/genética , RNA Bacteriano/química , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vacinas Atenuadas/imunologiaRESUMO
Francisella tularensis is a highly infectious pathogen that infects animals and humans, causing tularemia. The ability to replicate within macrophages is central for virulence and relies on expression of genes located in the Francisella pathogenicity island (FPI), as well as expression of other genes. Regulation of FPI-encoded virulence gene expression in F. tularensis involves at least four regulatory proteins and is not fully understood. Here we studied the RNA-binding protein Hfq in F. tularensis and particularly the role that it plays as a global regulator of gene expression in stress tolerance and pathogenesis. We demonstrate that Hfq promotes resistance to several cellular stresses (including osmotic and membrane stresses). Furthermore, we show that Hfq is important for the ability of the F. tularensis vaccine strain LVS to induce disease and persist in organs of infected mice. We also demonstrate that Hfq is important for stress tolerance and full virulence in a virulent clinical isolate of F. tularensis, FSC200. Finally, microarray analyses revealed that Hfq regulates expression of numerous genes, including genes located in the FPI. Strikingly, Hfq negatively regulates only one of two divergently expressed putative operons in the FPI, in contrast to the other known regulators, which regulate the entire FPI. Hfq thus appears to be a new pleiotropic regulator of virulence in F. tularensis, acting mostly as a repressor, in contrast to the other regulators identified so far. Moreover, the results obtained suggest a novel regulatory mechanism for a subset of FPI genes.
Assuntos
Proteínas de Bactérias/fisiologia , Francisella tularensis/fisiologia , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/fisiologia , Fatores de Virulência/biossíntese , Sequência de Aminoácidos , Animais , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Ordem dos Genes , Ilhas Genômicas , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Alinhamento de Sequência , Estresse Fisiológico , Análise de Sobrevida , Tularemia/microbiologia , Tularemia/patologia , VirulênciaRESUMO
Intracellular bacterial pathogens generally express chaperones such as Hsp100s during multiplication in host cells, allowing them to survive potentially hostile conditions. Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularaemia. The ability of F. tularensis to multiply and survive in macrophages is considered essential for its virulence. Although previous mutant screens in Francisella have identified the Hsp100 chaperone ClpB as important for intracellular survival, no detailed study has been performed. We demonstrate here that ClpB of F. tularensis live vaccine strain (LVS) is important for resistance to cellular stress. Promoter analysis shows that the transcriptional start is preceded by a sigma32-like promoter sequence and we demonstrate that expression of clpB is induced by heat shock. This indicates that expression of clpB is dependent on the heat-shock response mediated by sigma32, the only alternative sigma-factor present in Francisella. Our studies demonstrate that ClpB contributes to intracellular multiplication in vitro, but is not essential. However, ClpB is absolutely required for Francisella to replicate in target organs and induce disease in mice. Proteomic analysis of membrane-enriched fractions shows that five proteins are recovered at lower levels in the mutant strain. The crucial role of ClpB for in vivo persistence of Francisella may be linked to its assumed function in reactivation of aggregated proteins under in vivo stress conditions.
Assuntos
Proteínas de Bactérias/metabolismo , Francisella tularensis/metabolismo , Proteínas de Choque Térmico/metabolismo , Macrófagos/microbiologia , Adaptação Fisiológica/genética , Adaptação Fisiológica/fisiologia , Animais , Proteínas de Bactérias/genética , Eletroforese em Gel Bidimensional , Eletroporação , Feminino , Francisella tularensis/genética , Francisella tularensis/ultraestrutura , Proteínas de Choque Térmico/genética , Temperatura Alta , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Microscopia Eletrônica de Transmissão , Regiões Promotoras Genéticas/genética , Proteoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator sigma/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
Francisella tularensis is a highly virulent bacterium responsible for the zoonotic disease tularemia. It is a facultative intracellular pathogen that replicates in the cytoplasm of host cells, particularly in macrophages. Here we show that F. tularensis live vaccine strain (LVS) expresses a novel small RNA (sRNA), which modulates the virulence capacities of the bacterium. When this sRNA, designated FtrC (for Francisella tularensis RNA C), is expressed at high levels, F. tularensis replicates in macrophages less efficiently than the wild-type parent strain. Similarly, high expression of FtrC reduces the number of viable bacteria recovered from the spleen and liver of infected mice. Our data demonstrate that expression of gene FTL_1293 is regulated by FtrC. Furthermore, we show by in vitro gel shift assays that FtrC interacts specifically with FTL_1293 mRNA and that this happens independently of the RNA chaperone Hfq. Remarkably, FtrC interacts only with full-length FTL_1293 mRNA. These results, combined with a bioinformatic analysis, indicate that FtrC interacts with the central region of the mRNA and hence does not act by sterically hindering access of the ribosome to the mRNA. We further show that gene FTL_1293 is not required for F. tularensis virulence in vitro or in vivo, which indicates that another unidentified FtrC target modulates the virulence capacity of the bacterium.
Assuntos
Francisella tularensis/genética , Francisella tularensis/patogenicidade , RNA Bacteriano/genética , RNA não Traduzido/genética , Animais , Sequência de Bases , Feminino , Regulação Bacteriana da Expressão Gênica/genética , Espaço Intracelular/microbiologia , Macrófagos/citologia , Macrófagos/microbiologia , Camundongos , Dados de Sequência Molecular , Especificidade da EspécieRESUMO
Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularaemia. During its infectious cycle, F. tularensis is not only exposed to the intracellular environment of macrophages but also resides transiently in extracellular compartments, in particular during its systemic dissemination. The screening of a bank of F. tularensis LVS transposon insertion mutants on chemically defined medium (CDM) led us to identify a gene, designated trkH, encoding a homolog of the potassium uptake permease TrkH. Inactivation of trkH impaired bacterial growth in CDM. Normal growth of the mutant was only restored when CDM was supplemented with potassium at high concentration. Strikingly, although not required for intracellular survival in cell culture models, TrkH appeared to be essential for bacterial virulence in the mouse. In vivo kinetics of bacterial dissemination revealed a severe defect of multiplication of the trkH mutant in the blood of infected animals. The trkH mutant also showed impaired growth in blood ex vivo. Genome sequence analyses suggest that the Trk system constitutes the unique functional active potassium transporter in both tularensis and holarctica subspecies. Hence, the impaired survival of the trkH mutant in vivo is likely to be due to its inability to survive in the low potassium environment (1-5 mM range) of the blood. This work unravels thus the importance of potassium acquisition in the extracellular phase of the F. tularensis infectious cycle. More generally, potassium could constitute an important mineral nutrient involved in other diseases linked to systemic dissemination of bacterial pathogens.
Assuntos
Proteínas de Bactérias/fisiologia , Francisella tularensis/patogenicidade , Potássio/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genes Bacterianos , Camundongos , Dados de Sequência Molecular , Mutação , Homologia de Sequência de AminoácidosRESUMO
Listeria monocytogenes is a foodborne pathogen able to infect humans and many other mammalian species, leading to serious, often fatal disease. We have previously identified a five-gene locus in the genome of L. monocytogenes EGD-e which comprised three contiguous genes encoding paralogous type I signal peptidases. In the present study, we focused on the two distal genes of the locus (lmo1272 and lmo1273), encoding proteins sharing significant similarities with the YlqF and RnhB proteins, respectively, of Bacillus subtilis. lmo1273 could complement an Escherichia coli rnhA-rnhB thermosensitive growth phenotype, suggesting that it encodes a functional RNase H. Strikingly, inactivation of lmo1273 provoked a strong attenuation of virulence in the mouse model, and kinetic studies in infected mice revealed that multiplication of the lmo1273 mutant in target organs was significantly impaired. However, the mutation did not impair L. monocytogenes intracellular multiplication or cell-to-cell spread in cell culture models. Transcriptional profiles obtained with an lmo1273-overexpressing strain were compared to those of the wild-type strain, using microarray analyses. The data obtained suggest a pleiotropic regulatory role of Lmo1273 and possible links with amino acid uptake.
Assuntos
Proteínas de Bactérias/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Virulência , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Escherichia coli/genética , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Teste de Complementação Genética , Listeria monocytogenes/metabolismo , Listeriose/microbiologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano/genética , Ribonuclease H/genética , Ribonuclease H/metabolismoRESUMO
Francisella tularensis is a highly infectious bacterial pathogen, responsible for the zoonotic disease tularemia. We screened a bank of transposon insertion mutants of F. tularensis subsp. holarctica LVS for colony morphology alterations and selected a mutant with a transposon insertion in wbtA, the first gene of the predicted lipopolysaccharide O-antigen gene cluster. Inactivation of wbtA led to the complete loss of O antigen, conferred serum sensitivity, impaired intracellular replication, and severely attenuated virulence in the mouse model. Notably, this mutant afforded protection against a challenge against virulent LVS.
Assuntos
Francisella tularensis/imunologia , Francisella tularensis/patogenicidade , Genes Bacterianos , Antígenos O/genética , Antígenos O/imunologia , Animais , Elementos de DNA Transponíveis , Feminino , Francisella tularensis/genética , Camundongos , Camundongos Endogâmicos BALB C , Família Multigênica , Mutagênese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tularemia/imunologia , VirulênciaRESUMO
Molecular chaperones play an essential role in the folding of nascent chain polypeptides, as well as in the refolding and degradation of misfolded or aggregated proteins. They also assist in protein translocation and participate in stress functions. We identified a gene, designated tig, encoding a protein homologous to trigger factor (TF), a cytosolic ribosome-associated chaperone, in the genome of Listeria monocytogenes. We constructed a chromosomal Delta tig deletion and evaluated the impact of the mutation on bacterial growth in broth under various stress conditions and on pathogenesis. The Delta tig deletion did not affect cell viability but impaired survival in the presence of heat and ethanol stresses. We also identified the ffh gene, encoding a protein homologous to the SRP54 eukaryotic component of the signal recognition particle. However, a Delta ffh deletion was not tolerated, suggesting that Ffh is essential, as it is in Bacillus subtilis and Escherichia coli. Thus, although dispensable for growth, TF is involved in the stress response of L. monocytogenes. The Delta tig mutant showed no or very modest intracellular survival defects in eukaryotic cells. However, in vivo it showed a reduced capacity to persist in the spleens and livers of infected mice, revealing that TF has a role in the pathogenicity of L. monocytogenes.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/fisiologia , Listeria monocytogenes/patogenicidade , Virulência , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Células Cultivadas , Deleção Cromossômica , Etanol/farmacologia , Listeria monocytogenes/genética , Listeria monocytogenes/fisiologia , Listeriose , Camundongos , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Cloreto de Sódio/farmacologia , TemperaturaRESUMO
Listeria monocytogenes is a facultative intracellular gram-positive bacterium responsible for severe opportunistic infections in humans and animals. We had previously identified a gene encoding a putative UDP-N-acetylglucosamine 2-epimerase, a precursor of the teichoic acid linkage unit, in the genome of L monocytogenes strain EGD-e. This gene, now designated lmo2537, encodes a protein that shares 62% identity with the cognate epimerase MnaA of Bacillus subtilis and 55% identity with Cap5P of Staphylococcus aureus. Here, we addressed the role of lmo2537 in L. monocytogenes pathogenesis by constructing a conditional knockout mutant. The data presented here demonstrate that lmo2537 is an essential gene of L. monocytogenes that is involved in teichoic acid biogenesis. In vivo, the conditional mutant is very rapidly eliminated from the target organs of infected mice and thus is totally avirulent.
Assuntos
Genes Essenciais , Listeria monocytogenes/enzimologia , Listeria monocytogenes/patogenicidade , Ácidos Teicoicos/biossíntese , Fatores de Virulência/genética , Animais , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Linhagem Celular , Contagem de Colônia Microbiana , Citoplasma/microbiologia , Modelos Animais de Doenças , Listeria monocytogenes/genética , Listeria monocytogenes/ultraestrutura , Listeriose/microbiologia , Macrófagos/microbiologia , Camundongos , Microscopia Eletrônica de Transmissão , Mutação , Homologia de Sequência de Aminoácidos , Staphylococcus aureus , Fatores de Virulência/metabolismoRESUMO
Flagellar structures have been shown to participate in virulence in a variety of intestinal pathogens. Here, we have identified two potential flagellar genes of Listeria monocytogenes: lmo0713, encoding a protein similar to the flagellar basal body component FliF, and lmo0716, encoding a protein similar to FliI, the cognate ATPase energizing the flagellar export apparatus. Expression of fliF and fliI appears to be downregulated at 37 degrees C, like that of flaA, encoding flagellin. By constructing two chromosomal deletion mutants, we show that inactivation of either fliF or fliI (i) abolishes bacterial motility and flagella production, (ii) impairs adhesion and entry into nonphagocytic epithelial cells, and (iii) also reduces uptake by bone marrow-derived macrophages. However, the DeltafliF and DeltafliI mutations have only a minor impact on bacterial virulence in the mouse model, indicating that the flagellar secretion apparatus itself is not essential for survival in this animal model. Finally, among 100 human clinical isolates of L. monocytogenes tested, we found 20 strains still motile at 37 degrees C. Notably, all these strains adhered less efficiently than strain EGD-e to Caco-2 cells at 37 degrees C but showed no defect of intracellular multiplication. These data suggest that expression of the flagella at 37 degrees C might hinder optimal adhesion to epithelial cells but has no impact on intracytosolic survival of L. monocytogenes.
Assuntos
Proteínas de Bactérias/fisiologia , Flagelos/fisiologia , Listeria monocytogenes/fisiologia , Proteínas de Membrana/fisiologia , ATPases Translocadoras de Prótons/fisiologia , Animais , Aderência Bacteriana/genética , Células CACO-2 , Linhagem Celular Tumoral , Flagelos/química , Flagelos/genética , Flagelina/genética , Flagelina/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Cinética , Listeria monocytogenes/patogenicidade , Camundongos , Mutação , Processamento de Proteína Pós-Traducional , Análise de Sequência de DNA , TemperaturaRESUMO
In Listeria monocytogenes the promoter region of the svpA-srtB locus contains a well-conserved Fur box. We characterized the iron-regulation of this locus: real-time polymerase chain reaction analyses and anti-SvpA immunoblots showed that, in response to iron deprivation svpA transcription and SvpA production markedly increased (80-fold and 10-fold respectively), when initiated by either the addition of the iron chelator 2,2'-bipyridyl to BHI media, or by growth in iron-restricted minimal media. Green fluorescent protein (GFP) reporter constructs also showed increased activity of the svpA-srtB promoter in Escherichia coli (37-fold) and in L. monocytogenes (two- to threefold) when the bacteria were grown in iron-deficient conditions. A Deltafur mutant of L. monocytogenes constitutively synthesized SvpA, as well as GFP fused to the svpA-srtB promoter. Cellular fractionation data revealed that in iron-rich media wild-type SvpA was exclusively secreted to the culture supernatant. However, both the Deltafur derivative and wild-type L. monocytogenes grown in iron-deficient media anchored a fraction of the SvpA proteins (approximately 5%) to peptidoglycan, and produced a lower-molecular weight, wholly secreted form of SvpA. Together these data establish that iron availability controls transcription of the svpA-srtB locus (through Fur-mediated regulation), and attachment of SvpA to the cell wall (through SrtB-mediated covalent linkage). SvpA bears homology to IsdC, a haemin-binding protein of Staphylococcus aureus, and haemin bound to SvpA in solution. However, site-directed deletions of four structural genes and the promoter of the svpA-srtB locus did not impair haemin, haemoglobin or ferrichrome utilization in nutrition tests. We did not find strong evidence to support the notion that the svpA-srtB locus participates in haemin acquisition, as was reported for the homologous isd operon of S. aureus. Furthermore, the svpA-srtB mutant strains showed no significant attenuation of virulence in an intravenous mouse model system, but we found that the mutations reduced the persistence of L. monocytogenes in murine liver, spleen and intestines after oral administration.
Assuntos
Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Listeria monocytogenes/patogenicidade , Proteínas de Membrana/genética , Proteínas Repressoras/metabolismo , Aminoaciltransferases/metabolismo , Animais , Sequência de Bases , Células CACO-2 , Linhagem Celular , Meios de Cultura , Cisteína Endopeptidases , Modelos Animais de Doenças , Feminino , Deleção de Genes , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Listeriose/microbiologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Repressoras/genética , VirulênciaRESUMO
Listeria monocytogenes is a gram-positive facultative intracellular food-borne pathogen that can cause severe infections in humans and animals. We have recently adapted signature-tagged transposon mutagenesis (STM) to identify genes involved in the virulence of L. monocytogenes. A new round of STM allowed us to identify a new locus encoding a protein homologous to AgrA, the well-studied response regulator of Staphylococcus aureus and part of a two-component system involved in bacterial virulence. The production of several secreted proteins was modified in the agrA mutant of L. monocytogenes grown in broth, indicating that the agr locus influenced protein secretion. Inactivation of agrA did not affect the ability of the pathogen to invade and multiply in cells in vitro. However, the virulence of the agrA mutant was attenuated in the mouse (a 10-fold increase in the 50% lethal dose by the intravenous route), demonstrating for the first time a role for the agr locus in the virulence of L. monocytogenes.