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Listeria monocytogenes (Lm) is a facultative intracellular bacterial pathogen and the causative agent of listeriosis, a rare but fatal disease. During infection, Lm can traverse several physiological barriers; it can cross the intestine and placenta barrier and, in immunocompromised individuals, the blood-brain barrier. With the recent plethora of sequenced genomes available for Lm, it is clear that the complete repertoire of genes used by Lm to interact with its host remains to be fully explored. Recently, we focused on secreted Lm proteins because they are likely to interact with host cell components. Here, we investigated a putatively secreted protein of Lm, Lmo1656, that is present in most sequenced strains of Lm but absent in the nonpathogenic species Listeria innocua. lmo1656 gene is predicted to encode a small, positively charged protein. We show that Lmo1656 is secreted by Lm Furthermore, deletion of the lmo1656 gene (Δlmo1656) attenuates virulence in mice infected orally but not intravenously, suggesting that Lmo1656 plays a role during oral listeriosis. We identified sorting nexin 6 (SNX6), an endosomal sorting component and BAR domain-containing protein, as a host cell interactor of Lmol656. SNX6 colocalizes with WT Lm during the early steps of infection. This colocalization depends on Lmo1656, and RNAi of SNX6 impairs infection in infected tissue culture cells, suggesting that SNX6 is utilized by Lm during infection. Our results reveal that Lmo1656 is a novel secreted virulence factor of Lm that facilitates recruitment of a specific member of the sorting nexin family in the mammalian host.
Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Listeria monocytogenes/fisiologia , Listeriose/metabolismo , Nexinas de Classificação/metabolismo , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Feminino , Listeria monocytogenes/química , Listeriose/microbiologia , Camundongos Endogâmicos BALB C , Fatores de Virulência/químicaRESUMO
Listeria monocytogenes is responsible for gastroenteritis in healthy individuals and for a severe invasive disease in immunocompromised patients. Among the three identified L. monocytogenes evolutionary lineages, lineage I strains are overrepresented in epidemic listeriosis outbreaks, but the mechanisms underlying the higher virulence potential of strains of this lineage remain elusive. Here, we demonstrate that Listeriolysin S (LLS), a virulence factor only present in a subset of lineage I strains, is a bacteriocin highly expressed in the intestine of orally infected mice that alters the host intestinal microbiota and promotes intestinal colonization by L. monocytogenes, as well as deeper organ infection. To our knowledge, these results therefore identify LLS as the first bacteriocin described in L. monocytogenes and associate modulation of host microbiota by L. monocytogenes epidemic strains to increased virulence.
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Bacteriocinas/metabolismo , Microbioma Gastrointestinal , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Animais , Epidemias , Feminino , Interações Hospedeiro-Patógeno , Humanos , Listeria monocytogenes/patogenicidade , Listeriose/epidemiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , VirulênciaRESUMO
The DNA damage response (DDR) is an essential signaling pathway that detects DNA lesions, which constantly occur upon either endogenous or exogenous assaults, and maintains genetic integrity. An infection by an invading pathogen is one such assault, but how bacteria impact the cellular DDR is poorly documented. Here, we report that infection with Listeria monocytogenes induces host DNA breaks. Strikingly, the signature response to these breaks is only moderately activated. We uncover the role of the listerial toxin listeriolysin O (LLO) in blocking the signaling response to DNA breaks through degradation of the sensor Mre11. Knocking out or inactivating proteins involved in the DDR promotes bacterial replication showing the importance of this mechanism for the control of infection. Together, our data highlight that bacterial dampening of the DDR is critical for a successful listerial infection.
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Dano ao DNA , Replicação do DNA/genética , Listeria monocytogenes/genética , Listeriose/genética , Animais , Toxinas Bacterianas/farmacologia , Proteínas de Choque Térmico/farmacologia , Proteínas Hemolisinas/farmacologia , Humanos , Camundongos , Transdução de Sinais/genéticaRESUMO
During infection, pathogenic bacteria manipulate the host cell in various ways to allow their own replication, propagation and escape from host immune responses. Post-translational modifications are unique mechanisms that allow cells to rapidly, locally and specifically modify activity or interactions of key proteins. Some of these modifications, including phosphorylation and ubiquitylation, can be induced by pathogens. However, the effects of pathogenic bacteria on SUMOylation, an essential post-translational modification in eukaryotic cells, remain largely unknown. Here we show that infection with Listeria monocytogenes leads to a decrease in the levels of cellular SUMO-conjugated proteins. This event is triggered by the bacterial virulence factor listeriolysin O (LLO), which induces a proteasome-independent degradation of Ubc9, an essential enzyme of the SUMOylation machinery, and a proteasome-dependent degradation of some SUMOylated proteins. The effect of LLO on Ubc9 is dependent on the pore-forming capacity of the toxin and is shared by other bacterial pore-forming toxins like perfringolysin O (PFO) and pneumolysin (PLY). Ubc9 degradation was also observed in vivo in infected mice. Furthermore, we show that SUMO overexpression impairs bacterial infection. Together, our results reveal that Listeria, and probably other pathogens, dampen the host response by decreasing the SUMOylation level of proteins critical for infection.
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Listeria monocytogenes/patogenicidade , Listeriose/metabolismo , Listeriose/microbiologia , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Linhagem Celular , Células HeLa , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Camundongos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Fatores de Virulência/metabolismoRESUMO
The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5' and 3' untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.
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Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , RNA Bacteriano/genética , Transcrição Gênica/genética , Animais , Genes Bacterianos/genética , Genoma Bacteriano/genética , Intestinos/microbiologia , Camundongos , Fases de Leitura Aberta/genética , Óperon/genética , RNA Bacteriano/análise , Sequências Reguladoras de Ácido Ribonucleico/genética , Regiões não Traduzidas/genética , Virulência/genéticaRESUMO
Listeria monocytogenes is a foodborne pathogen that crosses the intestinal barrier and disseminates within the host. Here, we report a unique comprehensive analysis of the impact of two Lactobacillus species, Lactobacillus paracasei CNCM I-3689 and Lactobacillus casei BL23, on L. monocytogenes and orally acquired listeriosis in a gnotobiotic humanized mouse model. We first assessed the effect of treatment with each Lactobacillus on L. monocytogenes counts in host tissues and showed that each decreases L. monocytogenes systemic dissemination in orally inoculated mice. A whole genome intestinal transcriptomic analysis revealed that each Lactobacillus changes expression of a specific subset of genes during infection, with IFN-stimulated genes (ISGs) being the most affected by both lactobacilli. We also examined microRNA (miR) expression and showed that three miRs (miR-192, miR-200b, and miR-215) are repressed during L. monocytogenes infection. Treatment with each Lactobacillus increased miR-192 expression, whereas only L. casei association increased miR-200b and miR-215 expression. Finally, we showed that treatment with each Lactobacillus significantly reshaped the L. monocytogenes transcriptome and up-regulated transcription of L. monocytogenes genes encoding enzymes allowing utilization of intestinal carbon and nitrogen sources in particular genes involved in propanediol and ethanolamine catabolism and cobalamin biosynthesis. Altogether, these data reveal that the modulation of L. monocytogenes infection by treatment with lactobacilli correlates with a decrease in host gene expression, in particular ISGs, miR regulation, and a dramatic reshaping of L. monocytogenes transcriptome.
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Antibiose/fisiologia , Lactobacillus/fisiologia , Listeria monocytogenes/genética , Listeriose/genética , Animais , Expressão Gênica/efeitos dos fármacos , Genes Bacterianos/genética , Interações Hospedeiro-Patógeno/genética , Interferons/farmacologia , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Lactobacillus/classificação , Lacticaseibacillus casei/fisiologia , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Boca/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma/genéticaRESUMO
L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK(-) bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles.
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Autofagia , Proteínas de Bactérias/metabolismo , Listeria monocytogenes/metabolismo , Listeriose/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Fatores de Virulência/metabolismo , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Animais , Proteínas de Bactérias/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Listeria monocytogenes/genética , Listeriose/genética , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Fatores de Virulência/genéticaRESUMO
Listeria monocytogenes is an intracellular pathogen responsible for severe foodborne infections. It can replicate in both phagocytic and nonphagocytic mammalian cells. The infectious process at the cellular level has been studied extensively, but how the bacterium overcomes early host innate immune responses remains largely unknown. Here we show that InlC, a member of the internalin family, is secreted intracellularly and directly interacts with IKKα, a subunit of the IκB kinase complex critical for the phosphorylation of IκB and activation of NF-κB, the major regulator of innate immune responses. Infection experiments with WT Listeria or the inlC-deletion mutant and transfection of cells with InlC reveal that InlC expression impairs phosphorylation and consequently delays IκB degradation normally induced by TNF-α, a classical NF-κB stimulator. Moreover, infection of RAW 264.7 macrophages by the inlC mutant leads to increased production of proinflammatory cytokines compared with that obtained with the WT. Finally, in a peritonitis mouse model, we show that infection with the inlC mutant induces increased production of chemokines and increased recruitment of neutrophils in the peritoneal cavity compared with infection with WT. Together, these results demonstrate that InlC, by interacting with IKKα, dampens the host innate response induced by Listeria during the infection process.
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Proteínas de Bactérias/imunologia , Quinase I-kappa B/metabolismo , Imunidade Inata , Subunidades Proteicas/metabolismo , Animais , Linhagem Celular , Humanos , Quinase I-kappa B/genética , Listeria monocytogenes/imunologia , Listeria monocytogenes/patogenicidade , Camundongos , Regiões Promotoras Genéticas , Subunidades Proteicas/genética , Fator de Necrose Tumoral alfa/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND & AIMS: Mycobacterium bovis Bacillus Calmette-Guérin (BCG), killed by extended freeze-drying (EFD), induces secretion of interleukin-10 and reduces lung inflammation in a mouse model of asthma. We investigated the effects of EFD BCG in mouse models of inflammatory bowel disease. METHODS: EFD BCG was administered subcutaneously to mice with colitis induced by dextran sodium sulfate (DSS), oxazolone, or adoptive transfer of CD4(+)CD45RB(high)Foxp3(-) T cells from C57Bl/6 Foxp3GFP mice to RAG2(-/-) mice. RESULTS: EFD BCG, administered either before induction of DSS and oxazolone colitis or after development of acute or chronic DSS-induced colitis, reduced symptom scores, loss of body weight, and inflammation. Although transfer of CD4(+)CD45RB(high)Foxp3(-) cells induced colitis in RAG2(-/-) mice, administration of EFD BCG at the time of the transfer converted Foxp3(-) T cells to Foxp3(+) T cells and the mice did not develop colitis. EFD BCG protected mice from colitis via a mechanism that required expansion of T regulatory cells and production of interleukin-10 and transforming growth factor ß. EFD BCG activated the retinoid X receptor (RXR)-α-peroxisome proliferator-activated receptor (PPAR)-γ heterodimer, blocked translocation of nuclear factor κB to the nucleus, and reduced colonic inflammation; it did not increase the number of colon tumors that formed in mice with chronic DSS-induced colitis. CONCLUSIONS: EFD BCG controls severe colitis in mice by expanding T regulatory cell populations and PPAR-γ and might be developed to treat patients with inflammatory bowel disease.
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Vacina BCG/farmacologia , Colite/prevenção & controle , Colo/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Mycobacterium bovis , Linfócitos T Reguladores/metabolismo , Animais , Vacina BCG/administração & dosagem , Colite/induzido quimicamente , Colite/imunologia , Colo/patologia , Sulfato de Dextrana , Liofilização , Interleucina-1/sangue , Interleucina-10/metabolismo , Interleucina-6/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Oxazolona , PPAR gama/metabolismo , Peroxidase/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/sangue , Redução de PesoRESUMO
The high incidence of lung-damaging life-threatening respiratory infections in infants may be related to the immaturity of their immune systems. To determine whether lung immune features differ in early life compared with those in adulthood, whole lung as well as lung T lymphocyte and DC responses were investigated in BALB/c neonates versus adults. Higher expression of GATA-3 and rapid and sustained production of type 2 cytokines by lung explants after in vitro exposure to anti-CD3 was the hallmark of the neonatal period, suggestive of a Th2 bias. Neonatal lung GATA-3-producing cells were identified as CD3(+), CD4 and CD8 double-negative T lymphocytes, a subset found at a higher frequency in neonatal than adult lung. The neonatal lungs contained fewer conventional DCs, with a lower ratio of CD103(+) to CD11b(+) DCs, and a much lower number of plasmacytoid DCs in comparison with adult lungs. Yet, when stimulated in vivo by BCG, neonatal lung DCs matured and primed adult naïve CD4(+) T cells toward Th1 as efficiently as adult BCG-primed lung DCs. Conversely, both adult and neonatal BCG-primed lung DCs induced a Th2 cytokine response from neonatal naïve lymph node T cells, suggestive of an intrinsic feature of neonatal T lymphocytes.
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Citocinas/biossíntese , Células Dendríticas/imunologia , Pulmão/imunologia , Células Th2/imunologia , Fatores de Transcrição/biossíntese , Animais , Animais Recém-Nascidos , Antígenos CD/análise , Antígeno CD11b/análise , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citocinas/imunologia , Células Dendríticas/metabolismo , Fator de Transcrição GATA3/biossíntese , Cadeias alfa de Integrinas/análise , Pulmão/metabolismo , Ativação Linfocitária , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/imunologia , Células Th2/metabolismoRESUMO
Microbial pathogens have evolved mechanisms to overcome immune responses and successfully infect their host. Here, we studied how Listeria monocytogenes evades immune detection by peptidoglycan (PGN) modification. By analyzing L. monocytogenes muropeptides, we detected O-acetylated muramic acid residues. We identified an O-acetyltransferase gene, oatA, in the L. monocytogenes genome sequence. Comparison of PGN from parental and isogenic oatA mutant strains showed that the O-acetyltransferase OatA O-acetylates Listeria PGN. We also found that PGN O-acetylation confers resistance to different types of antimicrobial compounds targeting bacterial cell wall such as lysozyme, ß-lactam antibiotics, and bacteriocins and that O-acetylation is required for Listeria growth in macrophages. Moreover, oatA mutant virulence is drastically affected in mice following intravenous or oral inoculation. In addition, the oatA mutant induced early secretion of proinflammatory cytokines and chemokines in vivo. These results suggest an important role for OatA in limiting innate immune responses and promoting bacterial survival in the infected host.
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Acetiltransferases/imunologia , Citocinas/metabolismo , Listeria monocytogenes/imunologia , Listeriose/imunologia , Peptidoglicano/imunologia , Fatores de Virulência/imunologia , Acetilação , Acetiltransferases/genética , Animais , Linhagem Celular , Feminino , Humanos , Imunidade Inata , Dose Letal Mediana , Listeria monocytogenes/genética , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/patogenicidade , Listeriose/genética , Fígado/metabolismo , Fígado/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Ácidos Murâmicos/metabolismo , Peptidoglicano/química , Baço/microbiologia , Células Th1/metabolismo , Células Th2/metabolismo , Fatores de Virulência/genéticaRESUMO
Epidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) by Escherichia coli, a commensal of the digestive tract and a source of urinary tract pathogens. Bioinformatics analyses of a large collection of E. coli genomes from EnteroBase, enriched in clinical isolates of worldwide origins, suggest the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene, cnf1, is preferentially distributed in four common sequence types (ST) encompassing the pandemic E. coli MDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment, with known enhanced capacities to colonize the gastrointestinal tract. Statistical projections based on this dataset point to a global expansion of cnf1-positive multidrug-resistant ST131 strains from subclade H30Rx/C2, accounting for a rising prevalence of cnf1-positive strains in ST131. Despite the absence of phylogeographical signals, cnf1-positive isolates segregated into clusters in the ST131-H30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the same cnf1 allele. The suggested dominant expansion of cnf1-positive strains in ST131-H30Rx/C2 led us to uncover the competitive advantage conferred by cnf1 for gut colonization to the clinical strain EC131GY ST131-H30Rx/C2 versus cnf1-deleted isogenic strain. Complementation experiments showed that colon tissue invasion was compromised in the absence of deamidase activity on Rho GTPases by CNF1. Hence, gut colonization factor function of cnf1 was confirmed for another clinical strain ST131-H30Rx/C2. In addition, functional analysis of the cnf1-positive clinical strain EC131GY ST131-H30Rx/C2 and a cnf1-deleted isogenic strain showed no detectable impact of the CNF1 gene on bacterial fitness and inflammation during the acute phase of bladder monoinfection. Together these data argue for an absence of role of CNF1 in virulence during UTI, while enhancing gut colonization capacities of ST131-H30Rx/C2 and suggested expansion of cnf1-positive MDR isolates in subclade ST131-H30Rx/C2.
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Toxinas Bacterianas , Infecções por Escherichia coli , Proteínas de Escherichia coli , Microbioma Gastrointestinal , Antibacterianos/farmacologia , Toxinas Bacterianas/genética , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Humanos , Fatores de Virulência/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo , Proteínas rho de Ligação ao GTPRESUMO
The pathogenesis of chronic joint inflammation remains unclear, although the involvement of pathogen recognition receptors has been suggested recently. In the present article, we describe the role of two members of the NACHT-LRR (NLR) family, Nod1 (nucleotide-binding oligomerization domain) and Nod2 in a model of acute joint inflammation induced by intraarticular injection of Streptococcus pyogenes cell wall fragments. Here, we show that Nod2 deficiency resulted in reduced joint inflammation and protection against early cartilage damage. In contrast, Nod1 gene-deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage, consistent with a model in which Nod1 controls the inflammatory reaction. To explore whether the different function of Nod1 and Nod2 occurs also in humans, we exposed peripheral blood mononuclear cells (PBMCs) carrying either Nod1ins/del or Nod2fs mutation with SCW fragments in vitro. Production of both TNFalpha and IL-1beta was clearly impaired in PBMCs carrying the Nod2fs compared with PBMCs isolated from healthy controls. In line with results in Nod1 gene-deficient mice, PBMCs from individuals bearing a newly described Nod1 mutation produced enhanced levels of proinflammatory cytokines after 24-h stimulation with SCW fragments. These data indicate that the NLR family members Nod1 and Nod2 have different functions in controlling inflammation, and that intracellular Nod1-Nod2 interactions may determine the severity of arthritis in this experimental model. Whether a distorted balance between the function of Nod1 and/or Nod2 is involved in the pathogenesis of human autoinflammatory or autoimmune disease, such as rheumatoid arthritis, remains to be elucidated.
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Artrite Reumatoide/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Animais , Artrite Reumatoide/patologia , Linhagem Celular , Parede Celular/metabolismo , Regulação da Expressão Gênica , Humanos , Interleucina-1beta/biossíntese , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Proteína Adaptadora de Sinalização NOD1/deficiência , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Streptococcus , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The genome of the pathogenic bacterium Listeria monocytogenes contains a family of genes encoding proteins with a leucine-rich repeat domain. One of these genes, inlH, is a sigma(B)-dependent virulence gene of unknown function. Previously, inlH was proposed to be coexpressed with two adjacent internalin genes, inlG and inlE. Using tiling arrays, we showed that inlH expression is monocistronic and specifically induced in stationary phase as well as in the intestinal lumen of mice, independent of inlG and inlE expression. Consistent with inlH sigma(B)-dependent regulation, surface expression of the InlH protein is induced when bacteria are subjected to thermal, acidic, osmotic, or oxidative stress. Disruption of inlH increases the amount of the invasion protein InlA without changing inlA transcript level, suggesting that there is a link between inlH expression and inlA posttranscriptional regulation. However, in contrast to InlA, InlH does not contribute to bacterial invasion of cultured cells in vitro or of intestinal cells in vivo. Strikingly, the reduced virulence of inlH-deficient L. monocytogenes strains is accompanied by enhanced production of interleukin-6 (IL-6) in infected tissues during the systemic phase of murine listeriosis but not by enhanced production of any other inflammatory cytokine tested. Since InlH does not modulate IL-6 secretion in macrophages at least in vitro, it may play a role in other immune cells or contribute to a pathway that modulates survival or activation of IL-6-secreting cells. These results strongly suggest that InlH is a stress-induced surface protein that facilitates pathogen survival in tissues by tempering the inflammatory response.
Assuntos
Proteínas de Bactérias/fisiologia , Interleucina-6/antagonistas & inibidores , Listeria monocytogenes/patogenicidade , Listeriose/imunologia , Fatores de Virulência/fisiologia , Animais , Proteínas de Bactérias/genética , Sangue/microbiologia , Linhagem Celular , Contagem de Colônia Microbiana , Feminino , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Interleucina-6/imunologia , Listeria monocytogenes/imunologia , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Baço/microbiologia , Estresse Fisiológico , Virulência , Fatores de Virulência/biossíntese , Fatores de Virulência/genéticaRESUMO
Inactivation of the host GTPase RhoA by staphylococcal epidermal cell differentiation inhibitor (EDIN) exotoxins triggers the formation of large transcellular tunnels, named macroapertures, in endothelial cells. We used bioluminescent strains of Staphylococcus aureus to monitor the formation of infection foci during the first 24 h of hematogenous bacterial dissemination. Clinically derived EDIN-expressing S. aureus strains S25 and Xen36 produced many disseminated foci. EDIN had no detectable impact on infection foci in terms of histopathology or the intensity of emitted light. Moreover, EDIN did not modify the course of bacterial clearance from the bloodstream. In contrast, we show that EDIN expression promotes a 5-fold increase in the number of infection foci produced by Xen36. This virulence activity of EDIN requires RhoA ADP-ribosyltranferase activity. These results suggest that EDIN is a risk factor for S. aureus dissemination through the vasculature by virtue of its ability to promote the formation of infection foci in deep-seated tissues.
Assuntos
Bacteriemia/microbiologia , Bacteriemia/patologia , Proteínas de Bactérias/toxicidade , Staphylococcus aureus/patogenicidade , Fatores de Virulência/toxicidade , Animais , Feminino , Genes Reporter , Histocitoquímica , Humanos , Medições Luminescentes , Camundongos , Camundongos Endogâmicos BALB C , Microscopia , Staphylococcus aureus/isolamento & purificação , Imagem Corporal Total , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Listeria monocytogenes is a Gram-positive facultative intracellular pathogen which invades different cell types, including nonphagocytic cells, where it is able to replicate and survive. The different steps of the cellular infectious process have been well described and consist of bacterial entry, lysis of the endocytic vacuole, intracellular replication, and spreading to neighboring cells. To study the listerial infectious process, gentamicin survival assays, plaque formation, and direct microscopy observations are typically used; however, there are some caveats with each of these techniques. In this study we describe new single-cell techniques based on use of an array of integrative fluorescent plasmids (green, cyan, and yellow fluorescent proteins) to easily, rapidly, and quantitatively detect L. monocytogenes in vitro and in vivo. We describe construction of 13 integrative and multicopy plasmids which can be used for detecting intracellular bacteria, for measuring invasion, cell-to-cell spreading, and intracellular replication, for monitoring in vivo infections, and for generating transcriptional or translational reporters. Furthermore, we tested these plasmids in a variety of epifluorescence- and flow cytometry-based assays. We showed that we could (i) determine the expression of a particular promoter during the cell cycle, (ii) establish in one rapid experiment at which step in the cell cycle a particular mutant is defective, and (iii) easily measure the number of infected cells in vitro and in mouse organs. The plasmids that are described and the methods to detect them are new powerful tools to study host-Listeria interactions in a fast, robust, and high-throughput manner.
Assuntos
Técnicas Bacteriológicas/métodos , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Biologia Molecular/métodos , Coloração e Rotulagem/métodos , Animais , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Antibiotic resistance is a worldwide threat due to the decreasing supply of new antimicrobials. Novel targets and innovative strategies are urgently needed to generate pathbreaking drug compounds. NAD kinase (NADK) is essential for growth in most bacteria, as it supports critical metabolic pathways. Here, we report the discovery of a new class of antibacterials that targets bacterial NADK. We generated a series of small synthetic adenine derivatives to screen those harboring promising substituents in order to guide efficient fragment linking. This led to NKI1, a new lead compound inhibiting NADK that showed in vitro bactericidal activity against Staphylococcus aureus. In a murine model of infection, NKI1 restricted survival of the bacteria, including methicillin-resistant S. aureus. Collectively, these findings identify bacterial NADK as a potential drug target and NKI1 as a lead compound in the treatment of staphylococcal infections.
Assuntos
Antibacterianos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Adenina/química , Adenina/farmacologia , Animais , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Feminino , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Bibliotecas de Moléculas Pequenas , Staphylococcus aureus/enzimologia , Relação Estrutura-AtividadeRESUMO
The intestinal microbiota modulates host physiology and gene expression via mechanisms that are not fully understood. Here we examine whether host epitranscriptomic marks are affected by the gut microbiota. We use methylated RNA-immunoprecipitation and sequencing (MeRIP-seq) to identify N6-methyladenosine (m6A) modifications in mRNA of mice carrying conventional, modified, or no microbiota. We find that variations in the gut microbiota correlate with m6A modifications in the cecum, and to a lesser extent in the liver, affecting pathways related to metabolism, inflammation and antimicrobial responses. We analyze expression levels of several known writer and eraser enzymes, and find that the methyltransferase Mettl16 is downregulated in absence of a microbiota, and one of its target mRNAs, encoding S-adenosylmethionine synthase Mat2a, is less methylated. We furthermore show that Akkermansia muciniphila and Lactobacillus plantarum affect specific m6A modifications in mono-associated mice. Our results highlight epitranscriptomic modifications as an additional level of interaction between commensal bacteria and their host.
Assuntos
Adenosina/análogos & derivados , Ceco/metabolismo , Microbioma Gastrointestinal , Fígado/metabolismo , Adenosina/metabolismo , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Ceco/microbiologia , Feminino , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TranscriptomaRESUMO
Staphylococcus aureus is a common human cutaneous and nasal commensal and a major life-threatening pathogen. Adaptation to the different environments encountered inside and outside the host is a crucial requirement for survival and colonization. We identified and characterized a eukaryotic-like serine/threonine kinase with three predicted extracellular PASTA domains (SA1063, or Stk1) and its associated phosphatase (SA1062, or Stp1) in S. aureus. Biochemical analyses revealed that Stk1 displays autokinase activity on threonine and serine residues and is localized to the membrane. Stp1 is a cytoplasmic protein with manganese-dependent phosphatase activity toward phosphorylated Stk1. In-frame deletions of the stk1 and stp1 genes were constructed in S. aureus strain 8325-4. Phenotypic analyses of the mutants revealed reduced growth of the stk1 mutant in RPMI 1640 defined medium that was restored when adenine was added to the medium. Furthermore, the stk1 mutant displayed increased resistance to Triton X-100 and to fosfomycin, suggesting modifications in cell wall metabolism. The stk1 mutant was tested for virulence in a mouse pyelonephritis model and found to be strongly reduced for survival in the kidneys (approximately 2-log-unit decrease) compared to the parental strain. Renal histopathological analyses showed severe inflammatory lesions in mice infected with the parental S. aureus SH1000 strain, whereas the Deltastk1 mutant led to only minimal renal lesions. These results confirm the important role of Stk1 for full expression of S. aureus pathogenesis and suggest that phosphorylation levels controlled by stk1 are essential in controlling bacterial survival within the host.