RESUMO
Listeria monocytogenes is a pathogen causing serious or mortal infections in human risk populations. Its infectivity is in part due to its ability to infect diverse eukaryotic cells. Since several bacteria can enter into yeast cells, including Candida albicans, the aims of this work were to evaluate if L. monocytogenes was able to harbor, retaining its viability, within C. albicans cells and to evaluate the effect of temperature and an antibiotic as stressing factors in its rate of entry into yeast cells. Both microorganisms were co-incubated in BHI broth during 48 h and the entry of bacteria into yeast cells was evaluated at different times. Then, yeasts free of extracellular bacteria were obtained seeding samples of the co-culture on YGC agar, which contains chloramphenicol, to obtain extracellular bacteria-free yeasts. These extracellular bacteria free yeasts were used to search for bacterial DNA in total yeast DNA and to evaluate the viability of intra-yeast bacteria. Finally, the effect of temperature and of chloramphenicol as inducers of stress on the rate of bacterial entry into yeast cells were investigated. After co-culturing both microorganisms, wet mount optical microscopy showed the presence of moving bacteria within yeasts and transmission electron microscopy confirmed the presence of intra-yeast bacteria. PCR allowed to amplify L. monocytogenes iap gene in C. albicans total DNA obtained from yeasts free of extracellular bacteria. Moreover, the SYTO 9 green fluorescence observed in bacterial cells within vacuoles of yeasts suggests that intra-yeast bacteria remain viable. Furthermore, the entry of L. monocytogenes into yeasts cells was favored by the presence of stressing factors (chloramphenicol and temperature). Therefore, yeasts may be reservoirs of viable L. monocytogenes and might spread them to the following generations of yeasts.
Assuntos
Candida albicans/fisiologia , Reservatórios de Doenças/microbiologia , Listeria monocytogenes/fisiologia , Vacúolos/microbiologia , DNA Bacteriano/análiseRESUMO
Acanthamoeba castellanii is a free-living amoeba found mainly in humid environments and Arcobacter butzleri is an emerging zoonotic pathogen, both can establish in vitro endosymbiotic relationships in the absence of bacterial replication. We analyzed the localization of A. butzleri within A. castellanii establishing their association with endoplasmic reticulum vesicles and mitochondria. Through confocal microscopy, we observed that during the early stages of endosymbiosis, there is not colocalization between amoebic vacuoles containing A. butzleri and mitochondria or ER vesicles of A. castellanii. Considering that energy production of this bacterium occurs via metabolism of amino acids or the tricarboxylic acid cycle, these results contribute to explain the absence of bacterial replication, since A. butzleri would not have access to the nutrients found in endoplasmic reticulum vesicles and mitochondria. In addition, we observe that A. butzleri induces significantly the actin polymerization of A. castellanii during the early stages of endosymbiosis.
Assuntos
Acanthamoeba castellanii/microbiologia , Arcobacter/fisiologia , Simbiose , Vacúolos/microbiologiaRESUMO
Cyclic Adenosine 3',5'-monophosphate (cAMP) is a key second messenger known to directly regulate not only the protein kinase A (PKA) activity but also other important molecules such as the exchange protein activated by cAMP (EPAC), which is as a guanine nucleotide exchange factor (GEF) of the low molecular weight GTPase, Rap2. Coxiella burnetii is a Gram negative bacterium that survives and grows in a large Coxiella replicative vacuole (CRV), which displays lysosomal and autophagic features. In this report, we present evidence that both, EPAC and its downstream effector Rap2b, were recruited to the CRV. The transient over-expression of the Rap2b wt protein, but not its inactive mutant Rap2b ΔAAX, markedly inhibited the development of the large CRV. Additionally, Rap2b wtinhibited the fusion of early Coxiella phagosomes with the fully developed CRV, indicating that homotypic fusion events are altered in the presence of high levels of Rap2b wt. Likewise, the fusion of endosome/lysosomal compartments (heterotypic fusions) with the large CRV was also affected by the over-expression of this GTPase. Interestingly, cell overexpression of Rap2b wt markedly decreased the levels of the v-SNARE, Vamp7, suggesting that this down-regulation impairs the homotypic and heterotypic fusions events of the Coxiella vacuole.
Assuntos
Coxiella burnetii/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Febre Q/metabolismo , Vacúolos/microbiologia , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Células CHO , Chlorocebus aethiops , Cricetulus , AMP Cíclico/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Fusão de Membrana , Fagossomos/metabolismo , Fagossomos/microbiologia , Febre Q/microbiologia , Vacúolos/metabolismo , Células VeroRESUMO
Neurobrucellosis is an inflammatory disease caused by the invasion of Brucella spp. to the central nervous system (CNS). The pathogenesis of the disease is not well characterized; however, for Brucella to gain access to the brain parenchyma, traversing of the blood-brain barrier (BBB) must take place. To understand the CNS determinants of the pathogenesis of B. abortus, we have used the in vitro BBB model of human brain microvascular endothelial cells (HBMEC) to study the interactions between B. abortus and brain endothelial cells. In this study, we showed that B. abortus is able to adhere and invade HBMEC which was dependent on microtubules, microfilaments, endosome acidification and de novo protein synthesis. After infection, B. abortus rapidly escapes the endosomal compartment of HBMEC and forms a replicative Brucella-containing vacuole that involves interactions with the endoplasmic reticulum. Despite the ability of B. abortus to invade and replicate in HBMEC, the bacterium was unable by itself to traverse HBMEC, but could traverse polarized HBMEC monolayers within infected monocytes. Importantly, infected monocytes that traversed the HBMEC monolayer were a bacterial source for de novo infection of glial cells. This is the first demonstration of the mechanism whereby B. abortus is able to traverse the BBB and infect cells of the CNS. These results may have important implications in our understanding of the pathogenesis of neurobrucellosis.
Assuntos
Barreira Hematoencefálica/microbiologia , Brucella abortus/crescimento & desenvolvimento , Células Endoteliais/microbiologia , Leucócitos Mononucleares/microbiologia , Microvasos/microbiologia , Animais , Barreira Hematoencefálica/citologia , Brucella abortus/fisiologia , Brucelose/microbiologia , Retículo Endoplasmático/microbiologia , Endossomos/microbiologia , Células Endoteliais/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/citologia , Cultura Primária de Células , Transcitose/fisiologia , Vacúolos/microbiologiaRESUMO
Cryptococcus neoformans is a basidiomycetous yeast and the cause of cryptococcosis in immunocompromised individuals. The most severe form of the disease is meningoencephalitis, which is one of the leading causes of death in HIV/AIDS patients. In order to access the central nervous system, C. neoformans relies on the activity of certain virulence factors such as urease, which allows transmigration through the blood-brain barrier. In this study, we demonstrate that the calcium transporter Pmc1 enables C. neoformans to penetrate the central nervous system, because the pmc1 null mutant failed to infect and to survive within the brain parenchyma in a murine systemic infection model. To investigate potential alterations in transmigration pathways in these mutants, global expression profiling of the pmc1 mutant strain was undertaken, and genes associated with urease, the Ca2+ -calcineurin pathway, and capsule assembly were identified as being differentially expressed. Also, a decrease in urease activity was observed in the calcium transporter null mutants. Finally, we demonstrate that the transcription factor Crz1 regulates urease activity and that the Ca2+ -calcineurin signalling pathway positively controls the transcription of calcium transporter genes and factors related to transmigration.
Assuntos
Sistema Nervoso Central/microbiologia , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidade , Proteínas Fúngicas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Transporte Biológico/fisiologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/microbiologia , Encéfalo/metabolismo , Encéfalo/microbiologia , Calcineurina/metabolismo , Cálcio/metabolismo , Linhagem Celular , Criptococose/metabolismo , Criptococose/microbiologia , Modelos Animais de Doenças , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Meningoencefalite/metabolismo , Meningoencefalite/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Vacúolos/metabolismo , Vacúolos/microbiologia , Virulência/fisiologia , Fatores de Virulência/metabolismoRESUMO
A study with transmission electron microscopy of mycoplasma-contaminated HeLa cells using five cell donors referred to as donors A, B, C, D and E, observations are herein presented. Experiments performed with cells from donors B, C and D, revealed the presence of Mycoplasma hyorhinis after PCR and sequencing experiments. Bacteria probably originated from a cytoplasm with compacted tiny granular particles replacing the normal cytosol territories, or from the contact with the cytoplasm through a clear semi-solid material. The compact granularity (CG) of the cytoplasm was crossed by stripes of smooth and rough endoplasmic reticulum cisternae. Among apparently normal mitochondria, it was noted, in variable proportions, mitochondria with crista-delimited lucent central regions that expand to and occupied the interior of a crista-less organelle, which can undergo fission. Other components of the scenarios of mycoplasma-induced cell demolition are villus-like structures with associated 80-200 nm vesicles and a clear, flexible semi-solid, process-sensitive substance that we named jam-like material. This material coated the cytoplasmic surface, its recesses, irregular protrusions and detached cytoplasmic fragments. It also cushioned forming bacteria. Cyst-like structures were often present in the cytoplasm. Cells, mainly apoptotic, exhibiting ample cytoplasmic sectors with characteristic net-like profile due to adjoined vacuoles, as well as ovoid or elongated profiles, consistently appeared in all cells from the last four cell donors. These cells were named "modified host cells" because bacteria arose in the vacuoles. The possibility that, in some samples, there was infection and/or coinfection of the host cell by another organism(s) cannot be ruled out.
Assuntos
Citosol/microbiologia , Retículo Endoplasmático/microbiologia , Células HeLa/microbiologia , Mitocôndrias/microbiologia , Mycoplasma hyorhinis/isolamento & purificação , Vacúolos/microbiologia , Células Cultivadas , Citosol/patologia , DNA Bacteriano , Retículo Endoplasmático/patologia , Células HeLa/patologia , Humanos , Microscopia Eletrônica de Transmissão , Mitocôndrias/patologia , Reação em Cadeia da Polimerase , Estaurosporina/farmacologia , Vacúolos/patologiaRESUMO
Coxiella burnetii, the etiologic agent of Q fever, is a Gram-negative obligate intracellular bacterium. It has been previously described that both the endocytic and autophagic pathways contribute to the Coxiella replicative vacuole (CRV) generation. Galectins are ß-galactoside-binding lectins that accumulate in the cytosol before being secreted via a non-conventional secretory pathway. It has been shown that Galectin-3, -8, -9 monitor bacteria vacuolar rupture and endosomal and lysosomal loss of membrane integrity through binding of host glycans exposed in the cytoplasm after membrane damage. Using microinjection of fluorescence-coupled dextrans, a FRET assay, and galectins distribution, we demonstrate that Coxiella infection actually result in transient phagosomal/CRV membrane damage in a Dot/Icm-dependent manner. We also show the association of different adaptor molecules involved in autophagy and of LC3 to the limiting membrane of the CRV. Moreover, we show that upon autophagy inhibition, the proportion of CRVs labeled with galectins and less acidified increases which is associated with bacteria replication impairment. Based on these observations, we propose that autophagy can facilitate resealing of intracellular damaged membranes.
Assuntos
Autofagia/fisiologia , Coxiella burnetii/fisiologia , Citosol/metabolismo , Galectinas/metabolismo , Vacúolos/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Proteína Beclina-1/genética , Células CHO , Membrana Celular , Chlorocebus aethiops , Coxiella burnetii/genética , Coxiella burnetii/crescimento & desenvolvimento , Coxiella burnetii/patogenicidade , Cricetulus , Técnicas de Silenciamento de Genes , Células HeLa , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Viabilidade Microbiana , Fagossomos/metabolismo , Polissacarídeos/metabolismo , Febre Q/microbiologia , Febre Q/patologia , Células VeroRESUMO
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative pathogen that causes various host-specific diseases. During their life cycle, Salmonellae survive frequent exposures to a variety of environmental stresses, e.g. carbon-source starvation. The virulence of this pathogen relies on its ability to establish a replicative niche, named Salmonella-containing vacuole, inside host cells. However, the microenvironment of the SCV and the bacterial metabolic pathways required during infection are largely undefined. In this work we developed different biological probes whose expression is modulated by the environment and the physiological state of the bacterium. We constructed transcriptional reporters by fusing promoter regions to the gfpmut3a gene to monitor the expression profile of genes involved in glucose utilization and lipid catabolism. The induction of these probes by a specific metabolic change was first tested in vitro, and then during different conditions of infection in macrophages. We were able to determine that Entner-Doudoroff is the main metabolic pathway utilized by Salmonella during infection in mouse macrophages. Furthermore, we found sub-populations of bacteria expressing genes involved in pathways for the utilization of different sources of carbon. These populations are modified in presence of different metabolizable substrates, suggesting the coexistence of Salmonella with diverse metabolic states during the infection.
Assuntos
Adaptação Fisiológica , Citoplasma/microbiologia , Salmonella typhimurium/fisiologia , Vacúolos/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citometria de Fluxo , Regulação Bacteriana da Expressão Gênica , Macrófagos/microbiologia , Redes e Vias Metabólicas , Camundongos , Regiões Promotoras Genéticas , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , VirulênciaRESUMO
In this study we assessed the interaction of different strains of Bacillus cereus with murine peritoneal macrophages and cultured phagocytic cells (Raw 264.7 cells). Association, internalization, intracellular survival, routing of bacteria to different compartments and expression of MHCII were assessed in cells infected with different strains of B. cereus in vegetative form. Association values (adhering + internalized bacteria) and phagocytosis were higher for strain B10502 than those for strains 2 and M2. However, after 90 min interaction, intracellular survival was higher for strain 2 than for strains M2 and B10502. Acquisition of lysosomal markers by B. cereus containing vacuoles (BcCV), assessed by LAMP1 and Lysotracker labelling occurred shortly after internalization. The highest ratio of LAMP1(+)-BcCV was found for strain M2. This strain was able to survive longer than strain B10502 which routes to LAMP1 containing vacuoles to a lesser extent. In addition, strain M2 stimulated expression of MHCII by infected cells. Confocal analyses 60 or 90 min post-infection showed different percentages of co-localization of bacteria with Lysotracker. Results suggest strain-dependent interaction and intracellular killing of B. cereus by phagocytic cells. These findings could be relevant for the pathogenic potential of Bacillus cereus strains.
Assuntos
Bacillus cereus , Fagócitos/microbiologia , Animais , Proteína 1 de Membrana Associada ao Lisossomo , Lisossomos/microbiologia , Camundongos , Células RAW 264.7 , Vacúolos/microbiologiaRESUMO
Brucella abortus, the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCVs mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System (VirB) is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the VirB system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated proteins remain unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (BAB2_0123) is a B. abortus VirB-translocated effector protein recently identified by our group whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. In bone-marrow derived macrophages infected with B. abortus, ENO-1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO-1 depletion by siRNA impaired B. abortus intracellular replication in HeLa cells, confirming a role for α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus.
Assuntos
Proteínas de Bactérias/metabolismo , Biomarcadores Tumorais/metabolismo , Brucella abortus/metabolismo , Brucella abortus/fisiologia , Proteínas de Ligação a DNA/metabolismo , Interações Hospedeiro-Patógeno , Estilo de Vida , Fosfopiruvato Hidratase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sistemas de Secreção Tipo IV/metabolismo , Animais , Proteínas de Bactérias/genética , Biomarcadores Tumorais/isolamento & purificação , Brucella abortus/genética , Brucella abortus/crescimento & desenvolvimento , Brucelose/microbiologia , Sobrevivência Celular , Citoplasma/metabolismo , Citoplasma/microbiologia , DNA Bacteriano , Proteínas de Ligação a DNA/isolamento & purificação , Retículo Endoplasmático/microbiologia , Escherichia coli/genética , Células HeLa , Humanos , Imunoprecipitação/métodos , Macrófagos/microbiologia , Camundongos , Microscopia Confocal , Fosfopiruvato Hidratase/isolamento & purificação , Transporte Proteico , RNA Interferente Pequeno , Proteínas Supressoras de Tumor/isolamento & purificação , Sistemas de Secreção Tipo IV/genética , Vacúolos/microbiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207-238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207-238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Listeria monocytogenes/genética , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Fosfolipases Tipo C/genética , Vacúolos/microbiologia , Sequência de Aminoácidos , Proteínas de Bactérias/imunologia , Sítios de Ligação , Membrana Celular/imunologia , Membrana Celular/microbiologia , Membrana Celular/ultraestrutura , Citoplasma/imunologia , Citoplasma/microbiologia , Citoplasma/ultraestrutura , Deleção de Genes , Células HeLa , Humanos , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/imunologia , Proteínas de Membrana/imunologia , Metaloendopeptidases/imunologia , Óperon , Ligação Proteica , Fosfolipases Tipo C/imunologia , Vacúolos/imunologia , Vacúolos/ultraestruturaRESUMO
The survival of three Arcobacter butzleri strains inside Acanthamoeba castellanii was assessed using axenic cultures of A. castellanii that were inoculated with the tested strains and incubated at 26°C under aerobic conditions for 240h. The behavior of bacteria in contact with amoebae was monitored using phase contrast microscopy. The bacterial survival rate within amoebae was assessed through counting colony forming units, using the gentamicin protection assay. All A. butzleri strains were able to survive during 240h within the amoebae, thus suggesting that (i) A. butzleri resists the amoebic digestion processes at least for the analyzed time; (ii) that A. castellanii could serve as an environmental reservoir for this bacterium, probably acting as a transmission vehicle for A. butzleri.
Assuntos
Acanthamoeba castellanii/microbiologia , Arcobacter/fisiologia , Acanthamoeba castellanii/crescimento & desenvolvimento , Acanthamoeba castellanii/ultraestrutura , Aerobiose , Cultura Axênica , Reservatórios de Doenças , Microscopia de Contraste de Fase , Vacúolos/microbiologia , Vacúolos/ultraestrutura , Microbiologia da ÁguaRESUMO
Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance.
Assuntos
Autofagia , Bactérias/imunologia , Citoplasma/microbiologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Vacúolos/microbiologiaRESUMO
Shiga toxin-producing Escherichia coli (STEC) cause severe human infections and their virulence abilities are not fully understood. Cattle are a key reservoir, and the terminal rectum is the principal site of bacterial carriage. Most STEC possess a pathogenicity island termed the locus of enterocyte effacement (LEE). Nonetheless, LEE-negative STEC have been associated with disease. We found that invasion of LEE-positive and LEE-negative strains was higher for human enterocytic cell lines and for undifferentiated Caco-2 cells. Intracellular bacteria could be detected as early as 5 min after infection and transmission electron microscopy showed bacteria within membrane-bound vacuoles. STEC invasion depended on actin microfilaments and protein kinases. Scanning electron microscopy revealed that bacterial entry was not associated with membrane ruffling. Absence of macropinocytosis or actin rearrangement at the entry points suggests a zipper-like entry mechanism. Disruption of the tight junction by EGTA enhanced invasion of Caco-2 monolayers, and bacterial invasion mostly proceeded through the basolateral pole of enterocytes. STEC persisted within Caco-2 cells for up to 96 h without cell death and bacterial viability increased after 48 h, suggesting intracellular multiplication. The relatively harmless intracellular localization of STEC can be an efficient strategy to prevent its elimination from the bovine intestinal tract.
Assuntos
Endocitose , Células Epiteliais/microbiologia , Viabilidade Microbiana , Escherichia coli Shiga Toxigênica/fisiologia , Linhagem Celular , Humanos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Fatores de Tempo , Vacúolos/microbiologiaRESUMO
Coxiella burnetii is a Gram-negative intracellular bacterium. As previously described, both the endocytic and the autophagic pathways contribute to the maturation of Coxiella replicative vacuoles (CRVs). The large CRVs share the properties of both phagolysosomal and autophagolysosomal compartments. Vamp3, Vamp7 and Vamp8 are v-SNAREs involved in the endocytic pathway which participate mainly in the fusion between endosomes and lysosomes. In the present study we observed that Vamp7 interacts with C. burnetii at different infection times (1 h-48 h p.i.). We have determined that a truncated mutant of Vamp7 (Vamp7 NT) and a siRNA against this SNARE protein affects the optimal development of CRVs, suggesting that Vamp7 mediates fusion events that are required for the biogenesis of CRVs. Indeed, we have observed that overexpression of Vamp7 NT inhibited the heterotypic fusion with lysosomes and the homotypic fusion between individual Coxiella phagosomes and CRVs. Moreover, we have detected in the vacuole membrane, at different infection times, the Vamp7 partners (Vti1a and Vti1b). Interestingly, treatment with chloramphenicol reduced the colocalization between C. burnetii and Vamp7, Vti1a or Vti1b, indicating that the recruitment of these SNAREs proteins is a bacteria-driven process that favours the CRV biogenesis, likely by facilitating the interaction with the endolysosomal compartment.
Assuntos
Coxiella burnetii/patogenicidade , Endocitose/fisiologia , Proteínas SNARE/fisiologia , Vacúolos/microbiologia , Animais , Células CHO , Linhagem Celular , Cloranfenicol/farmacologia , Chlorocebus aethiops , Coxiella burnetii/fisiologia , Cricetinae , Cricetulus , Modelos Animais de Doenças , Células HeLa , Humanos , Proteínas R-SNARE/efeitos dos fármacos , Proteínas R-SNARE/fisiologia , RNA Interferente Pequeno/farmacologia , Proteínas SNARE/efeitos dos fármacos , Células VeroRESUMO
Haemophilus influenzae belongs to respiratory tract microbiota. We observed vacuoles formation in previous studies with H. influenzae culture supernatants, so in this work we characterised that cytotoxic effect. We observed an abundant production of acidic cytoplasmic vacuoles due to the presence of a "vacuolating factor" in H. influenzae supernatants which was characterised as thermolabile. Greatest vacuolating activity was observed when utilizing the fraction > 50 kDa. The presence of a large number of vacuoles in HEp-2 cells was verified by transmission electron microscopy and some vacuoles were identified with a double membrane and/or being surrounded by ribosomes. These results suggest similar behaviour to that of vacuolating effects described by autotransporter proteins an undescribed cytotoxic effect induced by H. influenzae.
Assuntos
Citotoxinas/toxicidade , Haemophilus influenzae/metabolismo , Vacúolos/microbiologia , Células Cultivadas , Citotoxinas/biossíntese , Haemophilus influenzae/ultraestrutura , Microscopia Eletrônica de Transmissão , Vacúolos/ultraestruturaRESUMO
The facultative intracellular bacterial pathogens Listeria monocytogenes and Salmonella enterica have evolved multiple strategies to invade a large panel of mammalian cells. These pathogens use the host cell actin system for invasion and became a paradigm for the study of host-pathogen interactions and bacterial adaptation to mammalian hosts. The key signaling component that these pathogens use to orchestrate actin remodeling is the Arp2/3 complex, which is related to polymerization of actin filaments. These bacterial pathogens are able to trigger distinct invasion mechanisms. On the one hand, L. monocytogenes invade a host cell in a way dependent on the specific interactions between bacterial and host cell proteins, which in turn activate the host cell actin polymerizing machinery that culminates with bacterial internalization. Also, Listeria escapes from the newly formed parasitophorous vacuole and moves among adjacent cells by triggering actin polymerization. On the other hand, Salmonella invades a host cell by delivering into the cytoplasm virulence factors which directly interact with host regulators of actin polymerization which leads to bacterial uptake. Moreover, Salmonella avoids vacuole lyses and modulates the early and late endosomal markers presented in the vacuole membrane. This mini-review focuses on the different pathways that L. monocytogenes and S. enterica activate to modulate the actin cytoskeleton in order to invade, to form the parasitophorous vacuole, and to migrate inside host cells.