RESUMO
Salmonella typhimurium is an intracellular bacterial pathogen that remains enclosed in vacuoles (SCV) upon entry into the host cell. In this study we have examined the intracellular trafficking route of S. typhimurium within epithelial cells. Indirect immunofluorescence analysis showed that bacteria initiated fusion with lysosomal membrane glycoprotein (lgp)-containing compartments approximately 15 min after bacterial internalization. This process was completed approximately 75 min later and did not require microtubules. Cation-independent (CI)- or cation-dependent (CD)-mannose 6-phosphate receptors (M6PRs) were not observed at detectable levels in SCV. Lysosomal enzymes showed a different distribution in SCV: lysosomal-acid phosphatase (LAP) was incorporated into these vacuoles with the same kinetics as lgps, while cathepsin D was present in a low proportion (approximately 30%) of SCV. Uptake experiments with fluid endocytic tracers such as fluorescein-dextran sulphate (F-DX) or horseradish-peroxidase (HRP) showed that after 2 h of uptake, F-DX was present in approximately 75% of lgp-containing vesicles in uninfected cells, while only approximately 15% of SCV contained small amounts of the tracer during the same uptake period. SCV also showed only partial fusion with HRP-preloaded secondary lysosomes, with approximately 30% of SCV having detectable amounts of HRP at 6 h after infection. These results indicate that SCV show limited accessibility to fluid endocytic tracers and mature lysosomes, and are therefore functionally separated from the endocytic route. Moreover, the unusual intracellular trafficking route of S. typhimurium inside epithelial cells has allowed us to establish the existence of two different lgp-containing vesicles in Salmonella-infected cells: one population is separated from the endocytic route, fusogenic with incoming SCV and may arise from a secretory pathway, while the second involves the classical secondary or mature lysosomes.
Assuntos
Membranas Intracelulares/fisiologia , Lisossomos/fisiologia , Glicoproteínas de Membrana/metabolismo , Receptor IGF Tipo 2/metabolismo , Salmonella typhimurium/fisiologia , Fosfatase Ácida/metabolismo , Animais , Endocitose , Epitélio/microbiologia , Epitélio/fisiologia , Imunofluorescência , Células HeLa , Humanos , Cinética , Lisossomos/metabolismo , Camundongos , Microscopia de Fluorescência , Microtúbulos/fisiologia , Vacúolos/fisiologiaRESUMO
Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.
Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano , Listeria monocytogenes/genética , Listeria/genética , Adaptação Fisiológica , Motivos de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Composição de Bases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cromossomos Bacterianos/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Transferência Genética Horizontal , Genes Bacterianos , Genômica , Listeria/química , Listeria/fisiologia , Listeria monocytogenes/química , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Análise de Sequência de DNA , Staphylococcus aureus/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Virulência/genéticaRESUMO
Many bacterial pathogens and eukaryotic parasites can enter mammalian cells and live intracellularly inside membrane-bound vacuoles. The intravacuolar lifestyle of these pathogens plays a key role in pathogenesis. Understanding the molecular basis of the development of these specialized intracellular compartments is critical to understanding how these organisms cause disease.
Assuntos
Fenômenos Fisiológicos Bacterianos , Parasitos/fisiologia , Vacúolos/microbiologia , Vacúolos/parasitologia , Animais , Eritrócitos/microbiologia , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Concentração de Íons de Hidrogênio , FagossomosRESUMO
Salmonella species proliferate within membrane-bound vacuoles of eukaryotic cells. Recent work has shown that macrophages are the main cell type supporting bacterial growth in vivo. In contrast, tissue culture models have traditionally described epithelial cells as the most permissive cells for bacterial growth. Unfortunately, no mechanism used by Salmonella to initiate growth within a vacuole has been characterised. Recently, it has been shown that Salmonella is capable of attenuating intracellular proliferation. This finding suggests that both the host and the pathogen contribute to a fine adjustment of the intracellular growth rate.
Assuntos
Células Epiteliais/microbiologia , Macrófagos/microbiologia , Infecções por Salmonella/microbiologia , Salmonella/crescimento & desenvolvimento , Animais , Células Cultivadas , Fibroblastos/microbiologia , Humanos , Rim/citologia , Camundongos , RatosRESUMO
Salmonella typhimurium is an intracellular pathogen capable of proliferating within vacuolar compartments of non-phagocytic eucaryotic cells. This process has been shown to be essential for virulence in the mouse typhoid model (Leung and Finlay, Proc. Natl. Acad. Sci. USA, 88, 11470-11474, 1990). Here we present evidence that certain non-phagocytic eucaryotic cell lines, such as 3T3 (mouse fibroblasts) and NRK (rat fibroblasts) cells, are not permissive for S. typhimurium intracellular proliferation. Moreover, viability of intracellular bacteria residing within both cell types notably decreases at late postinfection times (72 h). These results clearly demonstrate that non-phagocytic eucaryotic cells are capable of destroying intracellular S. typhimurium. Experimentation with 3T3 and NRK cell lines might provide an appropriate in vitro model for identifying new bacterial and/or eucaryotic factors regulating Salmonella intracellular proliferation within vacuoles of the host eucaryotic cell.
Assuntos
Células Eucarióticas/microbiologia , Salmonella typhimurium/crescimento & desenvolvimento , Células 3T3/imunologia , Células 3T3/microbiologia , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Células Eucarióticas/imunologia , Células Eucarióticas/ultraestrutura , Fibroblastos/imunologia , Fibroblastos/microbiologia , Gentamicinas/metabolismo , Células HeLa/imunologia , Células HeLa/microbiologia , Humanos , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Fagocitose/fisiologia , Coelhos , Ratos , Salmonella typhimurium/imunologia , Salmonella typhimurium/ultraestrutura , VirulênciaRESUMO
The fine structure of sacculi from Thermus thermophilus HB27, T. aquaticus YT-1 and Thermus ATCC27737 has been worked out by HPLC analysis and mass spectrometry techniques. The three microorganisms have a murein composition of the rare A3beta chemotype, but showed substantial differences in muropeptide composition. Phenylacetylated muropeptides,previously described in T. thermophilus HB8, were detected exclusively in T. thermophilus HB27. Murein from T. aquaticusYT-1 was devoid of D-Ala-D-Ala terminated muropeptides, which were, in contrast, abundant in T. thermophilus HB27 and Thermus ATCC27737. The significance of these findings is discussed.
Assuntos
Parede Celular/ultraestrutura , Peptidoglicano/química , Thermus/ultraestrutura , Parede Celular/química , Cromatografia Líquida de Alta Pressão , Peptídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Thermus/química , Thermus/classificaçãoRESUMO
Analysis of the functional role of penicillin-binding protein 1B (PBP1B) of Escherichia coli led us to find a new mutation able to suppress thermosensitive growth of the pbpB2158(Ts) mutant strain, which harbors a thermosensitive PBP3 protein only in the presence of a ponB+ background. The mutation, originally isolated in a strain with a high dosage of PBP1B, could also suppress the pbpB(Ts) phenotype when a single copy of the ponB gene was introduced. These results clearly give further support to the implication of PPB1B in the septation process in Escherichia coli.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/genética , Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase , Peptidil Transferases , D-Ala-D-Ala Carboxipeptidase Tipo Serina , Supressão Genética , Proteínas de Transporte/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/crescimento & desenvolvimento , Genótipo , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Proteínas de Ligação às Penicilinas , Fenótipo , TemperaturaRESUMO
Salmonella typhimurium multiplication inside eukaryotic host cells is critical for virulence. Salmonella typhimurium strain SL1344 appears as filaments upon growth in macrophages and MelJuSo cells, a human melanoma cell line, indicating a specific blockage in the bacterial cell division process. Several studies have investigated the host cell response impairing bacterial division. However, none looked at the bacterial factors involved in inhibition of Salmonella division inside eukaryotic cells. We show here that blockage in the bacterial division process is sulA-independent and takes place after FtsZ-ring assembly. Salmonella typhimurium genes in which mutations lead to filamentous growth within host cells were identified by a large scale mutagenesis approach on strain 12023, revealing bacterial functions crucial for cell division within eukaryotic cells. We finally demonstrate that SL1344 filamentation is a result of hisG mutation, requires the activity of an enzyme of the histidine biosynthetic pathway HisFH and is specific for the vacuolar environment.
Assuntos
Proteínas de Bactérias/metabolismo , Divisão Celular , Células Eucarióticas/microbiologia , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/citologia , Aminoidrolases/genética , Aminoidrolases/metabolismo , Animais , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Proteínas de Escherichia coli/metabolismo , Inibidores do Crescimento/metabolismo , Histidina/metabolismo , Humanos , Macrófagos/microbiologia , Camundongos , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , VirulênciaRESUMO
Salmonella species are intracellular facultative pathogens which survive within phagocytic cells such as macrophages and proliferate inside vacuoles of epithelial cells. Early reports suggested that the capacity for surviving within macrophages was due to the inhibitory effect on the phagosome-lysosome fusion event induced by intracellular Salmonella. However, recent cell biology data, obtained both with phagocytic and epithelial cells, have shown that Salmonella-containing phagosomes have large amounts of lysosomal membrane glycoproteins (lgp), major components of the lysosomal membrane. This apparent discrepancy has partly been clarified at least in epithelial cells: the Salmonella-containing phagosome fuses with lgp-rich compartment different from the classical mature lysosome, as they do not contain certain lysosomal enzymes and are not connected with the endocytic route. Therefore, Salmonella seems to use an alternative strategy not merely based on the inhibition of phagosome-lysosome fusion event. This strategy essentially involves acquisition of only certain lysosomal components to form a specialized phagosomal compartment in which to survive or proliferate intracellularly. These observations have also exemplified the potential use of intracellular bacterial pathogens as biological probes to understand normal biological aspects of the eukaryotic cell. The intracellular lifestyle of Salmonella will undoubtedly provide new insights into the process of lysosome biogenesis.
Assuntos
Células Eucarióticas/microbiologia , Lisossomos/microbiologia , Salmonella/fisiologia , Animais , Epitélio/microbiologia , Células Eucarióticas/ultraestrutura , Humanos , Lisossomos/enzimologia , Lisossomos/fisiologia , Fusão de Membrana , Fagocitose , Infecções por Salmonella/microbiologia , Salmonelose Animal/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologia , Vacúolos/microbiologia , VirulênciaRESUMO
Salmonella typhimurium induced massive uptake of extracellular fluid in epithelial cells in the form of macropinosomes. The appearance of macropinosomes in the infected cell was related to the induction of membrane ruffling during bacterial invasion. A noninvasive S. typhimurium invA mutant did not trigger such effects in the host cell. Similarly, S. typhimurium invA mutants that invaded via the invasin protein from Yersinia pseudotuberculosis or adhered to the host cell via the afimbrial AFA-I adhesin from Escherichia coli did not trigger formation of macropinosomes. In contrast to the formation of macropinosomes in macrophages, the appearance of macropinosomes in S. typhimurium-infected epithelial cells did not require microtubules. These data suggest that massive uptake of extracellular fluid in S. typhimurium-infected epithelial cells is an event related to the invasion mechanisms used by this pathogen.
Assuntos
Espaço Extracelular/metabolismo , Macrófagos/ultraestrutura , Fagossomos/metabolismo , Salmonella typhimurium/patogenicidade , Animais , Linhagem Celular , Humanos , Fator Estimulador de Colônias de Macrófagos/farmacologia , RatosRESUMO
Salmonella species penetrate (invade) and proliferate within non-phagocytic cells such as epithelial cells. These two processes are essential for Salmonella virulence and have been shown to occur in the intestinal epithelium and cultured epithelial cells. In recent years the signals that Salmonella transmits to the epithelial cell have begun to be elucidated. In this review, we summarize the most recent findings about the molecular nature of the interactions between Salmonella and epithelial cells. These studies reveal that Salmonella causes dramatic changes in the morphology of the host plasma membrane during bacterial invasion, and that this pathogen exploits other host structures such as actin filaments and lysosomes to trigger internalization and intracellular proliferation within non-phagocytic cells.
Assuntos
Epitélio/microbiologia , Salmonella/fisiologia , Animais , Divisão Celular , Membrana Celular/ultraestrutura , Células Cultivadas , Cobaias , Humanos , Mucosa Intestinal/citologia , Organelas/ultraestrutura , Fenótipo , Salmonella/patogenicidade , Infecções por Salmonella/microbiologia , VirulênciaRESUMO
Mutants of Salmonella typhimurium lacking DNA adenine methylase are attenuated for virulence in BALB/c mice. LD(50) values of a DNA adenine methylation (Dam)(-) mutant are at least 10(3)- to 10(4)-fold higher than those of the parental strain when administrated by oral or intraperitoneal routes. Dam(-) mutants are unable to proliferate in target organs but persist in low numbers in these locations. Efficient protection to challenge with the virulent parental strain is observed in mice infected with a Dam(-) mutant. Use of the ileal loop assay shows that Dam(-) mutants are less cytotoxic to M cells and fail to invade enterocytes. In the tissue culture model, lack of DNA adenine methylation causes reduced ability to invade nonphagocytic cells. In contrast, no effect is observed either in intracellular proliferation within nonphagocytic cells or in survival within macrophages. The invasion defect of Dam(-) mutants is correlated with a distinct pattern of secreted proteins, which is observed in both PhoP(+) and PhoP(-) backgrounds. Altogether, our observations suggest a multifactorial role of Dam methylation in Salmonella virulence.
Assuntos
Proteínas de Bactérias/metabolismo , Mucosa Intestinal/microbiologia , Salmonella typhimurium/enzimologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/fisiologia , Animais , Metilação de DNA , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , VirulênciaRESUMO
The VanX protein is a D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a D-Ala-D-Ala dipeptidase in the gram-negative species Salmonella enterica. The Salmonella enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes D-Ala-D-Ala, DL-Ala-DL-Phe, and D-Ala-Gly but not the tripeptides D-Ala-D-Ala-D-Ala and DL-Ala-DL-Lys-Gly or the dipeptides L-Ala-L-Ala, N-acetyl-D-Ala-D-Ala, and L-Leu-Pro. The Salmonella dipeptidase gene, designated pcgL, appears to have been acquired by horizontal gene transfer because pcgL-hybridizing sequences were not detected in related bacterial species and the G+C content of the pcgL-containing region (41%) is much lower than the overall G+C content of the Salmonella chromosome (52%). In contrast to wild-type Salmonella, a pcgL mutant was unable to use D-Ala-D-Ala as a sole carbon source. The pcgL gene conferred D-Ala-D-Ala dipeptidase activity upon Escherichia coli K-12 but did not allow growth on D-Ala-D-Ala. The PcgL protein localizes to the periplasmic space of Salmonella, suggesting that this dipeptidase participates in peptidoglycan metabolism.
Assuntos
Proteínas de Bactérias , Dipeptidases/fisiologia , Endopeptidases/fisiologia , Salmonella enterica/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA Bacteriano , Dipeptidases/genética , Dipeptidases/metabolismo , Cães , Endopeptidases/genética , Endopeptidases/metabolismo , Genes Bacterianos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Periplasma/enzimologia , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Frações Subcelulares , Especificidade por Substrato , VirulênciaRESUMO
Salmonella species invade and replicate within epithelial cells in membrane-bound vacuoles. In this report we show that upon infection of HeLa epithelial cells, Salmonella typhimurium residues in vacuoles that contain lysosomal membrane glycoproteins (lgps). Four to six hours after invasion, intracellular bacteria induce the formation of stable filamentous structures containing lgps that are connected to the bacteria-containing vacuoles. Formation of these lgp-rich structures requires viable intracellular bacteria and is blocked by inhibitors of vacuolar acidification. These structures are not present in uninfected cells or in cells infected with another invasive bacteria, Yersinia enterocolitica. Tracers added to the extracellular medium are not delivered to the Salmonella-induced filaments, suggesting that these structures are different from previously described tubular lysosomes. Initiation of intracellular bacterial replication correlates with formation of these lgp-containing filaments. Certain avirulent Salmonella mutants that are defective for intracellular replication fail to induce formation of these structures. These observations suggest that Salmonella-induced filaments containing lgps are linked to intracellular bacterial replication.
Assuntos
Epitélio/microbiologia , Lisossomos/ultraestrutura , Glicoproteínas de Membrana/metabolismo , Salmonella typhimurium/ultraestrutura , Compartimento Celular , Epitélio/ultraestrutura , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Membranas Intracelulares/ultraestrutura , Isoquinolinas/metabolismo , Nocodazol/farmacologia , Ovalbumina/metabolismo , Salmonella typhimurium/crescimento & desenvolvimentoRESUMO
We have examined the targeting of S. typhimurium-containing vacuoles to lysosomes after invasion of cultured HeLa epithelial cells. Our results show that intracellular bacteria colocalize with vacuoles containing lysosomal membrane glycoproteins (LGPs). Both human LGPs, hlamp-1 and hlamp-2, are present in S. typhimurium-containing vacuoles from approximately 2 h postinfection. At later times (4-6 h), long and stable filamentous structures with lysosomal markers appear connected to bacteria-containing vacuoles in infected cells. Viable intracellular bacteria are required for the formation of these structures, which are not detected in uninfected cells or in HeLa epithelial cells infected with another invasive bacteria, Yersinia enterocolitica. Kinetics analysis showed a strict correlation between the appearance of these LGP-rich filaments and the initiation of intracellular bacterial replication. Moreover, these structures are absent in epithelial cells infected with certain S. typhimurium intracellular replication-defective mutants. Additional data confirmed that an intact microtubule network and intravacuolar acidic pH are required to induce the formation of LGP-containing filamentous structures and that these structures are morphologically and functionally different from previously described tubular lysosomes.
Assuntos
Antígenos CD , Glicoproteínas de Membrana/análise , Salmonella typhimurium/crescimento & desenvolvimento , Animais , Epitélio/microbiologia , Epitélio/ultraestrutura , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Lisossomal , Camundongos , Vacúolos/microbiologia , Vacúolos/ultraestruturaRESUMO
The atp and fur genes are involved in the acid tolerance response of Salmonella typhimurium. An atp::Tn10 mutant was avirulent in the mouse typhoid model when assayed by oral and intraperitoneal routes. However, a fur mutant was completely virulent by the intraperitoneal route. No relevant differences in intracellular survival or invasion rates were observed for the two mutants in macrophages and epithelial cells. These data indicate that separate acid tolerance response genes may have different roles in S. typhimurium virulence.
Assuntos
Proteínas de Bactérias/metabolismo , Genes Bacterianos , ATPases Translocadoras de Prótons/metabolismo , Proteínas Repressoras/metabolismo , Salmonella typhimurium/patogenicidade , Animais , Cães , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Insercional , Salmonella typhimurium/genética , Febre Tifoide/microbiologiaRESUMO
Salmonella interact with eucaryotic membranes to trigger internalization into non-phagocytic cells. In this study we examined the distribution of host plasma membrane proteins during S. typhimurium invasion of epithelial cells. Entry of S. typhimurium into HeLa epithelial cells produced extensive aggregation of cell surface class I MHC heavy chain, beta 2-microglobulin, fibronectin-receptor (alpha 5 beta 1 integrin), and hyaluronate receptor (CD-44). Other cell surface proteins such as transferrin-receptor or Thy-1 were aggregated by S. typhimurium to a much lesser extent. Capping of these plasma membrane proteins was observed in membrane ruffles localized to invading S. typhimurium and in the area surrounding these structures. In contrast, membrane ruffling induced by epidermal growth factor only produced minor aggregations of surface proteins, localized exclusively in the membrane ruffle. This result suggests that extensive redistribution of these proteins requires a signal related to bacterial invasion. This bacteria-induced process was associated with rearrangement of polymerized actin but not microtubules, since preincubation of epithelial cells with cytochalasin D blocked aggregation of these proteins while nocodazole treatment did not. Of the host surface proteins aggregated by S. typhimurium, only class I MHC heavy chain was predominantly present in the bacteria-containing vacuoles. No extensive aggregation of host plasma membrane proteins was detected when HeLa epithelial cells were infected with invasive bacteria that do not induce membrane ruffling, including Yersinia enterocolitica, a bacterium that triggers internalization via binding to beta 1 integrin, and a S. typhimurium invasion mutant that utilizes the Yersinia-internalization route. In contrast to the situation with S. typhimurium, class I MHC heavy chain was not selectively internalized into vacuoles containing these other bacteria. Extensive aggregation of host plasma membrane proteins was also not observed when other S. typhimurium mutants that are defective for invasion were used. The amount of internalized host plasma membrane proteins in the bacteria-containing vacuoles decreased over time with all invasive bacteria examined, indicating that modification of the composition of these vacuoles occurs. Therefore, our data show that S. typhimurium induces selective aggregation and internalization of host plasma membrane proteins, processes associated with the specific invasion strategy used by this bacterium to enter into epithelial cells.
Assuntos
Adesinas Bacterianas , Microtúbulos/ultraestrutura , Salmonella typhimurium/patogenicidade , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Citocalasina D/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Células HeLa , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/ultraestrutura , Humanos , Receptores de Hialuronatos , Integrinas/metabolismo , Integrinas/ultraestrutura , Camundongos , Microtúbulos/efeitos dos fármacos , Mutagênese , Nocodazol/farmacologia , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/ultraestrutura , Receptores de Retorno de Linfócitos/metabolismo , Receptores de Retorno de Linfócitos/ultraestrutura , Salmonella typhimurium/fisiologia , Antígenos Thy-1/metabolismo , Antígenos Thy-1/ultraestrutura , Virulência/genética , Yersinia enterocolitica/patogenicidade , Yersinia pseudotuberculosis/genética , Microglobulina beta-2/metabolismo , Microglobulina beta-2/ultraestruturaRESUMO
Salmonella typhimurium has the capacity to enter into and multiply within epithelial cells. During the entire intracellular stage, bacteria are enclosed within a vacuole. To characterize the micro-environment of the bacteria-containing vacuoles, we have used a new method to measure the expression levels of several S. typhimurium genes in intracellular bacteria within Madin-Darby canine kidney (MDCK) epithelial cells. Our study was based on the determination of beta-galactosidase activity derived from lacZ transcriptional fusions using the highly sensitive substrate fluorescein-di-beta-D-galactoside (FDG). Expression of the iroA and mgtB genes (induced by Fe2+ and Mg2+ limitation respectively), and cadA (induced by pH 6.0 in the presence of lysine, with enhanced expression under anaerobiosis) were characterized at different post-infection times. High intracellular expression levels were detected for the iroA and mgtB genes, suggesting that the concentrations of free Fe2+ and Mg2+ in the vacuole may be low. cadA activity was detected only at early post-infection times (4 h), suggesting that the vacuole may have a mild-acidic pH, and oxygen and lysine present at this time. Globally, the results reported indicate that the use of a highly sensitive beta-galactosidase substrate can provide information about the micro-environment within which an intracellular pathogen, such as S. typhimurium, resides.
Assuntos
Epitélio/microbiologia , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/fisiologia , Vacúolos/microbiologia , Aerobiose , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Linhagem Celular , Cães , Epitélio/ultraestrutura , Concentração de Íons de Hidrogênio , Ferro/fisiologia , Rim , Lisina/metabolismo , Magnésio/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Salmonella typhimurium/genéticaRESUMO
The biological effects of bacterial lipopolysaccharide (LPS) on eucaryotic cells have traditionally been characterized following extracellular challenge of LPS on susceptible cells. In this study, we report the capacity of Salmonella typhimurium to release LPS once it is located in the intracellular environment of cultured epithelial cells. LPS is liberated from vacuolar compartments, where intracellular bacteria reside, to vesicles present in the host cell cytosol. The vesicle-associated LPS is detected in infected cells from the time when invading bacteria enter the host cell. Release of LPS is restricted to S. typhimurium-infected cells, with no LPS observed in neighboring uninfected cells, suggesting that dissemination of LPS occurs entirely within the intracellular environment of the infected cell. The amount of LPS present in host vesicles reaches a maximum when intracellular S. typhimurium cells start to proliferate, a time at which the entire host cell cytosol is filled with numerous vesicles containing LPS. All these data support the concept that intracellular bacterial pathogens might signal the host cell from intracellular locations by releasing bioactive bacterial components such as LPS.