RESUMO
Staphylococcus aureus is the primary etiological agent of several human diseases. S. aureus has classically been considered an extracellular pathogen; however, recent evidence indicates that S. aureus invades and persists in non-professional phagocytes. Experiments demonstrate that actin microfilaments, microtubules, receptor-mediated endocytosis, and protein tyrosine kinases play important roles in the uptake of S. aureus. Fibronectin-binding proteins and beta-integrins are implicated as critical cell surface molecules associated with internalization of S. aureus by non-phagocytic cells. Following invasion of eukaryotic cells, S. aureus induces the release of cytokines that have the potential to exacerbate disease and induce apoptosis. Finally, S. aureus has the ability to persist inside host cells as small colony variants, a phenotype associated with persistent and recurrent infections.
Assuntos
Células Eucarióticas/microbiologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/fisiopatologia , Staphylococcus aureus/patogenicidade , Animais , Endocitose , Células Eucarióticas/fisiologia , Humanos , Camundongos , Staphylococcus aureus/fisiologiaRESUMO
Staphylococcus aureus invades osteoblasts and can persist in the intracellular environment. The present study examined the role of osteoblast mitogen-activated protein kinase (MAPK) pathways in bacterial invasion. S. aureus infection of normal human and mouse osteoblasts resulted in an increase in the phosphorylation of the extracellular signal-regulated protein kinases (ERK 1 and 2). This stimulation of ERK 1 and 2 correlated with the time course of S. aureus invasion, and bacterial adherence induced the MAPK pathway. ERK 1 and 2 phosphorylation was time and dose dependent and required active S. aureus gene expression for maximal induction. The nonpathogenic Staphylococcus carnosus was also able to induce ERK 1 and 2 phosphorylation, albeit at lower levels than S. aureus. Phosphorylation of the stress-activated protein kinases was increased in both infected human and mouse osteoblasts; however, the p38 MAPK pathway was not activated in response to S. aureus. Finally, the transcription factor c-Jun, but not Elk-1 or ATF-2, was phosphorylated in response to S. aureus infection.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteoblastos/metabolismo , Osteoblastos/microbiologia , Staphylococcus aureus/patogenicidade , Animais , Aderência Bacteriana , Células Cultivadas , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismo , Staphylococcus aureus/crescimento & desenvolvimento , Raios UltravioletaRESUMO
Two common pathogens of bone, Staphylococcus aureus and Salmonella, were investigated for their ability to induce chemokine expression in bone-forming osteoblasts. Cultured mouse or human osteoblasts could rapidly respond to bacterial infection by upregulating the mRNA encoding the chemokine, monocyte chemoattractant protein-1 (MCP-1). This rapid induction occurred on infection with either the gram-positive pathogen, S. aureus, or the gram-negative pathogen, Salmonella. Increased mRNA expression translated into MCP-1 secretion by cultured mouse or human osteoblasts in response to viable bacteria, whereas UV-killed bacteria were less effective in stimulating chemokine secretion. There was a dose-response relationship observed between the amount of input bacteria and increases in MCP-1 secretion. Immunohistochemical staining of infected osteoblasts indicated that the majority of cells could express MCP-1, with some osteoblasts having a higher intensity of staining than others. Organ cultures of mouse calvaria (skullcap) bone showed increases in MCP-1 immunostaining following bacterial infection. The immunoreactive MCP-1 in infected calvaria localized to areas containing active osteoblasts. Taken together, these studies demonstrate a conserved osteoblast-derived MCP-1 response to two very different pathogens of bone.
Assuntos
Quimiocina CCL2/biossíntese , Osteoblastos/metabolismo , Osteoblastos/microbiologia , Salmonella enterica/imunologia , Staphylococcus aureus/imunologia , Animais , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismoRESUMO
Staphylococcus aureus and Salmonella enterica serovar Dublin invade osteoblasts and are causative agents of human bone disease. In the present study, we examined the ability of S. aureus and Salmonella serovar Dublin to induce the production of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by normal osteoblasts. Normal mouse and human osteoblasts were cocultured with S. aureus or Salmonella serovar Dublin at different multiplicities of infection. Following initial incubation and examination of TRAIL expression, extracellular bacteria were killed by the addition of media containing the antibiotic gentamicin. Lysates and conditioned media from osteoblast cultures were then collected at various times following invasion and analyzed. The results demonstrated that S. aureus and Salmonella serovar Dublin are potent inducers of TRAIL expression by osteoblasts. Mouse and human TRAIL mRNA expression was induced by bacterial infection and demonstrated a dose-dependent response. Analysis of kinetics suggested that TRAIL mRNA was induced within 30 min after exposure to bacteria and that its level of expression remained relatively constant over the time period examined. mRNA molecules encoding TRAIL receptors were constitutively expressed by osteoblasts. Furthermore, TRAIL protein was detected as early as 45 min and up to 24 h following infection. The quantity of TRAIL protein produced also increased in a dose-dependent manner. Collectively, these findings suggest a mechanism whereby bacterial pathogens mediate bone destruction via osteoblast apoptosis.
Assuntos
Apoptose , Glicoproteínas de Membrana/biossíntese , Osteoblastos/microbiologia , Salmonella enterica/patogenicidade , Staphylococcus aureus/patogenicidade , Fator de Necrose Tumoral alfa/biossíntese , Animais , Proteínas Reguladoras de Apoptose , Humanos , Camundongos , RNA Mensageiro/biossíntese , Receptores do Fator de Necrose Tumoral/biossíntese , Ligante Indutor de Apoptose Relacionado a TNFRESUMO
Staphylococcus aureus and Salmonella spp. are common causes of bone diseases; however, the immune response during such infections is not well understood. Colony-stimulating factors (CSF) have a profound influence on osteoclastogenesis, as well as the development of immune responses following infection. Therefore, we questioned whether interaction of osteoblasts with two very different bacterial pathogens could affect CSF expression by these cells. Cultured mouse and human osteoblasts were exposed to various numbers of S. aureus or Salmonella dublin bacteria, and a comprehensive analysis of granulocyte-macrophage (GM)-CSF, granulocyte (G)-CSF, macrophage (M)-CSF, and interleukin-3 (IL-3) mRNA expression and cytokine secretion was performed. Expression of M-CSF and IL-3 mRNAs by mouse osteoblasts was constitutive and did not increase significantly following bacterial exposure. In contrast, GM-CSF and G-CSF mRNA expression by mouse osteoblasts was dramatically upregulated following interaction with either viable S. aureus or Salmonella. This increased mRNA expression also translated into high levels of GM-CSF and G-CSF secretion by mouse and human osteoblasts following bacterial exposure. Viable S. aureus and Salmonella induced maximal levels of CSF mRNA expression and cytokine secretion compared to UV-killed bacteria. Furthermore, GM-CSF and G-CSF mRNA expression could be induced in unexposed osteoblasts separated by a permeable Transwell membrane from bacterially exposed osteoblasts. M-CSF secretion was increased in cultures of exposed human osteoblasts but not in exposed mouse osteoblast cultures. Together, these studies are the first to define CSF expression and suggest that, following bacterial exposure, osteoblasts may influence osteoclastogenesis, as well as the development of an immune response, via the production of these cytokines.
Assuntos
Fatores Estimuladores de Colônias/biossíntese , Osteoblastos/metabolismo , Salmonella/fisiologia , Staphylococcus aureus/fisiologia , Animais , Células Cultivadas , Fatores Estimuladores de Colônias/genética , Humanos , Camundongos , Osteoblastos/microbiologia , RNA Mensageiro/análiseRESUMO
OBJECTIVES: To determine the difference, if any, between the reduction of bacteria on contaminated normal skin and contaminated superficially abraded skin following standard surgical preparations at clinically relevant time points after injury. DESIGN: Prospective animal study. SETTING: Laboratory. SUBJECTS: Thirty-two New Zealand white rabbits. INTERVENTION: Two sites, two by two centimeters, one abraded and one nonabraded (control), were studied on each rabbit. Both were inoculated with encapsulated Staphylococcus aureus strain Wood 46. Four six-millimeter punch biopsies were obtained after inoculation, immediately before surgical scrub, and five minutes and then two hours after completion of the surgical scrub. The rabbits were divided into four cohort groups with surgical scrubs performed at six, twelve, twenty-four, and forty-eight hours after inoculation. Bacterial counts were determined. MAIN OUTCOME MEASUREMENTS: Numbers of bacteria on surgical sites. RESULTS: Before surgical preparation, the amount of bacteria on the normal skin (control sites) dropped significantly (p<0.02) except in the six-hour group (p<0.20). At the abraded skin sites, the bacteria flourished. The surgical scrub dropped bacterial counts at both the abraded and nonabraded skin sites significantly (p<0.05) except for the abraded site in the twenty-four-hour group (p<0.08). However in the twelve-, twenty-four-, and forty-eight-hour groups, the bacterial counts (colony-forming units) were still markedly elevated (>1x10(5) at abraded sites) when compared with the nonabraded skin sites (p<0.008) at the respective time intervals. Only at the six-hour interval were the bacterial counts reduced similarly at both the abraded and nonabraded skin sites. CONCLUSIONS: In a rabbit model the standard surgical preparation using povidone-iodine at six hours after inoculation is effective in reducing the bacterial count on abraded skin to that of surgically prepared nonabraded skin. Beyond that time, the standard surgical preparation is ineffective in reducing counts to those of nonabraded skin at similar time intervals.
Assuntos
Desinfecção das Mãos/normas , Pele/lesões , Pele/microbiologia , Staphylococcus aureus , Infecção da Ferida Cirúrgica/microbiologia , Ferimentos e Lesões/cirurgia , Animais , Contagem de Colônia Microbiana , Procedimentos Cirúrgicos Dermatológicos , Modelos Animais de Doenças , Estudos de Avaliação como Assunto , Feminino , Incidência , Masculino , Estudos Prospectivos , Coelhos , Valores de Referência , Sensibilidade e Especificidade , Infecção da Ferida Cirúrgica/epidemiologiaRESUMO
Staphylococcus aureus invades osteoblasts and is the primary cause of osteomyelitis. This study examined the ability of S. aureus to induce apoptosis in a mouse osteoblast cell line. The presence of intracellular S. aureus was demonstrated by transmission electron microscopy. Light microscopy was utilized to examine morphological changes in the osteoblasts following killing of extracellular bacteria. Cell rounding was observed, and dark centers due to condensation of chromatin were noted in cells in infected osteoblast cultures. DNA was isolated from infected osteoblast cultures, and electrophoresis revealed the laddering effect characteristic of cells undergoing apoptosis. Additionally, an in situ cell death detection assay was utilized to label apoptosis-induced DNA strand breaks. Apoptotic nuclei were present, providing further evidence that S. aureus induces apoptosis in osteoblasts.
Assuntos
Apoptose , Osteoblastos/microbiologia , Staphylococcus aureus/fisiologia , Staphylococcus aureus/patogenicidade , Células 3T3 , Animais , Fragmentação do DNA , Camundongos , Osteoblastos/citologia , Osteoblastos/ultraestruturaRESUMO
Staphlylococcus aureus is the primary pathogen associated with osteomyelitis, an acute and recurrent bone disease. Internalization of S. aureus by cultured embryonic chick calvarial osteoblasts has been observed. The purpose of this study was to demonstrate that internalization of bacteria by embryonic chick calvarial and tibial osteoblasts occurs in vivo. In initial experiments, 10(8) colony forming units (cfu) of S. aureus, strain UAMS-1 or Cowan 1, were injected subcutaneously under the scalp skin of 17 day chick embryos. After 45 min, calvariae were harvested and processed for transmission electron microscopy (TEM). In subsequent experiments, 10(9) cfu of UAMS-1 were injected into the allantoic sac of 17 day chick embryos via a small opening in the egg shell. After 48 h, calvariae and tibiae were harvested for TEM. S. aureus cells were found in approximately 14% of the calvarial osteoblasts after subcutaneous injection and in 11% of calvarial and tibial osteoblasts following intraallantoic injection. Endosomes were observed in some cells, but most bacteria internalized appeared to be free in the cytoplasm. Osteoblasts with as few as five bacteria had a greater loss of cytoplasmic integrity and a more heterochromatic nucleus than osteoblasts with fewer bacteria or than uninfected osteoblasts. S. aureus cells in calvariae and tibiae were also observed in the cytoplasm of approximately 4% of the osteocytes in mineralized bone matrix. Thus, internalization of S. aureus by osteoblasts in vivo augments the previous observation in vitro. This study has also shown that osteoblasts with few bacteria continue differentiating into osteocytes. Results of these experiments support the hypothesis that internalization of S. aureus by osteoblasts may play a role in the etiology of osteomyelitis.
Assuntos
Osteoblastos/microbiologia , Staphylococcus aureus/fisiologia , Alantoide/microbiologia , Animais , Células Cultivadas , Embrião de Galinha , Microscopia Eletrônica , Osteoblastos/ultraestrutura , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Staphylococcus aureus is the principal causative agent of the inflammatory bone disease osteomyelitis. Unfortunately, the pathogenesis of this often chronic infection is poorly understood and is complicated by the recent observation that bone-forming osteoblasts can harbor S. aureus. Such an infection presents a significant challenge for the host immune response, because osteoblasts are not known to initiate protective cell-mediated immune responses. Cultured mouse and human osteoblasts infected with S. aureus were found to express high levels of interleukin (IL)-6 and IL-12p75, on the basis of complementary investigations demonstrating both S. aureus-induced up-regulation of expression of IL-6 and IL-12p40 mRNA and secretion of IL-6 and IL-12p75 by these cells. Additionally, a quantitative bioassay demonstrated that IL-12p75 secreted after infection was biologically active. These studies are the first to demonstrate induced IL-12p75 expression by osteoblasts and suggest a previously unrecognized role for osteoblasts in initiating immune responses after S. aureus infection.
Assuntos
Interleucina-12/biossíntese , Interleucina-6/biossíntese , Osteoblastos/imunologia , Osteoblastos/microbiologia , Staphylococcus aureus/imunologia , Animais , Células Cultivadas , Humanos , Cinética , Camundongos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Staphylococcus aureus is a frequent cause of orthopedic infections in humans. The bacterium expresses several adhesins that facilitate bacterial binding to the bone matrix and to bone implant biomaterials coated with host plasma constituents. The relevant S. aureus adhesins are termed microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and specific MSCRAMMs are involved in bone and joint infections.
Assuntos
Adesinas Bacterianas/fisiologia , Aderência Bacteriana , Doenças Ósseas Infecciosas/microbiologia , Matriz Óssea/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Adesinas Bacterianas/genética , Animais , Materiais Biocompatíveis , Substitutos Ósseos , Humanos , Staphylococcus aureus/genéticaRESUMO
Staphylococcus aureus is a bacterial pathogen causing approximately 80% of all cases of human osteomyelitis. This bacterium can adhere to and become internalized by osteoblasts and previous studies indicate that osteoblasts are active in the internalization process. In the current study, we examined the roles of microfilaments, microtubules and clathrin-dependent receptor-mediated endocytosis in the internalization of S. aureus by MC3T3-E1 mouse osteoblast cells. Microfilament and microtubule polymerization was inhibited with cytochalasin D and colchicine. Clathrin-coated pit formation was examined by using the transaminase inhibitor, monodanslycadaverine. The results of this study indicate that mouse osteoblasts utilize actin microfilaments, microtubules and clathrin-coated pits in the internalization of S. aureus; however, microfilaments seem to play the most significant role in the invasion process.
Assuntos
Osteoblastos/microbiologia , Staphylococcus aureus/fisiologia , Animais , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Linhagem Celular , Sobrevivência Celular , Colchicina/farmacologia , Citocalasina D/farmacologia , Dimetil Sulfóxido/farmacologia , Gentamicinas/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologiaRESUMO
Intracellular polysaccharides (IPS) are glycogen-like storage polymers which contribute significantly to Streptococcus mutans-induced cariogenesis. We previously identified and cloned a locus from the S. mutans chromosome which is required for the accumulation of IPS. Sequencing of this locus revealed at least four contiguous open reading frames, all of which are preceded by a common promoter region and are transcribed in the same direction. Analysis of the amino acid sequence deduced from the first of these open reading frames (ORF1) revealed domains which are highly conserved among D-alanine-activating enzymes (DltA) in Lactobacillus rhamnosus (formerly Lactobacillus casei) and Bacillus subtilis. The deduced amino acid sequences derived from ORF2, -3, and -4 also exhibit extensive similarity to DltB, -C, and -D, respectively, in these microorganisms. However, Southern hybridization experiments indicate that this operon maps to a locus on the S. mutans chromosome which is separate from the glgP, glgA, and glgD genes, whose products are known mediators of bacterial IPS accumulation. We therefore assigned a new dlt designation to the locus which we had formerly called glg. We maintain that the dlt genes are involved in S. mutans IPS accumulation, however, since they complement a mutation in trans which otherwise renders S. mutans IPS deficient. In this study, we found that expression of the S. mutans dlt genes is growth phase dependent and is modulated by carbohydrates internalized via the phosphoenolpyruvate phosphotransferase system (PTS). We demonstrated that the S. mutans dlt genes are expressed constitutively when non-PTS sugars are provided as the sole source of carbohydrate. Consistent with a role for the PTS in dlt expression is a similar constitutive expression of the dlt genes in an S. mutans PTS mutant grown in a chemically defined medium supplemented with glucose. In summary, these findings support a novel role for the dlt gene products in S. mutans IPS accumulation and suggest that dlt expression in this oral pathogen is subject to complex mechanisms of control imposed by growth phase, dietary carbohydrate, and other factors present in the plaque environment.
Assuntos
Regulação Bacteriana da Expressão Gênica , Glicogênio/biossíntese , Óperon , Polissacarídeos Bacterianos/biossíntese , Streptococcus mutans/genética , Sequência de Aminoácidos , Sequência de Bases , Cárie Dentária/etiologia , Carboidratos da Dieta/farmacologia , Genes Bacterianos , Humanos , Dados de Sequência Molecular , RNA Bacteriano/análise , RNA Mensageiro/análise , Análise de Sequência de DNA , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/metabolismo , Streptococcus mutans/patogenicidadeRESUMO
The risk of bacteremia secondary to high pressure lavage of contaminated wounds was assessed. Twenty canines were divided randomly into four treatment groups. A 10-cm incision was made over the left shoulder of each dog. The deltoideus muscle was disrupted and traumatized. Groups A and B (n = 8) had wound contamination with 1.4 x 10(9) Staphylococcus aureus followed 75 minutes later by high pressure lavage or bulb syringe irrigation, respectively. Groups C and D (n = 2) had no contamination, followed by the same treatment. Bacterial counts were obtained before and after wound irrigation. Blood cultures were obtained before, during, and 15 minutes after irrigation. Positive control cultures were obtained during injection of bacteria into the antecubital vein. A detectable bacteremia did not occur during or after high pressure lavage or bulb syringe irrigation of acute contaminated wounds but did occur in 18 of 20 positive controls. Bacterial levels were reduced by an average of 70% +/- 10% by high pressure lavage and 44% +/- 50% by bulb irrigation. Reduction of wound bacteria was achieved more consistently with high pressure lavage than with bulb syringe irrigation.
Assuntos
Bacteriemia/etiologia , Irrigação Terapêutica/efeitos adversos , Infecção dos Ferimentos/terapia , Animais , Modelos Animais de Doenças , Cães , Estudos de Avaliação como Assunto , Pressão , Distribuição Aleatória , Irrigação Terapêutica/métodos , Infecção dos Ferimentos/complicaçõesRESUMO
Results from this laboratory have shown that bone metabolism is directly related to extracellular pH and that high concentrations of tobramycin released from impregnated polymethylmethacryrate (PMMA) beads has pH-dependent toxic effects on bone. In the present study, beneficial effects of calcium hydroxide-impregnated PMMA were investigated regarding tobramycin toxicity and bone metabolism in chick embryo tibiae in vitro. Also using Ca(OH)2 as a pH regulator, the antibiotic efficacy of tobramycin-impregnated PMMA was evaluated with respect to inhibition of Staphylococcus aureus growth. When Ca(OH)2 was added to PMMA beads containing tobramycin, the beads released hydroxyl and calcium ions into the culture medium and released more antibiotic than beads containing only tobramycin. Bone metabolism (glycolysis, total protein synthesis, and collagen synthesis) was enhanced by Ca(OH)2-impregnated beads with or without tobramycin. Additionally, bacterial growth was inhibited more strongly when S. aureus was incubated with tobramycin- and Ca(OH)2-impregnated PMMA disks than with disks containing only tobramycin. This study demonstrates the feasibility of adding Ca(OH)2 to tobramycin-impregnated PMMA beads as a regulator of local pH and a promoter of bone metabolism for protection of bone when high concentrations of tobramycin are used to treat osteomyelitis. It also suggests that lower concentrations of antibiotic may be effective if Ca(OH)2 and tobramycin are administered simultaneously.
Assuntos
Antibacterianos/toxicidade , Hidróxido de Cálcio/farmacologia , Cálcio/metabolismo , Tíbia/efeitos dos fármacos , Tobramicina/toxicidade , Animais , Embrião de Galinha , Meios de Cultura , Portadores de Fármacos , Concentração de Íons de Hidrogênio , Técnicas de Cultura de Órgãos , Osteomielite/tratamento farmacológico , Polimetil Metacrilato , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Tíbia/embriologia , Tíbia/metabolismo , Tobramicina/antagonistas & inibidoresRESUMO
In vivo expression of the virulence-associated fructosyltransferase gene (ftf) of Streptococcus mutans has been examined. S. mutans ftf expression is affected by both the specific carbohydrate consumed and the age of the host animal.
Assuntos
Regulação Enzimológica da Expressão Gênica , Hexosiltransferases/genética , Streptococcus mutans/genética , Fatores Etários , Animais , Carboidratos/farmacologia , Feminino , Masculino , Ratos , Ratos Sprague-Dawley , Fatores Sexuais , Streptococcus mutans/enzimologiaRESUMO
A fluoride-resistant Streptococcus mutans isolate NCH105 was characterized and compared with wild-type strain UA130. The growth and lactic acid production of strain NCH105 were found to be unaffected by the presence of fluoride at the initial medium pH values of 6.5 and 6. In addition, NCH105 was found to be capable of lowering the pH of the medium to approximately 5.5, which is the critical level where enamel demineralization begins in vivo. Lactic acid production, glucose uptake at pH 6.5 and 6, as well as ATPase activity at pH 5 were found to be unaffected by fluoride. Finally, strain NCH105 is capable of binding as well as the wild-type bacterium to artificial tooth pellicles. These results are unusual when compared with previously isolated fluoride-resistant mutants and suggest that NCH105 may have the ability to colonize the tooth surface and initiate dental caries.
Assuntos
Fluoretos/farmacologia , Streptococcus mutans/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Cárie Dentária/microbiologia , Resistência a Medicamentos , Glucose/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lactatos/metabolismo , Ácido Láctico , Streptococcus mutans/isolamento & purificação , Streptococcus mutans/metabolismoRESUMO
Staphylococcus aureus is the most common cause of both acute and chronic osteomyelitis; however, the pathogenesis of osteomyelitis is poorly understood. We investigated the ability of S. aureus to associate with chick osteoblasts in culture and have demonstrated internalization of bacteria by the osteoblasts. Two strains of S. aureus were examined that were ingested by osteoblasts to different extents, suggesting strain differences in uptake. Initial association of S. aureus strains with osteoblasts was independent of the presence of matrix collagen produced by the osteoblasts. Internalization of bacteria required live osteoblasts, but not live S. aureus, indicating osteoblasts are active in ingesting the organisms. The bacteria were not killed by the osteoblasts, since viable bacteria were cultured several hours after ingestion.
Assuntos
Osteoblastos/microbiologia , Staphylococcus aureus/patogenicidade , Animais , Aderência Bacteriana , Células Cultivadas , Galinhas , EndocitoseRESUMO
Three glucosyltransferases (GTFs), which catalyze the formation of water-insoluble adherent glucans, and fructosyltransferase (FTF), which synthesizes fructans, are believed to contribute to the pathogenic potential of Streptococcus mutans. Study of the regulation of expression of GTF and FTF has been difficult because of the complexity and number of exoenzymes produced by this bacterium. By using continuous chemostat culture to control environmental conditions, chloramphenicol acetyltransferase (CAT) operon fusions were utilized to measure transcriptional activity of the ftf and gtfBC gene promoters. Expression of these operon fusions was differentially regulated in response to culture pH and growth rate and during transition states between growth domains. Furthermore, the addition of sucrose to steady-state cultures resulted in significant increases in CAT specific activities for both fusions. In a few cases, GTF and FTF enzyme specific activities did not parallel those of the corresponding CAT fusion activities; this lack of correspondence was likely due to posttranscriptional events controlling enzyme secretion and enzyme activity, as well as to the differential expression of dextranase(s) and fructan hydrolase by S. mutans. These results clearly demonstrate that the extracellular polymer synthesis machinery of S. mutans is regulated in a complex manner. The use of operon fusions in combination with chemostat culture is a viable approach to analyzing gene expression in S. mutans and will be helpful in defining the molecular mechanisms underlying regulation of expression of virulence attributes under conditions that may more closely mimic those in dental plaque.