RESUMO
While many strains of Enterococcus faecalis have been reported to be capable of surviving within macrophages for extended periods, the exact mechanisms involved are largely unknown. In this study, we found that after phagocytosis by macrophages, enterococci-containing vacuoles resist acidification, and E. faecalis is resistant to low pH. Ultrastructural examination of the enterococci-containing vacuole by transmission electron microscopy revealed a single membrane envelope, with no evidence of the classical double-membraned autophagosomes. Western blot analysis further confirmed that E. faecalis could trigger inhibition of the production of LC3-II during infection. By employing cells transfected with RFP-LC3 plasmid and infected with GFP-labelled E. faecalis, we also observed that E. faecalis was not delivered into autophagosomes during macrophage infection. While these observations indicated no role for autophagy in elimination of intracellular E. faecalis, enhanced production of reactive oxygen species and nitric oxide were keys to this process. Stimulation of autophagy suppressed the intracellular survival of E. faecalis in macrophages in vitro and decreased the burden of E. faecalis in vivo. In summary, the results from this study offer new insights into the interaction of E. faecalis with host cells and may provide a new approach to treatment of enterococcal infections.
Assuntos
Enterococcus faecalis/patogenicidade , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Animais , Autofagia , Enterococcus faecalis/fisiologia , Concentração de Íons de Hidrogênio , Lisossomos , Camundongos , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Óxido Nítrico/metabolismo , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Vacúolos/metabolismo , Vacúolos/microbiologia , Vacúolos/ultraestruturaRESUMO
Apoptosis is an intrinsic immune defense mechanism in the host response to microbial infection. Not surprisingly, many pathogens have evolved various strategies to manipulate this important pathway to benefit their own survival and dissemination in the host during infection. To our knowledge, no attempts have been made to explore the host cell survival signals modulated by the bacterium Enterococcus faecalis. Here, we show for the first time that during early stages of infection, internalized enterococci can prevent host cell (RAW264.7 cells, primary macrophages, and mouse embryonic fibroblasts [MEFs]) apoptosis induced by a wide spectrum of proapoptotic stimuli. Activation of caspase 3 and cleavage of the caspase 3 substrate poly(ADP-ribose) polymerase were inhibited in E. faecalis-infected cells, indicating that E. faecalis protects macrophages from apoptosis by inhibiting caspase 3 activation. This antiapoptotic activity in E. faecalis-infected cells was dependent on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which resulted in the increased expression of the antiapoptotic factor Bcl-2 and decreased expression of the proapoptotic factor Bax. Further analysis revealed that active E. faecalis physiology was important for inhibition of host cell apoptosis, and this feature seemed to be a strain-independent trait among E. faecalis isolates. Employing a mouse peritonitis model, we also determined that cells collected from the peritoneal lavage fluid of E. faecalis-infected mice showed reduced levels of apoptosis compared to cells from uninfected mice. These results show early modulation of apoptosis during infection and have important implications for enterococcal pathogenesis.
Assuntos
Apoptose , Enterococcus faecalis/imunologia , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Macrófagos/fisiologia , Fosfatidilinositol 3-Quinase/metabolismo , Transdução de Sinais , Animais , Caspase 3/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/enzimologia , Fibroblastos/microbiologia , Fibroblastos/fisiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/patologia , Evasão da Resposta Imune , Macrófagos/enzimologia , Camundongos , Peritonite/microbiologia , Peritonite/patologiaRESUMO
EXECUTIVE SUMMARY The 2020-2021 AACP Research and Graduate Affairs Committee (RGAC) continued the work begun by the 2019-2020 RGAC to increase awareness of and capacity for implementation research to advance practice transformation in academic pharmacy. AACP President Anne Lin charged the RGAC with developing resources and programs for training faculty and graduate students in implementation science. The committee was further charged with developing a mechanism to pair pharmacy faculty and implementation experts on practice advancement projects. In its work, the committee focused on generating near-term opportunities for pharmacy practice faculty to pursue projects while developing programs that would support ongoing career development and future implementation practice and research by pharmacy faculty and trainees.
Assuntos
Educação em Farmácia , Farmácia , Docentes de Farmácia , Humanos , Ciência da Implementação , Faculdades de FarmáciaRESUMO
BACKGROUND: Coccidioidomycosis or Valley fever is caused by a highly virulent fungal pathogen: Coccidioides posadasii or immitis. Vaccine development against Coccidioides is of contemporary interest because a large number of relapses and clinical failures are reported with antifungal agents. An efficient Th1 response engenders protection. Thus, we have focused on developing a dendritic cell (DC)-based vaccine for coccidioidomycosis. In this study, we investigated the immunostimulatory characteristics of an intranasal primary DC-vaccine in BALB/c mouse strain that is most susceptible to coccidioidomycosis. The DCs were transfected nonvirally with Coccidioides-Ag2/PRA-cDNA. Expression of DC-markers, Ag2/PRA and cytokines were studied by flow cytometry, dot-immunoblotting and cytometric bead array methods, respectively. The T cell activation was studied by assessing the upregulation of activation markers in a DC-T cell co-culture assay. For trafficking, the DCs were co-transfected with a plasmid DNA encoding HSV1 thymidine kinase (TK) and administered intranasally into syngeneic mice. The trafficking and homing of TK-expressing DCs were monitored with positron emission tomography (PET) using 18F-FIAU probe. Based on the PET-probe accumulation in vaccinated mice, selected tissues were studied for antigen-specific response and T cell phenotypes using ELISPOT and flow cytometry, respectively. RESULTS: We found that the primary DCs transfected with Coccidioides-Ag2/PRA-cDNA were of immature immunophenotype, expressed Ag2/PRA and activated naïve T cells. In PET images and subsequent biodistribution, intranasally-administered DCs were found to migrate in blood, lung and thymus; lymphocytes showed generation of T effector memory cell population (T(EM)) and IFN-γ release. CONCLUSIONS: In conclusion, our results demonstrate that the intranasally-administered primary DC vaccine is capable of inducing Ag2/PRA-specific T cell response. Unique approaches utilized in our study represent an attractive and novel means of producing and evaluating an autologous DC-based vaccine.
Assuntos
Células Dendríticas/imunologia , Vacinas Fúngicas/administração & dosagem , Vacinas Fúngicas/imunologia , Imunização/métodos , Administração Intranasal , Animais , Arabinofuranosiluracila/análogos & derivados , Arabinofuranosiluracila/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Forma Celular , Células Cultivadas , Coccidioides/imunologia , Citocinas/metabolismo , DNA Complementar/genética , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Epitopos/imunologia , Memória Imunológica , Imunofenotipagem , Ativação Linfocitária/imunologia , Camundongos , Modelos Animais , Imagem Molecular , Plasmídeos/genética , Timidina Quinase/metabolismo , Distribuição Tecidual , Transfecção , Transgenes/genéticaRESUMO
Enterococcus faecalis has emerged as a prominent healthcare-associated pathogen frequently encountered in bacteremia, endocarditis, urinary tract infection, and as a leading cause of antibiotic-resistant infections. We recently demonstrated a capacity for high-level biofilm formation by a clinical E. faecalis isolate, E99. This high biofilm-forming phenotype was attributable to a novel locus, designated bee, specifying a pilus at the bacterial cell surface and localized to a large approximately 80 kb conjugative plasmid. To better understand the origin of the bee locus, as well as to potentially identify additional factors important to the biology and pathogenesis of strain E99, we sequenced the entire plasmid. The nucleotide sequence of the plasmid, designated pBEE99, revealed large regions of identity to the previously characterized conjugative plasmid pCF10. In addition to the bee locus, pBEE99 possesses an open reading frame potentially encoding aggregation substance, as well as open reading frames putatively encoding polypeptides with 60% to 99% identity at the amino acid level to proteins involved in regulation of the pheromone response and conjugal transfer of pCF10. However, strain E99 did not respond to the cCF10 pheromone in clumping assays. While pBEE99 was found to be devoid of any readily recognizable antibiotic resistance determinants, it carries two non-identical impB/mucB/samB-type genes, as well as genes potentially encoding a two-component bacteriocin similar to that encoded on pYI14. Although no bacteriocin activity was detected from an OG1RF transconjugant carrying pBEE99 against strain FA2-2, it was approximately an order of magnitude more resistant to ultraviolet radiation. Moreover, curing strain E99 of this plasmid significantly reduced its ability to survive UV exposure. Therefore, pBEE99 represents a novel conjugative plasmid that confers biofilm-forming and enhanced UV resistance traits that might potentially impact the virulence and/or fitness of E. faecalis.
Assuntos
Conjugação Genética/efeitos da radiação , Enterococcus faecalis/genética , Enterococcus faecalis/efeitos da radiação , Plasmídeos/genética , Tolerância a Radiação/genética , Raios Ultravioleta , Bacteriocinas/farmacologia , Sequência de Bases , Conjugação Genética/efeitos dos fármacos , Enterococcus faecalis/efeitos dos fármacos , Oligopeptídeos/genética , Fases de Leitura Aberta/genética , Feromônios/genética , Mapeamento Físico do Cromossomo , Tolerância a Radiação/efeitos da radiaçãoRESUMO
Enterococcus faecalis is a leading cause of nosocomial infections and is known for its ability to acquire and transfer virulence and antibiotic resistance determinants from other organisms. A 150-kb pathogenicity island (PAI) encoding several genes that contribute to pathogenesis was identified among antibiotic-resistant clinical isolates. In the current study, we examined the structure of the PAI in a collection of isolates from diverse sources in order to gain insight into its genesis and dynamics. Using multilocus sequence typing to assess relatedness at the level of strain background and microarray analysis to identify variations in the PAI, we determined the extent to which structural variations occur within the PAI and also the extent to which these variations occur independently of the chromosome. Our findings provide evidence for a modular gain of defined gene clusters by the PAI. These results support horizontal transfer as the mechanism for accretion of genes into the PAI and highlight a likely role for mobile elements in the evolution of the E. faecalis PAI.
Assuntos
Enterococcus faecalis/genética , Evolução Molecular , Variação Genética/genética , Ilhas Genômicas/genética , Enterococcus faecalis/classificação , Transferência Genética Horizontal/genética , Família Multigênica/genética , Análise de Sequência com Séries de Oligonucleotídeos , FilogeniaRESUMO
A gene encoding a putative AraC-type transcriptional regulator was identified on the 153-kb pathogenicity island (PAI) found among virulent Enterococcus faecalis strains. In an effort to understand the function of this regulator, designated PerA (for pathogenicity island-encoded regulator), we first examined the expression of the perA gene in the original PAI strain MMH594 and in an unrelated clinical isolate E99 by reverse transcription-PCR. Interestingly, expression analysis revealed no detectable perA transcript in MMH594, whereas a transcript was observed in strain E99. Nucleotide sequence analysis revealed that this altered expression between the two strains was attributable to the differential location of an IS1191 element within the putative promoter region upstream of the perA gene. In order to determine the role of this putative regulator in E. faecalis pathogenesis, a perA-deficient mutant was created in strain E99, and the wild-type and mutant pair were compared for phenotypic differences. In in vitro biofilm assays, the mutant strain showed a significantly higher level of growth medium-specific biofilm formation compared to the wild type. However, in a murine intraperitoneal infection model, the mutant strain was significantly less pathogenic. The mutant was also attenuated for survival within macrophages in vitro. These findings highlight the importance of PerA as a regulator of biofilm formation and survival within macrophages and is likely a regulator controlling determinants important to pathogenesis.
Assuntos
Fator de Transcrição AraC/genética , Enterococcus faecalis/genética , Enterococcus faecalis/patogenicidade , Ilhas Genômicas/genética , Infecções por Bactérias Gram-Positivas/genética , Animais , Sequência de Bases , Biofilmes , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Macrófagos/microbiologia , Macrófagos/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: To better understand the response of urinary epithelial (urothelial) cells to Enterococcus faecalis, a uropathogen that exhibits resistance to multiple antibiotics, a genome-wide scan of gene expression was obtained as a time series from urothelial cells growing as a layered 3-dimensional culture similar to normal urothelium. We herein describe a novel means of analysis that is based on deconvolution of gene variability into technical and biological components. RESULTS: Analysis of the expression of 21,521 genes from 30 minutes to 10 hours post infection, showed 9553 genes were expressed 3 standard deviations (SD) above the system zero-point noise in at least 1 time point. The asymmetric distribution of relative variances of the expressed genes was deconvoluted into technical variation (with a 6.5% relative SD) and biological variation components (>3 SD above the mode technical variability). These 1409 hypervariable (HV) genes encapsulated the effect of infection on gene expression. Pathway analysis of the HV genes revealed an orchestrated response to infection in which early events included initiation of immune response, cytoskeletal rearrangement and cell signaling followed at the end by apoptosis and shutting down cell metabolism. The number of poorly annotated genes in the earliest time points suggests heretofore unknown processes likely also are involved. CONCLUSION: Enterococcus infection produced an orchestrated response by the host cells involving several pathways and transcription factors that potentially drive these pathways. The early time points potentially identify novel targets for enhancing the host response. These approaches combine rigorous statistical principles with a biological context and are readily applied by biologists.
Assuntos
Mapeamento Cromossômico/métodos , Enterococcus faecalis/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Proteoma/fisiologia , Urotélio/metabolismo , Urotélio/microbiologia , Células Cultivadas , Células Epiteliais/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Variação Genética/genética , Humanos , Biologia de Sistemas/métodos , Urotélio/citologiaRESUMO
Enterococcal surface protein (Esp) is encoded on a pathogenicity island in Enterococcus faecalis and E. faecium and is involved in biofilm formation and binding to epithelial cells. In this study, using Esp-expressing E. faecalis MMH594 and its isogenic Esp-deficient strain, as well as purified Esp, we show that Esp is sufficient for activation of NF-κB and the subsequent production of pro-inflammatory cytokines IL-1ß and TNF-α in macrophages in vitro. In a mouse peritonitis model, we also show that mice infected with Esp-expressing E. faecalis showed comparatively higher levels of cytokines TNF-α, IL-1ß and IL-6 in peritoneal fluid, and IL-6 in serum. Moreover, neutrophil infiltration and tissue damage in the liver was higher in the mice infected with the Esp-expressing strain compared with mice infected with the Esp-deficient mutant. These results add Esp to the growing list of enterococcal virulence factors that can modulate inflammation during infection and has implications for enterococcal pathogenesis.
Assuntos
Proteínas de Bactérias/metabolismo , Enterococcus faecalis/imunologia , Infecções por Bactérias Gram-Positivas/imunologia , Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Peritonite/imunologia , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Proteínas de Membrana/genética , Camundongos , NF-kappa B/metabolismo , RNA Interferente Pequeno/genética , Deleção de Sequência/genética , Fatores de VirulênciaRESUMO
Enterococcal surface protein (Esp) is a cell wall-associated protein of Enterococcus faecalis that has been identified as a potential virulence factor. We used a mouse model to examine whether Esp facilitates intestinal colonization or translocation of E. faecalis to mesenteric lymph nodes. After clindamycin treatment, similar levels of high-density colonization were established after orogastric inoculation of an E. faecalis isolate containing the esp gene within a large pathogenicity island and an isogenic mutant created by allelic replacement of the esp gene with a chloramphenicol resistance cassette (P=0.7); translocation to mesenteric lymph nodes was detected in 3 of 12 (25%) mice in both groups. Isogenic mutants of FA2-2 (a plasmid-free derivative of E. faecalis strain JH2) with or without the esp gene failed to establish colonization of clindamycin-treated mice. These results suggest that Esp does not facilitate intestinal colonization or translocation of E. faecalis.
Assuntos
Proteínas de Bactérias/fisiologia , Clindamicina/farmacologia , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/patogenicidade , Linfonodos/microbiologia , Proteínas de Membrana/fisiologia , Animais , Clindamicina/uso terapêutico , Modelos Animais de Doenças , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , CamundongosRESUMO
Enterococcus faecalis is a commensal bacterium residing in the gastrointestinal tract of mammals, but in certain situations it is also an opportunistic pathogen which can cause serious disease. Macrophages have been shown to play a critical role in controlling infections by commensal enterococci and also have an important role in mediating chromosomal instability and promoting colon cancer during high-level enterococcal colonization in genetically susceptible mice. However, the molecular mechanisms involved in the interaction of macrophages with enterococci during infection are not fully understood. In this study, using BMDM and RAW264.7 macrophages we show that enterococcal infection activates ERK, JNK and p38 MAPK as well as NF-κB, and drives polarization of macrophages towards the M1 phenotype. Inhibition of NF-κB activation significantly reduced the expression of TNF-α and IL-1ß, as did the inhibition of ERK, JNK and p38 MAPK, although to differing extent. Enterococci-induced activation of these pathways and subsequent cytokine expression was contact dependent, modest compared to activation by E. coli and, required the adaptor protein MyD88. Phagocytosis of enterococci by macrophages was enhanced by preopsonization with E. faecalis antiserum and involved the ERK and JNK signaling pathways, with the adaptor protein MyD88 as an important mediator. This study of the interaction of macrophages with enterococci could provide a foundation for studying the pathogenesis of infection by this opportunistic pathogen and to developing new therapeutic approaches to combat enterococcal infection.
Assuntos
Citocinas/metabolismo , Enterococcus faecalis/patogenicidade , Infecções por Bactérias Gram-Positivas/imunologia , Macrófagos/fisiologia , Fator 88 de Diferenciação Mieloide/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Modelos Animais de Doenças , Enterococcus faecalis/imunologia , Enterococcus faecalis/isolamento & purificação , Infecções por Bactérias Gram-Positivas/genética , Infecções por Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Sistema de Sinalização das MAP Quinases , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , NF-kappa B/metabolismo , FagocitoseRESUMO
The potential virulence factors of enterococci include production of enterococcal surface protein (Esp), gelatinase, and hemolysin. Gelatinase- and hemolysin-producing strains of Enterococcus faecalis have been shown to be virulent in animal models of enterococcal infections. Esp production has been shown to enhance the persistence of E. faecalis in the urinary bladder. We determined the presence of the esp gene and production of gelatinase and hemolysin in 219 E. faecalis isolates from a larger prospective study of 398 patients with enterococcal bacteremia. Thirty-two percent of isolates carried the esp gene, 64% produced gelatinase, and 11% produced hemolysin. There was no significant association between 14-day mortality and any of the markers studied, singly or in combination.
Assuntos
Bacteriemia/mortalidade , Proteínas de Bactérias/metabolismo , Enterococcus faecalis/patogenicidade , Gelatinases/metabolismo , Proteínas Hemolisinas/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Bacteriemia/diagnóstico , Bacteriemia/metabolismo , Bacteriemia/microbiologia , Resistência a Medicamentos , Enterococcus faecalis/efeitos dos fármacos , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mortalidade , VirulênciaRESUMO
Twenty-nine Enterococcus faecalis isolates from patients with endocarditis or bacteremia or from stools of healthy volunteers were investigated for their ability to adhere to Int-407 and Girardi heart cell lines and for the presence of known enterococcal virulence factors. Eight strains (27.6%) adhered predominantly to Int-407 cells. The adherence of enterococci was enhanced by proteolytic digestion, suggesting that some cell binding components become surface-exposed after treatment with trypsin. The occurrence of known potential virulence factors of enterococci among these strains was determined and was as follows: enterococcal surface protein (72.4%), gelatinase (58.6%), aggregation substance (48.3%) and cytolysin (17.2%). Bacterial adherence was not significantly associated with any of these virulence factors.
Assuntos
Bacteriemia/microbiologia , Aderência Bacteriana , Endocardite Bacteriana/microbiologia , Enterococcus faecalis/patogenicidade , Fezes/microbiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Enterococcus faecalis/fisiologia , Humanos , Tripsina/metabolismo , VirulênciaRESUMO
Toll-like receptor signaling, mediated by functional Toll/interleukin-1 receptor (TIR) domains, plays a critical role in activating the innate immune response responsible for controlling and clearing infection. Bacterial protein mimics of components of this signaling pathway have been identified and function through inhibition of interactions between Toll-like receptors (TLRs) and their adaptor proteins, mediated by TIR domains. A previously uncharacterized gene, which we have named tcpF (for TIR domain-containing protein in E. faecalis) was identified in the genome of Enterococcus faecalis V583, and predicted to encode a protein resembling mammalian and bacterial TIR proteins. We overexpressed and purified TcpF from E. coli and found that the recombinant protein could bind to phosphatidylinositol phosphates in vitro, suggesting a mechanism by which TcpF may be anchored to the plasma membrane in close proximity to TIR domains of TLRs and adaptor proteins. Purified TcpF was also found to interact specifically with the TIR adaptor protein MyD88, and this interaction was dependent on the BB loop domain in the Box 2 region of TcpF. Despite no evidence of TcpF being a secreted protein, recombinant TcpF was effectively able to enter RAW264.7 cells in vitro although the mechanism by which this occurs remains to be determined. Overexpression of TcpF in mammalian cells suppressed the NF-κB activation induced by bacterial lipoteichoic acid. A mutant lacking the tcpF gene was attenuated for survival in macrophages, with increased ability to activate NF-κB compared to the wild type strain. Complementation in trans restored growth, and inhibition of NF-κB, to that of wild type levels. No appreciable difference in bacterial persistence, dissemination or pathogenesis was observed between the wild type and mutant in a mouse peritonitis model however, which suggested either a subtle role for TcpF or functional overlap with other redundant factor(s) in this virulence model.
Assuntos
Proteínas de Bactérias/fisiologia , Enterococcus faecalis/fisiologia , Fator 88 de Diferenciação Mieloide/fisiologia , NF-kappa B/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Linhagem Celular , Feminino , Infecções por Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Interações Hospedeiro-Patógeno , Lipopolissacarídeos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Dados de Sequência Molecular , Peritonite/metabolismo , Peritonite/microbiologia , Fagocitose , Estrutura Terciária de Proteína , Transdução de Sinais , Ácidos TeicoicosRESUMO
Enterococcus faecalis is a commensal organism of the gastrointestinal tract but can also cause serious opportunistic infections. In addition to high levels of antibiotic resistance, the ability to form biofilms on abiotic surfaces and on in-dwelling devices within the host complicates treatment strategies and successful outcomes of antibiotic therapy. Despite rapid advances made in recent years in understanding the genomics and virulence of this organism, much remains to be learned regarding the host response to enterococcal infections. In this study, we investigated the interaction of RAW264.7 macrophages and JAWS II dendritic cells with biofilm and planktonic E. faecalis, in vitro. Specifically, we compared phagocytosis, intracellular survival, secretion of proinflammatory cytokines, and the activation and maturation of phagocytes. Our results revealed that both macrophages and dendritic cells phagocytize biofilm mode cells at levels equal to or better than their planktonic counterparts. Internalized biofilm bacteria showed relatively greater survival at 24 h in macrophages than in dendritic cells and led to slightly higher expression of phagocyte activation markers. Macrophages infected with biofilm cells also secreted lower levels of proinflammatory cytokines studied. Overall, these results suggest that biofilm E. faecalis may be better adapted to overcome host defenses in vivo.
Assuntos
Biofilmes/crescimento & desenvolvimento , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Enterococcus faecalis/imunologia , Enterococcus faecalis/fisiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Animais , Linhagem Celular , Citocinas/metabolismo , Camundongos , Viabilidade MicrobianaRESUMO
Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections, traits facilitated by the ability to quickly acquire and transfer virulence determinants. A 150 kb pathogenicity island (PAI) comprised of genes contributing to virulence is found in many enterococcal isolates and is known to undergo horizontal transfer. We have shown that the PAI-encoded transcriptional regulator PerA contributes to pathogenicity in the mouse peritonitis infection model. In this study, we used whole-genome microarrays to determine the PerA regulon. The PerA regulon is extensive, as transcriptional analysis showed 151 differentially regulated genes. Our findings reveal that PerA coordinately regulates genes important for metabolism, amino acid degradation, and pathogenicity. Further transcriptional analysis revealed that PerA is influenced by bicarbonate. Additionally, PerA influences the ability of E. faecalis to bind to human platelets. Our results suggest that PerA is a global transcriptional regulator that coordinately regulates genes responsible for enterococcal pathogenicity.
Assuntos
Biofilmes , Plaquetas/metabolismo , Enterococcus faecalis/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas/genética , Infecções por Bactérias Gram-Positivas/genética , Regulon , Animais , Biomarcadores/metabolismo , Enterococcus faecalis/patogenicidade , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Peritonite/genética , Peritonite/microbiologia , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVES: To determine which basic and social science courses academic pharmacy administrators believe should be required for entry into the professional pharmacy program and what they believe should be the required length of preprofessional study. METHODS: An online survey was sent to deans of all colleges and schools of pharmacy in the United States. Survey respondents were asked to indicate their level of agreement as to whether the basic and social science courses listed in the survey instrument should be required for admission to the professional program. The survey instrument also included queries regarding the optimal length of preprofessional study, whether professional assessment testing should be part of admission requirements, and the respondents' demographic information. RESULTS: The majority of respondents strongly agreed that the fundamental coursework in the basic sciences (general biology, general chemistry, organic chemistry) and English composition should be required for entrance into the professional program. Most respondents also agreed that public speaking, ethics, and advanced basic science and math courses (physiology, biochemistry, calculus, statistics) should be completed prior to entering the professional program. The preprofessional requirements that respondents suggested were not necessary included many of the social science courses. Respondents were evenly divided over the ideal length for preprofessional pharmacy education programs. CONCLUSIONS: Although requirements for preprofessional admission have been changing, there is no consistent agreement on the content or length of the preprofessional program.
Assuntos
Academias e Institutos , Educação de Pós-Graduação em Farmácia , Docentes , Percepção , Critérios de Admissão Escolar , Faculdades de Farmácia , Ciências do Comportamento , Disciplinas das Ciências Biológicas , Química Farmacêutica , Teste de Admissão Acadêmica , Currículo , Correio Eletrônico , Guias como Assunto , Humanos , Matemática , Administração Farmacêutica , Ciências Sociais , Inquéritos e Questionários , Fatores de Tempo , Estados UnidosRESUMO
Enterococcus faecalis, a leading cause of nosocomial antibiotic resistant infections, frequently possesses a 150 kb pathogenicity island (PAI) that carries virulence determinants. The presence of excisionase and integrase genes, conjugative functions and multiple insertion sequence elements suggests that the PAI, or segments thereof, might be capable of horizontal transfer. In this report, the transfer of the E. faecalis PAI is demonstrated and a mechanism for transfer elucidated. In filter matings, chloramphenicol resistance was observed to transfer from strain MMH594b, a clinical isolate possessing the PAI tagged with a cat marker, to OG1RF (pCGC) with a frequency of 3.2 x 10(-10) per donor. Secondary transfer from primary transconjugant TCRFB1 to strain JH2SS in filter and broth matings occurred with a frequency of 1 and 2 x 10(-1) per donor respectively. Analysis of the transconjugants demonstrated that a 27,744 bp internal PAI segment was capable of excision and circularization in the donor, and is mobilized as a cointegrate with a pTEF1-like plasmid. High-frequency transfer also occurred from TCRFB1 to JH2SS during transient colonization of the mouse gastrointestinal tract. This is the first demonstration of the horizontal transfer of PAI-encoded virulence determinants in E. faecalis and has implications for genome evolution and diversity.
Assuntos
Proteínas de Bactérias/genética , Enterococcus faecalis/genética , Transferência Genética Horizontal , Ilhas Genômicas/genética , Fatores de Virulência/genética , Animais , Resistência ao Cloranfenicol/genética , Conjugação Genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Enterococcus faecalis/fisiologia , Trato Gastrointestinal/microbiologia , Genes Reporter , Camundongos , Modelos Animais , Plasmídeos/genética , Recombinação Genética , Estômago/microbiologiaRESUMO
Enterococci are opportunistic pathogens and among the leading causes of nosocomial infections. Enterococcus faecalis, the dominant species among infection-derived isolates, has recently been recognized as capable of forming biofilms on abiotic surfaces in vitro as well as on indwelling medical devices. A few bacterial factors known to contribute to biofilm formation in E. faecalis have been characterized. To identify additional factors which may be important to this process, we utilized a Tn917-based insertional mutagenesis strategy to generate a mutant bank in a high-biofilm-forming E. faecalis strain, E99. The resulting mutant bank was screened for mutants exhibiting a significantly reduced ability to form biofilms. One mutant, P101D12, which showed greater than 70% reduction in its ability to form biofilms compared to the wild-type parent, was further characterized. The single Tn917 insertion in P101D12 was mapped to a gene, bee-2, encoding a probable cell wall-anchored protein. Sequence information for the region flanking bee-2 revealed that this gene was a member of a locus (termed the bee locus for biofilm enhancer in enterococcus) comprised of five genes encoding three putative cell wall-anchored proteins and two probable sortases. Contour-clamped homogeneous electric field gel and Southern hybridization analyses suggested that the bee locus is likely harbored on a large conjugative plasmid. Filter mating assays using wild-type E99 or mutant P101D12 as a donor confirmed that the bee locus could transfer conjugally at high frequency to recipient E. faecalis strains. This represents the first instance of the identification of a mobile genetic element conferring biofilm-forming property in E. faecalis.
Assuntos
Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Enterococcus faecalis/fisiologia , Proteínas de Membrana/fisiologia , Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Southern Blotting , Mapeamento Cromossômico , Conjugação Genética , Cisteína Endopeptidases , Elementos de DNA Transponíveis , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Ágar , Enterococcus faecalis/genética , Transferência Genética Horizontal , Genes Bacterianos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Fenótipo , Plasmídeos/genética , Homologia de Sequência de AminoácidosRESUMO
Enterococcus faecalis isolates of porcine origin were screened for the presence of a previously identified pathogenicity island (PAI). By using the esp gene as a genetic marker for the presence of this PAI, 9 esp-positive and 10 esp-negative isolates of porcine origin were investigated by use of a designed oligonucleotide array. The results indicated the clustering of esp-positive strains by multilocus sequence typing (MLST), but surprisingly, all strains investigated contained parts of the PAI. None of the strains of animal origin investigated belonged to previously identified MLST complex 2, where most isolates from patients cluster. Five of the nine esp-positive E. faecalis isolates of animal origin belonged to the same PAI complex as human isolate MMH594 but differed in their sequence types, which strongly indicates the horizontal transfer of the PAI between enterococci of porcine and human origin.