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1.
Nat Commun ; 10(1): 3060, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31311921

RESUMO

Control of Streptococcus pneumoniae colonisation at human mucosal surfaces is critical to reducing the burden of pneumonia and invasive pneumococcal disease, interrupting transmission, and achieving herd protection. Here, we use an experimental human pneumococcal carriage model (EHPC) to show that S. pneumoniae colonisation is associated with epithelial surface adherence, micro-colony formation and invasion, without overt disease. Interactions between different strains and the epithelium shaped the host transcriptomic response in vitro. Using epithelial modules from a human epithelial cell model that recapitulates our in vivo findings, comprising of innate signalling and regulatory pathways, inflammatory mediators, cellular metabolism and stress response genes, we find that inflammation in the EHPC model is most prominent around the time of bacterial clearance. Our results indicate that, rather than being confined to the epithelial surface and the overlying mucus layer, the pneumococcus undergoes micro-invasion of the epithelium that enhances inflammatory and innate immune responses associated with clearance.


Assuntos
Portador Sadio/imunologia , Nasofaringe/imunologia , Infecções Pneumocócicas/imunologia , Mucosa Respiratória/imunologia , Streptococcus pneumoniae/imunologia , Adulto , Portador Sadio/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Voluntários Saudáveis , Humanos , Imunidade Inata , Masculino , Pessoa de Meia-Idade , Nasofaringe/microbiologia , Infecções Pneumocócicas/microbiologia , Mucosa Respiratória/microbiologia , Streptococcus pneumoniae/patogenicidade , Adulto Jovem
2.
Vet Microbiol ; 235: 80-85, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282382

RESUMO

Bovine respiratory disease complex is a major disease affecting the global cattle industry. Multiple infections by viruses and bacteria increase disease severity. Previously, we reported that bovine respiratory syncytial virus (BRSV) infection increases adherence of Pasteurella multocida to human respiratory and bovine kidney epithelial cells. To examine the interaction between the virus and bacteria in bovine respiratory cells, we generated respiratory epithelial cell lines from bovine trachea (bTEC), bronchus (bBEC), and lung (bLEC). Although all established cell lines were infected by BRSV and P. multocida susceptibility differed according to site of origin. The cells derived from the lower respiratory tract (bBEC and bLEC) were significantly more susceptible to BRSV than those derived from the upper respiratory tract (bTEC). Pre-infection of bBEC and bLEC with BRSV increased adherence of P. multocida; this was not the case for bTEC. These results indicate that BRSV may reproduce better in the lower respiratory tract and encourage adherence of bacteria. Thus, we identify one possible mechanism underlying severe pneumonia.


Assuntos
Coinfecção/veterinária , Células Epiteliais , Interações Microbianas , Infecções por Pasteurella/veterinária , Infecções por Vírus Respiratório Sincicial/veterinária , Animais , Complexo Respiratório Bovino/microbiologia , Complexo Respiratório Bovino/virologia , Brônquios/citologia , Brônquios/microbiologia , Brônquios/virologia , Bovinos , Linhagem Celular , Células Cultivadas , Coinfecção/microbiologia , Coinfecção/virologia , Citocinas/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/virologia , Pulmão/citologia , Pulmão/microbiologia , Pulmão/virologia , Infecções por Pasteurella/virologia , Pasteurella multocida/genética , Pasteurella multocida/isolamento & purificação , Infecções por Vírus Respiratório Sincicial/microbiologia , Vírus Sincicial Respiratório Bovino/genética , Vírus Sincicial Respiratório Bovino/isolamento & purificação , Traqueia/citologia , Traqueia/microbiologia , Traqueia/virologia
3.
Cell Physiol Biochem ; 53(1): 49-61, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169991

RESUMO

BACKGROUND/AIMS: The most prevalent infectious disease, chronic periodontitis which leads to alveolar bone destruction and subsequent tooth loss, develops due to proinflammatory cytokine production induced by periodontopathic bacteria. Chronic obstructive pulmonary disease (COPD), a non-infectious disease, is the third leading cause of death globally. This condition exacerbates frequently, and which is attributable to proinflammatory cytokine production induced by infection by respiratory microorganisms such as Streptococcus pneumoniae. Although a positive association has recently been revealed between chronic periodontitis and COPD, how periodontitis contributes to the pathogenesis of COPD remains unclear. Therefore, we hypothesized that some periodontopathic bacteria are involved in the exacerbation of COPD through the induction of proinflammatory cytokine production by respiratory epithelial cells. In this connection, COPD develops in the airways; however, because most periodontopathic bacteria are anaerobic, they are unlikely to exhibit stable virulence in the lower respiratory organs in humans. Hence, we aimed to elucidate whether exposure to heat-inactivated periodontopathic bacteria induces proinflammatory cytokine production by several human respiratory epithelial cell lines and in the lower respiratory organs and serum in mice. METHODS: Real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) were used to investigate in vitro induction by heat-inactivated periodontopathic bacteria and S. pneumoniae for mRNA expression and protein production of interleukin (IL)-8 and IL-6 by human respiratory epithelial cell lines. ELISA was also used to determine in vivo induction of cytokine production in the lower respiratory organs and serum of intratracheally heat-inactivated Fusobacterium nucleatum-inoculated mice. RESULTS: Some, but not all, periodontopathic bacteria, especially F. nucleatum, strongly induced IL-8 and IL-6 production by BEAS-2B bronchial epithelial cells. In addition, F. nucleatum induced IL-8 production by A549 alveolar epithelial cells as well as IL-8 and IL-6 production by Detroit 562 pharyngeal epithelial cells. Furthermore, F. nucleatum induced considerably higher cytokine production than S. pneumoniae. This was also observed in the entire lower respiratory organs and serum in mice. CONCLUSION: Exposure to increased number of F. nucleatum potentially induces proinflammatory cytokine production by human bronchial and pharyngeal epithelial cells, which may trigger exacerbation of COPD.


Assuntos
Fusobacterium nucleatum/patogenicidade , Interleucina-6/metabolismo , Sistema Respiratório/microbiologia , Animais , Brônquios/citologia , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Interleucina-6/sangue , Interleucina-6/genética , Interleucina-8/sangue , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sistema Respiratório/metabolismo , Streptococcus pneumoniae/patogenicidade
4.
Pol J Microbiol ; 68(2): 217-224, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250592

RESUMO

Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus, such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus. The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus, a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus, such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus. The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus, a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.


Assuntos
Infecções por Campylobacter/microbiologia , Campylobacter fetus/fisiologia , Endocitose , Células Epiteliais/microbiologia , Actinas/metabolismo , Animais , Antibacterianos/farmacologia , Bovinos , Doenças dos Bovinos , Células Cultivadas , Gentamicinas/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Confocal , Microscopia de Fluorescência , Modelos Biológicos
5.
J Dairy Sci ; 102(8): 6802-6819, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31202650

RESUMO

The process of fermentation contributes to the organoleptic properties, preservation, and nutritional benefits of food. Fermented food may interfere with pathogen infections through a variety of mechanisms, including competitive exclusion or improving intestinal barrier integrity. In this study, the effect of milk fermented with Lactococcus lactis ssp. cremoris JFR1 on Salmonella invasion of intestinal epithelial cell cultures was investigated. Epithelial cells (HT29-MTX, Caco-2, and cocultures of the 2) were treated for 1 h with Lactococcus lactis ssp. cremoris JFR1 fermented milk before infection with Salmonella enterica ssp. enterica Typhimurium. Treatment with fermented milk resulted in increased transepithelial electrical resistance, which remained constant for the duration of infection (up to 3 h), illustrating a protective effect. After gentamicin treatment to remove adhered bacterial cells, enumeration revealed a reduction in numbers of intracellular Salmonella. Quantitative reverse-transcription PCR data indicated a downregulation of Salmonella virulence genes hilA, invA, and sopD after treatment with fermented milk. Fermented milk treatment of epithelial cells also exhibited an immunomodulatory effect reducing the production of proinflammatory IL-8. In contrast, chemically acidified milk (glucono delta-lactone) failed to show the same effect on monolayer integrity, Salmonella Typhimurium invasion, and gene expression as well as immune modulation. Furthermore, an oppA knockout mutant of Salmonella Typhimurium infecting treated epithelial cells did not show suppressed virulence gene expression. Collectively, these results suggest that milk fermented with Lactococcus lactis ssp. cremoris JFR1 is effective in vitro in the reduction of Salmonella invasion into intestinal epithelial cells. A functional OppA permease in Salmonella is required to obtain the antivirulence effect of fermented milk.


Assuntos
Produtos Fermentados do Leite , Fermentação , Intestinos/microbiologia , Lactococcus lactis/metabolismo , Leite/fisiologia , Salmonella typhimurium/fisiologia , Animais , Reatores Biológicos , Células CACO-2 , Células Epiteliais/microbiologia , Expressão Gênica , Humanos , Fatores Imunológicos , Intestinos/citologia , Ácido Láctico/metabolismo , Leite/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Fatores de Virulência/genética
6.
Vet Res ; 50(1): 49, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221210

RESUMO

An ethanolic extract from Rhodomyrtus tomentosa leaves (RTL) was studied as a natural alternative to control Staphylococcus aureus, which is an important pathogen responsible for bovine mastitis. The minimal inhibitory concentrations (MICs) of the RTL extract and of rhodomyrtone, a pure compound isolated from the plant, were determined by a microdilution method. Rhodomyrtone and the RTL extract exhibited antibacterial activity against S. aureus, including its persistent phenotype (SCV: small-colony variant) and a biofilm hyperproducer strain, with MICs of 0.25-0.5 and 8-16 µg/mL, respectively. Time-kill kinetics showed a strong bactericidal activity for both the RTL extract- and rhodomyrtone-treated bacteria at 2 × MIC as early as 4 h post-exposure. An additive effect of the extract at 0.5 × MIC was observed in a combination with oxytetracycline or pirlimycin against S. aureus by showing a 64- to 128-fold reduction in antibiotic MICs. Moreover, the RTL extract significantly decreased the number of intracellular SCVs inside bovine mammary epithelial cells. However, the extract or its combination with pirlimycin only slightly improved the activity of pirlimycin against the bacterial colonization of mouse mammary glands. In vitro MICs determined in the presence of casein indicated that the limited activity of the RTL extract in the murine model of mastitis could be linked to neutralization of active components by milk proteins. While the RTL extract showed interesting antibacterial properties in vitro, to be considered as an alternative to antibiotics in dairy farms, formulation studies are needed to cope with the observed reduction of activity in vivo.


Assuntos
Antibacterianos/farmacologia , Mastite Bovina/tratamento farmacológico , Myrtaceae/química , Extratos Vegetais/farmacologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/efeitos dos fármacos , Animais , Bovinos , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Mastite Bovina/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Xantonas/farmacologia
7.
Microbiol Immunol ; 63(8): 293-302, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31209914

RESUMO

Antimicrobial peptides play important roles in the innate immune system of various organisms, and they may also be considered to prevent the organisms from infections. In particular, ß-defensins, mainly produced in epithelial cells, are recognized as one of the major antimicrobial peptides in mammals, including humans. In this study, we showed that Lactobacillus helveticus SBT2171 (LH2171), one of the several species of lactic acid bacteria, upregulates the production of ß-defensins in oral epithelial cells in vitro. Moreover, LH2171 reduced the increase of proinflammatory cytokine expression, induced by Porphyromonas gingivalis stimulation, in gingival epithelial cells. These data suggested that LH2171 suppresses P. gingivalis-induced inflammation by upregulating the expression of ß-defensins in gingival epithelial cells. We subsequently investigated the effects of LH2171 in vivo and revealed that ß-defensin expression was increased in the oral cavities of LH2171-fed mice. Furthermore, LH2171 decreased alveolar bone loss, gingival inflammation, and amounts of P. gingivalis-specific 16S ribosomal RNA in the gingiva of P. gingivalis-inoculated mice. Taken together, our results showed that LH2171 upregulates the expression of ß-defensins in oral cavity, thereby decreasing the number of P. gingivalis consequently ameliorating the experimental periodontal disease.


Assuntos
Células Epiteliais/metabolismo , Lactobacillus helveticus/fisiologia , Doenças Periodontais/prevenção & controle , Porphyromonas gingivalis/efeitos dos fármacos , Regulação para Cima , beta-Defensinas/metabolismo , beta-Defensinas/farmacologia , Perda do Osso Alveolar/prevenção & controle , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Feminino , Gengiva/metabolismo , Gengiva/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Doenças Periodontais/microbiologia , Porphyromonas gingivalis/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/genética
8.
World J Microbiol Biotechnol ; 35(6): 85, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134456

RESUMO

Surface properties like hydrophobicity, aggregation ability, adhesion to mucosal surfaces and epithelial cells and transit time are key features for the characterization of probiotic strains. In this study, we used two Lactobacillus paracasei subsp. paracasei strains (BGNJ1-64 and BGSJ2-8) strains which were previously described with very strong aggregation capacity. The aggregation promoting factor (AggLb) expressed in these strains showed high level of binding to collagen and fibronectin, components of extracellular matrix. The working hypothesis was that strains able to aggregate have an advantage to resist in intestinal tract. So, we assessed whether these strains and their derivatives (without aggLb gene) are able to bind or not to intestinal components and we compared the transit time of each strains in mice. In that purpose parental strains (BGNJ1-64 and BGSJ2-8) and their aggregation negative derivatives (BGNJ1-641 and BGSJ2-83) were marked with double antibiotic resistance in order to be tracked in in vivo experiments in mice. Comparative analysis of binding ability of WT and aggregation negative strains to different human intestinal cell lines and mucin revealed no significant difference among them, excluding involvement of AggLb in interaction with surface of intestinal cells and mucin. In vivo experiments showed that surviving and transit time of marked strains in mice did not drastically depend on the presence of the AggLb aggregation factor.


Assuntos
Moléculas de Adesão Celular/metabolismo , Células Epiteliais/microbiologia , Intestinos/microbiologia , Lactobacillus paracasei/crescimento & desenvolvimento , Lactobacillus paracasei/fisiologia , Ligação Proteica , Animais , Aderência Bacteriana/fisiologia , Células CACO-2 , Moléculas de Adesão Celular/fisiologia , Colágeno/metabolismo , Fibronectinas/metabolismo , Células HT29 , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucinas/metabolismo , Probióticos , Análise de Onda de Pulso , Propriedades de Superfície
9.
J Med Microbiol ; 68(6): 940-951, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31107199

RESUMO

PURPOSE: This study aimed to characterize 82 atypical enteropathogenic Escherichia coli (aEPEC) isolates, obtained from patients with diarrhea in Brazil, regarding their adherence patterns on HeLa cells and attaching and effacing (AE) lesion pathways. METHODOLOGY: The adherence and fluorescence-actin staining (FAS) assays were performed using HeLa cells. AE lesion pathways were determined through the detection of tyrosine residue 474 (Y474) phosphorylation in the Tir protein, after its translocation to host cells, and by PCR assays for tir genotyping and detection of Tir-cytoskeleton coupling protein (tccP) genes. RESULTS: Regarding the adherence pattern, determined in the presence of d-mannose, 12 isolates (14.6 %) showed the localized adherence (LA)-like pattern, 3 (3.7  %) the aggregative adherence pattern and 4 (4.9  %) a hybrid LA/diffuse adherence pattern. In addition, 36 (43.9  %) isolates displayed an undefined adherence, and 26 (31.7  %) were non-adherent (NA), while one (1.2 %) caused cell detachment. Among the 26 NA aEPEC isolates, 11 showed a type 1 pilus-dependent adherence in assays performed without d-mannose, while 15 remained NA. Forty-eight (58.5 %) aEPEC were able to trigger F-actin accumulation underneath adherent bacteria (FAS-positive), which is an important feature of AE lesions. The majority (58.3 %) of these used the Tir-Nck pathway, while 39.6  % may use both Tir-Nck and Tir-TccP pathways to induce AE lesions. CONCLUSION: Our results reveal the diversity of strategies used by aEPEC isolates to interact with and damage epithelial host cells, thereby causing diarrheal diseases.


Assuntos
Aderência Bacteriana , Escherichia coli Enteropatogênica/fisiologia , Infecções por Escherichia coli/microbiologia , Interações Hospedeiro-Patógeno , Actinas/metabolismo , Diarreia/microbiologia , Escherichia coli Enteropatogênica/genética , Escherichia coli Enteropatogênica/isolamento & purificação , Células Epiteliais/microbiologia , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/metabolismo , Genótipo , Células HeLa , Humanos , Fenótipo , Fosforilação , Receptores de Superfície Celular/metabolismo
10.
Nat Commun ; 10(1): 2297, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127085

RESUMO

Candida albicans is a fungal pathobiont, able to cause epithelial cell damage and immune activation. These functions have been attributed to its secreted toxin, candidalysin, though the molecular mechanisms are poorly understood. Here, we identify epidermal growth factor receptor (EGFR) as a critical component of candidalysin-triggered immune responses. We find that both C. albicans and candidalysin activate human epithelial EGFR receptors and candidalysin-deficient fungal mutants poorly induce EGFR phosphorylation during murine oropharyngeal candidiasis. Furthermore, inhibition of EGFR impairs candidalysin-triggered MAPK signalling and release of neutrophil activating chemokines in vitro, and diminishes neutrophil recruitment, causing significant mortality in an EGFR-inhibited zebrafish swimbladder model of infection. Investigation into the mechanism of EGFR activation revealed the requirement of matrix metalloproteinases (MMPs), EGFR ligands and calcium. We thus identify a PAMP-independent mechanism of immune stimulation and highlight candidalysin and EGFR signalling components as potential targets for prophylactic and therapeutic intervention of mucosal candidiasis.


Assuntos
Candida albicans/imunologia , Proteínas Fúngicas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Sacos Aéreos/microbiologia , Animais , Candida albicans/genética , Candida albicans/metabolismo , Candidíase/imunologia , Candidíase/microbiologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Receptores ErbB/genética , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/imunologia , Metaloproteinases da Matriz/imunologia , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Membrana Mucosa/imunologia , Membrana Mucosa/microbiologia , Faringite/imunologia , Faringite/microbiologia , Fosforilação , Peixe-Zebra
11.
MBio ; 10(3)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088918

RESUMO

Candida yeasts are common commensals that can cause mucosal disease and life-threatening systemic infections. While many of the components required for defense against Candida albicans infection are well established, questions remain about how various host cells at mucosal sites assess threats and coordinate defenses to prevent normally commensal organisms from becoming pathogenic. Using two Candida species, C. albicans and C. parapsilosis, which differ in their abilities to damage epithelial tissues, we used traditional methods (pathogen CFU, host survival, and host cytokine expression) combined with high-resolution intravital imaging of transparent zebrafish larvae to illuminate host-pathogen interactions at the cellular level in the complex environment of a mucosal infection. In zebrafish, C. albicans grows as both yeast and epithelium-damaging filaments, activates the NF-κB pathway, evokes proinflammatory cytokines, and causes the recruitment of phagocytic immune cells. On the other hand, C. parapsilosis remains in yeast morphology and elicits the recruitment of phagocytes without inducing inflammation. High-resolution mapping of phagocyte-Candida interactions at the infection site revealed that neutrophils and macrophages attack both Candida species, regardless of the cytokine environment. Time-lapse monitoring of single-cell gene expression in transgenic reporter zebrafish revealed a partitioning of the immune response during C. albicans infection: the transcription factor NF-κB is activated largely in cells of the swimbladder epithelium, while the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) is expressed in motile cells, mainly macrophages. Our results point to different host strategies for combatting pathogenic Candida species and separate signaling roles for host cell types.IMPORTANCE In modern medicine, physicians are frequently forced to balance immune suppression against immune stimulation to treat patients such as those undergoing transplants and chemotherapy. More-targeted therapies designed to preserve immunity and prevent opportunistic fungal infection in these patients could be informed by an understanding of how fungi interact with professional and nonprofessional immune cells in mucosal candidiasis. In this study, we intravitally imaged these host-pathogen dynamics during Candida infection in a transparent vertebrate model host, the zebrafish. Single-cell imaging revealed an unexpected partitioning of the inflammatory response between phagocytes and epithelial cells. Surprisingly, we found that in vivo cytokine profiles more closely match in vitro responses of epithelial cells rather than phagocytes. Furthermore, we identified a disconnect between canonical inflammatory cytokine production and phagocyte recruitment to the site of infection, implicating noncytokine chemoattractants. Our study contributes to a new appreciation for the specialization and cross talk among cell types during mucosal infection.


Assuntos
Candidíase/imunologia , Citocinas/imunologia , Células Epiteliais/imunologia , Imunidade Celular , Microscopia Intravital , Macrófagos/microbiologia , Animais , Candida albicans , Candida parapsilosis , Candidíase/microbiologia , Células Epiteliais/microbiologia , Imunidade nas Mucosas , Larva/microbiologia , Fagócitos/imunologia , Fagócitos/microbiologia , Análise de Célula Única , Peixe-Zebra/microbiologia
12.
Curr Top Microbiol Immunol ; 421: 209-227, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31123891

RESUMO

The ability of Helicobacter pylori to persist lifelong in the human gastric mucosa is a striking phenomenon. It is even more surprising since infection is typically associated with a vivid inflammatory response. Recent studies revealed the mechanism by which this pathogen inhibits the epithelial responses to IFN-γ and other central inflammatory cytokines in order to abolish an effective antimicrobial defense. The mechanism is based on the modification and depletion of cholesterol by the pathogen's cholesterol-α-glucosyltransferase. It abrogates the assembly of numerous cytokine receptors due to the reduction of lipid rafts. Particularly, the receptors for IFN-γ, IL-22, and IL-6 then fail to assemble properly and to activate JAK/STAT signaling. Consequently, cholesterol depletion prevents the release of antimicrobial peptides, including the highly effective ß-defensin-3. Intriguingly, the inhibition is spatially restricted to heavily infected cells, while the surrounding epithelium continues to respond normally to cytokine stimulation, thus providing a platform of the intense inflammation typically observed in H. pylori infections. It appears that pathogen and host establish a homeostatic balance between tightly colonized and rather inflamed sites. This homeostasis is influenced by the levels of available cholesterol, which potentially exacerbate H. pylori-induced inflammation. The observed blockage of epithelial effector mechanisms by H. pylori constitutes a convincing explanation for the previous failures of T-cell-based vaccination against H. pylori, since infected epithelial cells remain inert upon stimulation by effector cytokines. Moreover, the mechanism provides a rationale for the carcinogenic action of this pathogen in that persistent infection and chronic inflammation represent a pro-carcinogenic environment. Thus, cholesterol-α-glucosyltransferase has been revealed as a central pathogenesis determinant of H. pylori.


Assuntos
Colesterol/deficiência , Infecções por Helicobacter/sangue , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Colesterol/metabolismo , Células Epiteliais/microbiologia , Mucosa Gástrica/microbiologia , Glucosiltransferases/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/enzimologia , Humanos
13.
Int J Oncol ; 54(6): 2200-2210, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31081048

RESUMO

Helicobacter pylori (HP) is a pathogenic bacterium associated with chronic gastritis, gastric ulcer and gastric cancer. In the present study, the primary carcinogenesis process of normal gastric epithelial cells (GES­1) infected with HP was investigated. It was determined that infected gastric mucosal epithelial GES­1 cells secreted increased interleukin­8 (IL­8) and IL­23, and exhibited enhanced expression of inducible nitric oxide synthase and cyclooxygenase­2, inducing inflammatory reactions and resulting in apoptosis. The bacterial infection significantly increased the expression of carcinogenesis­associated genes, including p16, c­Myc, p53 and p21, as well as the expression of cell surface signaling molecules cluster of differentiation 44 (CD44) and CD54 in GES­1 cells or tissues of patients with gastritis and gastric cancer in vitro or in vivo. Simultaneously, the migration and invasion abilities of normal gastric epithelial GES­1 cells were increased following HP infection. These observations demonstrated that the inflammatory response of HP infection could cause normal gastric epithelial cells to undergo significant cancerous reactions, indicating that HP is a risk factor for gastric cancer.


Assuntos
Infecções por Helicobacter/complicações , Helicobacter pylori/imunologia , Neoplasias Gástricas/microbiologia , Estômago/citologia , Linhagem Celular , Movimento Celular , Células Epiteliais/citologia , Células Epiteliais/microbiologia , Infecções por Helicobacter/imunologia , Humanos , Interleucina-23/metabolismo , Interleucina-8/metabolismo , Invasividade Neoplásica , Óxido Nítrico Sintase Tipo II/metabolismo , Estômago/microbiologia , Estômago/patologia , Neoplasias Gástricas/imunologia
14.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30953429

RESUMO

Shigella is a genus of Gram-negative enteropathogens that have long been, and continue to be, an important public health concern worldwide. Over the past several decades, Shigella spp. have also served as model pathogens in the study of bacterial pathogenesis, and Shigella flexneri has become one of the best-studied pathogens on a molecular, cellular, and tissue level. In the arms race between Shigella and the host immune system, Shigella has developed highly sophisticated mechanisms to subvert host cell processes in order to promote infection, escape immune detection, and prevent bacterial clearance. Here, we give an overview of Shigella pathogenesis while highlighting innovative techniques and methods whose application has significantly advanced our understanding of Shigella pathogenesis in recent years.


Assuntos
Disenteria Bacilar/imunologia , Interações Hospedeiro-Patógeno/imunologia , Shigella/imunologia , Shigella/patogenicidade , Imunidade Adaptativa , Adesinas Bacterianas , Proteínas de Bactérias , Citosol/microbiologia , Disenteria Bacilar/microbiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Microbioma Gastrointestinal , Humanos , Evasão da Resposta Imune , Shigella flexneri/imunologia , Shigella flexneri/patogenicidade , Sistemas de Secreção Tipo III , Virulência , Fatores de Virulência/metabolismo
15.
Vet Microbiol ; 231: 154-159, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30955803

RESUMO

Pigs suffer enteritis induced by pathogenic bacteria infection and toxins in the moldy feed, which cause intestinal epithelial damage and diarrhea through the whole breeding cycle. Interleukin-22 (IL-22) plays a critical role in maintaining intestinal mucosal barrier function through repairing intestinal epithelial damage. However, little was known about the effects of IL-22 against apoptosis caused by toxins and infection of intestinal pathogens in the intestinal epithelium, especially in pigs. In this study, we had successfully used prokaryotic expression system to produce recombinant porcine interleukin-22. Meanwhile, purified rIL-22 could activate STAT3 signal pathway and have been demonstrated to be safe to IPEC-J2 cells by increasing E-cadherin expression, without proinflammatory cytokines changes. Furthermore, rIL-22 reversed apoptosis induced by deoxynivalenol (DON) and played a vital part in repairing the intestinal injury. We also found that rIL-22 stimulated epithelial cells to secrete pBD-1 against enterotoxigenic E. coli (ETEC) K88 infection, as well as alleviating apoptosis ratio. This study provided a theoretical basis for curing intestinal inflammation caused by ETEC infection and epithelial apoptosis induced by DON with rIL-22 in pigs.


Assuntos
Escherichia coli Enterotoxigênica/patogenicidade , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Interleucinas/farmacologia , Tricotecenos/efeitos adversos , Animais , Peptídeos Catiônicos Antimicrobianos/biossíntese , Apoptose , Linhagem Celular , Escherichia coli Enterotoxigênica/efeitos dos fármacos , Inflamação , Interleucinas/imunologia , Mucosa Intestinal/citologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Suínos
16.
Vet Microbiol ; 232: 22-29, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31030841

RESUMO

Chlamydia (C.) pecorum is an obligate intracellular bacterium that infects and causes disease in a broad range of animal hosts. Molecular studies have revealed that this pathogen is genetically diverse with certain isolates linked to different disease outcomes. Limited in vitro or in vivo data exist to support these observations, further hampering efforts to improve our understanding of C. pecorum pathogenesis. In this study, we evaluated whether genetically distinct C. pecorum isolates (IPA, E58, 1710S, W73, JP-1-751) display different in vitro growth phenotypes in different mammalian epithelial and immune cells. In McCoy cells, shorter lag phases were observed for W73 and JP-1-751 isolates. Significantly smaller inclusions were observed for the naturally plasmid-free E58 isolate. C. pecorum isolates of bovine (E58) and ovine origin (IPA, W73, JP-1-751) grew faster in bovine cells compared to a porcine isolate (1710S). C. pecorum isolates could infect but appear not able to complete their developmental cycle in bovine peripheral neutrophil granulocytes. All isolates, except 1710S, could multiply in bovine monocyte-derived macrophages. These results reveal potentially important phenotypic differences that will help to understand the pathogenesis of C. pecorum in vivo and to identify C. pecorum virulence factors.


Assuntos
Chlamydia/crescimento & desenvolvimento , Chlamydia/genética , Células Epiteliais/microbiologia , Granulócitos/microbiologia , Animais , Bovinos , Variação Genética , Camundongos , Filogenia , Células RAW 264.7 , Ovinos , Suínos
17.
Nat Commun ; 10(1): 1826, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015451

RESUMO

The bacterial pathogen Shigella flexneri causes 270 million cases of bacillary dysentery (blood in stool) worldwide every year, resulting in more than 200,000 deaths. A major challenge in combating bacillary dysentery is the lack of a small-animal model that recapitulates the symptoms observed in infected individuals, including bloody diarrhea. Here, we show that similar to humans, infant rabbits infected with S. flexneri experience severe inflammation, massive ulceration of the colonic mucosa, and bloody diarrhea. T3SS-dependent invasion of epithelial cells is necessary and sufficient for mediating immune cell infiltration and vascular lesions. However, massive ulceration of the colonic mucosa, bloody diarrhea, and dramatic weight loss are strictly contingent on the ability of the bacteria to spread from cell to cell. The infant rabbit model features bacterial dissemination as a critical determinant of S. flexneri pathogenesis and provides a unique small-animal model for research and development of therapeutic interventions.


Assuntos
Diarreia/patologia , Disenteria Bacilar/patologia , Hemorragia Gastrointestinal/patologia , Shigella flexneri/patogenicidade , Sistemas de Secreção Tipo III/imunologia , Animais , Animais Recém-Nascidos/microbiologia , Colo/microbiologia , Colo/patologia , Diarreia/microbiologia , Modelos Animais de Doenças , Disenteria Bacilar/microbiologia , Células Epiteliais/microbiologia , Feminino , Hemorragia Gastrointestinal/microbiologia , Células HT29 , Humanos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Gravidez , Coelhos
18.
Helicobacter ; 24(3): e12583, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30950121

RESUMO

BACKGROUND: Lack of a model that mirrors Helicobacter pylori-induced gastric mucosal inflammation has hampered investigation of early host-bacterial interactions. We used an ex vivo model of human stomach, gastric epithelial organoid monolayers (gastroid monolayers) to investigate interactions of H pylori infection and the apical junctional complex and interleukin-8 (IL-8) expression. METHOD: Morphology of human antral mucosal gastroid monolayers was evaluated using histology, immunohistochemical (IHC) staining, and transmission electron microscopy (TEM). Functional and gross changes in the apical junctional complexes were assessed using transepithelial electrical resistance (TEER), cytotoxicity assays, and confocal laser scanning microscopy. IL-8 expression was evaluated by real-time quantitative PCR and ELISA. RESULTS: When evaluated by IHC and TEM, the morphology of gastroid monolayers closely resembled in vivo human stomach. Following inoculation of H pylori, TEER transiently declined (up to 51%) in an H pylori density-dependent manner. TEER recovered by 48 hours post-infection and remained normal despite continued presence and replication of H pylori. Confocal scanning microscopy showed minimal disruption of zonula occludens-1 or E-cadherin structure. IL-8 production was unchanged by infection with either CagA-positive or CagA-negative H pylori and JNK and MEK inhibitors did not suppress IL-8 production, whereas p38 and IKK inhibitor significantly did. CONCLUSION: Human gastroid monolayers provide a model for experimental H pylori infection more consistent with in vivo human infections than seen with typical gastric epithelial cell lines. This ex vivo system should lead to better understanding of H pylori host-pathogen interactions.


Assuntos
Gastrite/patologia , Infecções por Helicobacter/patologia , Helicobacter pylori/fisiologia , Interações Hospedeiro-Patógeno , Interleucina-8/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Gastrite/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Humanos , Inflamação/microbiologia , Mutação , Estômago/microbiologia , Estômago/patologia , Junções Íntimas/metabolismo , Junções Íntimas/patologia
19.
Clin Lab ; 65(4)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30969081

RESUMO

BACKGROUND: Acinetobacter baumannii (Ab) is an important opportunistic pathogen in a hospital setting. The virulence varies among different clinical isolates, which can be measured by cytotoxicity assay or Galleria mellonella killing assay. However, these measurements are not suitable for routine clinical laboratory application. Thus, a putative marker for early prediction and intervention of these virulent isolates is desirable. METHODS: PgaB is part of the pgaABCD cluster, which is considered to be essential for the biofilm formation and pathogenesis of Ab. Sixty-five non-repetitive Ab clinical isolates were randomly collected from respiratory tract specimens in our hospital, which were pgaB positive as determined by PCR. The mRNA expression level of pgaB was measured by quantitative real-time PCR, with Taqman probe targeting the conserved region of pgaB. 16S rRNA was used as an internal control. The virulence was determined by the ability to kill A549 human lung epithelial cells with different MOIs and different time points. The virulence was also determined by Galleria mellonella killing assay and analyzed by Kaplan-Meier survival analysis. The statistical analysis was performed with SPSS 13.0 and Graphpad Prism 5.0. RESULTS: Sixty-five isolates were divided into pgaB high and low expression groups by the values of adjusted pgaB expression, the median values of which were 25.81 ± 6.34 (n = 41) and 4.16 ± 2.82 (n = 24), respectively (p < 0.05). The Ab virulence determined by cytotoxicity assay was significantly higher in the pgaB high expression group than in the low expression group (MOI = 100, incubated for 18 hours, p < 0.05). The killing rate of Galleria mellonella was significantly higher in the pgaB high expression group than in the low expression group (inoculum of 106 CFU/larva at 37°C, p < 0.05), with the median survival time of 40 hours and 75 hours, respectively. CONCLUSIONS: The adjusted expression level of pgaB was in agreement with the virulence of Ab in cytotoxicity assays and Galleria mellonella killing assay, thus it may be used potentially as a putative marker for early prediction of the virulence of Ab clinical isolates.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Amidoidrolases/genética , Amidoidrolases/metabolismo , Virulência , Células A549 , Infecções por Acinetobacter/mortalidade , Acinetobacter baumannii/patogenicidade , Técnicas de Tipagem Bacteriana , Biofilmes , China , Células Epiteliais/microbiologia , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética
20.
Microb Pathog ; 131: 15-21, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30930221

RESUMO

Staphylococcus aureus is a major pathogen of subclinical bovine mastitis that usually is chronic and recurrent, which has been related to its ability to internalize into bovine mammary epithelial cells (bMECs). Previously, we reported that short and medium fatty acids and cholecalciferol reduce S. aureus internalization into pretreated-bMECs with these molecules suggesting a role as immunomodulatory agents. Hence, we assessed the role of sodium butyrate (NaB), sodium octanoate (NaO) and cholecalciferol on S. aureus adhesin expression and its internalization into bMECs. S. aureus pre-treated 2 h with 0.5 mM or 2 mM NaB showed a reduction in internalization into bMECs (∼35% and ∼55%; respectively), which coincided with a down-regulated expression of clumping factor B (ClfB). Also, the S. aureus internalization reduction by 2 mM NaB (2 h) agreed with a down-regulated expression of sdrC. Moreover, the 2 mM NaB (24 h) pre-treatment induced bacterial internalization (∼3-fold), which was related with an up-regulation of spa, clfB and sdrC genes. Also, NaO (0.25 mM and 1 mM) only reduced S. aureus internalization when bacteria were grown 2 h with this molecule but there was no relationship with adhesin expression. In addition, cholecalciferol (50 nM) reduced bacteria internalization at similar levels (∼50%) when bacteria were grown 2 and 24 h in broth supplemented with this compound, which correlated with spa and sdrC mRNA expression down-regulated at 2 h, and fnba and clfB mRNA expression decreased at 24 h. In conclusion, our data support the fact that fatty acids and cholecalciferol regulate adhesin gene expression as well as bacteria internalization in nonprofessional phagocytic cells, which may lead to development of anti-virulence agents for control of pathogens.


Assuntos
Adesinas Bacterianas/genética , Células Epiteliais/imunologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glândulas Mamárias Animais/imunologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Adesinas Bacterianas/efeitos dos fármacos , Animais , Proteínas de Bactérias/genética , Ácido Butírico , Caprilatos/farmacologia , Bovinos , Linhagem Celular , Colecalciferol/farmacologia , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/microbiologia , Ácidos Graxos/farmacologia , Feminino , Gentamicinas/farmacologia , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Imunomodulação , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/imunologia , Mastite Bovina/microbiologia , Mastite Bovina/prevenção & controle , RNA Mensageiro/metabolismo , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , Fatores de Virulência/genética
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