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Melioidosis caused by Burkholderia pseudomallei is an infectious disease with a high mortality rate. In acute melioidosis, sepsis is a major cause of death among patients. Once the bacterium enters the bloodstream, immune system dysregulation ensues, leading to cytokine storms. In contrast to B. pseudomallei, a closely related but non-virulent strain B. thailandensis has rarely been reported to cause cytokine storms or death in patients. However, the mechanisms in which the virulent B. pseudomallei causes sepsis are not fully elucidated. It is well-documented that monocytes play an essential role in cytokine production in the bloodstream. The present study, therefore, determined whether there is a difference in the innate immune response to B. pseudomallei and B. thailandensis during infection of primary human monocytes and THP-1 monocytic cells by investigating pyroptosis, an inflammatory death pathway known to play a pivotal role in sepsis. Our results showed that although both bacterial species exhibited a similar ability to invade human monocytes, only B. pseudomallei can significantly increase the release of cytosolic enzyme lactate dehydrogenase (LDH) as well as the increases in caspase-1 and gasdermin D activations in both cell types. The results were consistent with the significant increase in IL-1ß and IL-18 production, key cytokines involved in pyroptosis. Interestingly, there was no significant difference in other cytokine secretion, such as IL-1RA, IL-10, IL-12p70, IL-15, IL-8, and IL-23 in cells infected by both bacterial species. Furthermore, we also demonstrated that ROS production played a crucial role in controlling pyroptosis activation during B. pseudomallei infection in primary human monocytes. These findings suggested that pyroptosis induced by B. pseudomallei in the human monocytes may contribute to the pathogenesis of sepsis in acute melioidosis patients.
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Burkholderia pseudomallei , Burkholderia , Melioidose , Monócitos , Piroptose , Sepse , Humanos , Burkholderia pseudomallei/imunologia , Burkholderia pseudomallei/fisiologia , Monócitos/imunologia , Monócitos/microbiologia , Melioidose/microbiologia , Melioidose/imunologia , Burkholderia/patogenicidade , Sepse/microbiologia , Sepse/imunologia , Citocinas/metabolismo , Células THP-1 , Imunidade Inata , Células CultivadasRESUMO
Melioidosis is a tropical infection caused by the intracellular pathogen Burkholderia pseudomallei, an underreported and emerging global threat. As melioidosis-associated mortality is frequently high despite antibiotics, novel management strategies are critically needed. Therefore, we sought to determine whether functional changes in the host innate and adaptive immune responses are induced during acute melioidosis and are associated with outcome. Using a unique whole blood stimulation assay developed for use in resource-limited settings, we examined induced cellular functional and phenotypic changes in a cohort of patients with bacteremic melioidosis prospectively enrolled within 24 h of positive blood culture and followed for 28 days. Compared to healthy controls, melioidosis survivors generated an IL-17 response mediated by Th17 cells and terminally-differentiated effector memory CD8+ T cells (P < .05, both), persisting to 28 days after enrolment. Furthermore, melioidosis survivors developed polyfunctional cytokine production in CD8+ T cells (P < .01). Conversely, a reduction in CCR6+ CD4+ T cells was associated with higher mortality, even after adjustments for severity of illness (P = 0.004). Acute melioidosis was also associated with a profound acute impairment in monocyte function as stimulated cytokine responses were reduced in classical, intermediate and non-classical monocytes. Impaired monocyte cytokine function improved by 28-days after enrolment. These data suggest that IL-17 mediated cellular responses may be contributors to host defense during acute melioidosis, and that innate immune function may be impaired. These insights could provide novel targets for the development of therapies and vaccine targets in this frequently lethal disease.
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Burkholderia pseudomallei , Linfócitos T CD8-Positivos , Melioidose , Células Th17 , Melioidose/imunologia , Melioidose/mortalidade , Melioidose/microbiologia , Humanos , Masculino , Feminino , Burkholderia pseudomallei/imunologia , Pessoa de Meia-Idade , Linfócitos T CD8-Positivos/imunologia , Células Th17/imunologia , Idoso , Adulto , Imunidade Celular , Interleucina-17/imunologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/sangue , Citocinas/imunologia , Estudos ProspectivosRESUMO
Extraintestinal pathogenic Escherichia coli (ExPEC), especially uropathogenic E. coli (UPEC) are responsible for urinary tract infections (UTIs), while diarrheagenic E. coli (DEC) cause foodborne illnesses. These pathogenic E. coli are a serious threat to human health and a public concern worldwide. However, the evidence on pork E. coli (PEC) harboring UPEC virulence-associated genes is currently limited. Therefore, this study aimed to determine the phylogroups, virulence genes, and their association between PEC and UPEC from UTI patients. In this study, 330 E. coli were obtained from archived stock culture isolated from pork (PEC; n = 165) and urine of patients with UTIs (UPEC; n = 165) during 2014-2022. Phylogroups, UPEC- and diarrheagenic E. coli (DEC) associated virulence genes were assessed using PCR assays. The results showed that phylogroups A (50.3%), and B1 (32.1%) were commonly found among PEC whereas phylogroups B2 (41.8%), and C (25.5%) were commonly detected in the UPEC. PEC and UPEC carried similar virulence-associated genes with different percentages. The most frequent UPEC virulence-associated gene among UPEC, and PEC strains was fimH, (93.3%, and 92.1%), followed by iucC (55.2%, and 12.7%), papC (21.8%, and 4.2%), afaC (22.4%, and 0%), hlyCA (17%, and 0.6%), cnf (16.4%, and 0.6%), and sfa/focDE (8.5%, and 4.8%). Additionally, 6 of 27 UPEC virulence-associated gene patterns were found in both PEC and UPEC strains regardless of phylogroups. Furthermore, the DEC virulence-associated genes were found in only 3 strains, one from PEC harboring eae, and two from UPEC carried fimH-bfpA or afaC-CVD432 indicating hybrid strains. Cluster analysis showed a relationship between PEC and UPEC strains and demonstrated that PEC harboring UPEC virulence-associated genes in pork may be associated with UPEC in humans. Food safety and hygiene practices during pork production chain are important procedures for minimizing cross-contamination of these strains that could be transmitted to the consumers.
Assuntos
Infecções por Escherichia coli , Filogenia , Infecções Urinárias , Fatores de Virulência , Infecções Urinárias/microbiologia , Humanos , Tailândia/epidemiologia , Animais , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/epidemiologia , Suínos , Fatores de Virulência/genética , Virulência/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/patogenicidade , Escherichia coli/isolamento & purificação , Escherichia coli/classificação , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/patogenicidade , Escherichia coli Uropatogênica/isolamento & purificação , Escherichia coli Uropatogênica/classificação , Variação GenéticaRESUMO
Pulmonary melioidosis is a severe tropical infection caused by Burkholderia pseudomallei and is associated with high mortality despite early antibiotic treatment. γδ T cells have been increasingly implicated as drivers of the host neutrophil response during bacterial pneumonia, but their role in pulmonary melioidosis is unknown. Here, we report that in patients with melioidosis, a lower peripheral blood γδ T cell concentration is associated with higher mortality even when adjusting for severity of illness. γδ T cells were also enriched in the lung and protected against mortality in a mouse model of pulmonary melioidosis. γδ T cell deficiency in infected mice induced an early recruitment of neutrophils to the lung, independent of bacterial burden. Subsequently, γδ T cell deficiency resulted in increased neutrophil-associated inflammation in the lung as well as impaired bacterial clearance. Additionally, γδ T cells influenced neutrophil function and subset diversity in the lung after infection. Our results indicate that γδ T cells serve a novel protective role in the lung during a severe bacterial pneumonia by regulating excessive neutrophil-associated inflammation.
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BACKGROUND: Cleistanthin A (CA), extracted from Phyllanthus taxodiifolius Beille, was previously reported as a potential V-ATPase inhibitor relevant to cancer cell survival. In the present study, ECDD-S16, a derivative of cleistanthin A, was investigated and found to interfere with pyroptosis induction via V-ATPase inhibition. OBJECTIVE: This study examined the ability of ECDD-S16 to inhibit endolysosome acidification leading to the attenuation of pyroptosis in Raw264.7 macrophages activated by both surface and endosomal TLR ligands. METHODS: To elucidate the activity of ECDD-S16 on pyroptosis-induced inflammation, Raw264.7 cells were pretreated with the compound before stimulation with surface and endosomal TLR ligands. The release of lactate dehydrogenase (LDH) was determined by LDH assay. Additionally, the production of cytokines and the expression of pyroptosis markers were examined by ELISA and immunoblotting. Moreover, molecular docking was performed to demonstrate the binding of ECDD-S16 to the vacuolar (V-)ATPase. RESULTS: This study showed that ECDD-S16 could inhibit pyroptosis in Raw264.7 cells activated with surface and endosomal TLR ligands. The attenuation of pyroptosis by ECDD-S16 was due to the impairment of endosome acidification, which also led to decreased Reactive Oxygen Species (ROS) production. Furthermore, molecular docking also showed the possibility of inhibiting endosome acidification by the binding of ECDD-S16 to the vacuolar (V-)ATPase in the region of V0. CONCLUSION: Our findings indicate the potential of ECDD-S16 for inhibiting pyroptosis and prove that vacuolar H+ ATPase is essential for pyroptosis induced by TLR ligands.
Assuntos
ATPases Vacuolares Próton-Translocadoras , Humanos , ATPases Vacuolares Próton-Translocadoras/metabolismo , Piroptose , Simulação de Acoplamento Molecular , InflamaçãoRESUMO
Melioidosis is an often fatal infection in tropical regions caused by an environmental bacterium, Burkholderia pseudomallei Current recommended melioidosis treatment requires intravenous ß-lactam antibiotics such as ceftazidime (CAZ), meropenem (MEM) or amoxicillin-clavulanic acid (AMC) and oral trimethoprim-sulfamethoxazole. Emerging antibiotic resistance could lead to therapy failure and high mortality. We performed a prospective multicentre study in northeast Thailand during 2015-2018 to evaluate antibiotic susceptibility and characterize ß-lactam resistance in clinical B. pseudomallei isolates. Collection of 1,317 B. pseudomallei isolates from patients with primary and relapse infections were evaluated for susceptibility to CAZ, imipenem (IPM), MEM and AMC. ß-lactam resistant isolates were confirmed by broth microdilution method and characterized by whole genome sequence analysis, penA expression and ß-lactamase activity. The resistant phenotype was verified via penA mutagenesis. All primary isolates were IPM-susceptible but we observed two CAZ-resistant and one CAZ-intermediate resistant isolates, two MEM-less susceptible isolates, one AMC-resistant and two AMC-intermediate resistant isolates. One of 13 relapse isolates was resistant to both CAZ and AMC. Two isolates were MEM-less susceptible. Strains DR10212A (primary) and DR50054E (relapse) were multi-drug resistant. Genomic and mutagenesis analyses supplemented with gene expression and ß-lactamase analyses demonstrated that CAZ-resistant phenotype was caused by PenA variants: P167S (N=2) and penA amplification (N=1). Despite the high mortality rate in melioidosis, our study revealed that B. pseudomallei isolates had a low frequency of ß-lactam resistance caused by penA alterations. Clinical data suggest that resistant variants may emerge in patients during antibiotic therapy and be associated with poor response to treatment.
RESUMO
Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei. This bacterium is able to survive and multiply inside the immune cells such as macrophages. It is well established that Toll-like receptors (TLRs), particularly surface TLRs such as TLR2, TLR4, and TLR5, play an essential role in defending against this bacterial infection. However, the involvement of endosomal TLRs in the infection has not been elucidated. In this study, we demonstrated that the number of intracellular bacteria is reduced in TLR9-depleted RAW264.7 cells infected with B. pseudomallei, suggesting that TLR9 is involved in intracellular bacterial killing in macrophages. As several reports have previously demonstrated that pyroptosis is essential for restricting intracellular bacterial killing, particularly in B. pseudomallei infection, we also observed an increased release of cytosolic enzyme lactate dehydrogenase (LDH) in TLR9-depleted cells infected with B. pseudomallei, suggesting TLR9 involvement in pyroptosis in this context. Consistently, the increases in caspase-11 and gasdermind D (GSDMD) activations, which are responsible for the LDH release, were also detected. Moreover, we demonstrated that the increases in pyroptosis and bacterial killing in B. pseudomallei-infected TLR9-depleted cells were due to the augmentation of the IFN-ß, one of the key cytokines known to regulate caspase-11. Altogether, this finding showed that TLR9 suppresses macrophage killing of B. pseudomallei by regulating pyroptosis. This information provides a novel mechanism of TLR9 in the regulation of intracellular bacterial killing by macrophages, which could potentially be leveraged for therapeutic intervention. IMPORTANCE Surface TLRs have been well established to play an essential role in Burkholderia pseudomallei infection. However, the role of endosomal TLRs has not been elucidated. In the present study, we demonstrated that TLR9 plays a crucial role by negatively regulating cytokine production, particularly IFN-ß, a vital cytokine to control pyroptosis via caspase-11 activation. By depletion of TLR9, the percentage of pyroptosis was significantly increased, leading to suppression of intracellular survival in B. pseudomallei-infected macrophages. These findings provide a new role of TLR9 in macrophages.
Assuntos
Burkholderia pseudomallei , Melioidose , Camundongos , Animais , Burkholderia pseudomallei/metabolismo , Receptor Toll-Like 9/metabolismo , Receptor 2 Toll-Like/metabolismo , Piroptose , Receptor 4 Toll-Like/metabolismo , Receptor 5 Toll-Like/metabolismo , Melioidose/metabolismo , Melioidose/microbiologia , Macrófagos , Linhagem Celular , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Citocinas/metabolismo , Caspases/metabolismo , Lactato Desidrogenases/metabolismoRESUMO
Vibrio parahaemolyticus is a Gram-negative, foodborne pathogenic bacterium that causes human gastroenteritis. This organism is ubiquitously present in the marine environment. Detection of V. parahaemolyticus in aquatic birds has been previously reported; however, the characterization of isolates of this bacterium recovered from these birds remains limited. The present study isolated and characterized V. parahaemolyticus from aquatic bird feces at the Bangpu Recreation Center (Samut Prakan province, Thailand) from 2016 to 2017, using multilocus sequence typing (MLST) and genome analysis. The results showed that V. parahaemolyticus was present in 34.9% (76/218) of the collected bird fecal samples. Among the ldh-positive V. parahaemolyticus isolates (n = 308), 1% (3/308) were positive for tdh, 1.3% (4/308) were positive for trh, and 0.3% (1/308) were positive for both tdh and trh. In turn, the MLST analysis revealed that 49 selected V. parahaemolyticus isolates resolved to 36 STs, 26 of which were novel (72.2%). Moreover, a total of 10 identified STs were identical to globally reported pathogenic strains (ST1309, ST1919, ST491, ST799, and ST2516) and environmental strains (ST1879, ST985, ST288, ST1925, and ST260). The genome analysis of isolates possessing tdh and/or trh (ST985, ST1923, ST1924, ST1929 and ST2516) demonstrated that the organization of the T3SS2α and T3SS2ß genes in bird fecal isolates were almost identical to those of human clinical strains posing public health concerns of pathogen dissemination in the recreational area. The results of this study suggest that aquatic birds are natural reservoirs of new strains with high genetic diversity and are alternative sources of potentially pathogenic V. parahaemolyticus in the marine environment. IMPORTANCE To our knowledge, infection of foodborne bacterium V. parahamolyticus occurs via the consumption of undercooked seafood contaminated with pathogenic strains. Aquatic bird is a neglectable source that can transmit V. parahaemolyticus along coastal areas. This study reported the detection of potentially pathogenic V. parahamolyticus harboring virulence genes from aquatic bird feces at the recreational center situated near the Gulf of Thailand. These strains shared identical genetic profile to the clinical isolates that previously reported in many countries. Furthermore, the strains from aquatic birds showed extremely high genetic diversity. Our research pointed out that the aquatic bird is possibly involved in the evolution of novel strains of V. parahaemolyticus and play a role in dissimilation of the potentially pathogenic strains across geographical distance.
Assuntos
Vibrioses , Vibrio parahaemolyticus , Animais , Aves/genética , Fezes , Humanos , Tipagem de Sequências Multilocus , Tailândia , Vibrioses/microbiologia , Vibrioses/veterinária , Vibrio parahaemolyticus/genética , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Rural areas usually show a higher prevalence of rickettsial infection than urban areas. However, information on the rickettsial infection status in urban settings (e.g., built-up areas and city parks) is still limited, particularly in the Bangkok metropolitan area. In this study, we performed a molecular rickettsial survey of spleen samples of small mammals caught in public parks and built-up areas of Bangkok. Out of 198 samples, the Rattus rattus complex was found to be most prevalent. The amplification of rickettsial gltA fragment gene (338 bp) by nested PCR assay revealed positive results in four samples, yielding a low prevalence of infection of 2.02%. DNA sequencing results confirmed that three samples were matched with Rickettsia typhi, and one was identified as R. felis. It is noteworthy that this is the first report of the occurrence of R. felis DNA in rodents in Southeast Asia.
RESUMO
Flavivirus reporters provide a robust tool for viral pathogenesis studies, anti-viral drug screening, disease diagnosis and functional antibody assays. In this study, we generated a luciferase-secreting, single-round reporter virus by replacing the capsid coding region in a DENV-2 genome with the secretory form of Lucia luciferase gene to produce infectious viral particles in a stable capsid-expressing mosquito cell line. Replication of the reporter virus in trans-complementing mosquito cells was sustained for up to two weeks. There were strong correlations between the extracellular luciferase activity and infectious reporter virus inocula upon infection of mosquito and mammalian cell lines with graded quantities of the reporter virus. A set of anti-E and anti-prM monoclonal antibodies affected the infectivity of reporter virus with similar dose-effect relationships as the parent virus. This simplified version of DENV-2 reporter provides a rapid and reliable method for the detection of neutralizing and infection-enhancing antibodies against dengue virus.
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Vírus da Dengue , Dengue , Flavivirus , Animais , Anticorpos Bloqueadores , Anticorpos Antivirais , Dengue/diagnóstico , Vírus da Dengue/genética , Luciferases/genéticaRESUMO
BACKGROUND: Melioidosis, infection caused by Burkholderia pseudomallei, is a common cause of sepsis with high associated mortality in Southeast Asia. Identification of patients at high likelihood of clinical deterioration is important for guiding decisions about resource allocation and management. We sought to develop a biomarker-based model for 28-day mortality prediction in melioidosis. METHODS: In a derivation set (Nâ =â 113) of prospectively enrolled, hospitalized Thai patients with melioidosis, we measured concentrations of interferon-γ, interleukin-1ß, interleukin-6, interleukin-8, interleukin-10, tumor necrosis factor-É, granulocyte-colony stimulating factor, and interleukin-17A. We used least absolute shrinkage and selection operator (LASSO) regression to identify a subset of predictive biomarkers and performed logistic regression and receiver operating characteristic curve analysis to evaluate biomarker-based prediction of 28-day mortality compared with clinical variables. We repeated select analyses in an internal validation set (Nâ =â 78) and in a prospectively enrolled external validation set (Nâ =â 161) of hospitalized adults with melioidosis. RESULTS: All 8 cytokines were positively associated with 28-day mortality. Of these, interleukin-6 and interleukin-8 were selected by LASSO regression. A model consisting of interleukin-6, interleukin-8, and clinical variables significantly improved 28-day mortality prediction over a model of only clinical variables [AUC (95% confidence interval [CI]): 0.86 (.79-.92) vs 0.78 (.69-.87); Pâ =â .01]. In both the internal validation set (0.91 [0.84-0.97]) and the external validation set (0.81 [0.74-0.88]), the combined model including biomarkers significantly improved 28-day mortality prediction over a model limited to clinical variables. CONCLUSIONS: A 2-biomarker model augments clinical prediction of 28-day mortality in melioidosis.
Assuntos
Citocinas/sangue , Melioidose , Adulto , Biomarcadores/sangue , Burkholderia pseudomallei , Humanos , Melioidose/diagnóstico , Melioidose/mortalidade , TailândiaRESUMO
Melioidosis is an often lethal tropical disease caused by the Gram-negative bacillus, Burkholderia pseudomallei. The study objective was to characterize transcriptomes in melioidosis patients and identify genes associated with outcome. Whole blood RNA-seq was performed in a discovery set of 29 melioidosis patients and 3 healthy controls. Transcriptomic profiles of patients who did not survive to 28 days were compared with patients who survived and healthy controls, showing 65 genes were significantly up-regulated and 218 were down-regulated in non-survivors compared to survivors. Up-regulated genes were involved in myeloid leukocyte activation, Toll-like receptor cascades and reactive oxygen species metabolic processes. Down-regulated genes were hematopoietic cell lineage, adaptive immune system and lymphocyte activation pathways. RT-qPCR was performed for 28 genes in a validation set of 60 melioidosis patients and 20 healthy controls, confirming differential expression. IL1R2, GAS7, S100A9, IRAK3, and NFKBIA were significantly higher in non-survivors compared with survivors (P < 0.005) and healthy controls (P < 0.0001). The AUROCC of these genes for mortality discrimination ranged from 0.80-0.88. In survivors, expression of IL1R2, S100A9 and IRAK3 genes decreased significantly over 28 days (P < 0.05). These findings augment our understanding of this severe infection, showing expression levels of specific genes are potential biomarkers to predict melioidosis outcomes.
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Biomarcadores/sangue , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Melioidose/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Melioidose/sangue , Melioidose/genética , Pessoa de Meia-Idade , Estudos Prospectivos , Análise de Sequência de RNA , Análise de SobrevidaRESUMO
Melioidosis is a tropical infectious disease caused by Burkholderia pseudomallei that results in high mortality. Hemolysin co-regulated protein 1 (Hcp1) and O-polysaccharide (OPS) are vaccine candidates and potential diagnostic antigens. The correlation of classes/subclasses of antibodies against these antigens with clinical characteristics of melioidosis patients is unknown. Antibodies in plasma samples from melioidosis patients and healthy donors were quantified by ELISA and compared with clinical features. In melioidosis patients, Hcp1 induced high IgG levels. OPS induced high IgG and IgA levels. The area under receiver operating characteristic curve (AUROCC) to discriminate melioidosis cases from healthy donors was highest for anti-Hcp1 IgG (0.92) compared to anti-Hcp1 IgA or IgM. In contrast, AUROCC for anti-OPS for IgG (0.91) and IgA (0.92) were comparable. Anti-Hcp1 IgG1 and anti-OPS IgG2 had the greatest AUROCCs (0.87 and 0.95, respectively) compared to other IgG subclasses for each antigen. Survivors had significantly higher anti-Hcp1 IgG3 levels than non-survivors. Male melioidosis patients with diabetes had higher anti-OPS IgA levels than males without diabetes. Thus, diverse and specific antibody responses are associated with distinct clinical characteristics in melioidosis, confirming the diagnostic utility of these responses and providing new insights into immune mechanisms.
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Proteínas Hemolisinas/imunologia , Melioidose/imunologia , Polissacarídeos/imunologia , Anticorpos Antibacterianos/sangue , Burkholderia pseudomallei/imunologia , Feminino , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Masculino , Melioidose/sangueRESUMO
Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging arboviruses that pose a worldwide threat to human health. Currently, neither vaccine nor antiviral treatment to control their infections is available. As the skin is a major viral entry site for arboviruses in the human host, we determined the global proteomic profile of CHIKV and ZIKV infections in human skin fibroblasts using Stable Isotope Labelling by Amino acids in Cell culture (SILAC)-based mass-spectrometry analysis. We show that the expression of the interferon-stimulated proteins MX1, IFIT1, IFIT3 and ISG15, as well as expression of defense response proteins DDX58, STAT1, OAS3, EIF2AK2 and SAMHD1 was significantly up-regulated in these cells upon infection with either virus. Exogenous expression of IFITs proteins markedly inhibited CHIKV and ZIKV replication which, accordingly, was restored following the abrogation of IFIT1 or IFIT3. Overexpression of SAMHD1 in cutaneous cells, or pretreatment of cells with the virus-like particles containing SAMHD1 restriction factor Vpx, resulted in a strong increase or inhibition, respectively, of both CHIKV and ZIKV replication. Moreover, silencing of SAMHD1 by specific SAMHD1-siRNA resulted in a marked decrease of viral RNA levels. Together, these results suggest that IFITs are involved in the restriction of replication of CHIKV and ZIKV and provide, as yet unreported, evidence for a proviral role of SAMHD1 in arbovirus infection of human skin cells.
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Vírus Chikungunya/fisiologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Pele/patologia , Replicação Viral/fisiologia , Zika virus/fisiologia , Linhagem Celular , Febre de Chikungunya/virologia , Humanos , Anotação de Sequência Molecular , Mapas de Interação de Proteínas , Proteólise , Regulação para Cima , Proteínas Virais Reguladoras e Acessórias/metabolismo , Infecção por Zika virus/virologiaRESUMO
Chikungunya virus (CHIKV) is an emerging arbovirus of the Togaviridae family that poses a present worldwide threat to human in the absence of any licensed vaccine or antiviral treatment to control viral infection. Here, we show that compounds interfering with intracellular cholesterol transport have the capacity to inhibit CHIKV replication in human skin fibroblasts, a major viral entry site in the human host. Pretreatment of these cells with the class II cationic amphiphilic compound U18666A, or treatment with the FDA-approved antidepressant drug imipramine resulted in a near total inhibition of viral replication and production at the highest concentration used without any cytotoxic effects. Imipramine was found to affect both the fusion and replication steps of the viral life cycle. The key contribution of cholesterol availability to the CHIKV life cycle was validated further by the use of fibroblasts from Niemann-Pick type C (NPC) patients in which the virus was unable to replicate. Interestingly, imipramine also strongly inhibited the replication of several Flaviviridae family members, including Zika, West Nile and Dengue virus. Together, these data show that this compound is a potential drug candidate for anti-arboviral treatment.
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Vírus Chikungunya/efeitos dos fármacos , Colesterol/metabolismo , Imipramina/farmacologia , Pele/virologia , Androstenos/farmacologia , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/virologia , Humanos , Doença de Niemann-Pick Tipo C/patologia , Pele/citologia , Pele/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for rapid bacterial identification. Studies of Burkholderia pseudomallei identification have involved small isolate numbers drawn from a restricted geographic region. There is a need to expand the reference database and evaluate B. pseudomallei from a wider geographic distribution that more fully captures the extensive genetic diversity of this species. Here, we describe the evaluation of over 650 isolates. Main spectral profiles (MSP) for 26 isolates of B. pseudomallei (N = 5) and other Burkholderia species (N = 21) were added to the Biotyper database. MALDI-TOF MS was then performed on 581 B. pseudomallei, 19 B. mallei, 6 B. thailandensis and 23 isolates representing a range of other bacterial species. B. pseudomallei originated from northeast and east Thailand (N = 524), Laos (N = 12), Cambodia (N = 14), Hong Kong (N = 4) and Australia (N = 27). All 581 B. pseudomallei were correctly identified, with 100% sensitivity and specificity. Accurate identification required a minimum inoculum of 5 x 107 CFU/ml, and identification could be performed on spiked blood cultures after 24 hours of incubation. Comparison between a dendrogram constructed from MALDI-TOF MS main spectrum profiles and a phylogenetic tree based on recA gene sequencing demonstrated that MALDI-TOF MS distinguished between B. pseudomallei and B. mallei, while the recA tree did not. MALDI-TOF MS is an accurate method for the identification of B. pseudomallei, and discriminates between this and other related Burkholderia species.
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Burkholderia pseudomallei/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ásia , Austrália , Burkholderia pseudomallei/genética , Meios de Cultura , Genes Bacterianos , Testes de Fixação do Látex , Tipagem de Sequências Multilocus , Filogenia , Especificidade da EspécieRESUMO
BACKGROUND: Identification of Burkholderia pseudomallei, the cause of melioidosis, using routine methods takes several days. Use of a monoclonal antibody-based immunofluorescent assay (IFA) on positive blood cultures may speed diagnosis. METHODS: We tested the diagnostic accuracy of the IFA on 545 blood cultures positive for Gram-negative organisms at Udon Thani Hospital, Thailand, between June 2015 and August 2016. RESULTS: Sensitivity of the IFA was 100% and specificity was 99.6%. The median decrease in time to pathogen identification between the IFA result and routine methods was 28 h (IQR 25-51), p<0.0001. CONCLUSIONS: The IFA accurately expedites the diagnosis of melioidosis.
Assuntos
Burkholderia pseudomallei , Melioidose , Hemocultura , Burkholderia pseudomallei/isolamento & purificação , Técnica Direta de Fluorescência para Anticorpo , Humanos , Melioidose/diagnósticoRESUMO
UNLABELLED: Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family, which includes dengue, West Nile, yellow fever, and Japanese encephalitis viruses, that causes a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here we examine the importance of human skin in the entry of ZIKV and its contribution to the induction of antiviral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, including DC-SIGN, AXL, Tyro3, and, to a lesser extent, TIM-1, permitted ZIKV entry, with a major role for the TAM receptor AXL. The ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing antibody and specific RNA silencing. ZIKV induced the transcription of Toll-like receptor 3 (TLR3), RIG-I, and MDA5, as well as several interferon-stimulated genes, including OAS2, ISG15, and MX1, characterized by strongly enhanced beta interferon gene expression. ZIKV was found to be sensitive to the antiviral effects of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes, whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy, and the specific autophagy inhibitor 3-methyladenine. The results presented herein permit us to gain further insight into the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging flavivirus. IMPORTANCE: Zika virus (ZIKV) is an arbovirus belonging to the Flaviviridae family. Vector-mediated transmission of ZIKV is initiated when a blood-feeding female Aedes mosquito injects the virus into the skin of its mammalian host, followed by infection of permissive cells via specific receptors. Indeed, skin immune cells, including dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells, were all found to be permissive to ZIKV infection. The results also show a major role for the phosphatidylserine receptor AXL as a ZIKV entry receptor and for cellular autophagy in enhancing ZIKV replication in permissive cells. ZIKV replication leads to activation of an antiviral innate immune response and the production of type I interferons in infected cells. Taken together, these results provide the first general insights into the interaction between ZIKV and its mammalian host.
Assuntos
Células Dendríticas/virologia , Flaviviridae/fisiologia , Queratinócitos/virologia , Internalização do Vírus , Replicação Viral , Aedes/virologia , Animais , Autofagia/imunologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Citocinas/biossíntese , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Células Dendríticas/imunologia , Fibroblastos/virologia , Flaviviridae/imunologia , Infecções por Flaviviridae/imunologia , Infecções por Flaviviridae/virologia , Células HEK293 , Receptor Celular 1 do Vírus da Hepatite A , Humanos , Insetos Vetores/virologia , Helicase IFIH1 Induzida por Interferon , Interferon beta/biossíntese , Interferon beta/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Resistência a Myxovirus/biossíntese , Fagossomos/imunologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos , Receptores Virais/genética , Receptores Virais/metabolismo , Pele/imunologia , Pele/virologia , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Receptor 3 Toll-Like/metabolismo , Receptor 7 Toll-Like/imunologia , Ubiquitinas/biossíntese , Células Vero , Receptor Tirosina Quinase AxlRESUMO
Arboviruses represent an emerging threat to human. They are transmitted to vertebrates by the bite of infected arthropods. Early transmission to vertebrates is initiated by skin puncture and deposition of virus in this organ. However, events at the bite site remain largely unknown. Here, we report that Chikungunya virus (CHIKV) and West Nile virus (WNV), despite belonging to distinct viral families, elicit a common antiviral signature in primary human dermal fibroblasts, attesting for the up regulation of interferon signaling pathways and leading to an increased expression of IFN-ß, interleukins and chemokines. Remarkably, CHIKV and WNV enhance IL-1ß expression and induce maturation of caspase-1, indicating the capacity of these pathogens to elicit activation of the inflammasome program in resident skin cells. CHIKV and WNV also induce the expression of the inflammasome sensor AIM2 in dermal fibroblasts, whereas inhibition of caspase-1 and AIM2 with siRNA interferes with both CHIKV- and WNV-induced IL-1ß production by these cells. Finally, inhibition of the inflammasome via caspase-1 silencing was found to enhance CHIKV replication in dermal fibroblasts. Together, these results indicate that the skin contributes to the pro-inflammatory and anti-viral microenvironment via the activation of the inflammasome in the early stages following infection with arboviruses.
Assuntos
Fibroblastos/imunologia , Fibroblastos/virologia , Inflamassomos/imunologia , Transdução de Sinais , Caspase 1/genética , Caspase 1/metabolismo , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Vírus Chikungunya/genética , Vírus Chikungunya/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Humanos , Inflamassomos/antagonistas & inibidores , Inflamassomos/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Interferons/genética , Interferons/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Regulação para Cima , Replicação Viral , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/fisiologiaRESUMO
Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses.