RESUMEN
GTPase high frequency of lysogenization X (HflX) is highly conserved in prokaryotes and acts as a ribosome-splitting factor as part of the heat shock response in Escherichia coli. Here we report that HflX produced by slow-growing Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a GTPase that plays a critical role in the pathogen's transition to a nonreplicating, drug-tolerant state in response to hypoxia. Indeed, HflX-deficient M. bovis BCG (KO) replicated markedly faster in the microaerophilic phase of a hypoxia model that resulted in premature entry into dormancy. The KO mutant displayed hallmarks of nonreplicating mycobacteria, including phenotypic drug resistance, altered morphology, low intracellular ATP levels, and overexpression of Dormancy (Dos) regulon proteins. Mice nasally infected with HflX KO mutant displayed increased bacterial burden in the lungs, spleen, and lymph nodes during the chronic phase of infection, consistent with the higher replication rate observed in vitro in microaerophilic conditions. Unlike fast growing mycobacteria, M. bovis BCG HlfX was not involved in antibiotic resistance under aerobic growth. Proteomics, pull-down, and ribo-sequencing approaches supported that mycobacterial HflX is a ribosome-binding protein that controls translational activity of the cell. With HflX fully conserved between M. bovis BCG and M. tuberculosis, our work provides further insights into the molecular mechanisms deployed by pathogenic mycobacteria to adapt to their hypoxic microenvironment.
Asunto(s)
Replicación del ADN , GTP Fosfohidrolasas/metabolismo , Hipoxia/genética , Hipoxia/metabolismo , Mycobacterium/genética , Mycobacterium/metabolismo , Animales , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Ratones , Mutación , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Ribosomas/metabolismoRESUMEN
Dengue is a prevalent mosquito-borne viral infection in the tropical and sub-tropical regions. Its potential to progress into severe, life-threatening disease, has pressed the research community to develop safe, effective and affordable antivirals. Metformin (MET), a first-line antidiabetic drug and indirect AMP-activated protein kinase (AMPK) activator, has recently emerged as a potential anti-DENV therapeutic candidate, based on some experimental evidence supporting anti-DENV activity in vitro and widely reported anti-inflammatory properties. Here, we examined MET in vitro activity against the four DENV serotypes and in two different mammalian cell lines. MET displayed a poor anti-DENV activity in BHK-21 cells with IC50 in the mM range, which was associated with increased p-AMPKα levels, thereby supporting that MET antiviral activity is mediated through AMPK activation. In contrast, MET exerted a pro-DENV activity in Vero cells that did not correlate with increased AMPK activation, suggesting AMPK-independent effects. Treatment with compound 991, a direct AMPK activator, led to reduced viral titers against all four serotypes and across both mammalian cell lines. In vivo, oral administration of MET did not reduce viremia titers in an asymptomatic mouse model, neither did it improve disease severity and progression in a mouse model of severe dengue. Instead, high dose regimen worsened disease outcome as evidenced by increased mortality, higher viremia and hyper-inflammation. Therefore, while AMPK may represent a potential host target, MET does not seem to hold great promise as a pan-serotype anti-dengue drug.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Metformina/farmacología , Replicación Viral/efectos de los fármacos , Animales , Chlorocebus aethiops , Dengue/tratamiento farmacológico , Dengue/virología , Virus del Dengue/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Células Vero , Viremia/tratamiento farmacológicoRESUMEN
Individuals with type 2 diabetes are at increased risk of acquiring melioidosis, a disease caused by Burkholderia pseudomallei infection. Although up to half of melioidosis patients have underlying diabetes, the mechanisms involved in this increased susceptibility are unknown. We found that B. pseudomallei-infected PBMCs from diabetic patients were impaired in IL-12p70 production, which resulted in decreased IFN-γ induction and poor bacterial killing. The defect was specific to the IL-12-IFN-γ axis. Defective IL-12 production was also observed during Mycobacterium tuberculosis infection, in which diabetes is likewise known to be a strong risk factor. In contrast, IL-12 production in diabetic cells was not affected upon Salmonella enterica infection or in response to TLR2, -3, -4, and -5 ligands. Poor IL-12 production correlated with a deficiency in intracellular reduced glutathione (GSH) concentrations in diabetic patients. Addition of GSH or N-acetylcysteine to PBMCs selectively restored IL-12 and IFN-γ production and improved bacterial killing. Furthermore, the depletion of GSH in mice led to increased susceptibility to melioidosis, reduced production of IL-12p70, and poorer disease outcome. Our data thus establish a link between GSH deficiency in diabetes and increased susceptibility to melioidosis that may open up new therapeutic avenues to protect diabetic patients against some intracellular bacterial pathogens.