(d) Kxd1- and Plekhm2-deficient BMDMs were infected with Alexa-546-labelled H37Rv or BJN at MOI of 10 for 15?min and chased for 1?h

(d) Kxd1- and Plekhm2-deficient BMDMs were infected with Alexa-546-labelled H37Rv or BJN at MOI of 10 for 15?min and chased for 1?h. positioning towards the perinuclear region and lysosomal delivery into its phagosome during autophagy induction by starvation to evade starvation-induced autophagic restriction. and more than 95% of tuberculosis-associated deaths occur in low- and middle-income countries2. The only available tuberculosis vaccine, dubbed BCG, can prevent severe tuberculosis in children but it does not protect against pulmonary tuberculosis in adults, while the emergence and rapid spread of multidrug-resistant and extensively drug-resistant strains have posed a serious threat to the global tuberculosis control program2. Difficulties in rapid diagnosis and inadequacy of drugs to treat drug-resistant tuberculosis are the main barriers for effective control3. Therefore, new drugs are urgently needed. Global distribution and emergence of drug-resistant tuberculosis are associated with genotypic variation4C8. The Beijing family, notorious for drug-resistance and hyper-virulence, now represents around 50% of the strains in East Asia and more than 13% of strains worldwide5, with an increasingly wide spread of the Beijing genotype4,9. The reasons for its high transmissibility are unclear. Previous studies showed that this Beijing genotype had greater ability to survive inside host macrophages, causing high bacterial load and greater mortality rates in animal PF429242 dihydrochloride models, with high acid-fast bacilli (AFB) smear-positive sputum in human patients6,10C14. However, the molecular mechanisms and factors underlying this increased ability of the Beijing genotype to survive in the host remain to be determined. Recently, we reported that Beijing strains had a previously unrecognised ability to evade elimination by host autophagy, an important innate immune mechanism against in host macrophages15. Autophagy is usually a conserved lysosomal-dependent degradation process that has been demonstrated to play a key role in defending against intracellular bacteria, viruses and protozoan parasites16C19. Autophagy of microbes is initiated by a stress signal such as starvation of the infected cells due to consumption of host cell amino acids by the pathogens and presence of pathogen-associated molecular patterns and host cytokines17,20,21. During autophagy, double-membrane autophagosomes engulf cytosolic substrates such as whole pathogens or their components and deliver them along microtubules to fuse with acidic lysosomes concentrating at the perinuclear region, resulting in the delivery of lysosomal hydrolases to digest the enclosed contents22. In the context of contamination, induction of autophagy by PF429242 dihydrochloride starvation or other autophagy inducers resulted in the death of intracellular reference strains such as H37Rv and strains belonging to the East African Indian genotype15,23C29, even though they have blocked the phagolysosome biogenesis and other host cell defence mechanisms30. Our previous study showed that unlike H37Rv and East African Indian strains, which were eliminated by starvation-induced autophagic Mouse monoclonal to FYN restriction in host macrophages dependent upon Beclin-1, the Beijing strains were able to resist starvation-induced autophagic control by host cells15. In the same study, we also revealed that evasion of the Beijing strains from starvation-induced autophagic restriction was not simply achieved by blocking the autophagy-mediated acidification of their phagosomes or by inhibiting the general autophagic flux in host cells but by blocking lysosomal delivery into their phagosomes upon autophagy induction by starvation15. We found that while lysosomes were delivered to the phagosomes of H37Rv and East African Indian strains upon autophagy induction by starvation dependent upon Beclin-1, lysosomal delivery to phagosomes of PF429242 dihydrochloride autophagy-resistant Beijing strains was inhibited15. However, the factors involved were unknown. In this study, we attempted to determine the host factors contributing to escape of the autophagy-resistant Beijing strain (BJN) from starvation-induced autophagic restriction by conducting RNA-Seq analyses of PF429242 dihydrochloride host macrophages infected with BJN compared to those infected with H37Rv upon autophagy induction by starvation. Our results identified several genes that were differentially regulated in BJN-infected macrophages during autophagy induction by starvation but not in H37Rv-infected cells. The findings were confirmed by qRT-PCR. In silico gene ontology (GO) analysis showed that several pathways were enriched in BJN-infected macrophages upon autophagy induction by starvation, including the lysosome localisation pathway. Two of the genes in this pathway, and that potentially could be targets for the discovery of new drugs against this emerging disease. Results Gene expression quantification Our previous study showed that Beijing strains were able to resist starvation-induced autophagic elimination in host macrophages but the factors and mechanisms involved were unclear15. We employed RNA-Seq technology to profile global gene expression levels in RAW264.7 macrophages infected with different mycobacteria induced to undergo autophagy.

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