Utilizing Autophagy To Control HIV And Tuberculosis
Creators: name: University Of California Office Of The President
Publisher: name: University of California Office of the President
publisherIdentifier: https://ror.org/00dmfq477
publisherIdentifierScheme: ROR
Year: 2024

description: In the proposed study we will ask the question whether it is possible to subvert macroautophagy (hereafter referred to as autophagy) to control HIV and Mycobacterium tuberculosis (Mtb) infection? In San Diego County, the Mtb case rate is more than twice as high as the rest of the US and the annual incidence of HIV-Mtb co-infection has remained stable despite the decline of both Mtb and HIV cases. Furthermore, the prevalence of multi-drug resistant (MDR)-Mtb and the extensively-drug resistant (XDR) Mtb is likely to increase. This, combined with the unwanted pharmacologic interactions between the drugs used to treat Mtb and HIV complicate therapy for persons co-infected, demonstrates a necessity to develop new drugs to combat HIV-Mtb co-infection. One mechanism by which Mtb and HIV subvert the immune system to establish a persistent and latent infection is through the inhibition of autophagy by blocking autophago/lysosome biogenesis, enabling their intracellular survival and persistence in macrophages. In the proposed study, we will examine the regulation of autophagy by HIV and Mtb, and on the survival and persistence of both pathogens within primary macrophages. We will also identify the molecular processes by which the active metabolite of vitamin D, interferon-gamma and interleukin-1-beta induce autophagy and inhibit HIV and Mtb infection of macrophages while developing our understanding of how the endo/lysosomal system of macrophages is converted into providing a niche environment for controlled replication by Mtb and HIV. We will also identify pharmacological agents that both induce autophagic flux and inhibit both HIV and Mtb in macrophages. The methodology that we will employ will involve the isolation and infection of macrophages with HIV and/or Mtb followed by treatment with the pharmacological agent being studied. HIV and Mtb replication will be monitored using conventional methods. The role of autophagy in altering HIV and Mtb infection will be assessed as part of each specific aim using: 1) immunoblotting to analyze the expression and turnover of LC3B-II, sequestosome 1, class 3 phosphoinositide-3 kinase-Beclin-1 and Atg12-Atg5 conjugation and mTOR phosphorylation; 2) qRT-PCR to assess transcriptional regulation of Beclin-1, ATG5 and LC3B; 3) flow cytometry to detect changes in LC3B lipidation; 4) fluorescence microscopy to detect changes in LC3B puncta formation and tandem mCherry-GFP-LC3B degradation; 5) flux measurements of protein degradation; and 6) measurements of IP3, cAMP, calcium flux and calpain inhibition. RNA interference, combined with chemical inhibitors, will be used to assess specific roles of autophagy proteins and immune modulators such as cathelicidin as well as monitoring downstream phosphorylation events using immunoblotting. The role of HIV proteins in modulating autophagy will be assessed using deletion mutants and recombinant proteins. We expected to elucidate how HIV modulates autophagy and, using this information, demonstrate that autophagy-inducing drugs inhibit both HIV and Mtb during co-infection and remain active against strains of MDR-Mtb and XDR-Mtb. Thus, results from this research will not only significantly contribute to the understanding of HIV-Mtb co-infection, but also have the potential to develop a novel approach to significantly improve the treatment of persons dually infected with HIV and Mtb.