Immunoevasion and immunosuppression of the macrophage by Mycobacterium tuberculosis.

By virtue of their position at the crossroads between the innate and adaptive immune response, macrophages play an essential role in the control of bacterial infections. Paradoxically, macrophages serve as the natural habitat to Mycobacterium tuberculosis (Mtb). Mtb subverts the macrophage's mechanisms of intracellular killing and antigen presentation, leading ultimately to the development of tuberculosis (TB) disease. Here, we describe mechanisms of Mtb uptake by the macrophage and address key macrophage functions that are targeted by Mtb-specific effector molecules enabling this pathogen to circumvent host immune response. The macrophage functions described in this review include fusion between phagosomes and lysosomes, production of reactive oxygen and nitrogen species, antigen presentation and major histocompatibility complex class II expression and trafficking, as well as autophagy and apoptosis. All these are Mtb-targeted key cellular pathways, normally working in concert in the macrophage to recognize, respond, and activate 'proper' immune responses. We further analyze and discuss major molecular interactions between Mtb virulence factors and key macrophage proteins and provide implications for vaccine and drug development.

Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium tuberculosis Growth and Infectivity.

Ergothioneine (EGT) is synthesized in mycobacteria, but limited knowledge exists regarding its synthesis, physiological role, and regulation. We have identified Rv3701c from Mycobacterium tuberculosis to encode for EgtD, a required histidine methyltransferase that catalyzes first biosynthesis step in EGT biosynthesis. EgtD was found to be phosphorylated by the serine/threonine protein kinase PknD. PknD phosphorylates EgtD both in vitro and in a cell-based system on Thr(213). The phosphomimetic (T213E) but not the phosphoablative (T213A) mutant of EgtD failed to restore EGT synthesis in a ΔegtD mutant. The findings together with observed elevated levels of EGT in a pknD transposon mutant during in vitro growth suggests that EgtD phosphorylation by PknD negatively regulates EGT biosynthesis. We further showed that EGT is required in a nutrient-starved model of persistence and is needed for long term infection of murine macrophages.

Glycogen synthase kinase 3 inhibition controls Mycobacterium tuberculosis infection.

Compounds targeting host control of infectious diseases provide an attractive alternative to antimicrobials. A phenotypic screen of a kinase library identified compounds targeting glycogen synthase kinase 3 as potent inhibitors of Mycobacterium tuberculosis (Mtb) intracellular growth in the human THP-1 cell line and primary human monocytes-derived macrophages (hMDM). CRISPR knockouts and siRNA silencing showed that GSK3 isoforms are needed for the growth of Mtb and that a selected compound, P-4423632 targets GSK3ß. GSK3 inhibition was associated with macrophage apoptosis governed by the Mtb secreted protein tyrosine phosphatase A (PtpA). Phospho-proteome analysis of macrophages response to infection revealed a wide array of host signaling and apoptosis pathways controlled by GSK3 and targeted by P-4423632. P-4423632 was additionally found to be active against other intracellular pathogens. Our findings strengthen the notion that targeting host signaling to promote the infected cell's innate antimicrobial capacity is a feasible and attractive host-directed therapy approach.

Inhibition of glycogen synthase kinase-3-beta (GSK3ß) blocks nucleocapsid phosphorylation and SARS-CoV-2 replication.

GSK3ß has been proposed to have an essential role in Coronaviridae infections. Screening of a targeted library of GSK3ß inhibitors against both SARS-CoV-2 and HCoV-229E to identify broad-spectrum anti-Coronaviridae inhibitors resulted in the identification of a high proportion of active compounds with low toxicity to host cells. A selected lead compound, T-1686568, showed low micromolar, dose-dependent activity against SARS-CoV-2 and HCoV-229E. T-1686568 showed efficacy in viral-infected cultured cells and primary 2D organoids. T-1686568 also inhibited SARS-CoV-2 variants of concern Delta and Omicron. Importantly, while inhibition by T-1686568 resulted in the overall reduction of viral load and protein translation, GSK3ß inhibition resulted in cellular accumulation of the nucleocapsid protein relative to the spike protein. Following identification of potential phosphorylation sites of Coronaviridae nucleocapsid, protein kinase substrate profiling assays combined with Western blotting analysis of nine host kinases showed that the SARS-CoV-2 nucleocapsid could be phosphorylated by GSK3ß and PKCa. GSK3ß phosphorylated SARS-CoV-2 nucleocapsid on the S180/S184, S190/S194 and T198 phospho-sites, following previous priming in the adjacent S188, T198 and S206, respectively. Such inhibition presents a compelling target for broad-spectrum anti-Coronaviridae compound development, and underlies the mechanism of action of GSK3ß host-directed therapy against this class of obligate intracellular pathogens.

Apoptosis assessment in high-content and high-throughput screening assays.

Here the authors describe the development of AUTOptosis, an economical and rapid apoptosis monitoring method suitable for high-content and high-throughput screening assays. AUTOptosis is based on the quantification of nuclei intensity via staining with Hoechst 33342. First, the authors calibrated the method using standard apoptosis inducers in multiple cell lines. Next, the authors validated the applicability of this approach to high-content screening using a small library of compounds and compared it with the terminal deoxynucleotidyl transferase dUTP nick end labeling gold standard. Finally, the authors demonstrated the specificity of the method by using AUTOposis to detect apoptosis triggered by Mycobacterium tuberculosis intracellular infections.