Mycobacterium tuberculosis (Mtb) is the causative agent of the disease tuberculosis (TB) and the leading cause of death worldwide from a bacterial infection. The success of Mtb stems from its ability to evade degradation by macrophages. Recent studies have revealed that macrophages clear microorganisms through two distinct lysosomal trafficking pathways that involve LC3-marked organelles (2, 3). Xenophagy is a process by which LC3-marked, double-membrane organelles capture and degrade invading microbes. LC3-associated phagocytosis (LAP) is similar to xenophagy, but does not involve a double membrane and requires NADPH oxidase and reactive oxygen species (ROS), which are not necessary for xenophagy. These lysosomal degradative pathways are activated by microbial ligands that stimulate pathogen recognition receptors (PRRs). The reason why Mtb, which activates numerous PRRs, fails to provoke substantive LC3-associated phagolysosomal trafficking is not understood. Our extensive preliminary data strongly suggest that CpsA, an uncharacterized protein secreted by Mtb, specifically blocks LAP. We hypothesize that CpsA interferes with the activation of NADPH oxidase, thereby blocking the generation of ROS and the LAP-mediated delivery of Mtb to the lysosome. Consistent with our hypothesis, we found that Mtb strains lacking cpsA exhibit dramatically enhanced colocalization with the LC3 marker of LAP and that they are highly attenuated in macrophages and mice. Moreover, NADPH oxidase and the proteins specifically required for LAP are necessary for macrophages to kill the ?cpsA mutant. CpsA contains a LytR-CpsA-Psr (LCP) domain, which is commonly found in Gram-positive organisms. In Streptococcus pneumoniae and Bacillus subtilis, the LCP domain binds phosphorylated polyisoprenoid lipids. We modeled the structure of Mtb CpsA using the crystal structures an S. pneumoniae LCP protein and found that all of the lipid phosphate-binding residues are conserved in Mtb CpsA. In addition, we found that CpsA can bind the human T-cell leukemia virus type I binding protein 1 (TAX1BP1), and nuclear dot protein 52 kDa (NDP52). TAX1BP1 and NDP52 are paralogs that are involved in linking bacterial cargo to the autophagy machinery. Thus, we hypothesize that the ability of CpsA to inhibit the NADPH oxidase and LAP depends upon binding lipid phosphate and host proteins TAX1BP1 and NDP52. To test our hypotheses, we will (1) study the pathway by which macrophages kill the ?cpsA mutant, (2) characterize the mechanism of action of the CpsA protein, and (3) evaluate the importance of this innate immune evasion mechanism in vivo. Combined, our studies will elucidate a novel mechanism of immune evasion by one of the most formidable pathogens. By studying the molecular mechanisms Mtb utilizes to sabotage host cellular functions, we will make fundamental observations that will aid in the development of better therapeutics and vaccines for Mtb.
Mycobacterium tuberculosis (Mtb)- the causative agent of tuberculosis (TB)- kills more people than any other bacteria. We found that an Mtb protein, CpsA, allows the bacteria to evade killing by host macrophages. We will characterize exactly how CpsA protects Mtb and assess its importance during infection in vivo. These studies will yield important insights into why humans fail to sterilize Mtb, enabling new therapies and vaccines for TB.
|Upadhyay, S; Mittal, E; Philips, J A (2018) Tuberculosis and the art of macrophage manipulation. Pathog Dis 76:|
|Köster, Stefan; Klevorn, Thais; Papavinasasundaram, Kadamba et al. (2018) Consequence of enhanced LC3-trafficking for a live, attenuated M. tuberculosis vaccine. Vaccine 36:939-944|
|Hsu, Fong-Fu (2018) Mass spectrometry-based shotgun lipidomics - a critical review from the technical point of view. Anal Bioanal Chem 410:6387-6409|
|Frankfater, Cheryl; Jiang, Xuntian; Hsu, Fong-Fu (2018) Characterization of Long-Chain Fatty Acid as N-(4-Aminomethylphenyl) Pyridinium Derivative by MALDI LIFT-TOF/TOF Mass Spectrometry. J Am Soc Mass Spectrom 29:1688-1699|
|Köster, Stefan; Upadhyay, Sandeep; Philips, Jennifer A (2018) Why macrophages cannot LAP up TB. Autophagy 14:552-554|
|Köster, Stefan; Upadhyay, Sandeep; Chandra, Pallavi et al. (2017) Mycobacterium tuberculosis is protected from NADPH oxidase and LC3-associated phagocytosis by the LCP protein CpsA. Proc Natl Acad Sci U S A 114:E8711-E8720|