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Updated: Feb 21, 2026
Preparation of Mycobacterium Tuberculosis Culture Filtrate to Understand TB Pathogenesis
Published on: March 28, 2025
Mycobacterium tuberculosis is protected from NADPH oxidase and LC3-associated phagocytosis by the LCP protein CpsA
Stefan Köster1, Sandeep Upadhyay2,3, Pallavi Chandra2,3
1Division of Infectious Diseases, Department of Medicine, New York University School of Medicine, New York, NY 10016.
Abstract:
Mycobacterium tuberculosis' success as a pathogen comes from its ability to evade degradation by macrophages. Normally macrophages clear microorganisms that activate pathogen-recognition receptors (PRRs) through a lysosomal-trafficking pathway called "LC3-associated phagocytosis" (LAP). Although Mtuberculosis activates numerous PRRs, for reasons that are poorly understood LAP does not substantially contribute to Mtuberculosis control. LAP depends upon reactive oxygen species (ROS) generated by NADPH oxidase, but Mtuberculosis fails to generate a robust oxidative response. Here, we show that CpsA, a LytR-CpsA-Psr (LCP) domain-containing protein, is required for Mtuberculosis to evade killing by NADPH oxidase and LAP. Unlike phagosomes containing wild-type bacilli, phagosomes containing the ΔcpsA mutant recruited NADPH oxidase, produced ROS, associated with LC3, and matured into antibacterial lysosomes. Moreover, CpsA was sufficient to impair NADPH oxidase recruitment to fungal particles that are normally cleared by LAP. Intracellular survival of the ΔcpsA mutant was largely restored in macrophages missing LAP components (Nox2, Rubicon, Beclin, Atg5, Atg7, or Atg16L1) but not in macrophages defective in a related, canonical autophagy pathway (Atg14, Ulk1, or cGAS). The ΔcpsA mutant was highly impaired in vivo, and its growth was partially restored in mice deficient in NADPH oxidase, Atg5, or Atg7, demonstrating that CpsA makes a significant contribution to the resistance of Mtuberculosis to NADPH oxidase and LC3 trafficking in vivo. Overall, our findings reveal an essential role of CpsA in innate immune evasion and suggest that LCP proteins have functions beyond their previously known role in cell-wall metabolism.
Insights
Mycobacterium tuberculosis evades macrophage killing using CpsA protein to block LC3-associated phagocytosis (LAP) and reactive oxygen species (ROS) production. This study identifies CpsA as crucial for tuberculosis immune evasion and survival.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Mycobacterium tuberculosis evades macrophage degradation, a key factor in its pathogenicity.
- LC3-associated phagocytosis (LAP) is a macrophage pathway for clearing microbes, but M. tuberculosis largely escapes it.
- M. tuberculosis fails to induce a strong oxidative burst, hindering its clearance via LAP.
Purpose of the Study:
- To investigate the role of CpsA in M. tuberculosis evasion of host immune responses.
- To determine if CpsA interferes with the LAP pathway and NADPH oxidase activity.
Main Methods:
- Compared phagosome maturation and immune marker recruitment for wild-type M. tuberculosis and a ΔcpsA mutant in macrophages.
- Assessed CpsA's ability to inhibit LAP in fungal particle clearance assays.
- Analyzed the intracellular survival of the ΔcpsA mutant in macrophages with genetic deficiencies in LAP or autophagy components.
- Evaluated the in vivo virulence of the ΔcpsA mutant in wild-type and knockout mice.
Main Results:
- Phagosomes with the ΔcpsA mutant, unlike wild-type, recruited NADPH oxidase, produced reactive oxygen species (ROS), and matured into antibacterial lysosomes.
- CpsA inhibited NADPH oxidase recruitment to fungal particles, demonstrating its role in blocking LAP.
- Intracellular survival of the ΔcpsA mutant was significantly restored in macrophages lacking LAP components but not canonical autophagy components.
- The ΔcpsA mutant showed impaired in vivo growth, which was partially restored in mice deficient in NADPH oxidase or key autophagy proteins.
Conclusions:
- CpsA is essential for Mycobacterium tuberculosis to evade killing by NADPH oxidase and the LAP pathway.
- CpsA actively inhibits the host's innate immune response, specifically the LAP-mediated clearance mechanism.
- This study reveals a novel role for LytR-CpsA-Psr (LCP) domain proteins in bacterial immune evasion beyond cell-wall metabolism.
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