Periodic Reporting for period 1 - MTB-DETOX (Molecular Mechanisms for Host-Mediated Metal Poisoning Detoxification in Mycobacterium tuberculosis)
Okres sprawozdawczy: 2022-09-01 do 2024-08-31
Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (Mtb), claims 1.5 million lives annually. Upon inhalation, Mtb reaches the alveoli of the lungs, where it is engulfed by macrophages and enclosed in phagosomes, specialized vacuoles designed to degrade pathogens. Inside the phagosome, Mtb is exposed to various stresses, including nutrient deprivation, hypoxia, reactive oxygen and nitrogen species, and acidification. Despite these hostile conditions, Mtb is an exceptionally resilient pathogen, having evolved mechanisms to survive and persist within its host.
In a seminal study (PMID: 21925112), our lab discovered the accumulation of zinc within macrophage phagosomes during Mtb infection, leading to the identification of a novel zinc detoxification system in Mtb. This system consists of a membrane pump, CtpC, and a previously unknown metallochaperone, PacL1, which is essential for stabilizing CtpC (PMID: 35961955). Our research demonstrated that the PacL1/CtpC system is critical for Mtb replication within macrophages, suggesting that bacterial metal detoxification mechanisms represent promising targets for novel drug development.
Mtb possesses two additional homologous systems to PacL1/CtpC, named PacL2/CtpG and PacL3/CtpV. While CtpV has been implicated in copper tolerance, the role of CtpG remains unclear. Furthermore, the biological functions of PacL2 and PacL3 in Mtb were completely unknown. The first objective of my project was to investigate the involvement of the PacL2/CtpG and PacL3/CtpV systems in metal detoxification and pathogen survival during infection, as well as to elucidate the specific roles of the uncharacterized PacL2 and PacL3 proteins.
Preliminary findings from our laboratory revealed that PacL1, PacL2, and PacL3 colocalize in dynamic patches at the plasma membrane, suggesting the existence of uncharted metal efflux platforms composed of multiple metal chaperones (PacL proteins) and efflux pumps (Ctp proteins). However, the structure, composition, and dynamics of these platforms, along with the potential impact of their formation on efflux efficiency, remained completely unknown. Therefore, the second objective of my project was to characterize the composition and dynamics of these uncharted structures and to explore the collaborative functions of the PacL1/CtpC, PacL2/CtpG, and PacL3/CtpV systems.
In a seminal study (PMID: 21925112), our lab discovered the accumulation of zinc within macrophage phagosomes during Mtb infection, leading to the identification of a novel zinc detoxification system in Mtb. This system consists of a membrane pump, CtpC, and a previously unknown metallochaperone, PacL1, which is essential for stabilizing CtpC (PMID: 35961955). Our research demonstrated that the PacL1/CtpC system is critical for Mtb replication within macrophages, suggesting that bacterial metal detoxification mechanisms represent promising targets for novel drug development.
Mtb possesses two additional homologous systems to PacL1/CtpC, named PacL2/CtpG and PacL3/CtpV. While CtpV has been implicated in copper tolerance, the role of CtpG remains unclear. Furthermore, the biological functions of PacL2 and PacL3 in Mtb were completely unknown. The first objective of my project was to investigate the involvement of the PacL2/CtpG and PacL3/CtpV systems in metal detoxification and pathogen survival during infection, as well as to elucidate the specific roles of the uncharacterized PacL2 and PacL3 proteins.
Preliminary findings from our laboratory revealed that PacL1, PacL2, and PacL3 colocalize in dynamic patches at the plasma membrane, suggesting the existence of uncharted metal efflux platforms composed of multiple metal chaperones (PacL proteins) and efflux pumps (Ctp proteins). However, the structure, composition, and dynamics of these platforms, along with the potential impact of their formation on efflux efficiency, remained completely unknown. Therefore, the second objective of my project was to characterize the composition and dynamics of these uncharted structures and to explore the collaborative functions of the PacL1/CtpC, PacL2/CtpG, and PacL3/CtpV systems.
Over the course of the two last years, we did not observe any involvement of the PacL3/CtpV system in metal intoxication tolerance in mycobacteria. However, my research has produced promising unpublished results. By generating deletion mutants of pacL2 and ctpG in Mtb, we discovered that the PacL2/CtpG system specifically confers tolerance to cadmium, but not to other metal ions such as copper, zinc, nickel, iron, or manganese. We also found that PacL2 is crucial for CtpG function, colocalizing with the pump in dynamic patches at the plasma membrane and ensuring its stability. Interestingly, using mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques on purified cytoplasmic domain of PacL2, we determined that, unlike PacL1, PacL2 does not bind metals, revealing a lack of metallochaperone activity.
My findings also revealed an unexpected collaboration between the PacL1/CtpC and PacL2/CtpG systems. We discovered that, in addition to conferring zinc tolerance, the PacL1/CtpC system also provides moderate cadmium tolerance to Mtb. Furthermore, we demonstrated that PacL1 and PacL2 physically interact, with both chaperones equally capable of stabilizing CtpG in membrane clusters, indicating a close functional interconnection between PacL1/CtpC and PacL2/CtpG systems. Using high-resolution microscopy (PALM), we thoroughly characterized these novel multi-metal efflux platforms. Our analysis revealed that 60% of cellular PacL2 is organized into an average of 13 membrane patches per cell, with patch sizes ranging from 10^5 nm³ to 10^8 nm³.
My findings also revealed an unexpected collaboration between the PacL1/CtpC and PacL2/CtpG systems. We discovered that, in addition to conferring zinc tolerance, the PacL1/CtpC system also provides moderate cadmium tolerance to Mtb. Furthermore, we demonstrated that PacL1 and PacL2 physically interact, with both chaperones equally capable of stabilizing CtpG in membrane clusters, indicating a close functional interconnection between PacL1/CtpC and PacL2/CtpG systems. Using high-resolution microscopy (PALM), we thoroughly characterized these novel multi-metal efflux platforms. Our analysis revealed that 60% of cellular PacL2 is organized into an average of 13 membrane patches per cell, with patch sizes ranging from 10^5 nm³ to 10^8 nm³.
My research has characterized atypical multi-metal efflux platforms in Mtb that plays a key role in both zinc and cadmium tolerance. While cadmium has no known biological functions in bacteria, it interacts with nucleic acids, competes with essential metal cofactors in proteins, and increases bacterial susceptibility to oxidative stress by triggering the production of reactive oxygen species. However, the potential role of cadmium in immune-mediated pathogen clearance remains unexplored. To address this, I secured a two-year postdoctoral fellowship from ANRS to investigate the PacL2/CtpG system in Mtb during infection, aiming to uncover the contribution of cadmium to macrophage-driven pathogen clearance. This research could potentially identify novel Mtb pathways that could serve as targets for new drugs to treat tuberculosis.