The human innate immunity factor apolipoprotein L-I (APOL1) protects against infection by several protozoan parasites, including Trypanosoma brucei brucei. Endocytosis and acidification of high-density lipoprotein (HDL)-associated APOL1 in trypanosome endosomes leads to eventual lysis of the parasite due to increased plasma membrane cation permeability, followed by colloid-osmotic swelling. It was previously shown that recombinant APOL1 inserts into planar lipid bilayers at acidic pH to form pH-gated non-selective cation channels that are opened upon pH neutralization. This corresponds to the pH changes encountered during endocytic-recycling, suggesting APOL1 forms a cytotoxic cation channel in the parasite plasma membrane. Currently, the mechanism and domains required for channel formation have yet to be elucidated, although a predicted Helix-Loop-Helix (H-L-H) was suggested to form pores by virtue of its similarity to bacterial pore-forming colicins. Here, we compare recombinant human and baboon APOL1 orthologs, along with inter-species chimeras and individual amino acid substitutions, to identify regions required for channel formation and pH gating in planar lipid bilayers. We found that while neutralization of glutamates within the H-L-H may be important for pH-dependent channel formation, there was no evidence of H-L-H involvement in either pH gating or ion selectivity. In contrast, we found two residues in the C-terminal domain (CTD), tyrosine-351 and glutamate-355, that influence pH gating properties, as well as a single residue, aspartate-348, which determines both cation selectivity and pH gating. These data point to the predicted transmembrane region closest to the APOL1 C-terminus as the pore-lining segment of this novel channel-forming protein.

Mycobacterium tuberculosis is a major human pathogen and the causative agent of tuberculosis disease. M. tuberculosis is able to persist in the face of host-derived antimicrobial molecules nitric oxide (NO) and copper (Cu). However, M. tuberculosis with defective proteasome activity is highly sensitive to NO and Cu, making the proteasome an attractive target for drug development. Previous work linked NO susceptibility with the accumulation of para -hydroxybenzaldehyde ( p HBA) in M. tuberculosis mutants with defective proteasomal degradation. In this study, we found that p HBA accumulation was also responsible for Cu sensitivity in these strains. We showed that exogenous addition of p HBA to wild-type M. tuberculosis cultures sensitized bacteria to Cu to a degree similar to that of a proteasomal degradation mutant. We determined that p HBA reduced the production and function of critical Cu resistance proteins of the r egulated i n c opper r epressor (RicR) regulon. Furthermore, we extended these Cu-sensitizing effects to an aldehyde that M. tuberculosis may face within the macrophage. Collectively, this study is the first to mechanistically propose how aldehydes can render M. tuberculosis susceptible to an existing host defense and could support a broader role for aldehydes in controlling M. tuberculosis infections. IMPORTANCE M. tuberculosis is a leading cause of death by a single infectious agent, causing 1.5 million deaths annually. An effective vaccine for M. tuberculosis infections is currently lacking, and prior infection does not typically provide robust immunity to subsequent infections. Nonetheless, immunocompetent humans can control M. tuberculosis infections for decades. For these reasons, a clear understanding of how mammalian immunity inhibits mycobacterial growth is warranted. In this study, we show aldehydes can increase M. tuberculosis susceptibility to copper, an established antibacterial metal used by immune cells to control M. tuberculosis and other microbes. Given that activated macrophages produce increased amounts of aldehydes during infection, we propose host-derived aldehydes may help control bacterial infections, making aldehydes a previously unappreciated antimicrobial defense.

Mycobacterium tuberculosis is a major human pathogen and the causative agent of tuberculosis disease. M. tuberculosis is able to persist in the face of host-derived antimicrobial molecules nitric oxide and copper. However, M. tuberculosis with defective proteasome activity is highly sensitive to nitric oxide and copper, making the proteasome an attractive target for drug development. Previous work linked nitric oxide susceptibility with the accumulation of para-hydroxybenzaldehyde in M. tuberculosis mutants with defective proteasomal degradation. In this study, we found that para-hydroxybenzaldehyde accumulation was also responsible for copper sensitivity in these strains. We showed that exogenous addition of para-hydroxybenzaldehyde to wild-type M. tuberculosis cultures sensitized bacteria to copper to a degree similar to that of a proteasomal degradation mutant. We determined that para-hydroxybenzaldehyde reduced the production and function of critical copper resistance proteins of the regulated in copper repressor (RicR) regulon. Further, we extended these Cu-sensitizing effects to an aldehyde that M. tuberculosis may face within the macrophage. Collectively, this study is the first to mechanistically propose how aldehydes can render M. tuberculosis susceptible to an existing host defense and could support a broader role for aldehydes in controlling M. tuberculosis infections.