Aldehyde accumulation in Mycobacterium tuberculosis with defective proteasomal degradation results in copper sensitivity

Abstract

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 (pHBA) in M. tuberculosis mutants with defective proteasomal degradation. In this study, we found that pHBA accumulation was also responsible for Cu sensitivity in these strains. We showed that exogenous addition of pHBA to wild-type M. tuberculosis cultures sensitized bacteria to Cu to a degree similar to that of a proteasomal degradation mutant. We determined that pHBA reduced the production and function of critical Cu resistance proteins of the regulated in copper repressor (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.

Keywords: Mycobacterium tuberculosis; aldehyde; copper; proteasome.

Fig 1

Copper (Cu) sensitivity of a Pup-proteasome system (PPS) mutant is suppressed by mutations in log or glpK. (A) Cu sensitivity assay shows a PPS M. tuberculosis mutant is hypersensitive to Cu. Bacteria were incubated for 10 days in the indicated CuSO₄ concentrations. “Input” (white bars) indicates CFU at the beginning of the experiment, and “0” indicates how many bacteria were present after 10 days of incubation without added CuSO₄ (striped bars). Bars represent mean with SD. “<LOD” indicates below the limit of detection (100 CFU). Significant differences were calculated, comparing CFU of strains to the first strain on the x-axis at the same CuSO₄ concentration using an unpaired t-test with *P < 0.05; ***P < 0.001. Unlabeled bars showed no significant differences. Right panel: Given the slower growth of the mpa mutant, we calculated the ratio of the mean output CFU to mean input CFU for 30 µM CuSO₄, which more accurately describes the survival of this strain in Cu. (B) Cu sensitivity assay performed as in (A) with previously characterized mpa strains suppressed for nitric oxide sensitivity. (C) Exogenous addition of para-hydroxybenzaldehyde (pHBA) sensitized wild-type (WT) M. tuberculosis to Cu. Cu sensitivity assays performed with WT M. tuberculosis incubated with or without 1.2 mM pHBA for 24 hours prior to addition of CuSO₄. For all data shown, experiments are representative of at least two independent experiments, each done in technical triplicate.

Fig 2

Volcano plot of significantly regulated genes in untreated versus para-hydroxybenzaldehyde-treated M. tuberculosis. x-axis: log₂ fold change; y-axis: negative log₁₀ P-value. Dotted horizontal line indicates cutoff for genes to be considered significantly differentially regulated (P < 0.01). Dotted vertical line indicates division between upregulated (black dots) and downregulated (blue dots) genes (log₂ fold change = 0). Among the labeled genes are the RicR regulon (Rv0846, mmcO, lpqS, Rv1706A, socAB, mymT, cysK2, Rv2963, ricR) and select members of the Zur regulon (rpmB2, rpsN2, rpmG1).

Fig 3

MmcO levels are reduced in a Pup-proteasome system mutant and in para-hydroxybenzaldehyde (pHBA)-treated M. tuberculosis. (A) M. tuberculosis strains indicated were grown in Sauton minimal media and treated with 50 µM CuSO₄ for 24 hours prior to collection of equivalent amounts of bacteria for whole-cell lysates and preparation for immunoblotting. Proteins were detected using polyclonal rabbit antibodies raised against MmcO or the β-subunit of the proteasome (PrcB) as a loading control. This experiment is a representative of three biological replicates. Below: quantification of band intensity from three independent experiments with MmcO levels normalized to PrcB levels before calculating the intensity ratio between each strain compared to the wild-type (WT) strain. Bars indicate mean with SD error bars. Significance was calculated using an unpaired t-test to WT with *P < 0.05; **P < 0.01. Unlabeled bars showed no significant differences. (B) Immunoblotting for MmcO in strains treated with or without 1.2 mM pHBA for 4 hours before treatment with CuSO₄. Below: quantification as in (A) of MmcO levels normalized to PrcB levels for each strain and condition, relative to MmcO levels in the WT strain treated with copper (Cu) only. Data are representative of two independent experiments. Bars indicate mean with SD error bars.

Fig 4

MymT activity is reduced in the presence of pHBA. (A) Cu sensitivity assay of a ricR mutant after preincubation with para-hydroxybenzaldehyde (pHBA). Data are representative of two independent experiments, each done in technical triplicate. (B) MymT Cu(I)-thiolate luminescence (excitation = 280 nm, emission = 595 nm, cutoff = 325 nm). Wild-type (WT) M. tuberculosis lysates exhibited a peak in luminescence in fraction 34, which was abolished in lysates of a mymT mutant. (C) Fractionated lysates from M. tuberculosis ricR mutant strain treated with or without pHBA. Inset: quantification of fold change between two experiments. Lower section: immunoblot for MymT of pooled fractions corresponding to the maximal fluorescence peak. Arrowhead indicates full-length MymT. (D) Fractionated M. tuberculosis lysates from WT, mpa, and mpa log strains. Inset: quantification of fold change between two independent experiments. All quantifications were analyzed for significance using an unpaired t-test with *P < 0.05; **P < 0.01. Unlabeled bars did not show significant differences.

Fig 5

Methylglyoxal sensitizes M. tuberculosis to copper (Cu) and disrupts MymT Cu binding. (A) Cu sensitivity assay with the addition of methylglyoxal (MG) to wild-type (WT) bacteria at day 0. Significant difference was calculated comparing with the first strain on the x-axis at the same CuSO₄ concentration using an unpaired t-test with ***P < 0.001. (B) MymT Cu(I)-thiolate luminescence with an M. tuberculosis ricR mutant treated with or without MG. Inset: quantification of peak fold change relative to untreated, analyzed for significance using a two-tailed t-test (P < 0.05). Data are representative of two experiments.

Fig 6

Proposed model of aldehyde sensitization of M. tuberculosis to copper (Cu). Left: in the absence of aldehyde, Cu(I) enters the cytoplasmic space through unknown transporters. Cu prevents RicR from binding to DNA, allowing expression of the RicR regulon genes, some of which mitigate Cu(I) toxicity, MymT sequesters Cu(I) to prevent it from damaging the cell, and MmcO is a periplasmic multicopper oxidase that oxidizes Cu(I) to less toxic Cu(II). Right: in the presence of aldehyde, RicR cannot release from DNA, even in the presence of Cu, leading to repression of the RicR regulon and the observed Cu sensitivity of aldehyde-treated and Pup-proteasome system mutant M. tuberculosis strains. Additionally, MymT is unable to bind Cu in the presence of aldehyde.