Effect of pH on the antimicrobial activity of the macrophage metabolite itaconate

Abstract

The production of itaconate by macrophages was only discovered in 2011. An increasing number of studies have since revealed essential biological functions for this small molecule, ranging from antimicrobial to immunomodulator. The antibacterial role of itaconate has however been questioned because the estimated concentration of itaconate in macrophages (low-millimolar) is lower than the minimum inhibitory concentration (MIC) of itaconate reported for several bacterial strains (low-to-mid-millimolar). We note that some of these investigations have tended to ignore the high acidity of this small diacid (pKas 3.85 and 5.45), thereby potentially biassing activity measurements. We measured the MIC of itaconate in Escherichia coli (not known to metabolize itaconate) and in Salmonella enterica serovar Typhimurium (known to metabolize itaconate) at varying pH values to probe the effect that pH has on itaconate toxicity. Herein, we demonstrate that the antimicrobial effect of itaconate is dependent upon the pH of the media and that itaconate does have antimicrobial activity at biologically relevant pH and concentrations. Under nutrient-poor conditions, the antimicrobial activity of itaconate in both E. coli and S. Typhimurium increased approximately 200-fold when the pH was dropped by one unit, whereas itaconate was not found to be toxic under nutrient rich conditions. Our results also reveal that the activity of itaconate is synergistic with acidity, yet is not a function of increased permeability with protonation. Similar experiments performed with succinate (a pKa-matched diacid) yielded drastically different results, consistent with a target-based mechanism of action for itaconate. Overall, our work shows the importance of controlling the pH when performing experiments with itaconic acid.

The Tumor Necrosis Factor Alpha and Interleukin 6 Auto-paracrine Signaling Loop Controls Mycobacterium avium Infection via Induction of IRF1/IRG1 in Human Primary Macrophages

“…However, the microbicidal activity of itaconate is most likely dependent on pH, since neutralization made it less efficient in inhibiting M. avium growth in culture. This was also recently suggested by others ( 42 , 43 ). There are also conflicting reports with regard to the membrane permeability of itaconate, and several derivatives have been made to facilitate cellular uptake ( 30 , 60 ).…”

“…6E ). It was recently shown that millimolar concentrations of itaconate acidify the medium but also that the bactericidal activity of itaconate is enhanced under acidic conditions, such as in the phagolysosome ( 42 , 43 ). In line with this, the neutralization of itaconate using NaOH reduced the growth-inhibitory effect of M. avium in liquid culture ( Fig.…”

Macrophages inhibit Coxiella burnetii by the ACOD1-itaconate pathway for containment of Q fever

“…Thus, ACOD1-mediated ITA generation appears to be required for curbing C. burnetii growth in macrophages. Since the first report of inhibition of M. tuberculosis by ITA (Michelucci et al, 2013), a similar effect has been shown for other intracellular bacteria, including M. avium (Gidon et al, 2021), L. pneumophila (Naujoks et al, 2016), Salmonella (S.) typhimurium (Chen et al, 2020), Brucella melitensis (Demars et al, 2021;Lacey et al, 2021) and F. tularensis (Jessop et al, 2020), but also for the extracellular bacteria Escherichia (E.) coli (Duncan et al, 2021) and Staphylococcus (S.) aureus (Singh et al, 2021). Remarkably, the ITA concentrations that we found to be required for inhibition of C. burnetii growth in macrophages or axenic cultures (0.6 -2.5 mM), were lower than those documented for the above-mentioned pathogens (> 5 mM).…”

“…For other bacteria, the mechanism of growth inhibition by ITA is unknown to date. Given the acidic conditions in the phagolysosome, the recent demonstration of synergy between ITA and low pH for blocking replication of E. coli and S. typhimurium (Duncan et al, 2021) is of interest.…”

Macrophages inhibit Coxiella burnetii by the ACOD1 ‐itaconate pathway for containment of Q fever

“…Thus, ACOD1‐mediated ITA generation appears to be required for curbing C. burnetii growth in macrophages. Since the first report of inhibition of M. tuberculosis by ITA (Michelucci et al , 2013 ), a similar effect has been shown for other intracellular bacteria, including M. avium (Gidon et al , 2021 ), L. pneumophila (Naujoks et al , 2016 ), Salmonella (S.) typhimurium (Chen et al , 2020 ), Brucella melitensis (Demars et al , 2021 ; Lacey et al , 2021 ) and F. tularensis (Jessop et al , 2020 ), but also for the extracellular bacteria Escherichia (E.) coli (Duncan et al , 2021 ) and Staphylococcus (S.) aureus (Singh et al , 2021 ). Remarkably, the ITA concentrations that we found to be required for inhibition of C. burnetii growth in macrophages or axenic cultures (0.6–2.5 mM), were lower than those documented for the above‐mentioned pathogens (>5 mM).…”