The Salmonella LysR family regulator, RipR, activates the SPI-13 encoded itaconate degradation cluster

Abstract

8Itaconate is a dicarboxylic acid that is able to inhibit the isocitrate lyase enzyme of the 9 bacterial glyoxylate shunt. Activated macrophage have been shown to produce itaconate, 10 suggesting that these immune cells may employ this metabolite as a weapon against invading 11 bacteria. Here we demonstrate that itaconate can exhibit bactericidal effects under acidic 12 conditions resembling the pH of a macrophage phagosome. In parallel, successful pathogens 13 including Salmonella have acquired a genetic operon encoding itaconate degradation proteins, 14 which are induced heavily in macrophage. We characterize the regulation of this operon by the 15 neighbouring gene, itaR, in specific response to itaconate. Moreover, we develop an itaconate 16 biosensor based on the operon promoter that can detect itaconate in a semi-quantitative 17 manner and, when combined with the itaR gene, is sufficient for itaconate-regulated expression 18 in E. coli. Using this biosensor with fluorescence microscopy, we observe bacteria responding to 19 itaconate in the phagosomes of macrophage and provide additional evidence that interferon-γ 20 stimulates macrophage itaconate synthesis and that mouse macrophage produce substantially 21 more itaconate than human cells. In summary, we examine the role of itaconate as an 22 antibacterial metabolite in mouse and human macrophage, characterize the regulation of 23 Salmonella's defense against it, and develop it as a convenient itaconate biosensor and 24 inducible promoter system. 25 26 Importance 27In response to invading bacteria, immune cells can produce a molecule called itaconate, 28 which can inhibit microbial metabolism. Here we show that itaconate can also directly kill 29 3 Salmonella when combined with moderate acidity, further supporting itaconate's role as an 30 antibacterial weapon. We also discover how Salmonella recognizes itaconate and activates a 31 defense to degrade it, and we harness this response to make a biosensor that detects the 32 presence of itaconate. This biosensor is versatile, working in Salmonella or harmless E. coli, and 33 can detect itaconate quantitatively in the environment and in immune cells. By understanding 34 how immune cells kill bacteria and how the microbes defend themselves, we can better 35 develop novel antibiotics to inhibit pathogens such as Salmonella. 36 37 42 Salmonella pathogenicity islands (SPI) that support its survival inside of a host organism. For 43 instance, Salmonella employs SPI-1 to invade non-phagocytic cells, and SPI-2 allows the bacteria 44 to survive intracellularly -including in macrophage -which is important for Salmonella 45 virulence (1-4). These traits allow Salmonella to invade the gut epithelium and induce intestinal 46 inflammation resulting in the characteristic gastroenteritis disease. Moreover, the induced 47 65 catalyzing the degradation of itaconate into pyruvate and acetyl-CoA (16). This operon is not 66 restricted to Yersinia and a variety of other pathogens encode homologs. These...

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

“…Bacterial growth is well known to be affected by pH [29][30][31], and failure to ensure a constant pH may considerably skew MIC measurements. Recent reports have also suggested that there may be synergy between pH and itaconate activity [32,33], although a systematic exploration of the phenomenon has yet to be reported. We elected to perform studies with Escherichia coli (a species that does not encode itaconate-degrading enzymes) and Salmonella enterica ser.…”

Itaconate: an antimicrobial metabolite of macrophages

“…It is well established that small organic acids are capable of inhibiting bacterial growth at low pH values, potentially via a "proton shuttle" effect. [69][70][71] With addition of an organic acid to growth medium, the pH decreases, favoring the protonated (uncharged) form of the acid, which may cross bacterial membranes more easily (Figure 3). Once inside the (less acidic) bacterial cytosol, the organic acid releases a proton, thereby reducing the cytosolic pH and causing toxicity.…”

“…However, organic acids have different toxicities that do not always correlate well with their respective pKa values, [72][73][74][75] implying that the exact molecular structure of the organic acid may lead to specific toxicity effects. Itaconate is known to have more potent antimicrobial activity at lower pH values, 60,71,76 and this has recently been investigated in more detail. 60 By comparing the MIC of itaconic acid with that of its sodium salt, Duncan et al demonstrated that the decrease in pH caused by itaconic acid addition to the growth medium increases the antimicrobial activity of itaconate against numerous bacterial species.…”

“…117 In S. Typhimurium, itaconate binds to the regulator protein RipR, which then forms a complex with the promoter of the rip operon (also known as itaconate response operon or IRO in this species). 71 This induces transcription of ict, ich, and ccl. Induction of the rip operon appears to be dependent only upon itaconate (or structural analogues such as mesaconate, citramalate, and methylsuccinate, albeit at a much lower level of induction), whether the growth medium is glucose-deprived or rich (e.g.…”

“…Lysogeny broth or glucose-containing minimal media). 71 It is not currently known if this regulatory mechanism of the itaconate degradation pathway is conserved across other itaconate-degrading organisms.…”