substantially more itaconate than the human THP-1 monocyte cell line. In summary, we 23 examine the role of itaconate as an antibacterial metabolite in mouse and human macrophage, 24 characterize the regulation of Salmonella's defense against it, and develop it as a convenient 25 itaconate biosensor and inducible promoter system. 26 27 Importance 28 In response to invading bacteria, immune cells can produce a molecule called itaconate, 29 which can inhibit microbial metabolism. Here we show that itaconate can also directly kill Introduction 39 The mammalian immune system includes a multitude of weapons to defend against 40 invading microbes and successful pathogens have evolved a plethora of mechanisms to evade, 41 manipulate, or even benefit from these immune responses. One such pathogen, Salmonella growth of, or kill, invading bacteria. 66 If itaconate is an immune-derived antibacterial metabolite then it follows logically that 67 successful pathogens must have methods to evade its effects. Indeed an operon has been 68 identified in Yersinia (ripABC for 'required for intracellular proliferation') that encodes three 69 enzymes catalyzing the ATP/succinyl-CoA-dependent degradation of itaconate into pyruvate 70 and acetyl-CoA 17 . The operon is not restricted to Yersinia and a variety of other bacteria 71 including Pseudomonas encode homologs or functional analogs of these enzymes. Several 72 lineages of Salmonella enterica harbor a cluster of genes (e.g. genes STM3120-STM3117 in S. 5 Typhimurium strain LT2) within SPI-13 that we refer to here as the 'itaconate response operon' 74 (IRO). Interestingly, the IRO genes of Salmonella have been shown to be induced heavily in 75 macrophage but not under any other condition tested, supporting a role in degrading 76 macrophage-produced itaconate [18] [19] [20] . High throughput screens have suggested that genes from 77 this operon are important for Salmonella survival in mice [21] [22] [23] . Furthermore, it has also been 78 shown that SPI-13 is present in many generalist S. enterica serovars but not in some human-79 restricted serovars of Salmonella (e.g. serovars Typhi and Paratyphi A, which encode SPI-8) 80 possibly due to reduced itaconate synthesis by human macrophages 24 . 81 In this work we show that itaconate is bactericidal at low but not neutral pH and 82 elucidate the regulation of the Salmonella IRO and its induction in mouse and human 83 macrophage. We show that the promoter of the IRO (P IRO ) is specifically induced by itaconate 84 and that the LysR family transcriptional regulator encoded by the upstream gene, STM3121 85 (which we propose to name ripR), is both necessary and sufficient for this induction. 86 Furthermore, using P IRO with a GFP reporter, we develop a semi-quantitative itaconate 87 biosensor and employ it to show that the IRO is induced heavily in the J774 mouse macrophage 88 cell line but requires interferon-γ (IFN-γ) stimulation to show a detectable response in the THP-1 89 human monocyte cell line. 
The Salmonella IRO is induced specifically by itaconate in a RipR-dependent manner 93 The Salmonella pathogenicity island-13 includes genes encoding RipC/Ccl (STM3120 in 94 strain LT2), RipB/Ich (STM3119) and RipA/Ict (STM3118), whose homologs have been 6 demonstrated to degrade itaconate into pyruvate and acetyl-CoA 17 . This operon, which we 96 refer to as the itaconate response operon (IRO), appears to also include the STM3117 gene 97 (encoding a predicted glyoxalase-domain containing protein) and is adjacent to the STM3121 98 (ripR) gene on the reverse DNA strand (Figure 2 ). 99 The IRO is strongly induced in cultured mouse macrophages but, according to 100 expression data from SalCom and other published work, is seemingly independent of known 101 virulence regulators (PhoP, RpoS, SsrA/B, OmpR, SlyA, etc.). We hypothesized that itaconate 102 may act as an inducer of IRO expression via the adjacent LysR-family regulator encoded by 103 STM3121. To assess this, we constructed a plasmid-borne fusion of the operon's promoter 104 (P IRO ) to superfolder GFP (sfGFP) as a reporter 25 . Indeed, we found that the P IRO promoter was 105 induced highly in the presence of itaconate and this response was entirely dependent on the 106 presence of RipR as neither a Salmonella ripR deletion mutant nor the same reporter plasmid in 107 E. coli K12 (which does not encode ripR) showed induction ( Figure 3A ). In contrast, when the 108 ripR gene was included on the reporter plasmid, itaconate-induced P IRO expression was restored 109 in both the ΔripR Salmonella strain and in E. coli. These data not only demonstrate that P IRO is 110 induced in response to itaconate, but also that the neighbouring gene, ripR, is both necessary 111 and sufficient for this induction. 112 To assess if the IRO promoter is induced specifically by itaconate, we examined 113 induction of the P IRO reporter plasmid in media supplemented with a panel of similar 114 metabolites. While mesaconate, citramalate and methylsuccinate (in order of induction 115 strength) slightly induced expression, induction by itaconate was drastically more pronounced, 116 suggesting that it is the principal inducer ( Figure 3B ). Notably, similar results were obtained in 7 complex media (LB) and in MOPS minimal media with either glucose or glycerol as a carbon 118 source, suggesting that the induction only requires itaconate and not additional factors in the 119 media ( Figure S2 ). Furthermore, induction by itaconate occurred in a dose dependent manner 120 indicating that the reporter can be used to semi-quantitatively assess itaconate concentrations 121 encountered by the bacteria ( Figure S3 ).
Itaconate import is independent of the dicarboxylate transporter DctA 124 Another important question was whether or not itaconate required the primary aerobic 125 dicarboxylate transporter, DctA, for import into the cell. We compared itaconate-mediated 126 induction of the P IRO -sfGFP reporter in wild-type or ΔdctA Salmonella. Interestingly, we found 127 that the IRO promoter was still heavily induced in the dctA mutant in the presence of itaconate 128 ( Figure S4 ). This demonstrates that the DctA dicarboxylate transporter is not required for 129 itaconate-mediated induction of P IRO , suggesting that itaconate import is independent of dctA. 132 Previous work has demonstrated that itaconate can inhibit the function of the 133 glyoxylate shunt enzyme, AceA, and act as a bacteriostatic agent when bacteria rely on carbon 134 sources such as acetate that require this pathway 11-13 . It has also been suggested that 135 itaconate can inhibit bacteria by influencing media pH and at least one publication has 136 demonstrated that it can have bactericidal activity 15,16 . To clarify this later phenotype we 137 hypothesized that the dicarboxylic acid chemistry of itaconate would allow it to act in a 138 bactericidal fashion at low pH by acting as a proton shuttle. In brief, the carboxyl groups of 8 itaconate (pK a = 5.5 and 3.8) protonate and lose their charge at lower pH allowing them to 140 traverse the bacterial membrane and release the protons in the more neutral pH of the 141 cytoplasm, thereby exacerbating acid stress ( Figure 1A ). 142 To emulate the intracellular conditions that Salmonella may encounter in a Salmonella 143 containing vacuole (SCV) of a macrophage we added itaconate to LPM media and then acidified 144 to pH 4.4, 5.0, or 5.8 to cover a range from the most acidic to more regular estimates of SCV pH 145 [26] [27] [28] . Indeed we found that wild-type Salmonella showed a 1000-fold decrease in survival after 146 3h hours at pH 4.4 with itaconate ( Figure 1B ). This lethality was alleviated at higher pH and also 147 occurred using a similar dicarboxylic acid, succinate. Importantly, the bactericidal effect was 148 also dependent on the presence of itaconate or succinate, as pH 4.4 LPM did not kill Salmonella 149 in the absence of a dicarboxylic acid ( Figure S1A ). Interestingly, deletion of the entire IRO 150 (STM3120-STM3117) or aceA had no effect, but deletion of the general stress response sigma The inhibitory effect of itaconate on AceA, its bactericidal activity under acidic 158 conditions, and the synthesis of itaconate in macrophage combine to support the concept that 159 these immune cells may be employing itaconate as an antibacterial compound. As a successful 160 pathogen, Salmonella has adapted to survive in activated macrophage and multiple previous 9 works have examined how IRO genes may influence Salmonella survival and virulence 21-23 . In 162 our hands, we found no significant reduction in survival of ΔIRO or ΔripR strains in the mouse 163 J774 macrophage cell line ( Figure S5 ). When the macrophages were pre-stimulated with IFN-γ, 164 there was a slight reduction in survival relative to wild-type, but this was not significant when 165 compared to an aceA mutant that showed no survival defect. In contrast, the growth of a phoP 166 deletion control strain was inhibited by macrophages even without IFN-γ. To examine itaconate levels encountered by intracellular bacteria inside macrophage, 176 we employed our P IRO -sfGFP reporter plasmid as a biosensor. By including a constitutively 177 expressed mCherry gene on the same plasmid, we could microscopically observe individual 178 bacteria inside macrophage and obtain semi-quantitative data by generating a GFP/mCherry 179 fluorescence ratio as an indicator of P IRO induction and accordingly itaconate levels. Using this 180 system, we observed strong induction of the P IRO promoter for wild-type Salmonella in J774 181 mouse macrophage and this signal was absent in the ΔripR control ( Figure 4) . Furthermore, the 182 response could also be observed in E. coli if RipR was encoded on the same plasmid, 10 demonstrating that bacteria that are poorly adapted to intracellular survival also encounter 184 itaconate in macrophage. 185 In contrast to the mouse cell line, unstimulated human THP-1 monocytes showed 186 negligible itaconate levels as very few of the bacterial reporters showed any green fluorescence 187 above background levels ( Figure 4) . The bacteria did express the constitutive mCherry and 188 could be observed in the macrophage, suggesting that the lack of green fluorescence was not 195 In this work we demonstrate that itaconate becomes bactericidal at acidic pH, 196 suggesting an additional mechanism for itaconate to act as an antibacterial metabolite beyond 197 inhibition of AceA. Thus, elevated itaconate levels in macrophage may act to inhibit bacterial 198 metabolism while also exacerbating acid stress on microbes in the phagosome. Protonation of 11 higher may contribute to why Salmonella manipulates the SCV to maintain a pH closer to 5.0 in 206 order to avoid this organic acid stress. Moreover, they imply that organic acids such as 207 itaconate and succinate may contribute to the antibacterial activity of acidified phagosomes. 208 The antibacterial potential of itaconate, its synthesis in activated macrophage, and the 209 localization of IRG1 to bacteria-containing vacuoles, support its potential role as a weapon 217 We find Salmonella responds to itaconate in vitro and intracellularly by strongly inducing 218 an operon encoding itaconate degradation proteins. This response is largely specific to 219 itaconate and is entirely dependent on the neighbouring gene (STM3121 in the prototypical 220 Salmonella enterica Sv. Typhimurium strain LT2), which we propose to rename ripR ('rip operon 221 regulator') to conform to earlier nomenclature. The ripR gene product is predicated to be a LysR 222 family transcriptional regulator, suggesting that itaconate induces IRO expression by interacting 12 requirement for growth on carbon sources independent of the glyoxylate shunt and also 228 growth at neutral or alkaline pH, as we demonstrate that itaconate is bactericidal under acidic 229 conditions. However, for many studies these conditions are met, adding P IRO to the repertoire 230 of available inducible promoter systems. shown below. Identities of ccl, ich, and ict are shown above as previously reported 17 . STM3117 18 appears to be encoded in the same operon however its function remains unknown. In this work 360 we designate the names 'ripR' for STM3121, 'itaconate response operon' (IRO) for STM3120-361 STM3117, and 'P IRO ' for the IRO promoter. (17). STM3117 appears to be encoded in the same operon however its function remains unknown. In this work we designate the names 'ripR' for STM3121, 'itaconate response operon' (IRO) for STM3120-STM3117, and 'P IRO ' for the IRO promoter.
Itaconate response operon (IRO) P IRO Figure 3 : RipR is necessary and sufficient to induce P IRO expression in response to itaconate. Expression of P IRO -sfGFP in wild-type (WT) or ripR knockout (∆ripR) Salmonella, or in E. coli. Figures show GFP fluorescence normalized to optical density at 600nm (OD 600 ) after 16h of growth in LB alone or supplemented with 0.2% itaconate (A) or similar metabolites (B). Data are the average of at least three biological replicates and error bars show one standard deviation. pP IRO , plasmidborne transcriptional fusion of P IRO to sfGFP; pRipR-P IRO , pP IRO with the ripR gene and its native promoter included on the plasmid. A Games-Howell ANOVA was conducted comparing with and without itaconate for each strain (A), or comparing WT to ΔripR (B) for each added metabolite. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Figure 4 , expression of P IRO -sfGFP in intracellular wild-type (WT) or ripR knockout (∆ripR) Salmonella containing the pICM-P IRO plasmid (constitutive mCherry expression). THP-1 macrophage were pre-treated with no stimulant, human IFN-γ, or IL-4 and IL-13 and infected with Salmonella for 8h. Data for WT Salmonella in THP-1 with no treatment or IFN-γ are shown in Figure 4 and are shown again here for comparison. As in Figure 4 , data shows relative fluorescence quantification of individual bacterial particles. Number of bacteria quantified is indicated above and totalled from at least three biological replicates. Boxes indicate first and third quartiles; central line, median; X, mean; whiskers, 95 th percentile; dots, all non-zero data points outside 95 th percentile. The y-axis changes scale at 1.5 to better show outliers. 