Multiple targets of nitric oxide in the tricarboxylic acid cycle of Salmonella enterica serovar typhimurium

Abstract

Host nitric oxide (NO⋅) production is important for controlling intracellular bacterial pathogens, including Salmonella enterica serovar Typhimurium, but the underlying mechanisms are incompletely understood. S. Typhmurium 14028s is prototrophic for all amino acids but cannot synthesize methionine (M) or lysine (K) during nitrosative stress. Here, we show that NO⋅-induced MK auxotrophy results from reduced succinyl-CoA availability as a consequence of NO⋅ targeting of lipoamide-dependent lipoamide dehydrogenase (LpdA) activity. LpdA is an essential component of the pyruvate and α-ketoglutarate dehydrogenase complexes. Additional effects of NO⋅ on gene regulation prevent compensatory pathways of succinyl-CoA production. Microarray analysis indicates that over 50% of the transcriptional response of S. Typhimurium to nitrosative stress is attributable to LpdA inhibition. Bacterial methionine transport is essential for virulence in NO⋅-producing mice, demonstrating that NO⋅-induced MK auxotrophy occurs in vivo. These observations underscore the importance of metabolic targets for antimicrobial actions of NO⋅.

Figure 1. NO· induces a transient MK auxotrophy in S. Typhimurium

A. Representative growth of S. Typhimurium strain 14028s in M9 minimal medium with glucose as the sole carbon source with or without amino acid supplements (AA, See Materials and Methods). Cells were inoculated at an OD₆₀₀ = 0.05 in the presence or absence of 2 mM SperNO (NO·). See also Figure S1. B. The lag phase associated with “drop-out” amino acid supplementation in the presence of 2 mM SperNO. Cells were inoculated in media as above, except that amino acid supplements lacked the indicated amino acid. Lag phase is depicted as mean time to ½ maximal OD₆₀₀ of four independent growth curves. Error bars represent standard error. See also Table S1.

Figure 2. The NO·-induced MK auxotrophy stems from insufficient levels of the TCA cycle intermediate succinyl-CoA

A. Biosynthetic pathway for the aspartate amino acid family. The TCA cycle intermediate succinyl-CoA is required only for Met and Lys biosynthesis. Oxa = oxaloacetate, Asp = aspartate/aspartyl, CHO = semialdehyde, HHDPA = dihydropicolinate, HHDPAHH = tetrahydropicolinate, Suc = succinate/succinyl, AKP = amino-ketopimelate, DAP = diaminopimelate. B. The lag phase associated with various amino acid mixtures with or without supplementation with exogenous succinyl-CoA (at 0.15%). Lag phase is depicted as mean time to ½ maximal OD₆₀₀ of four independent growth curves. Error bars represent standard error. See also Figure S2.

Figure 3. Only exogenous succinate is capable of replenishing succinyl-CoA and MK biosynthesis during nitrosative stress in S. Typhimurium

A. The TCA cycle and pyruvate node of S. Typhimurium. PDH, pyruvate dehydrogenase (aceEFlpdA); PPC, phosphoenolpyruvate carboxylase (ppc); POX, pyruvate oxidase (poxB); ACK, acetate kinase (ackA); PTA, phosphate acetyltransferase (pta); GLT, citrate synthase (gltA); ACN, aconitase (acnA acnB); ICD, isocitrate dehydrogenase (icdA); αKDH, α-ketoglutarate dehydrogenase (sucABlpdA); SUC, succinyl-CoA synthase (sucCD); SDH, succinate dehydrogenase (sdhCDAB); FRD, fumarate reductase (frdABCD); FUM, fumarase (fumA fumB fumC); MDH, malate dehydrogenase (mdh); ICL, isocitrate lyase (aceA); MS, malate synthase (aceB). B. Representative suppression of the NO·-induced auxotrophy is achieved by supplementation with 0.15% Na·succinate, but not equivalent amounts of Na·fumarate or Na·α-ketoglutarate. Although supplementation of NO·-exposed 14028s with either citrate or isocitrate has a general growth-promoting effect, cells still require both M and K for maximal growth (data not shown).

Figure 4. NO· targets lipoamide-dependent enzyme complexes

A. Enzyme activities in cell-free extracts with increasing concentrations of NO·. NO· was administered by the addition of ProliNO (t_1/2 = 1.8 s). Percent Activity is derived from three independent biological replicates normalized to unexposed cell-free extracts B. LpdA-lipoic acid interactions are sensitive to NO·. The oxidation of dihydrolipoamide by LpdA from cell free extracts of WT S. Typhimurium 14028s was monitored in the presence of increasing NO· concentrations. Inset: Specific activity of cell free extracts (nmol dihydrolipoic acid oxidized · mg protein⁻¹ · min⁻¹) with or without 100 μM NO· (administered by addition of ProliNO). One U of purified porcine LpdA, (LpdA^por; Calzymes, San Luis Obispo, CA) was used as a positive control See also Figure S3.

Figure 5. Transcriptional responses of ΔlpdA and NO·-exposed S. Typhimurium 14028s

Microarray analyses were performed on three biologically independent RNA samples extracted from ΔlpdA or WT S. Typhimurium in the presence/absense of exogenous NO· (1 mM SperNO). Substantial overlap was observed in the transcriptional responses to these conditions. For a complete list of genes showing altered regulation. See also files S1 and S2.

Figure 6. NO· creates a regulatory block that inhibits the reductive branch of the TCA cycle

A. Expression levels of sdhC or frdA as determined by Q-RT PCR of three independent RNA samples from WT and isogenic Δfnr S. Typhimurium strain 14028s either untreated (blue columns) or exposed to NO· (red columns). Transcript levels relative to rpoD were determined by a modified ΔΔC_t method as previously described. B. Expressing the reductive TCA cycle in the presence of NO· restores MK prototrophy to S. Typhimurium. The frdABCD genes from 14028s were cloned and expressed from an IPTG-inducible promoter (p_trc). IPTG was added at 100 μM for each growth curve. M9 Glc medium was either unsupplemented (None), supplemented with all 20 amino acids (All), or with a mixture of amino acids lacking both Met and Lys (−MK). pTrc99a was used as a vector control. A Δsdh mutation was introduced to prevent futile cycling by high expression of both SDH and FRD activities.

Figure 7. High affinity D, L-methionine transport is required for virulence

A. Female C3H/HeN mice were inoculated i.p. with 2500 cfu of wild-type S. Typhimurium strain 14028s (n = 19) or an isogenic ΔmetD mutant (n = 19). To inhibit murine iNOS activity and NO·-production, mice were administered L-NIL in their drinking water and infected with WT (n = 9) or ΔmetD mutant S. Typhimurium (n = 14). No more than 5 mice were inoculated per group on a given day, and no significant variation was observed between days within experimental groups. Survival plots were analyzed using a log rank test for equality of survival function. B. Female C3H/HeN mice were inoculated i.p. with 2000 cfu comprised of a 1:1 mixture of WT S. Typhimurium 14028s and either an isogenic ΔmetD mutant (n = 5) or the complemented ΔmetD mutant strain (C′) carrying plasmid pJK688 (metNIQ) (n = 5). Eight days post-infection, livers and spleens were harvested and plated to determine the ratios of WT to mutant CFU (Competitive Index = C.I.), as described in Experimental Procedures. P values were determined using the Wilcoxon Rank Sum Test. See also Figure S4.