The current RNA-seq study clarified the effects of yqiC during the colonization of Caco-2 cells on the expression of other Salmonella genes, particularly the negative regulation that occurs during pyrimidine and spermidine biosynthesis, osmoprotection, and DNA transcription and the positive regulation that occurs in ilvB operon, the tdc family, anaerobic dimethylsulfoxide reductase, the cytochrome c family, and NADH dehydrogenase. A few studies have reported the association of the aforementioned genes with bacterial colonization but not in ilvL (or ilvB operon) and dms genes. Early studies of E. coli and S. Typhimurium have revealed that carAB, pyrBI, pyrC, pyrD, pyrE, and pyrF are required for the biosynthesis of uridine monophosphate, which is the precursor of all pyrimidine nucleotides. The expression of pyr operons is repressed by nucleotides through the transcription attenuation control mechanism . The pyrE–deleted mutant exhibited a defect in the intestinal colonization of S. Typhimurium in chicks that cannot be restored by the salvage pathway, indicating the necessity of pyrE and de novo pyrimidine synthesis for colonization . Our RNA-seq results indicate yqiC inhibits the upregulation of pyrB, pyrI, pyrE, pyrD, and pyrC during S. Typhimurium colonization, and our GO analysis results indicate the involvement of pyrB in the cellular amino acid metabolic process and involvement of pyrI in metal ion binding. In addition, polyamines are essential for biofilm formation in E. coli. PotFGHI functions as a compensatory importer of spermidine when PotABCD is absent under biofilm-forming conditions . In the present study, potF was identified in 117 significantly upregulated genes and in the periplasmic space (through a GO analysis) after the depletion of yqiC in S. Typhimurium; this finding is consistent with the subcellular localization of YqiC . S. Typhimurium in chicken intestine lumen significantly upregulates the expression of the potFGHI operon . Therefore, potF can be negatively regulated by yqiC to affect Salmonella colonization. Moreover, the transient activation of tdcA in S. Typhimurium when bacterial growth shifted from aerobic to anaerobic growth; a tdcA mutation reduced the expression of the genes involved in flagellar biosynthesis, downregulated the expression of tdcBCDEG, and induced the expression of genes associated with energy metabolism, suggesting activation of carbon catabolism genes for cellular energy production before the full synthesis of ATP from anaerobic ETCs . In addition, our GO analysis of the tdc family revealed the involvement of tdcA in DNA-binding transcription factor activity and DNA-templated transcription, the involvement of tdcB in pyridoxal phosphate binding, and the involvement of tdcD in metal ion binding. The association of nrdD and nrfA with colonization was reported for other bacteria of the Enterobacteriaceae family than Salmonella. The knockout of nrdD attenuated the colonization of an adherent-invasive E. coli strain in murine gut mucosa , and a nrfA-disrupted mutant of Campylobacter jejuni significantly attenuated colonization in chicks . Our GO analysis revealed the involvement of nrfA in the five clusters, namely iron ion binding, heme binding, periplasmic space, microbial metabolism in diverse environments, and nitrogen metabolism.
The present study validated the findings of our previous study related to the characterization of non-SPI gene yqiC with respect to its role in regulating type 1 fimbriae, SPI-1 genes, and flagellin in S. Typihmurium SL1344 , which is similar to the phenotype of a SPI-19 locus SEN1005 in S. Enteritidis . The invH-mediated Sip effector proteins are important in early cecal inflammation by S. Typhimurium in mice colitis . Accordingly, sipA, sipC, and sipD were identified in the nine SPI-1 significantly downregulated genes of our RNA-seq analysis, emapplot analysis, and KEGG analysis. However, the role of yqiC in modulating SPI-2 genes is complex. Mutation in the SPI-2 gene hha induces no defect in S. Typhimurium colonization to the host gut , and this gene is not influenced by yqiC in Caco-2 cells by our RNA-seq analysis. In contrast to our previous finding regarding the downregulation of one representative SPI-2 gene sseB in ΔyqiC, the present RNA-seq revealed the diverse regulation of SPI-2 genes, including the downregulation of spvB, ttrS, and ttrA and upregulation of sseE and sscA; these findings suggest the presence of a complex mechanism involving the bidirectional regulation of yqiC and SPI-2 genes. In addition, the associations of SPI-3, SPI-4, SPI-5, and SPI-6 with bacterial colonization or with the intestinal lumen have been sporadically reported. The nonmotile and nonchemotactic S. Typhimurium in chicken intestinal lumen has been reported to exhibit the upregulation of SPI-3 (mgtC, rmbA, fidL, shdA, and misL) and SPI-5 (pipB) genes, suggesting a close physical interaction with the host during colonization . The SPI-3 gene-encoded MisL and the SPI-4 gene-encoded SiiC, SiiD, and SiiF assemble T1SS to secrete SiiE for the adhesion of Salmonella to intestinal epithelial cells during gut colonization [36, 37]. Similarly, a study of global transcriptomes revealed that the SPI-4 genes (siiABCDEF), the SPI-5 genes (sopB, pipB, and sigE), and the SPI-6 genes (sciJKNOR) are responsible for the colonization of S. enterica serovar Dublin in bovine mammary epithelial cells . The knockout of yqiC significantly downregulated the ydiA that encodes conserved hypothetical plasmid protein, suggesting that yqiC is required for expressing SPI-3 ydiA. Similar to the effect of cell association on SPI-4 and SPI-5 gene expression , colonization-associated yqiC significantly downregulated the SPI-4 gene siiD and the SPI-5 gene pipC. Although the SPI-6 genes sciJKNOR were downregulated in S. enterica serovar Dublin, we discovered that other SPI-6 genes safA and sciC were downregulated in S. Typhimurium after the depletion of yqiC and loss of colonization ability. Overall, the expression of type-1 fimbriae, SPI-1, and flagellin were regulated by yqiC, and several genes of SPI-2, -3, -4, -5, and -6 interacted with yqiC through unknown mechanisms that require further investigation.
To our knowledge, the biosynthesis of UQ-8 in E. coli requires the enzymes encoded by at least 15 ubi genes, including ubiC, ubiA, ubiD/X, ubiI, ubiB, ubiH, ubiE, ubiF, ubiG, ubiH, ubiJ, ubiK [7, 12, 39], ubiT, ubiU, and ubiV  through a novel oxygen-independent pathway. The biosynthesis of MK-8 in E. coli requires at least nine men genes, namely, menF, menD, menH, menC, menE, menB, menI, menA, and menG (also referred to as ubiE). The main difference between UQ and MK biosynthesis is that chorismate is converted into 4-hydroxybenzoate through UbiC for UQ synthesis and into isochorismate through MenF for MK synthesis [12, 41]. However, UQ and MK biosynthesis are not fully separate pathways. Required for the biosynthesis of both UQ and MK, UbiE (MenG), which is encoded by ubiE, is a nonspecific enzyme that can catalyze the C-methylation of 2-octaprenyl-6-methoxy-1,4-benzoquinol into 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinol in UQ biosynthesis; it also catalyzes the methylation of DMK-8 to MK-8 in the final step of MK biosynthesis in E. coli . In the E. coli strain MG1655 (alignment of yqiC is 77% identical to that of S. Typhimurium SL1344), the ubiI mutant had the highest correlation with the yqiC (also named ubiK) mutant that reduced UQ-8 to 18% and slightly increased MK-8 under aerobic conditions but was not detected under anaerobic conditions. In the S enterica strain 12,023 (alignment of yqiC is 75% identical to that of S. Typhimurium SL1344), the ubiK mutant also caused a 16-fold decrease in UQ-8, but no significant difference in MK-8 level was detected in the WT strain under aerobic conditions . However, our previous study reported that the absence of MK in S. Typhimurium SL1344 after the depletion of yqiC and the addition of MK reversed the effect of yqiC depletion on the expression of type-1 fimbrial, flagellar, SPI-1, and SPI-2 genes, which indicated the significant influence of MK on yqiC and its role as a upstream regulator of the virulence and ETC of S. Typhimurium . In the present study, yqiC was required for expressing menD. It requires more studies for validating whether yqiC serves as a regulator in the ETC through the modulation of men and/or ubi genes for maintaining the homeostasis between UQ and MK biosynthesis under various circumstances.
Studies have indicated the involvement of molybdenum, iron, and sulfur in bacterial virulence. Molybdoprotein oxidoreductase is an iron–sulfur cluster that is homologous to phs operon, which encodes thiosulfate reductase for thiosulfate reduction to contribute to the anaerobic energy metabolism in S. Typihmurium . The phylogenetic tree of the molybdenum subunits that form the dimethyl sulfoxide reductase superfamily in E coli includes ttr, dms, nar, and nap genes , which were present in our main emapplot network of ΔyqiC, indicating the close relationship of yqiC with molybdenum and iron–sulfur subunits. NarG is the only nitrate reductase for the colonization of E coli in mouse intestines . Under acute tolerance response, lacking of narZ encoding the nitrate reductase subunit NarZ results in S. Typhimurium deficiency and upregulation of dsrA encoding sRNA DsrA are associated with motility, adhesion, and invasion efficacy [46, 47], which were not found in our yqiC study in Caco-2 cells. However, napA disruption significantly attenuated the colonization by C. jejuni in the cecum of chickens . The napA mutant of S. Typhimurium exhibited a considerable growth defect in the low-nitrate colonic lumen of mice ; by contrast, the highest mortality rates of chickens challenged with mutants of S. gallinarum were associated with mutations in napA and narG, and additional attenuations were induced by a mutation in frdA and double mutations in dmsA and torC . The findings are consistent with our emapplot network of downregulation in napA, dmsA, torC, and narG after the mutation in yqiC that is associated with colonization and ETCs. In particular, during the early colonization of S. Typhimurium with Caco-2 cells, yqiC was required for expressing the six genes that encode dimethylsulfoxide reductase subunit A (napA), dimethylsulfoxide reductase subunit A (dmsA2, dmsA1, and dmsA), and two other unnamed genes (Fig. 3B). In addition, we discovered that yqiC downregulates ttrA to affect tetrathionate dehydrogenase. These four enzymes contribute to bacterial virulence and belong to the dimethyl sulfoxide reductase family; they have a subcellular location and exhibit a common structure comprising a Mo-containing subunit, an iron–sulfur protein, and a membrane-bound subunit with or without binding hemes .
The mechanisms involved cellular respiration in Salmonella virulence associated with bacterial colonization in hosts remain unclear. A cluster genes related to the carbohydrate metabolism and transportation required for intestinal colonization was identified using a library of targeted single-gene deletion mutants of S. Typhimurium inoculated in the ligated ileal loops of calves , and S. Typhimurium was revealed to use carbohydrates and their metabolites through the phosphoenolpyruvate-dependent phosphotransferase system . A study compared the global transcriptomes of highly pathogenic S. enterica serovar Dublin and the less pathogenic S. enterica serovar Cerro in their interactions with bovine mammary epithelial cells and identified the S. enterica genes responsible for Salmonella infection and colonization in cattle, including the genes associated with carbohydrate transport/metabolism, energy production/metabolism, and coenzyme transport/metabolism . A proteomic study of S. Typhimurium during the infection of HeLa epithelial cells revealed the preferential use of glycolysis, the pentose phosphate pathway, mixed acid fermentation, and nucleotide metabolism and the repression of the TCA cycle and aerobic and anaerobic respiration pathways . S. Typhimurium performs an incomplete TCA cycle in the anaerobic mammalian gut; however, a complete oxidative TCA cycle can be induced by inflammation-derived electron acceptors such that microbiota-derived succinate can be used as a carbon source during intestinal colonization . These findings are also reflected in our discovery of the decreased ATP production in S. Typhimurium after the deletion of yqiC, which is attributed to the complex role of yqiC in influencing the contribution of glycolysis, TCA cycles, and ETCs to the cell respiration that converts these nutrients into ATP.
The Seahorse XFp Analyzer was used to measure glycolysis and mitochondria respiration in the mammalian cells , and eukaryotic cells, including Caenorhabditis elegans (nematode) , Dictyostelium discoideum (amoeba) , Candida albicans , and Cryptococcus neoformans . Cellular respiration plays a similar role in mitochondrial respiration, and several studies have used the Seahorse XFp Analyzer to investigate mitochondria-absent prokaryotic bacteria such as E. coli, Staphylococcus aureus [24, 60] and Mycobacterium tuberculosis ; however, in this context, the research on Salmonella is limited. In the Seahorse Analyzer, ampicillin at a dose of 5 × MIC or 50 × MIC accelerates cellular respiration by increasing OCR, indicating the association of antibiotic efficacy and phenotypic resistance with cellular respiration in E. coli [24, 60]. By contrast, in our study, sublethal ampicillin did not have a considerable effect on the OCR and ECFR of S. Typhimurium or the phenotype of yqiC. A study that used the Seahorse Analyzer revealed that the iron–sulfur cluster biosynthesis protein SufT is required for glycolysis, oxidative phosphorylation, and survival in Mycobacterium tuberculosis after exposure to oxidative stress and nitric oxide ; this finding echoes our findings regarding the association of yqiC with electron transfer activity, iron–sulfur cluster assembly, and glycolysis in S. Typhimurium. Our RNA-seq analysis revealed the involvement of yqiC in energy and carbohydrate metabolism, and a series of experiments in the Seahorse XFp Analyzer further clarified how yqiC influences cellular respiration and glycolysis. Our cell phenotype energy test verified that yqiC influences cellular respiration more than glycolysis to maintain metabolic potential, which is achieved by inhibiting ATP synthase and uncoupling oxidative phosphorylation; this finding suggests that other metabolic pathways are responsible for increased oxygen consumption under energy stress. Furthermore, we revealed that yqiC is required for sufficient glycolysis and the maintenance of glycolytic capacity and glycolytic reserve. Therefore, the colonization-associated gene yqiC is expected to assist NTS in acquiring energy through cellular respiration and glycolysis to express NTS virulence, and oxygen consumption plays a major role in cellular respiration under energy stress conditions. Collectively, these findings are consistent with our previous findings regarding the phenotyping of yqiC (ubiK) as a regulator for the efficient aerobic biosynthesis of UQ and MK [4, 7]; however, the involved anaerobic effect requires further clarification.
S. Typhimurium and E. coli may differ with respect to the regulation of ETC complexes. The mutations in nuo and cyd operons suppressed the anaerobic growth of S. Typhimurium . In addition, mutations in the nuoG, nuoM, and nuoN of NDH-1 not only rescue motility, growth, and the rate of aerobic respiration but also use L-malate as the sole carbon source in a S. Typhimurium ubiA–ubiE mutant, suggesting that nuoG, nuoM, and nuoN suppress the electron flow activity of NDH-1 . Both ubiA and ubiE mutations do not lead to UQ biosynthesis and reduce the quinone pool, in which only ubiA mutations cause higher biosynthesis of MK than of DMK and only ubiE mutations deter the biosynthesis of UQ and MK while DMK biosynthesis continues to occur in S. Typhimurium ; this finding suggests that these nuo genes are negative regulators that influence the bridging roles of ubiA and ubiE, and ubiE in maintaining the equilibrium among UQ, MK, and DMK compositions in the total quinone pool. Researchers have explored the relationships of ETC complex genes with NTS growth. The S. gallinarum nuoG mutant was reported to be highly attenuated in the colonization that occurred in the caeca of chickens and the invasions that occurred in the liver or spleen of chickens . The S. Typhimurium genes involved in energy production and conversion (i.e., nuoJ, nuoI, napC, cyoD, frdD, nuoE, nuoF, cyoC, and cydA) were downregulated during colonization in chicken cecal lumen relative to their expression in broth cultures . By contrast, we examined the effects of yqiC on the expression of the five selected genes of the electron donating complexes NDH-1 (Nuo) and NDH-2 (Ndh), succinate dehydrogenase (SDH), and the electron accepting complexes cytochrome bo oxidases (Cyo) and cytochrome bd oxidases (Cyd) in the ETC of S. Typhimurium [13, 29] and the anaerobic effect on their expression. Our analysis indicated that yqiC depletion downregulated the expression of nuoE, ndh, sdhB, and cydA in both aerobic and anaerobic S. Typhimurium. However, yqiC depletion significantly upregulated the cyoC expression that was further reinforced by anaerobiosis, suggesting that yqiC is a suppressor of the expression of cyoC for receiving electrons in the ETC, particularly in anaerobic S. Typhimurium. This effect of yqiC depletion on the downregulation of nuoE, ndh, sdhB, and cydA and the upregulation of cyoC was reversed by S. Typhimurium colonization in Caco-2 cells with the significant upregulation of ndh and sdhB. The distinctive phenotype of the cyo genes from other ETC genes was also revealed in a study to exhibit cyo gene–involved cytochrome bo oxidase but not cytochrome bd-I and bd-II oxidases; therefore, it significantly contributes to the release of extracellular ATP in E coli and Salmonella and the survival of bacterial communities, playing roles in bacterial physiology other than that of an energy supplier . The exposure of S Typhimurium to anaerobiosis enhances virulence, adhesion to enterocytes and the penetration of mucus into host cells . Therefore, the modulation of yqiC in ETC complexes changes from downregulation to upregulation during colonization, and the unique expression of cyoC may play a role in the virulence of S. Typhimurium during its early interaction with intestinal epithelium.
The NADH/NAD+ ratio is a key metabolic marker of cellular state for balance in bacterial redox and for environmental adaptability, and a change in this ratio can influence metabolite distribution through the involvement of carbon sources under various oxidative states [25, 65]. Under aerobic conditions, E. coli uses the respiratory chain to oxidize NADH to NAD+ and channels redox energy to generate a proton gradient for ATP synthase. Anaerobically grown E. coli regenerates NAD+ from intermediates (e.g., pyruvate, oxaloacetic acids, malate, and acetyl-CoA) with NADH when no other electron acceptors (e.g., nitrate) are present [25, 65]. The NADH/NAD+ ratio is moderately adjusted by various carbon sources; the E. coli that is aerobically grown on acetate is an exception because it exhibits a considerably higher NADH/NAD+ ratio than that of glucose . In addition to the TCA cycle, the S. Typhimurium within epithelial cells can generate acetate and lactate under aerobic conditions through the overflow metabolism with the simultaneous synthesis of ATP and NADH . The total NADH/NAD+ intracellular pool is maintained in E. coli by NAD biosynthesis through the de novo pathway and by NAD recycling through the pyridine nucleotide salvage pathway. NAD does not limit metabolic rates because the generation of NADH (conversion of formate to CO2 and H2) and regeneration of NAD+ (efflux of succinate, ethanol, and lactate) can redistribute the metabolic fluxes in the central anaerobic metabolic pathway . At present, the contribution of ETC complexes to NADH/NAD+ metabolism in bacteria is poorly understood. NADH/NAD+ ratios increased when mutations occurred in two genes (nuo F and ndh) encoding NADH dehydrogenase and three genes (cydB, cyoB, and appB) encoding cytochrome oxidases in aerobic E. coli , indicating that the expression of these genes is responsible for the maintenance of a stabilized NADH/NAD+ ratio and that these enzymes can convert NADH to NAD+ in an ETC or either increase NADH or reduce NAD+ in other pathways (e.g., conversion of formate into CO2 , glycolysis, and the TCA cycle [16, 25, 65]). In addition, the NADH/NAD + ratios of aerobic E. coli are only approximately half of those of anaerobic E. coli , suggesting that NADH is a greater contributor than NAD+ to anaerobiosis.
In E. coli, the electron transfer in the respiratory chain blocked by bactericidal peptidoglycan recognition proteins (PRGPs) can suppress the NADH oxidoreductases NDH-1 and NDH-2, increase the NADH/NAD+ ratio after the supply of NADH from glycolysis and the TCA cycle is increased, divert electrons from NADH oxidoreductases to O2, and generate H2O2 to increase oxidative stress that kills bacteria . The diversion of electrons flow from formate dehydrogenase FDH-O, NDH-1, and NDH-2, and cytochrome bd-I with incomplete electron transfer from UQ-H2 or its malfunction can serve as another ETC component that enables the excessive production of H2O2 from O2 to induce oxidative stress . We demonstrated that in S. Typhimurium, yqiC is required for expressing nuoE, ndh, sdhB, and cydA in the ETC of aerobic and anaerobic grown S. Typhimurium and for expressing fdhF encoding formate dehydrogenase during the colonization in Caco-2 cells (Additional file 9: Table S9). However, the colonization in Caco-2 cells reversed the yqiC regulation in nuo, ndh, sdhB, and cyd and caused their expression to be repressed, suggesting the key role of yqiC in modulating ROS through these ETC components before and during colonization. We discovered that the repression of cyoC expression in cyoC in aerobic and anaerobic grown S. Typhimurium was stronger under anaerobic conditions than under aerobic conditions; however, this regulation was reduced by colonization (Additional file 9: Table S9). H2O2 is an ROS that is generated by oxidative stress inside the Salmonella-containing vacuoles that exist within phagocytes or exist intrinsically in bacteria because of the respiratory chain or indirect action of antibiotics . Most studies of ROS in S. Typhimurium have reported the ability of intracellular bacteria to survive in macrophages or neutrophils; however, few studies have studied ROS in bacteria that interact with intestinal epithelial cells. The deletion of the arcA of aerobic grown S. Typhimurium in vitro led to increased ROS production and an increased NADH/NAD+ ratio . In neutrophils and macrophages, S. Typhimurium arcA downregulates ompD and ompF in the presence of H2O2 in vitro . H2O2 stress increases the mRNA expression levels of porin-encoding ompX but not those of proteins, indicating the complex posttranscriptional regulation of ompX under oxidative stress . In the present study, yqiC had no effect on arcABC genes, but yqiC was required for expressing ompN, ompS, and ompW but not ompX and other omp genes after the infection of Caco-2 cells with S. Typhimurium (Additional file 10: Table S10). Moreover, ROS production can be bactericidal in host or can be used by S. Typhimurium to induce virulence genes for colonization . To induce virulence genes for colonization, inflammation-associated ROS production can generate tetrathionate as a respiratory electron pool through S. Typhimurium in an anaerobic environment (e.g., the gut) . In anaerobic respiration, S. enterica can be differentiated from E. coli by its use of tetrathionate and thiosulfate as electron acceptors for tetrathionate reduction and sulfide formation . In our study through RNA-seq analysis, emapplot of GO enrichment analysis, and KEGG pathways, yqiC was required for expressing ttrA and ttrS encoding SPI-2 T3SS effectors, and it is also involved in molybdopterin cofactor binding, iron–sulfur cluster binding, and metal ion binding (ttrA), sulfur metabolism and microbial metabolism in diverse environments (ttrC and ttrA), and the two-component system (ttrC, ttrA, and ttrS) after the priming of S. Typhimurium with Caco-2 cells. Collectively, our previous study revealed that yqiC and NADH dehydrogenase inhibitor rotenone are similar in terms of their effect on the expression of flagella and repression of type-1 fimbria ; it also indicated that yqiC is associated with ETC components and subsequent intrinsic ROS production such that it plays a key role in balancing oxidative stress and bacterial pathogenicity in S. Typhimurium.
The present study has several limitations. First, not all of the results obtained through the RNA-seq analysis were validated through qRT-PCR. Second, the knockout of specific genes of interest was not performed in S. Typhimurium. However, for the present study, a strict criterion for statistical significance was set, and the 20 most significantly regulated genes as identified through the RNA-seq analysis were validated.