Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy.

RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).

Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers.

RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). SIGNIFICANCE: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.

Tumour-selective activity of RAS-GTP inhibition in pancreatic cancer.

Broad-spectrum RAS inhibition has the potential to benefit roughly a quarter of human patients with cancer whose tumours are driven by RAS mutations1,2. RMC-7977 is a highly selective inhibitor of the active GTP-bound forms of KRAS, HRAS and NRAS, with affinity for both mutant and wild-type variants3. More than 90% of cases of human pancreatic ductal adenocarcinoma (PDAC) are driven by activating mutations in KRAS4. Here we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models. We observed broad and pronounced anti-tumour activity across models following direct RAS inhibition at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumour versus normal tissues. Treated tumours exhibited waves of apoptosis along with sustained proliferative arrest, whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC mouse model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumours identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identify a promising candidate combination therapeutic regimen to overcome monotherapy resistance.

Tumor-selective effects of active RAS inhibition in pancreatic ductal adenocarcinoma.

Broad-spectrum RAS inhibition holds the potential to benefit roughly a quarter of human cancer patients whose tumors are driven by RAS mutations. However, the impact of inhibiting RAS functions in normal tissues is not known. RMC-7977 is a highly selective inhibitor of the active (GTP-bound) forms of KRAS, HRAS, and NRAS, with affinity for both mutant and wild type (WT) variants. As >90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models, including human and murine cell lines, human patient-derived organoids, human PDAC explants, subcutaneous and orthotopic cell-line or patient derived xenografts, syngeneic allografts, and genetically engineered mouse models. We observed broad and pronounced anti-tumor activity across these models following direct RAS inhibition at doses and concentrations that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumor versus normal tissues. Treated tumors exhibited waves of apoptosis along with sustained proliferative arrest whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS inhibition in the setting of PDAC.

Abdominal obesity and digestive system cancer: a systematic review and meta-analysis of prospective studies.

BACKGROUND: The diagnostic criteria for abdominal obesity are usually waist circumference or waist-to-hip ratio. The magnitude of the risks for cancers of the digestive system and abdominal obesity is unknown. To assess whether abdominal obesity increases the risk of digestive cancer, we conducted a systematic review and meta-analysis of prospective cohort studies in a database. METHODS: PubMed, Embase, and Web of Science databases were searched from their inception to December 2022. The 9-star Newcastle Ottawa Scale was used to assess  study quality. Pooled relative risks and 95% confidence intervals were calculated using fixed or random effect models respectively. The stability of the results was explored by one-by-one exclusion. Subgroup analysis was conducted to explore sources of heterogeneity. Publication bias was evaluated by Begg's and Egger's tests. RESULTS: A total of 43 cohort studies were included. There were 42 and 31 studies in the meta-analysis of waist circumference and waist-to-hip ratio on digestive system cancer, respectively. The results of the meta-analysis revealed that the greater waist circumference and waist-to-hip ratio were correlated with increased incidence of digestive system cancers: waist circumference: RR 1.48, 95% CI 1.38-1.59, p < 0.001; waist-to-hip ratio: RR 1.33, 95% CI 1.28-1.38, p = 0.001. Subgroup analysis by cancer type showed that higher WC and WHR would increase the prevalence of LC, PC, GC, EC, and CRC. The sensitivity analysis was conducted by a one-by-one elimination method, and the results of the meta-analysis remained stable. It is proved that the results were robust by the trim-and-fill method. CONCLUSIONS: There was evidence to suggest that abdominal obesity increased the incidence of digestive cancer, it is necessary to take appropriate measures to reduce abdominal obesity. Waist circumference and waist-to-hip ratio may be better predictors of digestive system cancers. However, the association between waist circumference and digestive system cancer was greater, so more attention should be paid to measuring abdominal obesity with waist circumference.

Lactate promotes Salmonella intracellular replication and systemic infection via driving macrophage M2 polarization.

IMPORTANCE: The important enteropathogen Salmonella can cause lethal systemic infection via survival and replication in host macrophages. Lactate represents an abundant intracellular metabolite during bacterial infection, which can also induce macrophage M2 polarization. In this study, we found that macrophage-derived lactate promotes the intracellular replication and systemic infection of Salmonella. During Salmonella infection, lactate via the Salmonella type III secretion system effector SteE promotes macrophage M2 polarization, and the induction of macrophage M2 polarization by lactate is responsible for lactate-mediated Salmonella growth promotion. This study highlights the complex interactions between Salmonella and macrophages and provides an additional perspective on host-pathogen crosstalk at the metabolic interface.

Facile production of cellulose nanofibers from raw elephant grass by an aluminum chloride-enhanced acidic deep eutectic solvent.

To develop a greener and more efficient method for producing cellulose nanofibers (CNFs) from raw plants, an AlCl3-enhanced ternary deep eutectic solvent, DES2 (consisting of choline chloride, citric acid, and AlCl3·6H2O in a molar ratio of 1:0.4:0.08), was synthesized. Raw elephant grass (EG) was pretreated with DES2, followed by sodium chlorite (NaClO2) bleaching and ultrasonic disruption to extract high-performance CNFs. The DES2 and NaClO2 treatments effectively removed hemicellulose and lignin, achieving removal rates of 99.23 % and 99.62 %, respectively, while maintaining a cellulose content of 78.3 %. DES2 demonstrated easy recyclability and maintained excellent biomass pretreatment performance even after multiple cycles. Following a brief 30-min intermittent ultrasound treatment, the resulting CNFs demonstrated superior crystallinity, increased carboxyl content, and a narrower width distribution compared to CNFs obtained from AlCl3-free DES1. Optimized conditions at 110 °C yielded CNFs with 85.3 % crystallinity, 0.64 mmol/g carboxyl content, 5.15 nm width distribution, and excellent dispersion in water for at least six months. Additionally, CNFs enhanced the tensile strength of chia seed mucilage (CM) composite films, showing a significant improvement to 26.6 MPa, representing a 231.3 % increase over the control film. This study offers a promising approach for efficiently producing CNFs from raw plants.

Multi-omics-based identification of purple acid phosphatases and metabolites involved in phosphorus recycling in stylo root exudates.

Stylo (Stylosanthes guianensis) is a tropical forage and cover crop that possesses low phosphate (Pi) tolerance traits. However, the mechanisms underlying its tolerance to low-Pi stress, particularly the role of root exudates, remain unclear. This study employed an integrated approach using physiological, biochemical, multi-omics, and gene function analyses to investigate the role of stylo root exudates in response to low-Pi stress. Widely targeted metabolomic analysis revealed that eight organic acids and one amino acid (L-cysteine) were significantly increased in the root exudates of Pi-deficient seedlings, among which tartaric acid and L-cysteine had strong abilities to dissolve insoluble-P. Furthermore, flavonoid-targeted metabolomic analysis identified 18 flavonoids that were significantly increased in root exudates under low-Pi conditions, mainly belonging to the isoflavonoid and flavanone subclasses. Additionally, transcriptomic analysis revealed that 15 genes encoding purple acid phosphatases (PAPs) had upregulated expression in roots under low-Pi conditions. Among them, SgPAP10 was characterized as a root-secreted phosphatase, and overexpression of SgPAP10 enhanced organic-P utilization by transgenic Arabidopsis. Overall, these findings provide detailed information regarding the importance of stylo root exudates in adaptation to low-Pi stress, highlighting the plant's ability to release Pi from organic-P and insoluble-P sources through root-secreted organic acids, amino acids, flavonoids, and PAPs.

A noncoding single-nucleotide polymorphism at 8q24 drives IDH1-mutant glioma formation.

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase (IDH)-mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1R132H-driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

Collaborative Action of Microglia and Astrocytes Mediates Neutrophil Recruitment to the CNS to Defend against Escherichia coli K1 Infection.

Escherichia coli K1 is a leading cause of neonatal bacterial meningitis. Recruitment of neutrophils to the central nervous system (CNS) via local immune response plays a critical role in defense against E. coli K1 infection; however, the mechanism underlying this recruitment remains unclear. In this study, we report that microglia and astrocytes are activated in response to stimulation by E. coli K1 and/or E. coli K1-derived outer membrane vesicles (OMVs) and work collaboratively to drive neutrophil recruitment to the CNS. Microglial activation results in the release of the pro-inflammatory cytokine TNF-α, which activates astrocytes, resulting in the production of CXCL1, a chemokine critical for recruiting neutrophils. Mice lacking either microglia or TNF-α exhibit impaired production of CXCL1, impaired neutrophil recruitment, and an increased CNS bacterial burden. C-X-C chemokine receptor 2 (CXCR2)-expressing neutrophils primarily respond to CXCL1 released by astrocytes. This study provides further insights into how immune responses drive neutrophil recruitment to the brain to combat E. coli K1 infection. In addition, we show that direct recognition of E. coli K1 by microglia is prevented by the K1 capsule. This study also reveals that OMVs are sufficient to induce microglial activation.

Pan-cancer analysis of non-coding transcripts reveals the prognostic onco-lncRNA HOXA10-AS in gliomas.

Long non-coding RNAs (lncRNAs) are increasingly recognized as functional units in cancer and powerful biomarkers; however, most remain uncharacterized. Here, we analyze 5,592 prognostic lncRNAs in 9,446 cancers of 30 types using machine learning. We identify 166 lncRNAs whose expression correlates with survival and improves the accuracy of common clinical variables, molecular features, and cancer subtypes. Prognostic lncRNAs are often characterized by switch-like expression patterns. In low-grade gliomas, HOXA10-AS activation is a robust marker of poor prognosis that complements IDH1/2 mutations, as validated in another retrospective cohort, and correlates with developmental pathways in tumor transcriptomes. Loss- and gain-of-function studies in patient-derived glioma cells, organoids, and xenograft models identify HOXA10-AS as a potent onco-lncRNA that regulates cell proliferation, contact inhibition, invasion, Hippo signaling, and mitotic and neuro-developmental pathways. Our study underscores the pan-cancer potential of the non-coding transcriptome for identifying biomarkers and regulators of cancer progression.

Salmonella enterica Serovar Typhi Induces Host Metabolic Reprogramming to Increase Glucose Availability for Intracellular Replication.

Salmonella enterica serovar Typhi (S. Typhi) is a human-limited intracellular pathogen and the cause of typhoid fever, a severe systemic disease. Pathogen-host interaction at the metabolic level affects the pathogenicity of intracellular pathogens, but it remains unclear how S. Typhi infection influences host metabolism for its own benefit. Herein, using metabolomics and transcriptomics analyses, combined with in vitro and in vivo infection assays, we investigated metabolic responses in human macrophages during S. Typhi infection, and the impact of these responses on S. Typhi intracellular replication and systemic pathogenicity. We observed increased glucose content, higher rates of glucose uptake and glycolysis, and decreased oxidative phosphorylation in S. Typhi-infected human primary macrophages. Replication in human macrophages and the bacterial burden in systemic organs of humanized mice were reduced by either the inhibition of host glucose uptake or a mutation of the bacterial glucose uptake system, indicating that S. Typhi utilizes host-derived glucose to enhance intracellular replication and virulence. Thus, S. Typhi promotes its pathogenicity by inducing metabolic changes in host macrophages and utilizing the glucose that subsequently accumulates as a nutrient for intracellular replication. Our findings provide the first metabolic signature of S. Typhi-infected host cells and identifies a new strategy utilized by S. Typhi for intracellular replication.

High-resolution mass spectrometry-based methodology for the identification of the metabolites of pterostilbene produced by rat, dog and human hepatocytes.

Pterostilbene, a natural bibenzjyl compound, has been demonstrated to have pleiotropic anticancer effects against a variety of cancer types. The aim of this study was carried out to disclose the metabolic profiles of pterostilbene using rat, dog and human hepatocytes. Metabolites were characterized by ultra-high-performance liquid chromatography/quadrupole Orbitrap mass spectrometry with electrospray ionization interface operating in positive ion mode. The structures of the metabolites were proposed by accurate MS, MS/MS spectra and based on their fragmentation patterns. A total of 12 metabolites, including six new ones, were detected and identified. M10 and M12 were unambiguously identified as pinostilbene and 3'-hydroxy-pterostilbene, respectively, by using reference standards. Our results revealed that pterostilbene was metabolized through the following pathways: (a) hydroxylation to form 3'-hydroxy-pterostilbene (M12), which further undergoes glucuronidation (M9), demethylation (M7) and GSH conjugation through the ortho-quinone intermediate; (b) demethylation to produce desmethyl-pterostilbene (M10), which is further subject to glucuronidation (M4); (c) direct conjugation with glucuronide (M11); and (d) direct sulfation (M8). Among the tested species, no significant species difference was observed. The current study provides valuable information on the metabolism of pterostilbene, which is helpful for us to understand the action of this compound.

Salmonella Typhimurium reprograms macrophage metabolism via T3SS effector SopE2 to promote intracellular replication and virulence.

Salmonella Typhimurium establishes systemic infection by replicating in host macrophages. Here we show that macrophages infected with S. Typhimurium exhibit upregulated glycolysis and decreased serine synthesis, leading to accumulation of glycolytic intermediates. The effects on serine synthesis are mediated by bacterial protein SopE2, a type III secretion system (T3SS) effector encoded in pathogenicity island SPI-1. The changes in host metabolism promote intracellular replication of S. Typhimurium via two mechanisms: decreased glucose levels lead to upregulated bacterial uptake of 2- and 3-phosphoglycerate and phosphoenolpyruvate (carbon sources), while increased pyruvate and lactate levels induce upregulation of another pathogenicity island, SPI-2, known to encode virulence factors. Pharmacological or genetic inhibition of host glycolysis, activation of host serine synthesis, or deletion of either the bacterial transport or signal sensor systems for those host glycolytic intermediates impairs S. Typhimurium replication or virulence.

Downregulation of a novel flagellar synthesis regulator AsiR promotes intracellular replication and systemic pathogenicity of Salmonella typhimurium.

The intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) exploits host macrophage as a crucial survival and replicative niche. To minimize host immune response stimulated by flagellin, the expression of flagellar genes is downregulated during S. Typhimurium growth within host macrophages. However, the underlying mechanisms are largely unknown. In this study, we show that STM14_1285 (named AsiR), a putative RpiR-family transcriptional regulator, which is downregulated within macrophages as previously reported and also confirmed here, positively regulates the expression of flagellar genes by directly binding to the promoter of flhDC. By generating an asiR mutant strain and a strain that persistently expresses asiR gene within macrophages, we confirmed that the downregulation of asiR contributes positively to S. Typhimurium replication in macrophages and systemic infection in mice, which could be attributed to decreased flagellar gene expression and therefore reduced flagellin-stimulated secretion of pro-inflammatory cytokines IL-1ß and TNF-α. Furthermore, the acidic pH in macrophages is identified as a signal for the downregulation of asiR and therefore flagellar genes. Collectively, our results reveal a novel acidic pH signal-mediated regulatory pathway that is utilized by S. Typhimurium to promote intracellular replication and systemic pathogenesis by repressing flagellar gene expression.

SopA inactivation or reduced expression is selected in intracellular Salmonella and contributes to systemic Salmonella infection.

Pseudogenes are accumulated in host-restricted Salmonella enterica serovars, while pseudogenization is primarily regarded as a process that purges unnecessary genes from the genome. Here we showed that the inactivation of sopA, which encodes an effector of Salmonella Pathogenicity Island 1, in human-restricted S. enterica serovar Typhi (S. Ty) and Paratyphi A (S. PA) is under positive selection and aimed to reduce bacterial cytotoxicity toward host macrophages. Moreover, we found that the expression of sopA in Salmonella Typhimurium (S. Tm), a broad-host-range serovar which causes systemic disease in mice, was negatively regulated during mice infection and survival in murine macrophages. The sopA repression in S. Tm is mediated by IsrM, a small RNA absent from the genome of S. Ty and S. PA. Due to the lack of IsrM, sopA expression was unregulated in S. Ty and S. PA, which might have facilitated the convergent inactivation of sopA in these two serovars. In conclusion, our findings demonstrate that sopA inactivation or intracellular repression is the target of positive selection during the systemic infection caused by S. enterica serovars.

Biological mechanisms of cadmium accumulation in edible Amaranth (Amaranthus mangostanus L.) cultivars promoted by salinity: A transcriptome analysis.

Strategies to prevent cadmium (Cd) mobilization by crops under salinity conditions differs among distinct genotypes, but the biological mechanisms of Cd accumulation in different genotype crops promoted by salinity have remained scarce. In this study, we investigated the biological mechanisms of Cd accumulation in two quite different amaranth cultivars of low-Cd accumulator Quanhong (QH) and high-Cd accumulator Liuye (LY) in response to salt stress. Transcriptomes analysis was carried out on leaves and roots tissues of LY and QH grown with exchangeable Cd 0.27 mg kg-1 and salinity 3.0 g kg-1 treatment or control conditions, respectively. A total of 3224 differentially expressed genes (DEGs) in LY (1119 in roots, 2105 in leaves) and 848 in QH (207 in roots, 641 in leaves) were identified. Almost in each fold change category (2-25, 25-210, >210), the numbers of DEGs induced by salinity in LY treatments were much more than those in QH treatments, indicating that LY is more salt sensitive. Gene ontology (GO) analysis revealed that salinity stress promoted soil acidification and Cd mobilization in LY treatments through the enhancive expression of genes related to adenine metabolism (84-fold enrichment) and proton pumping ATPase (50-fold enrichment) in roots, and carbohydrate hydrolysis (2.5-fold enrichment) in leaves compared with that of whole genome, respectively. The genes expression of organic acid transporter (ALMT) was promoted by 2.71- to 3.94-fold in roots, facilitating the secretion of organic acids. Salt stress also inhibited the expression of key enzymes related to cell wall biosynthesis in roots, reducing the physical barriers for Cd uptake. All these processes altered in LY were more substantially compared with that of QH, suggesting that salt sensitive cultivars might accumulate more Cd and pose a higher health risk.

The putative transcriptional regulator STM14_3563 facilitates Salmonella Typhimurium pathogenicity by activating virulence-related genes.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important gram-negative intracellular pathogen that infects humans and animals. More than 50 putative regulatory proteins have been identified in the S. Typhimurium genome, but few have been clearly defined. In this study, the physiological function and regulatory role of STM14_3563, which encodes a ParD family putative transcriptional regulator in S. Typhimurium, were investigated. Macrophage replication assays and mice experiments revealed that S. Typhimurium showed reduced growth in murine macrophages and attenuated virulence in mice owing to deletion of STM14_3563 gene. RNA sequencing (RNA-Seq) data showed that STM14_3563 exerts wide-ranging effects on gene expression in S. Typhimurium. STM14_3563 activates the expression of several genes encoded in Salmonella pathogenicity island (SPI)-6, SPI-12, and SPI-13, which are required for intracellular replication of S. Typhimurium. Additionally, the global transcriptional regulator Fis was found to directly activate STM14_3563 expression by binding to the STM14_3563 promoter. These results indicate that STM14_3563 is involved in the regulation of a variety of virulence-related genes in S. Typhimurium that contribute to its growth in macrophages and virulence in mice.

PagR mediates the precise regulation of Salmonella pathogenicity island 2 gene expression in response to magnesium and phosphate signals in Salmonella Typhimurium.

To establish systemic infections, Salmonella enterica serovar Typhimurium (S. Typhimurium) requires Salmonella pathogenicity island 2 (SPI-2) to survive and replicate within macrophages. High expression of many SPI-2 genes during the entire intracellular growth period within macrophages is essential, as it contributes to the formation of Salmonella-containing vacuole and bacterial replication. However, the regulatory mechanisms underlying the sustained induction of SPI-2 within macrophages are not fully understood. Here, we revealed a time-dependent regulation of SPI-2 expression mediated by a novel regulator PagR (STM2345) in response to the low Mg2+ and low phosphate (Pi ) signals, which ensured the high induction of SPI-2 during the entire intramacrophage growth period. Deletion of pagR results in reduced bacterial replication in macrophages and attenuation of systemic virulence in mice. The effects of pagR on virulence are dependent on upregulating the expression of slyA, a regulator of SPI-2. At the early (0-4 hr) and later (after 4 hr) stage post-infection of macrophages, pagR is induced by the low Pi via PhoB/R two-component systems and low Mg2+ via PhoP/Q systems, respectively. Collectively, our findings revealed that the PagR-mediated regulatory mechanism contributes to the precise and sustained activation of SPI-2 genes within macrophages, which is essential for S. Typhimurium systemic virulence.

The LysR-type transcriptional regulator STM0030 contributes to Salmonella Typhimurium growth in macrophages and virulence in mice.

Salmonella enterica serovar Typhimurium (S. Tm) is a major intracellular pathogen that infects humans and animals, and its survival and growth in macrophages is essential for its pathogenicity. More than 50 putative regulatory proteins are encoded by the S. Tm genome, but the functions of these regulatory proteins in mediating S. Tm pathogenicity are largely unknown. In this study, we investigated the biological function of the STM0030 gene, which encodes a putative LysR-type transcriptional regulator. We found that STM0030 is upregulated 2.8-5.7-fold during S. Tm growth in macrophages. Further, mutating this gene decreased bacterial growth in macrophages and attenuated virulence in mice. RNA-sequencing to investigate the regulatory function of STM0030 in S. Tm revealed that 447 genes were differentially expressed between the mutant and the wild-type strains; 429 of these genes were downregulated, suggesting that STM0030 mainly acts as a transcriptional activator. Moreover, the expression of gluconate, maltose, and hexose-p transport genes, as well as allantoin utilization genes were downregulated in the STM0030 mutant; this might be associated with the observed decrease in intracellular replication and pathogenicity of the mutant. Our findings suggest that STM0030 is a new pathogenicity-associated regulatory protein that broadens our understanding of the virulence regulatory network of S. Tm.