Nucleolar c-Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence.

Pathogen type 3 secretion systems (T3SS) manipulate host cell pathways by directly delivering effector proteins into host cells. In Vibrio parahaemolyticus, the leading cause of bacterial seafood-borne diarrheal disease, we showed that a T3SS effector, VgpA, localizes to the host cell nucleolus where it binds Epstein-Barr virus nuclear antigen 1-binding protein 2 (EBP2). An amino acid substitution in VgpA (VgpAL10A ) did not alter its translocation to the nucleus but abolished the effector's capacity to interact with EBP2. VgpA-EBP2 interaction led to the re-localization of c-Myc to the nucleolus and increased cellular rRNA expression and proliferation of cultured cells. The VgpA-EBP2 interaction elevated EBP2's affinity for c-Myc and prolonged the oncoprotein's half-life. Studies in infant rabbits demonstrated that VgpA is translocated into intestinal epithelial cells, where it interacts with EBP2 and leads to nucleolar re-localization of c-Myc. Moreover, the in vivo VgpA-EBP2 interaction during infection led to proliferation of intestinal cells and heightened V. parahaemolyticus' colonization and virulence. These observations suggest that direct effector stimulation of a c-Myc controlled host cell growth program can contribute to pathogenesis.

Salmonella Enteritidis T1SS protein SiiD inhibits NLRP3 inflammasome activation via repressing the mtROS-ASC dependent pathway.

Inflammasome activation is an essential innate immune defense mechanism against Salmonella infections. Salmonella has developed multiple strategies to avoid or delay inflammasome activation, which may be required for long-term bacterial persistence. However, the mechanisms by which Salmonella evades host immune defenses are still not well understood. In this study, Salmonella Enteritidis (SE) random insertion transposon library was screened to identify the key factors that affect the inflammasome activation. The type I secretion system (T1SS) protein SiiD was demonstrated to repress the NLRP3 inflammasome activation during SE infection and was the first to reveal the antagonistic role of T1SS in the inflammasome pathway. SiiD was translocated into host cells and localized in the membrane fraction in a T1SS-dependent and partially T3SS-1-dependent way during SE infection. Subsequently, SiiD was demonstrated to significantly suppress the generation of mitochondrial reactive oxygen species (mtROS), thus repressing ASC oligomerization to form pyroptosomes, and impairing the NLRP3 dependent Caspase-1 activation and IL-1ß secretion. Importantly, SiiD-deficient SE induced stronger gut inflammation in mice and displayed NLRP3-dependent attenuation of the virulence. SiiD-mediated inhibition of NLRP3 inflammasome activation significantly contributed to SE colonization in the infected mice. This study links bacterial T1SS regulation of mtROS-ASC signaling to NLRP3 inflammasome activation and reveals the essential role of T1SS in evading host immune responses.

Salmonella Enteritidis antitoxin DinJ inhibits NLRP3-dependent canonical inflammasome activation in macrophages.

The inflammasome is a pivotal component of the innate immune system, acting as a multiprotein complex that plays an essential role in detecting and responding to microbial infections. Salmonella Enteritidis have evolved multiple mechanisms to regulate inflammasome activation and evade host immune system clearance. Through screening S. Enteritidis C50336ΔfliC transposon mutant library, we found that the insertion mutant of dinJ increased inflammasome activation. In this study, we demonstrated the genetic connection between the antitoxin DinJ and the toxin YafQ in S. Enteritidis, confirming their co-transcription. The deletion mutant ΔfliCΔdinJ increased cell death and IL-1ß secretion in J774A.1 cells. Western blotting analysis further showed elevated cleaved Caspase-1 product (p10 subunits) and IL-1ß secretion in cells infected with ΔfliCΔdinJ compared to cells infected with ΔfliC. DinJ was found to inhibit canonical inflammasome activation using primary bone marrow-derived macrophages (BMDMs) from Casp-/- C57BL/6 mice. Furthermore, DinJ specifically inhibited NLRP3 inflammasome activation, as demonstrated in BMDMs from Nlrp3-/- and Nlrc4-/- mice. Fluorescence resonance energy transfer (FRET) experiments confirmed the translocation of DinJ into host cells during infection. Finally, we revealed that DinJ could inhibit the secretion of IL-1ß and IL-18 in vivo, contributing to S. Enteritidis evading host immune clearance. In summary, our findings provide insights into the role of DinJ in modulating the inflammasome response during S. Enteritidis infection, highlighting its impact on inhibiting inflammasome activation and immune evasion.

The Salmonella T3SS1 effector IpaJ is regulated by ItrA and inhibits the MAPK signaling pathway.

Invasion plasmid antigen J (IpaJ) is a protein with cysteine protease activity that is present in Salmonella and Shigella species. Salmonella enterica serovar Pullorum uses IpaJ to inhibit the NF-κB pathway and the subsequent inflammatory response, resulting in bacterial survival in host macrophages. In the present study, we performed a DNA pull-down assay and EMSA and identified ItrA, a new DeoR family transcriptional regulator that could control the expression of IpaJ by directly binding to the promoter of ipaJ. The deletion of itrA inhibited the transcription of ipaJ in Salmonella. Tn-Seq revealed that two regulators of Salmonella pathogenicity island 1 (SPI-1), namely HilA and HilD, regulated the secretion of IpaJ. The deletion of hilA, hilD or SPI-1 inhibited the secretion of IpaJ in both cultured medium and Salmonella-infected cells. In contrast, the strain with the deletion of ssrB (an SPI-2 regulator-encoding gene) displayed normal IpaJ secretion, indicating that IpaJ is an effector of the SPI-1-encoded type III secretion system (T3SS1). To further demonstrate the role of IpaJ in host cells, we performed quantitative phosphoproteomics and compared the fold changes in signaling molecules in HeLa cells infected with wild-type S. Pullorum C79-13 with those in HeLa cells infected with the ipaJ-deleted strain C79-13ΔpSPI12. Both phosphoproteomics and Western blot analyses revealed that p-MEK and p-ERK molecules were increased in C79-13ΔpSPI12- and C79-13ΔpSPI12-pipaJ(C45A)-infected cells; and Co-IP assays demonstrated that IpaJ interacts with Ras to reduce its ubiquitination, indicating that IpaJ can inhibit the activation of the MAPK signaling pathway.

Retrospective analysis of preoperative application of triple-modal pre-rehabilitation on postoperative recovery of colorectal cancer patients.

PURPOSE: To retrospectively analyze the difference between triple-modal pre-rehabilitation and common treatment in patients with colorectal cancer (CRC). METHODS: A total of 145 patients with CRC diagnosed by pathology and admitted to our hospital for surgery between June 2020 and June 2022 were included in the study. All patients were divided into two groups: the triple-modal pre-rehabilitation group (pre-rehabilitation group) and the common treatment group. The triple-modal pre-rehabilitation strategy included exercise (3-5 times per week, with each session lasting more than 50 min), nutritional support, and psychological support. The study was designed to assess the potential of the pre-rehabilitation intervention to accelerate postoperative recovery by assessing the 6-min walk test, nutritional indicators, and HADS score before and after surgery. RESULTS: The pre-rehabilitation intervention did not reduce the duration of initial postoperative recovery or the incidence of postoperative complications, but it did increase the patients' exercise capacity (as determined by the 6-min walk test), with the pre-rehabilitation group performing significantly better than the common group (433.0 (105.0) vs. 389.0 (103.5), P < 0.001). The study also found that triple-modal pre-rehabilitation was beneficial for the early recovery of nutritional status in surgical patients and improved anxiety and depression in patients after surgery, especially in those who had not received neoadjuvant therapy. CONCLUSION: The triple-modal pre-rehabilitation strategy is of significant importance for reducing stress and improving the functional reserve of patients with colorectal cancer (CRC) during the perioperative period. The results of our study provide further support for the integration of the triple-modal pre-rehabilitation strategy into the treatment and care of CRC patients.

CCCTC-Binding Factor Mediates the Transcription of Insulin-Like Growth Factor Binding Protein 5 Through EZH2 in Ulcerative Colitis.

BACKGROUND: Ulcerative colitis (UC) features chronic, non-infectious inflammation of the colon. Insulin-like growth factor binding protein 5 (IGFBP5) has been indicated to be related to various inflammation-related diseases. However, its association with UC remains largely unclear. AIMS: Here, we investigate the role of IGFBP5 in colonic mucosal epithelial cell injury in UC. METHODS: Differentially expressed genes in the colonic tissues of UC mice were screened using the Gene Expression Omnibus database, and IGFBP5 was identified. UC mice were developed using dextran sulfate sodium, and IGFBP5 expression in the colonic tissues of UC mice was confirmed by immunohistochemistry and RT-qPCR. The effects of IGFBP5 in vivo and in vitro were investigated by intraperitoneal injection of adeno-associated virus into UC mice or by transfection with an IGFBP5 overexpression plasmid into lipopolysaccharide-treated colonic mucosal epithelial cells. The mechanisms causing IGFBP5 deletion in UC were predicted by bioinformatics analysis and ChIP-qPCR and verified by rescue experiments. RESULTS: IGFBP5 was reduced in UC. IGFBP5 impaired the NFκB pathway in the colonic tissue of UC mice and ameliorated inflammatory infiltration and colonic mucosal cell injury. IGFBP5 depletion was associated with H3K27me3 modification, which was induced by EZH2. CTCF was responsible for recruiting EZH2 to the promoter region of IGFBP5. CTCF inhibition repressed UC progression by reducing H3K27me3 modification via the discouragement of the enrichment of EZH2 in the IGFBP5 promoter. CONCLUSIONS: CTCF modulates H3K27me3 modification of the IGFBP5 promoter by recruiting EZH2, thereby downregulating IGFBP5 to accentuate colonic mucosal epithelial cell injury in UC mice.

Binding site profiles and N-terminal minor groove interactions of the master quorum-sensing regulator LuxR enable flexible control of gene activation and repression.

LuxR is a TetR family master quorum sensing (QS) regulator activating or repressing expression of hundreds of genes that control collective behaviors in Vibrios with underlying mechanism unknown. To illuminate how this regulator controls expression of various target genes, we applied ChIP-seq and DNase I-seq technologies. Vibrio alginolyticus LuxR controls expression of ∼280 genes that contain either symmetric palindrome (repDNA) or asymmetric (actDNA) binding motifs with different binding profiles. The median number of LuxR binding sites for activated genes are nearly double for that of repressed genes. Crystal structures of LuxR in complex with the respective repDNA and actDNA motifs revealed a new mode of LuxR DNA binding that involves contacts of its N-terminal extension to the minor groove. The N-terminal contacts mediated by Arginine-9 and Arginine-11 differ when LuxR binds to repDNA vs actDNA, leading to higher binding affinity at repressed targets. Moreover, modification of LuxR binding sites, binding profiles, and N-terminal extension have important consequences on QS-regulated phenotypes. These results facilitate fundamental understanding of the high flexibility of mechanisms of LuxR control of gene activation and repression in Vibrio QS, which may facilitate to design QS inhibiting chemicals that interfere with LuxR regulation to effectively control pathogens.

Comparative genomic analysis reveals the potential transmission of Vibrio parahaemolyticus from freshwater food to humans.

Vibrio parahaemolyticus is an increasingly important foodborne pathogen that cause acute gastroenteritis in humans. However, the prevalence and transmission of this pathogen in freshwater food remains unclear. This study aimed to determine the molecular characteristics and genetic relatedness of V. parahaemolyticus isolates obtained from freshwater food, seafood, environmental, and clinical samples. A total of 138 (46.6%) isolates were detected from 296 food and environmental samples, and 68 clinical isolates from patients. Notably, V. parahaemolyticus was more prevalent in freshwater food (56.7%, 85/150) than in seafood (38.8%, 49/137). Virulence phenotype analyses revealed that the high motility of isolates from freshwater food (40.0%) and clinical isolates (42.0%) was higher than that of isolates from seafood (12.2%), whereas the biofilm-forming capacity of freshwater food isolates (9.4%) was lower than that of seafood (22.4%) and clinical isolates (15.9%). Virulence genes analysis showed that 46.4% of the clinical isolates contained the tdh gene encoding thermostable direct hemolysin (TDH) and only two freshwater food isolates contained the trh gene encoding TDH-related hemolysin (TRH). Multilocus sequence typing (MLST) analysis divided the 206 isolates into 105 sequence types (STs), including 56 (53.3%) novel STs. ST2583, ST469, and ST453 have been isolated from freshwater food and clinical samples. Whole-genome sequence (WGS) analyses revealed that the 206 isolates were divided into five clusters. Cluster II contained isolates from freshwater food and clinical samples, whereas the other clusters contained isolates from seafood, freshwater food, and clinical samples. In addition, we observed that ST2516 had the same virulence pattern, with a close phylogenetic relationship to ST3. The increased prevalence and adaption of V. parahaemolyticus in freshwater food is a potential cause of clinical cases closely related to the consumption of V. parahaemolyticus contaminated freshwater food.

Transcriptome Analysis Reveals the Effect of Low NaCl Concentration on Osmotic Stress and Type III Secretion System in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a moderately halophilic foodborne pathogen that is mainly distributed in marine and freshwater environments. The transition of V. parahaemolyticus between aquatic ecosystems and hosts is essential for infection. Both freshwater and host environments have low salinity. In this study, we sought to further investigate the effects of low salinity (0.5% NaCl) on the fitness and virulence of V. parahaemolyticus. We found that V. parahaemolyticus could survive in Luria-Bertani (LB) and M9 mediums with different NaCl concentrations, except for the M9 medium containing 9% NaCl. Our results further showed that V. parahaemolyticus cultured in M9 medium with 0.5% NaCl had a higher cell density than that cultured at other NaCl concentrations when it entered the stationary phase. Therefore, we compared the transcriptomes of V. parahaemolyticus wild type (WT) cultured in an M9 medium with 0.5% and 3% NaCl at the stationary phase using RNA-seq. A total of 658 genes were significantly differentially expressed in the M9 medium with 0.5% NaCl, including regulators, osmotic adaptive responses (compatible solute synthesis systems, transporters, and outer membrane proteins), and virulence factors (T3SS1 and T6SS1). Furthermore, a low salinity concentration in the M9 medium induced the expression of T3SS1 to mediate the cytotoxicity of V. parahaemolyticus to HeLa cells. Similarly, low salinity could also induce the secretion of the T3SS2 translocon protein VPA1361. These factors may result in the high pathogenicity of V. parahaemolyticus in low-salinity environments. Taken together, these results suggest that low salinity (0.5% NaCl) could affect gene expression to mediate fitness and virulence, which may contribute to the transition of V. parahaemolyticus between aquatic ecosystems and the host.

New perspectives on the laser initiation for metal tetrazine complexes: a theoretical study.

In order to understand the relationship between laser initiation and charge transfer of metal tetrazine complexes (MTCs), several sets of MTCs with different metals and ligands were designed and their charge transfer (CT) characters were examined using a time-dependent density functional theory method (TD-DFT) in combination with UV-vis spectra, hole-electron distribution, interfragment charge transition, and transition density matrix analyses. Results show that Fe(II), Mn(II), and Cu(II) are suitable divalent transition metal cores in constructing the optical initiation tetrazine complexes. By replacing the divalent metal cores with a monovalent center, new sets of complexes are proved to possess metal-to-ligand charge transfer (MLCT) character and stronger absorption intensity in the near-infrared (NIR) region, which implies that monovalent MTCs are more in favor of low-energy laser initiation than divalent MTCs. Reasonable tuning of the structure of pyrazole substituent can expect to enhance the explosive performance while preserving the optical characteristics, which is an important design principle. This work thoroughly depicts the photoactive states for MTCs and gives a train of thought to explore new desirable laser initiation explosives.

Vibrio parahaemolyticus CadC regulates acid tolerance response to enhance bacterial motility and cytotoxicity.

Pathogens adapted to sub-lethal acidic conditions could increase the virulence and survival ability under lethal conditions. In the aquaculture industry, feed acidifiers have been used to increase the growth of aquatic animals. However, there is limited study on the effects of acidic condition on the virulence and survival of pathogens in aquaculture. In this study, we investigated the survival ability of Vibrio parahaemolyticus at lethal acidic pH (4.0) after adapted the bacteria to sub-lethal acidic pH (5.5) for 1 hr. Our results indicated that the adapted strain increased the survival ability at lethal acidic pH invoked by an inorganic (HCl) or organic (citric) acid. RNA-sequencing (RNA-seq) results revealed that 321 genes were differentially expressed at the sub-lethal acidic pH including cadC, cadBA and groES/groEL relating to acid tolerance response (ATR), as well as genes relating to outer membrane, heat-shock proteins, phosphotransferase system and flagella system. Quantitative real-time polymerase chain reaction (qRT-PCR) confirmed that cadC and cadBA were upregulated under sub-lethal acidic conditions. The CadC protein could directly regulate the expression of cadBA to modulate the ATR in V. parahaemolyticus. RNA-seq data also indicated that 113 genes in the CadC-dependent way and 208 genes in the CadC-independent way were differentially expressed, which were related to the regulation of ATR. Finally, the motility and cytotoxicity of the sub-lethal acidic adapted wild type (WT) were significantly increased compared with the unadapted strain. Our results demonstrated that the dietary acidifiers may increase the virulence and survival of V. parahaemolyticus in aquaculture.

Prevalence and Characteristics of Salmonella spp. from a Pig Farm in Shanghai, China.

Salmonella spp. is a major foodborne pathogen that is distributed among most pork production chains worldwide. This study aimed to investigate the dynamic changes in Salmonella spp. along the pig breeding process monthly from April 2018 to March 2019 in a pig farm in Shanghai, China, and identify the potential critical control points during the production. In total, 239 Salmonella spp. isolates were obtained from 1389 samples, in which Salmonella were detected from 26.3% (222/843) of fecal samples, 7.1% (17/240) of feed samples, and 0.0% (0/306) of both water and insect samples. Seven different serotypes were identified, with the predominant serotype being Salmonella Derby (21.8%), followed by Salmonella Typhimurium (18.8%), Salmonella Rissen (16.3%), Salmonella Mbandaka (12.6%), and Salmonella 1,4,[5],12:i:- (11.8%). Most probable number (MPN) analysis revealed that the load of Salmonella spp. gradually increased along the pig production chain, while the highest number of Salmonella spp. isolates was at the fattening stage (MPN value, 11-15 MPN/g). The pulsed-field gel electrophoresis showed that both Salmonella Typhimurium and Salmonella Derby isolates were grouped to six clusters. The antimicrobial resistance analyzed demonstrated that 80.0% of the isolates were of multidrug resistance and resistant to sulfamethoxazole (84.5%), lincomycin (89.4%), ampicillin (96.9%), oxytetracycline (93.8%), and tetracycline (95.1%). We further evaluated the Salmonella spp. Resistance to quaternary ammonium compounds (QACs) showed an increasing trend along with the testing period indicating that the use of QACs could induce the resistance of Salmonella spp. to QACs. Our study confirmed the dynamic changes in Salmonella spp. over time and space in this pig farm and identified feed and the fattening house as the key points for the prevention and control of Salmonella spp. contamination.

Essential role of Salmonella Enteritidis DNA adenine methylase in modulating inflammasome activation.

BACKGROUND: Salmonella Enteritidis (SE) is one of the major foodborne zoonotic pathogens of worldwide importance which can induce activation of NLRC4 and NLRP3 inflammasomes during infection. Given that the inflammasomes play an essential role in resisting bacterial infection, Salmonella has evolved various strategies to regulate activation of the inflammasome, most of which largely remain unclear. RESULTS: A transposon mutant library in SE strain C50336 was screened for the identification of the potential factors that regulate inflammasome activation. We found that T3SS-associated genes invC, prgH, and spaN were required for inflammasome activation in vitro. Interestingly, C50336 strains with deletion or overexpression of Dam were both defective in activation of caspase-1, secretion of IL-1ß and phosphorylation of c-Jun N-terminal kinase (Jnk). Transcriptome sequencing (RNA-seq) results showed that most of the differentially expressed genes and enriched KEGG pathways between the C50336-VS-C50336Δdam and C50336-VS-C50336::dam groups overlapped, which includes multiple signaling pathways related to the inflammasome. C50336Δdam and C50336::dam were both found to be defective in suppressing the expression of several anti-inflammasome factors. Moreover, overexpression of Dam in macrophages by lentiviral infection could specifically enhance the activation of NLRP3 inflammasome independently via promoting the Jnk pathway. CONCLUSIONS: These data indicated that Dam was essential for modulating inflammasome activation during SE infection, there were complex and dynamic interplays between Dam and the inflammasome under different conditions. New insights were provided about the battle between SE and host innate immunological mechanisms.

Skin-patch of Xin Huang Pian on relieving joint symptoms in patients with acute gouty arthritis: A Randomized, Double-Blind, Active-Controlled Trial.

AIMS: To evaluate the effectiveness and safety of Xin Huang Pian skin patches for patients with acute gouty arthritis. BACKGROUND: In China, patients with acute gouty arthritis benefit from skin patcheses with herbal medicines. But the clinical effects of skin patches with Xin Huang Pian are rarely reported. DESIGN: A Randomized, Double-Blind, Active-Controlled Trial. METHODS: The trial was performed from January 2015-December 2018 at the First Affiliated Hospital of Sun Yat-sen University in China. It was conducted with one intervention group (skin patches of Xin Huang Pian, N = 30) and one active control group (skin patches of Diclofenac Diethylamine Emulgel, N = 31). Participants and study investigators were both blinded to the treatment assignments. The primary outcomes were the improvement of joints' symptoms. The secondary outcomes were changes in white blood cells, erythrocyte sedimentation rate and C-reactive protein. RESULTS: Skin patches of Xin Huang Pian showed quick effect on decreasing joint pain at 3rd day of treatment. Wherever only at 7th day, Diclofenac Diethylamine Emulgel markedly lowered joint pain. Xin Huang Pian also showed superior effect than Diclofenac Diethylamine Emulgel on improving joint swelling and range of motion and decreasing the levels of C-reactive protein and erythrocyte sedimentation rate. No adverse reactions were observed in skin patches of Xin Huang Pian treatment. CONCLUSION: Skin patches of Xin Huang Pian appeared to be safe and efficacious for relieving joint symptoms in patients with acute gouty arthritis. The mechanism might be associated with the decreased levels of C-reactive protein and erythrocyte sedimentation rate. IMPACT: Skin-patcheses with Xin Huang Pian are more effective than Diclofenac Diethylamine Emulgel on improving joint pain, swelling and range of motion. Xin Huang Pian treatment showed superior effects compared with Diclofenac Diethylamine Emulgel on decreasing levels of C-reactive protein and erythrocyte sedimentation rate. Patients with acute gouty arthritis may benefit from skin patches of Xin Huang Pian for effective relief from joint pain and swelling. Chinese Clinical Trial Registration: ChiCTR-TRC-1300 4122.

Immunogenic potential and protective efficacy of a sptP deletion mutant of Salmonella Enteritidis as a live vaccine for chickens against a lethal challenge.

Salmonella Enteritidis (SE) is a highly adapted pathogen causing severe economic losses in the poultry industry worldwide. Chickens infected by SE are a major source of human food poisoning. Vaccination is an effective approach to control SE infections. This study evaluated the immunogenicity and protective efficacy of a SE sptP deletion mutant (C50336ΔsptP) as a live attenuated vaccine (LAV) candidate in chickens. 14 day-old specific pathogen-free (SPF) chickens were intramuscularly immunized with various doses of C50336ΔsptP. Several groups of chickens were challenged with the virulent wild-type SE strain Z-11 via the same route at 14 days post vaccination. Compared to the control group, the groups vaccinated with 1 × 106, 1 × 107 and 1 × 108 colony-forming units (CFU) of C50336ΔsptP exhibited no clinical symptoms after immunization. Only slight pathological changes occurred in the organs of the 1 × 109 CFU vaccinated group. C50336ΔsptP bacteria were cleared from the organs of immunized chickens within 14 days after vaccination. Lymphocyte proliferation and serum cytokine analyses indicated that significant cellular immune responses were induced after the vaccination of C50336ΔsptP. Compared to the control group, specific IgG antibody levels increased significantly in vaccinated chickens, and the levels increased markedly after the challenge. The 1 × 107, 1 × 108, and 1 × 109 CFU vaccinated chickens groups showed no clinical symptoms or pathological changes, and no death after the lethal challenge. Whereas severe clinical signs of disease and pathological changes were observed in the control group chickens after the challenge. These results suggest that a single dose of C50336ΔsptP could be an effective LAV candidate to against SE infection in chickens.

Alternative Sigma Factor RpoX Is a Part of the RpoE Regulon and Plays Distinct Roles in Stress Responses, Motility, Biofilm Formation, and Hemolytic Activities in the Marine Pathogen Vibrio alginolyticus.

Vibrio alginolyticus is one of the most abundant microorganisms in marine environments and is also an opportunistic pathogen mediating high-mortality vibriosis in marine animals. Alternative sigma factors play essential roles in bacterial pathogens in the adaptation to environmental changes during infection and the adaptation to various niches, but little is known about them for V. alginolyticus Our previous investigation indicated that the transcript level of the gene rpoX significantly decreased in an RpoE mutant. Here, we found that rpoX was highly expressed in response to high temperature and low osmotic stress and was under the direct control of the alternative sigma factor RpoE and its own product RpoX. Moreover, transcriptome sequencing (RNA-seq) results showed that RpoE and RpoX had different regulons, although they coregulated 105 genes at high temperature (42°C), including genes associated with biofilm formation, motility, virulence, regulatory factors, and the stress response. RNA-seq and chromatin immunoprecipitation sequencing (ChIP-seq) analyses as well as electrophoretic mobility shift assays (EMSAs) revealed the distinct binding motifs of RpoE and RpoX proteins. Furthermore, quantitative real-time reverse transcription-PCR (qRT-PCR) analysis also confirmed that RpoX can upregulate genes associated with flagella, biofilm formation, and hemolytic activities at higher temperatures. rpoX abrogation does not appear to attenuate virulence toward model fish at normal temperature. Collectively, data from this study demonstrated the regulatory cascades of RpoE and an alternative sigma factor, RpoX, in response to heat and osmotic stresses and their distinct and overlapping roles in pathogenesis and stress responses in the marine bacterium V. alginolyticusIMPORTANCE The alternative sigma factor RpoE is essential for the virulence of Vibrio alginolyticus toward marine fish, coral, and other animals in response to sea surface temperature increases. In this study, we characterized another alternative sigma factor, RpoX, which is induced at high temperatures and under low-osmotic-stress conditions. The expression of rpoX is under the tight control of RpoE and RpoX. Although RpoE and RpoX coregulate 105 genes, they are programming different regulatory functions in stress responses and virulence in V. alginolyticus These findings illuminated the RpoE-RpoX-centered regulatory cascades and their distinct and overlapping regulatory roles in V. alginolyticus, which facilitates unraveling of the mechanisms by which the bacterium causes diseases in various sea animals in response to temperature fluctuations as well as the development of appropriate strategies to tackle infections by this bacterium.

A σE-Mediated Temperature Gauge Controls a Switch from LuxR-Mediated Virulence Gene Expression to Thermal Stress Adaptation in Vibrio alginolyticus.

In vibrios, the expression of virulence factors is often controlled by LuxR, the master quorum-sensing regulator. Here, we investigate the interplay between LuxR and σE, an alternative sigma factor, during the control of virulence-related gene expression and adaptations to temperature elevations in the zoonotic pathogen Vibrio alginolyticus. An rpoE null V. alginolyticus mutant was unable to adapt to various stresses and was survival-deficient in fish. In wild type V. alginolyticus, the expression of LuxR-regulated virulence factors increased as the temperature was increased from 22°C to 37°C, but mutants lacking σE did not respond to temperature, indicating that σE is critical for the temperature-dependent upregulation of virulence genes. Further analyses revealed that σE binds directly to -10 and -35 elements in the luxR promoter that drive its transcription. ChIP assays showed that σE binds to the promoter regions of luxR, rpoH and rpoE at high temperatures (e.g., 30°C and 37°C). However, at higher temperatures (42°C) that induce thermal stress, σE binding to the luxR promoter decreased, while its binding to the rpoH and rpoE promoters was unchanged. Thus, the temperature-dependent binding of σE to distinct promoters appears to underlie a σE-controlled switch between the expression of virulence genes and adaptation to thermal stress. This study illustrates how a conserved temperature response mechanism integrates into quorum-sensing circuits to regulate both virulence and stress adaptation.

Construction of pSPI12-cured Salmonella enterica serovar Pullorum and identification of IpaJ as an immune response modulator.

In Salmonella, plasmids participate in many pathways involved in virulence, metabolism, and antibiotic resistance. To investigate the function of the ipaJ gene in a multi-copy plasmid pSPI12 prevalent in Salmonella enterica serovar Pullorum (S. Pullorum), we established a method to eliminate the plasmid and constructed the plasmid-cured bacteria C79-13-ΔpSPI12 by using the suicide vector pDM4. Briefly, a 500 bp fragment ipaJU from pSPI12 was cloned into pDM4 and transformed into S. Pullorum C79-13 by conjugative transfer. After homologous recombination, the suicide vector was inserted into pSPI12 to produce pSPI12-pDM4-ipaJU. Induction of the expression of the sacB gene in the suicide vector killed the bacteria harbouring plasmid, while the progeny losing the plasmid survived in the plate with sucrose. The plasmid-cured strain showed extremely decreased ability to infect chicken macrophage HD11 cells and LMH hepatic epithelial cells compared to wild type strain and complementary strain carrying ipaJ. Additionally, IFN-γ mRNA levels were up-regulated in HD11 cells or chicken spleens infected by plasmid-cured strain, but no difference was detected in IL-4 among the three strains. Transforming ipaJ into S. Enteritidis also decreased expression of proinflammatory cytokines in infected macrophages or chicken spleens compared to wild type strain. These results suggest that the ipaJ gene in pSPI12 is involved in S. Pullorum infection and that IpaJ protein modulates immune response.

Overexpression of S-Adenosyl-l-Methionine Synthetase 2 from Sugar Beet M14 Increased Arabidopsis Tolerance to Salt and Oxidative Stress.

The sugar beet monosomic addition line M14 is a unique germplasm that contains genetic materials from Beta vulgaris L. and Beta corolliflora Zoss, and shows tolerance to salt stress. Our study focuses on exploring the molecular mechanism of the salt tolerance of the sugar beet M14. In order to identify differentially expressed genes in M14 under salt stress, a subtractive cDNA library was generated by suppression subtractive hybridization (SSH). A total of 36 unique sequences were identified in the library and their putative functions were analyzed. One of the genes, S-adenosylmethionine synthetase (SAMS), is the key enzyme involved in the biosynthesis of S-adenosylmethionine (SAM), a precursor of polyamines. To determine the potential role of SAMS in salt tolerance, we isolated BvM14-SAMS2 from the salt-tolerant sugar beet M14. The expression of BvM14-SAMS2 in leaves and roots was greatly induced by salt stress. Overexpression of BvM14-SAMS2 in Arabidopsis resulted in enhanced salt and H2O2 tolerance. Furthermore, we obtained a knock-down T-DNA insertion mutant of AtSAMS3, which shares the highest homology with BvM14-SAMS2. Interestingly, the mutant atsam3 showed sensitivity to salt and H2O2 stress. We also found that the antioxidant system and polyamine metabolism play an important role in salt and H2O2 tolerance in the BvM14-SAMS2-overexpressed plants. To our knowledge, the function of the sugar beet SAMS has not been reported before. Our results have provided new insights into SAMS functions in sugar beet.

Chromatin Immunoprecipitation Sequencing Technology Reveals Global Regulatory Roles of Low-Cell-Density Quorum-Sensing Regulator AphA in the Pathogen Vibrio alginolyticus.

Quorum sensing (QS) is an important regulatory system in virulence expression and environmental adaptation in bacteria. The master QS regulators (MQSR) LuxR and AphA reciprocally control QS gene expression in vibrios. However, the molecular basis for the regulatory functions of AphA remains undefined. In this study, we characterized its regulatory roles in Vibrio alginolyticus, an important zoonotic pathogen causing diseases in marine animals as well as in humans. AphA is involved in the motility ability, biofilm formation, and in vivo survival of V. alginolyticus Specifically, AphA is expressed at low-cell-density growth phases. In addition, AphA negatively regulates the expression of the main virulence factor, alkaline serine protease (Asp), through LuxR. Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) detected 49 enriched loci harboring AphA-binding peaks across the V. alginolyticus genome. An AphA-specific binding motif was identified and further confirmed by electrophoretic mobility shift assay (EMSA) and mutagenesis analysis. A quantitative real-time PCR (qRT-PCR) assay further validated the regulation of AphA on these genes. AphA binds directly to the aphA promoter and negatively regulates its own expression. Moreover, AphA directly regulates genes encoding adenylate cyclase, anti-σD, FabR, and the small RNA CsrB, revealing versatile regulatory roles of AphA in its physiology and virulence. Furthermore, our data indicated that AphA modulates motility through the coordinated function of LuxR and CsrB. Collectively, the findings of this work contribute to better understanding of the regulatory roles of AphA in QS and non-QS genes. IMPORTANCE: In this work, we determined that AphA, the master regulator of QS at low cell density, plays essential roles in expression of genes associated with physiology and virulence in V. alginolyticus, a Gram-negative pathogen for humans and marine animals. We further uncovered that 49 genes could be directly regulated by AphA and a 19-bp consensus binding sequence was identified. Among the 49 genes, the QS and other non-QS-associated genes were identified to be regulated by AphA. Besides, the small RNA CsrB was negatively regulated by AphA, and AphA regulate motility abilities through both CsrB and LuxR. Taken together, the findings of this study improve our understanding of the complex regulation network of AphA and QS.