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MBio May 2024Bacterial enhancer-binding proteins (bEBPs) acquire a transcriptionally active state via phosphorylation. However, transcriptional activation by the dephosphorylated...
Bacterial enhancer-binding proteins (bEBPs) acquire a transcriptionally active state via phosphorylation. However, transcriptional activation by the dephosphorylated form of bEBP has been observed in DctD, which belongs to Group I bEBP. The formation of a complex between dephosphorylated DctD (d-DctD) and dephosphorylated IIA (d-IIA) is a prerequisite for the transcriptional activity of d-DctD. In the present study, characteristics of the transcriptionally active complex composed of d-IIA and phosphorylation-deficient DctD (DctD) of were investigated in its multimeric conformation and DNA-binding ability. DctD formed a homodimer that could not bind to the DNA. In contrast, when DctD formed a complex with d-IIA in a 1:1 molar ratio, it produced two conformations: dimer and dodecamer of the complex. Only the dodecameric complex exhibited ATP-hydrolyzing activity and DNA-binding affinity. For successful DNA-binding and transcriptional activation by the dodecameric d-IIA/DctD complex, extended upstream activator sequences were required, which encompass the nucleotide sequences homologous to the known DctD-binding site and additional nucleotides downstream. This is the first report to demonstrate the molecular characteristics of a dephosphorylated bEBP complexed with another protein to form a transcriptionally active dodecameric complex, which has an affinity for a specific DNA-binding sequence.IMPORTANCEResponse regulators belonging to the bacterial two-component regulatory system activate the transcription initiation of their regulons when they are phosphorylated by cognate sensor kinases and oligomerized to the appropriate multimeric states. Recently, it has been shown that a dephosphorylated response regulator, DctD, could activate transcription in a phosphorylation-independent manner in . The dephosphorylated DctD activated transcription as efficiently as phosphorylated DctD when it formed a complex with dephosphorylated form of IIA, a component of the glucose-phosphotransferase system. Functional mimicry of this complex with the typical form of transcriptionally active phosphorylated DctD led us to study the molecular characteristics of this heterodimeric complex. Through systematic analyses, it was surprisingly determined that a multimer constituted with 12 complexes gained the ability to hydrolyze ATP and recognize specific upstream activator sequences containing a typical inverted-repeat sequence flanked by distinct nucleotides.
Topics: Adenosine Triphosphate; Bacterial Proteins; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Phosphorylation; Protein Binding; Protein Multimerization; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Vibrio vulnificus
PubMed: 38564689
DOI: 10.1128/mbio.00330-24 -
BMC Veterinary Research Apr 2024Vibriosis is one of the most serious bacterial diseases and causes high morbidity and mortality among cultured sea breams. This study was undertaken to track the...
BACKGROUND
Vibriosis is one of the most serious bacterial diseases and causes high morbidity and mortality among cultured sea breams. This study was undertaken to track the surveillance of Vibrio infection and its correlation to environmental factors. A total of 115 gilthead sea breams were collected seasonally from a private earthen pond fish farm in the Shatta area of Damietta, Egypt from September 2022 to July 2023. Physicochemical parameters of water were analyzed, and heavy metal levels were measured. The fish samples were subjected to clinical, bacteriological, Enterobacterial Repetitive Intergenic Consensus (ERIC) fingerprinting, and hematoxylin and Eosin histopathological staining.
RESULTS
The results revealed significant variations in the water quality parameters over different seasons, in addition to an increase in heavy metals. Naturally infected fish showed external signs and postmortem lesions that were relevant to bacterial infection. Two dominant Vibrio subspecies of bacteria were identified: V. alginolyticus (205 isolates) and V. fluvialis (87 isolates). PCR confirmed the presence of V. alginolyticus using the species-specific primer collagenase at 737 bp. The highest prevalence of V. alginolyticus was detected during the summer season (57.72%), and the lowest prevalence was observed in autumn (39.75%). The correlation analysis revealed a positive relationship between V. alginolyticus and water temperature (r = 0.69). On the other hand, V. fluvialis showed a high prevalence during the autumn season (25.30%) and the lowest prevalence during the summer season (10.56%), where it was negatively correlated with water temperatures (r =-0.03). ERIC fingerprinting showed genetic variation within the Vibrio isolates. Antimicrobial susceptibility testing revealed sensitivity to ciprofloxacin and doxycycline, and resistance to amoxicillin and erythromycin. The multiple antibiotic resistance (MAR) index values for V. alginolyticus and V. fluvialis ranged from 0.3 to 0.7, with a multi-drug resistance pattern to at least three antibiotics. Histopathological alterations in the affected tissues revealed marked hemorrhage, vascular congestion, and hemosiderosis infiltration.
CONCLUSION
This study provides insights into the potential propagation of waterborne diseases and antibiotic resistance in the environment. Ensuring that the environment does not serve as a reservoir for virulent and contagious Vibrio species is a critical concern for regional aquaculture industries. Therefore, we recommend implementing environmental context-specific monitoring and surveillance tools for microbial resistance.
Topics: Animals; Sea Bream; Prevalence; Egypt; Drug Resistance, Bacterial; Vibrio; Anti-Bacterial Agents; Vibrio Infections; Genetic Variation
PubMed: 38561778
DOI: 10.1186/s12917-024-03978-0 -
Microorganisms Feb 2024Gut dysbiosis and subclinical intestinal damage are common in cirrhosis. The aim of this study was to examine the association of intestinal damage biomarkers (diamine...
Gut dysbiosis and subclinical intestinal damage are common in cirrhosis. The aim of this study was to examine the association of intestinal damage biomarkers (diamine oxidase [DAO], claudin 3, and intestinal fatty acid binding protein [I-FABP; FABP2]) with the state of the gut microbiota in cirrhosis. The blood levels of DAO were inversely correlated with blood levels of claudin 3, lipopolysaccharide (LPS), presepsin, TNF-α, and the severity of cirrhosis according to Child-Pugh scores. The blood level of I-FABP was directly correlated with the blood level of claudin 3 but not with that of DAO. Patients with small intestinal bacterial overgrowth (SIBO) had lower DAO levels than patients without SIBO. There was no significant difference in claudin 3 levels and I-FABP detection rates between patients with and without SIBO. The DAO level was directly correlated with the abundance of Akkermansiaceae, Akkermansia, Allisonella, Clostridiaceae, Dialister, Lactobacillus, Muribaculaceae, Negativibacillus, Ruminococcus, Thiomicrospiraceae, Verrucomicrobiae, and Verrucomicrobiota; and it was inversely correlated with the abundance of Anaerostipes, Erysipelatoclostridium, and Vibrio. The I-FABP level was directly correlated with Anaerostipes, Bacteroidia, Bacteroidota, Bilophila, Megamonas, and Selenomonadaceae; and it was inversely correlated with the abundance of Brucella, Pseudomonadaceae, Pseudomonas, and Vibrionaceae. The claudin 3 level was directly correlated with Anaerostipes abundance and was inversely correlated with the abundance of Brucella, Coriobacteriia, Eggerthellaceae, and Lactobacillus.
PubMed: 38543514
DOI: 10.3390/microorganisms12030463 -
PLoS Pathogens Mar 2024Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis in humans worldwide. The major virulence factor responsible for the enteropathogenicity of...
The read-through transcription-mediated autoactivation circuit for virulence regulator expression drives robust type III secretion system 2 expression in Vibrio parahaemolyticus.
Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis in humans worldwide. The major virulence factor responsible for the enteropathogenicity of this pathogen is type III secretion system 2 (T3SS2), which is encoded on the 80-kb V. parahaemolyticus pathogenicity island (Vp-PAI), the gene expression of which is governed by the OmpR-family transcriptional regulator VtrB. Here, we found a positive autoregulatory feature of vtrB transcription, which is often observed with transcriptional regulators of bacteria, but the regulation was not canonically dependent on its own promoter. Instead, this autoactivation was induced by heterogeneous transcripts derived from the VtrB-regulated operon upstream of vtrB. VtrB-activated transcription overcame the intrinsic terminator downstream of the operon, resulting in transcription read-through with read-in transcription of the vtrB gene and thus completing the autoregulatory loop for vtrB gene expression. The dampening of read-through transcription with an exogenous strong terminator reduced vtrB gene expression. Furthermore, a V. parahaemolyticus mutant with defects in the vtrB autoregulatory loop also showed compromises in T3SS2 expression and T3SS2-dependent cytotoxicity in vitro and enterotoxicity in vivo, indicating that this autoregulatory loop is essential for sustained vtrB activation and the consequent robust expression of T3SS2 genes for pathogenicity. Taken together, these findings demonstrate that the regulatory loop for vtrB gene expression based on read-through transcription from the upstream operon is a crucial pathway in T3SS2 gene regulatory network to ensure T3SS2-mediated virulence of V. parahaemolyticus.
Topics: Humans; Type III Secretion Systems; Virulence; Vibrio parahaemolyticus; Virulence Factors; Promoter Regions, Genetic; Bacterial Proteins; Vibrio Infections; Gene Expression Regulation, Bacterial
PubMed: 38536895
DOI: 10.1371/journal.ppat.1012094 -
Infection, Genetics and Evolution :... Jun 2024Non-O1/non-O139 Vibrio cholerae (NOVC) are ubiquitous in aquatic ecosystems. In rare cases, they can cause intestinal and extra-intestinal infections in human. This...
Non-O1/non-O139 Vibrio cholerae (NOVC) are ubiquitous in aquatic ecosystems. In rare cases, they can cause intestinal and extra-intestinal infections in human. This ability is associated with various virulence factors. The presence of NOVC in German North Sea and Baltic Sea was observed in previous studies. However, data on virulence characteristics are still scarce. Therefore, this work aimed to investigating the virulence potential of NOVC isolated in these two regions. In total, 31 NOVC strains were collected and subjected to whole genome sequencing. In silico analysis of the pathogenic potential was performed based on the detection of genes involved in colonization and virulence. Phenotypic assays, including biofilm formation, mobility and human serum resistance assays were applied for validation. Associated toxin genes (hlyA, rtxA, chxA and stn), pathogenicity islands (Vibrio pathogenicity island 2 (VPI-II) and Vibrio seventh pathogenicity island 2 (VSP-II)) and secretion systems (Type II, III and VI secretion system) were observed. A maximum likelihood analysis from shared core genes revealed a close relationship between clinical NOVCs published in NCBI and environmental strains from this study. NOVC strains are more mobile at 37 °C than at 25 °C, and 68% of the NOVC strains could form strong biofilms at both temperatures. All tested strains were able to lyse erythrocytes from both human and sheep blood. Additionally, one strain could survive up to 60% and seven strains up to 40% human serum at 37 °C. Overall, the genetic virulence profile as well as the phenotypic virulence characteristics of the investigated NOVC from the German North Sea and Baltic Sea suggest potential human pathogenicity.
Topics: Virulence Factors; Humans; Virulence; Vibrio cholerae non-O1; Germany; Genomic Islands; Biofilms; Phylogeny; North Sea; Vibrio cholerae; Cholera; Animals; Whole Genome Sequencing
PubMed: 38518953
DOI: 10.1016/j.meegid.2024.105587 -
PeerJ 2024The motility of species plays a pivotal role in their survival and adaptation to diverse environments and is intricately associated with pathogenicity in both humans...
The motility of species plays a pivotal role in their survival and adaptation to diverse environments and is intricately associated with pathogenicity in both humans and aquatic animals. Numerous mutant strains of have been generated using UV or EMS mutagenesis to probe flagellar motility using molecular genetic approaches. Identifying these mutations promises to yield valuable insights into motility at the protein structural physiology level. In this study, we determined the complete genomic structure of 4 reference specimens of laboratory . strains: a precursor strain, . 138-2, two strains showing defects in the lateral flagellum (VIO5 and YM4), and one strain showing defects in the polar flagellum (YM19). Subsequently, we meticulously ascertained the specific mutation sites within the 18 motility-deficient strains related to the polar flagellum (they fall into three categories: flagellar-deficient, multi-flagellar, and chemotaxis-deficient strains) by whole genome sequencing and mapping to the complete genome of parental strains VIO5 or YM4. The mutant strains had an average of 20.6 (±12.7) mutations, most of which were randomly distributed throughout the genome. However, at least two or more different mutations in six flagellar-related genes were detected in 18 mutants specifically selected as chemotaxis-deficient mutants. Genomic analysis using a large number of mutant strains is a very effective tool to comprehensively identify genes associated with specific phenotypes using forward genetics.
Topics: Animals; Humans; Vibrio alginolyticus; Mutation; Mutagenesis; Chemotaxis
PubMed: 38515459
DOI: 10.7717/peerj.17126 -
Frontiers in Immunology 2024Modern fish farming faces challenges in sourcing feed ingredients, most related with their prices, 21 availability, and specifically for plant protein sources,...
INTRODUCTION
Modern fish farming faces challenges in sourcing feed ingredients, most related with their prices, 21 availability, and specifically for plant protein sources, competition for the limited cultivation space for 22 vegetable crops. In that sense, halophytes have the added value of being rich in valuable bioactive compounds and salt tolerant. This study assessed the inclusion of non-food fractions of in European seabass diets.
METHODS
Different levels (2.5%, 5%, and 10%) were incorporated into seabass diets, replacing wheat meal (diets ST2.5, ST5, and ST10) or without inclusion (CTRL). Experimental diets were administered to seabass juveniles (8.62 ± 0.63 g) for 34 and 62 days and subsequent inflammatory responses to a heat-inactivated subsp. () were evaluated in a time-course manner (4, 24, 48, and 72 h after the challenge). At each sampling point, seabass haematological profile, plasma immune parameters, and head-kidney immune-related gene expression were evaluated.
RESULTS
After both feeding periods, most parameters remained unaltered by inclusion; nonetheless, seabass fed ST10 showed an upregulation of macrophage colony-stimulating factor 1 receptor 1 ( and cluster of differentiation 8 ( compared with those fed CTRL after 62 days of feeding. Regarding the inflammatory response, seabass fed ST10 showed lower plasma lysozyme levels than their counterparts fed ST2.5 and ST5 at 24 h following injection, while 4 h after the inflammatory stimulus, seabass fed ST10 presented higher numbers of peritoneal leucocytes than fish fed CTRL. Moreover, at 4 h, fish fed ST2.5, ST5, and ST10 showed a higher expression of interleukin 1β (), while fish fed ST5 showed higher levels of ornithine decarboxylase ( than those fed CTRL. An upregulation of macrophage colony-stimulating factor 1 receptor 1 () and glutathione peroxidase () was also observed at 72 h in fish fed ST10 or ST5 and ST10 compared with CTRL, respectively.
DISCUSSION
In conclusion, incorporating up to 10% of the non-food fraction in feed did not compromise seabass growth or immune status after 62 days, aligning with circular economy principles. However, inclusion improved the leucocyte response and upregulated key immune-related genes in seabass challenged with an inactivated pathogen.
Topics: Animals; Bass; Interleukin-1 Receptor-Like 1 Protein; Macrophage Colony-Stimulating Factor; Diet; Photobacterium
PubMed: 38500885
DOI: 10.3389/fimmu.2024.1342144 -
Applied Microbiology and Biotechnology Mar 2024ADP-activated β-D-manno-heptoses (ADP-β-D-manno-heptoses) are precursors for the biosynthesis of the inner core of lipopolysaccharide in Gram-negative bacteria....
ADP-activated β-D-manno-heptoses (ADP-β-D-manno-heptoses) are precursors for the biosynthesis of the inner core of lipopolysaccharide in Gram-negative bacteria. Recently, ADP-D-glycero-β-D-manno-heptose (ADP-D,D-manno-heptose) and its C-6'' epimer, ADP-L-glycero-β-D-manno-heptose (ADP-L,D-manno-heptose), were identified as potent pathogen-associated molecular patterns (PAMPs) that can trigger robust innate immune responses. Although the production of ADP-D,D-manno-heptose has been studied in several different pathogenic Gram-negative bacteria, current knowledge of ADP-β-D-manno-heptose biosynthesis in Vibrio strains remains limited. Here, we characterized the biosynthetic enzymes of ADP-D,D-manno-heptose and the epimerase that converts it to ADP-L,D-manno-heptose from Vibrio cholerae (the causative agent of pandemic cholera) and Vibrio parahaemolyticus (non-cholera pathogen causing vibriosis with clinical manifestations of gastroenteritis and wound infections) in comparison with their isozymes from Escherichia coli. Moreover, we discovered that β-D-mannose 1-phosphate, but not α-D-mannose 1-phosphate, could be activated to its ADP form by the nucleotidyltransferase domains of bifunctional kinase/nucleotidyltransferases HldE (from V. cholerae) and HldE (from V. parahaemolyticus). Kinetic analyses of the nucleotidyltransferase domains of HldE and HldE together with the E. coli-derived HldE were thus carried out using β-D-mannose 1-phosphate as a mimic sugar substrate. Overall, our works suggest that V. cholerae and V. parahaemolyticus are capable of synthesizing ADP-β-D-manno-heptoses and lay a foundation for further physiological function explorations on manno-heptose metabolism in Vibrio strains. KEY POINTS: • Vibrio strains adopt the same biosynthetic pathway as E. coli in synthesizing ADP-β-D-manno-heptoses. • HldEs from two Vibrio strains and E. coli could activate β-D-mannose 1-phosphate to ADP-β-D-mannose. • Comparable nucleotidyltransfer efficiencies were observed in the kinetic studies of HldEs.
Topics: Escherichia coli; Kinetics; Vibrio; Immunity, Innate; Nucleotidyltransferases
PubMed: 38498053
DOI: 10.1007/s00253-024-13108-3 -
Microbial Ecology Mar 2024In aquatic environments, Vibrio and cyanobacteria establish varying relationships influenced by environmental factors. To investigate their association, this study...
In aquatic environments, Vibrio and cyanobacteria establish varying relationships influenced by environmental factors. To investigate their association, this study spanned 5 months at a local shrimp farm, covering the shrimp larvae stocking cycle until harvesting. A total of 32 samples were collected from pond A (n = 6), pond B (n = 6), effluent (n = 10), and influent (n = 10). Vibrio species and cyanobacteria density were observed, and canonical correspondence analysis (CCA) assessed their correlation. CCA revealed a minor correlation (p = 0.847, 0.255, 0.288, and 0.304) between Vibrio and cyanobacteria in pond A, pond B, effluent, and influent water, respectively. Notably, Vibrio showed a stronger correlation with pH (6.14-7.64), while cyanobacteria correlated with pH, salinity (17.4-24 ppt), and temperature (30.8-31.5 °C), with salinity as the most influential factor. This suggests that factors beyond cyanobacteria influence Vibrio survival. Future research could explore species-specific relationships, regional dynamics, and multidimensional landscapes to better understand Vibrio-cyanobacteria connections. Managing water parameters may prove more efficient in controlling vibriosis in shrimp farms than targeting cyanobacterial populations.
Topics: Animals; Vibrio; Cyanobacteria; Ponds; Water; Aquaculture; Vibrio parahaemolyticus; Penaeidae
PubMed: 38488929
DOI: 10.1007/s00248-024-02356-5 -
Scientific Reports Mar 2024C-type cytochromes fulfil many essential roles in both aerobic and anaerobic respiration. Their characterization requires large quantities of protein which can be...
C-type cytochromes fulfil many essential roles in both aerobic and anaerobic respiration. Their characterization requires large quantities of protein which can be obtained through heterologous production. Heterologous production of c-type cytochromes in Escherichia coli is hindered since the ccmABCDEFGH genes necessary for incorporation of heme c are only expressed under anaerobic conditions. Different strategies were devised to bypass this obstacle, such as co-expressing the ccm genes from the pEC86 vector. However, co-expression methods restrict the choice of expression host and vector. Here we describe the first use of Vibrio natriegens V X2 for the recombinant production of difficult-to-express redox proteins from the extreme acidophile Acidithiobacillus ferrooxidans CCM4253, including three c-type cytochromes. Co-expression of the ccm genes was not required to produce holo-c-type cytochromes in V X2. E. coli T7 Express only produced holo-c-type cytochromes during co-expression of the ccm genes and was not able to produce the inner membrane cytochrome CycA. Additionally, V X2 cell extracts contained higher portions of recombinant holo-proteins than T7 Express cell extracts. All redox proteins were translocated to the intended cell compartment in both hosts. In conclusion, V. natriegens represents a promising alternative for the production of c-type cytochromes and difficult-to-express redox proteins.
Topics: Escherichia coli; Cell Extracts; Oxidation-Reduction; Cytochromes; Recombinant Proteins; Vibrio
PubMed: 38480761
DOI: 10.1038/s41598-024-54097-7