Effects of single and double deletion of Vibrio anguillarum histone-like protein HU subunits on biofilm formation and pathogenicity in flounder (Paralichthys olivaceus).

Transcriptomics and functional analysis reveal impaired motility, biofilm formation, invasion and intramacrophage survival of pimt gene-deleted strain of Salmonella Typhimurium.

Inside the host, Salmonella suffers but survives various stresses. Proteins are the prime targets of host inflammatory responses. Salmonella encodes two key protein repair enzymes, methionine sulfoxide reductase (Msr) and protein isoaspartate methyltransferase (PIMT), that reactivates damaged proteins without their translational synthesis. Under stress, the accumulation of L-isoaspartate (isoAsp) residues causes defects in protein shape and function, which lead to impaired bacterial survival. The PIMT converts the isoAsp residues into Asp residues. Earlier, we observed hypersensitivity of Δpimt mutant strain to various stresses and defective colonisation in mice and poultry. Here, we show that Δpimt mutant strain accumulates about 1.67-fold more intracellular ROS levels and higher protein aggregations. RNA-seq analysis of the pimt gene-deleted strain of S. typhimurium revealed differential expression of 2676 genes. Most of the downregulated genes are related to flagellar assembly, chemotaxis, fimbria formation, Salmonella pathogenicity island encoding type-3 secretion system, mainly implicated in motility, adhesion, invasion and intracellular survival inside intestinal epithelium, phagocytic cells, etc. These factors are known to be the major contributors for the virulence of S. typhimurium inside the host. The functional analysis revealed that the Δpimt mutant strain shows defective motility, reduced biofilm formation, and defective invasion and intramacrophage survival. Transcomplementation resulted in partial phenotype (like defective motility, biofilm formation and invasion and intramacrophage survival) restoration of Δpimt mutant strain. Interestingly, supplementation of Δpimt mutant strain culture with reduced glutathione (GSH) resulted in neutralization of ROS and rescued the defective motility and biofilm formation of the mutant strain.

YgeP, a global regulator within the ETT2 pathogenicity island, coordinates motility-biofilm balance and negatively controls serum resistance in avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is the primary pathogen causing avian colibacillosis, leading to significant economic losses in the global poultry industry. Its pathogenicity relies on a complex network of virulence factors, among which the type III secretion system 2 (ETT2) is a key component. However, unlike the classical T3SS, the ETT2 gene cluster in APEC commonly exhibits pseudogenization and deletions, suggesting that its function may have undergone adaptive remodeling. Notably, the YgeP gene located at the end of this pathogenicity island is highly conserved, implying that it may play an important role in the pathogenic process. To elucidate the function of YgeP, a member of this pathogenicity island, in APEC pathogenesis, a gene deletion mutant was constructed. The study confirmed that YgeP acts as a key global regulator, positively regulating bacterial motility by influencing the expression of flagellar synthesis-related genes. Conversely, YgeP negatively regulates biofilm formation and adhesion to chicken fibroblast cells. Additionally, YgeP differentially regulates bacterial tolerance to various environmental stresses, such as acid, alkali, heat, and oxidative stress. Mechanistically, YgeP negatively regulates serum resistance by inhibiting the Rcs phosphorelay system (manifested as upregulation of RcsA/B genes) and the expression of downstream capsular polysaccharide synthesis genes (e.g., wza, wzaA). Promoter activity assays further demonstrated that YgeP directly represses these target genes as a transcriptional repressor. Therefore, YgeP plays a central role in APEC pathogenesis by balancing two infection strategies: motility-mediated dissemination and colonization, and biofilm-mediated attachment and tolerance. Simultaneously, it negatively regulates key virulence traits, including serum resistance, during the early stages of infection.

flgK regulates food-related colonization and virulence potential of Vibrio mimicus in aquatic products.

Vibrio mimicus is a foodborne pathogen that contaminates aquatic products and causes gastroenteritis in humans. The flagellar protein FlgK is required for flagellar assembly and motility in several bacteria, but its role in V. mimicus remains unclear. In this study, we constructed an in-frame flgK deletion mutant and a complementary strain to investigate the biological functions of FlgK. Transmission electron microscopy revealed a complete loss of flagellar structures in the ΔflgK strain, which was restored upon complementation. Loss of flgK exerted no significant impact on bacterial growth yet abolished swimming motility entirely and diminished biofilm formation capacity. The ΔflgK strain exhibited significantly decreased adhesion to prawn, crayfish, plastic, glass, and stainless steel compared to the WT strain. Consistently, flgK deletion reduced colonization and pathogenicity in the prawn host. Transcriptomic analysis revealed extensive downregulation of flagellar biogenesis pathways accompanied by coordinated alterations in genes involved in transport processes and central metabolism in the ΔflgK strain. Collectively, these results demonstrate that flgK is indispensable for flagellar assembly, motility, and virulence in V. mimicus, and that its loss induces broad physiological adaptations that impair food-related persistence and host colonization, identifying flgK as a potential target for aquatic food safety interventions.

Phosphoenolpyruvate Carboxykinase Controls Edwardsiella tarda Virulence via Oxaloacetate

Understanding the metabolic basis of bacterial virulence is essential for developing anti-infective strategies. Phosphoenolpyruvate carboxykinase (PEPCK), encoded by pckA, is a key enzyme at the metabolic junction between the pyruvate cycle and the TCA cycle, but its role in virulence regulation remains largely unexplored. In this study, we generated a pckA deletion mutant, ΔpckA, in Edwardsiella tarda and investigated its impact on virulence, metabolism, and vaccine potential. The ΔpckA mutant exhibited increased antibiotic sensitivity, faster growth, reduced autoaggregation, and significantly attenuated virulence in a tilapia infection model. Quantitative proteomics revealed global downregulation of type III and type VI secretion systems (T3SS and T6SS) and upregulation of flagellar proteins. Mechanistically, accumulation of oxaloacetate (OAA) in the mutant suppressed T3SS/T6SS expression without affecting flagellar motility. Importantly, ΔpckA induced a robust immune response in zebrafish and conferred strong protection against lethal challenge, with relative percent survival values of 64% and 81.82% in zebrafish and tilapia, respectively. Taken together, our findings identify PEPCK as a critical metabolic regulator of E. tarda virulence and highlight ΔpckA as a promising live attenuated vaccine candidate.

Role of the Alkyl Hydroperoxide Reductase Subunit C1 Gene in Vibrio parahaemolyticus Against Hydrogen Peroxide

A haloarchaeal VapBC toxin-antitoxin system: regulatory mechanism and role in establishing population heterogeneity to facilitate survival of subpopulations under stress conditions

ABSTRACT Toxin-antitoxin (TA) systems are ubiquitously distributed in bacteria and archaea and have been implicated in various functions such as plasmid maintenance, phage defense, biofilm formation, stress response, and persistence, but their regulatory mechanisms and roles in haloarchaea are largely unknown. In this study, we found that four of the eight putative VapBC TA systems of Natrinema gari J7-2 are functional in strain J7-2 and Haloferax volcanii. Among them, the NgVapBC1 system is composed of the antitoxin NgVapB1 and the toxin NgVapC1, and the two proteins could form a NgVapBC1 complex. The NgVapBC1 system could be transcriptionally autoregulated, wherein a pseudo-palindromic sequence in the vapBC1 operon promoter acts as a negative cis-regulatory element, and the NgVapBC1 complex is a stronger repressor than NgVapB1. The qRT-PCR and in vivo toxicity analyses showed that strain J7-2 maintains a high vapB1/vapC1 mRNA ratio through truncation of the vapBC1 operon transcript within the toxin-coding region for preventing the synthesis of excess toxin NgVapC1 to release toxicity abnormally. Mutational analyses showed that differential start codon usage by vapB1 and vapC1 and a −4 nt overlap of their stop and start codons contribute to maintaining a higher vapB1/vapC1 mRNA ratio, and the −4 nt overlap-mediated translational coupling of vapB1 and vapC1 would enable both post-transcriptional and translational regulation of the NgVapBC1 system. The phenotypic comparison of strain J7-2 and its ΔvapBC1 mutant revealed that the NgVapBC1 system contributes to population heterogeneity of decline-phase rather than exponential-phase strain J7-2 and facilitates the survival of subpopulations under stress conditions. IMPORTANCE Although TA systems are regarded as versatile modulators of prokaryotic cell fate, their bona fide physiological roles remain elusive. Moreover, so far, there is no report on the regulatory mechanism and function of haloarchaeal VapBCs, which constitute the major group of TA systems in haloarchaea. Our results demonstrate that a VapBC system of the haloarchaeon Natrinema gari J7-2 is regulated at multiple levels and mediates the establishment of population heterogeneity, thereby facilitating the survival of the strain J7-2 population as a whole in diverse and changing environments. This study provides the first experimental evidence of the regulatory mechanism of the biological role of the VapBC system in conferring fitness advantages to haloarchaea. Although TA systems are regarded as versatile modulators of prokaryotic cell fate, their bona fide physiological roles remain elusive. Moreover, so far, there is no report on the regulatory mechanism and function of haloarchaeal VapBCs, which constitute the major group of TA systems in haloarchaea. Our results demonstrate that a VapBC system of the haloarchaeon Natrinema gari J7-2 is regulated at multiple levels and mediates the establishment of population heterogeneity, thereby facilitating the survival of the strain J7-2 population as a whole in diverse and changing environments. This study provides the first experimental evidence of the regulatory mechanism of the biological role of the VapBC system in conferring fitness advantages to haloarchaea.