TL;DR
The data obtained allows suggesting that SsrS and CsrC small RNAs played an important role in regulation of the cell switching to formation of biofilms, and hexuronates can be used as modeling agents in clinical practice.
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.
Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria, contributing to membrane integrity and environmental interactions. Genome-wide studies defining bacterial gene functions have been extensively performed in the model strain Escherichia coli K-12 which lacks O-antigen (OAg), and therefore does not produce smooth LPS (S-LPS). Consequently, the genetic requirements for S-LPS production in this model system remain incompletely defined. Here, a functional wbbL gene was introduced into the E. coli K-12 KEIO single-gene deletion mutant library to restore OAg synthesis, enabling genome-wide analysis of S-LPS production by screening with colicin E2 (ColE2) and validated with LPS silver staining. This identified 319 mutants with increased sensitivity to ColE2 in the presence of OAg, suggesting broader envelope-associated effects during screening. In addition, 27 mutants showed defects in S-LPS production, corresponding to genes involved in OAg biosynthesis, LPS core and sugar precursor synthesis, OAg ligation and regulation, and enterobacterial common antigen biosynthesis. A further 18 mutants initially appeared defective in S-LPS production but could not be validated upon reconstruction, and whole-genome sequencing revealed secondary mutations responsible for the observed phenotypes. This study provides a validated genetic framework for S-LPS production in E. coli K-12 and highlights the importance of rigorous validation in genome-wide screening approaches.
ABSTRACT Quorum sensing (QS) is widely involved in the regulation of the biological functions and pathogenic processes of bacteria in a cell density-dependent manner. The anthranilic acid signal is directly sensed by the receptor RaaR, a transcription regulator, to control the expression level of downstream target genes in Ralstonia solanacearum, thus affecting the QS signals generation and the bacterial virulence. Here, we reported that 4-Aminobenzoic acid (PABA), a structural analogue of anthranilic acid, not only significantly inhibited the production of extracellular polysaccharides (EPS), biofilm formation, and motility of R. solanacearum, but also reduced the expression level of QS signaling molecules. We also found that PABA can directly bind to RaaR and prevented it from interacting with the promoters of target genes associated with the QS system and virulence of R. solanacearum. Point mutations in RaaR at residues F137 and I270 abolished the binding between RaaR and PABA, indicating that these two amino acid residues are likely critical for the interaction between RaaR and PABA. These findings indicated that PABA could be a candidate inhibitor of anthranilic acid signaling to interfere with the QS systems of R. solanacearum to attenuate the virulence. Together, our work presented a non-antibiotic-based treatment strategy and provided a theoretical basis for the development of novel antibacterial treatment.
Uropathogenic Escherichia coli (UPEC) remains the most common etiologic agent of community-acquired urinary tract infections (UTIs), largely due to its extensive virulence arsenal and adaptive regulatory mechanisms. In this study, we examined the phenotypic regulation of major virulence factors—hemolysin production, biofilm, swarming motility, and adhesion—under exposure to sub-inhibitory concentrations of Ceftazidime, an extended-spectrum β-lactam antibiotic. The transcriptional response of the BarA/UvrY two-component regulatory system was also examined to unravel its role in response to antibiotic stress. Using an extensive range of microbiological, molecular, and phenotypic tests, we unveiled differential profiles of virulence traits. Exposure to sub-MICs Ceftazidime markedly diminished hemolytic activity, biofilm formation, and adhesive capacity, with swarming motility exhibiting almost complete inhibition. Moreover, quantitative real-time PCR (qRT-PCR) analysis revealed variable transcriptional responses of BarA and UvrY in the tested isolates, with the recovery of BarA expression noted after prolonged exposure to antibiotics. These findings emphasize the dynamic expression of UPEC's stress response mechanisms and outline the central role of Bar/UvrY in modulating pathoadaptive mechanisms under antibiotic stress.