Destruction of self-derived PAMP via T3SS2 effector VopY to subvert PAMP-triggered immunity mediates Vibrio parahaemolyticus pathogenicity.

Cyclic di-guanosine monophosphate (c-di-GMP) is a unique bacterial second messenger but is hijacked by host cells during bacterial infection as a pathogen-associated molecular pattern (PAMP) to trigger STING-dependent immune responses. Here, we show that upon infection, VopY, an effector of Vibrio parahaemolyticus, is injected into host cells by type III secretion system 2 (T3SS2), a secretion system unique to its pathogenic strains and indispensable for enterotoxicity. VopY is an EAL-domain-containing phosphodiesterase and is capable of hydrolyzing c-di-GMP. VopY expression in host cells prevents the activation of STING and STING-dependent downstream signaling triggered by c-di-GMP and, consequently, suppresses type I interferon immune responses. The presence of VopY in V. parahaemolyticus enables it to cause both T3SS2-dependent enterotoxicity and cytotoxicity. These findings uncover the destruction of self-derived PAMPs by injecting specific effectors to suppress PAMP-triggered immune responses as a unique strategy for bacterial pathogens to subvert immunity and cause disease.

Comparative in vitro and in silico study on the estrogenic effects of 2,2-bis(4-chlorophenyl)ethanol, 4,4'-dichlorobenzophenone and DDT analogs.

DDT and its transformation products (DDTs) are frequently detected in environmental and biological media. Research suggests that DDT and its primary metabolites (DDD and DDE) could induce estrogenic effects by disturbing estrogen receptor (ER) pathways. However, the estrogenic effects of DDT high-order transformation products, and the exact mechanisms underlying the differences of responses in DDT and its metabolites (or transformation products) still remain unknown. Here, besides DDT, DDD and DDE, we selected two DDT high-order transformation products, 2,2-bis(4-chlorophenyl) ethanol (p,p'-DDOH) and 4,4'-dichlorobenzophenone (p,p'-DCBP). We aim to explore and reveal the relation between DDTs activity and their estrogenic effects by receptor binding, transcriptional activity, and ER-mediated pathways. Fluorescence assays showed that the tested 8 DDTs bound to the two isoforms (ERα and ERß) of ER directly. Among them, p,p'-DDOH exhibited the highest binding affinity, with IC50 values of 0.43 µM and 0.97 µM to ERα and ERß, respectively. Eight DDTs showed different agonistic activity toward ER pathways, with p,p'-DDOH exhibiting the strongest potency. In silico studies revealed that the eight DDTs bound to either ERα or ERß in a similar manner to 17ß-estradiol, in which specific polar and non-polar interactions and water-mediated hydrogen bonds were involved. Furthermore, we found that 8 DDTs (0.0008-5 µM) showed distinct pro-proliferative effects on MCF-7 cells in an ER-dependent manner. Overall, our results revealed not only for the first time the estrogenic effects of two DDT high-order transformation products by acting on ER-mediated pathways, but also the molecular basis for differential activity of 8 DDTs.

Impacts of Ser/Thr Protein Kinase Stk1 on the Proteome, Twitching Motility, and Competitive Advantage in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium in the environment and a leading cause of nosocomial infections worldwide. Therefore, it is listed by the WHO as a human pathogen that urgently needs the development of new antibacterial drugs. Recent findings have demonstrated that eukaryote-type Ser/Thr protein kinases play a vital role in regulating various bacterial physiological processes by catalyzing protein phosphorylation. Stk1 has proven to be a Ser/Thr protein kinase in P. aeruginosa. However, the regulatory roles of Stk1 have not yet been revealed. Thus, we constructed a stk1 knockout mutant (∆stk1) from the P. aeruginosa PAO1 strain and employed a Tandem Mass Tag (TMT) labeling-based quantitative proteomic strategy to characterize proteome-wide changes in response to the stk1 knockout. In total, 620 differentially expressed proteins, among which 288 proteins were upregulated and 332 proteins were downregulated, were identified in ∆stk1 compared with P. aeruginosa PAO1. A detailed bioinformatics analysis of these differentially expressed proteins was performed, including GO annotation, protein domain profile, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, subcellular localization and enrichment analysis. Notably, the downregulation of type IV pilus-related proteins and upregulation of T6SS-H1-related proteins were found in the ∆stk1 strain, and the results were corroborated by quantitative PCR at the mRNA level. Further experiments confirmed that the loss of stk1 weakens bacterial twitching motility and promotes a growth competition advantage, which are, respectively, mediated by type IV pilus-related proteins and T6SS-H1-related proteins. These findings contribute to a better understanding of the physiological role of Stk1, and proteomic data will help further investigations of the roles and mechanisms of Stk1 in P. aeruginosa, although the detailed regulation and mechanism of Stk1 still need to be revealed.

Deletion of vp0057, a Gene Encoding a Ser/Thr Protein Kinase, Impacts the Proteome and Promotes Iron Uptake and Competitive Advantage in Vibrio parahaemolyticus.

The marine bacterial pathogen Vibrio parahaemolyticus is a major cause of food-borne gastroenteritis. Recent findings have demonstrated that protein phosphorylation is fundamental to the regulation of many physiological processes in pathogenic bacteria, including bacterial virulence. However, the underlying mechanisms remain to be completely clarified. Using bioinformatics analysis, we found that VP0057 may be a potential Ser/Thr protein kinase with phosphorylation activity. Thus, we constructed the vp0057-deletion mutant (Δvp0057) from the wild-type V. parahaemolyticus serotype O3:K6 and employed a mass spectrometry-based proteomic strategy to characterize proteome-wide changes in response to vp0057 deletion, owing to the potential roles of VP0057 in V. parahaemolyticus. One hundred ninety-seven differentially expressed proteins were identified in the Δvp0057 strain compared with the wild-type strain, among which 135 proteins were upregulated and 62 proteins were downregulated. Detailed annotation of these differentially expressed proteins was conducted. Notably, iron-related and T6SS1-related proteins were upregulated in the Δvp0057 strain, corroborating the results by quantitative PCR. Further experiments proved that vp0057 deletion promotes Fe2+ and Fe3+ uptake and provides a growth competition advantage, which is controlled by iron-related and T6SS1-related proteins, respectively. Although the regulatory roles and mechanisms of VP0057 remain to be revealed in V. parahaemolyticus, our systemic analysis of the protein profile of Δvp0057 provides a promising starting point for the intensive exploration of VP0057.

Regulatory Mechanisms and Promising Applications of Quorum Sensing-Inhibiting Agents in Control of Bacterial Biofilm Formation.

A biofilm is an assemblage of microbial cells attached to a surface and encapsulated in an extracellular polymeric substance (EPS) matrix. The formation of a biofilm is one of the important mechanisms of bacterial resistance, which not only leads to hard-to-control bacterial infections in humans and animals but also enables bacteria to be a major problem in various fields, such as food processing, wastewater treatment and metalworking. Quorum sensing (QS) is a bacterial cell-to-cell communication process that depends on the bacterial population density and is mediated by small diffusible signaling molecules called autoinducers (AIs). Bacteria use QS to regulate diverse arrays of functions, including virulence and biofilm formation. Therefore, the interference with QS by using QS inhibiting agents, including QS inhibitors (QSIs) and quorum quenching (QQ) enzymes, to reduce or even completely repress the biofilm formation of pathogenic bacteria appears to be a promising approach to control bacterial infections. In this review, we summarize the mechanisms of QS-regulating biofilm formation and QS-inhibiting agents that control bacterial biofilm formation, strategies for the discovery of new QS inhibiting agents, and the current applications of QS-inhibiting agents in several fields to provide insight into the development of effective drugs to control pathogenic bacteria.

Evaluation of the ß-barrel outer membrane protein VP1243 as a candidate antigen for a cross-protective vaccine against Vibrio infections.

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that causes acute gastroenteritis after the consumption of contaminated food, wound infection, and seizures. Antibiotic therapy is the main method for controlling Vibrio infections, which inevitably leads to drug resistance. Therefore, a vaccine is urgently needed to avoid this problem. Outer membrane proteins (OMPs) play a pivotal role in the interaction between the host immune system and bacteria. VP1243 is an OMP of V. parahaemolyticus, and it possessed immunogenicity in our previous study. The present study found that VP1243 was widely distributed, highly conserved and possessed similar surface epitopes among the major Vibrio species. The protein stimulated a strong antibody response and induced cross-reactive immune responses in V. parahaemolyticus, V. alginolyticus and V. vulnificus. Notably, it provided 100% immune protection against lethal challenges by the three Vibrio species in mice immunized with VP1243. Efficient clearance of cells of the three Vibrio bacterial species was observed in immunized mice. These findings provide solid evidence for VP1243 as a promising candidate for the development of a versatile vaccine to protect against Vibrio infections.