DctR contributes to the virulence of avian pathogenic Escherichia coli through regulation of type III secretion system 2 expression.

Pathogens could precisely alter their gene expression to facilitate their survival and successful infection. The LuxR family transcriptional regulator DctR (also known as YhiF) was shown to participate in the regulation of acid fitness and adhesion of enterohemorrhagic E. coli (EHEC) O157:H7. Avian pathogenic Escherichia coli (APEC) causes significant economic losses to the poultry industries and also potentially threatens human health. However, the effects of DctR on the fitness and virulence of APEC have not been investigated yet. To assess the function of DctR in APEC, the dctR gene mutant and complemented strains were constructed and biologically characterized. Our results show that inactivation of the dctR gene led to decreased biofilm formation, diminished serum resistance, reduced adherence capacity, attenuated colonization and virulence of APEC in ducks. The altered capacities of the mutant strain were restored by genetic complementation. In addition, we found that DctR positively regulates the expression of E. coli type III secretion system 2 (ETT2) core genes in APEC. The expression of the inflammatory cytokines interleukin (IL)-1ß and IL-8 were decreased in HD-11 macrophages infected with the mutant strain compared with the wild-type strain. These observations indicate that regulator DctR contributes to the virulence of APEC through regulation of ETT2 expression.

The Ferric Uptake Regulator Represses Type VI Secretion System Function by Binding Directly to the clpV Promoter in Salmonella enterica Serovar Typhimurium.

Type VI secretion systems (T6SSs) are highly conserved and complex protein secretion systems that deliver effector proteins into eukaryotic hosts or other bacteria. T6SSs are regulated precisely by a variety of regulatory systems, which enables bacteria to adapt to varied environments. A T6SS within Salmonella pathogenicity island 6 (SPI-6) is activated during infection, and it contributes to the pathogenesis, as well as interbacterial competition, of Salmonella enterica serovar Typhimurium (S. Typhimurium). However, the regulation of the SPI-6 T6SS in S. Typhimurium is not well understood. In this study, we found that the SPI-6 T6SS core gene clpV was significantly upregulated in response to the iron-depleted condition and during infection. The global ferric uptake regulator (Fur) was shown to repress the clpV expression in the iron-replete medium. Moreover, electrophoretic mobility shift and DNase I footprinting assays revealed that Fur binds directly to the clpV promoter region at multiple sites spanning the transcriptional start site. We also observed that the relieving of Fur-mediated repression on clpV contributed to the interbacterial competition activity and pathogenicity of S. Typhimurium. These findings provide insights into the direct regulation of Fur in the expression and functional activity of SPI-6 T6SS in S. Typhimurium and thus help to elucidate the mechanisms of bacterial adaptability and virulence.

The CpxR regulates type VI secretion system 2 expression and facilitates the interbacterial competition activity and virulence of avian pathogenic Escherichia coli.

Systemic infections caused by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide and are also potentially threatening to human health. Pathogens must be able to precisely modulate gene expression to facilitate their survival and the successful infection. The Cpx two-component signal transduction system (TCS) regulates surface structure assembly and virulence factors implicated in Gram-negative bacterial pathogenesis. However, the roles of the Cpx TCS in bacterial fitness and pathogenesis during APEC infection are not completely understood. Here, we show that the Cpx TCS response regulator CpxR is critical to the survival and virulence of APEC. Inactivation of cpxR leads to significant defects in the interbacterial competition activity, invasion and survival of APEC in vitro and in vivo. Moreover, activation of CpxR positive regulates the expression of the APEC type VI secretion system 2 (T6SS2). Further investigations revealed that phosphorylated CpxR directly bound to the T6SS2 hcp2B promoter region. Taken together, our results demonstrated that CpxR contributes to the pathogensis of APEC at least through directly regulating the expression and function of T6SS2. This study broadens understanding of the regulatory effect of Cpx TCS, thus elucidating the mechanisms through which Cpx TCS involved in bacterial virulence.

Harnessing noncanonical trans-cleavage characteristics of Cas12 and Cas13a to enhance CRISPR-based diagnostics.

Cas12 and Cas13 are extensively utilized in molecular diagnostics for their trans-cleavage activities, yet their activation characteristics remain partially understood. Here, we conduct an in-depth investigation of Cas12a, Cas12f1, and Cas13a, uncovering the characteristics of their trans-DNase and trans-RNase activities with noncanonical activators. Our findings reveal that DNA can serve as a direct target for CRISPR-Cas13a, markedly increasing the detection sensitivity for single-base mismatches. Moreover, the trans-cleavage activities of Cas12a and Cas13a can be activated by diverse RNA:DNA and RNA:RNA duplexes, respectively, indicating that the presence of stem-loop structures in crRNAs is not essential for their activation. Notably, Cas12f1, unlike Cas12a, exhibits intrinsic RNase activity independently of activation. Leveraging these insights, we have improved the accuracy of a dual-gene target detection approach that employs the CRISPR-Cas12f1 and Cas13a systems. Our research advances the understanding of the noncanonical activation characteristics of Cas12 and Cas13a, contributing to the field of CRISPR-based diagnostics.

Characterization of a Broad-Host-Range Lytic Phage SHWT1 Against Multidrug-Resistant Salmonella and Evaluation of Its Therapeutic Efficacy in vitro and in vivo.

Inappropriate use of antibiotics has accelerated to the emergence of multidrug-resistant bacteria, becoming a major health threat. Moreover, bacterial biofilms contribute to antibiotic resistance and prolonged infections. Bacteriophage (phage) therapy may provide an alternative strategy for controlling multidrug-resistant bacterial infections. In this study, a broad-host-range phage, SHWT1, with lytic activity against multidrug-resistant Salmonella was isolated, characterized and evaluated for the therapeutic efficacy in vitro and in vivo. Phage SHWT1 exhibited specific lytic activity against the prevalent Salmonella serovars, such as Salmonella Pullorum, Salmonella Gallinarum, Salmonella Enteritidis, and Salmonella Typhimurium. Morphological analysis showed that phage SHWT1 was a member of the family Siphoviridae and the order Caudovirales. Phage SHWT1 had a latent period of 5 min and burst size of ~150 plaque-forming units (PFUs)/cell. The phage was stable from pH 3-12 and 4-65°C. Phage SHWT1 also showed capacity to lyse Salmonella planktonic cells and inhibit the biofilm formation at optimal multiplicity of infection (MOI) of 0.001, 0.01, 0.1, and 100, respectively. In addition, phage SHWT1 was able to lyse intracellular Salmonella within macrophages. Genome sequencing and phylogenetic analyses revealed that SHWT1 was a lytic phage without toxin genes, virulence genes, antibiotic resistance genes, or significant genomic rearrangements. We found that phage SHWT1 could successfully protect mice against S. enteritidis and S. typhimurium infection. Elucidation of the characteristics and genome sequence of phage SHWT1 demonstrates that this phage is a potential therapeutic agent against the salmonellosis caused by multidrug-resistant Salmonella.