Porcine beta-defensin 2 (PBD-2) which is a member of the family of antimicrobial peptides, is widely expressed in pig organs with a broad spectrum of bactericidal activities confirmed in vitro. We previously demonstrated that transgenic (TG) pigs overexpressing PBD-2 could resist the infection by the porcine pathogen Actinobacillus pleuropneumoniae. In this study, the roles of PBD-2 in protecting against bacterial infection were further investigated. The biochemical indexes of the blood sample, body weights, histological morphologies, and weights of the organs of TG mice expressing PBD-2 were measured. Results confirmed that these mice showed normal physiological features. An assay of Salmonella Typhimurium infection was conducted on wild-type (WT) and TG mice. The TG mice possessed higher survival rate, less body weight loss, and pathological changes and smaller recovery rates of bacteria after infection with S. Typhimurium. The in vitro synthetic PBD-2 and the serum and tissue homogenates from the TG mice displayed a direct bactericidal activity. Moreover, PBD-2 could inhibit the release of the proinflammatory cytokines, including IL-6, TNF-α, IL-1β, and IL-12, in the TG mice infected with S. Typhimurium or treated with lipopolysaccharide (LPS). The WT mice treated with PBD-2 and S. Typhimurium or LPS showed reduced levels of proinflammatory cytokines. The mouse macrophage cell line RAW 264.7 which expressed PBD-2 was constructed to detect the signal pathways affected by PBD-2. The suppressing effect of PBD-2 on the release of the proinflammatory cytokines was confirmed using RAW 264.7 either expressing PBD-2 or supplemented with PBD-2. The promoter activity and mRNA level of NF-κB were detected, and PBD-2 was shown to significantly inhibit the activation of the NF-κB pathway induced by LPS. The direct interaction of PBD-2 with TLR4 was revealed by isothermal titration calorimetry and far-Western blot in vitro and the coimmunoprecipitation of PBD-2 with TLR4 on RAW 264.7 cells. This interaction indicates one reason for the interference of NF-κB activation. Overall, this study showed that PBD-2 protected against bacterial infection through a direct bactericidal activity and alleviated inflammation by interfering with the TLR4/NF-κB pathway.

The general secretory pathway (Sec) and twin-arginine translocase (Tat) operate in parallel to export proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Substrates are targeted to their respective machineries by N-terminal signal peptides that share a tripartite organization; however, Tat signal peptides harbor a conserved and almost invariant arginine pair that is critical for efficient targeting to the Tat machinery. Tat signal peptides interact with a membrane-bound receptor complex comprised of TatB and TatC components, with TatC containing the twin-arginine recognition site. Here, we isolated suppressors in the signal peptide of the Tat substrate, SufI, that restored Tat transport in the presence of inactivating substitutions in the TatC twin-arginine binding site. These suppressors increased signal peptide hydrophobicity, and copurification experiments indicated that they restored binding to the variant TatBC complex. The hydrophobic suppressors could also act in cis to suppress substitutions at the signal peptide twin-arginine motif that normally prevent targeting to the Tat pathway. Highly hydrophobic variants of the SufI signal peptide containing four leucine substitutions retained the ability to interact with the Tat system. The hydrophobic signal peptides of two Sec substrates, DsbA and OmpA, containing twin lysine residues, were shown to mediate export by the Tat pathway and to copurify with TatBC. These findings indicate that there is unprecedented overlap between Sec and Tat signal peptides and that neither the signal peptide twin-arginine motif nor the TatC twin-arginine recognition site is an essential mechanistic feature for operation of the Tat pathway. IMPORTANCE Protein export is an essential process in all prokaryotes. The Sec and Tat export pathways operate in parallel, with the Sec machinery transporting unstructured precursors and the Tat pathway transporting folded proteins. Proteins are targeted to the Tat pathway by N-terminal signal peptides that contain an almost invariant twin-arginine motif. Here, we make the surprising discovery that the twin arginines are not essential for recognition of substrates by the Tat machinery and that this requirement can be bypassed by increasing the signal peptide hydrophobicity. We further show that signal peptides of bona fide Sec substrates can also mediate transport by the Tat pathway. Our findings suggest that key features of the Tat targeting mechanism have evolved to prevent mistargeting of substrates to the Sec pathway rather than being a critical requirement for function of the Tat pathway.

The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system of Escherichia coli is made up of TatA, TatB, and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site-specific cross-linking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA–YFP fusion showed that the TatB F13Y substitution resulted in signal peptide-independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase. Significance The twin-arginine translocation (Tat) system transports folded proteins across the prokaryotic inner membrane and the thylakoid membrane of plant chloroplasts. Proteins are targeted to the Tat system by signal peptides containing a highly conserved twin arginine motif. We isolated suppressors in the TatB component that allowed a Tat substrate with a defective twin arginine motif to be transported. The strongest of these suppressors, TatB F13Y, resulted in the constitutive assembly of the Tat translocase in the absence of signal peptide binding. These results show that Tat signal peptides have two separable roles: they target their passenger proteins to the Tat machinery but they also trigger the assembly of the active Tat transporter.

Catecholamines are host stress hormones that can induce the growth of many bacteria by facilitating iron utilization and/or regulate the expression of virulence genes through specific hormone receptors. Whether these two responsive pathways are interconnected is unknown. In our previous study, it was found that catecholamines can regulate the expression of a great number of genes of Actinobacillus pleuropneumoniae, an important swine respiratory pathogen. However, bacterial growth was not affected by catecholamines in rich medium. In this study, it was discovered that catecholamines affected A. pleuropneumoniae growth in chemically defined medium (CDM). We found that serum inhibited A. pleuropneumoniae growth in CDM, while epinephrine, norepinephrine and dopamine promoted A. pleuropneumoniae growth in the CDM containing serum. The known bacterial hormone receptor QseC didn’t play roles in this process. Ion-supplementation and transcriptome analysis indicated that serum addition resulted in iron-restricted conditions which were alleviated by the addition of catecholamines. Transferrin, one of the components in serum, inhibited the growth of A. pleuropneumoniae in CDM, an effect reversed by addition of catecholamines in a TonB2-dependent manner. Our data demonstrate that catecholamines promote A. pleuropneumoniae growth by regulating iron-acquisition and metabolism, which is independent of the adrenergic receptor QseC.

Our previous study showed that the inactivation of the efflux pump TolC could abolish biofilm formation and curli production of extraintestinal pathogenic Escherichia coli (ExPEC) strain PPECC42 under hyper-osmotic conditions. In this study we investigated the role of OmpX in biofilm formation and curli production of ExPEC PPECC42. Our data showed that OmpX disruption or overexpression didn't significantly affect the biofilm formation and curli production of the wild-type strain. However, in the tolC-deleted mutant, overexpressing OmpX suppressed the effect of TolC inactivation on ExPEC biofilm formation and curli production under hyper-osmotic growth conditions. Real-time qRT-PCR confirmed that OmpX overexpression affected curli production by regulating the transcription of the curli biosynthesis-related genes in the ΔtolC strain. Our findings suggest that OmpX is involved in biofilm formation and curli production.