[Comparison of three methods for preparation of bacterial ghosts from avian pathogenic Escherichia coli].

Bacterial ghosts are bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology, which can be used as a new vaccine and delivery vector. In this study, a clinical isolate of avian pathogenic Escherichia coli (APEC) strain DE17 was used to prepare bacterial ghost through three different ways. The results showed that the cleavage efficiency of DE17 bacterial ghost was 99.9% with the lysis plasmid containing the PhiX174 lysis gene E. Scanning electron microscopy showed that transmembrane tunnels were formed in the middle or both ends of the cell envelope of DE17. Furthermore, the DE17 bacterial ghost was prepared with one of cell penetrating peptides (CPPs) named MAP (KLALKLALKALKAALKLA), which will completely inactivate DE17 (OD600=0.1) by 10 µmol/L MAP. The cell envelope showed a gully-like structure and obvious transmembrane tunnels were not found through the SEM. However, the DE17 could not be lysed by importing the lysis plasmid (pBV220-MAP), which was used to express MAP. The present study will benefit for research on bacterial ghost preparation methods and provide a reference for biosafety of bacterial ghost vaccines.

Different activated methyl cycle pathways affect the pathogenicity of avian pathogenic Escherichia coli.

The activated methyl cycle (AMC) regulates the cellular levels of S-adenosyl-l-homocysteine (SAH) in bacteria, which plays a crucial role in bacterial pathogenicity. There are two AMC pathways in bacteria: one is a two-step reaction pathway (named the LuxS/Pfs pathway) in which LuxS and Pfs catalyze the conversion of SAH to l-homocysteine and autoinducer-2 (AI-2), and the other is a one-step reaction (named the SahH pathway) mediated by S-adenosyl-l-homocysteine hydrolase (SahH), which completes this cycle without producing AI-2. In this study, we evaluated the effects of different AMC pathways on the pathogenicity of avian pathogenic Escherichia coli (APEC). The plasmid pSTV-sahH (containing the sahH gene of Pseudomonas aeruginosa) was transformed into the wild-type APEC strain DE17 (containing the LuxS/Pfs pathway) and the pfs mutant strain DE17Δpfs, which lacks the LuxS/Pfs pathway, to create the strains SahH-DE17Δpfs (containing the SahH pathway) and SahH-DE17 (containing the LuxS/Pfs and SahH pathways). The results showed that the different AMC pathways had different effects on the growth rate, AI-2 activity, and motility in APEC. Furthermore, we showed that the 50% lethal doses of the DE17Δpfs and SahH-DE17Δpfs strains were reduced by 650-fold and 52-fold, respectively, in ducklings, compared with that of the DE17 strain. The DE17Δpfs strain exhibited significantly reduced adherence and invasion (p<0.01). In addition, the DE17Δpfs and SahH-DE17Δpfs strains also showed reduced survival in vivo, as evidenced by significant (p<0.01) reductions in their bacterial loads in infected liver, spleen, kidney, and blood. This study suggests that different AMC pathways affect the pathogenesis of APEC.

The role of the ptsI gene on AI-2 internalization and pathogenesis of avian pathogenic Escherichia coli.

The LuxS/AI-2 quorum sensing mechanism can regulate the physiological functions of avian pathogenic Escherichia coli (APEC) through internalization of the small molecule autoinducer-2 (AI-2). The ptsI gene encodes enzyme I, which participates in the phosphotransferase system (PTS) that regulates the virulence and AI-2 internalization of bacteria. The aim of the present study was to determine the effect of ptsI on AI-2 internalization and other pathogenesis process in APEC using a ptsI mutant of the APEC strain DE17 (serotype O2), namely DE17ΔptsI. The results showed that deletion of the ptsI gene changed the rdar (red dry and rough) morphotype and decreased motility and biofilm formation in APEC (p < 0.05). Furthermore, scanning electron microscopy showed that the biofilm structure of DE17ΔptsI became sparse and more extracellular, as compared with the wild-type strain DE17. Moreover, AI-2 assay showed that AI-2 was internalized by DE17ΔptsI, while the recombinant PtsI protein had no AI-2 binding activity. Furthermore, deletion of the ptsI gene in APEC significantly increased adherence to DF-1 cells (p < 0.05). The 50% lethal dose of DE17ΔptsI was decreased by 17.8-fold and the bacterial loads of DE17ΔptsI were decreased by 13600-, 68.5-, 131-, and 3600-fold in the blood, liver, spleen, and kidney, respectively, as compared to the DE17. Moreover, histopathological analysis showed that the mutant DE17ΔptsI was associated with reduced pathological changes in the heart, liver, spleen, and kidney of ducklings, respectively, as compared to the wild-type strain DE17. The results of this study will benefit further studies on the functions of the ptsI in APEC.