Integrated dual RNA-seq and dual iTRAQ of infected tissue reveals the functions of a diguanylate cyclase gene of Pseudomonas plecoglossicida in host-pathogen interactions with Epinephelus coioides.

The interactions between host and pathogen is exceedingly complex, which involves alterations at multiple molecular layers. However, research to simultaneously monitor the alterations of transcriptome and proteome between a bacterial pathogen and aquatic animal host through integrated dual RNA-seq and dual iTRAQ of tissue during infection is currently lacking. The important role of a diguanylate cyclase gene (L321_RS15240) in pathogenicity of Pseudomonas plecoglossicida against Epinephelus coioides was suggested by previous dual RNA-seq of our lab. Then L321_RS15240-RNAi strains of P. plecoglossicida were constructed with pCM130/tac, and the mutant with the best silencing effect was selected for follow-up study. The RNAi of L321_RS15240 resulted in a significant decrease in bacterial virulence of P. plecoglossicida. The E. coioides spleens infected by wild type strain or L321_RS15240-RNAi strain of P. plecoglossicida were subjected to dual RNA-seq and dual iTRAQ, respectively. The results showed that: RNAi of L321_RS15240 led to 1)alterations of host transcriptome associated with complement and coagulation cascades, ribosome, arginine and proline metabolism, and oxidative phosphorylation; 2)high expression of host proteins which related to phagosome and metabolism responses (metabolism of glutathione, amino sugar and nucleotide sugar); 3)the highly differentially expression of host lncRNAs and miRNAs. The differentially expressed proteins and mRNAs of pathogen were different after infection, but the functions of these proteins and mRNAs were mainly related to metabolism and virulence. This study provides a new insight to comprehensively understand the gene functions of pathogens and hosts at multiple molecular layers during in vivo infection.

Characterization of Salmonella enterica derivatives harboring defined aroC and Salmonella pathogenicity island 2 type III secretion system (ssaV) mutations by immunization of healthy volunteers.

The attenuation and immunogenicity of two novel Salmonella vaccine strains, Salmonella enterica serovar Typhi (Ty2 Delta aroC Delta ssaV, designated ZH9) and S. enterica serovar Typhimurium (TML Delta aroC Delta ssaV, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 10(7), 10(8), or 10(9) CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 10(8) and two of three receiving 10(9) CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 10(8) and 10(9) CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 10(9) CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi.

Construction and characterization of genetically defined aro omp mutants of enterotoxigenic Escherichia coli and preliminary studies of safety and immunogenicity in humans.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea in travelers to countries where the disease is endemic and causes a major disease burden in the indigenous population, particularly children. We describe here the generation and preclinical characterization of candidate strains of ETEC which are intended to provide the basis of a live attenuated oral vaccine to prevent this disease. It has been shown previously that a spontaneously arising toxin-negative variant ETEC strain, E1392/75-2A, could confer 75% protection against challenge when administered to volunteers. Unfortunately this strain induced mild diarrhea in 15% of recipients. To eliminate the unacceptable reactogenicity of strain E1392/75-2A, it was further attenuated by introducing three different combinations of defined deletion mutations into the chromosome. A mouse intranasal model of immunization was developed and used to show that all of the strains were immunogenic. Immune responses against colonization factor antigens (CFAs) were particularly strong when the bacterial inocula were grown on "CFA agar," which induces strong expression of these antigens. Two of the strains were selected for a phase I dose escalation safety study with healthy adult volunteers. Freshly grown organisms were harvested from CFA agar plates and administered to volunteers as a suspension containing from 5 x 10(7) to 5 x 10(9) CFU. The vaccine was well tolerated at all doses and induced significant immune responses in all recipients at the highest dose of either strain. The results provide the basis for further clinical evaluation of these vaccine candidates.