The Pseudomonas syringae pathovars are composed of host-specific plant pathogens that characteristically elicit the defense-associated hypersensitive response (HR) in nonhost plants. P. s. pv. syringae 61 secretes an HR elicitor, harpinPss (HrpZPss), in a hrp-dependent manner. An internal fragment of the P. s. pv. syringae 61 hrpZ gene was used to clone the hrpZ locus from P. s. pv. glycinea race 4 (bacterial blight of soybean) and P. s. pv. tomato DC3000 (bacterial speck of tomato). DNA sequence analysis revealed that hrpZ is the second ORF in a polycistronic operon. The amino acid sequence identities of HrpZPss/HrpZPsg and HrpZPss/HrpZPst were 79 and 63%, respectively. Although none of the HrpZ proteins showed significant overall sequence similarity with other known proteins, HrpZPst contained a 24-amino acid sequence that is homologous with a region of the PopA1 elicitor protein of the tomato pathogen, Pseudomonas solanacearum GMI1000. hrpA, the upstream ORF, was highly divergent: The amino acid sequence identities of HrpAPss/HrpAPsg and HrpAPss/HrpAPst were 91 and 28%, respectively, and no HrpA sequence showed similarity to known proteins. In contrast, the predicted products of the downstream ORFs in P. s. pv. syringae and P. s. pv. tomato, hrpB, hrpC, hrpD, and hrpE showed varying levels of similarity to those of yscI, yscJ, yscK, and yscL. These are colinearly arranged genes in the virC locus of Yersinia spp., which are involved in the secretion of the Yop virulence proteins via the type III pathway. The similarity of the Ysc proteins was generally stronger in comparisons with the P. s. pv. tomato Hrp proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMedDate not supplied

Oral immunization with a live recombinant attenuated Salmonella typhimurium protects mice against Toxoplasma gondii.

The natural site of infection for T. gondii is the mucosal surface of the intestine, so the protective immunity obtained after natural infection with T. gondii points to the importance of developing a vaccine that stimulates mucosal defences. In this study, an aroA- and aroD- attenuated strain of Salmonella typhimurium (BRD509) has been used to deliver the recombinant eukaryotic plasmid pSAG(1-2)/CTA2/B expressing a multi-antigenic gene encoding SAG1 and SAG2 of T. gondii linked to A2/B subunits of cholera toxin as a candidate oral T. gondii vaccine. Immunoblot analysis showed compound gene expression in HeLa cells in vitro and intragastric immunization of mice with the recombinant salmonella resulted in the induction of humoral and Th1 type cellular immune responses and afforded protection against RH strain T. gondii challenge. Anti-T. gondii IgG values increased markedly in the BRD509/pSAG(1-2)-CTA2/B immunized group; these values were significantly higher than those in the negative controls (P = 0.008). With CTA2/B genetic adjuvant, the T. gondii-specific response was predominantly Th1, indicating that the CTA(2)/B genetic adjuvant was able to overcome the strong Th2-bias of the antigen (IgG2a >> IgG1). Antigen-specific T cell proliferative responses and CTL activity were significantly enhanced when cholera toxin CTA2/B genetic adjuvant was used (P = 0.009; P = 0.006). Culture supernatants from antigen-stimulated splenocytes from mice in these groups were also examined by ELISA for Th1- and Th2-type cytokines; mean IFN-gamma levels produced after oral immunization with BRD509/pSAG(1-2)-CTA2/B were about nine-fold higher than after immunization with BRD509/pSAG(1-2) (P = 0.007). On the other hand, the levels of IL-4 were low for all groups and no increase was seen in the presence of CTA2/B genetic adjuvant. When the immunized mice were intraperitoneally challenged with 10(3) tachyzoites of the highly virulent RH strain, the survival time of the mice immunized with BRD509/pSAG(1-2)-CTA2/B was markedly longer than other groups (P = 0.003) and a 40% survival rate was achieved. This is the first report that demonstrates that an oral attenuated salmonella DNA vaccine can induce protective immunity against the acute phase of T. gondii infection.

Adjuvants, Immunologic↗