Participation of the Salmonella OmpD porin in the infection of RAW264.7 macrophages and BALB/c mice.

Salmonella Typhimurium is the etiological agent of gastroenteritis in humans and enteric fever in mice. Inside these hosts, Salmonella must overcome hostile conditions to develop a successful infection, a process in which the levels of porins may be critical. Herein, the role of the Salmonella Typhimurium porin OmpD in the infection process was assessed for adherence, invasion and proliferation in RAW264.7 mouse macrophages and in BALB/c mice. In cultured macrophages, a ΔompD strain exhibited increased invasion and proliferation phenotypes as compared to its parental strain. In contrast, overexpression of ompD caused a reduction in bacterial proliferation but did not affect adherence or invasion. In the murine model, the ΔompD strain showed increased ability to survive and replicate in target organs of infection. The ompD transcript levels showed a down-regulation when Salmonella resided within cultured macrophages and when it colonized target organs in infected mice. Additionally, cultured macrophages infected with the ΔompD strain produced lower levels of reactive oxygen species, suggesting that down-regulation of ompD could favor replication of Salmonella inside macrophages and the subsequent systemic dissemination, by limiting the reactive oxygen species response of the host.

Salmonella Typhi OmpS1 and OmpS2 porins are potent protective immunogens with adjuvant properties.

Salmonella enterica serovar Typhi (S. Typhi) is the causal agent of typhoid fever, a disease that primarily affects developing countries. Various antigens from this bacterium have been reported to be targets of the immune response. Recently, the S. Typhi genome has been shown to encode two porins--OmpS1 and OmpS2--which are expressed at low levels under in vitro culture conditions. In this study, we demonstrate that immunizing mice with either OmpS1 or OmpS2 induced production of specific, long-term antibody titres and conferred protection against S. Typhi challenge; in particular, OmpS1 was more immunogenic and conferred greater protective effects than OmpS2. We also found that OmpS1 is a Toll-like receptor 4 (TLR4) agonist, whereas OmpS2 is a TLR2 and TLR4 agonist. Both porins induced the production of tumour necrosis factor and interleukin-6, and OmpS2 was also able to induce interleukin-10 production. Furthermore, OmpS1 induced the over-expression of MHC II molecules in dendritic cells and OmpS2 induced the over-expression of CD40 molecules in macrophages and dendritic cells. Co-immunization of OmpS1 or OmpS2 with ovalbumin (OVA) increased anti-OVA antibody titres, the duration and isotype diversity of the OVA-specific antibody response, and the proliferation of T lymphocytes. These porins also had adjuvant effects on the antibody response when co-immunized with either the Vi capsular antigen from S. Typhi or inactivated 2009 pandemic influenza A(H1N1) virus [A(H1N1)pdm09]. Taken together, the data indicate that OmpS1 and OmpS2, despite being expressed at low levels under in vitro culture conditions, are potent protective immunogens with intrinsic adjuvant properties.

Purification of MBP-EnvZ fusion proteins using an automated system.

Bacteria use two-component signal transduction systems to detect and respond to environmental changes. These systems have been studied systematically in Escherichia coli as a model organism. Most of the signal transduction systems present in E. coli are conserved in related pathogenic bacteria; however, differences in regulation by these systems have been reported from one bacterial species to another [Oropeza, R., and Calva, E. (2009). The cysteine 354 and 277 residues of Salmonella enterica serovar Typhi EnvZ are determinants of autophosphorylation and OmpR phosphorylation. FEMS Microbiol. Lett.292, 282-290]. Our laboratory has been interested in studying the OmpR/EnvZ two-component system in S. enterica. In S. enterica serovar Typhi (Typhi), it regulates the expression of the porin genes, namely ompC, ompF, ompS1, and ompS2. OmpR proteins are identical between E. coli and Typhi, but several differences exist between the EnvZ proteins. To define whether some differences in porin regulation are due to changes on EnvZ, we decided to overexpress and purify E. coli, Typhi, and S. enterica serovar Typhimurium (Typhimurium) EnvZ proteins fused to the maltose-binding protein (MBP) as a purification tag. Differences in the autophosphorylation level of these proteins were evidenced. Hence, considering the differences at the amino acid level between E. coli and Typhi EnvZ proteins, several mutations were introduced in the Typhi EnvZ protein in order to try to find the amino acids affecting the enzymatic activity of the protein. We found that Cys354 plays an important role in defining the enzymatic activity of this histidine kinase. Here, we report the automated purification of a collection of MBP-EnvZ fusions using a mini-chromatography commercial system, but adapting an amylose affinity column packed by ourselves.

The cysteine 354 and 277 residues of Salmonella enterica serovar Typhi EnvZ are determinants of autophosphorylation and OmpR phosphorylation.

An initial biochemical characterization of the Salmonella enterica serovar Typhi (S. Typhi) EnvZ sensor protein and several mutant derivatives was performed. Autophosphorylation levels were higher for Escherichia coli EnvZ, intermediate for S. enterica serovar Typhimurium EnvZ and very low for S. Typhi EnvZ, in spite of their high amino acid sequence identity. Consequently, OmpR phosphorylation was related to EnvZ autophosphorylation. Among the mutant derivatives, a C354G mutation in S. Typhi EnvZ resulted in a substantial increase in autophosphorylation, while mutation of its other cysteine residue at position 277 to L or S decreased the EnvZ autophosphorylation level. Upon heterodimerization, the S. Typhi C354G mutant complemented the wild type in vitro, increasing the EnvZ-P yield of both monomers, in accordance with the model where EnvZ autophosphorylation occurs in trans, indicating that dimer formation is a dynamic process. Hence, the C354 and the C277 residues are fundamental in determining the particular intrinsic biochemical characteristics of EnvZ.

Salmonella enterica serovar Typhimurium ompS1 and ompS2 mutants are attenuated for virulence in mice.

Salmonella enterica serovar Typhimurium mutants with mutations in the ompS1 and ompS2 genes, which code for quiescent porins, were nevertheless highly attenuated for virulence in a mouse model, indicating a role in pathogenesis. Similarly, a strain with a mutation in the gene coding for LeuO, a positive regulator of ompS2, was also attenuated.