Streptococcus suis is a gram-positive pathogen that causes serious diseases in pigs and, in some cases, humans. Three genes of the virulent S. suis 89/1591 strain, encoding putative divalent-cation-binding lipoproteins, were isolated based on information obtained from the draft annotation files of this organism's genome. The products of these genes, which are inducible by divalent-cation deprivation, were subsequently purified, and their immunogenic and protective abilities were analyzed. All three proteins (SsuiDRAFT 0103, SsuiDRAFT 0174, and SsuiDRAFT 1237) were found to be immunogenic, but only one of them (SsuiDRAFT 0103) induced a significant protective response (87.5%, P ‫؍‬ 0.01) against the same S. suis strain. Furthermore, the S. suis ssuiDRAFT 1240 gene (adcR), which encodes a predicted regulator of Zn 2؉ and/or Mn 2؉ uptake in streptococci, was cloned, and its protein product was purified. Electrophoretic mobility shift assays with purified S. suis AdcR protein showed experimentally, for the first time, that the AdcR DNA-binding sequence corresponds to the TTAACNRGTTAA motif. In addition, a requirement for either Zn 2؉ or Mn 2؉ , but not Fe 2؉ , to establish in vitro binding of AdcR to its target sequence and the ability of AdcR to bind the ssuiDRAFT 0103 and ssuiDRAFT 1237 gene promoters but not the promoter of the ssuiDRAFT 0174 gene were demonstrated. Taken together, these data suggest that SsuiDRAFT 0103 is a good candidate for vaccines against S. suis and support preliminary results indicating that bacterial envelope proteins involved in the uptake of divalent cations other than iron may be useful for protective purposes.

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Reverse transcriptase PCR analyses have demonstrated that open reading frames (ORFs) PM0298, PM0299, and PM0300 of the animal pathogen Pasteurella multocida constitute a single transcriptional unit. By cloning and overexpression studies in Escherichia coli cells, the product of ORF PM0300 was shown to bind hemoglobin in vitro; this ORF was therefore designated hgbA. In vitro and in vivo quantitative assays demonstrated that the P. multocida hgbA mutant bound hemoglobin to the same extent as the wild-type strain, although the adsorption kinetics was slightly slower for the hgbA cells. In agreement with this, the virulence of P. multocida hgbA cells was not affected, suggesting that other functional hemoglobin receptor proteins must be present in this organism. On the other hand, P. multocida mutants defective in PM0298 and PM0299 could be isolated only when a plasmid containing an intact copy of the gene was present in the cells, suggesting that these genes are essential for the viability of this bacterial pathogen. By adapting the recombinase-based expression technology in vivo to P. multocida, we also demonstrated that the transcriptional PM0298-PM0299-hgbA unit is iron regulated and that its expression is triggered in the first 2 h following infection in a mouse model. Furthermore, hybridization experiments showed that the hgbA gene is widespread in P. multocida strains regardless of their serotype or the animal from which they were isolated.

Many cell surface-associated, divalent cation-regulated proteins are immunogenic, and some of them confer protection against the bacterial species from which they are derived. In this work, two Streptococcus suis divalent cation uptake regulator genes controlling zinc/manganese and iron uptake (adcR and fur, respectively) were inactivated in order to study the protective capacities of their cell surface-associated proteins. The results obtained showed overexpression of a set of immunogenic proteins (including members of the pneumococcal histidine triad family previously reported to confer protection against streptococcal pathogens) in S. suis adcR mutant cell surface extracts. Likewise, genes encoding zinc transporters, putative virulence factors and a ribosomal protein paralogue related to zinc starvation appeared to be derepressed in this mutant strain. Moreover, protection assays in mice showed that although neither adcR-nor fur-regulated cell surface-associated proteins were sufficient to confer protection in mice, the combination of both adcR-and fur-regulated cell surface-associated proteins is able to confer significant protection (50 %, P50.038) against a challenge to mice vaccinated with them. INTRODUCTIONStreptococcus suis is an important pathogen that causes significant economic losses in the swine industry worldwide (Higgins et al., 1995). Infection caused by S. suis may vary from asymptomatic bacteraemia to fulminant systemic disease, with meningitis as the most outstanding feature (Higgins et al., 1995). It is also an important zoonotic agent for humans, who may acquire the infection through skin wounds upon contact with pigs and/or their by-products. Recently, during an outbreak in China, 215 human cases of S. suis infection were documented, and 39 of those patients died (Yu et al., 2006). More than 30 serotypes of S. suis, based on its capsular antigens, are currently known (Higgins et al., 1995). Serotype 2 is the most frequently isolated from diseased animals (Staats et al., 1997). Efforts to control infection are hampered by the lack of effective vaccines against S. suis. Furthermore, mechanisms involved in pathogenesis of this micro-organism are not yet completely understood (Gottschalk & Segura, 2000).Iron and zinc are essential components of many bacterial proteins either as structural or catalytic cofactors (Andrews et al., 2003;Babich & Stotzky, 1978). The concentration of both elements in the free state in the body fluids of mammals is extremely low in order to prevent bacterial proliferation. On the other hand, metals at high concentrations are toxic to micro-organisms. Toxicity occurs through the displacement of essential metals from their native binding sites or through ligand interactions (Babich & Stotzky, 1978; Chamnongpol et al., 2002). In addition, iron can be particularly toxic under aerobic conditions, interacting with either oxygen or oxygen-reduced species (Touati, 2000). Consequently, micro-organisms require homeostatic mechanisms to control intracellular metal levels.Fur (...