Streptococcus suis is an important pathogen that causes significant economical losses in the swine industry worldwide and it is also an important zoonotic agent. Although several approaches to develop either live or recombinant vaccines to prevent S. suis-mediated disease have been tested, efforts to control the infection are hampered by the lack of effective weapons against this pathogen. Different cell-wall-associated transporters involved in divalent-cation uptake, including ABC transporters, have been shown to be involved in bacterial virulence and have immunogenic properties against the bacterial species from which they are derived. Accordingly, several strategies have been developed to produce vaccines against this pathogenic bacterium. One of them involves overexpression on the bacterial cell surface of divalent-cation-uptake transporters induced by chelator agents or by the construction of deficient strains in the cation-uptake repressors. In this context, the aim of this work has been to study the S. suis-cation-uptake mechanisms and their role in virulence as well as their putative use as a tool to achieve broad protection against this pathogen. To achieve this purpose, several transporters involved in zinc and iron uptake and their putative regulators (AdcR and Fur, respectively) have been identified in silico. Furthermore, the S. suis adcR gene, which encodes a predicted regulator of Zn2+ and/or Mn2+ uptake in streptococci, was cloned and its protein product was purified. Footprinting and electrophoretic mobility shift assays with purified S. suis AdcR protein showed, for the first time, that the AdcR-DNA binding sequence corresponds to the TTAACNRGTTAA motif. In addition, the requirement for either Zn2+ or Mn2+ to establish in vitro binding of AdcR to its target sequence and the ability of AdcR to control the genes codifying the ABC-transporter components SsuiDRAFT 0103 and SsuiDRAFT 1237, involved in zinc and/or manganese uptake, were demonstrated. Besides, the S. suis fur gene, which encodes a predicted regulator of Fe2+ uptake, was cloned and its protein product was overexpressed in Escherichia coli. Electrophoretic mobility shift assays with crude extract of this E. coli strain showed that S. suis Fur protein controls the feoAB genes, involved in ferric uptake. In addition, both adcR and fur genes were deleted in a virulent S. suis strain in order to charecterize these regulons. Several cation-uptake transporters appeared desrepressed in the knockout strains when gene expression was compared with wild-type strain through real-time RT-PCR analyses. Accordingly, EMSA results showed that these regulators specifically bind to the promoter of these genes. Moreover, the absence of adcR and/or fur genes in pathogenic streptococci showed, for the first time, an important attenuation of its virulence in mice. Finally, immunogenic and protective analysis were carried out with the products of three genes encoding putative divalent-cation-binding lipoproteins of S. suis (SsuiDRAFT 0103, SsuiDRAFT 0174, and SsuiDRAFT 1237), being all of them immunogenic although only one (SsuiDRAFT 0103) induces a significant protective response against a virulent S. suis strain in mice. Moreover, the overexpressed cell wall-associated proteins Ssu0309 and Ssu1103 of the adcR mutant, were identified by mass spectrometry as putative virulence factors belonging to the Pht (Pneumococcal histidine triad) family. Likewise, protective abilities of mutant strains were analyzed showing that although mutant cells are not effective to confer protection in mice, the combination of adcR- and fur-regulated cell wall-associated proteins confers a significant protection against S. suis 89/1591 challenge to mice vaccinated with them.
Caracterización de los sistemas de captación de zinc y de hierro en Streptococcus suis: Potencial antigénico y protector
Aranda Rodríguez, J. (Author). 5 Dec 2008
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis