Lack of the Delta Subunit of RNA Polymerase Increases Virulence Related Traits of Streptococcus mutans

The delta subunit of the RNA polymerase, RpoE, maintains the transcriptional specificity in Gram-positive bacteria. Lack of RpoE results in massive changes in the transcriptome of the human dental caries pathogen Streptococcus mutans. In this study, we analyzed traits of the ΔrpoE mutant which are important for biofilm formation and interaction with oral microorganisms and human cells and performed a global phenotypic analysis of its physiological functions. The ΔrpoE mutant showed higher self-aggregation compared to the wild type and coaggregated with other oral bacteria and Candida albicans. It formed a biofilm with a different matrix structure and an altered surface attachment. The amount of the cell surface antigens I/II SpaP and the glucosyltransferase GtfB was reduced. The ΔrpoE mutant displayed significantly stronger adhesion to human extracellular matrix components, especially to fibronectin, than the wild type. Its adhesion to human epithelial cells HEp-2 was reduced, probably due to the highly aggregated cell mass. The analysis of 1248 physiological traits using phenotype microarrays showed that the ΔrpoE mutant metabolized a wider spectrum of carbon sources than the wild type and had acquired resistance to antibiotics and inhibitory compounds with various modes of action. The reduced antigenicity, increased aggregation, adherence to fibronection, broader substrate spectrum and increased resistance to antibiotics of the ΔrpoE mutant reveal the physiological potential of S. mutans and show that some of its virulence related traits are increased

Pathogen Proteins Eliciting Antibodies Do Not Share Epitopes with Host Proteins: A Bioinformatics Approach

The best way to prevent diseases caused by pathogens is by the use of vaccines. The advent of genomics enables genome-wide searches of new vaccine candidates, called reverse vaccinology. The most common strategy to apply reverse vaccinology is by designing subunit recombinant vaccines, which usually generate an humoral immune response due to B-cell epitopes in proteins. A major problem for this strategy is the identification of protective immunogenic proteins from the surfome of the pathogen. Epitope mimicry may lead to auto-immune phenomena related to several human diseases. A sequence-based computational analysis has been carried out applying the BLASTP algorithm. Therefore, two huge databases have been created, one with the most complete and current linear B-cell epitopes, and the other one with the surface-protein sequences of the main human respiratory bacterial pathogens. We found that none of the 7353 linear B-cell epitopes analysed shares any sequence identity region with human proteins capable of generating antibodies, and that only 1% of the 2175 exposed proteins analysed contain a stretch of shared sequence with the human proteome. These findings suggest the existence of a mechanism to avoid autoimmunity. We also propose a strategy for corroborating or warning about the viability of a protein linear B-cell epitope as a putative vaccine candidate in a reverse vaccinology study; so, epitopes without any sequence identity with human proteins should be very good vaccine candidates, and the other way around

Differential Localization of the Streptococcal Accessory Sec Components and Implications for Substrate Export

The accessory Sec system of Streptococcus gordonii is comprised of SecY2, SecA2, and five proteins (Asp1 through -5) that are required for the export of a serine-rich glycoprotein, GspB. We have previously shown that a number of the Asps interact with GspB, SecA2, or each other. To further define the roles of these Asps in export, we examined their subcellular localization in S. gordonii and in Escherichia coli expressing the streptococcal accessory Sec system. In particular, we assessed how the locations of these accessory Sec proteins were altered by the presence of other components. Using fluorescence microscopy, we found in E. coli that SecA2 localized within multiple foci at the cell membrane, regardless of whether other accessory Sec proteins were expressed. Asp2 alone localized to the cell poles but formed a similar punctate pattern at the membrane when SecA2 was present. Asp1 and Asp3 localized diffusely in the cytosol when expressed alone or with SecA2. However, these proteins redistributed to the membrane in a punctate arrangement when all of the accessory Sec components were present. Cell fractionation studies with S. gordonii further corroborated these microscopy results. Collectively, these findings indicate that Asp1 to -3 are not integral membrane proteins that form structural parts of the translocation channel. Instead, SecA2 serves as a docking site for Asp2, which in turn attracts a complex of Asp1 and Asp3 to the membrane. These protein interactions may be important for the trafficking of GspB to the cell membrane and its subsequent translocation