Streptococcus pneumoniae causes life-threatening infectious diseases in humans. It is responsible for high morbidity and mortality rates. Because of increasing resistance to beta-lactams, it is also becoming a public health problem in countries such as Norway and Spain. The specific function of each of the six penicillin-binding proteins (PBPs) produced by S. Pneumoniae remains poorly understood. To learn more, the partnership of the project aims at studying how decreased expression of individual PBPs can influence the expression of other surface proteins. They plan to use a collection of mutant strains in which the PBP-expression level can be regulated, and analyze their surface-attached proteins by protenomics. The planned experiments will also enable us to look for vaccine and diagnostic candidates that can be used in the fight against this pathogen. Norwegian University of Life Sciences has the collection of mutants needed for the execution of the project, and is very interested in understanding the mechanisms by which low-affinity PBPs help penomococci to survive in the presence of beta-lactams.
Summary of project results
Partners studied the effect of the absence of one non-essential PBPs (PBPl a) and low expression levels (depletion) of essential PBPs (PBP2x and PBP2b) on the surface-protein-profile by using a proteomics approach developed and successfully applied by the grantee in several Gram-positive pathogens. Bacterial cells are "shaved" with proteases and the resulting peptides are analyzed by liquid chromatography coupled to mass spectrometry. The comparison of the different mutants with their corresponding controls allowed partners to know the differential expression of surface proteins or clusters of proteins due to such mutations. The mutants were provided by the host group, which has developed a titratable expression/depletion system for proteins of interest. The application of the "shaving" proteomic approach did not produce any clear trend in the Pbp 1 a and the Pbp2x mutants, but a decrease in cell-wall attached proteins (those carrying the LPXTG anchoring motif) in the Pbp2b mutant. In addition, the project has tested the possible differences in the surface protein distributionj arrangement in other mutants: ComM, PcsB and the TarlJ system. ComM is a transmembrane protein responsible for competence-induced immunity. Partners have applied the "shaving" approach to this mutant and have found that when this protein is overexpressed, there is a clear reduction of most of identified lipoproteins. Whether these are the direct targets of the ComM protein or this is an indirect effect of such overexpression will be explored in future projects. The induction of the PcsB protein resulted in the overexpression of three proteins: Spr1875, Spr0096 and LytC. The two first ones are in the same operon as PcsB. Partners checked whether LytC interacted with PcsB by employing knock-out mutants, but no positive results were obtained. Finally, they checked for differences in a TarlJ mutant, a system responsible for teichoic acid synthesis and anchorage on the cell wall, but did not find any clear cluster of proteins that could be associated to such a mutation.
Summary of bilateral results
This project was planned as a series of exploratory experiments to check whether the "shaving" proteomic approach, developed by the grantee and which has been very successful for identifying in a fast and reliable way the set of surface proteins in many different biological systems, was useful for determining significant changes in clusters of proteins because of mutations in PBPs. Obviously, proteomics results must be validated by other biological experiments and approaches, so the results generated are being used by both groups (and mainly, that of ·Prof. Havarstein's group) to stlidy the participation of some of the identified changes in the phenomenon of pneumococcal compeTence. it is expected that some of the results obtained will be included in future publications, but ·the results must be validated. In addition to scientific publications, the University of Córdoba has broadcasted through its website the cooperation agreement with the Norwegian University of Life Sciences: http://www.uco.es!servicios
lcomunicacion / componentIk 2 ¡ item¡ 975 O 3-la-uco-entra-en-el-graqrama-n i ¡s-ciencia -ysostenibilidad The original idea of this project was to explore possible changes in the surface protein arrangement due to some protein mutations. If there were promising results, partners could prepare joint proposals for a EU project grant, within the Horizon 2020 scheme or some of the ERA-Net programs (e.g. Infect-ERA, former PathoGenomics). Once we have evaluated how promising the results are, we will make the most of them to try to get funding from some call.