CLPL AND CLPP ARE INVOLVED IN THE DEGRADATION OF SSRA-TAGGED PROTEINS OF Streptococcus pneumoniae

GERMAN E. PIÑAS; ANDREA G. ALBARRACIN ORIO; MELINA B. CIAN; PAULO R. CORTES; JOSE R. ECHENIQUE

Carlos Paz, Córdoba

Congreso; VI Congreso Argentino de Microbiología General; 2009

Sociedad Argentina de Microbiología General (SAMIGE)

S. pneumoniae is a major human pathogen that causes several diseases such as pneumonia, meningitis and bacteraemia. During the infection process, this bacterium is subjected to a number of hassle conditions, such as temperature, oxidative stress and low pH. In our laboratory, we found that acidic stress induces autolysis of S. pneumoniae. This phenomenon requires the participation of the acid-induced molecular chaperone, ClpL, the serine protease ClpP and the SsrA-tagging system, suggesting a direct interaction between them. It is known that SsrA-tagged proteins are sent to degradation by proteolytic complexes formed by Clp ATPases and ClpP in other bacteria. We decided to investigate if the removal of SsrA-tagged proteins is mediated by ClpL and ClpP in S. pneumoniae. For this purpose, we fused the gfp-mut3 and ssrA genes and we cloned this construction into a replicative vector for S. pneumoniae. These plasmids were used to transform the wt strain and the clpL and clpP mutants. The analysis of GFP expression by fluorescence microscopy, fluorometric quantification and western blot, revealed presence of GFP in clpL and clpP mutants harboring the plasmid pAT29-gfpmut3-ssrA, while it was almost undetectable in the wt strain (R801). To confirm these results and to rule out the possibility of a reporter-dependent effect, we fused the ssrA and gus genes, and glucuronidase activity was measured in the wt, clpL and clpP strains transformed with the plasmid pAT18-gusA-ssrA, and similar evidence was obtained. These experiments were performed in another genetic background, strain Cp1015, and we obtained the same results as with the R801 strain. The data presented in this work indicate that ClpL and ClpP are involved in the degradation in vivo of SsrA-tagged proteins in S. pneumoniae, and suggest that ClpL and ClpP are able to associate into a proteolytic complex that recognizes SsrA-tagged proteins.