Biogenesis of metallo-b-lactamases in Gram-negative bacteria

MORAN BARRIO J; LIMANSKY AS; VIALE AM.

Universidad Nacional de Rosario, Argentina

Congreso; Quinto Congreso Argentino de Microbiología General (SAMIGE); 2008

Sociedad Argentina de Microbiología General

The expression of periplasmic b-lactam degrading enzymes (b-lactamases) constitutes the most common mechanism of antibiotic resistance inGram-negative bacteria. The recent emergence of b-lactamases with the ability to degrade carbapenems, the last generation of b-lactam antibiotics, is now worrisome. Some of these novel enzymes possess one or two Zn(II) ions in the active site, and are known as metallo-b-lactamases (MbLs). MbLs have been described in nosocomil strains of many pathogens including Bacteroides fragilis, Pseudomonas aeruginosa, Aeromonas hydrophila, Serratia marcescens, Elizabethkingia meningoseptica, etc. and their dissemination due to genes carried on mobile DNA elements represents an acute problem worlwide.MbLs are synthesized as precursors in the cytoplasm, and must be therefore translocated across the inner cytoplasmic membrane by either the general secretory (Sec) pathway or the twin-arginine translocation (Tat) pathway to reach the final functional destination. The cytoplasmic traffic of the precursors must be coordinated with the secretion apparatus, a process in wich SecB and/or other molecular chaperones may participate. We have recently characterized the MbL GOB-18 from a clinical isolate of E. meningoseptica. This enzyme is the deepest branching member of the family and shows substitutions in amino acid positions highly conserved in other MbLs. Our in vitro studies showed tha GOB has a novel active site and is a broad spectrum enzyme maximally active with one equivalent of Zn(II). In the present work we studied the chaperone requirements for GOB biogenesis and secretion in E. coli. The extent of GOB secretion was analyzed by the ability to confer cefotaxime resistance to the cells as well as by immunoblot analysis of periplasmic contents in both wild-type cells and isogenic mutants deficient in components of different secretion pathways (Sec and Tat) or cytoplasmic chaperone systems (Hsp70, Hsp60 and TF). Our results indicate that the Sec machinery mediates the secretion of GOB in an extended conformation. In addition, we observed a fundamental role of the DnaK system in this process: DnaK itself was essential and the co-chaperone DjlA specifically participated in the secretion process. In addition, TF also participated, probably directing the precursor from the ribosome to the Sec machinery. On the contrary, GroEL/ES was dispensable for the secretion process. In summary, several interactions between different cytoplasmic chaperones and the Sec machinery are necessary for the secretion of a pathogenic factor such as an MbL in Gram-negative bacteria.