STRUCTURE AND FUNCTION OF THE ESKAPE PATHOGEN GROESL CHAPERONINS

Abstract

GroESL is an essential bacterial chaperone system comprising the homotetradecameric, double ring GroEL and the homoheptameric cochaperone GroES that is highly conserved from bacteria to man. This class of chaperonins (large, double ring chaperones are called chaperonins) uses ATP binding and hydrolysis to fold nascent or stress denatured polypeptides into their appropriate native conformations. E. coli GroESL has been shown to be the only chaperone system required for organismal growth and viability under all conditions and thus has been posited as a potential antibiotic target. To date, nearly all functional studies have been carried out in E. coli GroESL and it has been assumed this is a viable surrogate for other bacterial GroESLs. In this work, we show that despite high GroESL sequence identity between E. coli and the ESKAPE pathogens, there are distinct structural and functional differences that were previously unappreciated. This observation may have critical implications for the development of small molecule inhibitors to target GroESL folding function. Attempted expression of the GroESL chaperonin complex native to each ESKAPE pathogen in E. coli unveiled that only K. pneumonia, A. baumannii, and E. cloacae were capable of rescuing GroESL deficient LG6 (lac regulated chromosomal groESL). More thorough evaluation of these strains revealed that P. auruginosa, E. faecium, and S. aureus had a dominant negative effect when expressed in LG6. To circumvent this, we employed two distinct genetic strategies to generate an E. coli strain in which native GroESL could be replaced with ESKAPE GroEL without the potential for translation of both ESKAPE and E. coli GroEL: one with chromosomal GroEL removed, but contains a GroEL plasmid capable of negative selection, and the other where chromosomal E. coli groESL was replaced by ESKAPE groESL. We found that all of the ESKAPE chaperonins could compliment in the clean genetic background, except for S. aureus. Using a series of chimeras, we identified which GroEL domains from P. auruginosa and E. faecium may have been responsible for the observed dominant negative effect in the presence of E. coli GroEL. Coexpression of P. auruginosa/E.coli or E. faecium/E.coli GroEL produced mixed tetradecamers. The chaperonins generated by this method exhibited diminished ATPase activity, suggesting compromised chaperoning ability. The results of our studies suggest that the expression of GroESL in nonnative organisms may be affected by the formation of hypofunctional mixed rings as a consequence of allosteric differences which are to be further investigated.