Biophisical and biochemical characterization of plasmid-mediated colistin resistance determinat MCR-1

GINA DOTTA; ALEJANDRO J. VILA; PABLO E. TOMATIS

Leuven

Conferencia; 2018 MCAA Conference and General Assembly; 2018

MCAA

Polimyxin antibiotics (1), as colistin, are commonlyreferred as a last-chance to treat multidrug-resistantgram-negative infections. This positively chargedpeptide can binds to the negatively charged lipid Acore, disrupting the bacterial outer membrane (2).Unfortunately, in 2016 a transferable colistinresistance mechanism was reported, a plasmidencodedphosphoethanolamine transferase MCR thathas rapidly disseminated worldwide. MCR catalyzest h e t r a n s f e r o f p o s i t i v e l y c h a r g e dphosphoethanolamine onto lipid A, decreasing thenet negative charge and preventing colistin bindingand antibiotic success (3). It is a metalloenzymeembedded in the inner membrane, with the catalyticcenter in a periplasmic soluble domain (4). Recently,four structures of the soluble catalytic domain havebeen reported, depicting a zinc metalloprotein (5).Despite structural equivalence, the stoichiometry onmetal content is controversial. This information ispivotal for further mechanistic analysis to gaininformation for inhibitor design.Based in previous studies, we have cloned thecatalytic periplasmic domain in different expressionvectors. According to soluble protein yields, BL21(DE3) E. coli cells expressing the catalytic domain witha His 6x tag and a TEV protease recognition site wereselected for further protein production (6). Bacterialculture condition were tested with different Zn(II) ionconcentrations, in order to evaluate the metalcontent in the purified protein using PAR as a Znindicator. In all tested conditions, the catalytic domainwas able to take up to one Zn(II) ion equivalent (7).Metal quelator EDTA was used to obtain apo-proteinsderivatives, to further characterize the metal activesite using Co(II) as spectroscopic probe. CoCo andZnCo adduct were produced and the ligand fieldbands showed a tetrahedral geometry pattern (8).Secondary and tertiary structural features and relativestability of the apo and metal adducts were alsotested by circular dichroism spectroscopy (9).