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

PABLO E. TOMATIS; ALEJANDRO J. VILA

Congreso; Reunion Conjunta de Sociedades de Biociencias; 2017

Sociedad Argentina de Biofisica

Polimyxin antibiotics, as colistin, are commonly referred as a last-chance to treat multidrug-resistant gram-negative infections. This positively charged peptide can binds to the negatively charged lipid A core, disrupting the bacterial outer membrane. Unfortunately, in 2016 a transferable colistin resistance mechanism was reported, a plasmid-encoded phosphoethanolamine transferase MCR that has rapidly disseminated worldwide. MCR catalyzes the transfer of positively charged phosphoethanolamine onto lipid A, decreasing the net negative charge and preventing colistin binding and antibiotic success. It is a metalloenzyme embedded in the inner membrane, with the catalytic center in a periplasmic soluble domain. Recently, four structures of the soluble catalytic domain have been reported, depicting a zinc metalloprotein. Despite structural equivalence, the stoichiometry on metal content is controversial. This information is pivotal for further mechanistic analysis to gain information for inhibitor design. Based in previous studies, we have cloned the catalytic periplasmic domain in different expression vectors. According to soluble protein yields, BL21 (DE3) E. coli cells expressing the catalytic domain with a His 6x tag and a TEV protease recognition site were selected for further protein production. Bacterial culture condition were tested with different Zn(II) ion concentrations, in order to evaluate the metal content in the purified protein using PAR as a Zn indicator. In all tested conditions, the catalytic domain was able to take up to one Zn(II) ion equivalent.Metal quelator EDTA was used to obtain apo-proteins derivatives, to further characterize the metal active site using Co(II) as spectroscopic probe. CoCo and ZnCo adduct were produced and the ligand field bands showed a tetrahedral geometry pattern. Secondary and tertiary structural features and relative stability of the apo and metal adducts were also tested by circular dichroism spectroscopy.