The Molecular Mechanisms Underlying Hidden Phenotypic Variation among Metallo-β-Lactamases

Abstract

Genetic variation among orthologous genes has been largely formed through neutral genetic drift to maintain the same functional role. In some circumstances, however, this genetic variation can create critical phenotypic variation, particularly when genes are transferred to a new host by horizontal gene transfer (HGT). Unveiling "hidden phenotypic variation" through HGT is especially important for genes that confer resistance to antibiotics, which continue to disseminate to new organisms through HGT.Despite this biomedical importance, our understanding of the molecular mechanisms that underlie hidden phenotypic variation remains limited. Here we sought to determine the extent of hidden phenotypic variation in the B1 metallo-β-lactamase (MBL) family, as well as to determine its molecular basis by systematically characterizing eight MBL orthologs when they are expressed in three different organisms (E. coli, P. aeruginosa, and K. pneumoniae). We found that these MBLs confer diverse levels of resistance in each organism, which cannot be explained by variation in catalytic efficiency alone; rather, it is the combination of the catalytic efficiency and abundance of functional periplasmic enzyme that best predicts the observed variation in resistance. The level of functional periplasmic expression varied dramatically between MBL orthologs and between hosts. This was the result changes at multiple levels of each enzyme's functional: 1) the quantity of mRNA; 2) the amount of MBL expressed; and 3) the efficacy of functional enzyme translocation to the periplasm. Overall, we see that it is the interaction between each gene and the host's underlying cellular processes (transcription, translation, and translocation) that determines MBL genetic incompatibility thorough HGT. These hostspecific processes may constrain the effective spread and deployment of MBLs to certain host species, and could explain the current observed distribution bias..

Comprehensive exploration of the translocation, stability and substrate recognition requirements in VIM-2 lactamase

“…We uncover a lack of optimization in the signal peptide of VIM-2, that may be due to codon dependent RNA folding or incompatibility with the host translocation system. Such findings highlight the importance of genome and host context in resistance gene compatibility 23,60,65 . By performing DMS at various conditions, three different antibiotics, and two temperatures, we enhance the understanding of sequence-structure-function relationships by unveiling a set of mutations for protein stability, catalysis and substrate specificity of VIM-2.…”

“…The less than optimal residue level fitness of wtVIM-2 may be because we employ E. coli as a host while natural VIM variants are often found in Pseudomonas 62 , and/or the signal peptide is not selected to produce maximum expression in natural environments. It has been shown that different signal peptides produce variable expression levels and translocation rates for a given protein, both of which affect the final resistance, especially in different host organisms 23,57,58,[63][64][65] . Furthermore, the signal peptide is frequently mutated in naturally occurring VIM-type variants (see section on 'Natural VIM variation' below), suggesting changes in the signal peptide sequences may have played significant roles in dissemination of MBL genes to different hosts and adaptation to higher antibiotic concentrations.…”

“…All major MBLs have also been undergoing continual evolution; VIM-type MBLs have diversified up to 70 amino acid mutations (26% sequence difference) into over 50 isolated variants, and some variants seem to have developed new substrate specificity [9][10][11][12][13] . Much effort has been made to characterize the molecular mechanisms and identify key residues in several major MBLs using diverse biochemical and structural approaches [14][15][16][17][18][19][20][21][22][23][24] .…”

Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

“… 39 In addition, the expression of the same class B and D β-lactamases in different hosts exhibited a lack of correlation between MICs and the catalytic efficiency of these enzymes. 43 , 44 Hence, phenotypic variation can be due to differences in catalytic efficiency in the periplasmic conditions, but expression level, protein folding and translocation to the periplasm can also play a role. 43 …”

“… 43 , 44 Hence, phenotypic variation can be due to differences in catalytic efficiency in the periplasmic conditions, but expression level, protein folding and translocation to the periplasm can also play a role. 43 …”