A unique class of Zn2+-binding serine-based PBPs underlies cephalosporin resistance and sporogenesis in Clostridioides difficile

Abstract

Treatment with β-lactam antibiotics, particularly cephalosporins, is a major risk factor for Clostridioides difficile infection. These broad-spectrum antibiotics irreversibly inhibit penicillin-binding proteins (PBPs), which are serine-based enzymes that assemble the bacterial cell wall. However, C. difficile has four different PBPs (PBP1-3 and SpoVD) with various roles in growth and spore formation, and their specific links to β-lactam resistance in this pathogen are underexplored. Here, we show that PBP2 (known to be essential for vegetative growth) is the primary bactericidal target for β-lactams in C. difficile. PBP2 is insensitive to cephalosporin inhibition, and this appears to be the main basis for cephalosporin resistance in this organism. We determine crystal structures of C. difficile PBP2, alone and in complex with β-lactams, revealing unique features including ligand-induced conformational changes and an active site Zn2+-binding motif that influences β-lactam binding and protein stability. The Zn2+-binding motif is also present in C. difficile PBP3 and SpoVD (which are known to be essential for sporulation), as well as in other bacterial taxa including species living in extreme environments and the human gut. We speculate that this thiol-containing motif and its cognate Zn2+ might function as a redox sensor to regulate cell wall synthesis for survival in adverse or anaerobic environments.

A conserved zinc-binding site in Acinetobacter baumannii PBP2 required for elongasome-directed bacterial cell shape

“…In vitro data indicate that the zinc ion is required for protein stability, whereas metal deprivation does not preclude β-lactam binding, suggesting the Zn-depleted TPase site retains structural integrity to a certain extent. Our findings are in line with recent structures of Clostridium difficile bPBPs operative in PG synthesis during vegetative growth (PBP2) and sporogenesis (PBP3 and SpoVD), showing an analogous TPase Zn site that is critical for protein stability and affects β-lactam binding ( 47 , 48 ). It is worth noting that the Zn site in A. baumannii PBP2 (D-x14-D-x5-H-x12-C) and C. difficile bPBPs (D-x12-D-x9-H-x12-C in PBP2, C-x14-C-x5-H-x12-C in PBP3, and C-x12-C-x6-H-x12-C in SpoVD) can tolerate some variation in the metal ligation residues and their relative position in the polypeptide sequence.…”

“…Moreover, our bioinformatic analysis suggests that Zn-binding PBPs are present in aerobic bacteria and widespread in β- and γ-Proteobacteria. These results expand on previous findings of Zn-binding PBPs largely in anaerobic bacteria and the phylum Firmicutes ( 47 ) and together provide a broad picture of the zinc PBP distribution across the bacterial kingdom. The physiological relevance of the conserved Zn site in the bPBPs of A. baumannii and other selected bacteria has yet to be fully elucidated.…”

“…The A. baumannii pbp2 mutants are also sensitized to antibiotics that target divisome-associated PBP3, consistent with the absence of complementary PBP2 function ( 35 ). To give precedence to the functional role of the TPase zinc site, analogous changes in the β-hairpin region and SxN/D motif of B. subtilis SpoVD were shown to be detrimental to endospore cortex PG synthesis in vivo which, in light of recent evidence of zinc binding to SpoVD, is consistent with the essential nature of Zn coordination in the molecular mechanisms of PG synthesis in this and other numerous species ( 33 , 47 ). We note that a previous study by Hood et al.…”

Clostridioides difficileMreE (PBP2) variants facilitate clinical disease during cephalosporin exposures

“…The molecular mechanisms by which MreE mutations increase resistance to specific classes of beta-lactam antibiotics [7] [8,9] are as yet undefined and may include reduced binding affinity of affected antibiotic classes, in addition to interaction with other genomic loci that can produce higher levels of resistance, a finding suggested by the fact that transfer of the mreE alleles alone into susceptible strains did not elevate most cephalosporin MICs to the level of the original parent strain (Fig 2C). The gene-level variants also provide targets to aid in the detection of cephalosporin resistance, whether by strain genomic analyses or with targeted mreE probes to evaluate primary patient samples for resistant strains.…”

“…An intrinsic class D beta-lactamase providing degress of resistance across genomically diverse C. difficile has been identified [5,6]. Variants in the pathogen's penicillin-binding protein 2 (PBP2) have also been identified that have reduced binding-affinity to certain beta-lactam classes, including cephalosporins and carbapenems [7]. However, causative effects of these variants on phenotypic resistance in vitro and in vivo have not yet been defined, limiting our understanding of their role in patient disease [8][9][10].…”