Penicillin-Binding Proteins, β-Lactamases, and β-Lactamase Inhibitors in β-Lactam-Producing Actinobacteria: Self-Resistance Mechanisms

Abstract

The human society faces a serious problem due to the widespread resistance to antibiotics in clinical practice. Most antibiotic biosynthesis gene clusters in actinobacteria contain genes for intrinsic self-resistance to the produced antibiotics, and it has been proposed that the antibiotic resistance genes in pathogenic bacteria originated in antibiotic-producing microorganisms. The model actinobacteria Streptomyces clavuligerus produces the β-lactam antibiotic cephamycin C, a class A β-lactamase, and the β lactamases inhibitor clavulanic acid, all of which are encoded in a gene supercluster; in addition, it synthesizes the β-lactamase inhibitory protein BLIP. The secreted clavulanic acid has a synergistic effect with the cephamycin produced by the same strain in the fight against competing microorganisms in its natural habitat. High levels of resistance to cephamycin/cephalosporin in actinobacteria are due to the presence (in their β-lactam clusters) of genes encoding PBPs which bind penicillins but not cephalosporins. We have revised the previously reported cephamycin C and clavulanic acid gene clusters and, in addition, we have searched for novel β-lactam gene clusters in protein databases. Notably, in S. clavuligerus and Nocardia lactamdurans, the β-lactamases are retained in the cell wall and do not affect the intracellular formation of isopenicillin N/penicillin N. The activity of the β-lactamase in S. clavuligerus may be modulated by the β-lactamase inhibitory protein BLIP at the cell-wall level. Analysis of the β-lactam cluster in actinobacteria suggests that these clusters have been moved by horizontal gene transfer between different actinobacteria and have culminated in S. clavuligerus with the organization of an elaborated set of genes designed for fine tuning of antibiotic resistance and cell wall remodeling for the survival of this Streptomyces species. This article is focused specifically on the enigmatic connection between β-lactam biosynthesis and β-lactam resistance mechanisms in the producer actinobacteria.

Keywords: Streptomyces clavuligerus; antibiotic resistance; cephamycin C; clavulanic acid; clusters organization and evolution; penicillin-binding proteins; superclusters; β-lactamases.

Figure 3

Organization of the clavulanic acid gene clusters of different actinobacteria. The name of the strain is indicated in the left column. The genes are color-coded and their names are indicated. Genes in black color are not related to cephamycin or clavulanic acid biosynthesis. The initial search of CA gene clusters was made using the essential S. clavuligerus proteins GcaS and CeaS2 as probes. Subsequently, all the CA biosynthesis proteins were searched using the homologous proteins of S. clavuligerus as probe. The organization of the genes was obtained using the NCBI Genome Blast tool. The gene clusters of clavulanic acid in S. clavuligerus, S. jumonjinensis-S.katsurahamanus, S. flavogriseus, and Sacc. viridis have been previously reported [45,66,76]. The CA clusters of all other actinobacteria shown in Figure 3 are reported for the first time in this work.

Figure 1

Proposed model of the major resistance mechanisms to β-lactam antibiotics in β-lactam-producing S. clavuligerus. A S. clavuligerus cell (part of the mycelium) is shown in yellow color; the cell wall peptidoglycan is indicated in pink. PBPs (e.g., PBP74) are located in the cell wall (red circles). Cephamycin C encoded by the CFM-CA cluster is secreted through an efflux pump (purple ellipse). The secreted cephamycin C does not bind to the insensitive PBPs of S. clavuligerus (indicated by an X symbol). The β-lactamase Bla1 is secreted and retained in the cell wall matrix (blue pentagons). Clavulanic acid encoded by the CFM-CA cluster is also secreted (green circle), although the nature of the efflux mechanism is uncertain. The Bla1 β-lactamase inactivates the exogenous penicillin produced by filamentous fungi living in the same habitat (dark blue squares). The secreted clavulanic acid binds the Bla1 β-lactamase, modulating its activity. A similar mechanism occurs in other β-lactam-producing actinobacteria (see text for details).

Figure 2

Location of genes related to β-lactam resistance in β-lactam gene clusters. (A) Structure of cephamycin C (left side) and clavulanic acid (right side). (B) Location of genes encoding β-lactamases or PBPs in the cephamycin C gene clusters (purple), clavulanic acid gene clusters (green), and thienamycin gene cluster (pink) of S. clavuligerusm S, jumonjinensis, S. katsurahamanus, S. cattleya, and N. lactamdurans.