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. 2024 May 22;25(1):508.
doi: 10.1186/s12864-024-10410-2.

Potential involvement of beta-lactamase homologous proteins in resistance to beta-lactam antibiotics in gram-negative bacteria of the ESKAPEE group

Joyce de Souza  1 , Alexandre Zanatta Vieira  1 , Hellen Geremias Dos Santos  2 , Helisson Faoro  3   4
Affiliations

Potential involvement of beta-lactamase homologous proteins in resistance to beta-lactam antibiotics in gram-negative bacteria of the ESKAPEE group

Joyce de Souza et al. BMC Genomics. .

Abstract

Enzymatic degradation mediated by beta-lactamases constitutes one of the primary mechanisms of resistance to beta-lactam antibiotics in gram-negative bacteria. This enzyme family comprises four molecular classes, categorized into serine beta-lactamases (Classes A, C, and D) and zinc-dependent metallo-beta-lactamases (Class B). Gram-negative bacteria producing beta-lactamase are of significant concern, particularly due to their prevalence in nosocomial infections. A comprehensive understanding of the evolution and dissemination of this enzyme family is essential for effective control of these pathogens. In this study, we conducted the prospecting, phylogenetic analysis, and in silico analysis of beta-lactamases and homologous proteins identified in 1827 bacterial genomes with phenotypic data on beta-lactam resistance. These genomes were distributed among Klebsiella pneumoniae (45%), Acinetobacter baumannii (31%), Pseudomonas aeruginosa (14%), Escherichia coli (6%), and Enterobacter spp. (4%). Using an HMM profile and searching for conserved domains, we mined 2514, 8733, 5424, and 2957 proteins for molecular classes A, B, C, and D, respectively. This set of proteins encompasses canonical subfamilies of beta-lactamases as well as hypothetical proteins and other functional groups. Canonical beta-lactamases were found to be phylogenetically distant from hypothetical proteins, which, in turn, are closer to other representatives of the penicillin-binding-protein (PBP-like) and metallo-beta-lactamase (MBL) families. The catalytic amino acid residues characteristic of beta-lactamases were identified from the sequence alignment and revealed that motifs are less conserved in homologous groups than in beta-lactamases. After comparing the frequency of protein groups in genomes of resistant strains with those of sensitive ones applying Fisher's exact test and relative risk, it was observed that some groups of homologous proteins to classes B and C are more common in the genomes of resistant strains, particularly to carbapenems. We identified the beta-lactamase-like domain widely distributed in gram-negative species of the ESKAPEE group, which highlights its importance in the context of beta-lactam resistance. Some hypothetical homologous proteins have been shown to potentially possess promiscuous activity against beta-lactam antibiotics, however, they do not appear to expressly determine the resistance phenotype. The selective pressure due to the widespread use of antibiotics may favor the optimization of these functions for specialized resistance enzymes.

Keywords: Beta-lactamase; ESKAPEE; beta-lactam antibiotics; homologous protein; resistance.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Distribution of proteins by species. The sequence sets corresponding to each class were grouped according to the description of the protein product as follows: A A—Class A beta-lactamases; HpA—Hypothetical proteins. B MBL—Metallo-beta-lactamases; HpMBL—Hypothetical metal-binding enzymes; AHL—N-acyl homoserine lactone hydrolases; PQSE—Pseudomonas quinolone signal response proteins; B1—Subclass B1 beta-lactamases; HpB—Hypothetical proteins; B3—Subclass B3 beta-lactamases; AKS—Putative alkyl/aryl-sulfatase. C PEst—Putative esterases; C—Class C beta-lactamases; AmpH—D-alanyl-D-alanine-carboxypeptidase/endopeptidase; PBP—Penicillin-binding proteins superfamily; HpC—Hypothetical proteins; PPBP—Putative penicillin-binding proteins superfamily; PKS—Polyketide synthase modules and related proteins. D PBP2—Peptidoglycan D,D-transpeptidase; D—Class D beta-lactamases
Fig. 2
Fig. 2
Phylogenetic analysis of beta-lactamases and homologous proteins. Phylogenetic tree constructed using the Maximum Likelihood (ML) method. The scale represents one amino acid change per site per million years. The orange branches represent enzymes without functional classification, composed of sequences whose description includes the words hypothetical, putative or superfamily. Branches without species identification represent sequences from the Integrative beta-lactamase database or other enzyme families included in the phylogeny. A Phylogenetic tree for class A beta-lactamases (pfam domain 13,354) and homologous proteins that includes 19 proteins representing clusters prospected from the genomes and another 31 representatives of beta-lactamases families. B Phylogenetic tree for beta-lactamase class B (domain pfam00753) which includes 60 proteins representing clusters prospected from the genomes and 42 proteins representing families of beta-lactamases and different functional groups of Metallo-hydrolases—ARS: arylsulfatase, PTS: phytase, PCE: Phosphorylcholine esterase, PRNase: putative ribonuclease, Igni18: promiscuous ancestral enzyme, Hp: hypothetical protein, RNase: ribonuclease, GlyII: glyoxalases II, SDO: sulfur dioxygenase, MBLAC2: human metallo-beta-lactamase, AHL: N- acyl homoserine lactone hydrolases, MDP: methy-parathion hydrolase, PqsE: Pseudomonas quinolone signal response protein, AKS: alkylsulfatase, Chd: Chlorothalonyl dehalogenase, VarG: putative metallo-beta-lactamase. C Phylogenetic tree for beta-lactamase class C (domain pfam00144) which includes 49 proteins representing clusters prospected from the genomes and 19 proteins representing families of beta-lactamases and different functional groups of Serine-hydrolases—AmpH: carboxylpeptidase, PKS: polyketide synthase, PPBP: Putative penicillin binding proteins superfamily. D Phylogenetic tree for class D beta-lactamase (domain pfam00905) which includes 11 proteins representing clusters prospected from the genomes and other 13 families of beta-lactamases—PBP2: Peptidoglycan D, D-transpeptidase
Fig. 3
Fig. 3
Conservation of catalytic site motifs by class and phylogenetic group of beta-lactamases. Protein catalytic site motifs were recovered and divided according to phylogenetic grouping. A A: class A beta-lactamases, A2: beta-lactamases and hypothetical proteins from phylogenetic group A2. B B1/B2: beta-lactamases of subclasses B1 and B2, B3: beta-lactamases of subclass B3, VarG: putative metallo-beta-lactamases and chlorothalonyl dehalogenase, GlyII: hypothetical proteins, ribonuclease, glyoxalases II and sulfur dioxygenase, Human: hypothetical protein and Human MBL, AHL: hypothetical proteins and N-acyl homoserine lactone hydrolases, BHp: hypothetical proteins, AKS: hypothetical proteins and alkylsulfatase, PqsE: Pseudomonas quinolone signal response proteins, Others: other functional groups. C C—class C beta-lactamases, AmpH: hypothetical proteins and carboxylpeptidases, Est/PPBP: carboxylesterase VIII and putative penicillin binding proteins, EstI: hypothetical proteins and carboxylesterases VIII, EstII: hypothetical proteins and carboxylesterases VIII, CHp: proteins hypothetical, PKS: hypothetical proteins and polyketide synthases. D D: class D beta-lactamases, PBP2: penicillin binding proteins 2
Fig. 4
Fig. 4
Relative risk for the resistant R and R2 categories in relation to the sensitive category. Ip—imipenem, Mp—meropenem, CefT—ceftazidime, CefZ—cefazolin, Amp—ampicillin, Az—aztreonam. Comparisons that did not show statistical differences in the paired Fischer test were omitted. Positions where relative risk calculation was not possible due to frequency 0 in the sensitive category are indicated by a dash (-). beta-lactamases are marked in blue and homologous proteins are marked in orange. We highlight in bold comparisons with RR greater than 1
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