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

BACKGROUND: 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.

TgKDAC4: A Unique Deacetylase of Toxoplasma's Apicoplast.

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and causes toxoplasmosis infections, a disease that affects a quarter of the world's population and has no effective cure. Epigenetic regulation is one of the mechanisms controlling gene expression and plays an essential role in all organisms. Lysine deacetylases (KDACs) act as epigenetic regulators affecting gene silencing in many eukaryotes. Here, we focus on TgKDAC4, an enzyme unique to apicomplexan parasites, and a class IV KDAC, the least-studied class of deacetylases so far. This enzyme shares only a portion of the specific KDAC domain with other organisms. Phylogenetic analysis from the TgKDAC4 domain shows a putative prokaryotic origin. Surprisingly, TgKDAC4 is located in the apicoplast, making it the only KDAC found in this organelle to date. Transmission electron microscopy assays confirmed the presence of TgKDAC4 in the periphery of the apicoplast. We identified possible targets or/and partners of TgKDAC4 by immunoprecipitation assays followed by mass spectrometry analysis, including TgCPN60 and TgGAPDH2, both located at the apicoplast and containing acetylation sites. Understanding how the protein works could provide new insights into the metabolism of the apicoplast, an essential organelle for parasite survival.

Origin and evolution of nonulosonic acid synthases and their relationship with bacterial pathogenicity revealed by a large-scale phylogenetic analysis.

Nonulosonic acids (NulOs) are a group of nine-carbon monosaccharides with different functions in nature. N-acetylneuraminic acid (Neu5Ac) is the most common NulO. It covers the membrane surface of all human cells and is a central molecule in the process of self-recognition via SIGLECS receptors. Some pathogenic bacteria escape the immune system by copying the sialylation of the host cell membrane. Neu5Ac production in these bacteria is catalysed by the enzyme NeuB. Some bacteria can also produce other NulOs named pseudaminic and legionaminic acids, through the NeuB homologues PseI and LegI, respectively. In Opisthokonta eukaryotes, the biosynthesis of Neu5Ac is catalysed by the enzyme NanS. In this study, we used publicly available data of sequences of NulOs synthases to investigate its distribution within the three domains of life and its relationship with pathogenic bacteria. We mined the KEGG database and found 425 NeuB sequences. Most NeuB sequences (58.74 %) from the KEGG orthology database were classified as from environmental bacteria; however, sequences from pathogenic bacteria showed higher conservation and prevalence of a specific domain named SAF. Using the HMM profile we identified 13 941 NulO synthase sequences in UniProt. Phylogenetic analysis of these sequences showed that the synthases were divided into three main groups that can be related to the lifestyle of these bacteria: (I) predominantly environmental, (II) intermediate and (III) predominantly pathogenic. NeuB was widely distributed in the groups. However, LegI and PseI were more concentrated in groups II and III, respectively. We also found that PseI appeared later in the evolutionary process, derived from NeuB. We use this same methodology to retrieve sialic acid synthase sequences from Archaea and Eukarya. A large-scale phylogenetic analysis showed that while the Archaea sequences are spread across the tree, the eukaryotic NanS sequences were grouped in a specific branch in group II. None of the bacterial NanS sequences grouped with the eukaryotic branch. The analysis of conserved residues showed that the synthases of Archaea and Eukarya present a mutation in one of the three catalytic residues, an E134D change, related to a Neisseria meningitidis reference sequence. We also found that the conservation profile is higher between NeuB of pathogenic bacteria and NanS of eukaryotes than between NeuB of environmental bacteria and NanS of eukaryotes. Our large-scale analysis brings new perspectives on the evolution of NulOs synthases, suggesting their presence in the last common universal ancestor.

TgKDAC4: a unique deacetylase of toxoplasma's apicoplast

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and causes toxoplasmosis infections, a disease that affects a quarter of the world’s population and has no effective cure. Epigenetic regulation is one of the mechanisms controlling gene expression and plays an essential role in all organisms. Lysine deacetylases (KDACs) act as epigenetic regulators affecting gene silencing in many eukaryotes. Here, we focus on TgKDAC4, an enzyme unique to apicomplexan parasites, and a class IV KDAC, the least-studied class of deacetylases so far. This enzyme shares only a portion of the specific KDAC domain with other organisms. Phylogenetic analysis from the TgKDAC4 domain shows a putative prokaryotic origin. Surprisingly, TgKDAC4 is located in the apicoplast, making it the only KDAC found in this organelle to date. Transmission electron microscopy assays confirmed the presence of TgKDAC4 in the periphery of the apicoplast. We identified possible targets or/and partners of TgKDAC4 by immunoprecipitation assays followed by mass spectrometry analysis, including TgCPN60 and TgGAPDH2, both located at the apicoplast and containing acetylation sites. Understanding how the protein works could provide new insights into the metabolism of the apicoplast, an essential organelle for parasite survival.