May Sulfonamide Inhibitors of Carbonic Anhydrases from Mammaliicoccus sciuri Prevent Antimicrobial Resistance Due to Gene Transfer to Other Harmful Staphylococci?

Abstract

Mammaliicoccus sciuri, previously known as Staphylococcus sciuri, is a Gram-positive bacterium involved in gene transfer phenomena that confer resistance to multiple antibiotics. These plasmid-encoded genes can be easily transferred to other pathogenic staphylococci. Because antibiotic resistance is rising, inhibiting M. sciuri proliferation may be a credible strategy for restricting antimicrobial resistance gene transfer to other pathogenic bacteria. Recently, it has been shown that blocking bacterial carbonic anhydrases (CAs, EC 4.2.1.1), metalloenzymes sustaining bacterial metabolic activities, can reduce pathogen survival and fitness. Here, the recombinant M. sciuri γ-CA (MscCAγ) has been cloned and purified, utilizing the DNA recombinant technology. Its kinetic properties for the CO₂ hydration reaction, as well as the sulfonamide inhibition profile, were investigated and compared with those reported earlier for MscCAβ (previously described as SauBCA) and the two off-target human CA isoforms (hCA I and hCA II). The recombinant MscCAγ showed significant hydratase activity. Moreover, the MscCAγ sulfonamide inhibitory profile was different from that of MscCAβ, implying that a varied amino acid set typifies the catalytic pocket of the two enzymes. These differences provide additional evidence for the possibility of developing novel CA class-specific inhibitors.

Keywords: antibacterials; antimicrobial resistance; carbonic anhydrase; inhibitors; sulfonamides.

Figure 1

An alignment of the amino acid sequences of the γ-CAs from different bacterial sources, such as M. sciuri, M. vitulinus, M. stepanovicii, M. lentus, E. coli, P. gingivalis, and V. cholerae. The γ-CA hallmarks, such as the metal ion ligands, the residues which participate in a network of hydrogen bonds, and the proton shuttle residue, are shown in red bold, black bold, and violet bold, respectively. The asterisk (*) indicates identity at all aligned, the symbol (:) relates to conserved substitutions, while (.) means semi-conserved substitutions.

Figure 2

The phylogenetic tree was constructed using the software PhyML 3.0 and using α-, β-, γ-, and 𝜄-CAs identified in different bacterial species. Legend: CAMH_gamma, Methanosarcina thermophila TM-1, ACQ57353.1; CAM_gamma, Methanosarcina thermophila, WP_052721819.1; BglCA_gamma, Burkholderia gladioli, WP_013697304.1; PseCA_gamma, Pseudomonas sp., WP_010168409.1; VchCA_gamma, Vibrio cholerae, WP_000095101.1; Mstepanovicii_gamma, Mammaliicoccus stepanovicii, WP_095087344.1; Mlentus_gamma, Mammaliicoccus lentus, WP_257504613.1; Mvitulinus_gamma, Mammaliicoccus vitulinus, WP_103322725.1; MscCA_gamma, Mammaliicoccus sciuri, WP_049318612.1; PgiCA_gamma, Porphyromonas gingivalis, WP_004584482.1; EcoCA_gamma, Escherichia coli, MSL98108.1; HydCA_Iota Ephemeroptericola cinctiostellae, WP_114562659.1; BurCA_iota, Burkholderia territorii, WP_063553346.1; RalCA_Iota, Ralstonia sp., WP_089190700.1; ThiCA_iota, Thiotrichales bacterium, OYX05505.1; SacCA_Iota Saccharothrix sp., WP_053720260.1; CpoCA_beta, Candidatus Prometheoarchaeum syntrophicum, WP_147661847.1; Cab_beta, Methanothermobacter thermautotrophicus, 1G5C_A; NgonCA_alpha, Neisseria gonorrhoeae, WP_003688976.1; SsalCA_alpha, Streptococcus salivarius, WP_002888224.1; HpylCA_alpha, Helicobacter pylori, WP_010882609.1; SspCA_alpha, Sulfurihydrogenibium sp., HBT99398.1; BsuCA_beta, Brucella suis, AAN33967.1; BthCA_beta, Burkholderia thailandensis, WP_009893276.1; HpyCA_beta, Helicobacter pylori, BAF34127.1; LpnCA_beta, Legionella pneumophila, WP_011946835.1; EcoCA_beta, Escherichia Coli, WP_047081292.1; AbaCA_beta, Acinetobacter baumannii, WP_001141692.1; MinCA_beta, Myroides injenensis, WP_010254382.1; PgiCA_beta, Porphyromonas gingivalis, WP_012458351.1.

Figure 3

Schematic representation of the obtained construct for overexpressing the recombinant MscCAγ. The cloned nucleotide sequence (left) and the encoded amino acid sequence (right) are reported at the top of the figure. The bottom of the figure shows the construct with the T7 promoter to control the expression of the heterologous gene in E. coli, the initiation ATG in the N-terminal tag, the 6xHis tag, allowing the purification of the fusion protein with a metal-chelating resin, the gene of 507 bp, encoding for the MscCAγ, and the T7 transcription terminator, which permits an efficient transcription termination.

Figure 4

SDS-PAGE electropherogram (A), Western-Blot (B), and Protonogram (C). Legend: Lane 1, molecular markers, molecular mass values starting from the top: 150, 100, 75, 50, 37, 25, 20, and 15 kDa; Lane 2: cellular extract before induction with IPTG; Lane 3, MscCAγ overexpression induced by IPTG addition; Lane 4, MscCAγ band after affinity chromatography purification and detected by His-tag antibody; Lane 5, MscCAγ hydratase activity, which is denoted by the yellow band due to the CO₂ hydration reaction catalyzed by the enzyme on the polyacrylamide gel and responsible of the pH variation from 8.2 to the transition point of the dye; Lane 6, commercial bovine CA, used as positive controls.

Figure 5

The structures of sulfonamide, sulfamate and sulfamate CAIs (1–24 and AAZ-HCT) were investigated to inhibit bacterial and human CAs.