The genome exploration of microorganisms causing mammalian and non-mammalian infections as well as the genome of those considered not harmful evidenced genes encoding for an exciting class of enzymes that are involved in the metabolic balance of the bacterial carbon dioxide (CO₂), bicarbonate (HCO₃^–), and protons (H⁺) (Annunziato et al., 2016; Capasso and Supuran, 2016; Del Prete et al., 2016a, b; Ozensoy Guler et al., 2016), namely carbonic anhydrases (CAs, EC 4.2.1.1). They belong to a superfamily of metalloenzymes that catalyze the physiologically crucial reversible reaction of CO₂ hydration to HCO₃^– and H⁺, according to the following chemical reaction (Capasso and Supuran, 2015a):

Until now, eight CA classes indicated with α, β, γ, δ, ζ, η, θ, and ι have been described in all kingdoms of living organisms (Supuran and Capasso, 2017). All CA classes strictly conserve the CO₂ hydration and HCO₃^– dehydration mechanisms, showing an evident phenomenon of convergent evolution, having a very low sequence similarity, and different 3D molecular folds and structures (Supuran and Capasso, 2017). In Bacteria, four CA-classes (α, β, γ, and ι) regulate the CO₂ and HCO₃^– balance, being the only CA classes encoded by the bacterial genome (Capasso and Supuran, 2013, 2015b,c; Supuran and Capasso, 2015; Del Prete et al., 2020b). For enzyme catalysis, most of these CAs need Zn²⁺ as ion cofactor, which is coordinated by three amino acid residues from the protein backbone (Buzas and Supuran, 2016; Supuran, 2016e). The fourth metal ion ligand is a water molecule/hydroxide ion acting as the nucleophile in enzyme catalytic cycle (Carta et al., 2014). In particular, γ-CAs are Fe²⁺-dependent enzymes, but they are also active with bound Zn²⁺ or Co²⁺ ions; the last identified ι-CA class from the marine diatom Thalassiosira pseudonana prefers Mn²⁺ to Zn²⁺ as the ion cofactor. α-CAs are usually active as monomers or dimers; β-CAs are active only as dimers, tetramers, or octamers. The γ-CAs must be trimers for accomplishing their catalytic function (Di Fiore et al., 2013; De Simone et al., 2015; Ferraroni et al., 2015; Lomelino et al., 2016a). γ-CA monomers are characterized by a tandemly-repeated hexapeptide crucial for the left-hand fold of the trimeric β-helix structures (Fu et al., 2008). The X-ray structure of ι-CAs is not available at this moment. Intriguing, α- and ι-CAs catalyze also ester/thioester reactions (Supuran, 2016c; Jensen et al., 2019).

In 1979, examining the tissue samples from patients subjected to a gastric biopsy, the pathologist J. Robin Warren noted many spiral-shaped curved bacteria below the thick mucus layer, covering the stomach inner wall. Later, Warren associated this infection with chronic superficial gastritis, and the nested bacteria were identified as belonging to the genus Helicobacter (Warren, 2000, 2006). Today, we know that a high percentage of people infected with H. pylori have superficial chronic gastritis. Besides, if left untreated, both H. pylori infection and inflammation can persist for decades, and sometimes even for all the lifetime (Rahman et al., 2020). Over the years, it has learnt that H. pylori causes a chronic inflammatory process, the peptic ulcer in the stomach and duodenum, the portion of the small intestine that originates from the pylorus (Zhu et al., 2020). The infection, previously considered to be of metabolic origin, strongly increases the risk of neoplasms, such as adenocarcinomas and lymphomas. For example, it was discovered that some varieties of H. pylori have a 40 kb DNA insertion element called cag pathogenicity island (cag PAI), containing about 32 genes encoding the bacterial type IV secretion system (Noto and Peak, 2012). The cag system enables the transmission of bacterial effector molecules into the gastric epithelial cells of the host. Some H. pylori strains slowly inject into the gastric cells one of the virulent proteins, called CagA that can trigger severe gastritis atrophy, dysplasia, and gastric adenocarcinoma, when a comparison with counterpart strains lacking this component was done (Amieva et al., 2003; Ye et al., 2003).

Helicobacter pylori is a Gram-negative pathogenic neutralophilic bacterium with a metabolism harmonized for a neutral pH development, but it is adapted to live in the overly acidic gastrointestinal environment (Tarsia et al., 2018). H. pylori genome encodes for α- and β-CAs. The α-CA (hpβCA) has a periplasmic localization, while the β-CA (hpβCA) is localized in the cytoplasm. It was aforementioned that the activity of H. pylori CAs could be an additional adaptation of the bacterium in the high acidic gastrointestinal environment (pH in the range 1–2). Urease and CAs are the two enzymatic systems used by the microbe for growing in this extreme environment (Capasso and Supuran, 2015a). These enzymes regulate the bacterial pH value determining an increase in the cytoplasm through ammonia production (NH₃). Urea goes into the cytoplasm through the urea channel under acidic conditions, where the urease converts it into NH₃ and CO₂. In the cytoplasm, resulting CO₂ is then hydrated by β-CA, while the periplasmic α-CA hydrates the CO₂ diffused in the periplasm (Capasso and Supuran, 2015b). The produced ions (H⁺) by the CA-catalyzed reaction are used by NH₃ to form NH₄⁺ in the periplasm and cytoplasm, which neutralizes the entering acid in the above environments (Morishita et al., 2008). The combined action of urease and CAs result in the acid acclimatization of the pathogen within the stomach.

The bacterial extracellular vesicles (EVs) are generated in a budding manner similar to that of the yeasts (Kim et al., 2015). Gram-negative bacteria, differently from the Gram-positive bacteria, produce extracellular vesicles by pinching off the outer membrane and, for this reason, are defined with the acronym OMVs (Outer Membrane Vesicles) (Liu et al., 2018). Distinctive features of OMVs are the lipopolysaccharide (LPS) and encapsulate periplasmic components, which are absent in the Gram-positive EVs. The vesicles generated by Gram-positive bacteria could bring inside various molecules, including nucleic acids, proteins, lipids, viruses, enzymes, and toxins. Depending molecules contained inside, these vesicles can have variegated roles. For example, they are involved in horizontal gene transfer, antibiotic resistance, microbial survival, microbial competition, nutrient acquisition, health benefits for the host, microbial virulence, cell-cell communication among bacteria and hosts, and biofilm formation (Ronci et al., 2019). The H. pylori OMVs are implicated in biofilm formation, and the presence of DNA inside these vesicles appears to be involved in “joining” OMV–OMV and OMV–cell communications (Grande et al., 2012, 2015). As aforementioned, Ronci et al. used mass spectrometry to identify periplasmic β-CA in the H. pylori OMVs generated in vitro from the microbe both in its planktonic and biofilm phenotypes (Ronci et al., 2019). Besides, β-CA hydratase activity was determined using the protonography, a technique selective for the detection of CAs. As a result, it was demonstrated that the amount of the periplasmic β-CA was higher in the planktonic OMVs (pOMVs) than in the biofilm OMVs (bOMVs). Furthermore, it was observed that the content of β-CA increased in pOMVs over time.

Moreover, the biofilm phenotype, a complex structure in which bacteria adhere to a surface and are embedded in a self-produced EPS (extracellular polymeric substance) matrix, is a condition used by pathogenic bacteria to improve their survival, bacterial infection, and resistance to the effects of antimicrobial agents (Parsek and Singh, 2003; Grande et al., 2014). H. pylori tends to form a biofilm on human gastric mucosa (Yonezawa et al., 2015), and biofilm cells are more resistant to the effects of antimicrobial agents (Carron et al., 2006). Generally, the first-line therapy to eradicate H. pylori infection is based on a combination of drugs, such as proton pump inhibitor (PPIs), amoxicillin, clarithromycin (CAM) or metronidazole, and fluoroquinolones (Bang et al., 2020). Novel approaches to prevent biofilm formation and to treat infections by biofilm-forming bacteria are currently under development (Bjarnsholt et al., 2018). The identification of periplasmic β-CA in pOMVs and its specific inhibition with the classical CAIs might shed new light on this enzyme’s role in the H. pylori colonization, survival, persistence, and pathogenesis.

In the literature, it has been reported the possible existence of a correlation between intestinal microbiota (i.e., microbial populations living in the intestine) and various autoimmune diseases, such as systemic lupus erythematosus or autoimmune liver diseases (Manfredo Vieira et al., 2018). Kriegel and colleagues noted that the bacterium Enterococcus gallinarum, a very rare enterococcus, has been often identified in the intestinal flora and liver of patients with lupus; thus, it was considered as a trigger of the systemic lupus erythematosus (Manfredo Vieira et al., 2018). In experiments on mice, it was demonstrated that these bacteria can overcome the small intestine barrier and quickly reach the liver and other organs, particularly the spleen and lymph nodes. In this way, the bacterium triggers an inflammatory process, which allows the secretion of chemical messengers equal to those observed in subjects with lupus, inducing the proliferation of autoantibodies that also attack the cells of the organism. In contrast, some bacterial strains, which live within the microbiota, function as brakes against intestinal tumors (Zagato et al., 2020). For example, it has been observed the absence of Holdemanella biformis, a bacterium belonging to the family of Erysipelotrichaceae, in the microbiota of patients in an early stage of development of intestinal cancer. These bacteria have antitumor properties capable of blocking the uncontrolled proliferation of cells, which happens in the case of a lack of them in the gut (Zagato et al., 2020). Thus, it is reasonable to believe that these anti-tumorigenic bacteria have a strong diagnostic, therapeutic, and translational potential.

In H. pylori-infected individuals, the gastric microbiota is similar to that of the non-infected persons. In general, infected persons have 52.6% of Proteobacteria, 26.4% of Firmicutes, 12% of Bacteroidetes and 6.4% of Actinobacteria (Llorca et al., 2017; Ozbey et al., 2020). The resident gastric microflora may interfere with H. pylori’s proliferation and gut disease. For this reason, the pharmacological treatments for eradicating H. pylori from the gastric mucosa can be ameliorate using probiotics (Emara et al., 2014; Maccelli et al., 2020). The effect of new antimicrobial molecules is rarely evaluated; thus, the identification of new drugs that possess a selective toxicity between pathogens and some components of the human microbiota might represent an important step in the clinical field (Grande et al., 2020). Therefore, the identification of probiotic strains, which do not possess the CAs and constitute a significant component of the human microbiota (Martin et al., 2013) used in combination with innovative drugs, such as those coming from the modified scaffold of CAIs, might represent an innovative anti-H. pylori treatment. Not being affected by the inhibitor, the probiotic can exert a synergistic effect improving the antimicrobial action of the (bactericidal or bacteriostatic) CA inhibitors. Besides, the probiotic, by educating or stimulating the host immune system, could also contribute to the efficacy of the CA inhibitor. Thus, this combination might represent an innovative and successful strategy to fight infections without altering the normal microbiota.

At least four classes of CAs (α, β, γ, and ι) are present in Bacteria. During growth, microbes require CO₂ and HCO₃^– to support their metabolism, as well as H⁺ ions/bicarbonate for the balance of the pH value. CAs with their activity correctly balance the interconversion of inorganic these species. Thus, CAs play essential roles in the life cycle of pathogenic and non-pathogenic bacteria, and their inhibition prejudices the growth of the microbe. This paves the way for designing novel anti-infective drugs, which function differently from the standard antibiotics. The involvement of CAs in the lifecycle, pathogenicity, and virulence of several species (e.g., H. pylori, V. cholerae, B. suis, S. enterica, P. aeruginosa, and Enterococcus spp.) of human pathogens is not new, but only recently programs to develop agents that specifically and selectively inhibit these enzymes have been initiated. Presently, many bacterial species have been investigated for the presence of CAs belonging to all four classes mentioned above. Furthermore, many bacterial CAs have been prepared as recombinant enzymes and thoroughly characterized by a biochemical viewpoint and for their ability to be inhibited by various compounds. These enzymes were effectively inhibited by the classical CAIs, such as the sulfonamides and their derivatives, sulfamates, sulfamides, (in)organic anions, and some of them by dithiocarbamates as well as carboxylic acids. More exciting is the discovery that ethoxzolamide can kill H. pylori in vitro and in vivo, and that the bacterial resistance to this compound does not develop easily. The recent study on the efficacy of acetazolamide and some of its derivatives to act as inhibitors of vancomycin resistant enterococci (VRE) is a breakthrough in the field (Kaur et al., 2020). The drug design campaign reported in the same study led to the identification of sulfonamide derivatives, which seem to be orders of magnitude more efficient against VRE, when compared to the clinically used agent linezolid. Drug design campaigns were also useful in finding H. pylori-selective CAIs belonging to the sulfonamide class. Thus, it is not impossible to hypothesize that the field of CAIs as anti-infectives may lead to relevant developments in the near future and future dedicated studies are necessary in this context.

All authors contributed to the article and approved the submitted version.

RG and SC have a scientific collaboration with BioGaia AB (Stockholm, Sweden). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.