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Carbonic anhydrases (CAs, EC 220.127.116.11) are metalloenzymes which catalyze the hydration of carbon dioxide to bicarbonate and protons. Many pathogenic bacteria encode such enzymes belonging to the α-, β-, and/or γ-CA families. In the last decade, enzymes from some of these pathogens, including Legionella pneumophila, have been cloned and characterized in detail. These enzymes were shown to be efficient catalysts for CO2 hydration, with kcat values in the range of (3.4–8.3) × 105 s−1 and kcat/KM values of (4.7–8.5) × 107 M−1·s−1. In vitro inhibition studies with various classes of inhibitors, such as anions, sulfonamides and sulfamates, were also reported for the two β-CAs from this pathogen, LpCA1 and LpCA2. Inorganic anions were millimolar inhibitors, whereas diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid, and phenylarsonic acid were micromolar ones. The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide (KIs in the range of 40.3–90.5 nM). The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide, and dichlorophenamide (KIs in the range of 25.2–88.5 nM). Considering such preliminary results, the two bacterial CAs from this pathogen represent promising yet underexplored targets for obtaining antibacterials devoid of the resistance problems common to most of the clinically used antibiotics, but further studies are needed to validate them in vivo as drug targets.
Resistance to antibiotics belonging to all pharmacological classes is escalating and represents a worldwide problem [1,2,3,4,5,6,7,8,9,10]. Both Gram-negative and Gram-positive bacteria (such as Staphylococcus aureus, Mycobacterium tuberculosis, Helicobacter pylori, Brucella suis, Streptococcus pneumoniae, etc.) no longer respond to many clinically used such drugs belonging to several antibiotic classes [9,10,11,12]. Cloning of the genomes of many bacterial pathogens offers the possibility to explore alternative pathways for inhibiting virulence factors or proteins essential for their life cycles [8,13,14,15,16,17]. Among many such new possible drug targets explored recently, there is a class of enzymes catalyzing a simple but physiologically relevant process, i.e., the hydration carbon dioxide to bicarbonate and protons, generating thus a buffering weak base (bicarbonate) and a strong acid (H+) [18,19,20,21,22,23]. These metalloenzymes are the carbonic anhydrases (CAs, EC 18.104.22.168). Six genetically distinct CA families are known to date, the α-, β-, γ-, δ-, ζ- and η-CAs [18,24,25,26,27,28,29]. The α-, β-, δ- and η-CAs use Zn(II) ions at the active site, the γ-CAs are probably Fe(II) enzymes (but they are also active with bound Zn(II) or Co(II) ions), whereas the ζ-class uses Cd(II) or Zn(II) to perform the physiologic reaction catalysis [18,22,26,27].
The 3D fold of these enzyme classes are very different from each other (a nice example of convergent/divergent evolution ), as it is their oligomerization state: α-CAs are normally monomers and rarely dimers; β-CAs are dimers, tetramers, or octamers; γ-CAs are trimers , whereas the δ-, ζ- and η-CAs are probably monomers but in the case of the ζ-CA family, three slightly different active sites are present on the same protein backbone (which is thus a pseudotrimer, at least for the best investigated such enzyme, from the diatom Thalassiosira weissflogii) . Many representatives of all these enzyme classes have been crystallized and characterized in detail, except for representatives of the δ- and η-CAs, for which the exact structure is not known to date . The mammalian CAs and their inhibitors/activators have been thoroughly reviewed elsewhere [22,23,24] and these enzymes will be not discussed here, although they are off-targets when considering bacterial CAs as possible anti-infective drug targets [5,8].
These enzymes are ubiquitous: the α-CAs are present in vertebrates, protozoa, algae, and cytoplasm of green plants and in some Bacteria; the β-CAs are predominantly found in Bacteria, algae and chloroplasts of both mono- as well as dicotyledons, but also in many fungi and some Archaea [28,29,30,31,32]. In bacteria and fungi they are mostly homodimers/tetramers . The γ-CAs were found in Archaea and Bacteria, whereas the δ- and ζ-CAs seem to be present only in marine diatoms [28,29,30,31,32]. In most organisms, these enzymes are involved in crucial physiological processes connected with respiration and transport of CO2/bicarbonate, pH and CO2 homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions (such as gluconeogenesis, lipogenesis and ureagenesis), bone resorption, calcification, tumorigenicity, and many other physiologic or pathologic processes (thoroughly studied in vertebrates), whereas in algae, plants, and some bacteria they play an important role in photosynthesis and biosynthetic reactions. In diatoms δ- and ζ-CAs play a crucial role in carbon dioxide fixation [28,29].
Bacteria are ubiquitous microorganisms, with most available niches occupied by many members of this kingdom. Bacteria encode CAs from three classes, the α-, β-, and γ-CA class, thus suggesting that these enzymes play an important role in the prokaryotic physiology [5,8]. It has been demonstrated that these three families are genetically quite distinct between each other . Furthermore, the various members of the same class from diverse organisms possess very different biochemical properties, 3D folds of the protein backbone (for those enzymes for which the structure has been resolved by means of X-ray crystallography [5,20,22]), and susceptibility to be inhibited by various classes of CA inhibitors (CAIs), such as the sulfonamides and the inorganic anions, the most investigated such modulators of activity [8,33,34,35].
Legionella pneumophila is a Gram-negative bacterium causing Legionnaires’ disease or legionellosis, an often fatal pneumonia (mortality rate of 20%–50%), which was observed for the first time in July 1976, among the attendees of the 58th Annual Convention of the American Legion in Philadelphia [36,37,38,39]. There are many species of this bacterium, but only L. pneumophila and to a lower extent L. longbeachae provoke disease in humans (the last species is prevalent in Australia and New Zealand, whereas the first one in the other continents) . Legionellae are environmental bacteria and their natural host is the amoebae in which they replicate, but by accidentally infecting human macrophages they cause oportunistic infections [40,41]. The spread of legionellosis was favored ultimately by the development of artificial water systems for air conditioning, cooling towers, aerosolizing devices, etc. .
The mechanisms by which these bacteria infect their hosts include the formation of Legionella-containing vacuoles (LCVs) within the host cytoplasm, which requires the remodeling of the LCV surface and the hijacking of vesicles and organelles for effective infection . These processes are mainly controlled by the pH of these organelles, and as a consequence the pathogen, similar to other bacteria, fungi, or protozoa [5,8,18,19,20], evolved mechanisms of interfering with the pH. Both macrophages and amoebae use phagosomes to destroy foreign particles/pathogens, in which a highly acidic pH is crucial for the outcome of the process . Intracellular pathogens, such as L. pneumophila thus developed sophisticated strategies to evade their destruction by the acidic phagolysosomes [40,41]. Indeed, it has been demonstrated that L. pneumophila is able to maintain a neutral pH in its phagosome for at least 6 h, whereas vacuoles which did not contain the bacterium became highly acidic in 15 min after their formation . One of the proteins involved in this process is a vacuolar V-ATPase , but as for other organisms [5,8,18,19,20], the pH regulation is a complex process in which many other proteins are involved, among which the CAs (which generate protons and bicarbonate by the hydration of the highly available substrate CO2) [43,44]. Thus, the investigation of L. pneumophila CAs started to interest researchers due to the potential of such proteins to develop alternative antibiotics [43,44]. Here I shall review the developments in the field of the cloning, characterization, and inhibition of the two such enzymes from this pathogen.
The genome of Legionella pneumophilia subsp. pneumophila strain Philadelphia-1 has been cloned some years ago , and inspection of the genome revealed the presence of two putative such enzymes belonging to the β-class, denominated LpCA1 and LpCA2 [43,44]. They have been cloned as glutathione S-transferase (GST)-fusion proteins [43,44]. LpCA1 contains 245 amino acid residues and has a calculated molecular weight of 26.994 kDa, whereas LpCA2 is shorter, comprising 208 amino acid residues, with a theoretical molecular weight of 22.997 kDa.
We have aligned the amino acid sequences of LpCA1 and LpCA2 with those of other such enzymes characterized earlier (kinetically, and in some cases also by means of X-ray crystallography), such as HpyCA from Helicobacter pylori , BsuCA213 and BsuCA219 (from Brucella suis [46,47]) and stCA1 and stCA2 from the bacterial pathogen Salmonella typhimurium [48,49] (Figure 1). Data of Figure 1 show that, as all other bacterial β-CAs investigated so far, the two Legionella proteins LpCA1 and LpCA2 possess the amino acid residues crucial in the catalytic cycle of CO2 conversion to bicarbonate and protons: (i) the zinc coordinating amino acids, constituted by one His and two Cys residues, more precisely Cys90, His143, and Cys146 (LpCA1 numbering system); and (ii) the catalytic dyad constituted by residues Asp92 and Arg94, which is involved in the activation of the zinc-coordinated water molecule which leads to the formation of the nucleophilic, zinc-hydroxide species of the enzyme [43,44].
The measured CO2 hydrase activity of the two enzymes, LpCA1 and LpCA2 (Table 1), where the activity of LpCA1 and LpCA2 was compared to that of other α- and β-CAs from human (h), fungal (Cryptococcus neoformans , Candida albicans , Saccharomyces cerevisiae ), and bacterial (H. pylori  and B. suis [46,47]) sources, showed that both these enzymes possess a significant catalytic activity for the physiologic reaction catalyzed by these enzymes, i.e., hydration of carbon dioxide with formation of protons and bicarbonate. The first isoform, LpCA1 showed a moderate degree of activity with a kcat of 3.4 × 105 s−1 and kcat/Km of 4.7 × 107 M−1·s−1. This activity is in fact comparable to that of other α- and β-CAs, such as hCA I (a physiologically relevant human isoform) , Can2 from the fungal pathogen C. neoformans , and the bacterial pathogenic enzymes HpyCA and BsuCA219, from H. pylori and B. suis, respectively [45,46]. The second isoform, LpCA2, was on the other hand more active than LpCA1, with the folllowing kinetic parameters: kcat of 8.3 × 105 s−1 and kcat/Km of 8.5 × 107 M−1·s−1 (Table 1). This is a level of activity similar to that of the C. albicans and S. cerevisiae enzymes or the BsuCA213 isoform (all belonging to the β-CA family) and is only slightly lower compared to hCA II, a highly effective catalyst  for the CO2 hydration reaction (Table 1). It may also be observed that acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) AAZ, a clinically used drug, inhibited efficiently the two Legionella enzymes, with KIs in the range of 36–95 nM. The moderately active enzyme (LpCA1) was less sensitive to this compound compared to LpCA2 which showed a higher catalytic activity and also higher affinity for the sulfonamide inhibitor (Table 1).
We have also examined the phylogenetic relationship of the two new bacterial β-CAs, LpCA1 and LpCA2, with that of other such enzymes characterized earlier from various bacterial and fungal pathogens, as well as algae, plants, and Archaea (see Table 2 for the organisms included in the phylogenetic analysis). As seen from the dendrogram of Figure 2, the two Legionella enzymes are highly similar, clustering on their own branch at the upper part of the tree, with the next most similar enzyme being MinCA from the recently identified bacterial pathogen resistant to all known antibiotics Myroides injenesis (this β-CA was not yet cloned and characterized) . On the other hand, the other evolutionarily most related enzyme to LpCA1 and LpCA2 is AbaCA, from Acinetobacter baumanii, another not yet cloned or characterized β-CA. The remaining β-CAs from Figure 2 were less related to LpCA1 and LpCA2 compared to the previously discussed ones, and clustered all together on the lower branches of the tree. This includes two main branches, one constituted only by the archaeal enzyme cab (from Methanobacterium thermoautotrophicum) , whereas the remaining, rather ramificated, branch includes yeast/fungal (SceCA, DbrCA and SpoCA), bacterial (HpyCA, BsuCA), algal (CspCA and CreCA), and plant enzymes (VraCA, FbiCA, ZmaCA and AthCA), which are all more distantly related to the bacterial CAs from Legionella examined here (Figure 2).
Sulfonamides and their isosteres (sulfamates, sulfamides, etc.) constitute the main and most investigated family of CAIs, with several drugs used clinically for the last 60 years . Among them are first generation compounds such as acetazolamide AAZ, methazolamide MZA, ethoxzolamide EZA and dichlorophenamide DCP. Dorzolamide DZA and brinzolamide BRZ are topically-acting antiglaucoma agents belonging to the second generation of such drugs , whereas benzolamide BZA is an orphan drug belonging to this class of pharmacological agents [18,19,20,21,22]. Topiramate TPM (a sulfamate) and zonisamide ZNS (an aliphatic sulfonamide) are widely used antiepileptic drugs . Other compounds incorporating primary/secondary sulfamoyl moieties such as sulpiride SLP and indisulam IND were also shown to belong to this class of pharmacological agents together with the COX2 selective inhibitors celecoxib CLX and valdecoxib VLX, the antiepileptic sulthiame SLT, the sweetener saccharin SAC, and hydrochlorothiazide HCT, a diuretic agent —see Chart 1. Compound 1–24 are sulfonamides possessing a variety of scaffolds/substitution patterns, which were included in the study  in order to explore a wider chemical space for detecting effective inhibitors of these CAs, (Table 3).
Data of Table 3 show the inhibition of the two Legionella enzymes, LpCA1 and LpCA2 with this set of sulfonamides/sulfamates. For comparison reasons, the inhibition of the two human (h) offtarget isoforms hCA I and II (belonging to the α-CA family) as well as the bacterial β-CA from the pathogen H. pylori (HpyCA) with this set of 40 compounds, are also presented in Table 3. The following structure-activity relationships (SAR) were observed for the inhibition of LpCA1 and LpCA2 with these compounds:
Anions and other small molecules (e.g., sulfamide, sulfamate, phenylboronic acid, or phenylarsonic acid) represent a class of well-established CAIs, as they bind to the metal ion from the enzyme active site and impair catalysis [18,34,35]. An inhibition study of the two Legionella enzymes with a range of such inhibitors, including simple and complex inorganic anions as well as sulfamide, sulfamate, phenylboronic acid, and phenylarsonic acid was thus reported  (Table 4). The following has been noted regarding inhibition of the two enzymes by the anions and small molecules shown in Table 4.
A tentative catalytic/inhibition mechanism of the two LpCA1/2 enzymes is presented in Figure 3, based on crystallographic and kinetic data published for similar β-CAs [5,8,14,15,23,30], as the X-ray crystal structures of the two Legionella enzymes is not known at the moment.
The catalytically active species of the enzyme (A in Figure 3) has a hydroxide ion coordinated to the Zn(II), in addition to the protein ligands (which in Figure 3 are those from LpCA1, i.e., Cys90, His143 and Cys146). This is valid for all CAs belonging to the six different genetic families mentioned in the introduction. In the case of the α-CAs, the zinc hydroxide species is generated through a proton transfer process from the zinc coordinated water to the environment, assisted by the active site residue His64, acting as a proton shuttle . In the β-CAs the activation of the zinc-coordinated water molecule (shown in D in Figure 3) is more complex than for the α-CAs, as the catalytic dyad mentioned earlier (Asp-Arg) takes part in the process. Furthermore, the nature of the amino acid(s) acting as proton shuttles in the β-class enzymes is not completely elucidated . After the nucleophilic attack of the zinc hydroxide species of the enzyme on the bound CO2 (shown in B) bicarbonate is formed (C in Figure 3) which might be bidentately coordinated to the zinc ion (as in the α-CAs) . Inorganic anions are usually weak ligands of the zinc ion in metalloenzymes, so that species C is probably easily converted to species D, by a substitution reaction of the bicarbonate by means of a water molecule. This leads to liberation of the reaction product (bicarbonate) into solution, with generation of the acidic form of the enzyme, D, which is catalytically inactive. To generate the zinc hydroxide, catalytically effective species A, a proton transfer reaction must occur (Figure 3), probably assisted by an active site residue. In the β-CA from the alga Coccomyxa, it has been hypothesized that there are two residues participating in this process, Tyr88 and His92 . However, neither LpCA1 nor LpCA2 possess a sequence of Tyr and His residues close to each other as the algal enzyme, which leads the nature of the proton shuttling residue in these novel β-CAs unknown at this moment. The inhibition mechanism of LpCA1 and LpCA2 (shown in steps E and F in Figure 3) involves the inhibitor substituting the fourth, non-protein zinc ligand, leading to tetrahedral species, as those shown in E. Anions such as acetate bound to Can2 , as well as thiocyanate, azide, and phosphate bound to the Coccomyxa β-CA , were shown to possess this inhibition mechanism by means of high resolution X-ray crystallography. An alternative inhibition mechanism has been also reported again for the Coccomyxa β-CA for iodide , which was found anchored to the zinc-coordinated water molecule/hydroxide ion, as shown schematically in Figure 3, F. This mechanism was observed for phenols , polyamines , and sulfocoumarins  for the inhibition of α-CAs and may be more general than originally considered .
Legionella pneumophila is a Gram-negative bacterium causing Legionnaires' disease and infections caused by it are increasing all over the world. Two β-CAs were recently identified, cloned and purified in this pathogen (LpCA1 and LpCA2) [43,44] and considering the successful inhibition of growth of other bacteria when their CA was inhibited [57,58], the two enzymes were proposed as possible drug targets. LpCA1/LpCA2 efficiently catalyzed CO2 hydration to bicarbonate and protons, with kcat in the range of (3.4–8.3) × 105 s−1 and kcat/KM of (4.7–8.5) × 107 M−1·s−1, being thus among the very efficient bacterial CAs discovered and characterized so far. They were inhibited by many sulfonamides and sulfamates, the main class of CAIs, with several nanomolar inhibitors detected, although few studies are available in the literature to date . The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide (KIs in the range of 40.3–90.5 nM). The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide, and dichlorophenamide (KIs in the range of 25.2–88.5 nM). Inorganic anions were millimolar inhibitors, whereas diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid, and phenylarsonic acid were micromolar ones against both bacterial enzymes. As these enzymes may be involved in pH regulation in the phagosome during Legionella infection, their inhibition may lead to antibacterials with a novel mechanism of action, but in vivo studies are necessary in order to validate them definitively as drug targets.
Work from the author's laboratory was funded by several EU projects (DeZnIt, Metoxia, Gums & Joints and Dynano).
The author declares no conflict of interest.