Background/Objectives: Fusobacterium nucleatum and Akkermansia muciniphila are key components of the human microbiota, influencing health and disease. F. nucleatum is associated with colorectal cancer (CRC) and poor prognosis through its pro-inflammatory and pro-tumorigenic activity, whereas A. muciniphila is linked to metabolic benefits and anti-inflammatory effects. This study aimed to evaluate the immunomodulatory impact of protein extracts from these bacteria on peripheral T cell responses in healthy individuals and CRC patients. Methods: Peripheral blood mononuclear cells (PBMCs) were exposed to bacterial extracts, individually or in combination, and T cell subsets were analyzed by polychromatic flow cytometry. Results: In healthy donors, F. nucleatum increased Th0, Th2, and Tc9 cell frequencies while reducing Th1, Th1/Th17, and Treg cells. Conversely, A. muciniphila promoted a pro-inflammatory-associated T cell phenotype characterized by higher Th0, Th2, Th17, and Tc17 cells. Combined exposure enhanced Th0, Th17, and Tc17 cells while decreasing Th9 cells. In CRC patients, bacterial extracts induced no significant changes in T cell subsets. Conclusions: These findings indicate that F. nucleatum skews immune responses toward humoral and mucosal defense, whereas A. muciniphila enhances T cell polarization toward subsets usually associated with pro-inflammatory immune responses in healthy subjects. Further studies are needed to clarify their systemic immunological roles and interactions within the tumor microenvironment of CRC.

Carbonic anhydrases (CAs) form a widely distributed superfamily of metalloenzymes, characterized by considerable structural diversity. These enzymes catalyze the reversible conversion of carbon dioxide into bicarbonate and protons. While they all perform this fundamental reaction, CAs show notable structural plasticity, with eight classes (α, β, γ, δ, ζ, η, θ, and ι) that have evolved independently to optimize CO₂ hydration under a range of cellular, physiological, and ecological conditions. Their catalytic efficiency plays a crucial role in several biological processes, including acid-base balance, respiratory gas exchange, renal bicarbonate reabsorption, metabolic regulation, and photosynthetic carbon fixation. Isoform-specific expression allows CAs to act in multiple cellular contexts. They contribute to signaling pathways, help maintain pH balance, function within tissue-specific environments. When these enzymes are not properly regulated, they can be associated with a variety of health issues, including renal tubular acidosis, osteopetrosis, neurological disorders, and cancer progression. Evolution has shaped enzymes in different species to have similar active sites, flexible metal use, and varied protein assemblies, all helping them adapt to challenges like limited metal ions availability and changing CO₂ levels. CAs are not just important in biology. These metalloenzymes are also key targets for treating cancer, eye conditions, and infections. Besides, they show great promise as enzymes for capturing CO₂ and for other biotechnological applications. This chapter offers a thorough introduction to CAs, exploring their biology, how their structures and functions have evolved, and their potential applications, showing how these enzymes link the worlds of science, medicine, and the environment.

Malaria remains a major global health burden, compounded by increasing resistance to current therapies. Plasmodium falciparum carbonic anhydrase (PfaCA), a η-class metalloenzyme essential for the parasite metabolism and survival, has recently emerged as a promising antimalarial target. In this study, benzoxaborole 4 and its 6-substituted triazolyl derivatives (5-18) were evaluated for PfaCA inhibition and in vitro antiplasmodial activity against the P. falciparum 3D7 strain. Compounds 13 and 14 emerged as the most potent inhibitors (KI = 0.45 and 0.43 µM, respectively) of the enzyme, coupled with notable antiplasmodial activity (IC50 = 34 and 47 µM). Among the tested compounds, the ethyl ester 7 demonstrated the strongest antiplasmodial activity (IC50 = 2.5 µM). Molecular docking studies further supported the favorable binding of active derivatives within the PfaCA active site. These findings highlight the potential of benzoxaborole-based scaffolds as a new chemotype for antimalarial drug development.

Abstract Curcumin (CUR) is a natural polyphenol with recognized antibacterial and antioxidant properties, whose biomedical application is limited by poor aqueous solubility, low bioavailability, and rapid chemical degradation under physiological conditions. In this study, green and sustainable chitosan-fatty acid nanoparticles were developed through a mild cosolvent evaporation strategy to improve CUR stability and facilitate antibacterial activity while avoiding harsh preparation conditions. The obtained nanosystem showed that CUR encapsulation and formation of stable nanosized particles are suitable for aqueous dispersion and biological applications, since curcumin was efficiently transported into the intracellular compartment of Escherichia coli. This was used as Gram-negative bacterium to investigate the antibacterial activity of encapsulated CUR. In addition, the chitosan-based carrier effectively preserved the chemical integrity of CUR both during nanoparticle preparation and under physiological conditions. The combined effects of sustainable formulation, improved CUR stability, and antibacterial activity highlight the potential of these chitosan-based nanoparticles as effective delivery systems for poorly soluble bioactive molecules. Overall, the proposed strategy represents an environmentally friendly platform for the development of advanced antimicrobial nanomaterials based on natural polymers.

Malassezia spp. are lipophilic yeasts that colonize human and animal skin, being implicated in several dermatological and systemic disorders, including seborrheic dermatitis, pityriasis versicolor, and opportunistic fungemia in immunocompromised individuals. The increasing diffusion of resistant strains and the limited efficacy of conventional azole-based antifungal therapies have prompted the search for alternative treatment strategies. This review explores recent approaches to fighting Malassezia, focusing on both natural and synthetic compounds targeting key molecular pathways. Among the most innovative approaches are Malassezia carbonic anhydrases, essential enzymes involved in pH regulation and cellular metabolism, which represent promising and selective antifungal targets. The antifungal potential of plant-derived extracts rich in polyphenols, terpenoids, and flavonoids is also discussed, highlighting their ability to interfere with yeast growth and adhesion. Silver nanoparticles are examined as synergistic agents due to their antimicrobial properties and potential as carriers for natural bioactive compounds. Additional perspectives include the use of probiotics/postbiotics to restore skin microbiota balance, enzymes such as chitinase and chitosanase, and the inhibition of lipases, key enzymes in Malassezia lipid metabolism. Finally, the growing interest in antimicrobial peptides, both natural and synthetic, opens new avenues for targeted topical formulations. Overall, the integration of natural and synthetic approaches, supported by a deeper understanding of Malassezia’s molecular mechanisms, may promote the development of more effective and safer multifactorial therapies.

Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc-dependent metalloenzymes that catalyze the reversible hydration of carbon dioxide, a reaction essential for acid-base regulation, respiration, and metabolic integration in vertebrates. In animals, CA activity is exclusively encoded by the α-CA family, which has undergone extensive expansion during vertebrate evolution. This chapter presents a phylogenetic synthesis of vertebrate α-CAs, integrating sequence-based analyses with comparative genomics, structural constraints, and patterns of subcellular localization. Phylogenetic evidence indicates that the modern vertebrate CA repertoire originated early in the evolution of life, and was shaped by ancient whole-genome duplication events, followed by differential retention, divergence, and lineage-specific gene loss. Cytosolic, mitochondrial, membrane-associated, and secreted CAs form well-supported evolutionary lineages that primarily reflect diversification in regulation, cellular targeting and physiological roles, rather than changes in catalytic mechanism. Catalytically inactive CA-related proteins (CARPs) constitute a distinct evolutionary branch derived from gene duplication and functional divergence of catalytically active CAs. Molecular analyses reveal strong evolutionary constraints on residues essential for zinc coordination and proton transfer, whereas surface-exposed and regulatory regions show greater evolutionary flexibility. Comparative analyses across vertebrates highlight the broad conservation of major CA clades, together with lineage-specific differences in isoform repertoires. By embedding functional interpretation within a phylogenetic framework, this chapter provides an evolutionary basis for comparative and biomedical studies of vertebrate CAs.

We investigated the in vitro effects of nonsteroidal anti-inflammatory drug-carbonic anhydrase inhibitor hybrids (NSAID-CAIs) on β-carbonic anhydrases 1-3 expressed by Mycobacterium tuberculosis, as well as their antimycobacterial efficacy in a zebrafish infection model. The primary aims were to investigate whether these enzymes represent valid therapeutic targets and whether NSAID-CAIs constitute potential drug candidates for tuberculosis. In vitro inhibition activity against the carbonic anhydrases was evaluated with a CO2-hydration assay. Antimycobacterial properties of the NSAID-CAIs were assessed via screening assays based on bioluminescence and colony-forming units. Larval and adult zebrafish were used for in vivo experiments. NSAID-CAIs strongly inhibited Mycobacterium tuberculosis β-carbonic anhydrase 2 and human carbonic anhydrases IX and XII in vitro. They exhibited high efficacy against Mycobacterium marinum and attenuated Mycobacterium tuberculosis in biofilm environments, outperforming their coadministered constitutive fragments. Compounds 11, 19, and 24 showed bactericidal properties against Mycobacterium marinum, and 12 was able to potentiate the effects of rifampicin. The experimental data provides solid in vitro evidence for the use of NSAID-CAIs as antimycobacterial agents by means of Mycobacterium tuberculosis β-carbonic anhydrase inhibition along with the possible suppression of the host immune response through the induction of host-directed therapeutic effects.

γ-class carbonic anhydrases (γ-CAs) constitute one of the most ancient and structurally distinct CO₂-hydratase enzymes in prokaryotes, marked by metal flexibility and a catalytic mechanism compatible with anoxic, Fe2+-rich conditions inferred for the Archaean eon. Their widespread distribution across archaeal lineages, particularly methanogens, supports models proposing γ-CAs as an early-diverging carbonic anhydrase lineage. Two main types of γ-CAs are known: the Cam lineage relies on a canonical three-histidine metal-coordination cluster and a Glu-mediated proton shuttle. In contrast, the CamH subclass lacks this acidic residue, indicating a divergent proton-transfer mechanism. γ-CAs retain essential physiological roles in intracellular CO₂/HCO₃- homeostasis, pH regulation, and virulence control in several human pathogens, including Vibrio cholerae. Recent reports suggest that selected γ-CAs may exhibit silicase-like activity, indicating a potential catalytic plasticity that could expand the functional landscape of this enzyme class and suggest links between carbon metabolism and mineral-rich environments. Beyond their evolutionary significance, γ-CAs represent attractive biomedical targets; their total absence in vertebrates provides a clear pharmacological window for the development of selective antibacterials. In biotechnology, the intrinsic thermostability and metal tolerance of γ-CAs support applications in industrial CO₂ capture, synthetic biology, and mineral bioprocessing. This review synthesizes current structural, mechanistic, and functional knowledge on γ-CAs, highlights unresolved questions, particularly concerning catalytic promiscuity and the CamH subclass, and outlines future research directions to clarify their roles in enzyme evolution and modern microbial physiology.

Bacterial carbonic anhydrases (CAs) are essential for intracellular pH regulation, bicarbonate homeostasis, and energy metabolism, making them attractive antimicrobial targets. Here, building on evidence that acetazolamide (AZA) delivered via hyaluronic acid-palmitate (HA-PA) nanocarriers impairs Escherichia coli growth and its glucose uptake, we investigated the physiological roles of β- and γ-class CAs using sulphonamide inhibitors with distinct selectivity encapsulated in HA-PA nanomicelles to ensure intracellular delivery. AZA, a potent dual β/γ-CA inhibitor, ethoxzolamide (EZA), a selective β-CA inhibitor, and hydrochlorothiazide (HCT), a weaker inhibitor of both classes, were tested for effects on bacterial physiology. The nanoparticles reduced growth in a dose- and class-dependent manner, with AZA exerting the strongest activity, EZA intermediate inhibition, and HCT only modest effects at higher concentrations. Early metabolic responses assessed via intracellular ATP after three hours of exposure revealed an unexpected and reproducible ATP increase for all inhibitors relative to untreated cells, suggesting reduced ATP consumption in bicarbonate-dependent pathways. These findings provide indirect yet compelling evidence that β- and γ-class CAs influence bacterial energy homeostasis and support the rationale for CA inhibition as an antimicrobial strategy, while highlighting HA-PA carriers as effective systems for delivering CA inhibitors intracellularly and enhancing their functional activity in bacterial cells.

Carbonic anhydrases (CAs) are highly efficient metalloenzymes catalyzing the reversible conversion of carbon dioxide (CO2) into bicarbonate (HCO3-) and protons (H+). Over the past decades, CAs have been employed across diverse industrial and environmental applications, including carbon management and CO2 capture, chemical synthesis, biomineralization, water treatment, and renewable energy generation. This chapter provides a comprehensive overview of CA-based technologies, emphasizing their transition from laboratory research to pilot- and industrial-scale applications. Key topics covered include enzyme-assisted CO2 absorption, reactor and solvent design, enzyme immobilization, and hybrid systems integrating chemical, biological, or electrochemical conversion. The chapter also examines operational challenges, mitigation strategies, and future perspectives, highlighting how CAs can enable sustainable, low-energy, and value-added processes. It illustrates the integration of mechanistic insights, process design, and real-world examples, providing a valuable resource for both researchers and industrial practitioners.

An overview of carbonic anhydrases (CAs) in fungi and protozoa is provided, emphasizing their evolutionary significance, functional diversity, and implications for human health. CAs are metalloenzymes that catalyze the reversible hydration of carbon dioxide, playing crucial roles in cellular homeostasis, pH regulation, and metabolic adaptation. In fungi, α- and β-class CAs are predominant, facilitating growth and virulence, particularly in pathogenic species such as Candida spp., Cryptococcus neoformans and many others. Protozoa exhibit a broader range of CA classes, including the recently identified η-class in Plasmodium falciparum, which is vital for the survival of the parasite and presents a significant potential as a drug target. The evolutionary trajectories of CAs reflect adaptations to diverse ecological niches, with gene duplication leading to functional diversification. Understanding the biochemical properties and regulatory mechanisms of CAs in these organisms can lead to innovative therapeutic strategies against fungal and protozoan infections, highlighting their potential as drug and diagnostic targets.

Malaria parasites belonging to the genus Plasmodium encode for a carbonic anhydrase (CA, EC 4.2.1.1) originally considered to belong to the α-class, which has been investigated starting with 2004 as a potential antimalarial target, considering the observation that CA levels in red blood cells infected with these parasites are much higher compared to those of uninfected cells. In plasmodia, CA is involved in metabolic pathways leading to the biosynthesis of pyrimidines, which are scarcely present in the blood of infected hosts, making this enzyme crucial for the life cycle of the parasite in many intraerythrocytic stages of its development. It has been then shown in 2014 that P. falciparum CA (PfCA) belongs in fact to a new CA genetic class, the η-CA, characterized by a particular zinc ion coordination within the active site, with two histidine and a glutamine as protein ligands. A short, truncated and longer PfCA forms have been cloned and characterized in detail, being shown that they act as efficient catalysts for the hydration of CO2 to bicarbonate and protons, but neither of them were crystallized for the moment, and their 3D structure is not known. PfCA inhibition with anions, sulfonamides, phenols and coumarins has been investigated too, with many low nanomolar in vitro inhibitors being detected. Only for acetazolamide and an ureido-substituted benzenesulfonamide it has been demonstrated a potent growth inhibition of the pathogen in P. falciparum infected red blood cells. Although these results are encouraging but rather preliminary, η-CAs from malaria-producing protozoans and presumably other organisms encoding them, may be considered as innovative drug targets for obtaining anti-infectives with new mechanisms of action but these enzymes should be investigated in more details in order to better understand their structure and physiological/pathological roles.

The vital process of CO2 hydration in all living things is catalyzed by carbonic anhydrases (CAs, EC 4.2.1.1), which are actively involved in the control of numerous pathological and physiological situations. A number of coumarin-based sulfonamides (18) were examined as potential CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA I, hCA II, and the transmembrane hCA IX as well as against three fungal CAs: Malassezia globosa (MgCA), Malassezia pachydermatis (MpaCA), and Malassezia restricta (MreCA). Additionally, the binding mechanism of this family of chemicals within the active site of hCA IX was investigated using computational approaches. Compounds showed interesting inhibitory activity mainly against two fungal strains, Malassezia globosa (MgCA) and Malassezia pachydermatis (MpaCA), exceeding in the first case the activity of the reference drug, and in the second, 16 out of 18 showed Ki values lower than acetozolamide. Furthermore, four compounds were more potent against hCA I than AAZ and two against hCA II. Docking studies were used to investigate the binding mode of the most active compounds against human CA isoforms.

Antimicrobial resistance (AMR) is one of the world's most pressing health problems and requires immediate action from the scientific community. Pseudomonas aeruginosa is a particularly concerning bacterium due to its high level of resistance, especially among hospitalized and immunocompromised individuals. We recently investigated PsCA3, one of three P. aeruginosa β‑carbonic anhydrases (β-CAs), as a potential new pharmacological target for developing innovative antibacterial drugs. In this study, we applied a consensus structure-based virtual screening approach to select the most promising PsCA3 inhibitors from an in-house library of 607 small molecules. Twenty-one diverse compounds were selected and experimentally validated through enzyme inhibition assays, which assessed the ability of all ligands to block PsCA3 activity while sparing human α‑carbonic anhydrases (hCAs). Next, we focused on the benzoxazinone/dihydroquinolinone scaffold, testing fifteen additional analogues and expanding the set of assayed microbial β-CAs. Notably, we identified new, potent ligands based on unexplored scaffolds that can effectively target microbial β-CAs at micromolar/submicromolar concentrations with remarkable selectivity over human CAs.

Sordaria macrospora, a coprophylous fungus used for the last three decades as a model organism for studying fruiting body development of fungi, encodes for four carbonic anhydrases (CAs, EC 4.2.1.1), CAS1-CAS4. CAS1-CAS3 are β-CAs and were investigated in detail in the last years, whereas CAS4, an α-class enzyme, was less investigated. All of them are crucial for the fungus, as the mutant lacking the genes encoding for these four enzymes showed a drastically reduced vegetative growth rate compared to the wild type organism. CAS4 is a secreted protein, CAS2 is mitochondrial, whereas CAS1 and CAS3 are cytosolic enzymes. The catalytic activity of CAS1-CAS3 for the CO2 hydration reaction showed that all of them possess a significant activity, with CAS3 being the most effective catalyst. The X-ray crystal structures of CAS1 and CAS2 were also obtained, showing that the two enzymes are tetramers (dimers of dimers) with an open active site in the case of CAS1 and a closed one for CAS2, similar to other plant/fungal/bacterial β-CAs studied so far. Detailed anion and sulfonamide inhibition studies were reported for all three β-Cas, which led to the identification of several effective inhibitors. Potential biotechnological applications of these enzymes for carbon (CO2) capture are also discussed.

In this study, we provide the first evidence that sonepiprazole, a dopamine D4 receptor antagonist, acts as a potent inhibitor of human carbonic anhydrases (hCAs). Sonepiprazole exhibited significant inhibitory activity across the panel of catalytically active hCAs, with the exception of hCA IV, and hCA III. The most potent inhibition was observed against the brain-associated isoform hCA VII, with a K I of 2.9 nM. Insights from X-ray crystallographic structures of the complexes with hCA I, hCA II, and hCA XII revealed that the sulfonamide group of sonepiprazole coordinates the zinc ion in the active site, a typical interaction for this class of inhibitors. Despite the presence of isoform-specific residues at the rim of the active site pocket, these variations seem not to significantly impact the compound overall inhibition potency. These findings highlight a dual functionality of sonepiprazole as both a D4 receptor antagonist and a carbonic anhydrase inhibitor.

The biochemical complexity of cancers offers the possibility to repurpose known drugs for new therapeutic applications. In this study, we exploited the potential anticancer effects of a series of compounds structurally organized with the tumor-associated Carbonic Anhydrase (CA; EC 4.2.1.1) IX/XII inhibiting warhead and the antifungal drug Ketoconazole (KTZ), which was recently considered in oncology for its ability to suppress steroidogenesis as well as disruption of the biogenesis of extracellular vesicles (EVs). The data here reported gives the first line of evidence that the in vitro antiproliferative effects of such compounds on a panel of tumoral cell lines are ascribed to integration of the anti-CA IX/XII effect with the EV pathway disruption and thus might represent a promising dual-mechanism therapeutic approach.

Chitosan-based nanoparticles were prepared using an eco-friendly chemical procedure that conjugates natural fatty acids to the backbone of chitosan. This consists of reacting two molecules in the absence of a solvent and using microwaves to promote the chemical transformation. Both conditions make the whole chemical process more eco-compatible in terms of reagents and energy consumption. The chemical structure and the self-association behavior of chitosan–fatty acid conjugates were characterized by FT-IR, NMR, and dynamic light scattering. The conjugates displayed an enhanced solubility and efficient self-assembly in aqueous solution. The antimicrobial activity of the resulting nanoparticles was evaluated against Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). The micelles significantly inhibited E. coli growth (35–60%), even at relatively low concentrations, whereas negligible activity was observed against B. subtilis. The antibacterial efficacy appears to arise primarily from the ability of the developed nanostructured conjugates to perturb bacterial membranes. These results support the potential of chitosan–fatty acid conjugates as sustainable nanomaterials for biomedical applications, particularly as eco-friendly antimicrobial agents. Future work will evaluate their activity against other Gram-positive pathogens and explore their use in drug delivery.

Carbonic anhydrases (CAs) play an essential role in the physiology and survival of protozoan parasites. This study explores the biological functions, molecular features, and therapeutic potential of protozoan CAs, focusing on the α, β, and η classes. Emphasis is placed on the structural and functional divergences between protozoan and mammalian CAs, underscoring the opportunities for selective drug targeting. Key protozoan pathogens, including Toxoplasma gondii, Trypanosoma cruzi, Leishmania spp., Trichomonas vaginalis, Entamoeba histolytica and Plasmodium falciparum, are examined with respect to their CA classes, which are evaluated for their roles in parasite metabolism and as candidates for therapeutic intervention. The potential of CA inhibitors as novel antiparasitic agents was critically assessed. By integrating established findings with emerging data, this analysis offers a comprehensive framework for the strategic exploitation of protozoan CAs for the development of next generation antiparasitic therapies.