The first activation study of the β-carbonic anhydrases from the pathogenic bacteria Brucella suis and Francisella tularensis with amines and amino acids

Carbonic anhydrases (CAs) are essential metalloenzymes in nature, catalyzing the carbon dioxide reversible hydration into bicarbonate and proton. In humans, breathing and many other critical physiological processes depend on this enzymatic activity. The CA superfamily function and inhibition in pathogenic bacteria has recently been the object of significant advances, being demonstrated to affect microbial survival/virulence. Targeting bacterial CAs may thus be a valid alternative to expand the pharmacological arsenal against the emergence of widespread antibiotic resistance. Here, we report an extensive study on the inhibition profile of the recently discovered ι-CA class present in some bacteria, including Burkholderia territorii, namely BteCAι, using substituted benzene-sulfonamides and clinically licensed sulfonamide-, sulfamate- and sulfamide-type drugs. The BteCAι inhibition profile showed: (i) several benzene-sulfonamides with an inhibition constant lower than 100 nM; (ii) a different behavior with respect to other α, β and γ-CAs; (iii) clinically used drugs having a micromolar affinity. This prototype study contributes to the initial recognition of compounds which efficiently and selectively inhibit a bacterial member of the ι-CA class, for which such a selective inhibition with respect to other protein isoforms present in the host is highly desired and may contribute to the development of novel antimicrobials.

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Interplay between Two RND Systems Mediating Antimicrobial Resistance in Brucella suis

Journal of Bacteriology ◽

10.1128/jb.01198-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2530-2540 ◽

Cited By ~ 16

Author(s):

Fernando A. Martin ◽

Diana M. Posadas ◽

Mariela C. Carrica ◽

Silvio L. Cravero ◽

David O'Callaghan ◽

...

Keyword(s):

Crystal Violet ◽

Pathogenic Bacteria ◽

Regulatory Protein ◽

Efflux Pumps ◽

Reading Frame ◽

Membrane Fusion Protein ◽

Physiological Processes ◽

Defective Mutant ◽

Brucella Suis ◽

Rnd Efflux Pumps

ABSTRACT The RND-type efflux pumps are responsible for the multidrug resistance phenotype observed in many clinically relevant species. Also, RND pumps have been implicated in physiological processes, with roles in the virulence mechanisms of several pathogenic bacteria. We have previously shown that the BepC outer membrane factor of Brucella suis is involved in the efflux of diverse drugs, probably as part of a tripartite complex with an inner membrane translocase. In the present work, we characterize two membrane fusion protein-RND translocases of B. suis encoded by the bepDE and bepFG loci. MIC assays showed that the B. suis ΔbepE mutant was more sensitive to deoxycholate (DOC), ethidium bromide, and crystal violet. Furthermore, multicopy bepDE increased resistance to DOC and crystal violet and also to other drugs, including ampicillin, norfloxacin, ciprofloxacin, tetracycline, and doxycycline. In contrast to the ΔbepE mutant, the resistance profile of B. suis remained unaltered when the other RND gene (bepG) was deleted. However, the ΔbepE ΔbepG double mutant showed a more severe phenotype than the ΔbepE mutant, indicating that BepFG also contributes to drug resistance. An open reading frame (bepR) coding for a putative regulatory protein of the TetR family was found upstream of the bepDE locus. BepR strongly repressed the activity of the bepDE promoter, but DOC released the repression mediated by BepR. A clear induction of the bepFG promoter activity was observed only in the BepDE-defective mutant, indicating a regulatory interplay between the two RND efflux pumps. Although only the BepFG-defective mutant showed a moderate attenuation in model cells, the activities of both bepDE and bepFG promoters were induced in the intracellular environment of HeLa cells. Our results show that B. suis harbors two functional RND efflux pumps that may contribute to virulence.

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Carbonic Anhydrases: New Perspectives on Protein Functional Role and Inhibition in Helicobacter pylori

Frontiers in Microbiology ◽

10.3389/fmicb.2021.629163 ◽

2021 ◽

Vol 12 ◽

Author(s):

Cristina Campestre ◽

Viviana De Luca ◽

Simone Carradori ◽

Rossella Grande ◽

Vincenzo Carginale ◽

...

Keyword(s):

Helicobacter Pylori ◽

Pathogenic Bacteria ◽

Acid Tolerance ◽

Membrane Vesicles ◽

Research Field ◽

Outer Membrane Vesicles ◽

Bacterial Toxin ◽

Carbonic Anhydrases ◽

Ph Homeostasis ◽

H Pylori

Our understanding of the function of bacterial carbonic anhydrases (CAs, EC 4.2.1.1) has increased significantly in the last years. CAs are metalloenzymes able to modulate CO2, HCO3– and H+ concentration through their crucial role in catalysis of reversible CO2 hydration (CO2 + H2O ⇄ HCO3– + H+). In all living organisms, CA activity is linked to physiological processes, such as those related to the transport and supply of CO2 or HCO3–, pH homeostasis, secretion of electrolytes, biosynthetic processes and photosynthesis. These important processes cannot be ensured by the very low rate of the non-catalyzed reaction of CO2 hydration. It has been recently shown that CAs are important biomolecules for many bacteria involved in human infections, such as Vibrio cholerae, Brucella suis, Salmonella enterica, Pseudomonas aeruginosa, and Helicobacter pylori. In these species, CA activity promotes microorganism growth and adaptation in the host, or modulates bacterial toxin production and virulence. In this review, recent literature in this research field and some of the above-mentioned issues are discussed, namely: (i) the implication of CAs from bacterial pathogens in determining the microorganism growth and virulence; (ii) the druggability of these enzymes using classical CA inhibitors (CAIs) of the sulfonamide-type as examples; (iii) the role played by Helicobacter pylori CAs in the acid tolerance/adaptation of the microbe within the human abdomen; (iv) the role of CAs played in the outer membrane vesicles spawned by H. pylori in its planktonic and biofilm phenotypes; (v) the possibility of using H. pylori CAIs in combination with probiotic strains as a novel anti-ulcer treatment approach. The latter approach may represent an innovative and successful strategy to fight gastric infections in the era of increasing resistance of pathogenic bacteria to classical antibiotics.

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Activation of β- and γ-carbonic anhydrases from pathogenic bacteria with tripeptides

Journal of Enzyme Inhibition and Medicinal Chemistry ◽

10.1080/14756366.2018.1468530 ◽

2018 ◽

Vol 33 (1) ◽

pp. 945-950 ◽

Cited By ~ 14

Author(s):

Azzurra Stefanucci ◽

Andrea Angeli ◽

Marilisa Pia Dimmito ◽

Grazia Luisi ◽

Sonia Del Prete ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Carbonic Anhydrases

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Inhibition of β-carbonic anhydrases from Brucella suis with C-cinnamoyl glycosides incorporating the phenol moiety

Journal of Enzyme Inhibition and Medicinal Chemistry ◽

10.3109/14756366.2014.986120 ◽

2015 ◽

Vol 30 (6) ◽

pp. 1017-1020 ◽

Cited By ~ 9

Author(s):

Leonardo E. Riafrecha ◽

Daniela Vullo ◽

Safia Ouahrani-Bettache ◽

Stephan Köhler ◽

Pascal Dumy ◽

...

Keyword(s):

Carbonic Anhydrases ◽

Brucella Suis ◽

Phenol Moiety

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European brown hare as a potential source of zoonotic agents

Veterinární Medicína ◽

10.17221/2050-vetmed ◽

2008 ◽

Vol 52 (No. 10) ◽

pp. 451-456 ◽

Cited By ~ 11

Author(s):

F. Treml ◽

J. Pikula ◽

H. Bandouchova ◽

J. Horakova

Keyword(s):

Francisella Tularensis ◽

Environmental Changes ◽

The State ◽

The Other ◽

Brown Hare ◽

State Of Health ◽

European Brown Hare ◽

Potential Source ◽

Dramatic Decline ◽

Brucella Suis

There has recently been a growing interest in checking the state of health of European brown hares in hunting grounds because they are a susceptible bio-indicator of environmental changes and because of the dramatic decline in populations across Europe. A total of 1 051 (384, 302, and 365, respectively) blood sera were collected from hares during autumn hunting events and examined for tularaemia, brucellosis and leptospirosis in 2004, 2005 and 2006. Antibodies were found in 6.5, 1.6 and 7.5% of cases, respectively. Antibodies against Francisella tularensis, Brucella suis and leptospires were found in 7.9, 1.7 and 7.7% of females and 4.5, 1.4 and 7.9% of males, respectively. Higher seroprevalence of tularaemia was found in females (P = 0.05). Brucellosis was more prevalent in adult animals compared to subadult ones (P = 0.05). Only the L. grippotyphosa serotype was found and the titres mostly ranged from 100 to 400 (89.9%). Higher titres from 800 to 3 200 were found in the remaining 10.1% of the examined European brown hare sera. Antibodies against F. tularensis as well as B. suis were most frequent in low titres of 10 to 80. Higher titres were only exceptional. Confirming that hares are susceptible to various zoonotic agents, it is necessary to be careful when handling the animals killed. On the other hand, blood sera from hares may be used to survey the occurrence of natural nidi of zoonoses in hunting grounds.

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Activation studies of the β-carbonic anhydrases from Escherichia coli with amino acids and amines

Journal of Enzyme Inhibition and Medicinal Chemistry ◽

10.1080/14756366.2020.1781845 ◽

2020 ◽

Vol 35 (1) ◽

pp. 1379-1386

Author(s):

Alessio Nocentini ◽

Sonia Del Prete ◽

Margaret D. Mastrolorenzo ◽

William A. Donald ◽

Clemente Capasso ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Carbonic Anhydrases

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Carbonic anhydrase activators: Activation of the archaeal β-class (Cab) and γ-class (Cam) carbonic anhydrases with amino acids and amines

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2008.10.005 ◽

2008 ◽

Vol 18 (23) ◽

pp. 6194-6198 ◽

Cited By ~ 26

Author(s):

Alessio Innocenti ◽

Sabrina A. Zimmerman ◽

Andrea Scozzafava ◽

James G. Ferry ◽

Claudiu T. Supuran

Keyword(s):

Amino Acids ◽

Carbonic Anhydrase ◽

Carbonic Anhydrases

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The Human CXC Chemokine Granulocyte Chemotactic Protein 2 (GCP-2)/CXCL6 Possesses Membrane-Disrupting Properties and Is Antibacterial

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00028-08 ◽

2008 ◽

Vol 52 (7) ◽

pp. 2599-2607 ◽

Cited By ~ 40

Author(s):

Helena M. Linge ◽

Mattias Collin ◽

Pontus Nordenfelt ◽

Matthias Mörgelin ◽

Martin Malmsten ◽

...

Keyword(s):

Amino Acids ◽

Antibacterial Activity ◽

Pathogenic Bacteria ◽

Membrane Disruption ◽

Minimal Bactericidal Concentration ◽

Bacterial Surface ◽

Bacterial Membranes ◽

Chemotactic Protein ◽

Cxc Chemokine ◽

Α Helix

ABSTRACT Granulocyte chemotactic protein 2 (GCP-2)/CXCL6 is a CXC chemokine expressed by macrophages and epithelial and mesenchymal cells during inflammation. Through binding and activation of its receptors (CXCR1 and CXCR2), it exerts neutrophil-activating and angiogenic activities. Here we show that GCP-2/CXCL6 itself is antibacterial. Antibacterial activity against gram-positive and gram-negative pathogenic bacteria of relevance to mucosal infections was seen at submicromolar concentrations (minimal bactericidal concentration at which 50% of strains tested were killed, 0.063 ± 0.01 to 0.37 ± 0.03 μM). In killed bacteria, GCP-2/CXCL6 associated with bacterial surfaces, which showed membrane disruption and leakage. A structural prediction indicated the presence of three antiparallel NH2-terminal β-sheets and a short amphipathic COOH-terminal α-helix; the latter feature is typical of antimicrobial peptides. However, when the synthetic derivatives corresponding to the NH2-terminal (50 amino acids) and COOH-terminal (19 amino acids, corresponding to the putative α-helix) regions were compared, higher antibacterial activity was observed for the NH2-terminus-derived peptide, indicating that the holopeptide is necessary for full antibacterial activity. An artificial model of bacterial membranes confirmed these findings. The helical content of GCP-2/CXCL6 in the presence or absence of lipopolysaccharide or negatively charged membranes was studied by circular dichroism. As with many antibacterial peptides, membrane disruption by GCP-2/CXCL6 was dose-dependently reduced in the presence of NaCl, which, we here demonstrate, inhibited the binding of the peptide to the bacterial surface. Compared with CXC chemokines ENA-78/CXCL5 and NAP-2/CXCL7, GCP-2/CXCL6 showed a 90-fold-higher antibacterial activity. Taken together, GCP/CXCL6, in addition to its chemotactic and angiogenic properties, is likely to contribute to direct antibacterial activity during localized infection.

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