Specific targeting to the Mycobacterium tuberculosis P-type ATPase Membrane Transporter, CtpF, of antituberculous compounds obtained by structure-based design.

Background: The resurgence of Mycobacterium tuberculosis (Mtb) strains that resist anti-tuberculosis (anti-TB) drugs used currently stresses the search for more effective low-toxicity drugs against new targets. Due to their role in ion homeostasis and virulence, Mtb plasma membrane P-type ATPases are interesting anti-TB targets, in particular, the Ca2+ transporting P2-type ATPase CtpF which is involved in oxidative stress response and persistence. Methods: In this study, the effect on the transcription level of the ctpF gene and other Mtb P2-type ATPases of two anti-Mtb hits was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Both anti-Mtb hits ZINC14541509 and ZINC63908257 had been previously identified using pharmacophore-based virtual screening and MM-GBSA binding free energy. In addition, the bacterial activity of both compounds on Mycobacterium bovis was evaluated to see whether or not there is an effect on other mycobacteria of the Mtb complex. Results: qRT-PCR experiments showed that the ctpF transcription level was significantly higher in the presence of both compounds, especially ZINC14541509, strongly suggesting that CtpF may be a specific target of the selected compound. Conclusions: ZINC14541509 should be considered as an alternative for the structural-based design of novel anti-TB drugs.

The ctpF Gene Encoding a Calcium P-Type ATPase of the Plasma Membrane Contributes to Full Virulence of Mycobacterium tuberculosis.

Identification of alternative attenuation targets of Mycobacterium tuberculosis (Mtb) is pivotal for designing new candidates for live attenuated anti-tuberculosis (TB) vaccines. In this context, the CtpF P-type ATPase of Mtb is an interesting target; specifically, this plasma membrane enzyme is involved in calcium transporting and response to oxidative stress. We found that a mutant of MtbH37Rv lacking ctpF expression (MtbΔctpF) displayed impaired proliferation in mouse alveolar macrophages (MH-S) during in vitro infection. Further, the levels of tumor necrosis factor and interferon-gamma in MH-S cells infected with MtbΔctpF were similar to those of cells infected with the parental strain, suggesting preservation of the immunogenic capacity. In addition, BALB/c mice infected with Mtb∆ctpF showed median survival times of 84 days, while mice infected with MtbH37Rv survived 59 days, suggesting reduced virulence of the mutant strain. Interestingly, the expression levels of ctpF in a mouse model of latent TB were significantly higher than in a mouse model of progressive TB, indicating that ctpF is involved in Mtb persistence in the dormancy state. Finally, the possibility of complementary mechanisms that counteract deficiencies in Ca2+ transport mediated by P-type ATPases is suggested. Altogether, our results demonstrate that CtpF could be a potential target for Mtb attenuation.

Interactive web-based tool for evaluating the spread of bovine tuberculosis and brucellosis in Colombia / Herramienta interactiva basada en la web para evaluar la propagación de la tuberculosis bovina y la brucelosis en Colombia / Ferramenta interativa baseada na web para avaliar a disseminação da tuberculose e brucelose bovina na Colômbia

Abstract Background: Bovine tuberculosis (BTB) and brucellosis are associated with devastating losses in the livestock sector in Colombia and even in developed countries. Real-time disease surveillance is a key strategy to control and eradicate infectious disease outbreaks. Objective: To design an epidemiological tool for monitoring BTB and brucellosis in Colombia. Methods: An interactive platform for disease mapping of BTB and brucellosis during an observation period between years 2004 and 2019 was designed. Results: Our analysis showed that the provinces of Cundinamarca and Valle del Cauca are regions affected by BTB and brucellosis epidemics, respectively (p<0.001). Furthermore, increased case detection of BTB was reported in 2012 and brucellosis in 2019 (p<0.001). Conclusions: This epidemiological platform allows tracking BTB and tuberculosis hotspots, identifying trends over time, and provides useful information to animal health authorities for designing new strategies in control programs.

Resumen Antecedentes: La tuberculosis bovina (TBB) y la brucelosis están asociadas con problemas persistentes en la ganadería Colombiana e incluso en los países desarrollados. La vigilancia de enfermedades en tiempo real es una estrategia clave para controlar y erradicar brotes de enfermedades infecciosas. Objetivo: Diseñar una herramienta epidemiológica para monitorear TBB y brucelosis en Colombia. Métodos: Se diseñó un panel de control interactivo para el mapeo de ambas enfermedades durante el periodo de observación entre los años 2004 y 2019. Resultados: El análisis de la herramienta mostró que las Provincias de Cundinamarca y Valle del Cauca han sido áreas epidémicas para TBB y brucelosis, respectivamente (p<0,001). Además, se encontró un aumento de la detección de casos de TBB en 2012 y de brucelosis durante 2019 (p<0,001). Conclusiones: Este panel epidemiológico permite el seguimiento de puntos críticos de TBB y tuberculosis, identificando sus tendencias a lo largo del tiempo, y proporciona información útil para las autoridades de sanidad animal que diseñan nuevas estrategias para los programas de control.

Resumo Antecedentes: A tuberculose bovina (TBB) e a brucelose estão associadas a problemas persistentes no campo da pecuária na Colômbia e até em países desenvolvidos. Portanto, a vigilância de doenças em tempo real é uma estratégia essencial para controlar e erradicar surtos de doenças infecciosas. Objetivo: Projetar uma ferramenta epidemiológica para monitorar a TB e a brucelose na Colômbia. Métodos: Um painel de controle interativo foi projetado para o mapeamento de ambas as doenças entre 2004 e 2019 como período de observação. Resultados: A análise da ferramenta mostrou que as Províncias de Cundinamarca e Valle del Cauca foram áreas epidêmicas para TBB e brucelose, respectivamente (p<0,001). Além disso, foi encontrado um aumento na detecção de casos em 2012 para TBB e brucelose durante 2019 (p<0,001). Conclusões: Esse painel epidemiológico poderia permitir o monitoramento de pontos críticos dessas doenças, identificando tendências ao longo do tempo, fornecendo informações úteis para as autoridades de saúde animal que elaboram novas estratégias para programas de controle.

Phenolic profile, in vitro antimicrobial activity and antioxidant capacity of Vaccinium meridionale swartz pomace.

Berries of Vaccinium meridionale Swartz contain a variety of phytochemicals, which are believed to account for their bioactive properties. The potential of Vaccinium meridionale Swartz pomace as a source of bioactive compounds was investigated. The dietary fiber (DF) content was assessed by the AOAC method, phenolic compounds were characterized and quantified via HPLC-PDA and UPLC-QTOF-MS. The in vitro antibacterial activity was tested against Gram-positive and Gram-negative bacteria. The antioxidant properties were assessed by the ORAC and the ABTS assays. The DF content was 52.4 ± 3.7%, phenolic compounds comprised anthocyanins (ACNs) (747.6 ± 167.5 mg cyanidin-3-glucoside/100 g FW), hydroxycinammic acids (HCAs) (229.2 ± 68.4 mg chlorogenic acid equivalents/100 g FW), flavonols (335.0 ± 139.5 rutin equivalents/100 g FW), and procyanidins (PACs) (140.9 ± 33.3 mg cocoa procyanidin equivalents/100 g FW). Staphylococcus aureus was more sensitive than E. coli. The ORAC value was 250.0 ± 32.0 µmol TE/g fresh weight (FW). Results suggest that the residue from V. meridionale S. can be utilized to obtain valuable nutraceuticals for the development of functional foods.

CtpB is a plasma membrane copper (I) transporting P-type ATPase of Mycobacterium tuberculosis.

BACKGROUND: The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS: In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma membrane showed features of high affinity/slow turnover ATPases, with enzymatic parameters KM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS: The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.

Identification of Mycobacterium tuberculosis CtpF as a target for designing new antituberculous compounds.

The emergence of tuberculosis (TB) produced by multi-drug resistance (MDR) and extensively-drug resistance (XDR) Mycobacterium tuberculosis (Mtb), encourages the development of new antituberculous compounds, as well as the identification of novel drug targets. In this regard, plasma membrane P-type ATPases are interesting targets because they play a crucial role in ion homeostasis and mycobacterial survival. We focused on Mtb CtpF, a calcium P-type ATPase that responds to a broad number of intraphagosomal conditions, as a novel target. In this study, we evaluated the capacity of cyclopiazonic acid (CPA), a well-known inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), to inhibit the ATPase activity of CtpF and the Mtb growth demonstrating that CtpF is a druggable target. A homology modeling of CtpF was generated for molecular docking studies of CtpF with CPA and key pharmacophoric features were identified, which were used to perform a pharmacophore-based virtual screening of the ZINC database, and to identify CtpF inhibitor candidates. Molecular docking-based virtual screening and MM-BGSA calculations of candidates allowed identifying six compounds with the best binding energies. The compounds displayed in vitro minimum inhibitory concentrations (MIC) ranging from 50 to 100 µg/mL, growth inhibitions from 29.5 to 64.0% on Mtb, and inhibitions of Ca2+-dependent ATPase activity in Mtb membrane vesicles (IC50) ranging from 4.1 to 35.8 µM. The compound ZINC63908257 was the best candidate by displaying a MIC of 50 µg/mL and a Ca2+ P-type ATPase inhibition of 45% with IC50 = 4.4 µM. Overall, the results indicate that CtpF is a druggable target for designing new antituberculous compounds.

CtpB is a plasma membrane copper (I) transporting P-type ATPase of Mycobacterium tuberculosis

BACKGROUND: The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS: In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma mem-brane showed features of high affinity/slow turnover ATPases, with enzymatic parametersKM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS: The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.

The P-type ATPase CtpG preferentially transports Cd2+ across the Mycobacterium tuberculosis plasma membrane.

P1B-type ATPases are involved in heavy metal transport across the plasma membrane. Some Mycobacterium tuberculosis P-type ATPases are induced during infection, suggesting that this type of transporter could play a critical role in mycobacterial survival. To date, the ion specificity of M. tuberculosis heavy metal-transporting P1B-ATPases is not well understood. In this work, we observed that, although divalent heavy metal cations such as Cu2+, Co2+, Ni2+, Zn2+ Cd2+ and Pb2+ stimulate the ATPase activity of the putative P1B-type ATPase CtpG in the plasma membrane, whole cells of M. smegmatis expressing CtpG only tolerate high levels of Cd2+ and Cu2+. As indicator of the catalytic constant, Michaelis-Menten kinetics showed that CtpG embedded in the mycobacterial cell membrane has a V max/K m ratio 7.4-fold higher for Cd2+ than for Cu2+ ions. Thus, although CtpG can accept different substrates in vitro, this P-type ATPase transports Cd2+ more efficiently than other heavy metal cations across the mycobacterial plasma membrane.

In silico approaches and chemical space of anti-P-type ATPase compounds for discovering new antituberculous drugs.

Tuberculosis (TB) is one of the most important public health problems around the world. The emergence of multi-drug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis strains has driven the finding of alternative anti-TB targets. In this context, P-type ATPases are interesting therapeutic targets due to their key role in ion homeostasis across the plasma membrane and the mycobacterial survival inside macrophages. In this review, in silico and experimental strategies used for the rational design of new anti-TB drugs are presented; in addition, the chemical space distribution based on the structure and molecular properties of compounds with anti-TB and anti-P-type ATPase activity is discussed. The chemical space distribution compared to public compound libraries demonstrates that natural product libraries are a source of novel chemical scaffolds with potential anti-P-type ATPase activity. Furthermore, compounds that experimentally display anti-P-type ATPase activity belong to a chemical space of molecular properties comparable to that occupied by those approved for oral use, suggesting that these kinds of molecules have a good pharmacokinetic profile (drug-like) for evaluation as potential anti-TB drugs.

Pma1 is an alkali/alkaline earth metal cation ATPase that preferentially transports Na(+) and K(+) across the Mycobacterium smegmatis plasma membrane.

Mycobacterium smegmatis Pma1 is the orthologue of M. tuberculosis P-type ATPase cation transporter CtpF, which is activated under stress conditions, such as hypoxia, starvation and response to antituberculous and toxic substances. The function of Pma1 in the mycobacterial processes across the plasma membrane has not been characterised. In this work, bioinformatic analyses revealed that Pma1 likely contains potential sites for, Na(+), K(+) and Ca(2+) binding and transport. Accordingly, RT-qPCR experiments showed that M. smegmatis pma1 transcription is stimulated by sub-lethal doses of Na(+), K(+) and Ca(2+); in addition, the ATPase activity of plasma membrane vesicles in recombinant Pma1-expressing M. smegmatis cells is stimulated by treatment with these cations. In contrast, M. smegmatis cells homologously expressing Pma1 displayed tolerance to high doses of Na(+) and K(+) but not to Ca(2+) ions. Consistently, the recombinant protein Km embedded in plasma membrane demonstrated that Ca(2+) has more affinity for Pma1 than Na(+) and K(+) ions; furthermore, the estimation of Vmax/Km suggests that Na(+) and K(+) ions are more efficiently translocated than Ca(2+). Thus, these results strongly suggest that Pma1 is a promiscuous alkali/alkaline earth cation ATPase that preferentially transports Na(+) and/or K(+) across the mycobacterial plasma membrane.

CtpA, a putative Mycobacterium tuberculosis P-type ATPase, is stimulated by copper (I) in the mycobacterial plasma membrane.

The transport of heavy-metal ions across the plasma membrane is essential for mycobacterial intracellular survival; in this context, P-type ATPases are pivotal for maintenance of ionic gradients and the plasma membrane homeostasis of mycobacteria. To date, the copper ion transport that is mediated by P-type ATPases in mycobacteria is poorly understood. In this work, the ion-specific activation of CtpA, a putative plasma membrane Mycobacterium tuberculosis P-type ATPase, with different heavy-metal cations was assessed. Mycobacterium smegmatis mc(2)155 cells heterologously expressing the M. tuberculosis ctpA gene displayed an increased tolerance to toxic levels of the Cu(2+) ion (4 mM) compared to control cells, suggesting that CtpA is possibly involved in the copper detoxification of mycobacterial cells. In contrast, the tolerance of M. smegmatis recombinant cells against other heavy-metal divalent cations, such as Co(2+), Mn(2+), Ni(2+) and Zn(2+), was not detected. In addition, the ATPase activity of plasma membrane vesicles that were obtained from M. smegmatis cells expressing CtpA was stimulated by Cu(+) (4.9 nmol of Pi released/mg of protein.min) but not by Cu(2+) ions; therefore, Cu(2+) reduction to Cu(+) inside mycobacterial cells is suggested. Finally, the plasma membrane vesicles of M. smegmatis that were enriched with CtpA exhibited an optimal activity at 37 °C and pH 7.9; the apparent kinetic parameters of the enzyme were a K(1/2) of 4.68 × 10(-2) µM for Cu(+), a Vmax of 10.3 U/mg of protein, and an h value of 1.91.

The DosR dormancy regulator of Mycobacterium tuberculosis stimulates the Na(+)/K (+) and Ca (2+) ATPase activities in plasma membrane vesicles of mycobacteria.

The latency global regulator DosR regulon of Mycobacterium tuberculosis, which is stimulated by hypoxia, comprises approximately fifty genes including ctpF (Rv1997), which encodes a putative alkali/alkaline earth ion transporter of the plasma membrane. In this work, the influence of hypoxia and M. tuberculosis DosR on the ATPase activity of mycobacterial plasma membrane was assessed. We performed bioinformatic analyses which indicated that the pma1 gene product is the M. smegmatis ortholog of the M. tuberculosis cation transporter CtpF. In addition, a possible Na(+), K(+) and/or Ca(2+) pumping mediated by Pma1 was also predicted. Enzymatic analyses indicated that the basal ATPase activity of plasma membrane vesicles from M. smegmatis cells cultured under hypoxia and over-expressing DosR, decreased 30 and 40 % respectively in comparison to oxygenated cells. In contrast, the specific Na(+)/K(+) and Ca(2+) ATPase activities of the plasma membrane increased 2.8- and 3.5-fold, respectively, under hypoxia, similar to that observed for cells over-expressing the DosR regulator. In agreement, RT-qPCR experiments demonstrated that the transcription level of the pma1 gene increased under hypoxia at levels similar to that of M. smegmatis cells over-expressing the M. tuberculosis DosR regulator. The entire findings suggest that hypoxia stimulates Na(+)/K(+) and Ca(2+) ATPase activities in the mycobacterial plasma membrane, and this is possibly mediated by the dormancy regulator DosR.