Characterization of the MmpL4/MmpS4 efflux system in Mycobacterium bovis and Mycobacterium tuberculosis

Mycobacterium tuberculosis posee varias proteínas de la familia MmpL (Mycobacterial Membrane Protein Large) que pertenecen a la superfamilia de transportadores RND (Resistance Nodulation Division). El sistema de eflujo MmpL4/MmpS4 (Rv0450c y Rv0451c) puede contribuir a su virulencia y posiblemente al transporte de lípidos, además de al transporte de sideróforos y/o de otros compuestos. El objetivo principal de esta tesis ha sido la caracterización de este sistema de eflujo en M. bovis y M. tuberculosis. En primer lugar, hemos construido una cepa mutante de M. tuberculosis en la que se ha delecionado el gen rv0450c, una cepa mutante en la que se han delecionado los genes rv0450c y rv0451c, y cepas derivadas de las anteriores, en la que los genes wild-type complementan la deleción. Además, se ha generado una cepa que sobreexpresa los genes rv0450c y rv0451c. Con estas cepas se han realizado diversos ensayos encaminados a identificar la función de este sistema de eflujo. En cuanto a los experimentos in vitro, los ensayos de susceptibilidad a antibióticos indican que el sistema de eflujo MmpL4/MmpS4 en M. tuberculosis no estaría implicado en la resistencia intrínseca a fármacos, pero sí se ha puesto en evidencia su capacidad para transportar bromuro de etidio. Además, hemos determinado que la deleción del sistema de eflujo MmpL4/MmpS4 en M. tuberculosis no afecta la capacidad de la bacteria para crecer en medios deficientes en hierro, probablemente debido a que las bacterias todavía conservan el sistema de eflujo MmpL5/MmpS5 que compensa las deleciones realizadas. Sin embargo, la sobreexpresión del sistema de eflujo MmpL4/MmpS4 en M. tuberculosis provoca cambios en la morfología de las colonias y disminuye su velocidad de crecimiento, lo que sugiere que la estructura y/o composición de la membrana se ha visto alterada de forma importante. Las cepas de M. tuberculosis con alteraciones en la expresión del sistema de eflujo MmpL4/MmpS4, tanto por sobreexpresión como por deleción, se vieron afectadas en su capacidad para sobrevivir en el interior de macrófagos, presentando una menor citotoxicidad que la cepa parental H37Rv. Estos resultados indican que el sistema de eflujo MmpL4/MmpS4 es necesario para la virulencia de M. tuberculosis, y que su nivel de expresión es crítico. La deleción del gen mmpL4 en M. tuberculosis ha provocado la alteración de los niveles de expresión de otros genes que codifican transportadores de la misma familia, entre ellos mmpL5/mmpS5 y mmpL10. A nivel fenotípico, no pudo observarse ninguna variación significativa en la producción de los principales lípidos de M. tuberculosis causados por la pérdida del gen mmpL4 o de ambos genes mmpL4 y mmpS4, ni tampoco por la sobreexpresión de los genes mmpL4 y mmpS4. Con el análisis proteómico de las cepas con alteraciones en la expresión de estos genes, o de los mutantes complementados, se encontraron cambios relevantes en los niveles de proteínas relacionadas con el metabolismo de ácidos grasos. En M. bovis, se observó que la sobreexpresión del sistema de eflujo MmpL4/MmpS4 provocó alteraciones en la producción de PDIM, un lípido importante para la virulencia de las micobacterias

Structure-function analysis of MmpL7-mediated lipid transport in mycobacteria

Mycobacterial membrane protein Large (MmpL7) is a Resistance-Nodulation-Division (RND) family transporter required for the export of the virulence lipid, phthiocerol dimycocerosate (PDIM), in Mycobacterium tuberculosis. Using a null mutant of the related, vaccine strain Mycobacterium bovis BCG, we show that MmpL7 is also involved in the transport of the structurally related phenolic glycolipid (PGL), which is also produced by the hypervirulent M. tuberculosis strain HN878, but absent in M. tuberculosis H37Rv. Furthermore, we generated an in silico model of M. tuberculosis MmpL7 that revealed MmpL7 as a functional outlier within the MmpL-family, missing a canonical proton-relay signature sequence, suggesting that it employs a yet-unidentified mechanism for energy coupling for transport. In addition, our analysis demonstrates that the periplasmic porter domain 2 insert (PD2-insert), which doesn't share any recognisable homology, is highly alpha-helical in nature, suggesting an organisation similar to that seen in the hopanoid PD3/4 domains. Using the M. bovis BCG mmpL7 mutant for functional complementation with mutated alleles of mmpL7, we were able to identify residues present in the transmembrane domains TM4 and TM10, and the PD2 domain insert that play a crucial role in PDIM transport, and in certain cases, biosynthesis of PDIM

The mycobacterial desaturase DesA2 is associated with mycolic acid biosynthesis

Mycolic acids are critical for the survival and virulence of Mycobacterium tuberculosis, the causative agent of tuberculosis. Double bond formation in the merochain of mycolic acids remains poorly understood, though we have previously shown desA1, encoding an aerobic desaturase, is involved in mycolic acid desaturation. Here we show that a second desaturase encoded by desA2 is also involved in mycolate biosynthesis. DesA2 is essential for growth of the fast-growing Mycobacterium smegmatis in laboratory media. Conditional depletion of DesA2 led to a decrease in mycolic acid biosynthesis and loss of mycobacterial viability. Additionally, DesA2-depleted cells also accumulated fatty acids of chain lengths C19-C24. The complete loss of mycolate biosynthesis following DesA2 depletion, and the absence of any monoenoic derivatives (found to accumulate on depletion of DesA1) suggests an early role for DesA2 in the mycolic acid biosynthesis machinery, highlighting its potential as a drug target

Prevalence of Toxoplasma gondii in Endangered Wild Felines (Felis silvestris and Lynx pardinus) in Spain

The wildcat (Felis silvestris) and the Iberian lynx (Lynx pardinus) are important species in Spain, considered as near-threatened and endangered, respectively. Both can be infected by Toxoplasma gondii, a parasite that can cause morbidity and mortality in transplacentally-infected or immunocompromised mammals. The data on the prevalence of this parasite in wild populations of these species in Spain are outdated. The objective of this study was to update information and evaluate the role of these felines in parasite epidemiology and the potential impact of the parasite on their conservation. Blood and fecal samples were collected from captured animals, as well as the tongue, diaphragm, and spleen, from animals killed in road accidents in central Spain. An indirect fluorescent antibody test (IFAT) was used to detect parasite antibodies in serum, microscopy and molecular analysis were used to detect oocysts in feces, and molecular analysis was used to determine the existence of tissue cysts. Seroprevalence was 85% in wildcats and 45% in lynx, and parasite DNA was detected in the feces of one wildcat and in tissue samples from 10 wildcats and 11 Iberian lynxes. These results highlight the epidemiological importance and high risk of T. gondii infection in animals and humans in the studied areas. Considering feline susceptibility to infection, monitoring programs are needed to assess the health status of wild felines.This research was partially funded by Alfonso X el Sabio Foundation, project number 1.010.119 and Health Research Fund, Instituto de Salud Carlos III, Ministry of Science and Innovation, project FIS AESI PI21CIII/00031.S

Evaluation of critical parameters in the hollow-fibre system for tuberculosis: A case study of moxifloxacin

AimsThe hollow‐fibre system for tuberculosis (HFS‐TB) is a preclinical model qualified by the European Medicines Agency to underpin the anti‐TB drug development process. It can mimic in vivo pharmacokinetic (PK)–pharmacodynamic (PD) attributes of selected antimicrobials, which could feed into in silico models to inform the design of clinical trials. However, historical data and published protocols are insufficient and omit key information to allow experiments to be reproducible. Therefore, in this work, we aim to optimize and standardize various HFS‐TB operational procedures.MethodsFirst, we characterized bacterial growth dynamics with different types of hollow‐fibre cartridges, Mycobacterium tuberculosis strains and media. Second, we mimicked a moxifloxacin PK profile within hollow‐fibre cartridges, in order to check drug–fibres compatibility. Lastly, we mimicked the moxifloxacin total plasma PK profile in human after once daily oral dose of 400 mg to assess PK–PD after different sampling methods, strains, cartridge size and bacterial adaptation periods before drug infusion into the system.ResultsWe found that final bacterial load inside the HFS‐TB was contingent on the studied variables. Besides, we demonstrated that drug–fibres compatibility tests are critical preliminary HFS‐TB assays, which need to be properly reported. Lastly, we uncovered that the sampling method and bacterial adaptation period before drug infusion significantly impact actual experimental conclusions.ConclusionOur data contribute to the necessary standardization of HFS‐TB experiments, draw attention to multiple aspects of this preclinical model that should be considered when reporting novel results and warn about critical parameters in the HFS‐TB currently overlooked

Path-seq identifies an essential mycolate remodeling program for mycobacterial host adaptation

The success of Mycobacterium tuberculosis (MTB) stems from its ability to remain hidden from the immune system within macrophages. Here, we report a new technology (Path-seq) to sequence miniscule amounts of MTB transcripts within up to million-fold excess host RNA Using Path-seq and regulatory network analyses, we have discovered a novel transcriptional program for in vivo mycobacterial cell wall remodeling when the pathogen infects alveolar macrophages in mice. We have discovered that MadR transcriptionally modulates two mycolic acid desaturases desA1/desA2 to initially promote cell wall remodeling upon in vitro macrophage infection and, subsequently, reduces mycolate biosynthesis upon entering dormancy. We demonstrate that disrupting MadR program is lethal to diverse mycobacteria making this evolutionarily conserved regulator a prime antitubercular target for both early and late stages of infection

MadR mediates acyl CoA-dependent regulation of mycolic acid desaturation in mycobacteria.

Mycobacterium tuberculosis has a lipid-rich cell envelope that is remodeled throughout infection to enable adaptation within the host. Few transcriptional regulators have been characterized that coordinate synthesis of mycolic acids, the major cell wall lipids of mycobacteria. Here, we show that the mycolic acid desaturase regulator (MadR), a transcriptional repressor of the mycolate desaturase genes desA1 and desA2, controls mycolic acid desaturation and biosynthesis in response to cell envelope stress. A madR-null mutant of M. smegmatis exhibited traits of an impaired cell wall with an altered outer mycomembrane, accumulation of a desaturated α-mycolate, susceptibility to antimycobacterials, and cell surface disruption. Transcriptomic profiling showed that enriched lipid metabolism genes that were significantly down-regulated upon madR deletion included acyl-coenzyme A (aceyl-CoA) dehydrogenases, implicating it in the indirect control of β-oxidation pathways. Electromobility shift assays and binding affinities suggest a unique acyl-CoA pool-sensing mechanism, whereby MadR is able to bind a range of acyl-CoAs, including those with unsaturated as well as saturated acyl chains. MadR repression of desA1/desA2 is relieved upon binding of saturated acyl-CoAs of chain length C16 to C24, while no impact is observed upon binding of shorter chain and unsaturated acyl-CoAs. We propose this mechanism of regulation as distinct to other mycolic acid and fatty acid synthesis regulators and place MadR as the key regulatory checkpoint that coordinates mycolic acid remodeling during infection in response to host-derived cell surface perturbation