RESUMEN

Purpose: Tuberculosis (TB) remains a major health threat worldwide, and the spread of drug-resistant (DR) TB impedes the reduction of the global disease burden. Ebselen (EbSe) targets bacterial thioredoxin reductase (bTrxR) and causes an imbalance in the redox status of bacteria. Previous work has shown that the synergistic action of bTrxR and sensitization to common antibiotics by EbSe is a promising strategy for the treatment of DR pathogens. Thus, we aimed to evaluate whether EbSe could enhance anti-TB drugs against Mycobacterium marinum (M. marinum) which is genetically related to Mycobacterium tuberculosis (Mtb) and resistant to many antituberculosis drugs. Methods: Minimum inhibitory concentrations (MIC) of isoniazid (INH), rifampicin (RFP), and streptomycin (SM) against M. marinum were determined by microdilution. The Bliss Independence Model was used to determine the adjuvant effects of EbSe over the anti-TB drugs. Thioredoxin reductase activity was measured using the DTNB assay, and its effects on bacterial redox homeostasis were verified by the elevation of intracellular ROS levels and intracellular GSH levels. The adjuvant efficacy of EbSe as an anti-TB drug was further evaluated in a mouse model of M. marinum infection. Cytotoxicity was observed in the macrophage cells Raw264.7 and mice model. Results: The results reveal that EbSe acts as an antibiotic adjuvant over SM on M. marinum. EbSe + SM disrupted the intracellular redox microenvironment of M. marinum by inhibiting bTrxR activity, which could rescue mice from the high bacterial load, and accelerated recovery from tail injury with low mammalian toxicity. Conclusion: The above studies suggest that EbSe significantly enhanced the anti-Mtb effect of SM, and its synergistic combination showed low mammalian toxicity in vitro and in vivo. Further efforts are required to study the underlying mechanisms of EbSe as an antibiotic adjuvant in combination with anti-TB drug MS.

Asunto(s)

Homeostasis , Isoindoles , Pruebas de Sensibilidad Microbiana , Compuestos de Organoselenio , Oxidación-Reducción , Estreptomicina , Compuestos de Organoselenio/farmacología , Compuestos de Organoselenio/química , Isoindoles/farmacología , Animales , Ratones , Homeostasis/efectos de los fármacos , Estreptomicina/farmacología , Antituberculosos/farmacología , Antituberculosos/química , Mycobacterium marinum/efectos de los fármacos , Azoles/farmacología , Azoles/química , Relación Dosis-Respuesta a Droga , Antibacterianos/farmacología , Antibacterianos/química , Relación Estructura-Actividad , Estructura Molecular , Ratones Endogámicos BALB C

RESUMEN

Mycobacterium marinum infection often affects the extremities, causing single or multiple skin lesions. With the improvement of molecular detection technology and the clinical application of NGS pathogen detection, the diagnosis rate of Mycobacterium marinum skin disease is gradually increasing. This article reported the case of a 54-year-old man who was stung by a marine fish and gradually developed swelling and nodules on his right hand and right upper limb. He was diagnosed with Mycobacterium marinum infection by detection of the tuberculosis resistance gene dissolution curve of the pus and the identification of the bacteria. Oral rifampicin combined with clarithromycin and minocycline was given for anti-infection treatment. During follow-up, the abscesses and nodules gradually shrank and eventually disappeared. By presenting the diagnosis and treatment of this case, the understanding of this disease among clinicians can be improved to avoid misdiagnosis and missed diagnosis.

Asunto(s)

Infecciones por Mycobacterium no Tuberculosas , Mycobacterium marinum , Humanos , Persona de Mediana Edad , Masculino , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/aislamiento & purificación , Enfermedades Cutáneas Bacterianas/diagnóstico , Enfermedades Cutáneas Bacterianas/microbiología , Enfermedades Cutáneas Bacterianas/tratamiento farmacológico , Claritromicina/uso terapéutico , Rifampin/uso terapéutico , Antibacterianos/uso terapéutico

RESUMEN

PE/PPE proteins secreted by the ESX-5 type VII secretion system constitute a major protein repertoire in pathogenic mycobacteria and are essential for bacterial survival, pathogenicity, and host-pathogen interaction; however, little is known about their expression and secretion. The scarcity of arginine and lysine residues in PE/PPE protein sequences and the high homology of their N-terminal domains limit protein identification using classical trypsin-based proteomic methods. This study used endoproteinase AspN and trypsin to characterize the proteome of Mycobacterium marinum. Twenty-seven PE/PPE proteins were uniquely identified in AspN digests, especially PE_PGRS proteins. These treatments allowed the identification of approximately 50% of the PE/PPE pool encoded in the genome. Moreover, EspG5 pulldown assays retrieved 44 ESX-5-associated PPE proteins, covering 85% of the PPE pool in the identified proteome. The identification of PE/PE_PGRS proteins in the EspG5 interactome suggested the presence of PE-PPE pairs. The correlation analysis between protein abundance and phylogenetic relationships found potential PE/PPE pairs, indicating the presence of multiple PE/PE_PGRS partners in one PPE. We validated that EspG5 interacted with PPE31 and PPE32 and mapped critical residues for complex formation. The modified proteomic platform increases the coverage of PE/PPE proteins and elucidates the expression and localization of these proteins.

Asunto(s)

Proteínas Bacterianas , Mycobacterium marinum , Proteoma , Mycobacterium marinum/metabolismo , Mycobacterium marinum/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteoma/metabolismo , Proteómica/métodos , Filogenia , Sistemas de Secreción Tipo VII/metabolismo , Sistemas de Secreción Tipo VII/genética , Especificidad por Sustrato

Asunto(s)

Infecciones por Mycobacterium no Tuberculosas , Mycobacterium marinum , Humanos , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Masculino , Enfermedades Cutáneas Bacterianas/microbiología , Enfermedades Cutáneas Bacterianas/patología , Enfermedades Cutáneas Bacterianas/tratamiento farmacológico , Enfermedades Cutáneas Bacterianas/diagnóstico , Femenino , Antibacterianos/uso terapéutico , Persona de Mediana Edad

RESUMEN

Tuberculosis (TB) remains a major public health challenge, with research on new anti-TB drugs crucial for global TB elimination efforts. Here, we report a novel class of anti-TB agents. Especially, compounds 5b and 5j exhibited the highest activity [minimum inhibitory concentration (MIC) H37Rv: 0.16 and 0.12 µg/mL]. Chiral resolution was performed on compounds 5b and 5j; the isomers were evaluated for their activity and safety, confirming that the R-isomer 5bb and 5jb displayed significant anti-TB activity (MIC H37Rv: 0.03-0.06 µg/mL; MDR-Mtb: 0.125-0.06 µg/mL) and low hERG toxicity. Further evaluations on 5bb and 5jb demonstrated good metabolic stability, favorable kinetic parameters and oral bioavailability (F: 56.7 and 63.8%, respectively). The results of in vivo activity assessment indicate that 5bb and 5jb exhibit protective and therapeutic effects on zebrafish larvae and adult zebrafish infected with Mycobacterium marinum. Based on these results, compounds 5bb and 5jb are considered promising candidates for further in-depth studies.

Asunto(s)

Antituberculosos , Diseño de Fármacos , Pruebas de Sensibilidad Microbiana , Pez Cebra , Antituberculosos/farmacología , Antituberculosos/síntesis química , Antituberculosos/farmacocinética , Antituberculosos/química , Animales , Relación Estructura-Actividad , Mycobacterium tuberculosis/efectos de los fármacos , Pirimidinas/farmacología , Pirimidinas/síntesis química , Pirimidinas/química , Pirimidinas/farmacocinética , Humanos , Mycobacterium marinum/efectos de los fármacos , Estructura Molecular

RESUMEN

Serine protease inhibitors (serpins) constitute the largest family of protease inhibitors expressed in humans, but their role in infection remains largely unexplored. In infected macrophages, the mycobacterial ESX-1 type VII secretion system permeabilizes internal host membranes and causes leakage into the cytosol of host DNA, which induces type I interferon (IFN) production via the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) surveillance pathway, and promotes infection in vivo. Using the Mycobacterium marinum infection model, we show that ESX-1-mediated type I IFN signaling in macrophages selectively induces the expression of serpina3f and serpina3g, two cytosolic serpins of the clade A3. The membranolytic activity of ESX-1 also caused leakage of cathepsin B into the cytosol where it promoted cell death, suggesting that the induction of type I IFN comes at the cost of lysosomal rupture and toxicity. However, the production of cytosolic serpins suppressed the protease activity of cathepsin B in this compartment and thus limited cell death, a function that was associated with increased bacterial growth in infected mice. These results suggest that cytosolic serpins act in a type I IFN-dependent cytoprotective feedback loop to counteract the inevitable toxic effect of ESX-1-mediated host membrane rupture. IMPORTANCE: The ESX-1 type VII secretion system is a key virulence determinant of pathogenic mycobacteria. The ability to permeabilize host cell membranes is critical for several ESX-1-dependent virulence traits, including phagosomal escape and induction of the type I interferon (IFN) response. We find that it comes at the cost of lysosomal leakage and subsequent host cell death. However, our results suggest that ESX-1-mediated type I IFN signaling selectively upregulates serpina3f and serpina3g and that these cytosolic serpins limit cell death caused by cathepsin B that has leaked into the cytosol, a function that is associated with increased bacterial growth in vivo. The ability to rupture host membranes is widespread among bacterial pathogens, and it will be of interest to evaluate the role of cytosolic serpins and this type I IFN-dependent cytoprotective feedback loop in the context of human infection.

Asunto(s)

Proteínas Bacterianas , Citosol , Interferón Tipo I , Macrófagos , Mycobacterium marinum , Serpinas , Animales , Femenino , Ratones , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Muerte Celular , Citosol/microbiología , Citosol/metabolismo , Retroalimentación Fisiológica , Interacciones Huésped-Patógeno , Interferón Tipo I/metabolismo , Macrófagos/microbiología , Ratones Endogámicos C57BL , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/patogenicidad , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Serpinas/metabolismo , Serpinas/genética , Transducción de Señal , Sistemas de Secreción Tipo VII/metabolismo , Sistemas de Secreción Tipo VII/genética

RESUMEN

BACKGROUND: In December 2023, our hospital confirmed a case of finger infection with Mycobacterium marinum. The patient sought medical attention at our hospital due to a hard scratch on her left middle finger, which was red, swollen, and ulcerated for one month. PHYSICAL EXAMINATION: A lesion of approximately 1.5 cm x 2 cm in the patient's left middle finger, surrounded by redness and swelling, unclear boundaries, surface rupture, partial scabbing, and no tenderness during compression. She was treated at the previous clinic, common infectious diseases were considered, and was given intravenous infusion treatment: cefotaxime and clarithromycin, and erythromycin ointment was applied externally. She came to our hospital after poor treatment results. The patient has had hypertension for 3 years, no other systemic diseases, no similar medical history among family members, no history of drug or food allergies. METHODS: Clean the wound and remove the scab from the affected area, and use a surgical blade to scrape off necrotic tissue. Send the scraped tissue for pathogen testing: tissue bacterial culture+identification (matrix assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF), tissue acid fast staining, and tissue metagenomic next-generation sequencing (mNGS). Other auxiliary examinations: blood routine, urine routine, blood fat, liver function, and kidney function. RESULTS: Tissue bacterial culture+identification: growth of Mycobacterium marinum; Acid fast staining of tissue: positive; Tissue mNGS: Mycobacterium marinum. Clinical treatment plan: clarithromycin 0.5 g bid po+rifampicin 0.45 g qd po+5-aminolevulinic acid photodynamic therapy (ALA-PDT) qw+boric acid wash wet compress tid. After 14 days of treatment, the area of redness and swelling significantly decreased, and the degree of redness and swelling was significantly reduced compared to admission. The degree of ulcer edge protrusion was also reduced compared to admission. There was a small amount of exudation from the wound, and no necrotic tissue was observed. The patient improved and was discharged. CONCLUSIONS: This article reports a case of finger infection with Mycobacterium marinum. Mycobacterium marinum was quickly and accurately identified by mNGS, and reasonable treatment measures were adopted clinically. The patient improved and was discharged. This study has important reference significance for the clinical diagnosis and treatment of Mycobacterium infection. In addition, mNGS as a novel detection method has considerable prospects for rapid diagnosis of pathogens.

Asunto(s)

Secuenciación de Nucleótidos de Alto Rendimiento , Infecciones por Mycobacterium no Tuberculosas , Mycobacterium marinum , Humanos , Femenino , Mycobacterium marinum/aislamiento & purificación , Mycobacterium marinum/genética , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Metagenómica/métodos , Antibacterianos/uso terapéutico , Antibacterianos/administración & dosificación , Dedos/microbiología , Persona de Mediana Edad

RESUMEN

Paradoxical reactions occur when an infection has acute worsening in response to antibiotic therapy. Here, we describe a patient with chronic cutaneous ulcerative lymphangitis that acutely worsened following initiation of antibiotic therapy. The infection was caused by Mycobacterium marinum, a species which has not previously been associated with paradoxical reaction in immunocompetent persons. In this case report, we describe our patient's diagnosis and management, review the management of Mycobacterium marinum infection, and discuss paradoxical reactions in mycobacterial disease.

Asunto(s)

Antibacterianos , Infecciones por Mycobacterium no Tuberculosas , Mycobacterium marinum , Humanos , Persona de Mediana Edad , Antibacterianos/uso terapéutico , Linfangitis/microbiología , Linfangitis/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Mycobacterium marinum/aislamiento & purificación

RESUMEN

Mycobacterium tuberculosis is a pathogenic mycobacterium that causes tuberculosis. Tuberculosis is a significant global health concern that poses numerous clinical challenges, particularly in terms of finding effective treatments for patients. Throughout evolution, host immune cells have developed cell-autonomous defence strategies to restrain and eliminate mycobacteria. Concurrently, mycobacteria have evolved an array of virulence factors to counteract these host defences, resulting in a dynamic interaction between host and pathogen. Here, we review recent findings, including those arising from the use of the amoeba Dictyostelium discoideum as a model to investigate key mycobacterial infection pathways. D. discoideum serves as a scalable and genetically tractable model for human phagocytes, providing valuable insights into the intricate mechanisms of host-pathogen interactions. We also highlight certain similarities between M. tuberculosis and Mycobacterium marinum, and the use of M. marinum to more safely investigate mycobacteria in D. discoideum.

Asunto(s)

Dictyostelium , Interacciones Huésped-Patógeno , Mycobacterium marinum , Tuberculosis , Dictyostelium/microbiología , Mycobacterium marinum/patogenicidad , Tuberculosis/microbiología , Tuberculosis/patología , Tuberculosis/inmunología , Humanos , Modelos Biológicos , Animales , Infecciones por Mycobacterium no Tuberculosas

RESUMEN

Tuberculosis is a major global health problem and is one of the top 10 causes of death worldwide. There is a pressing need for new treatments that circumvent emerging antibiotic resistance. Mycobacterium tuberculosis parasitises macrophages, reprogramming them to establish a niche in which to proliferate, therefore macrophage manipulation is a potential host-directed therapy if druggable molecular targets could be identified. The pseudokinase Tribbles1 (Trib1) regulates multiple innate immune processes and inflammatory profiles making it a potential drug target in infections. Trib1 controls macrophage function, cytokine production, and macrophage polarisation. Despite wide-ranging effects on leukocyte biology, data exploring the roles of Tribbles in infection in vivo are limited. Here, we identify that human Tribbles1 is expressed in monocytes and is upregulated at the transcript level after stimulation with mycobacterial antigen. To investigate the mechanistic roles of Tribbles in the host response to mycobacteria in vivo, we used a zebrafish Mycobacterium marinum (Mm) infection tuberculosis model. Zebrafish Tribbles family members were characterised and shown to have substantial mRNA and protein sequence homology to their human orthologues. trib1 overexpression was host-protective against Mm infection, reducing burden by approximately 50%. Conversely, trib1 knockdown/knockout exhibited increased infection. Mechanistically, trib1 overexpression significantly increased the levels of proinflammatory factors il-1ß and nitric oxide. The host-protective effect of trib1 was found to be dependent on the E3 ubiquitin kinase Cop1. These findings highlight the importance of Trib1 and Cop1 as immune regulators during infection in vivo and suggest that enhancing macrophage TRIB1 levels may provide a tractable therapeutic intervention to improve bacterial infection outcomes in tuberculosis.

Asunto(s)

Péptidos y Proteínas de Señalización Intracelular , Proteínas Serina-Treonina Quinasas , Pez Cebra , Animales , Humanos , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Macrófagos/microbiología , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/genética , Mycobacterium marinum , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pez Cebra/microbiología , Masculino , Femenino

RESUMEN

INTRODUCTION: Mycobacterium marinum infection rarely occurs and has atypical symptoms. It is challenging to distinguish disseminated M. marinum infection from multifocal dermatosis caused by other factors clinically. CASE PRESENTATION: Herein, we reported a 68-year-old male patient with Human Immunodeficiency Virus (HIV) who presented redness and swelling in his left hand after being stabbed by marine fish for over 2 months. Mycobacterium tuberculosis infection was considered according to biochemical and pathological examinations, while empirical anti-infection treatment was ineffective. RESULTS: The metagenomic next-generation sequencing (mNGS) detected a large amount of M. marinum sequences, and the patient was finally diagnosed with M. marinum infection. After one month of combination therapy with ethambutol, rifabutin, moxifloxacin, and linezolid, the swelling disappeared significantly. In this case, the successful application of mNGS in diagnosing and treating M. marinum infection has improved the understanding of the microbe both in the laboratory and clinically, especially in patients with HIV. CONCLUSIONS: For diseases with atypical symptoms or difficulty in determining the pathogens, mNGS is suggested in clinical procedures for rapid and accurate diagnosis and treatment.

Asunto(s)

Infecciones por VIH , Infecciones por Mycobacterium no Tuberculosas , Mycobacterium marinum , Humanos , Masculino , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Anciano , Mycobacterium marinum/aislamiento & purificación , Mycobacterium marinum/genética , Infecciones por VIH/complicaciones , Secuenciación de Nucleótidos de Alto Rendimiento , Metagenómica , Etambutol/uso terapéutico , Antibacterianos/uso terapéutico

RESUMEN

Developing effective tuberculosis drugs is hindered by mycobacteria's intrinsic antibiotic resistance because of their impermeable cell envelope. Using benzothiazole compounds, we aimed to increase mycobacterial cell envelope permeability and weaken the defenses of Mycobacterium marinum, serving as a model for Mycobacterium tuberculosis Initial hit, BT-08, significantly boosted ethidium bromide uptake, indicating enhanced membrane permeability. It also demonstrated efficacy in the M. marinum-zebrafish embryo infection model and M. tuberculosis-infected macrophages. Notably, BT-08 synergized with established antibiotics, including vancomycin and rifampicin. Subsequent medicinal chemistry optimization led to BT-37, a non-toxic and more potent derivative, also enhancing ethidium bromide uptake and maintaining synergy with rifampicin in infected zebrafish embryos. Mutants of M. marinum resistant to BT-37 revealed that MMAR_0407 (Rv0164) is the molecular target and that this target plays a role in the observed synergy and permeability. This study introduces novel compounds targeting a new mycobacterial vulnerability and highlights their cooperative and synergistic interactions with existing antibiotics.

Asunto(s)

Benzotiazoles , Sinergismo Farmacológico , Mycobacterium marinum , Pez Cebra , Animales , Benzotiazoles/farmacología , Mycobacterium marinum/efectos de los fármacos , Antituberculosos/farmacología , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Humanos , Antibacterianos/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Macrófagos/metabolismo , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Rifampin/farmacología

RESUMEN

Vitamin B12 (B12) serves as a critical cofactor within mycobacterial metabolism. While some pathogenic strains can synthesize B12 de novo, others rely on host-acquired B12. In this investigation, we studied the transport of vitamin B12 in Mycobacterium marinum using B12-auxotrophic and B12-sensitive strains by deleting metH or metE, respectively. These two enzymes rely on B12 in different ways to function as methionine synthases. We used these strains to select mutants affecting B12 scavenging and confirmed their phenotypes during growth experiments in vitro. Our analysis of B12 uptake mechanisms revealed that membrane lipids and cell wall integrity play an essential role in cell envelope transport. Furthermore, we identified a potential transcription regulator that responds to B12. Our study demonstrates that M. marinum can take up exogenous B12 and that altering mycobacterial membrane integrity affects B12 uptake. Finally, during zebrafish infection using B12-auxotrophic and B12-sensitive strains, we found that B12 is available for virulent mycobacteria in vivo.IMPORTANCEOur study investigates how mycobacteria acquire essential vitamin B12. These microbes, including those causing tuberculosis, face challenges in nutrient uptake due to their strong outer layer. We focused on Mycobacterium marinum, similar to TB bacteria, to uncover its vitamin B12 absorption. We used modified strains unable to produce their own B12 and discovered that M. marinum can indeed absorb it from the environment, even during infections. Changes in the outer layer composition affect this process, and genes related to membrane integrity play key roles. These findings illuminate the interaction between mycobacteria and their environment, offering insights into combatting diseases like tuberculosis through innovative strategies. Our concise research underscores the pivotal role of vitamin B12 in microbial survival and its potential applications in disease control.

Asunto(s)

Membrana Externa Bacteriana , Mycobacterium marinum , Vitamina B 12 , Pez Cebra , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Vitamina B 12/metabolismo , Animales , Pez Cebra/microbiología , Membrana Externa Bacteriana/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Permeabilidad de la Membrana Celular , Transporte Biológico , Membrana Celular/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología

RESUMEN

Mycobacterium tuberculosis (Mtb), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The melH gene in Mtb and Mycobacterium marinum (Mm) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that melH encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of melH in Mm resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of whiB3, a universal stress sensor. The absence of melH also resulted in reduced intracellular levels of NAD+, NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon source dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of melH in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria. IMPORTANCE: This study unveils the pivotal role played by the melH gene in Mycobacterium tuberculosis and in Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para-hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH. Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.

Asunto(s)

Proteínas Bacterianas , Cisteína , Metabolismo Energético , Glicopéptidos , Homeostasis , Mycobacterium tuberculosis , Oxidación-Reducción , Estrés Oxidativo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Antituberculosos/farmacología , Ergotioneína/metabolismo , Inositol/metabolismo , Mycobacterium marinum/efectos de los fármacos , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Eliminación de Gen

RESUMEN

The mycobacterial cell envelope is a major virulence determinant in pathogenic mycobacteria. Specific outer lipids play roles in pathogenesis, modulating the immune system and promoting the secretion of virulence factors. ESX-1 (ESAT-6 system-1) is a conserved protein secretion system required for mycobacterial pathogenesis. Previous studies revealed that mycobacterial strains lacking the outer lipid PDIM have impaired ESX-1 function during laboratory growth and infection. The mechanisms underlying changes in ESX-1 function are unknown. We used a proteo-genetic approach to measure phthiocerol dimycocerosate (PDIM)- and phenolic glycolipid (PGL)-dependent protein secretion in M. marinum, a non-tubercular mycobacterial pathogen that causes tuberculosis-like disease in ectothermic animals. Importantly, M. marinum is a well-established model for mycobacterial pathogenesis. Our findings showed that M. marinum strains without PDIM and PGL showed specific, significant reductions in protein secretion compared to the WT and complemented strains. We recently established a hierarchy for the secretion of ESX-1 substrates in four (I-IV) groups. Loss of PDIM differentially impacted secretion of Group III and IV ESX-1 substrates, which are likely the effectors of pathogenesis. Our data suggest that the altered secretion of specific ESX-1 substrates is responsible for the observed ESX-1-related effects in PDIM-deficient strains.IMPORTANCEMycobacterium tuberculosis, the cause of human tuberculosis, killed an estimated 1.3 million people in 2022. Non-tubercular mycobacterial species cause acute and chronic human infections. Understanding how these bacteria cause disease is critical. Lipids in the cell envelope are essential for mycobacteria to interact with the host and promote disease. Strains lacking outer lipids are attenuated for infection, but the reasons are unclear. Our research aims to identify a mechanism for attenuation of mycobacterial strains without the PDIM and PGL outer lipids in M. marinum. These findings will enhance our understanding of the importance of lipids in pathogenesis and how these lipids contribute to other established virulence mechanisms.

Asunto(s)

Proteínas Bacterianas , Glucolípidos , Mycobacterium marinum , Factores de Virulencia , Mycobacterium marinum/patogenicidad , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Glucolípidos/metabolismo , Virulencia , Lípidos , Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/genética

RESUMEN

Bacterial pathogens use protein secretion systems to transport virulence factors and regulate gene expression. Among pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum, the ESAT-6 system 1 (ESX-1) secretion is crucial for host interaction. Secretion of protein substrates by the ESX-1 secretion system disrupts phagosomes, allowing mycobacteria cytoplasmic access during macrophage infections. Deletion or mutation of the ESX-1 system attenuates mycobacterial pathogens. Pathogenic mycobacteria respond to the presence or absence of the ESX-1 system in the cytoplasmic membrane by altering transcription. Under laboratory conditions, the EspM repressor and WhiB6 activator control transcription of specific ESX-1-responsive genes, including the ESX-1 substrate genes. However, deleting the espM or whiB6 gene does not phenocopy the deletion of the ESX-1 substrate genes during macrophage infection by M. marinum. In this study, we identified EspN, a critical transcription factor whose activity is masked by the EspM repressor under laboratory conditions. In the absence of EspM, EspN activates transcription of whiB6 and ESX-1 genes during both laboratory growth and macrophage infection. EspN is also independently required for M. marinum growth within and cytolysis of macrophages, similar to the ESX-1 genes, and for disease burden in a zebrafish larval model of infection. These findings suggest that EspN and EspM coordinate to counterbalance the regulation of the ESX-1 system and support mycobacterial pathogenesis.IMPORTANCEPathogenic mycobacteria, which are responsible for tuberculosis and other long-term diseases, use the ESX-1 system to transport proteins that control the host response to infection and promote bacterial survival. In this study, we identify an undescribed transcription factor that controls the expression of ESX-1 genes and is required for both macrophage and animal infection. However, this transcription factor is not the primary regulator of ESX-1 genes under standard laboratory conditions. These findings identify a critical transcription factor that likely controls expression of a major virulence pathway during infection, but whose effect is not detectable with standard laboratory strains and growth conditions.

Asunto(s)

Mycobacterium marinum , Mycobacterium tuberculosis , Tuberculosis , Sistemas de Secreción Tipo VII , Animales , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Tipo VII/genética , Sistemas de Secreción Tipo VII/metabolismo , Pez Cebra , Tuberculosis/microbiología , Mycobacterium tuberculosis/metabolismo , Mycobacterium marinum/metabolismo

RESUMEN

Surface lipids on pathogenic mycobacteria modulate infection outcomes by regulating host immune responses. Phenolic glycolipid (PGL) is a host-modulating surface lipid that varies among clinical Mycobacterium tuberculosis strains. PGL is also found in Mycobacterium marinum, where it promotes infection of zebrafish through effects on the innate immune system. Given the important role this lipid plays in the host-pathogen relationship, tools for profiling its abundance, spatial distribution, and dynamics are needed. Here, we report a strategy for imaging PGL in live mycobacteria using bioorthogonal metabolic labeling. We functionalized the PGL precursor p-hydroxybenzoic acid (pHB) with an azide group (3-azido pHB). When fed to mycobacteria, 3-azido pHB was incorporated into the cell surface, which could then be visualized via the bioorthogonal conjugation of a fluorescent probe. We confirmed that 3-azido pHB incorporates into PGL using mass spectrometry methods and demonstrated selectivity for PGL-producing M. marinum and M. tuberculosis strains. Finally, we applied this metabolic labeling strategy to study the dynamics of PGL within the mycobacterial membrane. This new tool enables visualization of PGL that may facilitate studies of mycobacterial pathogenesis.

Asunto(s)

Mycobacterium marinum , Mycobacterium tuberculosis , Animales , Glucolípidos/metabolismo , Factores de Virulencia/metabolismo , Pez Cebra , Mycobacterium tuberculosis/metabolismo , Mycobacterium marinum/metabolismo

RESUMEN

With the emergence of drug-resistant strains, the treatment of tuberculosis (TB) is becoming more difficult and there is an urgent need to find new anti-TB drugs. Mycobacterium marinum, as a model organism of Mycobacterium tuberculosis, can be used for the rapid and efficient screening of bioactive compounds. The 14-membered resorcylic acid lactones (RALs) have a wide range of bioactivities such as antibacterial, antifouling and antimalarial activity. In order to further study their bioactivities, we initially constructed a 14-membered RALs library, which contains 16 new derivatives. The anti-M. marinum activity was evaluated in vitro. Derivatives 12, 19, 20 and 22 exhibited promising activity with MIC90 values of 80, 90, 80 and 80 µM, respectively. The preliminary structure-activity relationships showed that the presence of a chlorine atom at C-5 was a key factor to improve activity. Further studies showed that 12 markedly inhibited the survival of M. marinum and significantly reduced the dosage of positive drugs isoniazid and rifampicin when combined with them. These results suggest that 12 is a bioactive compound capable of enhancing the potency of existing positive drugs, and its effective properties make it a very useful leads for future drug development in combating TB resistance.

Asunto(s)

Antimaláricos , Mycobacterium marinum , Anticuerpos , Antituberculosos , Lactonas

RESUMEN

The cytochrome P450 family of heme metalloenzymes (CYPs) catalyse important biological monooxygenation reactions. Mycobacterium marinum contains a gene encoding a CYP105Q4 enzyme of unknown function. Other members of the CYP105 CYP family have key roles in bacterial metabolism including the synthesis of secondary metabolites. We produced and purified the cytochrome P450 enzyme CYP105Q4 to enable its characterization. Several nitrogen-donor atom-containing ligands were found to bind to CYP105Q4 generating type II changes in the UV-vis absorbance spectrum. Based on the UV-vis absorbance spectra none of the potential substrate ligands we tested with CYP105Q4 were able to displace the sixth distal aqua ligand from the heme, though there was evidence for binding of oleic acid and amphotericin B. The crystal structure of CYP105Q4 in the substrate-free form was determined in an open conformation. A computational structural similarity search (Dali) was used to find the most closely related characterized relatives within the CYP105 family. The structure of CYP105Q4 enzyme was compared to the GfsF CYP enzyme from Streptomyces graminofaciens which is involved in the biosynthesis of a macrolide polyketide. This structural comparison to GfsF revealed conformational changes in the helices and loops near the entrance to the substrate access channel. A disordered B/C loop region, usually involved in substrate recognition, was also observed.

Asunto(s)

Mycobacterium marinum , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Estructura Secundaria de Proteína , Macrólidos/química , Macrólidos/metabolismo , Hemo/química , Cristalografía por Rayos X

RESUMEN

Retinoic acid inducible gene I (Rig-I) is a cytosolic pattern recognition receptor canonically described for its important role in sensing viral RNAs. Increasingly, bacterially-derived RNA from intracellular bacteria such as Mycobacterium tuberculosis, have been shown to activate the same host Rig-I/Mitochondrial antiviral sensing protein (MAVS) signaling pathway to drive a type-I interferon response that contributes to bacterial pathogenesis in vivo. In M. tuberculosis, this response is mediated by the protein secretion system SecA2, but little is known about whether this process is conserved in other pathogenic mycobacteria or the mechanism by which these nucleic acids gain access to the host cytoplasm. Because the M. tuberculosis and M. marinum SecA2 protein secretion systems share a high degree of genetic and functional conservation, we hypothesized that Rig-I/MAVS activation and subsequent induction of IFN-ß secretion by host macrophages will also be conserved between these two mycobacterial species. To test this, we generated a ΔsecA2 M. marinum strain along with complementation strains expressing either the M. marinum or M. tuberculosis secA2 genes. Our results suggest that the ΔsecA2 strain has a growth defect in vitro but not in host macrophages. These intracellular growth curves also suggested that the calculation applied to estimate the number of bacteria added to macrophage monolayers in infection assays underestimates bacterial inputs for the ΔsecA2 strain. Therefore, to better examine secreted IFN-ß levels when bacterial infection levels are equal across strains we plated bacterial CFUs at 2hpi alongside our ELISA based infections. This enabled us to normalize secreted levels of IFN-ß to a standard number of bacteria. Applying this approach to both WT and MAVS-/- bone marrow derived macrophages we observed equal or higher levels of secreted IFN-ß from macrophages infected with the ΔsecA2 M. marinum strain as compared to WT. Together our findings suggest that activation of host Rig-I/MAVS cytosolic sensors and subsequent induction of IFN-ß response in a SecA2-dependent manner is not conserved in M. marinum under the conditions tested.

Asunto(s)

Mycobacterium marinum , Mycobacterium tuberculosis , Tuberculosis , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium marinum/genética , Transducción de Señal , Macrófagos/metabolismo , Proteína 58 DEAD Box/metabolismo , Tuberculosis/patología