Mycobacterium tuberculosis Reactivates HIV-1 via Exosome-Mediated Resetting of Cellular Redox Potential and Bioenergetics

ABSTRACT The synergy between Mycobacterium tuberculosis and human immunodeficiency virus-1 (HIV-1) interferes with therapy and facilitates the pathogenesis of both human pathogens. Fundamental mechanisms by which M. tuberculosis exacerbates HIV-1 infection are not clear. Here, we show that exosomes secreted by macrophages infected with M. tuberculosis, including drug-resistant clinical strains, reactivated HIV-1 by inducing oxidative stress. Mechanistically, M. tuberculosis-specific exosomes realigned mitochondrial and nonmitochondrial oxygen consumption rates (OCR) and modulated the expression of host genes mediating oxidative stress response, inflammation, and HIV-1 transactivation. Proteomics analyses revealed the enrichment of several host factors (e.g., HIF-1α, galectins, and Hsp90) known to promote HIV-1 reactivation in M. tuberculosis-specific exosomes. Treatment with a known antioxidant—N-acetyl cysteine (NAC)—or with inhibitors of host factors—galectins and Hsp90—attenuated HIV-1 reactivation by M. tuberculosis-specific exosomes. Our findings uncover new paradigms for understanding the redox and bioenergetics bases of HIV-M. tuberculosis coinfection, which will enable the design of effective therapeutic strategies. IMPORTANCE Globally, individuals coinfected with the AIDS virus (HIV-1) and with M. tuberculosis (causative agent of tuberculosis [TB]) pose major obstacles in the clinical management of both diseases. At the heart of this issue is the apparent synergy between the two human pathogens. On the one hand, mechanisms induced by HIV-1 for reactivation of TB in AIDS patients are well characterized. On the other hand, while clinical findings clearly identified TB as a risk factor for HIV-1 reactivation and associated mortality, basic mechanisms by which M. tuberculosis exacerbates HIV-1 replication and infection remain poorly characterized. The significance of our research is in identifying the role of fundamental mechanisms such as redox and energy metabolism in catalyzing HIV-M. tuberculosis synergy. The quantification of redox and respiratory parameters affected by M. tuberculosis in stimulating HIV-1 will greatly enhance our understanding of HIV-M. tuberculosis coinfection, leading to a wider impact on the biomedical research community and creating new translational opportunities.

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Mycobacterium tuberculosis reactivates HIV via exosomes mediated resetting of cellular redox potential and bioenergetics

10.1101/629048 ◽

2019 ◽

Author(s):

Priyanka Tyagi ◽

Virender Kumar Pal ◽

Sandhya Srinivasan ◽

Amit Singh

Keyword(s):

Oxidative Stress ◽

Mycobacterium Tuberculosis ◽

Consumption Rate ◽

Host Factors ◽

Drug Resistant ◽

Human Pathogens ◽

Mitochondrial Oxygen Consumption ◽

Cellular Redox ◽

Expression Of Genes ◽

Hiv 1

AbstractThe synergy betweenMycobacterium tuberculosis(Mtb) and HIV-1 interferes with therapy and facilitates pathogenesis of both human pathogens. Fundamental mechanisms by whichMtbexacerbates HIV-1 are not clear. Here, we show that exosomes secreted by macrophages infected withMtb, including drug-resistant clinical strains, reactivate HIV-1 by inducing oxidative stress. Mechanistically,Mtb-specific exosomes realign mitochondrial and non-mitochondrial oxygen consumption rate (OCR) and modulates the expression of genes mediating oxidative stress response, inflammation, and HIV-1 transactivation. Proteomics revealed the enrichment of several host factors (e.g.,HIF-1α, galectins, Hsp90) known to promote HIV-1 reactivation in theMtb-specific exosomes. Treatment with a known antioxidant, N-acetyl cysteine, or with the inhibitors of host factors galectins and Hsp90 attenuated HIV-1 reactivation byMtb-specific exosomes. Our findings uncovered new paradigms for understanding the redox and bioenergetics basis of HIV-TB co-infection, which will enable the design of effective therapeutic strategies.

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A Novel Tuberculosis DNA Vaccine in an HIV-1 p24 Protein Backbone Confers Protection against Mycobacterium tuberculosis and Simultaneously Elicits Robust Humoral and Cellular Responses to HIV-1

Clinical and Vaccine Immunology ◽

10.1128/cvi.05700-11 ◽

2012 ◽

Vol 19 (5) ◽

pp. 723-730 ◽

Cited By ~ 4

Author(s):

Xiaoman Li ◽

Wei Xu ◽

Sidong Xiong

Keyword(s):

Mycobacterium Tuberculosis ◽

T Cell ◽

Dna Vaccine ◽

Cellular Response ◽

Mycobacterial Infection ◽

T Cell Epitopes ◽

Content Type ◽

P24 Protein ◽

Elevated Frequency ◽

Hiv 1

ABSTRACTTuberculosis (TB) caused byMycobacterium tuberculosisremains a major infectious disease worldwide. Moreover, latentM. tuberculosisinfection is more likely to progress to active TB and eventually leads to death when HIV infection is involved. Thus, it is urgent to develop a novel TB vaccine with immunogenicity to bothM. tuberculosisand HIV. In this study, four uncharacterized T cell epitopes from MPT64, Ag85A, Ag85B, and TB10.4 antigens ofM. tuberculosiswere predicted, and HIV-1-derived p24, an immunodominant protein that can induce protective responses to HIV-1, was used as an immunogenic backbone.M. tuberculosisepitopes were incorporated separately into the gene backbone of p24, forming a pP24-Mtb DNA vaccine. We demonstrated that pP24-Mtb immunization induced a strongM. tuberculosis-specific cellular response as evidenced by T cell proliferation, cytotoxicity, and elevated frequency of gamma interferon (IFN-γ)-secreting T cells. Interestingly, a p24-specific cellular response and high levels of p24-specific IgG were also induced by pP24-Mtb immunization. When the protective effect was assessed after mycobacterial challenge, pP24-Mtb vaccination significantly reduced tissue bacterial loads and profoundly attenuated the mycobacterial infection-related lung inflammation and injury. Our findings demonstrated that the pP24-Mtb tuberculosis vaccine confers effective protection against mycobacterial challenge with simultaneously elicited robust immune responses to HIV-1, which may provide clues for developing novel vaccines to prevent dual infections.

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Mycobacterium tuberculosis Rv0991c Is a Redox-Regulated Molecular Chaperone

mBio ◽

10.1128/mbio.01545-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Samuel H. Becker ◽

Kathrin Ulrich ◽

Avantika Dhabaria ◽

Beatrix Ueberheide ◽

William Beavers ◽

...

Keyword(s):

Oxidative Stress ◽

Quality Control ◽

Mycobacterium Tuberculosis ◽

Molecular Chaperone ◽

Protein Quality ◽

Protein Quality Control ◽

Cellular Protein ◽

Content Type ◽

Hsp70 Chaperone

ABSTRACT The bacterial pathogen Mycobacterium tuberculosis is the leading cause of death by an infectious disease among humans. Here, we describe a previously uncharacterized M. tuberculosis protein, Rv0991c, as a molecular chaperone that is activated by oxidation. Rv0991c has homologs in most bacterial lineages and appears to function analogously to the well-characterized Escherichia coli redox-regulated chaperone Hsp33, despite a dissimilar protein sequence. Rv0991c is transcriptionally coregulated with hsp60 and hsp70 chaperone genes in M. tuberculosis, suggesting that Rv0991c functions with these chaperones in maintaining protein quality control. Supporting this hypothesis, we found that, like oxidized Hsp33, oxidized Rv0991c prevents the aggregation of a model unfolded protein in vitro and promotes its refolding by the M. tuberculosis Hsp70 chaperone system. Furthermore, Rv0991c interacts with DnaK and can associate with many other M. tuberculosis proteins. We therefore propose that Rv0991c, which we named “Ruc” (redox-regulated protein with unstructured C terminus), represents a founding member of a new chaperone family that protects M. tuberculosis and other species from proteotoxicity during oxidative stress. IMPORTANCE M. tuberculosis infections are responsible for more than 1 million deaths per year. Developing effective strategies to combat this disease requires a greater understanding of M. tuberculosis biology. As in all cells, protein quality control is essential for the viability of M. tuberculosis, which likely faces proteotoxic stress within a host. Here, we identify an M. tuberculosis protein, Ruc, that gains chaperone activity upon oxidation. Ruc represents a previously unrecognized family of redox-regulated chaperones found throughout the bacterial superkingdom. Additionally, we found that oxidized Ruc promotes the protein-folding activity of the essential M. tuberculosis Hsp70 chaperone system. This work contributes to a growing body of evidence that oxidative stress provides a particular strain on cellular protein stability.

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Clade-Specific Virulence Patterns of Mycobacterium tuberculosis Complex Strains in Human Primary Macrophages and Aerogenically Infected Mice

mBio ◽

10.1128/mbio.00250-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 81

Author(s):

Norbert Reiling ◽

Susanne Homolka ◽

Kerstin Walter ◽

Julius Brandenburg ◽

Lisa Niwinski ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

High Growth ◽

Careful Consideration ◽

Model Systems ◽

Human Pathogens ◽

Simple Correlation ◽

Content Type ◽

Cytokine Induction ◽

Host Interaction ◽

Specific Virulence

ABSTRACT In infection experiments with genetically distinct Mycobacterium tuberculosis complex (MTBC) strains, we identified clade-specific virulence patterns in human primary macrophages and in mice infected by the aerosol route, both reflecting relevant model systems. Exclusively human-adapted M. tuberculosis lineages, also termed clade I, comprising “modern” lineages, such as Beijing and Euro-American Haarlem strains, showed a significantly enhanced capability to grow compared to that of clade II strains, which include “ancient” lineages, such as, e.g., East African Indian or M. africanum strains. However, a simple correlation of inflammatory response profiles with strain virulence was not apparent. Overall, our data reveal three different pathogenic profiles: (i) strains of the Beijing lineage are characterized by low uptake, low cytokine induction, and a high replicative potential, (ii) strains of the Haarlem lineage by high uptake, high cytokine induction, and high growth rates, and (iii) EAI strains by low uptake, low cytokine induction, and a low replicative potential. Our findings have significant implications for our understanding of host-pathogen interaction and factors that modulate the outcomes of infections. Future studies addressing the underlying mechanisms and clinical implications need to take into account the diversity of both the pathogen and the host. IMPORTANCE Clinical strains of the Mycobacterium tuberculosis complex (MTBC) are genetically more diverse than previously anticipated. Our analysis of mycobacterial growth characteristics in primary human macrophages and aerogenically infected mice shows that the MTBC genetic differences translate into pathogenic differences in the interaction with the host. Our study reveals for the first time that “TB is not TB,” if put in plain terms. We are convinced that it is very unlikely that a single molecular mechanism may explain the observed effects. Our study refutes the hypothesis that there is a simple correlation between cytokine induction as a single functional parameter of host interaction and mycobacterial virulence. Instead, careful consideration of strain- and lineage-specific characteristics must guide our attempts to decipher what determines the pathological potential and thus the outcomes of infection with MTBC, one of the most important human pathogens.

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Glutamate Racemase Is the Primary Target of β-Chloro-d-Alanine in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01249-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 6091-6099 ◽

Cited By ~ 23

Author(s):

Gareth A. Prosser ◽

Anne Rodenburg ◽

Hania Khoury ◽

Cesira de Chiara ◽

Steve Howell ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Early Stage ◽

Human Pathogens ◽

Intact Cells ◽

Content Type ◽

Peptidoglycan Biosynthesis ◽

Glutamate Racemase ◽

Starting Point ◽

Effective Drugs

ABSTRACTThe increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except inMycobacterium tuberculosis, where it has so far been underexploited.d-Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within thed-alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies ofM. tuberculosis. During our studies on alternative inhibitors of thed-alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor β-chloro–d-alanine (BCDA) is a very poor inhibitor of recombinantM. tuberculosisAlr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit thed-alanine pathway in intact cells, consistent with its poorin vitroactivity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI inM. tuberculosisand thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development.

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Antioxidant nanozyme counteracts HIV-1 by modulating intracellular redox potential

10.1101/2020.08.15.252098 ◽

2020 ◽

Author(s):

Shalini Singh ◽

Sourav Ghosh ◽

Virender Kumar Pal ◽

MohamedHusen Munshi ◽

Raju S Rajmani ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Mycobacterium Tuberculosis ◽

Glutathione Peroxidase ◽

Transgenic Mice ◽

Redox Potential ◽

Vanadium Pentoxide ◽

Immune Cells ◽

Human Pathogens ◽

Immunodeficiency Virus ◽

Hiv 1

AbstractReactive oxygen species (ROS) regulates the proliferation of human immunodeficiency virus (HIV-1) and Mycobacterium tuberculosis (Mtb) inside the infected immune cells. However, the application of this knowledge to develop therapeutic strategies remained unsuccessful due to unfavorable consequences of manipulating cellular antioxidant systems that respond to ROS. Here, we show that vanadium pentoxide (V2O5) nanosheets functionally mimic the activity of natural glutathione peroxidase (GPX) to mitigate ROS associated with HIV-1 infection without triggering detrimental changes in cellular physiology. Using genetic reporters of glutathione (GSH) redox potential (EGSH; Grx1-roGFP2) and H2O2 (Orp1-roGFP2), we showed that V2O5-nanosheets catalyze GSH-dependent neutralization of ROS in HIV-1 infected cells. Notably, V2O5-nanosheets uniformly blocked HIV-1 reactivation, multiplication, and impaired survival of drug-resistant Mtb during HIV-TB co-infection. Mechanistically, V2O5-nanosheets suppressed HIV-1 by affecting the expression of pathways coordinating redox balance, virus transactivation (e.g., NF-κB and FOS), inflammation, and apoptosis. Importantly, a combination of V2O5-nanosheets with a pharmacological inhibitor of NF-κB (BAY11-7082) abrogated activation of HIV-1 from latency. Lastly, V2O5-nanosheets counteracted ROS, disease pathophysiology, and virus expression in HIV-1 transgenic mice. Our data successfully revealed the usefulness of V2O5-nanosheets against human pathogens and suggest nanozymes as future platforms to develop interventions against infectious diseases.Significance StatementRedox stress, such as those caused by the deregulation of the antioxidant glutathione, promotes the multiplication of human immunodeficiency virus-1 (HIV-1) and Mycobacterium tuberculosis (Mtb). Here we present a vanadium pentoxide (V2O5)-based antioxidant nanozyme that targets cells infected with HIV-1. The nanozyme, by mimicking the activity of glutathione peroxidase, reprograms redox signaling to subvert HIV-1 from monocytes, lymphocytes, and HIV-1 transgenic mice. Treatment with nanozyme bolsters the antiviral potential of immune cells by reducing the expression of genes involved in virus activation, inflammation, and apoptosis. The nanozyme also inhibited the proliferation of Mtb, which is a major cause of lethality in HIV patients. These V2O5-based nanozymes may be applied to numerous human pathogens where redox signaling contributes to disease progression.

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Nucleoside and Nucleotide Reverse Transcriptase Inhibitors Induce Aging by Inhibiting Telomerase Function

Journal of Clinical Research in HIV AIDS and Prevention ◽

10.14302/issn.2324-7339.jcrhap-19-3070 ◽

2019 ◽

Vol 3 (4) ◽

pp. 20-27

Author(s):

Shweta Singh ◽

Bechan Sharma ◽

Nikhat Jamal Siddiqi

Keyword(s):

Oxidative Stress ◽

Cell Division ◽

Adverse Effects ◽

Negative Influence ◽

Host Factors ◽

Adverse Impact ◽

Reverse Transcriptase Inhibitors ◽

Homologous Chromosomes ◽

Eukaryotic Chromosome ◽

Hiv 1

The telomeres existing at the end of the eukaryotic chromosome, play an important role in localization, pairing of homologous chromosomes during cell division and synapsis formation, while telomerase is involved in maintenance of the telomere length. The application of antiHIV-1 molecules particularly NRTIs have been shown to interfere with telomerase function thereby inducing aging processes. Since the application of these molecules has already indicated production of oxidative stress and toxicity in AIDS patients, their adverse impact on telomerase function may further worsen the situation. In addition, the negative influence of antiHIV-1 regimens on certain host factors involved in telomerase function may enhance aging. HAART changes the landscape of the disease by progressively decreasing the progression of HIV-1, but exerts prolonged adverse effects on the telomerase function. Though there is no exact information available on this issue, intensive efforts are needed to explore regulation of telomerase expression in HIV infected individuals and particularly those receiving antiretrovirals.

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rBCG30-Induced Immunity and Cross-Protection against Mycobacterium leprae Challenge Are Enhanced by Boosting with the Mycobacterium tuberculosis 30-Kilodalton Antigen 85B

Infection and Immunity ◽

10.1128/iai.01499-13 ◽

2014 ◽

Vol 82 (9) ◽

pp. 3900-3909 ◽

Cited By ~ 11

Author(s):

Thomas P. Gillis ◽

Michael V. Tullius ◽

Marcus A. Horwitz

Keyword(s):

Mycobacterium Tuberculosis ◽

Immune Responses ◽

Mycobacterium Bovis ◽

Protective Efficacy ◽

Mycobacterium Leprae ◽

Secretory Protein ◽

Cross Protection ◽

Content Type ◽

Antigen 85B ◽

The One

ABSTRACTLeprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimalMycobacterium bovisBCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG againstMycobacterium tuberculosisandMycobacterium bovischallenge in animal models, for efficacy againstMycobacterium lepraechallenge in a murine model of leprosy. rBCG30 overexpresses theM. tuberculosis30-kDa major secretory protein antigen 85B, which is 85% homologous with theM. lepraehomolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viableM. lepraeinto each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating theM. lepraebacteria per footpad. Both BCG and rBCG30 induced significant protection againstM. lepraechallenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purifiedM. tuberculosisorM. lepraeantigen 85B also induced protection againstM. lepraechallenge but less so than BCG or rBCG30. Notably, boosting rBCG30 withM. tuberculosisantigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection againstM. lepraechallenge. Thus, rBCG30, a vaccine that induces improved protection againstM. tuberculosis, induces cross-protection againstM. lepraethat is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy.

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Biochemical and Mutational Studies of the Bacillus cereus CECT 5050T Formamidase Support the Existence of a C-E-E-K Tetrad in Several Members of the Nitrilase Superfamily

Applied and Environmental Microbiology ◽

10.1128/aem.00312-11 ◽

2011 ◽

Vol 77 (16) ◽

pp. 5761-5769 ◽

Cited By ~ 13

Author(s):

Pablo Soriano-Maldonado ◽

Ana Isabel Martínez-Gómez ◽

Montserrat Andújar-Sánchez ◽

José L. Neira ◽

Josefa María Clemente-Jiménez ◽

...

Keyword(s):

Helicobacter Pylori ◽

Sequence Analysis ◽

Bacillus Cereus ◽

The Other ◽

Human Pathogens ◽

Purification And Characterization ◽

Content Type ◽

The One ◽

Derivatives Of

ABSTRACTFormamidases (EC 3.5.1.49) are poorly characterized proteins. In spite of this scarce knowledge, ammonia has been described as playing a central role in the pathogenesis of human pathogens such asHelicobacter pylori, for which formamidase has been shown to participate in the nitrogen metabolic pathway. Sequence analysis has revealed that at least two different groups of formamidases are classified as EC 3.5.1.49: on the one hand, the derivatives of the FmdA-AmdA superfamily, which are the best studied to date, and on the other hand, the derivatives ofHelicobacter pyloriAmiF. Here we present the cloning, purification, and characterization of a recombinant formamidase fromBacillus cereusCECT 5050T (BceAmiF), the second member of the AmiF subfamily to be characterized, showing new features of the enzyme further supporting its relationship with aliphatic amidases. We also present homology modeling-based mutational studies confirming the importance of the Glu140 and Tyr191 residues in the enzymatic activities of the AmiF family. Moreover, we can conclude that a second glutamate residue is critical in several members of the nitrilase superfamily, meaning that what has consistently been identified as a C-E-K triad is in fact a C-E-E-K tetrad.

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Genomic variant-identification methods may alter Mycobacterium tuberculosis transmission inferences

Microbial Genomics ◽

10.1099/mgen.0.000418 ◽

2020 ◽

Vol 6 (8) ◽

Author(s):

Katharine S. Walter ◽

Caroline Colijn ◽

Ted Cohen ◽

Barun Mathema ◽

Qingyun Liu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Sequence Data ◽

Phylogenetic Reconstruction ◽

Variant Calling ◽

Genomic Variation ◽

Real Sequence ◽

Human Pathogens ◽

Content Type ◽

Identification Approach ◽

Variant Identification

Pathogen genomic data are increasingly used to characterize global and local transmission patterns of important human pathogens and to inform public health interventions. Yet, there is no current consensus on how to measure genomic variation. To test the effect of the variant-identification approach on transmission inferences for Mycobacterium tuberculosis, we conducted an experiment in which five genomic epidemiology groups applied variant-identification pipelines to the same outbreak sequence data. We compared the variants identified by each group in addition to transmission and phylogenetic inferences made with each variant set. To measure the performance of commonly used variant-identification tools, we simulated an outbreak. We compared the performance of three mapping algorithms, five variant callers and two variant filters in recovering true outbreak variants. Finally, we investigated the effect of applying increasingly stringent filters on transmission inferences and phylogenies. We found that variant-calling approaches used by different groups do not recover consistent sets of variants, which can lead to conflicting transmission inferences. Further, performance in recovering true variation varied widely across approaches. While no single variant-identification approach outperforms others in both recovering true genome-wide and outbreak-level variation, variant-identification algorithms calibrated upon real sequence data or that incorporate local reassembly outperform others in recovering true pairwise differences between isolates. The choice of variant filters contributed to extensive differences across pipelines, and applying increasingly stringent filters rapidly eroded the accuracy of transmission inferences and quality of phylogenies reconstructed from outbreak variation. Commonly used approaches to identify M. tuberculosis genomic variation have variable performance, particularly when predicting potential transmission links from pairwise genetic distances. Phylogenetic reconstruction may be improved by less stringent variant filtering. Approaches that improve variant identification in repetitive, hypervariable regions, such as long-read assemblies, may improve transmission inference.

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