Biology subjects

Rajmani, R. S.

Publications and source records attributed to Rajmani, R. S..

27 records · Page 2Linked to original sources

bioRxiv2022

Moxifloxacin-mediated killing of Mycobacterium tuberculosis involves respiratory downshift, reductive stress, and ROS accumulation

Moxifloxacin is central to treatment of multidrug-resistant tuberculosis. Effects of moxifloxacin on Mycobacterium tuberculosis redox state were explored to identify strategies for increasing lethality and reducing the prevalence of extensively resistant tuberculosis. A non-invasive redox biosensor and an ROS-sensitive dye revealed that moxifloxacin induces oxidative stress correlated with M. tuberculosis death. Moxifloxacin lethality was mitigated by supplementing bacterial cultures with an ROS scavenger (thiourea), an iron chelator (bipyridyl), and, after drug removal, an antioxidant enzyme (catalase). Lethality was also reduced by hypoxia and nutrient starvation. Moxifloxacin increased the expression of genes involved in the oxidative stress response, iron-sulfur cluster biogenesis, and DNA repair. Surprisingly, and in contrast with Escherichia coli studies, moxifloxacin decreased expression of genes involved in respiration, suppressed oxygen consumption, increased the NADH/NAD+ ratio, and increased the labile iron pool in M. tuberculosis. Lowering the NADH/NAD+ ratio in M. tuberculosis revealed that NADH-reductive stress facilitates an iron-mediated ROS surge and moxifloxacin lethality. Treatment with N-acetyl cysteine (NAC) accelerated respiration and ROS production, increased moxifloxacin lethality, and lowered the mutant prevention concentration. Moxifloxacin induced redox stress in M. tuberculosis inside macrophages, and co-treatment with NAC potentiated the anti-mycobacterial efficacy of moxifloxacin during nutrient starvation, inside macrophages, and in mice where NAC restricted the emergence of resistance. Thus, oxidative stress, generated in a novel way, contributes to moxifloxacin-mediated killing of M. tuberculosis. The results open a way to make fluoroquinolones more effective anti-tuberculosis agents and provide a mechanistic basis for NAC-mediated enhancement of fluoroquinolone lethality in vitro and in vivo. Author Summary A new paradigm was revealed for stress-mediated bacterial death in which moxifloxacin treatment of M. tuberculosis decreases respiration rate (respiration increases in E. coli). Although moxifloxacin-induced, ROS-mediated bacterial death was observed, it derived from elevated levels of NADH and iron, a phenomenon not seen with antibiotic-treated E. coli. Nevertheless, stimulation of respiration and ROS by N-acetyl cysteine (NAC) enhanced moxifloxacin-mediated killing of M. tuberculosis, thereby reinforcing involvement of ROS in killing. NAC stimulation of moxifloxacin-mediated killing of M. tuberculosis and restriction of the emergence of resistance in a murine model of infection emphasize the importance of lethal action against pathogens. The work, plus published benefits of NAC to TB patients, encourage studies of NAC-based enhancement of fluoroquinolones.

microbiology↗

bioRxiv2022

A natural broad-spectrum inhibitor of enveloped virus entry, effective against SARS-CoV-2 and Influenza A Virus in preclinical animal models.

The COVID-19 pandemic has highlighted the need for novel antivirals for pandemic management and preparedness. Targeting host processes that are co-opted by viruses is an attractive strategy for developing antivirals with a high resistance barrier. Picolinic acid (PA) is a byproduct of tryptophan metabolism, endogenously produced in humans and other mammals. Here we report broad-spectrum antiviral effects of PA against enveloped viruses, including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), Influenza A virus (IAV), Flaviviruses, Herpes Simplex Virus, and Human Parainfluenza Virus. We further demonstrate using animal models that PA is effective against SARS-CoV-2 and IAV, especially as an oral prophylactic. The mode of action studies revealed that PA inhibits viral entry of enveloped viruses, primarily by interfering with viral-cellular membrane fusion, inhibiting virus-mediated syncytia formation, and dysregulating cellular endocytosis. Overall, our data establish PA as a broad-spectrum antiviral agent, with promising preclinical efficacy against pandemic viruses SARS-CoV-2 and IAV.

microbiology↗

bioRxiv2021

PRMT5 epigenetically regulates the E3 ubiquitin ligase ITCH to influence lipid accumulation during mycobacterial infection

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), triggers enhanced accumulation of lipids to generate foamy macrophages (FMs). This process has been often attributed to the surge in the expression of lipid influx genes with a concomitant decrease in those involved in lipid efflux genes. Here, we define an Mtb-orchestrated modulation of the ubiquitination mechanism of lipid accumulation markers to enhance lipid accretion during infection. We find that Mtb infection represses the expression of the E3 ubiquitin ligase, ITCH, resulting in the sustenance of key lipid accrual molecules viz. ADRP and CD36, that are otherwise targeted by ITCH for proteasomal degradation. In line, overexpressing ITCH in Mtb-infected cells was found to suppress Mtb-induced lipid accumulation. Molecular analyses including loss-of-function and ChIP assays demonstrated a role for the concerted action of the transcription factor YY1 and the arginine methyl transferase PRMT5 in restricting the expression of Itch gene by conferring repressive symmetrical H4R3me2 marks on its promoter. Consequently, siRNA-mediated depletion of YY1 or PRMT5 rescued ITCH expression, thereby compromising the levels of Mtb-induced ADRP and CD36 and limiting FM formation during infection. Accumulation of lipids within the host has been implicated as a pro-mycobacterial process that aids in pathogen persistence and dormancy. In our study, perturbation of PRMT5 enzyme activity resulted in compromised lipid levels and reduced mycobacterial survival in primary murine macrophages (ex vivo) and in a therapeutic mouse model of TB infection (in vivo). These findings provide new insights on the role of PRMT5 and YY1 in augmenting mycobacterial pathogenesis. Thus, we posit that our observations could help design novel adjunct therapies and combinatorial drug regimen for effective anti-TB strategies. Author SummaryMycobacterium tuberculosis generates lipid-laden cells (foamy macrophages-FMs) that offer a favorable shelter for its persistence. During infection, we observe a significant reduction in the expression of the E3 ubiquitin ligase, ITCH. This repression allows the sustenance of key lipid accretion molecules (ADRP and CD36), by curbing their proteasomal degradation. Further, we show the repression of ITCH to be dependent on the concerted action of the bifunctional transcription factor, YY1 and the arginine methyl transferase, PRMT5. NOTCH signaling pathway was identified as a master-regulator of YY1 expression. In vitro and in vivo analyses revealed the significance of PRMT5 in regulating FM formation and consequently mycobacterial burden.

microbiology↗

bioRxiv2021

Preexisting mycobacterial infection modulates Candida albicans-driven pyroptosis

Active tuberculosis patients are at high risk of co-infection with opportunistic fungal pathogen C. albicans. However, the molecular mechanisms that orchestrate pathogenesis of Mycobacterium tuberculosis (Mtb)-C. albicans co-infection remains elusive. In the current study, we utilise a mouse model to demonstrate that Mtb promotes macrophage environment conducive for C. albicans survival. Mtb-dependent PKC{zeta}-WNT signalling axis induces expression of an E3 ubiquitin ligase, COP1. A secondary infection of C. albicans in such Mtb-infected macrophages causes COP1 to mediate the proteasomal degradation of IRF9, a cardinal factor that we identified to arbitrate an inflammatory programmed cell death, pyroptosis. In vivo experiments mimicking a preexisting Mtb infection demonstrate that inhibition of pyroptosis in mice results in increased C. albicans burden and aberrant lung tissue architecture, leading to increased host mortality. Together, our study reveals the crucial role of pyroptosis regulation for manifesting a successful C. albicans-Mtb co-infection.

microbiology↗

bioRxiv2021

G9a and Sirtuin6 epigenetically modulate host cholesterol accumulation to facilitate mycobacterial survival

Cholesterol derived from the host milieu forms a critical factor for mycobacterial pathogenesis. However, the molecular circuitry co-opted by Mycobacterium tuberculosis (Mtb) to accumulate cholesterol in host cells remains obscure. Here, we report that a functional amalgamation of WNT-responsive histone modifiers G9a (H3K9 methyltransferase) and Sirt6 (H3K9 deacetylase) orchestrate cholesterol build-up in in-vitro and in-vivo models of Mtb infection. Mechanistically, G9a, along with SREBP2, drives the expression of cholesterol biosynthesis and uptake genes; while Sirt6 represses the genes involved in cholesterol efflux. The accumulated cholesterol promotes the expression of antioxidant genes leading to reduced oxidative stress, thereby supporting Mtb survival. In corroboration, loss-of-function of G9a in vitro and in vivo by pharmacological inhibition; or utilization of BMDMs derived from Sirt6 KO mice or in vivo infection in Sirt6 heterozygous mice; hampers host cholesterol accumulation and restricts Mtb burden. These findings shed light on the novel roles of G9a and Sirt6 during Mtb infection and highlight the previously unknown contribution of host cholesterol in potentiating anti-oxidative responses for aiding Mtb survival.

microbiology↗

bioRxiv2020

Aging associated altered response to intracellular bacterial infections and its implication on the host

The effects of senescence and aging on geriatric diseases has been well explored but how these influence infections in the elderly have been scarcely addressed. Here, we show that several innate immune responses are elevated in senescent cells and old mice, allowing them to promptly respond to bacterial infections. We have identified higher levels of iNOS as a crucial host response and show that p38 MAPK in senescent cells acts as a negative regulator of iNOS transcription. In old mice, however the ability to impede bacterial proliferation does not correlate with increased survival as elevated immune responses persist unabated eventually affecting the host. The use of anti-inflammatory drugs that could consequently be recommended also decreases iNOS disarming the host of a critical innate immune response. Overall, our study highlights that infection associated mortality in the elderly is not merely an outcome of pathogen load but is also influenced by the hosts ability to resolve inflammation induced damage. Summary statementUsing cellular models and old mice we demonstrate the effect of aging on host response to bacterial infections. Aged systems mount a more effective anti-bacterial innate immune response but its persistence results in mortality of the host.

microbiology↗

bioRxiv2020

Antioxidant nanozyme counteracts HIV-1 by modulating intracellular redox potential

Reactive 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-{kappa}B and FOS), inflammation, and apoptosis. Importantly, a combination of V2O5-nanosheets with a pharmacological inhibitor of NF-{kappa}B (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.

bioengineering↗

bioRxiv2020

Nitric oxide-inducible Fe-S cluster biogenesis enables Mycobacterium tuberculosis to persist in mice

The persistence of Mycobacterium tuberculosis (Mtb) is a major problem in managing tuberculosis (TB). Host-generated nitric oxide (NO) is perceived as one of the signals by Mtb to reprogram metabolism and respiration for persistence. However, the mechanisms involved in NO sensing and reorganizing Mtbs physiology are not fully understood. Since NO damages Fe-S clusters of essential enzymes, the mechanism(s) involved in regulating iron-sulfur (Fe-S) cluster biogenesis could help Mtb persist in host tissues. Here, we show that a transcription factor SufR (Rv1460) senses NO via its 4Fe-4S cluster and promotes persistence of Mtb by mobilizing the Fe-S cluster biogenesis system; suf operon (Rv1460-Rv1466). Analysis of anaerobically purified SufR by UV-visible spectroscopy, circular dichroism, and iron-sulfide estimation confirms the presence of a 4Fe-4S cluster. Atmospheric O2 and H2O2 gradually degrade the 4Fe-4S cluster of SufR. Furthermore, electron paramagnetic resonance (EPR) analysis demonstrates that NO directly targets SufR 4Fe-4S cluster by forming a protein-bound dinitrosyl-iron-dithiol complex. DNase I footprinting, gel-shift, and in vitro transcription assays confirm that SufR directly regulates the expression of the suf operon in response to NO. Consistent with this, RNA- sequencing of Mtb{Delta}sufR demonstrates deregulation of the suf operon under NO stress. Strikingly, NO inflicted irreversible damage upon Fe-S clusters to exhaust respiratory and redox buffering capacity of Mtb {Delta}sufR. Lastly, Mtb{Delta} sufR failed to recover from a NO-induced non-growing state and displayed persistence defect inside immune-activated macrophages and murine lungs in a NO-dependent manner. Data suggest that SufR is a sensor of NO that supports persistence by reprogramming Fe-S cluster metabolism and bioenergetics. HighlightsO_LIMycobacterium tuberculosis (Mtb) induces the expression of suf operon for Fe-S cluster biogenesis in response to nitric oxide (NO). C_LIO_LIWe found that a transcription factor SufR senses NO via its 4Fe-4S cluster and regulates the expression of the suf operon for Fe-S cluster biogenesis. C_LIO_LISufR-regulated Fe-S cluster biogenesis confers respiratory and redox features that promote recovery of Mtb from NO stress. C_LIO_LISufR activity is required to support the NO-dependent persistence of Mtb in macrophages and mice. C_LI

microbiology↗

bioRxiv2017

Efficacy of the β-lactam\β-lactamase inhibitor combination is linked to WhiB4 mediated changes in redox physiology of Mycobacterium tuberculosis

AimsInhibition of {beta}-lactamase by clavulanate (Clav) sensitizes multi-and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains towards {beta}-lactams such as amoxicillin (Amox). However, the underlying mechanism of how Mtb responds to Amox-Clav combination (Augmentin; AG) is not characterized.\n\nResultsWe integrated global expression profiling with the protein-protein interaction landscape and generated a genome-scale network of Mtb in response to AG. In addition to specific targets (e.g., peptidoglycan biosynthesis and {beta}-lactamase), the response to AG was also centered on redox-balance, central carbon metabolism (CCM), and respiration in Mtb. We discovered that AG modulates superoxide levels, NADH/NAD+ balance and mycothiol redox potential (EMSH) of Mtb. Higher intra-mycobacterial EMSH potentiates mycobactericidal efficacy of AG, whereas lower EMSH induces tolerance. Further, Mtb responds to AG via a redox-sensitive transcription factor, WhiB4. Mtb{Delta}whiB4 displayed higher expression of genes involved in {beta}-lactam resistance along with those mediating respiration, CCM and redox balance. Moreimportantly, WhiB4 binds to the promoter regions and represses transcription of genes involved in {beta}-lactamase expression in a redox-dependent manner. Lastly, while Mtb{Delta}whiB4 maintained internal EMSH, exhibited greater {beta}-lactamase activity and displayed AG-tolerance, overexpression of WhiB4 induced oxidative shift in EMSH and repressed {beta}-lactamase activity to aggravate AG-mediated killing of drug-sensitive and -resistant strains of Mtb.\n\nInnovation and ConclusionsThis work demonstrate that efficacy of {beta}-lactam\\{beta}-lactamase inhibitor combination can be attenuated by elevating mycobacterial antioxidant capabilities and potentiated by impairing redox buffering capacity of Mtb. The functional linkage between {beta}-lactams, redox balance, and WhiB4 can be exploited to potentiate AG action against drug-resistant Mtb.

microbiology↗