Comprehensive polyphenolic profiling, antioxidant, antimicrobial, and cytotoxicity studies of Rhynchostylis retusa (L.) Blume: a medicinal orchid.

Rhynchostylis retusa (L.) Blume, commonly known as the Foxtail orchid, has garnered worldwide attention for its diverse medicinal properties. In this study, root extract and its fractions were evaluated for total polyphenols, flavonoids, targeted polyphenols, and antioxidant potential. The antimicrobial activity was assessed against Gram-positive and Gram-negative bacterial strains while cytotoxicity was assessed using the A549 and HCT-116 cell lines. The investigations showed that chloroform and ethyl acetate are the most effective solvents for fractionation of polyphenols from the parent extract. These fractions also exhibited strong antioxidant and cytotoxic potentials. The chloroform fraction showed maximum cell death of 87.35 and 92.36% in A549 and HCT- 116 cell lines respectively. All samples showed growth inhibition against bacterial strains except the n-hexane fraction, whereas the n-butanol fraction showed comparable antimicrobial activity with the tetracycline standard. The possible health benefits and thereby, application of R. retusa were thus revealed in this investigation.

Biapenem, a Carbapenem Antibiotic, Elicits Mycobacteria Specific Immune Responses and Reduces the Recurrence of Tuberculosis.

Tuberculosis (TB) still tops the list of global health burdens even after COVID-19. However, it will sooner transcend the current pandemic due to the prevailing risk of reactivation of latent TB in immunocompromised individuals. The indiscriminate misuse and overuse of antibiotics have resulted in the emergence of deadly drug-resistant variants of Mycobacterium tuberculosis (M.tb). This study aims to characterize the functionality of the carbapenem antibiotic-Biapenem (BPM) in generating long-lasting immunity against TB. BPM treatment significantly boosted the activation status of the innate immune arm-macrophages by augmenting p38 signaling. Macrophages further primed and activated the adaptive immune cells CD4+ and CD8+ T-cells in the lung and spleen of the infected mice model. Furthermore, BPM treatment significantly amplified the polarization of T lymphocytes toward inflammatory subsets, such as Th1 and Th17. The treatment also helped generate a long-lived central memory T-cell subset. The generation of central memory T lymphocyte subset upon BPM treatment in the murine model led to a significant curtailing in the recurrence of TB due to reactivation and reinfection. These results suggest the potentiality of BPM as a potent adjunct immunomodulator to improve host defense against M.tb by enriching long-term protective memory cells. IMPORTANCE Tuberculosis (TB) caused by Mycobacterium tuberculosis (M.tb) tops the list of infectious killers around the globe. The emergence of drug-resistant variants of M.tb has been a major hindrance toward realizing the "END TB" goal. Drug resistance has amplified the global burden toward the quest for novel drug molecules targeting M.tb. Host-directed therapy (HDT) offers a lucrative alternative to tackle emerging drug resistance and disease relapse by strengthening the host's immunity. Through our present study, we have tried to characterize the functionality of the carbapenem antibiotic-Biapenem (BPM). BPM treatment significantly augmented long-lasting immunity against TB by boosting the innate and adaptive immune arms. The generation of long-lived central memory T lymphocyte subset significantly improved the disease outcome and provided sterilizing immunity in the murine model of TB. The present investigation's encouraging results have helped us depict BPM as a potent adjunct immunomodulator for treating TB.

Berberine governs NOTCH3/AKT signaling to enrich lung-resident memory T cells during tuberculosis.

Stimulation of naïve T cells during primary infection or vaccination drives the differentiation and expansion of effector and memory T cells that mediate immediate and long-term protection. Despite self-reliant rescue from infection, BCG vaccination, and treatment, long-term memory is rarely established against Mycobacterium tuberculosis (M.tb) resulting in recurrent tuberculosis (TB). Here, we show that berberine (BBR) enhances innate defense mechanisms against M.tb and stimulates the differentiation of Th1/Th17 specific effector memory (TEM), central memory (TCM), and tissue-resident memory (TRM) responses leading to enhanced host protection against drug-sensitive and drug-resistant TB. Through whole proteome analysis of human PBMCs derived from PPD+ healthy individuals, we identify BBR modulated NOTCH3/PTEN/AKT/FOXO1 pathway as the central mechanism of elevated TEM and TRM responses in the human CD4+ T cells. Moreover, BBR-induced glycolysis resulted in enhanced effector functions leading to superior Th1/Th17 responses in human and murine T cells. This regulation of T cell memory by BBR remarkably enhanced the BCG-induced anti-tubercular immunity and lowered the rate of TB recurrence due to relapse and re-infection. These results thus suggest tuning immunological memory as a feasible approach to augment host resistance against TB and unveil BBR as a potential adjunct immunotherapeutic and immunoprophylactic against TB.

Withaferin A Protects against Primary and Recurrent Tuberculosis by Modulating Mycobacterium-Specific Host Immune Responses.

The fate of Mycobacterium tuberculosis infection is governed by immune signaling pathways that can either eliminate the pathogen or result in tuberculosis (TB). Anti-TB therapy (ATT) is extensive and is efficacious only against active, drug-sensitive strains of M. tuberculosis. Due to severe side effects, ATT often causes impairment of host immunity, making it imperative to use novel immunotherapeutics for better clinical outcomes. In this study, we have explored the immunomodulatory potential of withaferin A (WA) as an immunotherapeutic against TB. Here, we demonstrate that WA can constrain intracellular drug-sensitive and -resistant strains of M. tuberculosis by augmenting host immune responses. We also established the potential of WA treatment in conjunction with isoniazid. We show that WA directs the host macrophages toward defensive M1 polarization and enhances TH1 and TH17 immune responses against M. tuberculosis infection. The reduced bacterial burden upon T cell adoptive transfer further corroborated the augmented T cell responses. Interestingly, WA stimulated the generation of T cell memory populations by instigating STAT signaling, thereby reducing the rate of TB recurrence due to reactivation and reinfection. We substantiate the prospects of WA as a potent adjunct immunomodulator that enriches protective memory cells by prompting STAT signaling and improves host defense against M. tuberculosis. IMPORTANCE Despite being extensive, conventional antituberculosis therapy (ATT) is barely proficient in providing sterile immunity to tuberculosis (TB). Failure to constrain the escalating global TB burden due to the emergence of drug-resistant bacterial strains and immune dampening effects of ATT necessitates adjunct immunotherapeutics for better clinical outcomes. We evaluated the prospects of withaferin A (WA), an active constituent of Withania somnifera, as an adjunct immunomodulator against diverse M. tuberculosis strains. WA efficiently restricts the progression of TB by stimulating antimycobacterial host responses, protective immune signaling, and activation of diverse immune cell populations. Protective effects of WA can be attributed to the enrichment of memory T cells by induction of STAT signaling, thereby enhancing resistance to reinfections and reactivation of disease. We ascertained the immunotherapeutic potential of WA in boosting host immune responses against M. tuberculosis.

The 1, 2-ethylenediamine SQ109 protects against tuberculosis by promoting M1 macrophage polarization through the p38 MAPK pathway.

Directly Observed Treatment Short-course (DOTs), is an effective and widely recommended treatment for tuberculosis (TB). The antibiotics used in DOTs, are immunotoxic and impair effector T cells, increasing the risk of re-infections and reactivation. Multiple reports suggest that addition of immune-modulators along with antibiotics improves the effectiveness of TB treatment. Therefore, drugs with both antimicrobial and immunomodulatory properties are desirable. N1-(Adamantan-2-yl)-N2-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]ethane-1,2-diamine (SQ109) is an asymmetric diamine derivative of adamantane, that targets Mycobacterial membrane protein Large 3 (MmpL3). SQ109 dissipates the transmembrane electrochemical proton-gradient necessary for cell-wall biosynthesis and bacterial activity. Here, we examined the effects of SQ109 on host-immune responses using a murine TB model. Our results suggest the pro-inflammatory nature of SQ109, which instigates M1-macrophage polarization and induces protective pro-inflammatory cytokines through the p38-MAPK pathway. SQ109 also promotes Th1 and Th17-immune responses that inhibit the bacillary burden in a murine model of TB. These findings put forth SQ109 as a potential-adjunct to TB antibiotic therapy.

The Indigenous Volatile Inhibitor 2-Methyl-2-butene Impacts Biofilm Formation and Interspecies Interaction of the Pathogenic Mucorale Rhizopus arrhizus.

2-Methyl-2-butene has recently been reported to be a quorum-based volatile self-inhibitor of spore germination and growth in pathogenic Mucorale Rhizopus arrhizus. The present study aimed to elucidate if this compound can influence R. arrhizus biofilm formation and interspecies interaction. The compound was found to significantly decrease R. arrhizus biofilm formation (p < 0.001), with nearly 25% and 50% lesser biomass in the biofilms cultured with exposure to 4 and 32 µg/ml of 2-methyl-2-butene, respectively. The growth of pre-formed biofilms was also impacted, albeit to a lesser extent. Additionally, 2-methyl-2-butene was found to self-limit R. arrhizus growth during interspecies interaction with Staphylococcus aureus and was detected at a substantially greater concentration in the headspace of co-cultures (2338.75 µg/ml) compared with monocultures (69.52 µg/ml). Some of the C5 derivatives of this compound (3-methyl-1-butanol, 2-methyl-2-butanol, and 3-methyl-1-butyne) were also observed to partially mimic its action, such as inhibition of spore germination, but did not impact R. arrhizus biofilm formation. Finally, the treated R. arrhizus displayed changes in fungal morphology suggestive of cytoskeletal alterations, such as filopodia formation, blebs, increased longitudinal folds and/or corrugations, and finger-like and sheet-like surface protrusions, depending upon the concentration of the compound(s) and the planktonic or biofilm growth mode.

Epigenetic code during mycobacterial infections: therapeutic implications for tuberculosis.

Epigenetics involves changing the gene function without any change in the sequence of the genes. In the case of tuberculosis (TB) infections, the bacilli, Mycobacterium tuberculosis (M.tb), uses epigenetics as a tool to protect itself from the host immune system. TB is a deadly disease-causing maximum death per year due to a single infectious agent. In the case of TB, there is an urgent need for novel host-directed therapies which can effectively target the survival and long-term persistence of the bacteria without developing drug resistance in the bacterial strains while also reducing the duration and toxicity associated with the mainstream anti-TB drugs. Recent studies have suggested that TB infection has a significant effect on the host epigenome thereby manipulating the host immune response in the favor of the pathogen. M.tb alters the activation status of key genes involved in the immune response against TB to promote its survival and subvert the antibacterial strategies of the host. These changes are reversible and can be exploited to design very efficient host-directed therapies to fight against TB. This review has been written with the purpose of discussing the role of epigenetic changes in TB pathogenesis and the therapeutic approaches involving epigenetics, which can be utilized for targeting the pathogen.

Correction: Luteolin-mediated Kv1.3 K+ channel inhibition augments BCG vaccine efficacy against tuberculosis by promoting central memory T cell responses in mice.

[This corrects the article DOI: 10.1371/journal.ppat.1008887.].

Nasal Microbiota Imbalance as a Contributory Link in the Emergence of COVID-19 Associated Mucormycosis.

COVID-19 associated mucormycosis (CAM) is being reported at an elevated frequency and has been declared an ongoing epidemic in India. A huge diabetic population, inappropriate corticosteroid usage and environmental mucoralean spore count, along with COVID-19 associated glycemic imbalance, hypoxemia, increased iron levels, vascular endothelial injury, as well as the immunosuppressive impact are being considered as important risk factors for CAM. The present viewpoint aims to discuss the plausible role of another important facet, the nasal microbiota imbalance, in the emergence of mucormycosis under the prevailing COVID-19 pandemic conditions.

Advances in adjunct therapy against tuberculosis: Deciphering the emerging role of phytochemicals.

Eastern countries are a major source of medicinal plants, which set up a rich source of ethnopharmacologically known medicines used in the treatment of various diseases. These traditional medicines have been known as complementary, alternative, or nonconventional therapy across globe for ages. Tuberculosis (TB) poses a huge global burden and leads to maximum number of deaths due to an infectious agent. Treatment of TB using Directly Observed Treatment Short-course (DOTS) therapy comprises multiple antibiotics is quite lengthy and causes serious side-effects in different organs. The length of the TB treatment leads to withdrawal from the patients, which paves the way for the emergence of drug resistance in the bacterial population. These concerns related to therapy need serious and immediate interventions. Traditional medicines using phytochemicals has shown to provide tremendous potential in TB treatment, mainly in the eradication of Mycobacterium tuberculosis (M.tb), increasing natural immunity, and managing the side effects of anti-TB drugs. This review describes the antituberculosis potential of selected ethnopharmacologically important phytochemicals as potential immune-modulator and as an adjunct-therapy in TB. This review will be a useful reference for researchers working on ethnopharmacology and will open the door for the discovery of novel agents as an adjunct-therapy to tuberculosis.

Luteolin-mediated Kv1.3 K+ channel inhibition augments BCG vaccine efficacy against tuberculosis by promoting central memory T cell responses in mice.

Despite the availability of multiple antibiotics, tuberculosis (TB) remains a major health problem worldwide, with one third of the population latently infected and ~2 million deaths annually. The only available vaccine for TB, Bacillus Calmette Guérin (BCG), is ineffective against adult pulmonary TB. Therefore, alternate strategies that enhance vaccine efficacy are urgently needed. Vaccine efficacy and long-term immune memory are critically dependent on central memory T (TCM) cells, whereas effector memory T (TEM) cells are important for clearing acute infections. Recently, it has been shown that inhibition of the Kv1.3 K+ ion channel, which is predominantly expressed on TEM but not TCM cells, profoundly enhances TCM cell differentiation. We exploited this phenomenon to improve TCM:TEM cell ratios and protective immunity against Mycobacterium tuberculosis infection in response to BCG vaccination of mice. We demonstrate that luteolin, a plant-derived Kv1.3 K+ channel inhibitor, profoundly promotes TCM cells by selectively inhibiting TEM cells, and significantly enhances BCG vaccine efficacy. Thus, addition of luteolin to BCG vaccination may provide a sustainable means to improve vaccine efficacy by boosting host immunity via modulation of memory T cell differentiation.

[6]-Gingerol exhibits potent anti-mycobacterial and immunomodulatory activity against tuberculosis.

The currently available anti-tuberculosis treatment (ATT) comprises exclusively of anti-bacterial drugs, is very lengthy, has adverse side effects on the host and leads to the generation of drug-resistant variants. Therefore, a combination therapy directed against the pathogen and the host is required to counter tuberculosis (TB). Here we demonstrate that [6]-Gingerol, one of the most potent and pharmacologically active ingredients of ginger restricted mycobacterial growth inside the lungs, spleen and liver of mice infected with Mycobacterium tuberculosis (Mtb). The spleen of [6]-Gingerol treated mice displayed increased expression of pro-inflammatory cytokines and enhanced Th1/Th17 responses confirming the immunomodulatory action of [6]-Gingerol. Finally, [6]-Gingerol displayed an excellent potential as an adjunct drug, along with front line anti-TB drug isoniazid. Interestingly, [6]-Gingerol displayed stark anti-tubercular activity against dormant/starved bacilli and drug-resistant variants of Mtb. Taken together, these results indicate strong prospects of [6]-Gingerol as an adjunct anti-mycobacterial and immunomodulatory drug for the treatment of drug-susceptible and drug-resistant strains of TB.

Tuberculosis vaccine: A journey from BCG to present.

Tuberculosis (TB) is the leading cause of death worldwide due to an infectious disease, causing around 1.6 million deaths each year. This situation has become more complicated by the emergence of drug-resistant Mycobacterium tuberculosis (M.tb) and HIV-TB co-infection, which has significantly worsened TB prognosis and treatment. Despite years of intensive research, Bacille Calmette-Guerin (BCG) remains the only licensed vaccine and has variable efficacy. It provides protection against childhood TB but is not effective in adult pulmonary TB. As a result of intense research in understanding TB vaccinology, there are many new vaccine candidates in clinical development and many more in pre-clinical trials which aim either to replace or boost BCG vaccine. This review discusses the history of BCG vaccine development and summarizes limitations of the current vaccine strategy and recent advances in improving BCG immunization along with other new vaccines in clinical trials which are promising candidates for the future tuberculosis vaccinology program.

Facially Amphiphilic Cholic Acid-Lysine Conjugates as Promising Antimicrobials.

The emergence of multidrug-resistant microbes is a significant health concern posing a constant need for new antimicrobials. Membrane-targeting antibiotics are promising candidates with reduced ability of microbes to develop resistance. In the present investigation, the principal reason behind choosing cholic acid as the crucial scaffold lies in the fact that it has a facially amphiphilic nature, which provides ample opportunity to refine the amphiphilicity by linking the amino acid lysine. A total of 16 novel amphipathic cholic acid derivatives were synthesized by sequentially linking lysine to C3-ß-amino cholic acid methyl ester to maintain the hydrophobic/hydrophilic balance, which could be the essential requirement for the antimicrobial activity. Among the synthesized conjugates, a series with fluorenyl-9-methoxycarbonyl moiety attached to cholic acid via lysine linker showed promising antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Candida albicans. A pronounced effect of increase in lysine residues was noted on the observed activity. The lead compounds were found to be active against drug-resistant bacterial and fungal clinical isolates and also improved the efficacy of antifungal agents amphotericin B and voriconazole. Membrane-permeability studies demonstrated the ability of these compounds to induce membrane damage in the tested microbes. The active conjugates did not show any hemolytic activity and were also found to be nontoxic to the normal cells as well as the examined cancer cell lines. The observed antimicrobial activity was attributed to the facial amphiphilic conformations, hydrophobic/hydrophilic balance, and the overall charge on the molecules.

Antagonistic Interaction of Staphylococcus aureus and Staphylococcus epidermidis with Rhizopus arrhizus Mediated by Phenol Soluble Modulins and Organic Acids.

Rhizopus arrhizus (R. arrhizus) is a common causative agent of mucormycosis that usually enters the human body through the respiratory tract and skin. Both these sites harbor staphylococci as a part of the normal microflora, indicating the possibility of interspecies interactions. We aimed to elucidate this interaction and identify the molecular mechanisms involved. Both Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) substantially hindered R. arrhizus radial growth, spore germination, and liquid culture biomass. Secreted components in the stationary-phase supernatant were responsible for this activity. The active components, based on molecular weight-based fractionation, mass spectrometry, and ion exclusion chromatography, were identified as a truncated version of phenol soluble modulin α2 (Δ1Δ2PSMα2) and PSMα3 in S. aureus, PSMδ in S. epidermidis, and organic acids in both the species. Exposure to the phenol soluble modulins (PSMs) extensively damaged the fungal spores and pre-existing hyphae, leading to bleb formation, shriveling, hyphal shrinkage, and cell distortion.

Medically important interactions of staphylococci with pathogenic fungi.

Staphylococci are common inhabitants at several human body sites and are also implicated in infections either as primary or opportunistic pathogens. These bacteria can thus both contribute to the host defense being a part of the commensalistic microbiota or synergize with the other microbes during the infection process. Among fungi, staphylococci interact synergistically with Candida spp. and Aspergillus fumigatus, and antagonistically with Cryptococcus neoformans and Trichosporon asahii. These interactions are highly dynamic and are orchestrated by a multitude of microbial and host factors. During such cross-talks, staphylococci can modulate the virulence, immune response or drug resistance of the coexisting microbe(s), thereby influencing the infection course, disease severity, treatment strategy and the clinical outcome.

Relevance of antifungal penetration in biofilm-associated resistance of Candida albicans and non-albicans Candida species.

The role of penetration limitation in Candida biofilm-associated antifungal resistance remains unclear. Most of the previous work has been done on Candida albicans, although non-albicans (NAC) species are also implicated in invasive candidiasis and the biofilm matrix has been shown to vary amongst different species. Only a few studies have evaluated clinical isolates. This study aimed to determine the relevance of penetration limitation in the antifungal resistance of biofilms formed by C. albicans and NAC clinical isolates, using an agar disk diffusion assay. The penetration of posaconazole and amphotericin B through the biofilms was significantly reduced. Fluconazole, voriconazole and caspofungin showed a superior penetration capacity in C. albicans, Candida tropicalis and Candida parapsilosis biofilms, but exhibited inter-species and strain/isolate variation. Candida krusei biofilms were the most resilient to antifungal permeation. All of the antifungal drugs failed to kill the biofilm cells, independent of penetration, suggesting that the other factors contribute markedly to the recalcitrance of the biofilms.

Role of biofilm morphology, matrix content and surface hydrophobicity in the biofilm-forming capacity of various Candida species.

The present study aimed to evaluate the role of biofilm morphology, matrix content and surface hydrophobicity in the biofilm-forming capacity of Candida albicans and non-albicans Candida (NAC) spp. Biofilm formation was determined by microtitre plate assay and bright-field and scanning electron microscopy. The matrix carbohydrates, proteins and e-DNA were quantified by phenol-sulfuric acid, bicinchoninic acid and UV spectroscopy, respectively. Specific glycosyl residues were detected by dot blot. The cell-surface hydrophobicity was determined by hydrocarbon adhesion assay. Candida tropicalis was found to exhibit the highest adherence to polystyrene. It formed dense biofilms with extensive pseudohyphae and hyphal elements, high hydrophobicity and the greatest amount of matrix carbohydrates, proteins and e-DNA. C. albicans displayed higher adherence and a complex biofilm morphology with larger aggregates than Candida parapsilosis and Candida krusei, but had lower matrix content and hydrophobicity. Thus, the combinatorial effect of increased filamentation, maximum matrix content and high hydrophobicity contributes to the enhanced biofilm-forming capacity of C. tropicalis.