Identification of genes associated with persistence in Mycobacterium smegmatis.

The prevalence of bacterial persisters is related to their phenotypic diversity and is responsible for the relapse of chronic infections. Tolerance to antibiotic therapy is the hallmark of bacterial persistence. In this study, we have screened a transposon library of Mycobacterium smegmatis mc2155 strain using antibiotic tolerance, survival in mouse macrophages, and biofilm-forming ability of the mutants. Out of 10 thousand clones screened, we selected ten mutants defective in all the three phenotypes. Six mutants showed significantly lower persister abundance under different stress conditions. Insertions in three genes belonging to the pathways of oxidative phosphorylation msmeg_3233 (cydA), biotin metabolism msmeg_3194 (bioB), and oxidative metabolism msmeg_0719, a flavoprotein monooxygenase, significantly reduced the number of live cells, suggesting their role in pathways promoting long-term survival. Another group that displayed a moderate reduction in CFU included a glycosyltransferase, msmeg_0392, a hydrogenase subunit, msmeg_2263 (hybC), and a DNA binding protein, msmeg_2211. The study has revealed potential candidates likely to facilitate the long-term survival of M. smegmatis. The findings offer new targets to develop antibiotics against persisters. Further, investigating the corresponding genes in M. tuberculosis may provide valuable leads in improving the treatment of chronic and persistent tuberculosis infections.

The Pharmacological Implications of Flavopiridol: An Updated Overview.

Flavopiridol is a flavone synthesized from the natural product rohitukine, which is derived from an Indian medicinal plant, namely Dysoxylum binectariferum Hiern. A deeper understanding of the biological mechanisms by which such molecules act may allow scientists to develop effective therapeutic strategies against a variety of life-threatening diseases, such as cancer, viruses, fungal infections, parasites, and neurodegenerative diseases. Mechanistic insight of flavopiridol reveals its potential for kinase inhibitory activity of CDKs (cyclin-dependent kinases) and other kinases, leading to the inhibition of various processes, including cell cycle progression, apoptosis, tumor proliferation, angiogenesis, tumor metastasis, and the inflammation process. The synthetic derivatives of flavopiridol have overcome a few demerits of its parent compound. Moreover, these derivatives have much improved CDK-inhibitory activity and therapeutic abilities for treating severe human diseases. It appears that flavopiridol has potential as a candidate for the formulation of an integrated strategy to combat and alleviate human diseases. This review article aims to unravel the potential therapeutic effectiveness of flavopiridol and its possible mechanism of action.

Antibody engineering and its therapeutic applications.

As a natural function, antibodies defend the host from infected cells and pathogens by recognizing their pathogenic determinants. Antibodies (Abs) gained wide acceptance with an enormous impact on human health and have predominantly captured the arena of bio-therapeutics and bio-diagnostics. The scope of Ab-based biologics is vast, and it is likely to solve many unmet clinical needs in future. The majority of attention is now devoted to developing innovative technologies for manufacturing and engineering Abs, better suited to satisfy human needs. The advent of Ab engineering technologies (AET) led to phenomenal developments leading to the generation of Abs-/Ab-derived molecules with desirable functional properties proportional to their expanding requirements. Evolution brought by AET, from the naturally occurring Ab forms to several advanced Ab formats and derivatives, was much needed as it is of great interest to the pharmaceutical industry. Thus, numerous advancements in AET have propelled success in therapeutic Ab development, along with the potential for ever-increasing improvements. Unique characteristics of Abs, such as its diversity, specificity, structural integrity and an array of possible applications, together inspire continuous innovation in the field. Overall, the AET could assist in conquer of several limitations of Abs in terms of their applicability in the field of therapeutics, diagnostics and research; AET has so far led to the production of next-generation Abs, which have revolutionized these arenas. Here in this review, we discuss the various distinguished engineering platforms for Ab development and the progress in modern therapeutics by the so-called "next-generation Abs."

Biosensors for the detection of Mycobacterium tuberculosis: a comprehensive overview.

Tuberculosis (TB) infection is one of the leading causes of death in the world. According to WHO reports 2019, the average rate of decrease in global TB incidences was only 1.6% per year from 2000 to 2018, besides that the global decline in TB deaths was just 11%. Therefore, the dire need for early detection of the pathogen for the successful diagnosis of TB seems justified. Mycobacterium tuberculosis secretory proteins have gained more attention as TB biomarkers, for the early diagnosis and treatment of TB. Here in this review, we elaborate on the recent advancements made in the field of piezoelectric, magnetic, optical, and electrochemical biosensors, in addition to listing their merits and setbacks. Additionally, this review also discusses the construction of biosensors through modern integrated technologies, such as combinations of analytical chemistry, molecular biology, and nanotechnology. Integrated technologies enhance the detection for perceiving highly selective, specific, and sensitive signals to detect M. tuberculosis. Furthermore, this review highlights the recent challenges and scope of improvement in numerous biosensors developed for rapid, specific, selective, and sensitive detection of tuberculosis to reduce the TB burden and successful treatment.

Insights into the molecular determinants involved in Mycobacterium tuberculosis persistence and their therapeutic implications.

The establishment of persistent infections and the reactivation of persistent bacteria to active bacilli are the two hurdles in effective tuberculosis treatment. Mycobacterium tuberculosis, an etiologic tuberculosis agent, adapts to numerous antibiotics and resists the host immune system causing a disease of public health concern. Extensive research has been employed to combat this disease due to its sheer ability to persist in the host system, undetected, waiting for the opportunity to declare itself. Persisters are a bacterial subpopulation that possesses transient tolerance to high doses of antibiotics. There are certain inherent mechanisms that facilitate the persister cell formation in Mycobacterium tuberculosis, some of those had been characterized in the past namely, stringent response, transcriptional regulators, energy production pathways, lipid metabolism, cell wall remodeling enzymes, phosphate metabolism, and proteasome protein degradation. This article reviews the recent advancements made in various in vitro persistence models that assist to unravel the mechanisms involved in the persister cell formation and to hunt for the possible preventive or treatment measures. To tackle the persister population the immunodominant proteins that express specifically at the latent phase of infection can be used for diagnosis to distinguish between the active and latent tuberculosis, as well as to select potential drug or vaccine candidates. In addition, we discuss the genes engaged in the persistence to get more insights into resuscitation and persister cell formation. The in-depth understanding of persistent cells of mycobacteria can certainly unravel novel ways to target the pathogen and tackle its persistence.

Zur: Zinc-Sensing Transcriptional Regulator in a Diverse Set of Bacterial Species.

Zinc (Zn) is the quintessential d block metal, needed for survival in all living organisms. While Zn is an essential element, its excess is deleterious, therefore, maintenance of its intracellular concentrations is needed for survival. The living organisms, during the course of evolution, developed proteins that can track the limitation or excess of necessary metal ions, thus providing survival benefits under variable environmental conditions. Zinc uptake regulator (Zur) is a regulatory transcriptional factor of the FUR superfamily of proteins, abundant among the bacterial species and known for its intracellular Zn sensing ability. In this study, we highlight the roles played by Zur in maintaining the Zn levels in various bacterial species as well as the fact that in recent years Zur has emerged not only as a Zn homeostatic regulator but also as a protein involved directly or indirectly in virulence of some pathogens. This functional aspect of Zur could be exploited in the ventures for the identification of newer antimicrobial targets. Despite extensive research on Zur, the insights into its overall regulon and its moonlighting functions in various pathogens yet remain to be explored. Here in this review, we aim to summarise the disparate functional aspects of Zur proteins present in various bacterial species.

Delineation of the Residues of Bacillus anthracis Zinc Uptake Regulator Protein Directly Involved in Its Interaction with Cognate DNA.

Zinc uptake regulator (Zur) is a negative transcriptional regulator of bacteria that belongs to the FUR superfamily of proteins and regulates zinc (Zn) homeostasis under extreme Zn conditions. The Zur protein of Bacillus anthracis (BaZur) was though characterized previously, but the residues of this transcriptional regulator, crucial for binding to the consensus Zur box in the cognate DNA, remain unexplored. In this study, we reveal the essential residues of the protein that govern the specific interaction with the cognate DNA, through mutational and binding studies. In silico predicted model of the BaZur protein with the promoter region of one of the regulon candidates was utilized to identify specific residues of the N-terminal domain (NTD), constituting the DNA-binding recognition helix. Our results suggest that two phenylalanine residues, a non-polar aliphatic leucine and a positively charged arginine residue of NTD, are predominantly involved in DNA binding of BaZur. Among these, the arginine residue (Arg58) is conserved among all the Zur proteins and the two Phe residues, namely Phe53 and Phe63, are conserved in the Zur proteins of Staphylococcus aureus and Listeria monocytogenes. Taken together, the current study represents an in-depth investigation into the key DNA-binding residues involved in the BaZur-DNA interaction.

Anthrax prevention through vaccine and post-exposure therapy.

INTRODUCTION: Vaccines and therapeutic antibodies are the most crucial components of anthrax prophylaxis (pre- and post-exposure) and treatment. The improvement in the availability and safety profile of vaccines and the therapeutic antibodies has helped immensely in reducing the worldwide burden of anthrax. AREAS COVERED: Current recommendations for anthrax prophylaxis and control, vaccines and therapeutic antibodies, the recent endeavors, particularly, made after 2010 toward making them safer and more efficacious along with our opinion on its future course. Primarily, PubMed and Europe PMC were searched to cover the recent developments in the above-indicated areas. EXPERT OPINION: Some key existing lacunae in our understanding of the working of biologicals-based anthrax-control measures, i.e., vaccines and therapeutic antibodies, should be addressed to improve their overall stability, safety profile, and efficacy. The identification of novel inhibitors targeting different key-molecules and vital-steps contributing to the overall anthrax pathophysiology could make a difference in anthrax control.

Identification, Functional Characterization, and Regulon Prediction of the Zinc Uptake Regulator (zur) of Bacillus anthracis - An Insight Into the Zinc Homeostasis of the Pathogen.

Zinc has an abounding occurrence in the prokaryotes and plays paramount roles including catalytic, structural, and regulatory. Zinc uptake regulator (Zur), a Fur family transcriptional regulator, is connoted in maintaining zinc homeostasis in the pathogenic bacteria by binding to zinc and regulating the genes involved in zinc uptake and mobilization. Zinc homeostasis has been marginally scrutinized in Bacillus anthracis, the top-rated bio-terror agent, with no decipherment of the role of Zur. Of the three Fur family regulators in B. anthracis, BAS4181 is annotated as a zinc-specific transcriptional regulator. This annotation was further substantiated by our stringent computational and experimental analyses. The residues critical for zinc and DNA binding were delineated by homology modeling and sequence/structure analysis. ba zur existed as a part of a three-gene operon. Purified BaZur prodigiously existed in the dimeric form, indicated by size exclusion chromatography and blue native-polyacrylamide gel electrophoresis (PAGE). Computational and manual strategies were employed to decipher the putative regulon of ba zur, comprising of 11 genes, controlled by six promoters, each harboring at least one Zur box. The DNA binding capability of the purified BaZur to the upstream regions of the ba zur operon, yciC, rpmG, znuA, and genes encoding a GTPase cobalamine synthesis protein and a permease was ascertained by electrophoretic mobility shift assays. The regulon genes, implicated in zinc uptake and mobilization, were mostly negatively regulated by BaZur. The ba zur expression was downregulated upon exposure of cells to an excess of zinc. Conversely, it exhibited a marked upregulation under N, N, N', N'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) mediated zinc-depleted environment, adding credence to its negative autoregulation. Moreover, an increase in the transcript levels of the regulon genes znuA, rpmG, and yciC upon exposure of cells to TPEN connoted their role in combating hypo-zincemic conditions by bringing about zinc uptake and mobilization. Thus, this study functionally characterizes Zur of B. anthracis and elucidates its role in maintaining zinc homeostasis.

Basigin Interacts with Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 as a Second Erythrocyte Receptor to Promote Parasite Growth.

Elucidating the molecular mechanisms of the host-parasite interaction during red cell invasion by Plasmodium is important for developing newer antimalarial therapeutics. Recently, we have characterized a Plasmodium vivax tryptophan-rich antigen PvTRAg38, which is expressed by its merozoites, binds to host erythrocytes, and interferes with parasite growth. Interaction of this parasite ligand with the host erythrocyte occurs through its two regions present at amino acid positions 167-178 (P2) and 197-208 (P4). Each region recognizes its own erythrocyte receptor. Previously, we identified band 3 as the chymotrypsin-sensitive erythrocyte receptor for the P4 region, but the other receptor, binding to P2 region, remained unknown. Here, we have identified basigin as the second erythrocyte receptor for PvTRAg38, which is resistant to chymotrypsin. The specificity of interaction between PvTRAg38 and basigin was confirmed by direct interaction where basigin was specifically recognized by P2 and not by the P4 region of this parasite ligand. Interaction between P2 and basigin is stabilized through multiple amino acid residues, but Gly-171 and Leu-175 of P2 were more critical. These two amino acids were also critical for parasite growth. Synthetic peptides P2 and P4 of PvTRAg38 interfered with the parasite growth independently but had an additive effect if combined together indicating involvement of both the receptors during red cell invasion. In conclusion, PvTRAg38 binds to two erythrocyte receptors basigin and band 3 through P2 and P4 regions, respectively, to facilitate parasite growth. This advancement in our knowledge on molecular mechanisms of host-parasite interaction can be exploited to develop therapeutics against P. vivax malaria.

Identification, Functional Characterization and Regulon Prediction of a Novel Two Component System Comprising BAS0540-BAS0541 of Bacillus anthracis.

Two component systems (TCSs) can be envisaged as complex molecular devices that help the bacteria to sense its environment and respond aptly. 41 TCSs are predicted in Bacillus anthracis, a potential bioterrorism agent, of which only four have been studied so far. Thus, the intricate signaling network contributed by TCSs remains largely unmapped in B. anthracis and needs comprehensive exploration. In this study, we functionally characterized one such system composed of BAS0540 (Response regulator) and BAS0541 (Histidine kinase). BAS0540-BAS0541, the closest homolog of CiaRH of Streptococcus in B. anthracis, forms a functional TCS with BAS0541 displaying autophosphorylation and subsequent phosphotransfer to BAS0540. BAS0540 was also found to accept phosphate from physiologically relevant small molecule phosphodonors like acetyl phosphate and carbamoyl phosphate. Results of qRT-PCR and immunoblotting demonstrated that BAS0540 exhibits a constitutive expression throughout the growth of B. anthracis. Regulon prediction for BAS0540 in B. anthracis was done in silico using the consensus DNA binding sequence of CiaR of Streptococcus. The predicted regulon of BAS0540 comprised of 23 genes, which could be classified into 8 functionally diverse categories. None of the proven virulence factors were a part of the predicted regulon, an observation contrasting with the regulon of CiaRH in Streptococci. Electrophoretic mobility shift assay was used to show direct binding of purified BAS0540 to the upstream regions of 5 putative regulon candidates- BAS0540 gene itself; a gene predicted to encode cell division protein FtsA; a self-immunity gene; a RND family transporter gene and a gene encoding stress (heat) responsive protein. A significant enhancement in the DNA binding ability of BAS0540 was observed upon phosphorylation. Overexpression of response regulator BAS0540 in B. anthracis led to a prodigious increase of ~6 folds in the cell length, thereby conferring it a filamentous phenotype. Furthermore, the sporulation titer of the pathogen also decreased markedly by ~16 folds. Thus, this study characterizes a novel TCS of B. anthracis and elucidates its role in two of the most important physiological processes of the pathogen: cell division and sporulation.