Hemant Joshi

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.

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.

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

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.

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.

*These authors contributed equally to this work Purpose: The objective of this research was to generate a tool for the first-line detection of fungal infection in plants. Chitin is one of the unique fungal cell wall polysaccharide which is naturally deacetylated to chitosan upon infection. It is said to be involved in the fungal cell wall modulation and plant-pathogen communication. Therefore, detection of chitosan could be potentially helpful in the detection of fungal contamination. Methods: Five different phytopathogenic fungi strains were used for the study. Polyclonal sera were raised in the mice against Trimethylchitosan nanoparticles to generate an enhanced humoral immune response and generate a rich and heterogeneous repertoire of antibodies. The binding affinity of the sera with fungal cell wall was analyzed by ELISA, Langmuir isotherm, confocal microscopy and ITC (Isothermal Calorimetry). Results: The raised polyclonal sera could detect chitosan in the fungal cell wall, as analyzed with the different techniques. However, the detection specificity varied among the strains in proportion to the chitin content of their cell wall. Fusarium oxysporum was detected with the highest affinity while Trichoderma reesei was detected with the least affinity by ELISA. Adsorption isotherm, as well as ITC, revealed the specific and high binding capacity. Confocal microscopy also confirmed the detection of all strains used in the study. Conclusion: This novel technique employing TMC nanoparticulate system could be potentially used as a source to raise sera against chitosan in an inexpensive and less laborious manner. Rapid detection of fungal contamination by the polyclonal antibodies could help in devising a quick solution. The polyclonal sera are expected to detect a span of epitopes and provide precise detection. The detection system could be advanced for future applications such as food quality control, crop protection, and human fungal infection detection and treatment.