Reetika Chaurasia

Tuberculosis (TB) is one of the deadliest infectious diseases globally, ranking as 13th leading cause of mortality and morbidity. According to the Global Tuberculosis Report 2022, TB claimed the lives of 1.6 million people worldwide in 2021. Among the casualties, 1 870 000 individuals with HIV co-infections contributed to 6.7% of the total fatalities, accounting TB as the second most lethal infectious disease following COVID-19. In the quest to identify biomarkers for disease progression and anti-TB therapy, microRNAs (miRNAs) have gained attention due to their precise regulatory role in gene expression in disease stages and their ability to distinguish latent and active TB, enabling the development of early TB prognostic signatures. miRNAs are stable in biological fluids and therefore will be useful for non-invasive and broad sample collection. However, their inherent lack of specificity and experimental variations may lead to false-positive outcomes. These limitations can be overcome by integrating standard protocols with machine learning, presenting a novel tool for TB diagnostics and therapeutics. This review summarizes, discusses and highlights the potential of miRNAs as a biomarker, particularly their differential expression at disease stages. The review assesses the advantages and obstacles associated with miRNA-based diagnostic biomarkers in pulmonary TB and facilitates rapid, point-of-care testing.

Background: Tuberculosis is a serious life-threatening disease among the top global health challenges and rapid and effective diagnostic biomarkers are vital for early diagnosis especially given the increasing prevalence of multidrug resistance. Methods: Two human whole blood microarray datasets, GSE42826 and GSE42830 were retrieved from publicly available gene expression omnibus (GEO) database. Deregulated genes (DEGs) were identified using GEO2R online tool and Gene Ontology (GO), protein-protein interaction (PPI) network analysis was performed using Metascape and STRING databases. Significant genes (n = 8) were identified using T-test/ANOVA and Molecular Complex Detection (MCODE) score ≥10, which was validated in GSE34608 dataset. The diagnostic potential of three biomarkers was assessed using Area Under Curve (AUC) of Receiver Operating Characteristic (ROC) plot. The transcriptional levels of these genes were also examined in a separate dataset GSE31348, to monitor the patterns of variation during tuberculosis treatment. Results: A total of 62 common DEGs (57 upregulated, 7 downregulated genes) were identified in two discovery datasets. GO functions and pathway enrichment analysis shed light on the functional roles of these DEGs in immune response and type-II interferon signaling. The genes in Module-1 (n = 18) were linked to innate immune response, interferon-gamma signaling. The common genes (n = 8) were validated in GSE34608 dataset, that corroborates the results obtained from discovery sets. The gene expression levels demonstrated responsiveness to Mtb infection during anti-TB therapy in GSE31348 dataset. In GSE34608 dataset, the expression levels of three specific genes, GBP5, IFITM3, and EPSTI1, emerged as potential diagnostic makers. In combination, these genes scored remarkable diagnostic performance with 100% sensitivity and 89% specificity, resulting in an impressive Area Under Curve (AUC) of 0.958. However, GBP5 alone showed the highest AUC of 0.986 with 100% sensitivity and 89% specificity. Conclusions: The study presents valuable insights into the critical gene network perturbed during tuberculosis. These genes are determinants for assessing the effectiveness of an anti-TB response and distinguishing between active TB and healthy individuals. GBP5, IFITM3 and EPSTI1 emerged as candidate core genes in TB and holds potential as novel molecular targets for the development of interventions in the treatment of TB.

Tuberculosis (TB) remains a significant global health concern, necessitating accurate diagnosis and treatment monitoring. Extracellular vesicles (EVs), including exosomes, play crucial roles in disease progression, with their associated genes serving as potential biomarkers and therapeutic targets. Leveraging publicly available RNA-Seq datasets of TB patients and healthy controls (HCs), to identify differentially expressed genes (DEGs) and their associated protein–protein interaction networks and immune cell profiles, the common EV-related DEGs were identified and validated in the GSE42830 and GSE40553 datasets. We have identified nine common EV-related DEGs (SERPINA1, TNFAIP6, MAPK14, STAT1, ITGA2B, VAMP5, CTSL, CEACAM1, and PLAUR) upregulated in TB patients. Immune cell infiltration analysis revealed significant differences between TB patients and HCs, highlighting increased proportions of various immune cells in TB patients. These DEGs are involved in crucial cellular processes and pathways related to exocytosis and immune response regulation. Notably, VAMP5 exhibited excellent diagnostic performance (AUC—0.993, sensitivity—93.8%, specificity—100%), with potential as a novel biomarker for TB. The EV-related genes can serve as novel potential biomarkers that can distinguish between TB and HCs. VAMP5, which functions in exosome biogenesis and showed significant upregulation in TB, can be targeted for therapeutic interventions and treatment outcomes.

Recombinant fluorescent fusion proteins are fundamental to advancing many aspects of protein science. Such proteins are typically used to enable the visualization of functional proteins in experimental systems, particularly cell biology. An important problem in biotechnology is the production of functional, soluble proteins. Here we report the use of mCherry-fusions of soluble, cysteine-rich, Leptospira-secreted exotoxins in the PF07598 gene family, the so-called virulence modifying (VM) proteins. The mCherry fusion proteins facilitated the visual detection of pink colonies of the VM proteins (LA3490 and LA1402) and following them through lysis and sequential chromatography steps. CD-spectroscopy analysis confirmed the stability and robustness of the mCherry-fusion protein, with a structure comparable to AlphaFold structural predictions. LA0591, a unique member of the PF07598 gene family that lacks N-terminal ricin B-like domains, was produced without mCherry tag that strengthens the recombinant protein production protocol without fusion protein as well. The current study provides the approaches for the synthesis of 50–125 kDa soluble, cysteine-rich, high-quality fast protein liquid chromatography (FPLC)-purified protein, with and without a mCherry tag. The use of mCherry-fusion proteins enables a streamlined, efficient process of protein production and qualitative and quantitative downstream analytical and functional studies. Approaches for troubleshooting and optimization were evaluated to overcome difficulties in recombinant protein expression and purification, demonstrating biotechnology utility in accelerating recombinant protein production.

Background Recombinant fluorescent fusion proteins are fundamental to advancing many aspects of protein science. Such proteins are typically used to enable the visualization of functional proteins in experimental systems, particularly cell biology. An important problem in biotechnology is the production of functional, soluble proteins. Here we report the use of mCherry-fusions of soluble, cysteine-rich, Leptospira-secreted exotoxins in the PF07598 gene family, the so-called virulence modifying (VM) proteins. Results The mCherry fusion proteins facilitated the production of the VM proteins (LA3490 and LA1402) by enabling the visual detection of pink colonies and following them through lysis and sequential chromatography steps. CD-spectroscopy analysis confirmed the stability and robustness of the mCherry-fusion protein, with a structure comparable to AlphaFold structural predictions. LA0591, a unique member of the PF07598 gene family that lacks N-terminal ricin B-like domains, was produced as a tagless protein that strengthens the recombinant protein production protocol. The current study provides the approaches for the synthesis of 50-125 kDa soluble, cysteine-rich, high-quality mCherry tagged or tagless fast protein liquid chromatography (FPLC)-purified protein. Conclusions The use of mCherry-fusion proteins enables a streamlined, efficient process of protein production and qualitative and quantitative downstream analytical and functional studies. Approaches for troubleshooting and optimization were systemically evaluated to overcome difficulties in recombinant protein expression and purification, demonstrating biotechnology utility in accelerating recombinant protein production.

Mechanisms of leptospirosis pathogenesis remain unclear despite the identification of a number of potential leptospiral virulence factors. We recently demonstrated potential mechanisms by which the virulence-modifying (VM) proteins—defined as containing a Domain of Unknown function (DUF1561), encoded by the PF07598 gene family—found only in group 1 pathogenic Leptospira—might mediate the clinical pathogenesis of leptospirosis. VM proteins belongs to classical AB toxin paradigm though have a unique AB domain architecture, unlike other AB toxins such as diphtheria toxin, pertussis toxin, shiga toxin, or ricin toxin which are typically encoded by two or more genes and self-assembled into a multi-domain holotoxin. Leptospiral VM proteins are secreted R-type lectin domain-containing exotoxins with discrete N-terminal ricin B-like domains involved in host cell surface binding, and a C-terminal DNase/toxin domain. Here we use the artificial intelligence-based AlphaFold algorithm and other computational tools to predict and elaborate on details of the VM protein structure-function relationship. Comparative AlphaFold and CD-spectroscopy defined the consistent secondary structure (Helix and ß-sheet) content, and the stability of the functional domains were further supported by molecular dynamics simulation. VM proteins comprises distinctive lectic family (QxW)₃ motifs, the Mycoplasma CARDS toxin (D3 domain, aromatic patches), C-terminal similarity with mammalian DNase I. In-silico study proposed that Gln412, Gln523, His533, Thr59 are the high binding energy or ligand binding residues plausibly anticipates in the functional activities. Divalent cation (Mg⁺²-Gln412) and phosphate ion (PO₄]⁻³-Arg615) interaction further supports the functional activities driven by C-terminal domain. Computation-driven structure-function studies of VM proteins will guide experimentation towards mechanistic understandings of leptospirosis pathogenesis, which underlie development of new therapeutic and preventive measures for this devastating disease.

Leptospirosis is an emerging infectious disease, with increasing frequency and severity of outbreaks, a changing epidemiology of populations at risk, and the emergence of new strains, serovars, serogroups, and species. Virulence-modifying (VM) proteins encoded by the PF07598 gene family are hypothesized to be Leptospira-secreted exotoxins that mediate the molecular and cellular pathogenesis of severe and fatal leptospirosis. If confirmed experimentally, this concept could revolutionize the treatment, diagnosis, prognosis, and vaccine-mediated prevention of leptospirosis by enabling a novel array of targeted interventions. VM proteins, as with other bacterial-secreted protein exotoxins, mediate their virulence effects by attaching to eukaryotic cells, competing with other microorganisms for limited resources in environmental niches, directly intoxicating target cells, and disrupting their function in the mammalian host. In contrast with the most pathogenic group of Lept ospira, particularly L. interrogans, whose genomes contain 12–15 PF07598 paralogs, strains of the livestock and human pathogen L. borgpetersenii have two PF07598 paralogs. Given the possible non-environmentally mediated transmission of some L. borgpetersenii strains and the much smaller number of VM proteins in this species, their role in infection and disease may well differ from other leptospiral species. Comparison of VM proteins from different clades of pathogenic Leptospira may deepen our understanding of leptospirosis’s pathogenesis, leading to novel approaches to ameliorating Leptospira infection in humans and animals.

Sphingomyelinases produced by the pathogenic members of the genusLeptospiraare implicated in the haemorrhagic manifestations seen in the severe form of leptospirosis. With multiple sphingomyelinase genes present in the genome of pathogenicLeptospira, much remains to be understood about these molecules. They include factors regulating their expression, post-translational modifications, and release of the biologically active forms of these molecules. In this study, serovar Pomona was chosen as it is reported to express high levels of sphingomyelinase that explained the haemolytic activity seen in experimental animals infected with this pathogen. Here, we demonstrate the cytotoxicity of a 42 kDa sphingomyelinase secreted byLeptospira interrogansserovar Pomona strain Pomona upon infecting Vero cells. This sphingomyelinase detected using specific anti-sphingomyelinase antibodies, exhibited haemolytic and sphingomyelinase activities that caused host-cell damage evident from the confocal images and scanning electron micrographs. The implications of these findings and the detection of a 42 kDa sphingomyelinase in the urine of human patients with leptospirosis in our earlier study is discussed with an emphasis on the potential of these sphingomyelinases as candidate markers for the early diagnosis of leptospirosis.

Leptospirosis is a globally important neglected zoonotic disease subject to both small scale outbreaks and weather-driven, large-scale epidemics. Due to gaps in our understanding of Leptospira biology, pathogenetic mechanisms of leptospirosis remain largely unknown. Previous data suggest that a gene family, PF07598, unique amongst most known bacterial pathogens and encoding so-called “Virulence-Modifying (VM)” proteins, are important virulence determinants. Here, we show that VM proteins are potent cytotoxins, sharing a distinct domain organization while exhibiting varied mechanisms of cellular toxicity. Structural homology searches using Phyre2 suggest that VM proteins are novel R-type lectins containing an N-terminal ricin B chain-like domain. As is known for native ricin B-chain, recombinant full-length rLA3490 (most highly up-regulated in vivo ) and an N-terminal fragment, t3490 , containing a partial ricin B-domain, bound to asialofetuin and directly competed for asialofetuin binding with recombinant ricin B chain. While t3490 bound to the HeLa cell surface but was neither internalized nor cytotoxic, rLA3490 bound to the HeLa cell surface, was rapidly internalized, translocated to the nucleus inducing chromosomal fragmentation, and was rapidly cytolethal, providing strong evidence that Leptospira VM proteins are bona fide cytotoxins. Because monoclonal antibodies impeding cell entry or intracellular trafficking of ricin holotoxin clearly mitigate its toxicity, that VM proteins share binding and intracellular trafficking mechanisms suggests that anti-VM-protein antibody-based (anti-toxin) therapeutics could ameliorate severe complications of leptospirosis thereby improving prognosis. As most VM proteins are restricted to high-virulence Leptospira species with some, e.g., LA3490, being exceptionally potent, their level in serum might be a potentially useful indicator of a poor prognosis, thus identifying high risk patients. Author Summary: The PF07598 gene family encoding Virulence-Modifying (VM) proteins in pathogenic Leptospira species is associated with severe manifestations of leptospirosis. Structural homology searches indicate that VM proteins contain an N-terminal ricin B chain-like domain, biochemically confirmed in asialofetuin binding and competitive-binding assays suggesting that VM proteins bind to terminal galactosyl residues of this model ricin B domain binding protein. The leptospiral N-terminal ricin B chain-like domain mediated VM protein binding to HeLa cells. Full-length recombinant protein rapidly led to cell death. Amino acid conservation among PF07598 family members at the N-terminal ricin B chain-like domain suggests that VM protein levels in serum might be a useful biomarker for quickly identifying at-risk patients, and that novel “anti-toxin”-based therapeutics could ameliorate severe complications of leptospirosis, both of which remain to be explored.