Exploring the Role of Extracellular Vesicles in the Pathogenesis of Tuberculosis.

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.

Production and Purification of Cysteine-Rich Leptospiral Virulence-Modifying Proteins with or Without mCherry Fusion.

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.

mCherry Fusion Proteins Facilitate Production of Recombinant, Cysteine-Rich Leptospira interrogans Proteins in Escherichia coli.

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.

Vaccination With Leptospira interrogans PF07598 Gene Family-Encoded Virulence Modifying Proteins Protects Mice From Severe Leptospirosis and Reduces Bacterial Load in the Liver and Kidney.

The molecular and cellular pathogenesis of leptospirosis remains poorly understood. Based on comparative bacterial genomics data, we recently identified the hypothetical PF07598 gene family as encoding secreted exotoxins (VM proteins) that mediate cytotoxicity in vitro. To address whether VM proteins mediate in vivo leptospirosis pathogenesis, we tested the hypothesis that VM protein immunization of mice would protect against lethal challenge infection and reduce bacterial load in key target organs. C3H/HeJ mice were immunized with recombinant E. coli-produced, endotoxin-free, leptospiral VM proteins (derived from L. interrogans serovar Lai) in combination with the human-compatible adjuvant, glucopyranoside lipid A/squalene oil-in-water. Mice receiving full length recombinant VM proteins were protected from lethal challenge infection by L. interrogans serovar Canicola and had a 3-4 log10 reduction in bacterial load in the liver and kidney. These experiments show that immunization with recombinant VM proteins prevents leptospirosis clinical pathogenesis and leads to markedly reduced key target organ infection in this animal model. These data support the role of leptospiral VM proteins as virulence factors and suggest the possibility that a VM protein-based, serovar-independent, pan-leptospirosis vaccine may be feasible.

Pathogenic Leptospira Evolved a Unique Gene Family Comprised of Ricin B-Like Lectin Domain-Containing Cytotoxins.

Leptospirosis is a globally important neglected zoonotic disease. Previous data suggest that a family of virulence-modifying (VM) proteins (PF07598) is a distinctive feature of group I pathogenic Leptospira that evolved as important virulence determinants. Here, we show that one such VM protein, LA3490 (also known as Q8F0K3), is expressed by Leptospira interrogans serovar Lai, as a secreted genotoxin that is potently cytotoxic to human cells. Structural homology searches using Phyre2 suggested that VM proteins are novel R-type lectins containing tandem N-terminal ricin B-chain-like ß-trefoil domains. Recombinant LA3490 (rLA3490) and an N-terminal fragment, t3490, containing only the predicted ricin B domain, bound to the terminal galactose and N-acetyl-galactosamine residues, asialofetuin, and directly competed for asialofetuin-binding sites with recombinant ricin B chain. t3490 alone was sufficient for binding, both to immobilized asialofetuin and to the HeLa cell surface but was neither internalized nor cytotoxic. Treatment of HeLa cells with rLA3490 led to cytoskeleton disassembly, caspase-3 activation, and nuclear fragmentation, and was rapidly cytolethal. rLA3490 had DNase activity on mammalian and bacterial plasmid DNA. The combination of cell surface binding, internalization, nuclear translocation, and DNase functions indicate that LA3490 and other VM proteins evolved as novel forms of the bacterial AB domain-containing toxin paradigm.

A Phase 2 Randomized, Double-Blind, Placebo-controlled Trial of Oral Camostat Mesylate for Early Treatment of COVID-19 Outpatients Showed Shorter Illness Course and Attenuation of Loss of Smell and Taste.

Importance: Early treatment of mild SARS-CoV-2 infection might lower the risk of clinical deterioration in COVID-19. Objective: To determine whether oral camostat mesylate would reduce upper respiratory SARS-CoV-2 viral load in newly diagnosed outpatients with mild COVID-19, and would lead to improvement in COVID-19 symptoms. Design: From June, 2020 to April, 2021, we conducted a randomized, double-blind, placebo-controlled phase 2 trial. Setting: Single site, academic medical center, outpatient setting in Connecticut, USA. Participants: Of 568 COVID-19 positive potential adult participants diagnosed within 3 days of study entry and assessed for eligibility, 70 were randomized and 498 were excluded (198 did not meet eligibility criteria, 37 were not interested, 265 were excluded for unknown or other reasons). The primary inclusion criteria were a positive SARS-CoV-2 nucleic acid amplification result in adults within 3 days of screening regardless of COVID-19 symptoms. Intervention: Treatment was 7 days of oral camostat mesylate, 200 mg po four times a day, or placebo. Main Outcomes and Measures: The primary outcome was reduction of 4-day log10 nasopharyngeal swab viral load by 0.5 log10 compared to placebo. The main prespecified secondary outcome was reduction in symptom scores as measured by a quantitative Likert scale instrument, Flu-PRO-Plus modified to measure changes in smell/taste measured using FLU-PRO-Plus. Results: Participants receiving camostat had statistically significant lower quantitative symptom scores (FLU-Pro-Plus) at day 6, accelerated overall symptom resolution and notably improved taste/smell, and fatigue beginning at onset of intervention in the camostat mesylate group compared to placebo. Intention-to-treat analysis demonstrated that camostat mesylate was not associated with a reduction in 4-day log10 NP viral load compared to placebo. Conclusions and relevance: The camostat group had more rapid resolution of COVID-19 symptoms and amelioration of the loss of taste and smell. Camostat compared to placebo was not associated with reduction in nasopharyngeal SARS-COV-2 viral load. Additional clinical trials are warranted to validate the role of camostat mesylate on SARS-CoV-2 infection in the treatment of mild COVID-19. Trial registration: Clinicaltrials.gov, NCT04353284 (04/20/20)(https://clinicaltrials.gov/ct2/show/NCT04353284?term=camostat+%2C+yale&draw=2&rank=1).

Comparison of the PF07598-Encoded Virulence-Modifying Proteins of L. interrogans and L. borgpetersenii.

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.

In silico prediction of molecular mechanisms of toxicity mediated by the leptospiral PF07598 gene family-encoded virulence-modifying proteins.

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.

Cytotoxicity of the 42 kDa SMase C sphingomyelinase secreted by Leptospira interrogans serovar Pomona on Vero cells.

Sphingomyelinases produced by the pathogenic members of the genus Leptospira are implicated in the haemorrhagic manifestations seen in the severe form of leptospirosis. With multiple sphingomyelinase genes present in the genome of pathogenic Leptospira, 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 by Leptospira interrogans serovar 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.