Leptospiral lipopolysaccharide mediated Hog1 phosphorylation in Saccharomyces cerevisiae directs activation of autophagy.

Cells have developed a variety of mechanisms to counteract stress to give a specific and adaptive response. Yeast Hog1 is a homolog to mammalian p38, which is a mitogen-activated protein kinase. In this work, we analyze the Hog1 signaling during the induction of leptospiral LPS (100 ng/mL) and the hyperosmotic element NaCl (0.8 M). After the addition of stress elements, the stress-activated protein kinase was phosphorylated within 30 min of exposure and led to the expression of various genes responsible for cell survival. We found that leptospiral lipopolysaccharide mediated Hog1 phosphorylation leads to activation of autophagy-related genes phosphorylation; thereby cells encounter and digest the metabolic waste or organelles for their energy during starvation. And, the wild-type cells accumulate lipid droplets and trigger vacuole calcium release, to maintain cell survival. Loss of Hog1 leads to shrinkage in the cell wall, condensation of the cytoplasmic part, and high-level ROS production. This led to the Hog1 mutant cell death under LPS treatment or stress condition. The phosphorylation of stress-activated kinase during exposure to leptospiral LPS provides insight and knowledge about the organization of cellular metabolic products and cell survival during stress conditions and identifies the pathogenic mechanisms of leptospirosis.

Electrochemical biosensor for serogroup specific diagnosis of leptospirosis.

A problem with the current leptospirosis diagnostic methods is the low sensitivity and specificity during the acute phase of illness. Rapid point-of-care (POC) assays with minimal sample utilization and low cost are desired in clinical practice. Here, we report for the first time lipopolysaccharide (LPS) based electrochemical biosensor that offers a rapid, highly sensitive, serogroup specific diagnosis of leptospirosis during the acute stage of infection and also to distinguish from other flu like infections. The proposed sensor is fabricated by the immobilization of LPS onto dodecanethiol (DT) modified gold electrode. Monolayer of DT is attached through covalent bond (Au-S) interaction onto the gold electrode. Thus, leptospiral antibodies from the human serum samples bind to the LPS present on self-assembled monolayer (SAM) of DT and showed a higher RCT value compared to SAM. The detection limit of the developed LPS sensor is estimated to be 100 nM. This biosensor is the first electrochemical sensing platform used for detection of LPS from Leptospira spp. This method is completely a solution-based diagnostic method and therefore it is rapid, simple, and sensitive; thus establishing a key technology towards a useful POC diagnostic strategy in serogroup level and hence an alternative to MAT.

Construction of Genomic Library and Screening of Edwardsiella tarda Immunogenic Proteins for Their Protective Efficacy Against Edwardsiellosis.

Edwardsiella tarda is a severe aquaculture pathogen that can infect many hosts including humans, animals, and fish. Timely diagnosis and treatment are crucial for the control of edwardsiellosis in the aqua industry. By using rabbit polyclonal antibody, an expression gene library of virulent Edwardsiella tarda strain ED-BDU 1 isolated in south India was constructed and screened. The identified immune expressive proteins were characterized, and the corresponding coding sequences were cloned, expressed, and the purified recombinant proteins were used as antigens. The identified immunoreactive proteins namely HflC, HflK, and YhcI were studied for their immune protective potential in vivo by challenge experiments. The protective efficacy of HflC, HflK, and YhcI showed that the clearance of Edwardsiella from the host with ~ 60% survivability. Further, the immunoreactive proteins induce a strong immune response upon infection and elicit the significant production of IL-10, IFN-γ, Th1, and Th2 mediated mRNA expression and were therefore effective in vaccine production for edwardsiellosis.

Prevalence of leptospirosis among animal herds of north eastern provinces of India.

Serum samples from 840 animals were examined for Leptospira spp. antibodies by microscopic agglutination test (MAT) to assess the risk factors and the prevalence of leptospirosis among animal herds of Assam, Meghalaya, Mizoram the north eastern (NER) provinces. They were compared with Tamilnadu (TN) the southern province of India for the serovar and risk factor inconsistency. Serovar Ballum was reported to be prevalent (28.1 %) in Assam and Grippotyphosa (16.1 %) in Tamilnadu. The overall seropositivity observed was 36.8 %(206/560) from NER and 30.7 %(86/280) from TN. In this study, the higher seroprevalence was observed in pigs (42.6 %), cattle (39.8 %) and goats (26 %) in NER. Cattle (36.4 %) and goat (17.6 %) showed higher prevalence in TN. The presence of rodents in pig herds was found to be significant (P = 0.0088) in NER and it was for cattle in TN (P = 0.0063). We suggest that a program of rodent control should be included in the flock management practices aiming to reduce transmission of the leptospires.

Biochemical analysis of leptospiral LPS explained the difference between pathogenic and non-pathogenic serogroups.

Lipopolysaccharide (LPS) is the major surface antigen of Leptospira. In this study, the genes involved in the LPS biosynthesis were analyzed and compared by bioinformatics tools. Also, the chemical composition analysis of leptospiral lipopolysaccharides (LPS) extracted from 5 pathogenic serovars like Autumnalis, Australis, Ballum, Grippotyphosa, Pomona, and the nonpathogenic serovar Andamana was performed. Methods used were Limulus amebocyte lysate assay (LAL), gas chromatography-mass spectrometry (GC-MS), fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance spectroscopy (NMR). LAL assay showed a significantly higher level of endotoxicity among pathogenic serovars (~0.490 EU/mL) than that of nonpathogenic Andamana (~0.102 EU/mL). FAMES analysis showed the presence of palmitic acid (C16:0), hydroxy lauric acid (3-OH-C12:0), and oleic acid (C18:0). Palmitoleic acid (C16: 1), and 3- hydroxy palmitate (3-OH-C16:0) was detected only in pathogenic serovars. In contrast myristoleic acid (C14:1) and stearic acid (C18:0) were present in Andamana. FTIR analysis revealed C-O-C stretch of esters, 3°ROH functional groups and carbohydrate vibration range were similar among pathogenic serovars. The NMR analysis reveals similarity for 6 deoxy sugars and methyl groups of Autumnalis, Australis, and Ballum. Further, the presence of palmitoleic acid and 3-hydroxy palmitate may be the significant pathogen-associated predisposing factor. This mediates high osmolarity glycerol (HOG) mediated stress response in leptospiral LPS mediated pathogenesis.

Leptospiral protein LIC11334 display an immunogenic peptide KNSMP01.

Leptospirosis is considered as a neglected tropical disease which is caused by pathogenic Leptospira spp. The precise mechanisms of leptospirosis pathogenesis are unclear and hence, the progress in development of treatment modalities has been dismal. The present study aimed to identify novel virulent factors of leptospires to understand the disease pathogenesis and to develop treatment modalities. Leptospira interrogans contains two chromosomes and encodes for ~3703 genes, but the functions of several open reading frames have not yet been explored. Among them, novel virulent associated leptospiral proteins (LIC11334, LIC11542, LIC11436, LIC11120 and LIC12539) were identified using VirulentPredict and the antigenicity of these targets was explored by VaxiJen server. Domain architecture of the pathogen specific proteins revealed that LIC11334 had potential to evoke significant immune response against leptospiral infection and LIC11436 contains four folds of immunoglobulin-like domain and plays a vital role in pathogenesis. Therefore, B-cell epitopes were predicted and the epitope of high virulence (and VaxiJen score from LIC11334) was chemically synthesized as peptide (KNSMP01) and labeled with Biotin (Biotin-SGSGEVENPDPKVAQEC). Binding affinity of KNSMP01 with MHC molecules was predicted and the molecule was discovered to have potential to elicit both humoral and cell mediated immune responses and found to interact with host components via hydrophobic interaction, hydrogen bonding and salt bridges. Rabbit antisera was raised against KNSMP01 and found to elicit antigenicity using Western, ELISA and dot blot assays. In silico and in vitro experiments show KNSMP01 to be a promising immunogen and may be a better vaccine candidate for leptospirosis.

MicroRNAs Regulated by the LPS/TLR2 Immune Axis as Bona Fide Biomarkers for Diagnosis of Acute Leptospirosis.

Leptospirosis remains a significant human health issue due to its systemic complications. Therefore, biomarkers that are more effective are urgently needed for the early diagnosis of leptospirosis. MicroRNAs (miRNAs) are evolutionarily conserved regulatory RNAs that have shown the potential to be used as biomarkers for diagnosis, prognosis, and therapy of infectious diseases. In this study, we performed an unbiased screen using the miRNome miRNA array to identify circulating miRNAs with the potential to serve as authentic biomarkers for early diagnosis of leptospirosis. Because leptospiral lipopolysaccharide (LPS) is the predominant leptospiral antigen and plays a vital role in immunological and biological activities, we used LPS treated and untreated in vitro (THP1 cells) and in vivo (BALB/c mice) surrogate models to identify the LPS-specific miRNAs. Differential expression analysis revealed 18 miRNAs to be associated strongly with LPS stimulation in THP1 cells. Of these, three (miR-let-7b-5p, miR-144-3p, and miR-21-5p) were observed to be present at increased levels in vivo The identified miRNAs were validated for their biomarker potential using serum samples from leptospirosis-negative patients and patients with confirmed cases of leptospirosis. Identified miRNAs were able to discriminate the acute leptospiral infection from other febrile diseases with a test sensitivity and specificity of 93.2% and 88.19%, respectively. Gene functional enrichment and protein-protein interaction (PPI) network analysis revealed that the identified miRNAs play important roles in disease signal transduction, signaling by interleukins, the stress-activated protein kinase signaling cascade, the mitogen-activated protein kinase (MAPK) signaling pathway, and the cellular response to a transforming growth factor ß (TGF-ß) stimulus with a notable interconnection between these biological processes.IMPORTANCE Here, we used miRNAs that are differentially regulated by the LPS/TLR2 immune axis to devise a miRNA-based diagnosis for leptospirosis. The study established the role of the circulating stable miRNAs (miR-21-5p, miR-144-3p, and miR-let-7b-5p) as an early diagnostic marker for leptospirosis. These miRNAs can be used to diagnose acute leptospirosis and also to differentiate leptospiral infection from other bacterial and spirochetal infections, as proved by the use of human clinical samples. Thus, our findings indicate that miRNAs can play a crucial role in the diagnosis of infectious diseases, like leptospirosis, that are generally misdiagnosed.

Silver enhanced nano-gold dot blot immunoassay for leptospirosis.

Leptospirosis is a widespread zoonotic disease and lacks in efficient diagnostic tools. In the present study, a nanogold based dot blot immunoassay was developed and evaluated for the detection of leptospirosis in human urine samples. This method was found to be rapid (<4 h) with higher sensitivity (>4.2-14.6%) than horse radish peroxidase (HRP) conjugated dot blot assay.