Leptospiral imelysin (LIC_10713) is secretory, immunogenic and binds to laminin, fibronectin, and collagen IV.

Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira. Early and accurate diagnosis is the prime step in managing the disease. Secretory proteins of Leptospira remain distinguished for diagnosis due to their availability as soluble proteins in the serum and their interaction with the host immune response due to their extracellular presence. This study presents the cloning, expression, purification, and characterization of imelysin or LruB (LIC_10713), a putative leptospiral protein. We report that the localization of imelysin showed its presence in the inner membrane and in the culture supernatant. The imelysin was upregulated under in vitro physiological conditions of infection. The LIC_10713 interacted significantly with laminin, fibronectin, collagen type I, and collagen type IV in a dose-dependent manner. Phylogenetic analysis showed that LIC_10713 is predominately found in the pathogenic species of Leptospira, and the GxHxxE motif of imelysin-like proteins is represented as the amino acid sequence GWHAIE. Also, immunoglobulins in leptospirosis-infected patients recognize recombinant-LIC_10713 with 100% specificity and 90.9% sensitivity. The secretion nature, abundance, upregulation, binding to ECM components, and immunogenicity determine LIC_10713 as an important molecule that can be used as an anti-leptospirosis measure. KEY POINTS: • The imelysin-like protein (LIC_10713) of Leptospira is a secretory protein • The protein LIC_10713 can bind ECM molecules • The LIC_10713 is mainly found in pathogenic leptospires • The anti-LIC_10713 antibody from human serum can detect the r-LIC_10713.

Osteoporosis and its Association with Vitamin D Receptor, Oestrogen α Receptor, Parathyroid Receptor and Collagen Type I alpha Receptor Gene Polymorphisms with Bone Mineral Density: A Pilot Study from South Indian Postmenopausal Women of Tamil Nadu.

The involvement of many putative genetic factors makes osteoporosis a complex disease. With increasing longevity of the Indian population, it's now being realized that, as within the West, osteoporotic fractures are also a significant explanation for morbidity and mortality in postmenopausal women. Studies have suggested that the genetic component liable for bone mass could be linked to single nucleotide polymorphisms. Therefore, this study is aimed to research the role of seven gene polymorphisms previously associated with bone phenotype in a cohort of postmenopausal South Indian women from Tamil Nadu. The subjects for the study (n = 300) included 100 osteoporotic women (age 59.3 ± 9.26), 100 osteopenic women (age 55.6 ± 8.17) and 100 non-osteoporotic women as controls (age 55.4 ± 8.85).Genetic polymorphisms were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism. Case-control genetic association analysis of BsmI of the VDR and BstBI of the PTH gene showed a significant allelic association with low bone mineral density amongst the osteoporotic postmenopausal women. The association of BMD with the VDR gene polymorphisms revealed that the average BMD in the BsmI polymorphism with the recessive genotype GG in osteoporotic women was significantly reduced compared with the average BMD in osteoporotic women with AA and AG genotypes. In the BstBI polymorphism, the BMD in the osteoporotic subjects were significantly lower in the AA group than in the GA and GG groups. These results provide evidence for an independent association between BMD and rs1544410 in VDR and rs6254 in PTH and may contribute in being a possible genetic marker for predicting the disease susceptibility in the population tested.

Comparative study of potyvirid NIa proteases and their cleavage sites.

Nuclear inclusion a protease (NIaPro), a major protease of potyvirids, processes its cognate viral polyprotein at distinct cleavage sites. Although Potyviridae is the largest family of the realm Riboviria, the individual NIaPro enzymes and their cleavage sites are believed to be species-specific. In the present study, the NIaPro amino acid sequences of 165 potyvirids of 10 genera and their 1154 cleavage sites were compared to understand their genus/species-specificity and functional regulation. Of these, the NIaPro of macluraviruses, maintains a constant length of 217 amino acids, while those of other genera allow variation. In particular, poaceviruses exhibited a broad range of NIaPro amino acid sequence lengths. Alignment of 162 NIaPro amino acid sequences showed that the N- and C-terminal regions allow variations, while the central region, with the catalytic triad and S1 subsite, are highly conserved. NIaPro cleavage sites are composed of seven amino acids (heptapeptide) denoted as P6-P5-P4-P3-P2-P1/P1'. A survey of 1154 cleavage sites showed that the P1 position is predominantly occupied by Gln/Glu, as is seen in picornaviruses. The P6 (Glu), P4 (Val/Cys/Gln), P2 (His/Tyr/Leu), and P1' (Ser/Ala/Gly/Met) positions are predominantly occupied by genus-specific residues, while P5 and P3 are not genus-specific. The 6K2-VPg and VPg-NIaPro junctions possess Glu at the P1 position in order to maintain latency.

Novel approaches on identification of conserved miRNAs for broad-spectrum Potyvirus control measures.

Potyviridae comprises more than 200 ssRNA viruses, many of which have a broad host range and geographical distributions. Potyvirids (members of Potyviridae) infect several economically important plants such as saffron, cardamom, cucumber, pepper, potato, tomato, yam, etc. Cumulatively, potyvirids cause a substantial economic loss. The major bottleneck in developing an efficient antiviral strategy is that viruses quickly evade host immunity owing to their higher mutation and recombination rates. Due to this reason, the emergence of newer and improved broad-spectrum approaches to combat viral infections is essential. The use of microRNA's (miRNA) to circumvent viral infection against animal viruses has been successfully employed. Fewer studies reported the development of efficient miRNA-based antivirus resistant strategies against plant viruses and none focused on multiple virus resistance. We focused on potyviruses since studies are limited and identification of conserved miRNAs among various host plants would be an initiative to design broad-spectrum antivirus strategies. In this study, we predicted evolutionarily conserved miRNAs by BLAST searching of reported miRNAs from 15 plants against the GSS and EST sequences of banana. A total of nine miRNAs were predicted and screened in nine diverse potyvirids' hosts (Banana, Tomato, Green gram, Jasmine, Chilli, Coriander, Onion, Rose and Colocasia) belonging to eight different orders (Zingiberales, Solanales, Fabales, Lamiales, Apiales, Asperagales, Rosales and Alismatales). Results suggested that miR168 and miR162 are conserved among all the selected plants. This comprehensive study laid the foundations to design broad-spectrum antivirus resistance using miRNAs. To conclude miR168 and miR162 are conserved among many plants and play a crucial role in evading virus infection which could be used as a potential candidate for developing antiviral strategies against potyvirid infections.

Triton X-114 Fractionated Subcellular Proteome of Leptospira interrogans Shows Selective Enrichment of Pathogenic and Outer Membrane Proteins in the Detergent Fraction.

The Triton X-114-based solubilization and temperature-dependent phase separation of proteins is used for subcellular fractionation where, aqueous, detergent, and pellet fractions represents cytoplasmic, outer membrane (OM), and inner membrane proteins, respectively. Mass spectrometry-based proteomic analysis of Triton X-114 fractions of proteomic analysis of Leptospira interrogans identified 2957 unique proteins distributed across the fractions. The results are compared with bioinformatics predictions on their subcellular localization and pathogenic nature. Analysis of the distribution of proteins across the Triton X-114 fractions with the predicted characteristics is performed based on "number" of unique type of proteins, and "quantity" which represents the amount of unique protein. The highest number of predicted outer membrane proteins (OMPs) and pathogenic proteins are found in aqueous and pellet fractions, whereas detergent fraction representing the OM has the highest quantity of OMPs and pathogenic proteins though lower in number than the aqueous and pellet fractions. This leaves the possibility of an upsurge in pathogenic proteins and OMPs on the OM under pathogenic conditions suggesting their potential use to combat leptospirosis. Further, the Triton X-114 subcellular fractions are more correlated to enrichment of pathogenic proteins predicted by MP3 software than predicted localization.

Hubei poty-like virus 1 is likely an interspecies recombinant of sugarcane mosaic virus and putative bean yellow mosaic virus.

In 2016, Shi et al. (Nature 540:539-543. https://doi.org/10.1038/nature20167 2016) reported Hubei poty-like virus 1 (HuPLV1) to be a new member of the family Potyviridae. At that time, its polyprotein shared the highest sequence identity (80%) with sugarcane mosaic virus (SCMV). A year later, two isolates of SCMV from Canna sp. whose sequences were submitted to the GenBank database showed 91% identity to the HuPLV1 polyprotein sequence. Based on the species demarcation criteria for the family Potyviridae, HuPLV1 should possibly be considered an isolate of SCMV. To resolve this taxonomical inconsistency, we carried out a phylogenetic and recombination analysis and found that HuPLV1 is an interspecies recombinant of SCMV and bean yellow mosaic virus (BYMV).

The first complete genomic sequence of cardamom mosaic virus, a member of the genus Macluravirus (family Potyviridae).

The complete genome sequence of the KS isolate of cardamom mosaic virus (CdMV) was determined using transcriptome sequencing data from CdMV-infected Elettaria cardamomum as well as from overlapping cDNA clones made from RNA extracted from viral particles. The viral genome consists of 8249 nucleotides (nt) and encodes a large polyprotein of 2636 amino acids (aa). The polyprotein of CdMV shared 48.9%-67.4% aa sequence identity with other reported macluraviruses. Similar to the other members of genus Macluravirus, the genome of CdMV lacks the P1 coding region and the N-terminus of the HC-Pro coding region. The putative small open reading frame, PIPO, embedded within the P3 cistron, is preceded by a C(A)6 motif instead of G(A)6. Phylogenetic analysis based on the complete genome sequence aided the grouping of CdMV along with all other macluraviruses and showed that it is closely related to alpinia oxyphylla mosaic virus (AloMV). Among CdMV isolates, the KS isolate is most similar to the Appangala isolate based on disease symptoms and phylogeny.

Proteomic approach and expression analysis revealed the differential expression of predicted leptospiral proteases capable of ECM degradation.

Leptospira, the causative agent of leptospirosis is known to have many proteases with potential to degrade extracellular matrix. However, a multipronged approach to identify, classify, characterize and elucidate their role has not been attempted. Our proteomic approach using high-resolution LC-MS/MS analysis of Triton X-114 fractions of Leptospira interrogans resulted in the identification of 104 proteases out of 130 proteases predicted by MEROPS. In Leptospira approximately 3.5% of the genome complements for proteases, which include catalytic types of metallo-, serine-, cysteine-, aspartic-, threonine- and asparagine- peptidases. Comparison of proteases from different serovars revealed that M04, M09B, M14A, M75, M28A, A01 and U73 protease families are exclusively present in pathogenic form. The M23 and S33 protease families are represented with >14 members in Leptospira. The differential expression under physiological temperature (37 °C) and osmolarity (300 mOsM) showed that proteases belonging to the catalytic type of Metallo-peptidases are upregulated significantly in pathogenic conditions. In silico prediction and characterization of the proteases revealed that several proteases are membrane anchored and secretory, classical as well as non-classical system. The study demonstrates the diversity and complexity of proteases, while maintaining conservation across the serovars in Leptospira and their differential expression under pathogenic conditions.

Wuhan poty-like virus 1 is a possible member of the genus Macluravirus.

We have analysed the genome sequence of Wuhan poty-like virus 1 (WuPLV1), reported as an unclassified RNA virus in GenBank (Accession no: KX884573.1). Based on the polyprotein sequence identity (ranging from 55.2 to 71.1%), with classifiable members of the Macluravirus genus of the plant virus family Potyviridae, we suggest that WuPLV1 represents a possible new species of Macluravirus, although the virus was isolated from the Chinese land snail Mastigeulota kiangsinensis, which is not known to be a host or vector of macluraviruses.

Identification of sigma factor 54-regulated small non-coding RNAs by employing genome-wide and transcriptome-based methods in rhizobium strains.

Rhizobium-legume symbiosis is considered as the major contributor of biological nitrogen fixation. Bacterial small non-coding RNAs are crucial regulators in several cellular adaptation processes that occur due to the changes in metabolism, physiology, or the external environment. Identifying and analysing the conditional specific/sigma factor-54 regulated sRNAs provides a better understanding of sRNA regulation/mechanism in symbiotic association. In the present study, we have identified sigma factor 54-regulated sRNAs from the genome of six rhizobium strains and from the RNA-seq data of free-living and symbiotic conditions of Bradyrhizobium diazoefficiens USDA 110 to identify the novel putative sRNAs that are over expressed during the regulation of nitrogen fixation. A total of 1351 sRNAs were predicted from the genome of six rhizobium strains and 1375 sRNAs were predicted from the transcriptome data of B. diazoefficiens USDA 110. Analysis of target mRNA for these novel sRNAs was inferred to target several nodulation and nitrogen fixation genes including nodC, nodJ, nodY, nodJ, nodM, nodW, nodZ, nifD, nifN, nifQ, fixK, fixL, fdx, nolB, and several cytochrome proteins. In addition, sRNAs of B. diazoefficiens USDA 110 which targeted the regulatory genes of nitrogen fixation were confirmed by wet-lab experiments with semi-quantitative reverse transcription polymerase chain reaction. Predicted target mRNAs were functionally classified based on the COG analysis and GO annotations. The genome-wide and transcriptome-based integrated methods have led to the identification of several sRNAs involved in the nodulation and symbiosis. Further validation of the functional role of these sRNAs can help in exploring the role of sRNAs in nitrogen metabolism during free-living and symbiotic association with legumes. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03394-x.

Novel interactions of cardamom mosaic virus VPg with cardamom histones H3 and H4.

The host genome targeting potyviral proteins is sparsely reported. Viral genome-linked protein (VPg) is a multifaceted protein known for its interactions with a suite of host proteins, guides essential viral life cycle processes such as genome replication, translation, genome packing, and antiviral defence. Besides, VPg also plays a crucial role in assisting the transport of nuclear inclusion a protease (NIa protease) into the host nucleus. Apart from that, the role of VPg in the nucleus of the cognate host is not clear. Although NIa protease has been reported for DNase activity, the molecular mechanisms underlying host genome accessibility are not yet understood completely. Here, we employed yeast two hybrid assays to test the cardamom histones H3 and H4 interaction with the VPg and NIa protease of macluravirus cardamom mosaic virus (CdMV). Although CdMV NIa protease has the putative histone-binding ER motif of MYST histone acetyltransferase, it did not interact with host histones H3 and H4. Surprisingly, CdMV VPg displayed strong interaction with histone proteins H3 and H4. Leucine prototrophy and ß-galactosidase assays were performed which validated VPg interaction with histones. To the best of our knowledge, this study is the first report for the multipartnered potyvirid protein VPg interaction with host histones H3 and H4.

Genome of a known but distinct begomovirus associated with a novel satellite molecule infecting a new host bitter gourd (Momordica charantia).

Coccinia mosaic Virdhunagar virus (KY860899), Tomato leaf curl New Delhi virus (KY860898) and Tomato leaf curl Virdhunagar alphasatellite (KY848691) were found to be associated with leaf curl disease in Momordica charantia (bitter gourd). The complete nucleotide sequence of Coccinia mosaic Virdhunagar virus showed 82% identity with Coccinia mosaic Tamil Nadu virus (KM244719), whereas Tomato leaf curl New Delhi virus was 96% identical to Tomato leaf curl New Delhi virus (KP868764) and Tomato leaf curl Virdhunagar alphasatellite illustrated 81% similarity with Tomato leaf curl New Delhi alphasatellite (JQ041697). Phylogenetic and RDP analysis revealed the proximity of these begomoviruses with other monopartite begomoviruses and alphasatellites already reported from India. As per the threshold criteria laid down by International Committee on Taxonomy of Viruses for species demarcation in begomoviruses and satellite molecules, the identified virus isolates, Coccinia mosaic Virdhunagar virus and Tomato leaf curl Virdhunagar alphasatellite are proposed as new species. To the best of our knowledge, this is the first ever account of mixed infection of begomoviruses in Momordica charantia, a vegetable crop commonly cultivated throughout India.

Prediction and identification of novel sRNAs involved in Agrobacterium strains by integrated genome-wide and transcriptome-based methods.

Small RNAs (sRNAs) are a class of gene regulators in bacteria, playing a central role in their response to environmental changes. Bioinformatic prediction facilitates the identification of sRNAs expressed under different conditions. We propose a novel method of prediction of sRNAs from the genome of Agrobacterium based on a positional weight matrix of conditional sigma factors. sRNAs predicted from the genome are integrated with the virulence-specific transcriptome data to identify putative sRNAs that are overexpressed during Agrobacterial virulence induction. A total of 384 sRNAs are predicted from transcriptome data analysis of Agrobacterium fabrum and 100-500 sRNAs from the genome of different Agrobacterial strains. In order to refine our study, a final set of 10 novel sRNAs with best features across different replicons targeting virulence genes were experimentally identified using semi-quantitative polymerase chain reaction. Since Ti plasmid plays a major role in virulence, out of 10 sRNAs across the replicons, 4 novel sRNAs differentially expressed under virulence induced and non-induced conditions are predicted to be present in the Ti plasmid T-DNA region flanking virulence-related genes like agrocinopine synthase, indole 3-lactate synthase, mannopine synthase and tryptophan monooxygenase. Further validation of the function of these sRNAs in conferring virulence would be relevant to explore their role in Agrobacterium-mediated plant transformation.

Molecular modeling and in-silico engineering of Cardamom mosaic virus coat protein for the presentation of immunogenic epitopes of Leptospira LipL32.

Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particles. In this study, the structure of CdMV CP was predicted and used as a platform to display epitopes of the most abundant surface-associated protein, LipL32 of Leptospira at C, N, and both the termini of CdMV CP. In silico, we have mapped sequential and conformational B-cell epitopes from the crystal structure of LipL32 of Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 using IEDB Elipro, ABCpred, BCPRED, and VaxiJen servers. Our results show that the epitopes displayed at the N-terminus of CdMV CP are promising vaccine candidates as compared to those displayed at the C-terminus or at both the termini. LipL32 epitopes, EP2, EP3, EP4, and EP6 are found to be promising B-cell epitopes for vaccine development. Based on the type of amino acids, length, surface accessibility, and docking energy with CdMV CP model, the order of antigenicity of the LipL32 epitopes was found to be EP4 > EP3 > EP2 > EP6.