Genome mining of Fusarium reveals structural and functional diversity of pectin lyases: a bioinformatics approach.

Pectin lyase (PNL) is an important enzyme of the pectinases group which degrades pectin polymer to 4,5-unsaturated oligogalacturonides by a unique ß-elimination mechanism and is used in several industries. The existence of multigene families of pectin lyases has been investigated by mining microbial genomes. In the present study, 52 pectin lyase genes were predicted from sequenced six species of Fusarium, namely F. fujikuroi, F. graminearum, F. proliferatum, F. oxysporum, F. verticillioides and F. virguliforme. These sequences were in silico characterized for several physico-chemical, structural and functional attributes. The translated PNL proteins showed variability with 344-1142 amino acid residues, 35.44-127.41 kDa molecular weight, and pI ranging from 4.63 to 9.28. The aliphatic index ranged from 75.33 to 84.75. Multiple sequence alignment analysis showed several conserved amino acid residues and five distinct groups marked as I, II, III, IV, and V were observed in the phylogenetic tree. The Three-dimensional Structure of five of these PNLs, each representing a distinct group of phylogenetic trees was predicted using I-TASSER Server and validated. The pectin lyase proteins of Fusarium species revealed close similarity with pectin lyase of Aspergillus niger PelA(1IDJ) and PelB(1QCX). Diversity in the structural motifs was observed among Fusarium species with 2 ß-sheets, 1 ß-hairpin, 7-12 ß bulges, 18-25 strands, 6 -11 helices, 1 helix-helix interaction, 32-49 ß turns, 2-6 γ turns and 2- 3 disulfide bonds. The unique Pec_lyase domain was uniformly observed among all PNL proteins confirming its identity. The genome-wide mining of Fusarium species was attempted to provide the diversity of PNL genes, which could be explored for diverse applications after performing cloning and expression studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03333-w.

Visceral leishmaniasis: A novel nuclear envelope protein 'nucleoporins-93 (NUP-93)' from Leishmania donovani prompts macrophage signaling for T-cell activation towards host protective immune response.

The shift of macrophage and T-cell repertoires towards proinflammatory cytokine signalling ensures the generation of host-protective machinery that is otherwise compromised in cases of the intracellular Leishmania parasite. Different groups have attempted to restore host protective immunity. These vaccine candidates showed good responses and protective effects in murine models, but they generally failed during human trials. In the present study, we evaluated the effect of 97 kDa recombinant nucleoporin-93 of Leishmania donovani (rLd-NUP93) on mononuclear cells in healthy and treated visceral leishmaniasis (VL) patients and on THP-1 cell lines. rLd-NUP93 stimulation increased the expression of the early lymphocyte activation marker CD69 on CD4+ and CD8+ T cells. The expression of the host protective pro-inflammatory cytokines IFN-γ, IL-12 and TNF-α was increased, with a corresponding down-regulation of IL-10 and TGF-ß upon rLd-NUP93 stimulation. This immune polarization resulted in the up-regulation of NF-κB p50 with scant expression of SMAD-4. Augmenting lymphocyte proliferation upon priming with rLd-NUP93 ensured its potential for activation and generation of strong T-cell mediated immune responses. This stimulation extended the leishmanicidal activity of macrophages by releasing high amounts of reactive oxygen species (ROS). Further, the leishmanicidal activity of macrophages was intensified by the elevated production of nitric oxide (NO). The fact that this antigen was earlier reported in circulating immune complexes of VL patients highlights its antigenic importance. In addition, in silico analysis suggested the presence of MHC class I and II-restricted epitopes that proficiently trigger CD8+ and CD4+ T-cells, respectively. This study reported that rLd-NUP93 was an effective immunoprophylactic agent that can be explored in future vaccine design.

Genome-Wide Assessment of Polygalacturonases-Like (PGL) Genes of Medicago truncatula, Sorghum bicolor, Vitis vinifera and Oryza sativa Using Comparative Genomics Approach.

The polygalacturonases (PG) is one of the important members of pectin-degrading glycoside hydrolases of the family GH28. In plants, PG represents multigene families associated with diverse processes. In the present study, an attempt has been made to investigate the diversity of PG genes among monocots and dicots with respect to phylogeny, gene duplication and subcellular localization to get an insight into the evolutionary and functional attributes. The genome-wide assessment of Medicago truncatula, Vitis vinifera Sorghum bicolor, and Oryza sativa L. ssp. japonica genomes revealed 53, 49, 38 and 35 PG-like (PGL) genes, respectively. The predominance of glyco_hydro_28 domain, hydrophilic nature and genes with multiple introns were uniformly observed. The subcellular localization showed the presence of signal sequences targeting the secretory pathways. The phylogenetic tree constructed marked uniformity with three distinct clusters for each plant irrespective of the variability in the genome sizes. The site-specific selection pressure analysis based on K a/K s values showed predominance of purifying selection pressures among different groups identified in these plants. The functional divergence analysis revealed significant site-specific selective constraints. Results of site-specific selective pressure analysis throw light on the functional diversity of PGs in various plant processes and hence its constitutive nature. These findings are further strengthened by functional divergence analysis which reveals functionally diverse groups in all the four species representing monocots and dicots. The outcome of the present work could be utilized for deciphering the novel functions of PGs in plants.

Co-factor-independent phosphoglycerate mutase of Leishmania donovani modulates macrophage signalling and promotes T-cell repertoires bearing epitopes for both MHC-I and MHC-II.

Immunoactivation depends upon the antigen potential to modulate T-cell repertoires. The present study has enumerated the effect of 61 kDa recombinant Leishmania donovani co-factor-independent phosphoglycerate mutase (rLd-iPGAM) on mononuclear cells of healthy and treated visceral leishmaniasis subjects as well as on THP-1 cell line. rLd-iPGAM stimulation induced higher expression of interleukin-1ß (IL-1ß) in the phagocytic cell, its receptor and CD69 on T-cell subsets. These cellular activations resulted in upregulation of host-protective cytokines IL-2, IL-12, IL-17, tumour necrosis factor-α and interferon-γ, and downregulation of IL-4, IL-10 and tumour growth factor-ß. This immune polarization was also evidenced by upregulation of nuclear factor-κ light-chain enhancer of activated B cells p50 and regulated expression of suppressor of mother against decapentaplegic protein-4. rLd-iPGAM stimulation also promoted lymphocyte proliferation and boosted the leishmaniacidal activity of macrophages by upregulating reactive oxygen species. It also induced 1·8-fold higher release of nitric oxide (NO) by promoting the transcription of inducible nitric oxide synthase gene. Besides, in silico analysis suggested the presence of major histocompatibility complex class I and II restricted epitopes, which can proficiently trigger CD8+ and CD4+ cells, respectively. This study reports rLd-iPGAM as an effective immunoprophylactic agent, which can be used in future vaccine design.

Differential gene expression in tomato fruit and Colletotrichum gloeosporioides during colonization of the RNAi-SlPH tomato line with reduced fruit acidity and higher pH.

BACKGROUND: The destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi-SlPH tomato lines was sequenced and gene-expression patterns were compared. RESULTS: C. gloeosporioides inoculation of the RNAi-SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi-SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi-SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. Comparison of C. gloeosporioides gene expression during colonization of the WT and RNAi-SlPH lines showed that the fungus upregulates ammonia and nitrogen transport and the gamma-aminobutyric acid metabolic process during colonization of the WT, while on the RNAi-SlPH tomato, it mainly upregulates the nitrate metabolic process. CONCLUSIONS: Modulation of tomato acidity and pH had significant phenotypic effects on C. gloeosporioides development. The fungus showed increased colonization on the neutral RNAi-SlPH fruit, and limited colonization on the WT acidic fruit. The change in environmental pH resulted in different defense responses for the two tomato lines. Interestingly, the WT line showed upregulation of jasmonate pathways and glutamate accumulation, supporting the reduced symptom development and increased ammonia accumulation, as the fungus might utilize glutamate to accumulate ammonia and increase environmental pH for better expression of pathogenicity factors. This was not found in the RNAi-SlPH line which downregulated sugar metabolism and upregulated the phenylpropanoid pathway, leading to host susceptibility.

Identification of B-cell Epitope of Leishmania donovani and its application in diagnosis of visceral leishmaniasis.

Diagnosis of visceral leishmaniasis (VL) is often hindered by cross-reactions with antigens from other related parasite infections. This study aimed to develop an immunochromatographic test (ICT) which can detect the antigen present in circulating immune complexes (CICs) of VL patients using B-cell epitope-specific antibodies. MS analysis of six immunoreactive 2DE spots revealed two epitopes i.e. RFFVQGDGIGQHSLQEALERR (P1) and RRVAVLVLLDRL (P2) (From a hypothetical protein [Acc No: XP_003861458.1]). The epitope conservancy analysis suggested that the linear epitope (P1P2) is 97-100% conserved among Leishmania species and diverged from Homo sapiens (61% query coverage and 80% identity). Further, immunoinformatics analysis of hydrophilicity and flexibility confirmed the antigenicity of the peptide fragment. The linear epitope (P1P2) was synthesized (98% purity) and the purity was confirmed by high-performance liquid chromatography and MS. The indirect Enzyme linked immunosorbent assay results confirmed the presence of the corresponding antibody in VL patient's sera but not in those of healthy and other diseases. The result demonstrated a sensitivity 90%; Se Cl95% (82.16-96.27)% and specificity 100%; Sp Cl95% (84.56-100)% which indicated the possibility to be used as a diagnostic tool. Sensitivity, specificity, and diagnostic efficiency of colloidal gold conjugated anti-P1P2 antibody ICT strip was 100, 95.2, and 96.7%, respectively, which is slightly better as compared to other ICT for VL. Though, our result indicated the utility of anti-P1P2 antibody to detect CICs epitopes, a large-scale inspection in endemic and non-endemic area and in different ethnic population is needed for its validation and authentication.

Cation-Stress-Responsive Transcription Factors SltA and CrzA Regulate Morphogenetic Processes and Pathogenicity of Colletotrichum gloeosporioides.

Growth of Colletotrichum gloeosporioides in the presence of cation salts NaCl and KCl inhibited fungal growth and anthracnose symptom of colonization. Previous reports indicate that adaptation of Aspergillus nidulans to salt- and osmotic-stress conditions revealed the role of zinc-finger transcription factors SltA and CrzA in cation homeostasis. Homologs of A. nidulans SltA and CrzA were identified in C. gloeosporioides. The C. gloeosporioides CrzA homolog is a 682-amino acid protein, which contains a C2H2 zinc finger DNA-binding domain that is highly conserved among CrzA proteins from yeast and filamentous fungi. The C. gloeosporioides SltA homolog encodes a 775-amino acid protein with strong similarity to A. nidulans SltA and Trichoderma reesei ACE1, and highest conservation in the three zinc-finger regions with almost no changes compared to ACE1 sequences. Knockout of C. gloeosporioides crzA (ΔcrzA) resulted in a phenotype with inhibited growth, sporulation, germination and appressorium formation, indicating the importance of this calciu006D-activated transcription factor in regulating these morphogenetic processes. In contrast, knockout of C. gloeosporioides sltA (ΔsltA) mainly inhibited appressorium formation. Both mutants had reduced pathogenicity on mango and avocado fruit. Inhibition of the different morphogenetic stages in the ΔcrzA mutant was accompanied by drastic inhibition of chitin synthase A and B and glucan synthase, which was partially restored with Ca2+ supplementation. Inhibition of appressorium formation in ΔsltA mutants was accompanied by downregulation of the MAP kinase pmk1 and carnitine acetyl transferase (cat1), genes involved in appressorium formation and colonization, which was restored by Ca2+ supplementation. Furthermore, exposure of C. gloeosporioides ΔcrzA or ΔsltA mutants to cations such as Na+, K+ and Li+ at concentrations that the wild type C. gloeosporioides is not affected had further adverse morphogenetic effects on C. gloeosporioides which were partially or fully restored by Ca2+. Overall results suggest that both genes modulating alkali cation homeostasis have significant morphogenetic effects that reduce C. gloeosporioides colonization.