Potential Involvement of the South American Lungfish Intelectin-2 in Innate-Associated Immune Modulation.

Intelectins belong to a family of lectins with specific and transitory carbohydrate interaction capabilities. These interactions are related to the activity of agglutinating pathogens, as intelectins play a significant role in immunity. Despite the prominent immune defense function of intelectins, limited information about its structural characteristics and carbohydrate interaction properties is available. This study investigated an intelectin transcript identified in RNA-seq data obtained from the South American lungfish (Lepidosiren paradoxa), namely LpITLN2-B. The structural analyses predicted LpITLN2-B to be a homo-trimeric globular protein with the fibrinogen-like functional domain (FReD), exhibiting a molecular mass of 57 kDa. The quaternary structure is subdivided into three monomers, A, B, and C, and each domain comprises 11 ß-sheets: an anti-parallel ß-sheet, a ß-hairpin, and a disordered ß-sheet structure. Molecular docking demonstrates a significant interaction with disaccharides rather than monosaccharides. The preferential interaction with disaccharides highlights the potential interaction with pathogen molecules, such as LPS and Poly(I:C). The hemagglutination assay inhibited lectins activity, especially maltose and sucrose, highlighting lectin activity in L. paradoxa samples. Overall, our results show the potential relevance of LpITLN2-B in L. paradoxa immune defense against pathogens.

Characterization of a Molecularly Engineered Banlec-Type Lectin (rBTL).

Lectins are proteins that reversibly bind to carbohydrates and are commonly found across many species. The Banana Lectin (BanLec) is a member of the Jacalin-related Lectins, heavily studied for its immunomodulatory, antiproliferative, and antiviral activity. In this study, a novel sequence was generated in silico considering the native BanLec amino acid sequence and 9 other lectins belonging to JRL. Based on multiple alignment of these proteins, 11 amino acids of the BanLec sequence were modified because of their potential for interference in active binding site properties resulting in a new lectin named recombinant BanLec-type Lectin (rBTL). rBTL was expressed in E. coli and was able to keep biological activity in hemagglutination assay (rat erythrocytes), maintaining similar structure with the native lectin. Antiproliferative activity was demonstrated on human melanoma lineage (A375), evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT). rBTL was able to inhibit cellular growth in a concentration-dependent manner, in an 8-h incubation, 12 µg/mL of rBTL led to a 28.94% of cell survival compared to cell control with 100%. Through a nonlinear fit out log-concentration versus biological response, an IC50% of 3.649 µg/mL of rBTL was determined. In conclusion, it is possible to state that the changes made to the rBTL sequence maintained the structure of the carbohydrate-binding site without changing specificity. The new lectin is biologically active, with an improved carbohydrate recognition spectrum compared to nBanLec, and can also be considered cytotoxic for A375 cells.

A fibrinogen-related Lectin from Echinometra lucunter represents a new FReP family in Echinodermata phylum.

Fibrinogen-related proteins (FREPs) have been identified in several animals. They are involved in the body's defense, acting as mediators of phagocytosis. Ficolins and intelectins are some of the most studied Fibrinogen-related Domain (FReD)-containing lectins. In this work, we have isolated a singular FReD-containing lectin, which cannot be classified as ficolin or intelectin. ELL (Echinometra lucunter lectin) was isolated from coelomic plasma by affinity chromatography on xanthan gum. Primary structure was determined by tandem mass spectrometry. Moreover, antimicrobial activity of ELL was evaluated against planktonic cells and biofilm of Escherichia coli, Staphylococcus aureus and S. epidermidis. ELL showed hemagglutinating activity in Ca2+ presence, which was inhibited by glycoprotein mucin and thyroglobulin. Complete amino acid sequence consisted of 229 residues, including a FReD in the N-terminal. Searches for similarity found that ELL was very close to putative proteins from Strongylocentrotus purpuratus. ELL showed moderate similarity with uncharacterized sea stars proteins and protochordate intelectins. ELL was able to inhibit the planktonic growth of the Gram-positive bacteria and significantly reduce the biofilm formation of all bacteria tested. In conclusion, we identified a new type of FReP-containing lectin with some structural and functional conservation towards intelectins.

Evaluation of the PEΔIII-LC3-KDEL3 Chimeric Protein of Entamoeba histolytica-Lectin as a Vaccine Candidate against Amebic Liver Abscess.

Entamoeba histolytica is an intestinal parasite that causes dysentery and amebic liver abscess. E. histolytica has the capability to invade host tissue by union of virulence factor Gal/GalNAc lectin; this molecule induces an adherence-inhibitory antibody response as well as to protect against amebic liver abscess (ALA). The present work showed the effect of the immunization with PEΔIII-LC3-KDEL3 recombinant protein. In vitro, this candidate vaccine inhibited adherence of E. histolytica trophozoites to HepG2 cell monolayer, avoiding the cytolysis, and in a hamster model, we observed a vaccine-induced protection against the damage to tissue liver and the inhibition of uncontrolled inflammation. PEΔIII-LC3-KDEL3 reduced the expression of TNF-α, IL-1ß, and NF-κB in all immunized groups at 4- and 7-day postinfection. The levels of IL-10, FOXP3, and IFN-γ were elevated at 7 days. The immunohistochemistry assay confirmed this result, revealing an elevated quantity of +IFN-γ cells in the liver tissue. ALA formation in hamsters immunized was minimal, and few trophozoites were identified. Hence, immunization with PEΔIII-LC3-KDEL3 herein prevented invasive amebiasis, avoided an acute proinflammatory response, and activated a protective response within a short time. Finally, this recombinant protein induced an increase of serum IgG.

Ficolin-3 in chronic Chagas disease: Low serum levels associated with the risk of cardiac insufficiency.

AIMS: To investigate whether FCN3 polymorphisms and circulating ficolin-3 levels were associated with clinical forms of chronic Chagas disease (CD) and to assess their potential use as biomarkers for the disease or its severity. METHODS AND RESULTS: FCN3 polymorphisms (g.1637delC (rs532781899) in exon 5; g.3524_3532insTATTTGGCC (rs28362807) in intron 5 and g.4473C > A) (rs4494157) in intron 7) were determined in 178 chronic CD patients (65 asymptomatic, 68 cardiac, 21 digestive and 24 cardiodigestive), and 285 healthy controls by sequence-specific PCR. Ficolin-3 serum levels, measured by ELISA in 80 patients and 80 controls, did not differ between groups. On the other hand, ficolin-3 levels were positively correlated with left ventricular ejection fraction (P = .002; r = .5), with lower levels associated with increased risk of cardiac insufficiency (P = .033; OR 7.21, 95%IC 1.17-44.4). Ficolin-3 levels were positively correlated with ficolin-2 (P = .021; r = .63), and negatively with MBL (P = .002; r = -.36) and pentraxin-3 (P = .04; r = -.32) levels. No significant results were observed for the investigated FCN3 polymorphisms and CD. The g.1637del/1637C heterozygotes presented lower ficolin-3 levels than g.1637C/1637C homozygotes in the control group (P = .023). CONCLUSION: Low ficolin-3 levels may play a role in the pathophysiology of cardiac insufficiency associated with CD.

Bioaccessibility and In Vitro Intestinal Permeability of a Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) Using the Everted Intestine Assay.

Tepary bean (Phaseolus acutifolius) lectins exhibit differential in vitro and in vivo biological effects, but their gastrointestinal interactions and digestion have not yet been assessed. This work aimed to evaluate the changes of a recombinant Tepary bean lectin (rTBL-1) through an in vitro and ex vivo gastrointestinal process. A polyclonal antibody was developed to selectively detect rTBL-1 by Western blot (WB) and immunohistochemical analysis. Everted gut sac viability was confirmed until 60 min, where protein bioaccessibility, apparent permeability coefficient, and efflux ratio showed rTBL-1 partial digestion and absorption. Immunoblot assays suggested rTBL-1 internalization, since the lectin was detected in the digestible fraction. The immunohistochemical assay detected rTBL-1 presence at the apical side of the small intestine, potentially due to the interaction with the intestinal cell membrane. The in silico interactions between rTBL-1 and some saccharides or derivatives showed high binding affinity to sialic acid (-6.70 kcal/mol) and N-acetylglucosamine (-6.10 kcal/mol). The ultra-high-performance liquid chromatography-electron spray ionization-quantitative time-of-flight coupled to mass spectrometry (UHPLC-ESI-QTOF/MS) analysis showed rTBL-1 presence in the gastric content and the non-digestible fraction after intestinal simulation conditions. The results indicated that rTBL-1 partially resisted the digestive conditions and interacted with the intestinal membrane, whereas its digestion allowed the absorption or internalization of the protein or the derivative peptides. Further purification of digestion samples should be conducted to identify intact rTBL-1 protein and digested peptides to assess their physiological effects.

A rhamnose-binding lectin from Rhodnius prolixus and the impact of its silencing on gut bacterial microbiota and Trypanosoma cruzi.

Lectins are ubiquitous proteins involved in the immune defenses of different organisms and mainly responsible for non-self-recognition and agglutination reactions. This work describes molecular and biological characterization of a rhamnose-binding lectin (RBL) from Rhodnius prolixus, which possesses a 21 amino acid signal peptide and a mature protein of 34.6 kDa. The in-silico analysis of the primary and secondary structures of RpLec revealed a lectin domain fully conserved among previous insects studied. The three-dimensional homology model of RpLec was similar to other RBL-lectins. Docking predictions with the monosaccharides showed rhamnose and galactose-binding sites comparable to Latrophilin-1 and N-Acetylgalactosamine-binding in a different site. The effects of RpLec gene silencing on levels of infecting Trypanosoma cruzi Dm 28c and intestinal bacterial populations in the R. prolixus midgut were studied by injecting RpLec dsRNA into the R. prolixus hemocoel. Whereas T. cruzi numbers remained unchanged compared with the controls, numbers of bacteria increased significantly. The silencing also induced the up regulation of the R. prolixus defC (defensin) expression gene. These results with RpLec reveal the potential importance of this little studied molecule in the insect vector immune response and homeostasis of the gut bacterial microbiota.

Ficolin-3 in rheumatic fever and rheumatic heart disease.

Rheumatic fever (RF) and chronic rheumatic heart disease (RHD) are complications of oropharyngeal infection caused by Streptococcus pyogenes. Despite the importance of the complement system against infections and autoimmunity diseases, studies on the role of the lectin pathway in RF and RHD are scarce. Thus, our aim was to evaluate the association of ficolin-3 serum levels, FCN3 polymorphisms and haplotypes with the susceptibility to RF and RHD. We investigated 179 patients with a history of RF (126 RHD and 53 RF only) and 170 healthy blood donors as control group. Ficolin-3 serum concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Three FCN3 single nucleotide polymorphisms (SNPs rs532781899, rs28362807 and rs4494157) were genotyped through the sequence-specific PCR method. Lower ficolin-3 serum levels were observed in RF patients when compared to controls (12.81 µg/mL vs. 18.14 µg/mL respectively, p < 0.0001, OR 1.22 [1.12-1.34]), and in RHD in comparison to RF only (RFo) (12.72 µg/mL vs. 14.29 µg/mL respectively, p = 0.016, OR 1.38 [1.06-1.80]). Low ficolin-3 levels (<10.7 µg/mL) were more common in patients (39.5 %, 30/76) than controls (20.6 %, 13/63, p = 0.018, OR = 2.51 [1.14-5.31]), and in RHD (44.4 %, 28/63) than RFo (15.4 %, 2/13, p = 0.007, OR = 3.08 [1.43-6.79]). On the other hand, FCN3 polymorphism/haplotypes were not associated with ficolin-3 serum levels or the disease. Low ficolin-3 levels might be associated with RF, being a potential marker of disease progression.

Ficolin-1 and ficolin-3 polymorphisms and susceptibility to rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease, which compromises the synovial membrane resulting in chronic inflammation. Ficolins are key proteins of the lectin pathway of complement able to recognize pathogen-associated molecular patterns, apoptotic cells, and cellular debris mediating the clearance by phagocytes. High ficolin-1 and ficolin-3 levels have been observed in RA patients, however, the influence of polymorphisms in the FCN1 gene in RA is not completely established, while no study evaluated FCN3 gene polymorphisms in RA to date. We investigated the influence of FCN1 and FCN3 gene polymorphisms in the susceptibility and clinical presentation of RA. A total of 148 patients with RA and up to 160 controls from Southern Brazil were genotyped by sequence-specific PCR (PCR-SSP) for five FCN1 promoter polymorphisms (rs2989727, rs10120023, rs17039495, rs10117466, and rs10858293) and three FCN3 gene variants (rs532781899, rs28362807, and rs4494157). The FCN1 g.-542GG (rs10120023) genotype and g.-542G allele, were associated with increased susceptibility to RA (p = .025, OR = 1.69 [1.07-2.69]; p = .041, OR = 1.47 [1.02-2.12], respectively) and related to decreased FCN1 gene expression in whole blood (p < .00001), according to gene expression databases. In addition, the FCN1 AAGAG haplotype was more prevalent in rheumatoid factor seronegative in comparison to seropositive patients (p = .006, OR = 0.042 [0.002-0.80]). There was no association of FCN3 polymorphisms with the susceptibility or clinical characteristics of RA. Our results indicate that the FCN1 rs10120023 [g.-542G>A] polymorphism in the promoter region might contribute to RA susceptibility, probably by impacting FCN1 gene expression.

Paracoccin: Purification and Validation of Its Lectin and Enzymatic Properties.

Studies on the effects of components derived from the human pathogenic fungi Paracoccidioides brasiliensis have identified paracoccin (PCN), as a bifunctional protein with lectin (GlcNAc-binding) and enzymatic (chitinase) activities, able to induce modulation of host immune response. Endogenous PCN acts as a fungal virulence factor, whereas exogenous purified PCN, administered to the host, confers protective immunity in a murine model of paracoccidioidomycosis. The immunomodulation induced by purified-PCN injection has characterized it as an agent applicable in the therapy and vaccine against paracoccidioidomycosis. This section describes methods for PCN purification and validation of its lectin and enzymatic activities. It includes detailed protocols to obtain homogeneous PCN from P. brasiliensis yeasts, as well as to purify recombinant PCN from transformed heterologous microorganisms.

Nephropathy in Hypertensive Animals Is Linked to M2 Macrophages and Increased Expression of the YM1/Chi3l3 Protein.

Macrophages contribute to a continuous increase in blood pressure and kidney damage in hypertension, but their polarization status and the underlying mechanisms have not been clarified. This study revealed an important role for M2 macrophages and the YM1/Chi3l3 protein in hypertensive nephropathy in a mouse model of hypertension. Bone marrow cells were isolated from the femurs and tibia of male FVB/N (control) and transgenic hypertensive animals that overexpressed the rat form of angiotensinogen (TGM(rAOGEN)123, TGM123-FVB/N). The cells were treated with murine M-CSF and subsequently with LPS+IFN-γ to promote their polarization into M1 macrophages and IL-4+IL-13 to trigger the M2 phenotype. We examined the kidneys of TGM123-FVB/N animals to assess macrophage polarization and end-organ damage. mRNA expression was evaluated using real-time PCR, and protein levels were assessed through ELISA, CBA, Western blot, and immunofluorescence. Histology confirmed high levels of renal collagen. Cells stimulated with LPS+IFN-γ in vitro showed no significant difference in the expression of CD86, an M1 marker, compared to cells from the controls or the hypertensive mice. When stimulated with IL-4+IL-13, however, macrophages of the hypertensive group showed a significant increase in CD206 expression, an M2 marker. The M2/M1 ratio reached 288%. Our results indicate that when stimulated in vitro, macrophages from hypertensive mice are predisposed toward polarization to an M2 phenotype. These data support results from the kidneys where we found an increased infiltration of macrophages predominantly polarized to M2 associated with high levels of YM1/Chi3l3 (91,89%), suggesting that YM1/Chi3l3 may be a biomarker of hypertensive nephropathy.

Variability in Genomic and Virulent Properties of Porphyromonas gingivalis Strains Isolated From Healthy and Severe Chronic Periodontitis Individuals.

Porphyromonas gingivalis has been extensively associated with both the onset and progression of periodontitis. We previously isolated and characterized two P. gingivalis strains, one from a patient exhibiting severe chronic periodontitis (CP3) and another from a periodontally healthy individual (H3). We previously showed that CP3 and H3 exhibit differences in virulence since H3 showed a lower resistance to cationic peptides compared with CP3, and a lower ability to induce proliferation in gingival epithelial cells. Here, we aimed to determine whether differences in virulence between these two strains are associated with the presence or absence of specific genes encoding virulence factors. We sequenced the whole genomes of both P. gingivalis CP3 and H3 and conducted a comparative analysis regarding P. gingivalis virulence genetic determinants. To do so, we performed a homology search of predicted protein sequences in CP3 and H3 genomes against the most characterized virulence genes for P. gingivalis available in the literature. In addition, we performed a genomic comparison of CP3 and H3 with all the 62 genomes of P. gingivalis found in NCBI's RefSeq database. This approach allowed us to determine the evolutionary relationships of CP3 and H3 with other virulent and avirulent strains; and additionally, to detect variability in presence/absence of virulence genes among P. gingivalis genomes. Our results show genetic variability in the hemagglutinin genes. While CP3 possesses one copy of hagA and two of hagC, H3 has no hagA and only one copy of hagC. Experimentally, this finding is related to lower in vitro hemmaglutination ability of H3 compared to CP3. Moreover, while CP3 encodes a gene for a major fimbrium subunit FimA type 4 (CP3_00160), H3 possess a FimA type 1 (H3_01400). Such genetic differences are in agreement with both lower biofilm formation ability and less intracellular invasion to oral epithelial cells exhibited by H3, compared with the virulent strain CP3. Therefore, here we provide new results on the genome sequences, comparative genomics analyses, and phenotypic analyses of two P. gingivalis strains. The genomics comparison of these two strains with the other 62 genomes included in the analysis provided relevant results regarding genetic determinants and their association with P. gingivalis virulence.

Transcriptomics of atopy and atopic asthma in white blood cells from children and adolescents.

Early allergic sensitisation (atopy) is the first step in the development of allergic diseases such as atopic asthma later in life. Genes and pathways associated with atopy and atopic asthma in children and adolescents have not been well characterised.A transcriptome-wide association study (TWAS) of atopy and atopic asthma in white blood cells (WBCs) or whole blood was conducted in a cohort of 460 Puerto Ricans aged 9-20 years (EVA-PR study) and in a cohort of 250 Swedish adolescents (BAMSE study). Pathway enrichment and network analyses were conducted to further assess top findings, and classification models of atopy and atopic asthma were built using expression levels for the top differentially expressed genes (DEGs).In a meta-analysis of the study cohorts, both previously implicated genes (e.g. IL5RA and IL1RL1) and genes not previously reported in TWASs (novel) were significantly associated with atopy and/or atopic asthma. Top novel genes for atopy included SIGLEC8 (p=8.07×10-13), SLC29A1 (p=7.07×10-12) and SMPD3 (p=1.48×10-11). Expression quantitative trait locus analyses identified multiple asthma-relevant genotype-expression pairs, such as rs2255888/ALOX15 Pathway enrichment analysis uncovered 16 significantly enriched pathways at adjusted p<0.01, including those relevant to T-helper cell type 1 (Th1) and Th2 immune responses. Classification models built using the top DEGs and a few demographic/parental history variables accurately differentiated subjects with atopic asthma from nonatopic control subjects (area under the curve 0.84).We have identified genes and pathways for atopy and atopic asthma in children and adolescents, using transcriptome-wide data from WBCs and whole blood samples.

ConA-Like Lectins: High Similarity Proteins as Models to Study Structure/Biological Activities Relationships.

Lectins are a widely studied group of proteins capable of specific and reversible binding to carbohydrates. Undoubtedly, the best characterized are those extracted from plants of the Leguminosae family. Inside this group of proteins, those from the Diocleinae subtribe have attracted attention, in particular Concanavalin A (ConA), the best-studied lectin of the group. Diocleinae lectins, also called ConA-like lectins, present a high similarity of sequence and three-dimensional structure and are known to present inflammatory, vasoactive, antibiotic, immunomodulatory and antitumor activities, among others. This high similarity of lectins inside the ConA-like group makes it possible to use them to study structure/biological activity relationships by the variability of both carbohydrate specificity and biological activities results. It is in this context the following review aims to summarize the most recent data on the biochemical and structural properties, as well as biological activities, of ConA-like lectins and the use of these lectins as models to study structure/biological activity relationships.

Epithelial Cells Expressing EhADH, An Entamoeba histolytica Adhesin, Exhibit Increased Tight Junction Proteins.

In Entamoeba histolytica, the EhADH adhesin together with the EhCP112 cysteine protease, form a 124 kDa complex named EhCPADH. This complex participates in trophozoite adherence, phagocytosis and cytolysis of target cells. EhCPADH and EhCP112 are both involved on epithelium damage, by opening tight junctions (TJ) and reaching other intercellular junctions. EhADH is a scaffold protein belonging to the ALIX family that contains a Bro1 domain, expresses at plasma membrane, endosomes and cytoplasm of trophozoites, and is also secreted to the medium. Contribution of EhADH to TJ opening still remains unknown. In this paper, to elucidate the role of EhADH on epithelium injury, we followed two strategies: producing a recombinant protein (rEhADH) and transfecting the ehadh gene in MDCK cells. Results from the first strategy revealed that rEhADH reached the intercellular space of epithelial cells and co-localized with claudin-1 and occludin at TJ region; later, rEhADH was mainly internalized by clathrin-coated vesicles. In the second strategy, MDCK cells expressing EhADH (MDCK-EhADH) showed the adhesin at plasma membrane. In addition, MDCK-EHADH cells exhibited adhesive features, producing epithelial aggregation and adherence to erythrocytes, as described in trophozoites. Surprisingly, the adhesin expression produced an increase of claudin-1, occludin, ZO-1 and ZO-2 at TJ, and also the transepithelial electric resistance (TEER), which is a measure of TJ gate function. Moreover, MDCK-EhADH cells resulted more susceptible to trophozoites attack, as showed by TEER and cytopathic experiments. Overall, our results indicated that EhADH disturbed TJ from the extracellular space and also intracellularly, suggesting that EhADH affects by itself TJ proteins, and possibly synergizes the action of other parasite molecules during epithelial invasion.

Chromatin Loop Formation Induced by a Subtelomeric Protosilencer Represses EPA Genes in Candida glabrata.

Adherence, an important virulence factor, is mediated by the EPA (Epithelial Adhesin) genes in the opportunistic pathogen Candida glabrata Expression of adhesin-encoding genes requires tight regulation to respond to harsh environmental conditions within the host. The majority of EPA genes are localized in subtelomeric regions regulated by subtelomeric silencing, which depends mainly on Rap1 and the Sir proteins. In vitro adhesion to epithelial cells is primarily mediated by Epa1. EPA1 forms a cluster with EPA2 and EPA3 in the right telomere of chromosome E (E-R). This telomere contains a cis-acting regulatory element, the protosilencer Sil2126 between EPA3 and the telomere. Interestingly, Sil2126 is only active in the context of its native telomere. Replacement of the intergenic regions between EPA genes in E-R revealed that cis-acting elements between EPA2 and EPA3 are required for Sil2126 activity when placed 32 kb away from the telomere (Sil@-32kb). Sil2126 contains several putative binding sites for Rap1 and Abf1, and its activity depends on these proteins. Indeed, Sil2126 binds Rap1 and Abf1 at its native position and also when inserted at -32 kb, a silencing-free environment in the parental strain. In addition, we found that Sil@-32kb and Sil2126 at its native position can physically interact with the intergenic regions between EPA1-EPA2 and EPA2-EPA3 respectively, by chromosome conformation capture assays. We speculate that Rap1 and Abf1 bound to Sil2126 can recruit the Silent Information Regulator complex, and together mediate silencing in this region, probably through the formation of a chromatin loop.

Molecular characterization of the silencing complex SIR in Candida glabrata hyperadherent clinical isolates.

An important virulence factor for the fungal pathogen Candida glabrata is the ability to adhere to the host cells, which is mediated by the expression of adhesins. Epa1 is responsible for ∼95% of the in vitro adherence to epithelial cells and is the founding member of the Epa family of adhesins. The majority of EPA genes are localized close to different telomeres, which causes transcriptional repression due to subtelomeric silencing. In C. glabrata there are three Sir proteins (Sir2, Sir3 and Sir4) that are essential for subtelomeric silencing. Among a collection of 79 clinical isolates, some display a hyperadherent phenotype to epithelial cells compared to our standard laboratory strain, BG14. These isolates also express several subtelomeric EPA genes simultaneously. We cloned the SIR2, SIR3 and SIR4 genes from the hyperadherent isolates and from the BG14 and the sequenced strain CBS138 in a replicative vector to complement null mutants in each of these genes in the BG14 background. All the SIR2 and SIR4 alleles tested from selected hyper-adherent isolates were functional and efficient to silence a URA3 reporter gene inserted in a subtelomeric region. The SIR3 alleles from these isolates were also functional, except the allele from isolate MC2 (sir3-MC2), which was not functional to silence the reporter and did not complement the hyperadherent phenotype of the BG14 sir3Δ. Consistently, sir3-MC2 allele is recessive to the SIR3 allele from BG14. Sir3 and Sir4 alleles from the hyperadherent isolates contain several polymorphisms and two of them are present in all the hyperadherent isolates analyzed. Instead, the Sir3 and Sir4 alleles from the BG14 and another non-adherent isolate do not display these polymorphisms and are identical to each other. The particular combination of polymorphisms in sir3-MC2 and in SIR4-MC2 could explain in part the hyperadherent phenotype displayed by this isolate.

Sickening or Healing the Heart? The Association of Ficolin-1 and Rheumatic Fever.

Rheumatic fever (RF) and its subsequent progression to rheumatic heart disease (RHD) are chronic inflammatory disorders prevalent in children and adolescents in underdeveloped countries, and a contributing factor for high morbidity and mortality rates worldwide. Their primary cause is oropharynx infection by Streptococcus pyogenes, whose acetylated residues are recognized by ficolin-1. This is the only membrane-bound, as well as soluble activator molecule of the complement lectin pathway (LP). Although LP genetic polymorphisms are associated with RF, FCN1 gene's role remains unknown. To understand this role, we haplotyped five FCN1 promoter polymorphisms by sequence-specific amplification in 193 patients (138 with RHD and 55, RF only) and 193 controls, measuring ficolin-1 serum concentrations in 78 patients and 86 controls, using enzyme-linked immunosorbent assay (ELISA). Patients presented lower ficolin-1 serum levels (p < 0.0001), but did not differ according to cardiac commitment. Control's genotype distribution was in the Hardy-Weinberg equilibrium. Four alleles (rs2989727: c.-1981A, rs10120023: c.-542A, rs10117466: c.-144A, and rs10858293: c.33T), all associated with increased FCN1 gene expression in whole blood or adipose subcutaneous tissue (p = 0.000001), were also associated with increased protection against the disease. They occur within the *3C2 haplotype, associated with an increased protection against RF (OR = 0.41, p < 0.0001) and with higher ficolin-1 levels in patient serum (p = 0.03). In addition, major alleles of these same polymorphisms comprehend the most primitive *1 haplotype, associated with increased susceptibility to RF (OR = 1.76, p < 0.0001). Nevertheless, instead of having a clear-cut protective role, the minor c.-1981A and c.-144A alleles were also associated with additive susceptibility to valvar stenosis and mitral insufficiency (OR = 3.75, p = 0.009 and OR = 3.37, p = 0.027, respectively). All associations were independent of age, sex or ethnicity. Thus, minor FCN1 promoter variants may play a protective role against RF, by encouraging bacteria elimination as well as increasing gene expression and protein levels. On the other hand, they may also predispose the patients to RHD symptoms, by probably contributing to chronic inflammation and tissue injury, thus emphasizing the dual importance of ficolin-1 in both conditions.

Molecular characterization of transport lectin vesicular integral membrane protein 36 kDa (VIP36) in the life cycle of Schistosoma mansoni.

VIP36 is a protein described as an L-type lectin in animals, responsible for the intracellular transport of glycoproteins within the secretory pathway, and also localized on the plasma membrane. Schistosoma mansoni has a complex system of vesicles and protein transport machinery to the cell surface. The excreted/secreted products of the larvae and eggs are known to be exposed to the host immune system. Hence, characterizing the role and action of SmVIP36 in the S. mansoni life cycle is important for a better understanding of the parasite-host relationship. To this purpose, we firstly performed in silico analysis. Analysis of SmVIP36 in silico revealed that it contains a lectin leg-like domain with a jellyroll fold as seen by its putative 3D tertiary structure. Additionally, it was also observed that its CRD contains calcium ion-binding amino acids, suggesting that the binding of SmVIP36 to glycoproteins is calcium-dependent. Finally, we observed that the SmVIP36 predicted amino acid sequence relative to its orthologs was conserved. However, phylogenetic analysis revealed that SmVIP36 follows species evolution, forming a further cluster with its definitive host Homo sapiens. Moreover, q-PCR analysis in the S. mansoni life cycle points to a significant increase in gene expression in the eggs, schistosomulae, and female adult stages. Similarly, protein expression increased in eggs, cercariae, schistosomulae, and adult worm stages. These results suggest that SmVIP36 might participate in the complex secretory activity within the egg envelope and tegument proteins, both important for the stages of the parasite that interact with the host.

A L-type lectin gene is involved in the response to hormonal treatment and water deficit in Volkamer lemon.

Combination of biotic and abiotic stress is a major challenge for crop and fruit production. Thus, identification of genes involved in cross-response to abiotic and biotic stress is of great importance for breeding superior genotypes. Lectins are glycan-binding proteins with a functions in the developmental processes as well as in the response to biotic and abiotic stress. In this work, a lectin like gene, namely ClLectin1, was characterized in Volkamer lemon and its expression was studied in plants exposed to either water stress, hormonal elicitors (JA, SA, ABA) or wounding to understand whether this gene may have a function in the response to multiple stress combination. Results showed that ClLectin1 has 100% homology with a L-type lectin gene from C. sinensis and the in silico study of the 5'UTR region showed the presence of cis-responsive elements to SA, DRE2 and ABA. ClLectin1 was rapidly induced by hormonal treatments and wounding, at local and systemic levels, suggesting an involvement in defence signalling pathways and a possible role as fast detection biomarker of biotic stress. On the other hand, the induction of ClLectin1 by water stress pointed out a role of the gene in the response to drought. The simultaneous response of ClLectin1 expression to water stress and SA treatment could be further investigated to assess whether a moderate drought stress may be useful to improve citrus performance by stimulating the SA-dependent response to biotic stress.