Antimony resistance and gene expression in Leishmania: spotlight on molecular and proteomic aspects.

Leishmaniasis is a vector-borne parasitic disease caused by Leishmania parasites with a spectrum of clinical manifestations, ranging from skin lesions to severe visceral complications. Treatment of this infection has been extremely challenging with the concurrent emergence of drug resistance. The differential gene expression and the discrepancies in protein functions contribute to the appearance of 2 distinct phenotypes: resistant and sensitive, but the current diagnostic tools fail to differentiate between them. The identification of gene expression patterns and molecular mechanisms coupled with antimony (Sb) resistance can be leveraged to prompt diagnosis and select the most effective treatment methods. The present study attempts to use comparative expression of Sb resistance-associated genes in resistant and sensitive Leishmania, to disclose their relative abundance in clinical or in vitro selected isolates to gain an understanding of the molecular mechanisms of Sb response/resistance. Data suggest that the analysis of resistance gene expression would verify the Sb resistance or susceptibility only to a certain extent; however, none of the individual expression patterns of the studied genes was diagnostic as a biomarker of Sb response of Leishmania. The findings highlighted will be useful in bridging the knowledge gap and discovering innovative diagnostic tools and novel therapeutic targets.

Treatment of Cutaneous Leishmaniasis and Insights into Species-Specific Responses: A Narrative Review.

Cutaneous leishmaniasis (CL) is a complex skin infection that has imposed a heavy burden on many developing countries and is caused by more than 20 Leishmania species. This disease is predominantly associated with disfiguring scars and major social stigma upon infection. The severity of the disease seemingly depends on many factors including the species of parasite, the host, region of endemicity, socio-economic status and the accessibility to health facilities. Despite myriad studies that have been performed on current and novel therapies, the treatment outcomes of CL remain contentious, possibly because of the knowledge gaps that still exist. The differential responses to the current CL therapies have become a major drawback in disease control, and the dearth of information on critical analyses of outcomes of such studies is a hindrance to the overall understanding. On the basis of currently available literature on treatment outcomes, we discuss the most effective doses, drug susceptibilities/resistance and treatment failures of the Leishmania genus for both monotherapy and combination therapy. This review focuses on the available treatment modalities for CL caused by different Leishmania species, with insights into their species-specific efficacies, which would inform the selection of appropriate drugs for the treatment and control of leishmaniasis.

Molecular characterization and expressional analysis of two poly (ADP-ribose) polymerase (PARP) domain-containing Gig2 isoforms in rockfish (Sebastes schlegelii) and their antiviral activity against viral hemorrhagic septicemia virus.

Remedies toward sustainable aquaculture rely upon research that unveils the molecular mechanisms behind host immunity and their interactions with pathogens. Antiviral defense is a major innate immune response in fish. The antiviral protein GCHV-induced gene-2 (Gig2), a member of the interferon-stimulated gene (ISG), was identified and characterized from rockfish (Sebastes schlegelii). Gig2 exists in two isoforms, namely, SsGig2-I1 and SsGig2-I2, in rockfish with lengths of 163 and 223 bp, respectively. Bioinformatic analysis indicated the availability of poly (ADP-ribose) polymerase domain in both proteins, and 51.3% identity and 71.3% similarity between both isoforms were observed. The basal expression pattern revealed the highest tissue-specific expression in rockfish gills for both isoforms. The immune challenge experiment disclosed a distinctive and strong expression of each transcript in the presence of poly I:C. Both isoforms are localized in the endoplasmic reticulum. Interferon (IFN) pathway gene analysis revealed no significant upregulation of IFN related genes. Viral hemorrhagic septicemia virus (VHSV) gene expression analysis revealed strong downregulation of viral transcripts after 48 h of infection in the presence of Gig2 isoforms. Collectively, these results indicate the protective role of Gig2 in rockfish against VHSV infection and help broaden our understanding of the innate immunity of fish.

A copper-zinc-superoxide dismutase (CuZnSOD) from redlip mullet, Liza haematocheila: Insights to its structural characteristics, immune responses, antioxidant activity, and potent antibacterial properties.

Copper-zinc superoxide dismutase (CuZnSOD) is a nuclear-encoded metalloenzyme responsible for scavenging harmful reactive oxygen species (ROS). In this study, the CuZnSOD homolog from redlip mullet (Liza haematochelia) (MuCuZnSOD) was structurally and functionally characterized to evaluate its antioxidant capacity, antibacterial properties, and protective level in various pathogenic stress conditions. Structural characteristics of MuCuZnSOD were evaluated using different bioinformatics tools. Pairwise sequence comparison and evolutionary tree structure revealed that the MuCuZnSOD sequence was closely related to the CuZnSOD sequence of Oplegnathus fasciatus with a 94.2% sequence identity. Sequence alignment analysis indicated that the CuZnSOD domain was well conserved. The highest transcriptional expression of MuCuZnSOD was identified in the blood. Immune challenge with lipopolysaccharide (LPS), Lactococcus garvieae, and polyinosinic-polycytidylic acid (poly I:C) exhibited an increased MuCuZnSOD mRNA expression in the blood and liver. Transfected green fluorescent protein-fused MuCuZnSOD was localized in the cytoplasm. Recombinant MuCuZnSOD (rMuCuZnSOD) was overexpressed in a bacterial system. The rMuCuZnSOD possessed significant antioxidant properties as determined by conventional xanthine oxidase assay. The optimum pH and temperature of rMuCuZnSOD were found to be pH 9 and 25 °C, respectively. rMuCuZnSOD enzyme activity increased in a concentration-dependent manner. Treatment with potassium cyanide highly inhibited the rMuCuZnSOD activity. rMuCuZnSOD possessed a significant peroxidation activity in the presence of HCO3- ions as demonstrated by the increased viability in cells treated with the enzyme in the presence of hydrogen peroxide. Antibacterial assays showed that rMuCuZnSOD had significant growth-inhibitory effects on both gram-positive and gram-negative bacteria. Collectively, our findings demonstrate that MuCuZnSOD is an essential antioxidant protein, which regulates the host defense mechanisms and innate immunity under oxidative stress.

Transcription profile, NF-ĸB promoter activation, and antiviral activity of Amphiprion clarkii Akirin-2.

Animal defense system constitutes a series of distinct mechanisms that specifically defend against microbial invasion. Understanding these complex biological mechanisms is of paramount importance for implementing disease prevention strategies. In this study, the transcription factor, Akirin-2 was identified from ornamental fish Amphiprion clarkii and its involvement in immune response was characterized. A. clarkii Akirin-2 (AcAkirin-2) was identified as a highly conserved protein with two nuclear localization signals. In-vitro localization analysis in fathead minnow cells revealed that AcAkirin-2 is strictly localized to the nucleus. With regard to tissue-specific expression without immune challenge, AcAkirin-2 expression was highest in the brain and lowest in the liver. Immune challenge experiments revealed that AcAkirin-2 expression was the strongest in response to poly I:C. Overexpression of AcAkirin-2 alone did not enhanced NF-ĸB activity significantly in HEK293T cells; however, it significantly enhanced NF-ĸB activity in the presence of poly I:C. AcAkirin-2-mediated expression of antiviral genes was analyzed using qPCR in mullet kidney cells and plaque assay was performed to decipher the involvement of AcAkirin-2 in antiviral immunity. AcAkirin-2 overexpression significantly enhanced the expression of Viperin but not of Mx. Plaque assays revealed the ability of AcAkirin-2 to enervate VHSV titers. Taken together, this study unveiled the involvement of AcAkirin-2 in NF-ĸB-mediated transcription of antiviral genes.

Transcriptional modifications and the cytoprotective, DNA protective, and wound healing effects of peroxiredoxin-1 from Sebastes schlegelii.

Peroxiredoxins are a group of thiol-specific antioxidant proteins that take six isoforms in vertebrates and allow the innate immune system to sense and detoxify reactive oxygen species. In this study, we identified and characterized the perxiredoxin-1 (SsPrdx1) cDNA sequence from the rockfish, Sebastes schlegelii. In silico analysis revealed that SsPrdx1 contained a 594 bp long open reading frame (ORF) encoding a protein of 198 amino acids, with a predicted molecular weight and theoretical isoelectric point of 21.97 kDa and 6.30, respectively. The SsPrdx1 gene comprised six exons linked by five introns, while peroxiredoxin signature motifs were found in the highly conserved third, fourth, and fifth exons. Phylogenetic analysis and sequence alignment suggested that SsPrdx1 is evolutionarily conserved and that its most closely related counterpart is Salarias fasciatus. Recombinant SsPrdx1 (rSsPrdx1) displayed supercoiled DNA protection and insulin disulfide reduction activities in a concentration-dependent manner, while cells transiently transfected with pcDNA3.1 (+)/SsPrdx1 exhibited significant cytoprotective effects under oxidative stress and wound healing activity. SsPrdx1 transcripts were constitutively expressed under normal physiological conditions, with the highest expression observed in the blood. Moreover, SsPrdx1 expression increased in the blood, spleen, and liver following immune provocation by LPS, poly I:C, and Streptococcus iniae injection. Thus, this study provides insights into the role of SsPrdx1 in rockfish immune protection.

Role of rockfish (Sebastes schlegelii) glutaredoxin 1 in innate immunity, and alleviation of cellular oxidative stress: Insights into localization, molecular characteristics, transcription, and function.

Glutaredoxins are a group of heat stable oxidoreductases ubiquitously found in prokaryotes and eukaryotes. They are widely known for GSH (glutathione)-dependent protein disulfide reduction and cellular redox homeostasis. This study was performed to identify and characterize rockfish (Sebastes schlegelii) glutaredoxin 1 (SsGrx1) at molecular, transcriptional, and functional levels. The coding sequence of SsGrx1 was 318 bp in length and encoded a protein containing 106 amino acids. The molecular weight and theoretical isoelectric point of the putative SsGrx1 protein were 11.6 kDa and 6.71 kDa, respectively. The amino acid sequence of SsGrx1 comprised a CPYC redox active motif surrounded by several conserved GSH binding sites. The modeled protein structure was found to consist of five α-helices and four ß-sheets, similar to human Grx1. SsGrx1 showed a tissue specific expression in all the tissues tested, with the highest expression in the kidney. Immune stimulation by lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (polyI:C), and Streptococcus iniae (S. iniae) could significantly modulate the SsGrx1 expression pattern in the blood and gills. Analysis of its subcellular localization disclosed that SsGrx1 was prominently localized in the cytosol. Recombinant SsGrx1 (rSsGrx1) exhibited significant activity in insulin disulfide reduction assay and HED (ß-Hydroxyethyl Disulfide) assay. Furthermore, transient overexpression of SsGrx1 in FHM (fathead minnow) cells significantly enhanced cell survival upon H2O2-induced apoptosis. Collectively, our findings strongly suggest that SsGrx1 plays a crucial role in providing rockfish immune protection against pathogens and oxidative stress.

Molecular, transcriptional and functional delineation of Galectin-8 from black rockfish (Sebastes schlegelii) and its potential immunological role.

Galectins are ß-galactoside-binding lectins, which are involved in pattern recognition, cell adhesion, and stimulation of the host innate immune responses against microbial pathogens. In spite of several functional studies on different galectins isolated from vertebrates and invertebrates, this is the first report to present functional studies for galectin-8 from the marine teleost tissues. In the present study, we characterized galectin-8 homolog from black rockfish (Sebastes schlegelii), in molecular and functional aspects. Rockfish galectin-8 (SsGal8) was found to consist of a 969 bp long open reading frame (ORF), encoding a protein of 322 amino acids and the predicted molecular weight was 35.82 kDa. In silico analysis of SsGal8 revealed the presence of two carbohydrate binding domains (CRDs), at both N and C-termini and a linker peptide of 40 amino acids, in between the two domains. As expected, the phylogenetic tree categorized SsGal8 as a tandem-repeat galectin, and ultimately positioned it in the sub-clade of fish galectin-8. rSsGal8 was able to strongly agglutinate fish erythrocytes and the inhibition of agglutination was successfully exhibited by lactose and d-galactose. Bacterial agglutination assay resulted in agglutination of both Gram (+) and Gram (-) bacteria, including Escherichia coli, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus parauberis, Lactococcus garvieae, Streptococcus iniae and Vibrio tapetis. The tissue distribution analysis based on qPCR assays, revealed a ubiquitous tissue expression of SsGal8 for the examined rockfish tissues, with the most pronounced expression in blood, followed by brain, intestine, head kidney and kidney. Furthermore, the mRNA transcription level of SsGal8 was significantly up-regulated in spleen, liver and head kidney, upon immune challenges with Streptococcus iniae, LPS and poly I:C, in a time dependent manner. Taken together, these findings strongly suggest the contribution of SsGal8 in regulating innate immune responses to protect the rockfish from bacterial infections.