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1.
BMC Genomics ; 25(1): 313, 2024 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-38532318

RESUMEN

BACKGROUND: Effective vector control is key to malaria prevention. However, this is now compromised by increased insecticide resistance due to continued reliance on insecticide-based control interventions. In Kenya, we have observed heterogenous resistance to pyrethroids and organophosphates in Anopheles arabiensis which is one of the most widespread malaria vectors in the country. We investigated the gene expression profiles of insecticide resistant An. arabiensis populations from Migori and Siaya counties in Western Kenya using RNA-Sequencing. Centers for Disease Control and Prevention (CDC) bottle assays were conducted using deltamethrin (DELTA), alphacypermethrin (ACYP) and pirimiphos-methyl (PMM) to determine the resistance status in both sites. RESULTS: Mosquitoes from Migori had average mortalities of 91%, 92% and 58% while those from Siaya had 85%, 86%, and 30% when exposed to DELTA, ACYP and PMM, respectively. RNA-Seq analysis was done on pools of mosquitoes which survived exposure ('resistant'), mosquitoes that were not exposed, and the insecticide-susceptible An. arabiensis Dongola strain. Gene expression profiles of resistant mosquitoes from both Migori and Siaya showed an overexpression mainly of salivary gland proteins belonging to both the short and long form D7 genes, and cuticular proteins (including CPR9, CPR10, CPR15, CPR16). Additionally, the overexpression of detoxification genes including cytochrome P450s (CYP9M1, CYP325H1, CYP4C27, CYP9L1 and CYP307A1), 2 carboxylesterases and a glutathione-S-transferase (GSTE4) were also shared between DELTA, ACYP, and PMM survivors, pointing to potential contribution to cross resistance to both pyrethroid and organophosphate insecticides. CONCLUSION: This study provides novel insights into the molecular basis of insecticide resistance in An. arabiensis in Western Kenya and suggests that salivary gland proteins and cuticular proteins are associated with resistance to multiple classes of insecticides.


Asunto(s)
Anopheles , Insecticidas , Malaria , Compuestos Organotiofosforados , Piretrinas , Animales , Insecticidas/farmacología , Resistencia a los Insecticidas/genética , Anopheles/genética , Kenia , Mosquitos Vectores , Glutatión Transferasa , Perfilación de la Expresión Génica , Proteínas y Péptidos Salivales/genética , Glándulas Salivales
2.
Mol Biol Evol ; 40(10)2023 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-37804524

RESUMEN

Herbivorous insects such as whiteflies, planthoppers, and aphids secrete abundant orphan proteins to facilitate feeding. Yet, how these genes are recruited and evolve to mediate plant-insect interaction remains unknown. In this study, we report a horizontal gene transfer (HGT) event from fungi to an ancestor of Aleyrodidae insects approximately 42 to 190 million years ago. BtFTSP1 is a salivary protein that is secreted into host plants during Bemisia tabaci feeding. It targets a defensive ferredoxin 1 in Nicotiana tabacum (NtFD1) and disrupts the NtFD1-NtFD1 interaction in plant cytosol, leading to the degradation of NtFD1 in a ubiquitin-dependent manner. Silencing BtFTSP1 has negative effects on B. tabaci feeding while overexpressing BtFTSP1 in N. tabacum benefits insects and rescues the adverse effect caused by NtFD1 overexpression. The association between BtFTSP1 and NtFD1 is newly evolved after HGT, with the homologous FTSP in its fungal donor failing to interact and destabilize NtFD1. Our study illustrates the important roles of horizontally transferred genes in plant-insect interactions and suggests the potential origin of orphan salivary genes.


Asunto(s)
Áfidos , Hemípteros , Animales , Ferredoxinas/metabolismo , Plantas/metabolismo , Hemípteros/genética , Nicotiana/genética , Nicotiana/metabolismo , Áfidos/metabolismo , Proteínas y Péptidos Salivales/genética
3.
Arch Insect Biochem Physiol ; 116(2): e22123, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38860775

RESUMEN

Aphids are sap-sucking insects responsible for crop losses and a severe threat to crop production. Proteins in the aphid saliva are integral in establishing an interaction between aphids and plants and are responsible for host plant adaptation. The cotton aphid, Aphis gossypii (Hemiptera: Aphididae) is a major pest of Gossypium hirsutum. Despite extensive studies of the salivary proteins of various aphid species, the components of A. gossypii salivary glands are unknown. In this study, we identified 123,008 transcripts from the salivary gland of A. gossypii. Among those, 2933 proteins have signal peptides with no transmembrane domain known to be secreted from the cell upon feeding. The transcriptome includes proteins with more comprehensive functions such as digestion, detoxification, regulating host defenses, regulation of salivary glands, and a large set of uncharacterized proteins. Comparative analysis of salivary proteins of different aphids and other insects with A. gossypii revealed that 183 and 88 orthologous clusters were common in the Aphididae and non-Aphididae groups, respectively. The structure prediction for highly expressed salivary proteins indicated that most possess an intrinsically disordered region. These results provide valuable reference data for exploring novel functions of salivary proteins in A. gossypii with their host interactions. The identified proteins may help develop a sustainable way to manage aphid pests.


Asunto(s)
Áfidos , Proteínas de Insectos , Glándulas Salivales , Transcriptoma , Animales , Áfidos/genética , Áfidos/metabolismo , Glándulas Salivales/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Proteínas de Insectos/química , Proteínas y Péptidos Salivales/genética , Proteínas y Péptidos Salivales/metabolismo , Gossypium/genética , Gossypium/metabolismo , Perfilación de la Expresión Génica
4.
Pestic Biochem Physiol ; 205: 106118, 2024 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-39477579

RESUMEN

Etofenprox is a pyrethroid insecticide that acts on the nervous system of insects. Due to its low toxicity to aquatic animals, it is permitted for use in controlling insect pests in rice fields. The brown planthopper (BPH), Nilaparvata lugens, a significant piercing-sucking pest feeding on rice exclusively, secretes various salivary components when feeding. Salivary proteins are essential for BPH feeding, but their response to etofenprox is not well understood. The application of etofenprox down-regulated the expression of 21 salivary protein genes, among which 9 genes (NlShpa, Salivap 3, CA, NlSEF1, Nl12, NlHSC70-3, NlSP1, NlG14, and NlDNAJB9) showed significant differences. Most differentially expressed genes are found important for BPH physiological processes, except Nl12. Here we found that silencing Nl12 impeded ovary development, thereby inhibiting oocyte formation. The potential explanation was that Nl12 was highly expressed in both salivary gland and ovary, and the ovary development abnormality may be due to the direct effect from expression reduction in ovary and/or indirect influence from expression reduction in salivary gland. Altogether, our findings provide a new insight into the mechanism of action of etofenprox on insect pests and explain part of the reason why etofenprox does not stimulate reproduction in BPH.


Asunto(s)
Regulación hacia Abajo , Fertilidad , Hemípteros , Insecticidas , Piretrinas , Proteínas y Péptidos Salivales , Animales , Hemípteros/efectos de los fármacos , Hemípteros/genética , Piretrinas/farmacología , Piretrinas/toxicidad , Fertilidad/efectos de los fármacos , Insecticidas/farmacología , Insecticidas/toxicidad , Regulación hacia Abajo/efectos de los fármacos , Proteínas y Péptidos Salivales/genética , Proteínas y Péptidos Salivales/metabolismo , Femenino , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Ovario/efectos de los fármacos , Ovario/metabolismo
5.
Int J Mol Sci ; 25(9)2024 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-38731798

RESUMEN

Aphids are insect pests that suck phloem sap and introduce salivary proteins into plant tissues through saliva secretion. The effector of salivary proteins plays a key role in the modulation of host plant defense responses and enhancing aphid host adaptation. Based on previous transcriptome sequencing results, a candidate effector cyclin-dependent kinase-like (CDK) was identified from the grain aphid Sitobion avenae. In this study, the function of SaCDK in wheat defense response and the adaptation of S. avenae was investigated. Our results showed that the transient overexpression of SaCDK in tobacco Nicotiana benthamiana suppressed cell death triggered by mouse pro-apoptotic protein-BAX or Phytophthora infestans PAMP-INF1. SaCDK, delivered into wheat cells through a Pseudomonas fluorescens-mediated bacterial type III secretion system, suppressed callose deposition in wheat seedlings, and the overexpression of SaCDK in wheat significantly decreased the expression levels of salicylic acid and jasmonic acid signaling pathway-related genes phenylalanine ammonia lyase (PAL), pathogenesis-related 1 protein (PR1), lipoxygenase (LOX) and Ω-3 fatty acid desaturase (FAD). In addition, aphid bioassay results showed that the survival and fecundity of S. avenae were significantly increased while feeding on the wheat plants carrying SaCDK. Taken together, our findings demonstrate that the salivary protein SaCDK is involved in inhibiting host defense response and improving its host adaptation, which lays the foundation to uncover the mechanism of the interaction of cereal aphids and host plants.


Asunto(s)
Áfidos , Triticum , Animales , Áfidos/fisiología , Triticum/parasitología , Triticum/genética , Triticum/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Proteínas y Péptidos Salivales/genética , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Adaptación Fisiológica , Enfermedades de las Plantas/parasitología , Regulación de la Expresión Génica de las Plantas , Nicotiana/parasitología , Nicotiana/genética , Ciclopentanos/metabolismo , Oxilipinas
6.
Pestic Biochem Physiol ; 197: 105645, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-38072520

RESUMEN

RNA interference (RNAi) is a promising tool for pest control and relies on sequence-specific gene silencing. Salivary proteins are cooperatively secreted into plants to guarantee the feeding of aphids; thus they have potential to develop as selective targets for RNAi-based pest control strategy. For this purpose, we firstly analyzed 18 salivary proteomes of various aphid species, and these salivary proteins can be mainly categorized into seven functional groups. Secondly, we created a work-flow for fusion dsRNA design that can target multiple genes but were selectively safe to beneficial insects. Based on this approach, seven fusion dsRNAs were designed to feed the green peach aphid, which induced a significant reduction in aphid fitness. Among them, ingestion of dsperoxidase induced the highest mortality in aphids, which was also significantly higher than that of traditional dsRNAs in targeting three peroxidases separately. In addition, dsperoxidase-fed green peach aphids triggered the highest H2O2 content of host plants as well as the attraction to natural enemies (ladybeetle and parasitic wasp) but repellent to other control aphids. Our results indicate that the fusion dsRNA design approach can improve aphid control capacity, and the fusion dsRNA targeting salivary protein-encoding genes can enhance the direct and indirect defenses of host plants, thus providing a new strategy for RNAi-based aphid control.


Asunto(s)
Áfidos , Animales , Interferencia de ARN , Áfidos/genética , Áfidos/metabolismo , Peróxido de Hidrógeno/metabolismo , Silenciador del Gen , ARN Bicatenario/genética , Proteínas y Péptidos Salivales/genética , Proteínas y Péptidos Salivales/metabolismo
7.
Int J Mol Sci ; 24(19)2023 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-37834461

RESUMEN

Saliva houses over 2000 proteins and peptides with poorly clarified functions, including proline-rich proteins, statherin, P-B peptides, histatins, cystatins, and amylases. Their genes are poorly conserved across related species, reflecting an evolutionary adaptation. We searched the nucleotide substitutions fixed in these salivary proteins' gene loci in modern humans compared with ancient hominins. We mapped 3472 sequence variants/nucleotide substitutions in coding, noncoding, and 5'-3' untranslated regions. Despite most of the detected variations being within noncoding regions, the frequency of coding variations was far higher than the general rate found throughout the genome. Among the various missense substitutions, specific substitutions detected in PRB1 and PRB2 genes were responsible for the introduction/abrogation of consensus sequences recognized by convertase enzymes that cleave the protein precursors. Overall, these changes that occurred during the recent human evolution might have generated novel functional features and/or different expression ratios among the various components of the salivary proteome. This may have influenced the homeostasis of the oral cavity environment, possibly conditioning the eating habits of modern humans. However, fixed nucleotide changes in modern humans represented only 7.3% of all the substitutions reported in this study, and no signs of evolutionary pressure or adaptative introgression from archaic hominins were found on the tested genes.


Asunto(s)
Hominidae , Proteínas y Péptidos Salivales , Humanos , Animales , Proteínas y Péptidos Salivales/genética , Histatinas , Proteoma , Nucleótidos
8.
J Biol Chem ; 297(1): 100865, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34118237

RESUMEN

During feeding, a tick's mouthpart penetrates the host's skin and damages tissues and small blood vessels, triggering the extrinsic coagulation and lectin complement pathways. To elude these defense mechanisms, ticks secrete multiple anticoagulant proteins and complement system inhibitors in their saliva. Here, we characterized the inhibitory activities of the homologous tick salivary proteins tick salivary lectin pathway inhibitor, Salp14, and Salp9Pac from Ixodesscapularis in the coagulation cascade and the lectin complement pathway. All three proteins inhibited binding of mannan-binding lectin to the polysaccharide mannan, preventing the activation of the lectin complement pathway. In contrast, only Salp14 showed an appreciable effect on coagulation by prolonging the lag time of thrombin generation. We found that the anticoagulant properties of Salp14 are governed by its basic tail region, which resembles the C terminus of tissue factor pathway inhibitor alpha and blocks the assembly and/or activity of the prothrombinase complex in the same way. Moreover, the Salp14 protein tail contributes to the inhibition of the lectin complement pathway via interaction with mannan binding lectin-associated serine proteases. Furthermore, we identified BaSO4-adsorbing protein 1 isolated from the tick Ornithodoros savignyi as a distant homolog of tick salivary lectin pathway inhibitor/Salp14 proteins and showed that it inhibits the lectin complement pathway but not coagulation. The structure of BaSO4-adsorbing protein 1, solved here using NMR spectroscopy, indicated that this protein adopts a noncanonical epidermal growth factor domain-like structural fold, the first such report for tick salivary proteins. These data support a mechanism by which tick saliva proteins simultaneously inhibit both the host coagulation cascade and the lectin complement pathway.


Asunto(s)
Proteínas de Artrópodos/ultraestructura , Interacciones Huésped-Patógeno/genética , Lectinas/genética , Proteínas y Péptidos Salivales/ultraestructura , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Coagulación Sanguínea/genética , Vasos Sanguíneos/parasitología , Vasos Sanguíneos/patología , Lectina de Unión a Manosa de la Vía del Complemento/genética , Ixodes/patogenicidad , Ixodes/ultraestructura , Lectinas/ultraestructura , Espectroscopía de Resonancia Magnética , Conformación Proteica , Saliva/química , Saliva/metabolismo , Proteínas y Péptidos Salivales/química , Proteínas y Péptidos Salivales/genética , Trombina/genética , Garrapatas/genética , Garrapatas/patogenicidad
9.
Mol Ecol ; 31(15): 4162-4175, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35661311

RESUMEN

Few studies have examined tick proteomes, how they adapt to their environment, and their roles in the parasite-host interactions that drive tick infestation and pathogen transmission. Here we used a proteomics approach to screen for biologically and immunologically relevant proteins acting at the tick-host interface during tick feeding and, as proof of principle, measured host antibody responses to some of the discovered candidates. We used a label-free quantitative proteomic workflow to study salivary proteomes of (i) wild Ixodes ricinus ticks fed on different hosts, (ii) wild or laboratory ticks fed on the same host, and (iii) adult ticks cofed with nymphs. Our results reveal high and stable expression of several protease inhibitors and other tick-specific proteins under different feeding conditions. Most pathways functionally enriched in sialoproteomes were related to proteolysis, endopeptidase, and amine-binding activities. The generated catalogue of tick salivary proteins enabled the selection of six candidate secreted immunogenic peptides for rabbit immunizations, three of which induced strong and durable antigen-specific antibody responses in rabbits. Furthermore, rabbits exposed to ticks mounted immune responses against the candidate peptides/proteins, confirming their expression at the tick-vertebrate interface. Our approach provides insights into tick adaptation strategies to different feeding conditions and promising candidates for developing antitick vaccines or markers of exposure of vertebrate hosts to tick bites.


Asunto(s)
Proteínas de Artrópodos , Ixodes , Animales , Proteínas de Artrópodos/genética , Ixodes/genética , Proteoma/genética , Proteoma/metabolismo , Proteómica/métodos , Conejos , Proteínas y Péptidos Salivales/genética , Proteínas y Péptidos Salivales/metabolismo , Vertebrados
10.
J Exp Bot ; 73(22): 7477-7487, 2022 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-36056768

RESUMEN

The brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is a serious insect pest on rice. It uses its stylet to collect sap by penetrating the phloem and at the same time it delivers saliva into the host plant, which can trigger a reaction. The molecular mechanisms by which BPH salivary proteins result in plant responses are poorly understood. In this study, we screened transcriptomic data from different BPH tissues and found a protein specific to the salivary gland, NlG14, that could induce cell death in plants. We determined that NlG14 is uniquely found in the insect family Delphacidae. Detailed examination of N. lugens showed that NlG14 was mainly localized in the A-follicle of the principal gland of the salivary gland, and that it was secreted into rice plants during feeding. Knockdown of NlG14 resulted in significant nymph mortality when BPH was fed on either rice plants or on an artificial diet. Further analysis showed that NlG14 triggered accumulation of reactive oxygen species, cell death, callose deposition, and activation of jasmonic acid signaling pathways in plants. Transient expression of NlG14 in Nicotiana benthamiana decreased insect feeding and suppressed plant pathogen infection. Thus, NlG14, an essential salivary protein of N. lugens, acted as a potential herbivore-associated molecular pattern to enhance plant resistance to both insects and plant pathogens by inducing multiple plant defense responses. Our findings provide new insights into the molecular mechanisms of insect-plant interactions and offer a potential target for pest management.


Asunto(s)
Proteínas y Péptidos Salivales , Proteínas y Péptidos Salivales/genética
11.
Proc Natl Acad Sci U S A ; 116(28): 13873-13878, 2019 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-31221752

RESUMEN

Hematophagous organisms produce a suite of salivary proteins which interact with the host's coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure-activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.


Asunto(s)
Anticoagulantes/química , Proteínas de Insectos/química , Proteínas y Péptidos Salivales/química , Trombina/química , Secuencia de Aminoácidos/genética , Coagulación Sanguínea/genética , Biología Computacional , Biblioteca de Genes , Humanos , Proteínas de Insectos/genética , Procesamiento Proteico-Postraduccional/genética , Proteínas y Péptidos Salivales/genética , Relación Estructura-Actividad , Trombina/antagonistas & inhibidores , Trombina/genética , Tirosina/química
12.
Int J Mol Sci ; 23(24)2022 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-36555254

RESUMEN

Hard ticks feed for several days or weeks on their hosts and their saliva contains thousands of polypeptides belonging to dozens of families, as identified by salivary transcriptomes. Comparison of the coding sequences to protein databases helps to identify putative secreted proteins and their potential functions, directing and focusing future studies, usually done with recombinant proteins that are tested in different bioassays. However, many families of putative secreted peptides have a unique character, not providing significant matches to known sequences. The availability of the Alphafold2 program, which provides in silico predictions of the 3D polypeptide structure, coupled with the Dali program which uses the atomic coordinates of a structural model to search the Protein Data Bank (PDB) allows another layer of investigation to annotate and ascribe a functional role to proteins having so far being characterized as "unique". In this study, we analyzed the classification of tick salivary proteins under the light of the Alphafold2/Dali programs, detecting novel protein families and gaining new insights relating the structure and function of tick salivary proteins.


Asunto(s)
Ixodidae , Garrapatas , Animales , Garrapatas/genética , Garrapatas/metabolismo , Saliva/metabolismo , Ixodidae/metabolismo , Proteínas y Péptidos Salivales/genética , Proteínas y Péptidos Salivales/metabolismo , Transcriptoma , Proteínas de Artrópodos/metabolismo
13.
J Biol Chem ; 295(42): 14367-14378, 2020 10 16.
Artículo en Inglés | MEDLINE | ID: mdl-32817341

RESUMEN

Ticks, as blood-sucking parasites, have developed a complex strategy to evade and suppress host immune responses during feeding. The crucial part of this strategy is expression of a broad family of salivary proteins, called Evasins, to neutralize chemokines responsible for cell trafficking and recruitment. However, structural information about Evasins is still scarce, and little is known about the structural determinants of their binding mechanism to chemokines. Here, we studied the structurally uncharacterized Evasin-4, which neutralizes a broad range of CC-motif chemokines, including the chemokine CC-motif ligand 5 (CCL5) involved in atherogenesis. Crystal structures of Evasin-4 and E66S CCL5, an obligatory dimeric variant of CCL5, were determined to a resolution of 1.3-1.8 Å. The Evasin-4 crystal structure revealed an L-shaped architecture formed by an N- and C-terminal subdomain consisting of eight ß-strands and an α-helix that adopts a substantially different position compared with closely related Evasin-1. Further investigation into E66S CCL5-Evasin-4 complex formation with NMR spectroscopy showed that residues of the N terminus are involved in binding to CCL5. The peptide derived from the N-terminal region of Evasin-4 possessed nanomolar affinity to CCL5 and inhibited CCL5 activity in monocyte migration assays. This suggests that Evasin-4 derivatives could be used as a starting point for the development of anti-inflammatory drugs.


Asunto(s)
Quimiocina CCL5/antagonistas & inhibidores , Proteínas y Péptidos Salivales/química , Garrapatas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Quimiocina CCL5/metabolismo , Cristalografía por Rayos X , Humanos , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Proteínas y Péptidos Salivales/genética , Proteínas y Péptidos Salivales/metabolismo
14.
Protein Expr Purif ; 177: 105750, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32920041

RESUMEN

Cutaneous leishmaniasis is a parasitic and neglected tropical disease transmitted by the bites of sandflies. The emergence of cutaneous leishmaniasis in areas of war, conflict, political instability, and climate change has prompted efforts to develop a preventive vaccine. One vaccine candidate antigen is PpSP15, a 15 kDa salivary antigen from the sandfly Phlebotomus papatasi that facilitates the infection of the Leishmania parasite and has been shown to induce parasite-specific cell-mediated immunity. Previously, we developed a fermentation process for producing recombinant PpSP15 in Pichia pastoris and a two-chromatographic-step purification process at 100 mL scale. Here we expand the process design to the 10 L scale and examine its reproducibility by performing three identical process runs, an essential transition step towards technology transfer for pilot manufacture. The process was able to reproducibly recover 81% of PpSP15 recombinant protein with a yield of 0.75 g/L of fermentation supernatant, a purity level of 97% and with low variance among runs. Additionally, a freeze-thaw stability study indicated that the PpSP15 recombinant protein remains stable after undergoing three freeze-thaw cycles, and an accelerated stability study confirmed its stability at 37 °C for at least one month. A research cell bank for the expression of PpSP15 was generated and fully characterized. Collectively, the cell bank and the production process are ready for technology transfer for future cGMP pilot manufacturing.


Asunto(s)
Proteínas de Insectos/inmunología , Leishmania/inmunología , Vacunas contra la Leishmaniasis/inmunología , Phlebotomus/química , Proteínas y Péptidos Salivales/inmunología , Animales , Clonación Molecular , Femenino , Fermentación , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Leishmania/química , Vacunas contra la Leishmaniasis/genética , Vacunas contra la Leishmaniasis/metabolismo , Leishmaniasis Cutánea/prevención & control , Peso Molecular , Phlebotomus/fisiología , Estabilidad Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Proteínas y Péptidos Salivales/genética , Proteínas y Péptidos Salivales/metabolismo
15.
Malar J ; 20(1): 11, 2021 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-33407511

RESUMEN

BACKGROUND: The invasion of the mosquito salivary glands by Plasmodium sporozoites is a critical step that defines the success of malaria transmission and a detailed understanding of the molecules responsible for salivary gland invasion could be leveraged towards control of vector-borne pathogens. Antibodies directed against the mosquito salivary gland protein SGS1 have been shown to reduce Plasmodium gallinaceum sporozoite invasion of Aedes aegypti salivary glands, but the specific role of this protein in sporozoite invasion and in other stages of the Plasmodium life cycle remains unknown. METHODS: RNA interference and CRISPR/Cas9 were used to evaluate the role of A. aegypti SGS1 in the P. gallinaceum life cycle. RESULTS: Knockdown and knockout of SGS1 disrupted sporozoite invasion of the salivary gland. Interestingly, mosquitoes lacking SGS1 also displayed fewer oocysts. Proteomic analyses confirmed the abolishment of SGS1 in the salivary gland of SGS1 knockout mosquitoes and revealed that the C-terminus of the protein is absent in the salivary gland of control mosquitoes. In silico analyses indicated that SGS1 contains two potential internal cleavage sites and thus might generate three proteins. CONCLUSION: SGS1 facilitates, but is not essential for, invasion of A. aegypti salivary glands by P. gallinaceum and has a dual role as a facilitator of parasite development in the mosquito midgut. SGS1 could, therefore, be part of a strategy to decrease malaria transmission by the mosquito vector, for example in a transgenic mosquito that blocks its interaction with the parasite.


Asunto(s)
Aedes/genética , Proteínas de Insectos/genética , Plasmodium gallinaceum/fisiología , Proteínas y Péptidos Salivales/genética , Aedes/parasitología , Secuencia de Aminoácidos , Animales , Femenino , Tracto Gastrointestinal/parasitología , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Mosquitos Vectores/genética , Mosquitos Vectores/parasitología , Glándulas Salivales/parasitología , Proteínas y Péptidos Salivales/química , Proteínas y Péptidos Salivales/metabolismo , Alineación de Secuencia , Esporozoítos/fisiología
16.
Cell Mol Biol (Noisy-le-grand) ; 67(2): 89-94, 2021 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-34817334

RESUMEN

Erectile dysfunction (ED) is one of the most common sexual disorders in men. During the past 30 years, there has been no new drug development for ED. Thus, exploring the genetic basis of ED deserves further study, in hope of developing new pharmacological treatments for ED. In this study, Real-Time PCR analysis was used to assess the expression of androgen regulatory protein (Andpro) and pyruvate dehydrogenase kinase 4 (Pdk4) genes in ED. For this purpose, the experiment was performed on 20 men with severe ED and 20 potent men. IIEF-15 was used to determine the ED severity. The study was conducted in the Department of Sexual Medicine of the Kermanshah University of Medical Sciences, Kermanshah, Iran. The EDTA-Na vacuum blood tube was taken from ED patients and controls. Informed consent was obtained from all participants. After blood sampling, RNA was extracted from whole blood. Then cDNA was synthesized. The gene expression was analyzed through the qPCR method. The ß-actin was used as a reference gene. To further study these two proteins, their three-dimensional structures were predicted through I-TASSER. Compared with controls, in ED patients, the expression of the Andpro gene decreased, while the expression of the Pdk4 gene increased (p<0.01). Predicting the structure of the protein showed that Pyruvate Dehydrogenase Kinase 4 had a double subunit and androgen-regulated protein had a single subunit.


Asunto(s)
Disfunción Eréctil , Regulación de la Expresión Génica , Proteínas Quinasas , Proteínas y Péptidos Salivales , Adulto , Humanos , Masculino , Persona de Mediana Edad , Algoritmos , Secuencia de Aminoácidos , Biología Computacional/métodos , Disfunción Eréctil/genética , Disfunción Eréctil/metabolismo , Disfunción Eréctil/patología , Modelos Moleculares , Conformación Proteica , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas y Péptidos Salivales/química , Proteínas y Péptidos Salivales/genética , Proteínas y Péptidos Salivales/metabolismo , Índice de Severidad de la Enfermedad
17.
Future Oncol ; 17(17): 2209-2223, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-33593085

RESUMEN

Background: We describe the first studies investigating a role for opiorphin genes (PROL1, SMR3A and SMR3B) in prostate cancer (PrCa). Materials & methods: Databases and PrCa tissue arrays were screened for opiorphin expression. Xenografted tumor growth of human PrCa cells overexpressing PROL1 was compared with controls in nude mice. Modulated gene expression by overexpression of PROL1 was determined by RNA sequencing. Results: PrCa is associated with overexpression of opiorphin genes. Xenografted androgen-sensitive PrCa cells overexpressing PROL1 developed into tumors in castrated male mice (in contrast to parental cells). PROL1 overexpression modulates expression of genes in angiogenesis, steroid and hypoxic response pathways. Conclusions: Opiorphins promote the development of androgen-insensitive PrCa and activate pathways that potentially overcome the hypoxic barrier generated during tumor growth.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica , Oligopéptidos/metabolismo , Neoplasias de la Próstata/patología , Proteínas y Péptidos Salivales/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Humanos , Masculino , Ratones , Ratones Desnudos , Invasividad Neoplásica , Oligopéptidos/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas y Péptidos Salivales/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
18.
BMC Genomics ; 21(1): 331, 2020 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-32349672

RESUMEN

BACKGROUND: Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. RESULTS: We annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. CONCLUSIONS: Here we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.


Asunto(s)
Genoma , Hirudo medicinalis/genética , Proteínas y Péptidos Salivales/genética , Animales , Anticoagulantes/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hirudo medicinalis/metabolismo , Sanguijuelas/clasificación , Sanguijuelas/genética , Sanguijuelas/metabolismo , Proteómica , Saliva/metabolismo , Proteínas y Péptidos Salivales/metabolismo
19.
Rheumatology (Oxford) ; 59(4): 742-753, 2020 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-31377809

RESUMEN

OBJECTIVES: Xerostomia in SS patients has been associated with low quality and quantity of salivary mucins, which are fundamental for the hydration and protection of the oral mucosa. The aim of this study was to evaluate if cytokines induce aberrant mucin expression and whether tauroursodeoxycholic acid (TUDCA) is able to counteract such an anomaly. METHODS: Labial salivary glands from 16 SS patients and 15 control subjects, as well as 3D acini or human submandibular gland cells stimulated with TNF-α or IFN-γ and co-incubated with TUDCA, were analysed. mRNA and protein levels of Mucin 1 (MUC1) and MUC7 were determined by RT-qPCR and western blot, respectively. Co-immunoprecipitation and immunofluorescence assays for mucins and GRP78 [an endoplasmic reticulum (ER)-resident protein] were also performed. mRNA levels of RelA/p65 (nuclear factor-κB subunit), TNF-α, IL-1ß, IL-6, SEL1L and EDEM1 were determined by RT-qPCR, and RelA/p65 localization was evaluated by immunofluorescence. RESULTS: MUC1 is overexpressed and accumulated in the ER of labial salivary gland from SS patients, while MUC7 accumulates throughout the cytoplasm of acinar cells; however, MUC1, but not MUC7, co-precipitated with GRP78. TUDCA diminished the overexpression and aberrant accumulation of MUC1 induced by TNF-α and IFN-γ, as well as the nuclear translocation of RelA/p65, together with the expression of inflammatory and ER stress markers in 3D acini. CONCLUSION: Chronic inflammation alters the secretory process of MUC1, inducing ER stress and affecting the quality of saliva in SS patients. TUDCA showed anti-inflammatory properties decreasing aberrant MUC1 accumulation. Further studies are necessary to evaluate the potential therapeutic effect of TUDCA in restoring glandular homeostasis in SS patients.


Asunto(s)
Células Acinares/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Mucina-1/efectos de los fármacos , Glándulas Salivales Menores/efectos de los fármacos , Síndrome de Sjögren/metabolismo , Glándula Submandibular/efectos de los fármacos , Ácido Tauroquenodesoxicólico/farmacología , Xerostomía/metabolismo , Células Acinares/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/genética , Femenino , Proteínas de Choque Térmico/efectos de los fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Inmunoprecipitación , Técnicas In Vitro , Interferón gamma/farmacología , Interleucina-1beta/efectos de los fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Mucina-1/genética , Mucina-1/metabolismo , Mucinas/efectos de los fármacos , Mucinas/genética , Mucinas/metabolismo , Proteínas/efectos de los fármacos , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Glándulas Salivales Menores/metabolismo , Proteínas y Péptidos Salivales/efectos de los fármacos , Proteínas y Péptidos Salivales/genética , Proteínas y Péptidos Salivales/metabolismo , Síndrome de Sjögren/genética , Glándula Submandibular/citología , Glándula Submandibular/metabolismo , Factor de Transcripción ReIA/efectos de los fármacos , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Xerostomía/genética
20.
Int J Med Sci ; 17(12): 1733-1743, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32714076

RESUMEN

Xerostomia (dry mouth) is a significant age-related condition. Meanwhile, cordycepin, the natural therapeutic agent, has demonstrated an anti-aging effect. Therefore, the present study aimed to investigate the preventive effects of cordycepin on secretory function in an in vitro model of hydrogen peroxide (H2O2)-induced salivary hypofunction. After being exposed to H2O2, human submandibular gland (HSG) cells were treated with various concentrations of cordycepin (6.25-50 µM) for 24, 48, and 72h. To evaluate cell proliferation and reactive oxygen species (ROS) generation, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and 2, 7'-dichlorodihydrofluorescein diacetate assays were performed. The amylase activity was kinetically measured by 2-chloro-p-nitrophenol linked with maltotrioside. The expression of salivary, antioxidant and apoptotic markers at mRNA and protein levels were performed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence analysis, respectively. We demonstrated that cordycepin (6.25-25 µM) contributed to significant increases in expression of the salivary marker genes, alpha-amylase 1 (AMY1A) and aquaporin-5 (AQP5), and in amylase secretion without changes in cell viability. Under oxidative stress, HSG cells showed remarkable dysfunction. Cordycepin rescued the protective effects partially by decreasing ROS generation and restoring the expression of the salivary proteins, AMY and AQP5 via anti-oxidant and anti-apoptotic activity. In addition, the amount of amylase that was secreted from HSG cells cultured in cordycepin was increased. In conclusion, cordycepin demonstrated a protective effect on H2O2 -induced HSG cells by decreasing ROS generation and upregulating the salivary function markers, AMY1A and AQP5, at both the transcriptional and translational levels.


Asunto(s)
Acuaporina 5/genética , Desoxiadenosinas/farmacología , alfa-Amilasas Salivales/genética , Xerostomía/tratamiento farmacológico , Línea Celular , Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/toxicidad , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Saliva/efectos de los fármacos , Saliva/metabolismo , Proteínas y Péptidos Salivales/genética , Glándula Submandibular/efectos de los fármacos , Glándula Submandibular/patología , Xerostomía/inducido químicamente , Xerostomía/patología
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