Dataset Information

Qualitative and quantitative proteomics informed by transcriptomics insight into the proteome of Ornithodoros erraticus adult tick saliva.

ABSTRACT: Anti-tick vaccines have proved to be an effective and sustainable method for the control of tick infestations and tick-borne diseases with clear advantages over the application of chemical acaricides (Šmit and Postma, 2016; de la Fuente, 2018; Ndawula and Tabor, 2020). Moreover, the efficacy of acaricide application against Ornithodoros ticks is seriously limited owing to their endophilic/nidicolous life style, which make these ticks less accessible to the chemical acaricides (Astigaraga et al., 1995). Success in tick vaccine development is largely dependent on identification of new and highly protective tick antigens. Searching of new candidate protective antigens is currently being approached among tick molecules that play important biological functions at the tick-host interface, and more precisely among the salivary and intestinal proteins involved in biological processes specifically evolved by ticks to adapt to haematophagy (de la Fuente et al., 2016; Oleaga et al., 2021; Pérez-Sánchez et al., 2021). Accordingly, next-generation sequencing (NGS) and high-throughput proteomics technologies are been used to explore the transcriptome and proteome of the salivary glands/saliva and midguts of an increasing number of tick species and obtain the corresponding sialomes and mialomes (Chmelař et al., 2016; Almeida-Martins et al., 2020; Mans et al., 2020; Oleaga et al., 2021). These studies have identified a wealth of tick molecules related to tick haematophagy, tick-host interplay and pathogen transmission, which can then be scrutinized and filtered in vaccinomics pipelines for selecting candidate protective antigens (Chmelař et al., 2016; Maruyama et al., 2017; Antunes et al., 2018; de la Fuente et al., 2018; Ren et al., 2019; Couto et al., 2021). Similarly, we were also interested in characterizing the O. erraticus sialome. As far as O. erraticus saliva must contain all the bioactive molecules that the tick need to successfully feed, decoding its composition will lead to the discovery of new antigen targets for developing vaccines for the control and prevention of O. erraticus infestations and the diseases it transmits. Accordingly, the objective of the present work was to obtain the proteome of the saliva of O. erraticus adult ticks. For this, we have used a proteomics informed by transcriptomics approach to analyse female and male saliva separately using two different mass spectrometry approaches: liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-dependent acquisition (DDA) mode, and Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH MS). SWATH MS is a specific variant of data-independent acquisition (DIA) methods that combines deep proteome coverage capabilities with quantitative consistency and accuracy (Ludwig et al., 2018). Here we reported the identification of 387 non-redundant proteins in the saliva of O. erraticus adult ticks as well as a qualitative and quantitative comparison of the saliva protein composition between both sexes. The integration of O. erraticus sialoproteomic and sialotranscriptomic datasets facilitate a better understanding of the physiology of feeding in O. erraticus and will drive the discovery of new and more effective antigen targets for development of anti-tick vaccines.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Ornithodoros Erraticus

TISSUE(S): Saliva

SUBMITTER: Luz Valero

LAB HEAD: Ricardo Pérez-Sánchez

PROVIDER: PXD027367 | Pride | 2022-02-17

REPOSITORIES: Pride

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A proteomics informed by transcriptomics insight into the proteome of Ornithodoros erraticus adult tick saliva.

Pérez-Sánchez Ricardo R Carnero-Morán Angel A Luz Valero M M Oleaga Ana A

Parasites & vectors 20220103 1

<h4>Background</h4>The argasid tick Ornithodoros erraticus is the main vector of tick-borne human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin. The prevention and control of these diseases would greatly benefit from the elimination of O. erraticus populations, and anti-tick vaccines are envisaged as an effective and sustainable alternative to chemical acaricide usage for tick control. Ornithodoros erraticus saliva contains bioactive proteins that play essential ...[more]

PMID: 34980218

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Project description:Ticks are obligate blood feeding ectoparasites that transmit a wide variety of pathogenic microorganisms to their vertebrate hosts. Amblyomma sculptum is vector of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, the most lethal rickettsiosis that affects humans. It is known that the transmission of pathogens by ticks is mainly associated with the physiology of the feeding process. Pathogens that are acquired with the blood meal must first colonize the tick gut and later the salivary glands in order to be transmitted during a subsequent blood feeding via saliva. Tick saliva contains a complex mixture of bioactive molecules with anticlotting, antiplatelet aggregation, vasodilatory, anti-inflammatory, and immunomodulatory properties to counteract both the hemostasis and defense mechanisms of the host. Besides facilitating tick feeding, the properties of saliva may also benefits survival and establishment of pathogens in the host. In the current study, we compared the sialotranscriptome of unfed A. sculptum ticks and those fed for 72 h on rabbits using next generation RNA sequencing (RNA-seq). The total of reads obtained were assembled in 9,560 coding sequences (CDSs) distributed in different functional classes. CDSs encoding secreted proteins, including lipocalins, mucins, protease inhibitors, glycine-rich proteins, metalloproteases, 8.9 kDa superfamily members, and immunity-related proteins were mostly upregulated by blood feeding. Selected CDSs were analyzed by real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR), corroborating the transcriptional profile obtained by RNA-seq. Finally, high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis revealed 124 proteins in saliva of ticks fed for 96-120 h. The corresponding CDSs of 59 of these proteins were upregulated in salivary glands of fed ticks. To the best of our knowledge, this is the first report on the proteome of A. sculptum saliva. The functional characterization of the identified proteins might reveal potential targets to develop vaccines for tick control and/or blocking of R. rickettsii transmission as well as pharmacological bioproducts with antihemostatic, anti-inflammatory and antibacterial activities.

Project description:The NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to screen the resulting mRNA harvested from ticks treated with the tick Ubiquitin-63E 600bp dsRNA in comparison to controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A stringent bioinformatics analysis was undertaken to predict off target effects (OTE) resulting from the in silico dicing of the 600bp Ubiquitin-63E dsRNA which identified 10 down (including TC6372) and 13 up-regulated ESTs within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196bp and 109bp dsRNAs, and a cocktail of short hairpin RNAs targeting Ubiquitin-63E demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative real time PCR analysis confirmed TC6372 and predicted off target differential expression. Our study demonstrates the effectiveness and specificity for utilising ~100bp dsRNA products for targeted tick gene knockdown with nil identified off targets. The microarray was conducted by NimbleGen Systems Inc following the method reported by Saldivar [Saldivar L et al., Insect Mol Biol 2008, 17(6):597-606]. Briefly, Custom high-density single channel oligonucleotide arrays were constructed by NimbleGen Systems Inc. using 13 601 of the 13 643 members of BmiGI Version 2 with 14 perfect match 50-mer probes per BmiGI target. No mismatched probes were included on the arrays, although probes with randomly generated sequences were included. These random sequence probes were designed to match the melting temperature (Tm) of the other probes on the array and to reflect the distribution of non-specific signal intensities for binding events to probes with approximately the same composition as the perfect match probes but with random sequences. The associated GEO dataset can be found online at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL6373. Each microarray chip includes two in-slide replicates (spot replicates). In this experiment two biological replicates for each sample were hybridized to assess variability between individual samples. Each pooled sample consisted of 30 female adult ticks (N strain) as the source of RNA. Four technical replicates were processed, i.e. repeated measurements of the same pooled R. microplus mRNA, consisting of two chip replicates with two spot replicates. The chip replicates were hybridized on different dates to different chips, while spot replicates indicate the same probe was spotted on different locations on the chip. Four R. microplus samples were hybridized to separate microarrays at NimbleGen Systems Inc. (Madison, WI, USA). Data Processing: Statistical analysis Following array hybridization the raw intensity values were background corrected using convolution and normalized using quantile normalization to adjust for technical sources of variation. Final log2 expression intensities were generated using the Robust Multichip Average (RMA) algorithm [Irizarry et al.,Biostatistics 2003, 4:249-264]; [Groeneveld E, et al.,Proceedings 6th World Cong Genet Appl Livest Prod,: 1998; Armidale, NSW, Australia; 455-458] implemented in R. Differential expression was tested on the RMA normalized intensities using a mixed model of the form yijkr=?+BSrk+Gi+GTij+Eijkr where yijkr are the log2 RMA normalized signal intensities; i =gene, j=treatment, r=block, k=slide/array, the main fixed effect is BSrk (block by slide interaction), Gi is the main random effect of the gene; GTij is the random interaction term of the gene by treatment; E is just the error term; and normal assumptions for the random effects – iid are assumed. The model was fitted using VCE4.0 [Groeneveld E, et al., 1998]. Differentially expressed (DE) probes were considered as those which were three or more standard deviations away from the mean (p-value <0.001). The contrast of interest was the 600pb dsRNA microinjected ticks (R. microplus Ubiquitin-63E homologue as per Kurscheid et al 2009, BMC Molecular Biology 10:26) compared to normal untreated controls in order to examine the knockdown effect associated with phenotype. Kurscheid et al 2009 reported a strong knockdown phenotype following dsRNA knockdown and the subsequent differentially expressed ESTs were examined in this array experiment. The putative identities of the lists of up and down regulated tick sequences (DE probes) were then subsequently analysed using bioinformatic tools to predict function (described below). Bioinfomatics: Nimblegen tick microarray differentially expressed probes with fold changes >1.5 and p-value <0.001 were screened against the following databases on the CCG [Centre for Comparative Genomics: http://ccg.murdoch.edu.au], Grendel HPC system [Hunter A, et al.,Australasian Workshop on Grid Computing and e-Research 2005:2-3]; NCBI protein (nr and patent) [National Centre for Biotechnology Information: http://www.ncbi.nlm.nih.gov], String [Mering von C., et al.,Nucleic Acids Research 2006:00: D01-D05], COG [Tatusov R, et al., BMC Bioinformatics 2003, 4], tigr_bmigi.062608 [Wang et al.,BMC Genomics 8:368], NCBI Conserved Domain database [Goonesekere N, et al., Proteins 2008, 71(2):910-919], and KEGG (Kanehisa & Goto 2000 Nucleic Acids Res. 28, 27-30; Kanehisa et al 2006 Nucleic Acids Research 34, D354-D357; Kanehisa et al 2010 Nucleic Acids Research 38, D355-D360). 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Dataset Information

Qualitative and quantitative proteomics informed by transcriptomics insight into the proteome of Ornithodoros erraticus adult tick saliva.

Publications

A proteomics informed by transcriptomics insight into the proteome of Ornithodoros erraticus adult tick saliva.

Similar Datasets

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