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Disruption of blood meal-responsive serpins prevents Ixodes scapularis from feeding to repletion.

ABSTRACT: Serine protease inhibitors (serpins) are thought to mediate the tick's evasion of the host's serine protease-mediated defense pathways such as inflammation and blood clotting. This study describes characterization and target validation of 11 blood meal-responsive serpins that are associated with nymph and adult Ixodes scapularis tick feeding as revealed by quantitative (q)RT-PCR and RNAi silencing analyses. Given the high number of targets, we used combinatorial (co) RNAi silencing to disrupt candidate serpins in two groups (G): seven highly identical and four non-identical serpins based on amino acid identities, here after called GI and GII respectively. We show that injection of both GI and GII co-dsRNA into unfed nymph and adult I. scapularis ticks triggered suppression of cognate serpin mRNA. We show that disruption of GII, but not GI serpins significantly reduced feeding efficiency of both nymph and adult I. scapularis ticks. Knockdown of GII serpin transcripts caused significant respective mortalities of ?40 and 71% of nymphal and adult ticks that occurred within 24-48?h of attachment. This is significant, as the observed lethality preceded the tick feeding period when transmission of tick borne pathogens is predominant. We suspect that some of the GII serpins (S9, S17, S19 and S32) play roles in the tick detachment process in that upon detachment, mouthparts of GII co-dsRNA injected were covered with a whitish gel-like tissue that could be the tick cement cone. Normally, ticks do not retain tissue on their mouthparts upon detachment. Furthermore, disruption of GII serpins reduced tick blood meal sizes and the adult tick's ability to convert the blood meal to eggs. We discuss our data with reference to tick feeding physiology and conclude that some of the GII serpins are potential targets for anti-tick vaccine development.

SUBMITTER: Bakshi M

PROVIDER: S-EPMC5857477 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Disruption of blood meal-responsive serpins prevents Ixodes scapularis from feeding to repletion.

Bakshi Mariam M Kim Tae Kwon TK Mulenga Albert A

Ticks and tick-borne diseases 20180110 3

Serine protease inhibitors (serpins) are thought to mediate the tick's evasion of the host's serine protease-mediated defense pathways such as inflammation and blood clotting. This study describes characterization and target validation of 11 blood meal-responsive serpins that are associated with nymph and adult Ixodes scapularis tick feeding as revealed by quantitative (q)RT-PCR and RNAi silencing analyses. Given the high number of targets, we used combinatorial (co) RNAi silencing to disrupt ca ...[more]

PMID: 29396196

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Project description:Ixodes scapularis is the most medically important tick species and transmits five of the 14 reportable human tick borne disease (TBD) agents in the USA. This study describes LC-MS/MS identification of 582 tick- and 83 rabbit proteins in saliva of I. scapularis ticks that fed for 24, 48, 72, 96, and 120 h, as well as engorged but not detached (BD), and spontaneously detached (SD). The 582 tick proteins include proteases (5.7%), protease inhibitors (7.4%), unknown function proteins (22%), immunity/antimicrobial (2.6%), lipocalin (3.1%), heme/iron binding (2.6%), extracellular matrix/ cell adhesion (2.2%), oxidant metabolism/ detoxification (6%), transporter/ receptor related (3.2%), cytoskeletal (5.5%), and housekeeping-like (39.7%). Notable observations include: (i) tick saliva proteins of unknown function accounting for >33% of total protein content, (ii) 79% of proteases are metalloproteases, (iii) 13% (76/582) of proteins in this study were found in saliva of other tick species and, (iv) ticks apparently selectively inject functionally similar but unique proteins every 24 h, which we speculate is the tick's antigenic variation equivalent strategy to protect important tick feeding functions from host immune system. The host immune responses to proteins present in 24 h I. scapularis saliva will not be effective at later feeding stages. Rabbit proteins identified in our study suggest the tick's strategic use of host proteins to modulate the feeding site. Notably fibrinogen, which is central to blood clotting and wound healing, was detected in high abundance in BD and SD saliva, when the tick is preparing to terminate feeding and detach from the host. A remarkable tick adaptation is that the feeding lesion is completely healed when the tick detaches from the host. Does the tick concentrate fibrinogen at the feeding site to aide in promoting healing of the feeding lesion? Overall, these data provide broad insight into molecular mechanisms regulating different tick feeding phases. These data set the foundation for in depth I. scapularis tick feeding physiology and TBD transmission studies.

Project description:BACKGROUND: Recent studies of the tick saliva transcriptome have revealed the profound role of salivary proteins in blood feeding. Kunitz/BPTI proteins are abundant in the salivary glands of ticks and perform multiple functions in blood feeding, such as inhibiting blood coagulation, regulating host blood supply and disrupting host angiogenesis. However, Kunitz/BPTI proteins in soft and hard ticks have different functions and molecular mechanisms. How these differences emerged and whether they are associated with the evolution of long-term blood feeding in hard ticks remain unknown. RESULTS: In this study, the evolution, expansion and expression of Kunitz/BPTI family in Ixodes scapularis were investigated. Single- and multi-domain Kunitz/BPTI proteins have similar gene structures. Single-domain proteins were classified into three groups (groups I, II and III) based on their cysteine patterns. Group I represents the ancestral branch of the Kunitz/BPTI family, and members of this group function as serine protease inhibitors. The group I domain was used as a module to create multi-domain proteins in hard ticks after the split between hard and soft ticks. However, groups II and III, which evolved from group I, are only present and expanded in the genus Ixodes. These lineage-specific expanded genes exhibit significantly higher expression during long-term blood feeding in Ixodes scapularis. Interestingly, functional site analysis suggested that group II proteins lost the ability to inhibit serine proteases and evolved a new function of modulating ion channels. Finally, evolutionary analyses revealed that the expansion and diversification of the Kunitz/BPTI family in the genus Ixodes were driven by positive selection. CONCLUSIONS: These results suggest that the differences in the Kunitz/BPTI family between soft and hard ticks may be linked to the evolution of long-term blood feeding in hard ticks. In Ixodes, the lineage-specific expanded genes (Group II and III) lost the ancient function of inhibiting serine proteases and evolved new functions to adapt to long-term blood feeding. Therefore, these genes may play a profound role in the long-term blood feeding of hard ticks. Based our analysis, we propose that the six genes identified in our study may be candidate target genes for tick control.

Project description:Ixodid ticks are vectors of human diseases such as Lyme disease, babesiosis, anaplasmosis, and tick-borne encephalitis. These diseases cause significant morbidity and mortality worldwide and are transmitted to humans during tick feeding. The tick-host-pathogen interface is a complex environment where host responses are modulated by the molecules in tick saliva to enable the acquisition of a blood meal. Disruption of host responses at the site of the tick bite may also provide an advantage for pathogens to survive and replicate. Thus, the molecules in tick saliva not only aid the tick in securing a nutrient-rich blood meal, but can also enhance the transmission and acquisition of pathogens. To investigate the effect of feeding and flavivirus infection on the salivary gland transcript expression profile in ticks, a first-generation microarray was developed using ESTs from a cDNA library derived from Ixodes scapularis salivary glands. When the salivary gland transcript profile in ticks feeding over the course of 3 days was compared to that in unfed ticks, a dramatic increase in transcripts related to metabolism was observed. Specifically, 578 transcripts were up-regulated compared to 151 down-regulated transcripts in response to feeding. When specific time points post attachment were analyzed, a temporal pattern of gene expression was observed. When Langat virus-infected ticks were compared to mock-infected ticks, transcript expression changes were observed at all 3 days of feeding. Differentially regulated transcripts include putative secreted proteins, lipocalins, Kunitz domain-containing proteins, anti-microbial peptides, and transcripts of unknown function. These studies identify salivary gland transcripts that are differentially regulated during feeding or in the context of flavivirus infection in Ixodes scapularis nymphs, a medically important disease vector. Further analysis of these transcripts may identify salivary factors that affect the transmission or replication of tick-borne flaviviruses.

Project description:Introduction: Ixodes scapularis ticks are hematophagous arthropods capable of transmitting many infectious agents to humans. The process of blood feeding is an extended and continuous interplay between tick and host responses. While this process has been studied extensively in vitro, no global understanding of the host response to ticks has emerged. To address this issue, we measured skin-specific expression of 233 discrete genes at 8 time points during primary and secondary infestations of mice with pathogen-free I. scapularis nymphs. Selected results were then validated at the mRNA and protein levels. Results: Primary infestation was characterized by the late induction of an innate immune response. Lectin pattern recognition receptors, cytokines, and chemokines were upregulated consistent with increased neutrophil and macrophage migration. Gene ontology and pathway analyses of downregulated genes suggested inhibition of gene transcription and Th17 immunity. During the secondary infestation, additional genes were modulated suggesting a broader involvement of immune cells including CD8 and CD4 positive T lymphocytes. The cytokine response showed a mixed Th1/Th2 profile with a potential for T regulatory cell activity. Key gene ontology clusters observed during the secondary infestation were cell migration and activation. Matrix metalloproteinases were upregulated, apoptosis-related genes were differentially modulated, and immunoreceptor signaling molecules were upregulated. In contrast, transcripts related to mitogenic, WNT, Hedgehog, and stress pathways were downregulated. Conclusions: Our results support a model of tick feeding where lectin pattern recognition receptors orchestrate an innate inflammatory response during primary infestation that primes a mixed Th1/Th2 response upon secondary exposure. Tick feeding inhibits gene transcription and Th17 immunity. Salivary molecules may also inhibit upregulation of mitogenic, WNT, Hedgehog, and stress pathways and enhance the activity of T regulatory cells, production of IL-10, and suppressors of cytokine signaling molecules (SOCS). This study provides the first comprehensive transcriptional analysis of the host response at the tick bite site and suggests both a potential model of the host cutaneous response and candidate genes for further description and investigation. Ear biopsies from BALB/cJ mice infested with Ixodes scapularis nymphs were assayed at 12, 48, 72, and 96 hours after infestation during a primary and secondary exposure. 3 mice were measured at each time point. Controls were 3 similarly housed but tick-free mice.

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Disruption of blood meal-responsive serpins prevents Ixodes scapularis from feeding to repletion.

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Disruption of blood meal-responsive serpins prevents Ixodes scapularis from feeding to repletion.

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