Tick saliva and its importance for tick feeding and pathogen transmission

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

The accomplishments of the Tick-Pathogen Unit are: 1- We conducted a comprehensive longitudinal RNA-sequencing of Ixodes scapularis tick midguts before, during, and after feeding. By collecting ticks in different feeding stages (unfed, slow feeding, rapid feeding, and early post-detached), we obtained a comprehensive overview of the transcripts present in each stage and the dynamic transcriptional changes that occur between them. This provides valuable insights into tick physiology. Additionally, through unsupervised clustering, we identified transcripts with similar patterns and stage-specific sequences. These findings serve as a foundation for selecting targets in the development of anti-tick control strategies and facilitate a better understanding of how blood feeding and pathogen infection impact tick physiology. 2- We performed transcriptomic analyses of immature stages of the soft tick Ornithodoros hermsi, the vector of Borrelia hermsii in the U.S. A total of 45 libraries and 2.5 billion of reads were generated, which were used for de novo transcriptome assembly, gene expression and annotation analyses. By comparing the transcriptomes of unfed versus fed (6h, 12h, 24h and 5d) groups, we observed that most of the O. hermsi genes are up-regulated for unfed larvae and nymphs (1st and 2nd stage). It seems that the core of genes needed for blood feeding and digestion is transcribed before taking a blood meal. Since this soft tick complete a blood meal within minutes, this finding may give a glimpse on the genetic strategy behind the rapid blood feeder ticks. The RNA-seq data provided a panoramic view of the transcriptomic profile of O. hermsi immature ticks. Further studies targeting tissue specific transcriptomes would give us better snapshots of the core genes driving the blood feeding and digestion of rapid-feeder ticks. 3- We performed the characterization of persulcatin, a new multifunctional protein identified from Ixodes persulcatus ticks. The recombinant persulcatin was purified and found to be a 25 kDa acidic protein with two Kunitz-type domains. Persulcatin is expressed in salivary glands and is a classical tight-binding competitive inhibitor of serine proteases, targeting plasmin (Ki: 28 nM) and thrombin (Ki: 115 nM). It blocks plasmin generation on keratinocytes and inhibits its migration, matrix protein degradation, down-regulates MMP-2 and -9, and causes a delay in blood coagulation, endothelial cell activation, and thrombin-induced fibrinocoagulation. It interacts with exosite I of thrombin and reduces thrombin-induced endothelial cell permeability by inhibiting VE-cadherin disruption. The multifaceted roles of persulcatin as an inhibitor and modulator within the thrombin and plasminogen-plasmin system not only unveils new insights into the intricate mechanisms governing wound healing but also provides a fresh perspective on the intricate interactions between ticks and their host organisms. 4- We identified a new mechanism by which a specific plasmatic protease contributes to wound healing. We demonstrated for the first time that keratinocyte surface support high molecular weight-dependent pre-kallikrein activation and kinin generation. This event was modulated and completely blocked by a tick salivary inhibitor that binds to keratinocyte surface through a specific phospholipid. This unique mechanism allows kallikrein inhibition locally at the keratinocyte membrane, reducing cell migration and wound closure induced by the protease. The inhibitor also reduced intracellular nitric oxide formation by a mechanism independent of NOS, bradykinin receptor B2, and the reactive oxygen species generated by the protease. The keratinocyte migration seems to be modulated by the inhibitor through a pathway dependent of EGF receptor phosphorylation, ERK1/2 and paxillin activation, which ultimately coordinates MMP2 secretion, actin polymerization and stress fiber formation. These results highlighted interesting new strategies used by tick salivary proteins to avoid host skin barriers against blood feeding. 5- In collaboration with colleagues from the Czech Republic, we performed the characterization of Iripin-1, a new anti-inflammatory tick serpin from Ixodes ricinus ticks. Iripin-1 inhibited primarily trypsin and further exhibited weaker inhibitory activity against kallikrein, matriptase, and plasmin. In the mouse model of acute peritonitis, Iripin-1 enhanced the production of the anti-inflammatory cytokine IL-10 and chemokines involved in neutrophil and monocyte recruitment, including MCP-1/CCL2, a potent histamine-releasing factor. Despite increased chemokine levels, the migration of neutrophils and monocytes to inflamed peritoneal cavities was significantly attenuated following Iripin-1 administration. Based on the results of in vitro experiments, immune cell recruitment might be inhibited due to Iripin-1-mediated reduction of the expression of chemokine receptors in neutrophils and adhesion molecules in endothelial cells. Decreased activity of serine proteases in the presence of Iripin-1 could further impede cell migration to the site of inflammation. Finally, we determined the tertiary structure of native Iripin-1 at 2.10 resolution by employing the X-ray crystallography technique. In conclusion, this study suggests that Iripin-1 facilitates I. ricinus feeding by attenuating the host's inflammatory response at the tick attachment site. 6- In collaboration with colleagues from the Texas A&M University, we performed a study describing the identification and characterization of proteins that form the inner core Ixodes scapularis tick attachment cement layer. The inner core cement layer completes formation by 24 h of tick attachment. Thus, we used laser-capture microdissection to isolate cement from cryosections of 6 h and 24 h tick attachment sites and to distinguish between early and late inner core cement proteins. LC-MS/MS analysis identified 138 tick cement proteins (TCPs) of which 37 and 35 were unique in cement of 6 and 24 h attached ticks respectively. We grouped TCPs in 14 functional categories: cuticular protein (16%), tick specific proteins of unknown function, cytoskeletal proteins, and enzymes (13% each), enzymes (10%), antioxidant, glycine rich, scaffolding, heat shock, histone, histamine binding, proteases and protease inhibitors, and miscellaneous (3-6% each). Gene ontology analysis confirm that TCPs are enriched for bio adhesive properties. Our data offer insights into tick cement bonding patterns and set the foundation for understanding the molecular basis of I. scapularis tick cement formation.