Albert Mulenga

Proteomic analysis of cattle tick Rhipicephalus (Boophilus) microplus saliva: a comparison between partially and fully engorged females.

The cattle tick Rhipicephalus (Boophilus) microplus is one of the most harmful parasites affecting bovines. Similarly to other hematophagous ectoparasites, R. microplus saliva contains a collection of bioactive compounds that inhibit host defenses against tick feeding activity. Thus, the study of tick salivary components offers opportunities for the development of immunological based tick control methods and medicinal applications. So far, only a few proteins have been identified in cattle tick saliva. The aim of this work was to identify proteins present in R. microplus female tick saliva at different feeding stages. Proteomic analysis of R. microplus saliva allowed identifying peptides corresponding to 187 and 68 tick and bovine proteins, respectively. Our data confirm that (i) R. microplus saliva is complex, and (ii) that there are remarkable differences in saliva composition between partially engorged and fully engorged female ticks. R. microplus saliva is rich mainly in (i) hemelipoproteins and other transporter proteins, (ii) secreted cross-tick species conserved proteins, (iii) lipocalins, (iv) peptidase inhibitors, (v) antimicrobial peptides, (vii) glycine-rich proteins, (viii) housekeeping proteins and (ix) host proteins. This investigation represents the first proteomic study about R. microplus saliva, and reports the most comprehensive Ixodidae tick saliva proteome published to date. Our results improve the understanding of tick salivary modulators of host defense to tick feeding, and provide novel information on the tick-host relationship.

Functional Characterization and Novel Rickettsiostatic Effects of a Kunitz-Type Serine Protease Inhibitor from the Tick Dermacentor variabilis

ABSTRACT Here we report the novel bacteriostatic function of a five-domain Kunitz-type serine protease inhibitor (KPI) from the tick Dermacentor variabilis. As ticks feed, they release anticoagulants, anti-inflammatory and immunosuppressive molecules that mediate the formation of the feeding lesion on the mammalian host. A number of KPIs have been isolated and characterized from tick salivary gland extracts. Interestingly, we observe little D. variabilis KPI gene expression in the salivary gland and abundant expression in the midgut. However, our demonstration of D. variabilis KPIs anticoagulant properties indicates that D. variabilis KPI may be important for blood meal digestion in the midgut. In addition to facilitating long-term attachment and blood meal acquisition, gene expression studies of Drosophila, legumes, and ticks suggest that KPIs play some role in the response to microbial infection. Similarly, in this study, we show that challenge of D. variabilis with the spotted fever group rickettsia, Rickettsia montanensis, results in sustained D. variabilis KPI gene expression in the midgut. Furthermore, our in vitro studies show that D. variabilis KPI limits rickettsial colonization of L929 cells (mouse fibroblasts), implicating D. variabilis KPI as a bacteriostatic protein, a property that may be related to D. variabilis KPIs trypsin inhibitory capability. This work suggests that anticoagulants play some role in the midgut during feeding and that D. variabilis KPI may be involved as part of the ticks defense response to rickettsiae.

Differential Expression of Genes in Uninfected and Rickettsia-Infected Dermacentor variabilis Ticks as Assessed by Differential-Display PCR

ABSTRACT Ticks serve as both the vector and the reservoir for members of the spotted fever group rickettsiae. The molecular interaction(s) that results from this close relationship is largely unknown. To identify genetic factors associated with the tick response to rickettsial infection, we utilized differential-display PCR. The majority of upregulation appeared in the infected tissue. We cloned and sequenced 54 differentially expressed transcripts and compared the sequences to those in the GenBank database. Nine of the 54 clones were assigned putative identities and included a clathrin-coated vesicle ATPase, peroxisomal farnesylated protein, Ena/vasodilator-stimulated phosphoprotein-like protein, α-catenin, tubulin α-chain, copper-transporting ATPase, salivary gland protein SGS-3 precursor, glycine-rich protein, and Dreg-2 protein. Confirmation of the rickettsial influence on the differential expression in the ovaries for a number of these clones was demonstrated by semiquantitative reverse transcription-PCR and Northern blot analyses, resulting in confirmation of six out of nine and three out of four assessed clones, respectively. Further characterization of the clones identified tissue-dependent expression in the midguts and salivary glands. The potential roles of these molecules in the maintenance and transmission of rickettsiae are discussed.

Amblyomma americanum (L.) (Acari: Ixodidae) tick salivary gland serine protease inhibitor (serpin) 6 is secreted into tick saliva during tick feeding

SUMMARY In order to successfully feed and transmit disease agents, ticks are thought to inject serine protease inhibitors (serpins) into the host to modulate host defense responses to tick feeding, such as inflammation, the complement activation pathway and blood coagulation. In this study, we show that Amblyomma americanum (Aam) serpin (S) 6 is putatively injected into the host during tick feeding, in that the antibody to recombinant (r) AamS6 specifically reacted with the expected ∼43/45 kDa AamS6 protein band on western blots of pilocarpine-induced tick saliva. Additionally, antibodies to tick saliva proteins that were generated by repeated 48 h infestations of rabbits with adult A. americanum specifically reacted with rAamS6. We speculate that AamS6 is associated with regulating events at the start of the tick feeding process, as temporal and spatial RT-PCR and western blot analyses revealed that both AamS6 mRNA and protein are strongly expressed during the first 24–72 h of feeding time before starting to fade from 96 h. The AamS6 protein has an apparently slow turnover rate in that, although the injection of AamS6 dsRNA into unfed ticks triggered complete disruption of the AamS6 mRNA by the 48 h feeding time point, western blot analysis of protein extracts of the same animals showed that the AamS6 protein that may have been expressed prior to disruption of the AamS6 mRNA was not depleted. We speculate that the presence of the AamS6 protein in ticks despite the complete disruption of the AamS6 mRNA explains the observation that RNAi-mediated silencing of the AamS6 mRNA did not affect the ability of A. americanum ticks to attach onto host skin, successfully feed and lay eggs. These findings are discussed in regards to advances in the molecular biology of ticks.

Ixodes scapularis tick serine proteinase inhibitor (serpin) gene family; annotation and transcriptional analysis

BackgroundSerine proteinase inhibitors (Serpins) are a large superfamily of structurally related, but functionally diverse proteins that control essential proteolytic pathways in most branches of life. Given their importance in the biology of many organisms, the concept that ticks might utilize serpins to evade host defenses and immunizing against or disrupting their functions as targets for tick control is an appealing option.ResultsA sequence homology search strategy has allowed us to identify at least 45 tick serpin genes in the Ixodes scapularis genome that are structurally segregated into 32 intronless and 13 intron-containing genes. Nine of the intron-containing serpins occur in a cluster of 11 genes that span 170 kb of DNA sequence. Based on consensus amino acid residues in the reactive center loop (RCL) and signal peptide scanning, 93% are putatively inhibitory while 82% are putatively extracellular. Among the 11 different amino acid residues that are predicted at the P1 sites, 16 sequences possess basic amino acid (R/K) residues. Temporal and spatial expression analyses revealed that 40 of the 45 serpins are differentially expressed in salivary glands (SG) and/or midguts (MG) of unfed and partially fed ticks. Ten of the 38 serpin genes were expressed from six to 24 hrs of feeding while six and fives genes each are predominantly or exclusively expressed in either MG and SG respectively.ConclusionGiven the diversity among tick species, sizes of tick serpin families are likely to be variable. However this study provides insight on the potential sizes of serpin protein families in ticks. Ticks must overcome inflammation, complement activation and blood coagulation to complete feeding. Since these pathways are regulated by serpins that have basic residues at their P1 sites, we speculate that I. scapularis may utilize some of the serpins reported in this study to manipulate host defense. We have discussed our data in the context of advances on the molecular physiology of I. scapularis. Although the paper is descriptive, this study provides the first step toward a comprehensive understanding of serpins in tick physiology.

Amblyomma americanum tick saliva serine protease inhibitor 6 is a cross-class inhibitor of serine proteases and papain-like cysteine proteases that delays plasma clotting and inhibits platelet aggregation.

We previously demonstrated that Amblyomma americanum tick serine protease inhibitor 6 (AamS6) was secreted into the host during tick feeding and that both its mRNA and protein were ubiquitously and highly expressed during the first 3 days of tick feeding. This study demonstrates that AamS6 is a cross‐class inhibitor of both serine‐ and papain‐like cysteine proteases that has apparent antihaemostatic functions. Consistent with the typical inhibitory serpin characteristics, enzyme kinetics analyses revealed that Pichia pastoris‐expressed recombinant (r) AamS6 reduced initial velocities of substrate hydrolysis (V0) and/or maximum enzyme velocity (Vmax) of trypsin, chymotrypsin, elastase, chymase, and papain in a dose–response manner. We speculate that rAamS6 inhibited plasmin in a temporary fashion in that while rAamS6 reduced V0 of plasmin by up to ∼53%, it had no effect on Vmax. Our data also suggest that rAmS6 has minimal or no apparent effect on V0 or Vmax of thrombin, factor Xa, and kallikrein. We speculate that AamS6 is apparently involved in facilitating blood meal feeding in that various amounts of rAamS6 reduced platelet aggregation by up to ∼47% and delayed plasma clotting time in the recalcification time assay by up to ∼210 s. AamS6 is most likely not involved with the ticks evasion of the hosts complement defense mechanism, in that rAamS6 did not interfere with the complement activation pathway. Findings in this study are discussed in the context of expanding our understanding of tick proteins that control bloodmeal feeding and hence tick‐borne disease transmission by ticks.

Deorphanization and target validation of cross-tick species conserved novel Amblyomma americanum tick saliva protein

We previously identified a cross-tick species conserved tick feeding stimuli responsive Amblyomma americanum (Aam) AV422 gene. This study demonstrates that AamAV422 belongs to a novel group of arthropod proteins that is characterized by 14 cysteine amino acid residues: C(23)-X7/9-C(33)-X23/24-C(58)-X8-C(67)-X7-C(75)-X23-C(99)-X15-C(115)-X10-C(126)-X24/25/33-C(150)C(151)-X7-C(159)-X8-C(168)-X23/24-C(192)-X9/10-C(202) predicted to form seven disulfide bonds. We show that AamAV422 protein is a ubiquitously expressed protein that is injected into the host within the first 24h of the tick attaching onto the host as revealed by Western blotting analyses of recombinant (r)AamAV422, tick saliva and dissected tick organ protein extracts using antibodies to 24 and 48 h tick saliva proteins. Native AamAV422 is apparently involved with mediating tick anti-hemostasis and anti-complement functions in that rAamAV422 delayed plasma clotting time in a dose responsive manner by up to ≈ 160 s, prevented platelet aggregation by up to ≈ 16% and caused ≈ 24% reduction in production of terminal complement complexes. Target validation analysis revealed that rAamAV422 is a potential candidate for a cocktail or multivalent tick vaccine preparation in that RNA interference (RNAi)-mediated silencing of AamAV422 mRNA caused a statistically significant (≈ 44%) reduction in tick engorgement weights, which is proxy for amounts of ingested blood. We speculate that AamAV422 is a potential target antigen for development of the highly desired universal tick vaccine in that consistent with high conservation among ticks, antibodies to 24h Ixodes scapularis tick saliva proteins specifically bound rAamAV422. We discuss data in this study in the context of advancing the biology of tick feeding physiology and discovery of potential target antigens for tick vaccine development.

Ixodes scapularis Tick Saliva Proteins Sequentially Secreted Every 24 h during Blood Feeding

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 ticks 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 ticks 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.

A blood meal-induced Ixodes scapularis tick saliva serpin inhibits trypsin and thrombin, and interferes with platelet aggregation and blood clotting

Ixodes scapularis is a medically important tick species that transmits causative agents of important human tick-borne diseases including borreliosis, anaplasmosis and babesiosis. An understanding of how this tick feeds is needed prior to the development of novel methods to protect the human population against tick-borne disease infections. This study characterizes a blood meal-induced I. scapularis (Ixsc) tick saliva serine protease inhibitor (serpin (S)), in-house referred to as IxscS-1E1. The hypothesis that ticks use serpins to evade the hosts defense response to tick feeding is based on the assumption that tick serpins inhibit functions of protease mediators of the hosts anti-tick defense response. Thus, it is significant that consistent with hallmark characteristics of inhibitory serpins, Pichia pastoris-expressed recombinant IxscS-1E1 (rIxscS-1E1) can trap thrombin and trypsin in SDS- and heat-stable complexes, and reduce the activity of the two proteases in a dose-responsive manner. Additionally, rIxscS-1E1 also inhibited, but did not apparently form detectable complexes with, cathepsin G and factor Xa. Our data also show that rIxscS-1E1 may not inhibit chymotrypsin, kallikrein, chymase, plasmin, elastase and papain even at a much higher rIxscS-1E1 concentration. Native IxscS-1E1 potentially plays a role(s) in facilitating I. scapularis tick evasion of the hosts hemostatic defense as revealed by the ability of rIxscS-1E1 to inhibit adenosine diphosphate- and thrombin-activated platelet aggregation, and delay activated partial prothrombin time and thrombin time plasma clotting in a dose-responsive manner. We conclude that native IxscS-1E1 is part of the tick saliva protein complex that mediates its anti-hemostatic, and potentially inflammatory, functions by inhibiting the actions of thrombin, trypsin and other yet unknown trypsin-like proteases at the tick-host interface.

A family of serine protease inhibitors (serpins) in the cattle tick Rhipicephalus (Boophilus) microplus

Proteins belonging to the serine protease inhibitor (serpin) superfamily play essential roles in many organisms. In arthropods these proteins are involved in innate immune system, morphogenesis and development. In mammals serpins regulate pathways that are essential to life such as blood coagulation, fibrinolysis, inflammation and complement activation, some of which are considered the hosts first line of defense to hematophagous and/or blood dueling parasites. Thus, it is hypothesized that ticks use serpins to evade host defense, facilitating parasitism. This study describes eighteen full-length cDNA sequences encoding serpins identified in Rhipicephalus (Boophilus) microplus, here named RmS 1-18 (R. microplus serpin). Spatial and temporal transcriptional profiling demonstrated that R. microplus serpins are transcribed during feeding, suggesting their participation in tick physiology regulation. We speculate that the majority of R. microplus serpins are conserved in other ticks, as indicated by phylogeny analysis. Over half of the 18 RmSs are putatively functional in the extracellular environment, as indicated by putative signal peptides on 11 of 18 serpins. Comparative modeling and structural-based alignment revealed that R. microplus serpins in this study retain the consensus secondary of typical serpins. This descriptive study enlarges the knowledge on the molecular biology of R. microplus, an important tick species.