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

Mulenga, Albert; Khumthong, Rabuesak; Chalaire, K. C.

doi:10.1186/1471-2164-10-217

Cited by 66 publications

(91 citation statements)

References 68 publications

(115 reference statements)

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“…PCR products were electrophoresed on a 2% agarose gel containing 1 mg ml -1 ethidium bromide. The tick actin gene (Mulenga et al, 2009) was used for PCR template load control. To determine mRNA abundance, densitograms of amplified PCR bands were determined using the web based ImageJ software (http://rsb.info.nih.gov/ij/).…”

Section: Expression Analyses By Rt-pcrmentioning

confidence: 99%

Silencing of threeAmblyomma americanum(L.) insulin-like growth factor binding protein-related proteins prevents ticks from feeding to repletion

Mulenga

Khumthong

2010

Journal of Experimental Biology

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SUMMARYThe insulin-like growth factor (IGF) binding proteins (IGFBP) family is the regulatory arm of the IGF signaling system that control mitogenic and anabolic actions of IGF peptide hormones. This study describes cloning and biological characterization of three Amblyomma americanum (L.) (Aam) proteins that show amino-terminal sequence and secondary structure similarity to the IGFBP superfamily. The three molecules here provisionally identified as AamIGFBP-rP1 and short (S) and long (L) AamIGFBP-rP6 are expressed in multiple tick organs and are responsive to tick feeding activity with the former being upregulated and the latter being downregulated. We show that they regulate tick physiological functions that may be related to A. americanum tick feeding success as revealed by RNAi-mediated dual silencing of AamIGFBP-rP6S and AamIGFBP-rP6L or AamIGFBP-rP1 alone, which caused a reduction in blood meal size compared to the controls. Additionally, in the case of AamIGFBP-rP1 silencing, 47% of ticks died while attempting to feed and those that did survive and spontaneously detached from the host failed to lay eggs. Although AamIGFBP-rP6S and AamIGFBP-rP6L show overall identities of 49% and 59%, respectively, to Rhipicephalus microplus C protein, the identity level jumps to ~84% when the comparison is restricted to first 70 amino acids of the mature protein. Similarly, the AamIGFBP-rP1 mature protein is ~72%, 87%, 88% and 92% identical to that of Ixodes scapularis S, R. microplus, R. appendiculatus N and A. variegatum F, respectively. The observed across-tick-species conservation suggests that the three molecules (AamIGFBP-rP1, AamIGFBP-rP6S and AamIGFBP-rP6L) represent target for development of vaccines to protect animals against multiple tick species. The data are discussed with reference to advances in tick molecular biology and the potential of the three proteins as targets for immunizing animals against tick feeding.

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Section: Expression Analyses By Rt-pcrmentioning

confidence: 99%

Silencing of threeAmblyomma americanum(L.) insulin-like growth factor binding protein-related proteins prevents ticks from feeding to repletion

Mulenga

Khumthong

2010

Journal of Experimental Biology

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“…In previous studies the basis for this assumption has been the expression of K. C. Chalaire and others Mr (kDa) RP 1 putatively extracellular serpin mRNA in tick salivary glands (Prevot et al, 2007;Prevot et al, 2006;Mulenga et al, 2003;Mulenga et al, 2008;Mulenga et al, 2009). In this study, the observed specific reactivity of the antibody to rAamS6 with the native AamS6 protein band on the western blot of pilocarpine-induced tick saliva, coupled with antibodies to tick saliva proteins specifically binding to rAamS6, strongly suggests that the AamS6 protein is injected into the host during tick feeding.…”

Section: Discussionmentioning

confidence: 99%

“…In hypothesizing that tick-encoded serpins play key role(s) in regulating the evasion of host defense reactions by ticks (Prevot et al, 2007;Mulenga et al, 2001a;Mulenga et al, 2003;Mulenga et al, 2008, Mulenga et al, 2009, the underlying assumption is that ticks inject serpins into the host during tick feeding. In previous studies the basis for this assumption has been the expression of K. C. Chalaire and others Mr (kDa) RP 1 putatively extracellular serpin mRNA in tick salivary glands (Prevot et al, 2007;Prevot et al, 2006;Mulenga et al, 2003;Mulenga et al, 2008;Mulenga et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

“…The extracted total RNA was subjected to a two-step semiquantitative RT-PCR using specific PCR primers targeting the AamS6 specific and AamS6/S17 domains (shown above). Tick actin PCR primers (Mulenga et al, 2009) were used for sample load controls. Samples from the PCR reactions (10ml) were analyzed by electrophoresis along with a 1Kb DNA ladder on a 2% agarose gel containing 1mgml -1 ethidium bromide to qualitatively corroborate silencing.…”

Section: Rna Interference (Rnai)-mediated Gene Silencingmentioning

confidence: 99%

“…Given that the host's primary lines of defense to tick feeding activity, inflammation and blood coagulation, are serine protease-mediated pathways that are regulated by serpins (Huntington, 2006;Gettins, 2002), it has been hypothesized that ticks may use serpins to evade the host's immune response (Mulenga et al, 2001a). An increasing number of serpin-encoding cDNAs have been identified in several tick species of medical and veterinary importance, including A. americanum (Mulenga et al, 2007), Haemaphysalis longicornis (Mulenga et al, 2001b;Imamura et al, 2005;Sugino et al, 2003), Ixodes ricinus (Prevot et al, 2006), I. scapularis (Mulenga et al, 2009) and Rhiphicephalus appendiculatus Imamura et al, 2006;Imamura et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

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

Chalaire

Kim

Garcia-Rodriguez

et al. 2011

Journal of Experimental Biology

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SUMMARYIn 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.

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Molecular characterization of serine protease inhibitor isoform 3, SmSPI, from Schistosoma mansoni

et al. 2016

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Serine protease inhibitors, known as serpins, are pleiotropic regulators of endogenous and exogenous proteases, and molecule transporters. They have been documented in animals, plants, fungi, bacteria, and viruses; here, we characterize a serpin from the trematode platyhelminth Schistosoma mansoni. At least eight serpins have been found in the genome of S. mansoni, but only two have characterized molecular properties and functions. Here, the function of S. mansoni serpin isoform 3 (SmSPI) was analyzed, using both computational and molecular biological approaches. Phylogenetic analysis showed that SmSPI was closely related to Schistosoma haematobium serpin and Schistosoma japonicum serpin B10. Structure determined in silico confirmed that SmSPI belonged to the serpin superfamily, containing nine α-helices, three β-sheets, and a reactive central loop. SmSPI was highly expressed in schistosomules, predominantly in the head gland, and in adult male and female with intensive accumulation on the spines, which suggests that it may have a role in facilitating intradermal and intravenous survival. Recombinant SmSPI was overexpressed in Escherichia coli; the recombinant protein was of the same size (46 kDa) as the native protein. Immunological analysis suggested that mice infected with S. mansoni responded to rSmSPI at 8 weeks postinfection (wpi) but not earlier. The inhibitory activity of rSmSPI was specific to chymotrypsin but not trypsin, neutrophil elastase, and porcine pancreatic elastase. Elucidating the biological and physiological functions of SmSPI as well as other serpins will lead to further understanding of host-parasite interaction machinery that may provide novel strategies to prevent and control schistosomiasis in the future.

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

Cited by 66 publications

References 68 publications

Silencing of threeAmblyomma americanum(L.) insulin-like growth factor binding protein-related proteins prevents ticks from feeding to repletion

Silencing of threeAmblyomma americanum(L.) insulin-like growth factor binding protein-related proteins prevents ticks from feeding to repletion

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

Molecular characterization of serine protease inhibitor isoform 3, SmSPI, from Schistosoma mansoni

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