Peter J. Waniek Ulrike B. Hendgen-CottaPia Stock Christoph Mayer Astrid H. KollienGunter A. Schaub
Serine proteinases of the human body louse (Pediculus humanus):sequence characterization and expression patterns
Received: 29 June 2005 / Accepted: 1 July 2005 / Published online: 7 October 2005 Springer-Verlag 2005
Abstract After the previous characterization of onetrypsin gene (Try1) of the human body louse Pediculushumanus, genes encoding a second trypsin (Try2) and achymotrypsin (Chy1) have been cloned using degenerateserine proteinase primers and 5- and 3-RACE, andsequenced. The deduced 259 and 267 amino acid se-quences of Try2 and Chy1 show an identity of 33%40% to trypsinogens and chymotrypsinogens of otherinsects. Considering previously published partial se-quences, P. humanus possesses at least one Try1 gene,five variants/isoforms of Try2 and six variants/isoformsof Chy1. The genomic DNA of Try2 contains three in-trons and Chy1 contains five introns. Using wholemount in situ hybridization, gene expression of Try1,Try2 and Chy1 has been localized not only in the dis-tensible anterior region of the midgut of lice but some-times also in the area following the distensible region.The Try2 gene was always expressed at much lowerlevels than Try1 or Chy1. This lower expression, theconstitutive expression of Try1 and Chy1 at 1, 2, 6, 12and 24 h after feeding of adults and the regional differ-ences have been verified in quantitative real-time PCR.
The human body louse, Pediculus humanus, and thehuman head louse, Pediculus capitis, are cosmopolitanectoparasites and have been a hygiene problem forthousands of years (Mumcuoglu and Zias 1988;Mumcuoglu et al. 2003). Additionally, P. humanus is
the vector of three pathogenic bacteria, Rickettsiaprowazekii, Borrelia (syn. Spirochaeta) recurrentis andBartonella (syn. Rickettsia, syn. Rochalimaea) quintana,which cause louse-borne epidemic typhus, relapsingfever and trench fever, respectively (Fulton and Smith1960; Maurin and Raoult 1996; Schaub, 2001; Four-nier et al. 2002). In the United States alone, at least100 million dollars are spent annually on head licecontrol (Jones and English 2003; Hipolito et al. 2001).Mainly, insecticides are used but the appearance ofresistance against pediculocides and their potentialtoxicity to the host (Elgart 1999) necessitates investi-gation into other approaches. One way to controlectoparasites is the immunization of the host againstkey antigens of the parasite, which are usually notinoculated into the host, e.g. digestive enzymes (Eastet al. 1993; Willadsen and Billingsley 1996; Wang andNuttal 1999).
Despite the significance of human lice, knowledgeabout their digestive physiology is weak. In contrast toother blood-sucking insects, lice stay continuously ontheir host and ingest small but frequent blood meals,several times per day. The blood is stored and digestedrapidly in the distensible anterior region of the midgut(Buxton 1947; Lehane 1991). Among haematophagousinsects, lice have the fastest digestion, as they need about4 h6 h for a blood meal and, like the majority of theseinsects, they use several alkaline digestive proteases(Lehane 1991, 1994; Vaughan and Azad 1993). In en-zyme assays, only a trypsin and a leucine aminopepti-dase have been discovered, the latter having beenpartially purified and characterized (Borovsky andSchlein 1988; Ochanda et al. 1998, 2000).
At the molecular level, an analysis of a cDNA libraryshowed 40 clones of 115810 bp representing putativedigestion-associated proteins (Pedra et al. 2003). Twoclones belong to ATP-dependent peptidases (414 and712 bp), one clone of 728 bp to a cysteine endopepti-dase, one clone of 754 bp to a cathepsin-like cysteineproteinase, 12 clones to trypsin (533810 bp) and 22clones to chymotrypsin (115806 bp); two other clones
P. J. Waniek U. B. Hendgen-Cotta P. Stock C. MayerA. H. Kollien G. A. Schaub (&)Department of Special Zoology, Ruhr-University,44780 Bochum, GermanyE-mail: firstname.lastname@example.orgTel.: +49-234-3224587Fax: +49-234-3214787
Parasitol Res (2005) 97: 486500DOI 10.1007/s00436-005-1463-y
of 691 and 710 bp were classified to belong to differentserine proteinases. In a parallel investigation of trypsinsand chymotrypsins of P. humanus, one 759 bp sequenceencoding one trypsin-like proteinase (Try1) had beencompletely sequenced and characterized (Kollien et al.2004a).
Serine proteinases are a superfamily of proteins withvarious functions and a widespread occurrence in viru-ses, bacteria and eukaryotes (Kraut 1977; Rawlings andBarrett 1994). This family includes many different en-zymes, e.g. trypsin, chymotrypsin, subtilisin and serinecarboxypeptidase (Rawlings and Barrett 1994). Onecharacteristic of serine proteinases is the active triadformed by His, Asp and Ser (Rawlings and Barrett1994). In addition, vertebrate trypsins and chymotryp-sins have eight Cys residues for the formation of disul-phide bridges, whereas in invertebrates these enzymespossess only three bridges formed by six cysteine resi-dues (Davis et al. 1985). The surface of the substrate-binding pocket is made up by three amino acid residues,Asp, Gly and Gly for trypsins and mainly Ser, Gly andGly for chymotrypsin (Kraut 1977; Hedstrom, 2002).The amino acid residue at the bottom of the substrate-binding pocket, the Asp of trypsin, which is replaced bySer in chymotrypsin (Kraut 1977; Lehane et al. 1998;Zhu and Baker 2000) causes the difference in the sub-strate specifity of both proteinases. Whereas trypsinspreferentially hydrolyze peptide bonds following a lysineor an arginine, chymotrypsins cleave after aromaticamino acid residues (Kraut 1977).
Often trypsins together with chymotrypsins are in-volved in protein digestion (Zhu and Baker 2000; Ra-malho-Ortigao et al. 2003). They are the most abundantdigestive proteases in several Lepidoptera, Coleoptera,Siphonaptera and blood sucking Diptera (Noriega et al.1996; Gaines et al. 1999; Zhu and Baker 2000; Yan et al.2001; Hegedus et al. 2003). Other important tasks arereorganization of proteins during developmental pro-cesses of insects and proenzyme activation cleavage(Indrasith et al. 1988; Hong and Hashimoto 1996; Jiet al. 2004). The proenzyme of trypsin is mainly acti-vated by a tryptic cleavage (Davis et al. 1985). However,an unusual activation by an enzyme with chymotrypticactivity was verified for Try1 of P. humanus (Kollienet al. 2004a). Therefore, we focussed on chymotrypsin,initially in parallel with the investigation of Pedra et al.(2003), but then considering their sequences. In thepresent study, we have completely sequenced the cDNAencoding a second trypsin-like and a chymotrypsin-likeproteinase and their respective genomic DNA. In addi-tion, we have localized the site of expression in themidgut using whole mount in situ hybridization. By real-time PCR, we have characterized the expression levels inunfed first instars and in adult lice at different times afterfeeding. Thereby, we showed for the first time that notonly the distensible anterior region of the midgut of licebut also the subsequent part of the narrow posteriorregion is engaged in the digestion of blood via trypsinsand chymotrypsins.
Materials and methods
Pediculus humanus were obtained from the Umweltb-undesamt Berlin. This strain is adapted to feeding onrabbits (Culpepper 1948) and reared at 311C and70%80% relative humidity and on a 1/23 h light/darkcycle (Habedank and Schrader 2001). In Bochum, theselice were maintained at 261C, 60%70% relativehumidity and 18/6 h light/dark cycle and were fed daily,except the weekends, on the arm of volunteers. Firstinstar larvae were collected after hatching and adult liceat different times after feeding. All were killed byimmersion in liquid nitrogen and stored at 80C.
RNA and genomic DNA isolation
Total RNA was isolated from P. humanus homogenatesusing the RNeasy Mini Kit (Qiagen, Hilden, Germany)following the manufacturers protocol. One adult louseor 20 unfed first instars weighed about 1 mg and con-tained an average amount of 10 ng polyA+ mRNA(Kollien et al. 2004a). Genomic DNA of unfed first in-star larvae was isolated using the Genomic DNA Puri-fication kit (Fermentas, St. Leon-Rot, Germany)following the manufacturers protocol.
Primer selection, PCR, cloning and sequencing
The following degenerate primers were designedaccording to the amino acid residues flanking the His andSer of the active site of serine proteinases (Sakanari et al.1989; Elvin et al. 1993): SP-F (5-TGGGT(A/C/G/T)GT(A/C/G/T)AC(A/C/G/T)GC(A/C/G/T)GC(A/C/G/T)CA(T/C)TG-3) and SP-R (5-A(A/G)(A/C/G/T)GG(A/C/G/T)CC(A/C/G/T)CC(A/C/G/T)(G/C)(A/T)(A/G)T-C(A/C/G/T)CC-3). Details of the PCR amplificationhave been published previously (Kollien et al. 2004b).Only the annealing parameters in a Hybaid (Omni Gene)thermal cycler (Hybaid, Ashford, Middx, UK) differed(54.2, 55.5, 56.9, 58.4, 59.3 and 59.8C).
To amplify the chymotrypsin sequence the specificoligonucleotides Chy-For1 (5-GGATCCATGAAAGGTTTTTTTGCTCTTT-3) and Chy-Rev1 (5-AAGCTTGTCAGTGTAGTCAGAATTGTC-3) weredesigned according to partial sequences published byPedra et al. (2003), including BamHI and HindIIIrestriction sites. Total RNA, isolated 3 h after feeding,was used as template for the reverse transcription withan oligo-dT18VN primer. The amplified product of thepredicted size, approximately 470 bp, was cloned intopGEM T-Easy vector (Promega, Madison, WI, USA)following the manufacturers instructions. Sequenceanalysis by MWG-BIOTECH (Ebersberg, Germany)showed that a chymotrypsin sequence had been cloned.
To exclude Try1-cDNA, which contains an EcoRIrestriction site at the positions 373378 in the cDNAsequence, the PCR product was digested with EcoRI,separated on a 1% agarose-gel, and again a 470 bpfragment was cloned (