Characterization of nit sheath protein functions and transglutaminase-mediated cross-linking in the human head louse, Pediculus humanus capitis.

BACKGROUND: Head louse females secrete liquid glue during oviposition, which is solidified to form the nit sheath over the egg. Recently, two homologous proteins, named louse nit sheath protein (LNSP) 1 and LNSP 2, were identified as adhesive proteins but the precise mechanism of nit sheath solidification is unknown. METHODS: We determined the temporal transcriptome profiles of the head louse accessory glands plus oviduct, from which putative major structural proteins and those with functional importance were deduced. A series of RNA interference (RNAi) experiments and treatment of an inhibitor were conducted to elucidate the function and action mechanism of each component. RESULTS: By transcriptome profiling of genes expressed in the louse accessory glands plus uterus, the LNSP1 and LNSP2 along with two hypothetical proteins were confirmed to be the major structural proteins. In addition, several proteins with functional importance, including transglutaminase (TG), defensin 1 and defensin 2, were identified. When LNSP1 was knocked down via RNA interference, most eggs became nonviable via desiccation, suggesting its role in desiccation resistance. Knockdown of LNSP2, however, resulted in oviposition failure, which suggests that LNSP2 may serve as the basic platform to form the nit sheath and may have an additional function of lubrication. Knockdown of TG also impaired egg hatching, demonstrating its role in the cross-linking of nit sheath proteins. The role of TG in cross-linking was further confirmed by injecting or hair coating of GGsTop, a TG inhibitor. CONCLUSIONS: Both LNSP1 and LNSP2 are essential for maintaining egg viability besides their function as glue. The TG-mediated cross-linking plays critical roles in water preservation that are essential for ensuring normal embryogenesis. TG-mediated cross-linking mechanism can be employed as a therapeutic target to control human louse eggs, and any topically applied TG inhibitors can be exploited as potential ovicidal agents.

First evidence of blaNDM-1 and blaOXA-23 carbapenemase genes in human body lice infesting a second-hand T-shirt in a street market in Italy.

BACKGROUND: The spread of carbapenems resistance is a public health concern. The main group of carbapenemases encoding the ß-lactamases activity (bla genes) is the Metallo-ß-lactamases (MBLs). METHODS: The presence of carbapenemase blaOXA-23-like, blaOXA-40-like, blaOXA-51-like, blaOXA-58-like, and blaNDM-1 genes was screened by real time PCR in 26 Pediculus humanus insects identified from second-hand clothes in a local market in Central Italy. Bacteria diversity was also characterized through shotgun metagenomic amplification for a deep sequencing of the host-associated bacterial microbiomes. RESULTS: The blaOXA-23 and blaNDM-1 carbapenemases genes were found and metagenomic analysis showed a great presence of Acinetobacter species. CONCLUSIONS: These results suggest a new potential transmission path for carbapenemase gene spread through bacteria ingested by insects infesting humans.

Structure of PINK1 in complex with its substrate ubiquitin.

Autosomal-recessive juvenile Parkinsonism (AR-JP) is caused by mutations in a number of PARK genes, in particular the genes encoding the E3 ubiquitin ligase Parkin (PARK2, also known as PRKN) and its upstream protein kinase PINK1 (also known as PARK6). PINK1 phosphorylates both ubiquitin and the ubiquitin-like domain of Parkin on structurally protected Ser65 residues, triggering mitophagy. Here we report a crystal structure of a nanobody-stabilized complex containing Pediculus humanus corporis (Ph)PINK1 bound to ubiquitin in the 'C-terminally retracted' (Ub-CR) conformation. The structure reveals many peculiarities of PINK1, including the architecture of the C-terminal region, and reveals how the N lobe of PINK1 binds ubiquitin via a unique insertion. The flexible Ser65 loop in the Ub-CR conformation contacts the activation segment, facilitating placement of Ser65 in a phosphate-accepting position. The structure also explains how autophosphorylation in the N lobe stabilizes structurally and functionally important insertions, and reveals the molecular basis of AR-JP-causing mutations, some of which disrupt ubiquitin binding.

Identification and characterization of an esterase involved in malathion resistance in the head louse Pediculus humanus capitis.

Enhanced malathion carboxylesterase (MCE) activity was previously reported to be involved in malathion resistance in the head louse Pediculus humanus capitis (Gao et al., 2006 [8]). To identify MCE, the transcriptional profiles of all five esterases that had been annotated to be catalytically active were determined and compared between the malathion-resistant (BR-HL) and malathion-susceptible (KR-HL) strains of head lice. An esterase gene, designated HLCbE3, exhibited approximately 5.4-fold higher transcription levels, whereas remaining four esterases did not exhibit a significant increase in their transcription in BR-HL, indicating that HLCbE3 may be the putative MCE. Comparison of the entire cDNA sequences of HLCbE3 revealed no sequence differences between the BR-HL and KR-HL strains and suggested that no single nucleotide polymorphism is associated with enhanced MCE activity. Two copies of the HLCbE3 gene were observed in BR-HL, implying that the over-transcription of HLCbE3 is due to the combination of a gene duplication and up-regulated transcription. Knockdown of HLCbE3 expression by RNA interference in the BR-HL strain led to increases in malathion susceptibility, confirming the identity of HLCbE3 as a MCE responsible for malathion resistance in the head louse. Phylogenetic analysis suggested that HLCbE3 is a typical dietary esterase and belongs to a clade containing various MCEs involved in malathion resistance.

[Pyrethroid resistance mechanisms in the body lice Pediculus humanus humanus L.: detoxification enzyme systems].

Synergists (piperonyl butoxide, MGK-264 (N-octyl bicycloheptene dicarboximide), DEF (S, S, S, tributyl phosphorotrithioate), and diethylmaleate) were used to investigate the metabolic resistance of body lice to permethrin. Instances of the synergistic activity ofpermethrin when combined with enzyme system inhibitors have been identified. The synergic ratios were 3.3 to 52.0 for monooxygenase inhibitors; 5.2 to 7.4 for esterases, and about 4 for glutathione-S-transferases. Since the use of the synergists did not lead to full inhibition of resistance, it was concluded that the lice had also a kdr-like type of resistance.

Insecticide resistance in head lice: clinical, parasitological and genetic aspects.

Insecticide treatment resistance is considered to be a major factor in the increasing number of infestations by head lice. The large insecticide selection pressure induced by conventional topical pediculicides has led to the emergence and spread of resistance in many parts of the world. Possible mechanisms of resistance include accelerated detoxification of insecticides by enzyme-mediated reduction, esterification, oxidation that may be overcome by synergistic agents such as piperonyl butoxide, alteration of the binding site, e.g. altered acetylcholinesterase or altered nerve voltage-gated sodium channel, and knockdown resistance (kdr). Clinical, parasitological and molecular data on resistance to conventional topical pediculicides show that treatments with neurotoxic insecticides have suffered considerable loss of activity worldwide. In particular, resistance to synthetic pyrethroids has become prominent, probably because of their extensive use. As other treatment options, including non-insecticidal pediculicides such as dimeticone, are now available, the use of older insecticides, such as lindane and carbaryl, should be minimized, owing to their loss of efficacy and safety concerns. The organophosphorus insecticide malathion remains effective, except in the UK, mostly in formulations that include terpineol.

Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations.

Missense mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene cause autosomal-recessive Parkinson's disease. To date, little is known about the intrinsic catalytic properties of PINK1 since the human enzyme displays such low kinase activity in vitro. We have discovered that, in contrast to mammalian PINK1, insect orthologues of PINK1 we have investigated-namely Drosophila melanogaster (dPINK1), Tribolium castaneum (TcPINK1) and Pediculus humanus corporis (PhcPINK1)-are active as judged by their ability to phosphorylate the generic substrate myelin basic protein. We have exploited the most active orthologue, TcPINK1, to assess its substrate specificity and elaborated a peptide substrate (PINKtide, KKWIpYRRSPRRR) that can be employed to quantify PINK1 kinase activity. Analysis of PINKtide variants reveal that PINK1 phosphorylates serine or threonine, but not tyrosine, and we show that PINK1 exhibits a preference for a proline at the +1 position relative to the phosphorylation site. We have also, for the first time, been able to investigate the effect of Parkinson's disease-associated PINK1 missense mutations, and found that nearly all those located within the kinase domain, as well as the C-terminal non-catalytic region, markedly suppress kinase activity. This emphasizes the crucial importance of PINK1 kinase activity in preventing the development of Parkinson's disease. Our findings will aid future studies aimed at understanding how the activity of PINK1 is regulated and the identification of physiological substrates.

Decreased detoxification genes and genome size make the human body louse an efficient model to study xenobiotic metabolism.

The human body louse, Pediculus humanus humanus, has one of the smallest insect genomes, containing ∼10 775 annotated genes. Annotation of detoxification [cytochrome P450 monooxygenase (P450), glutathione-S-transferase (GST), esterase (Est) and ATP-binding cassette transporter (ABC transporter)] genes revealed that they are dramatically reduced in P. h. humanus compared to other insects except for Apis mellifera. There are 37 P450, 13 GST and 17 Est genes present in P. h. humanus, approximately half the number found in Drosophila melanogaster and Anopheles gambiae. The number of putatively functional ABC transporter genes in P. h. humanus and Ap. mellifera are the same (36) but both have fewer than An. gambiae (44) or Dr. melanogaster (65). The reduction of detoxification genes in P. h. humanus may be a result of this louse's simple life history, in which it does not encounter a wide variety of xenobiotics. Neuronal component genes are highly conserved across different insect species as expected because of their critical function. Although reduced in number, P. h. humanus still retains at least a minimum repertoire of genes known to confer metabolic or toxicokinetic resistance to xenobiotics (eg Cyp3 clade P450s, Delta GSTs, B clade Ests and B/C subfamily ABC transporters), suggestive of its high potential for resistance development.

Activity of increased specific and non-specific esterases and glutathione transferases associated with resistance to permethrin in pediculus humanus capitis (phthiraptera: pediculidae) from Argentina.

Enhanced metabolism by oxidative enzymes is a major cause of pyrethroid resistance in insects. In this work, we evaluated the role of specific and non-specific esterases in head louse populations from Buenos Aires with different levels of resistance to permethrin. As esterase activity is substrate-dependent, four different esters were used as unspecific substrates in order to obtain a better characterization of the possible role of these enzymes in the resistance phenomenon. The unspecific substrates were phenylthioacetate, 1- and 2-naphtyl-acetate, and p-nitrophenyl acetate. A 7-coumaryl permethrate was synthesized and used as a specific substrate to measure pyrethroid esterases by a very sensitive microfluorometric method. The results on pyrethroid esterase activity obtained with this substrate showed that these enzymes contribute to the detoxifying activity in resistant populations, although no correlation was found between pyrethroid esterase activity and resistance ratios. In this study, we established that the activity of esterase against specific and non-specific substrates is increased in pyrethroid-resistant populations of head lice from Buenos Aires. Also, dichlorodiphenyltrichloroethane (DDT) resistance values demonstrated that there is a DDT cross-resistance phenomenon in pyrethroid-resistant head louse populations and suggested that an alteration in the receptor of the nervous system (kdr gen) is a key factor of the resistance phenomena in these head louse populations.

Direct evidence of the cyclooxygenase pathway of prostaglandin synthesis in arthropods: genetic and biochemical characterization of two crustacean cyclooxygenases.

Prostaglandins, well-known lipid mediators in vertebrate animals, have also shown to play certain regulatory roles in insects and other arthropods acting on reproduction, immune system and ion transport. However, knowledge of their biosynthetic pathways in arthropods is lacking. In the present study, we report the cloning and expression of cyclooxygenase (COX) from amphipod crustaceans Gammarus spp and Caprella spp. The amphipod COX proteins contain key residues shown to be important for cyclooxygenase and peroxidase activities. Differently from all other known cyclooxygenases the N-terminal signal sequence of amphipod enzymes is not cleaved during protein expression in mammalian cells. The C-terminus of amphipod COX is shorter than that of mammalian isoforms and lacks the KDEL(STEL)-type endoplasmic reticulum retention/retrieval signal. Despite that, amphipod COX proteins are N-glycosylated and locate similarly to the vertebrate COX on the endoplasmic reticulum and nuclear envelope. Both amphipod COX mRNAs encode functional cyclooxygenases that catalyze the transformation of arachidonic acid into prostaglandins. Using bioinformatic analysis we identified a COX-like gene from the human body louse Pediculus humanus corporis genome that encodes a protein with about 30% sequence identity with human COX-1 and COX-2. Although the COX gene is known to be absent from genomes of Drosophila sp., Aedes aegypti, Bombyx mori, and other insects, our studies establish the existence of the COX gene in certain lineages within the insect world.

Metalloproteases and egg-hatching in Pediculus humanus, the body (clothes) louse of humans (Phthiraptera: Insecta).

To investigate the biochemical components of egg-hatch in the body louse, Pediculus humanus, egg-shell-washings (ESW) were collected during the first 2 h post-hatching and analysed by gelatin SDS-PAGE. These ESW contained proteases with molecular mass in the range of 25-100 kDa; the most abundant proteases were approximately 25 kDa. The 3 main regions of protease activity in the one-dimensional gelatin SDS-PAGE gels resolved to at least 23 distinct regions of protease activity when analysed by two-dimensional gelatin SDS-PAGE, with iso-electric points spread over the entire 3 to 10 pH range. Mechanistic characterization indicated that the ESW contained proteases of the metallo-class, inhibited by both 1,10-phenanthroline and EDTA. Several protease inhibitors were tested for their ability to inhibit louse egg-hatch in vitro. The metalloprotease inhibitor 1,10-phenanthroline and the aminopeptidase inhibitor bestatin significantly inhibited (P<0.05) louse egg-hatch (100% and 58%, respectively). The presence of metalloproteases at the time of egg-hatch and the inhibition of egg-hatch in P. humanus by metalloprotease inhibitors suggests a crucial role for these proteases in the hatching of this medically important parasite.

Serine proteinases of the human body louse (Pediculus humanus): sequence characterization and expression patterns.

After the previous characterization of one trypsin gene (Try1) of the human body louse Pediculus humanus, genes encoding a second trypsin (Try2) and a chymotrypsin (Chy1) have been cloned using degenerate serine proteinase primers and 5'- and 3'-RACE, and sequenced. The deduced 259 and 267 amino acid sequences of Try2 and Chy1 show an identity of 33%-40% to trypsinogens and chymotrypsinogens of other insects. Considering previously published partial sequences, P. humanus possesses at least one Try1 gene, five variants/isoforms of Try2 and six variants/isoforms of Chy1. The genomic DNA of Try2 contains three introns and Chy1 contains five introns. Using whole mount in situ hybridization, gene expression of Try1, Try2 and Chy1 has been localized not only in the distensible anterior region of the midgut of lice but sometimes also in the area following the distensible region. The Try2 gene was always expressed at much lower levels than Try1 or Chy1. This lower expression, the constitutive expression of Try1 and Chy1 at 1, 2, 6, 12 and 24 h after feeding of adults and the regional differences have been verified in quantitative real-time PCR.

Increased monooxygenase activity associated with resistance to permethrin in Pediculus humanus capitis (Anoplura: Pediculidae) from Argentina.

We studied the profile of permethrin resistance in populations of head lice infesting children 6-12 yr old in schools and their homes in and around Buenos Aires, Argentina. Five permethrin-resistant populations with different levels of resistance were collected: Hogar Loyola (HL), Republica de Turquia (RT), Hogar Mitre (HM), Guardia de Honor (GH), and Ricardo Guiraldes (RG). One susceptible population, Bandera Argentina (BA), also was collected. Their level of resistance was evaluated, and results showed resistance ratios of 13 for HL, 16 for RT, 22 for HM, 61 for GH, and 69 for RG. To elucidate the possible involvement of the cytochrome P450 monooxygenase system in conferring permethrin resistance, ethoxycoumarin-O-deethylase (ECOD) activity was measured in abdomens of individual third instars and adults by using a fluorometric assay. The ECOD activity was lower in the susceptible BA population (4.7 ng per louse) than in the resistant ones (13.7 ng per louse for RG, 12.3 ng per louse for GH, 8.6 ng per louse for RT, and 8.2 ng per louse for HL). ECOD activity was significantly correlated with the level of resistance in the field populations (r = 0.97, P = 0.0009), suggesting a role for cytochrome monooxygenase P450 system in permethrin resistance by head louse, Pediculus humanus capitis De Geer.

Evidence from mitochondrial DNA that head lice and body lice of humans (Phthiraptera: Pediculidae) are conspecific.

The specific status of the head and body lice of humans has been debated for more than 200 yr. To clarify the specific status of head and body lice, we sequenced 524 base pairs (bp) of the cytochrome oxidase I (COI) gene of 28 head and 28 body lice from nine countries. Ten haplotypes that differed by 1-5 bp at 11 nucleotide positions were identified. A phylogeny of these sequences indicates that these head and body lice are not from reciprocally monophyletic lineages. Indeed, head and body lice share three of the 10 haplotypes we found. F(ST) values and exact tests of haplotype frequencies showed significant differences between head and body lice. However, the same tests also showed significant differences among lice from different countries. Indeed, more of the variation in haplotype frequencies was explained by differences among lice from different countries than by differences between head and body lice. Our results indicate the following: (1) head and body lice do not represent reciprocally monophyletic lineages and are conspecific; (2) gene flow among populations of lice from different countries is limited; and (3) frequencies of COI haplotypes can be used to study maternal gene flow among populations of head and body lice and thus transmission of lice among their human hosts.

Isoenzymes of human lice: pediculus humanus and P. capitis.

Human lice (Phthiraptera: Pediculidae) from Africa, America and Europe were electrophoresed for 28 enzymes, with special interest in metabolic factors likely to be involved with insecticide resistance. Zymogram profiles of the body louse (Pediculus humanus L. from France and U.S.A.) and the head louse (P. capitis DeGeer from France, Madagascar, Mali & Senegal) were compared. Only esterase two enzymes, phosphoglucomutase (Pgm) and 3 (Est-3), showed electrophoretic variation. In our starch gel electrophoresis conditions, P. humanus showed three electromorphs of Pgm migrating anodally 6, 11 and 16 mm (designated alleles a, b, c, respectively). Of the putative Pgm alleles, b and c occurred in all samples of both species of lice, whereas allele a was found only in P. humanus lab strain from U.S.A. Esterase 3 had four electromorphs migrating 23, 26, 30 and 35 mm (designated alleles a, b, c and d). Among putative Est alleles, a was found only in P. capitis from Bamako (all 14 specimens aa homozygotes), allele d was found only in P. capitis from Dakar (39% frequency), whereas Est-3 alleles b and c showed apparently balanced polymorphism in all samples of both P. humanus and P. capitis except that from Bamako. Despite the limited amount of isoenzyme variation detected (only 2/31 polymorphic loci), divergences of Est-3 and Pgm among Pediculus populations may be relevant to their biosystematics and resistance.