Characterization of the human head louse nit sheath reveals proteins with adhesive property that show no resemblance to known proteins

Abstract

Human head and body lice attach their eggs respectively to human hair or clothing by female lice secreted glue that hardens into a nit sheath that protects the egg. In this study, a series of experiments were conducted to characterize the glue-like material of the nit sheath. Fourier transform infrared spectroscopy on embryo-cleared nit showed proteinaceous amide I bands. With this result, we determined the amino acid composition of the nit sheath proteins and performed similarity search against the protein products of the body louse genome to identify the candidate nit sheath proteins. The identified two homologous proteins newly named as louse nit sheath protein (LNSP) 1 and LNSP2 are composed of three domains of characteristic repeating sequences. The N-terminal and middle domains consist of tandem two-residue repeats of Gln-Ala and Gly-Ala, respectively, which are expected to fold into β-strands and may further stack into β-sheets, whereas the C-terminal domain contains multiple consecutive Gln residues. Temporal and spatial transcription profiling demonstrated that both LNSP1 and LNSP2 are most predominantly expressed in the accessory gland of females of egg-laying stage, supporting that they indeed encode the nit sheath proteins. Further adhesive property of recombinant partial LNSP1 suggests that both LNSP1 and LNSP2 may act as glues.

Figure 1

FTIR analysis of the head louse nit. (A) FTIR micro-spectroscopic analysis was performed on a single nit devoid of the embryo, which supported that all the peaks are of protein origin. (B) The deconvoluted amide I bands are centered around 1610 cm⁻¹, 1626 cm⁻¹, 1651 cm⁻¹, and 1691 cm⁻¹, however they could not be used to determine the exact secondary structure content of the nit protein.

Figure 2

Amino acid composition analysis of the head louse nits. The amino acid composition analysis was performed on the nits that are devoid of the embryos. The elution volumes and the areas under the amino acid peaks of the sample chromatogram were compared to that of the standards to determine the amino acid types and the relative mole percentage of the amino acids composing the nit. The contents of cysteine and tryptophan were undetermined due to the harsh HCl hydrolysis condition.

Figure 3

Bioinformatic search of candidate protein. Root sum squares (R) of the 18 amino acid (excluding cysteine and tryptophan) offsets between the amino acids of 10,773 proteins encoded by body louse genome and the amino acid analysis result of the nit sheath were calculated. The R values ranged from 0.083 to 0.506, and the four candidate proteins with lowest R (shown in arrows) were identified as the candidate proteins of the nit. The values on the horizontal axis are from the order of body louse genes listed in NCBI.

Figure 4

Protein sequence alignment of candidate louse nit sheath proteins (LNSP1 for protein product of PHUM595880, and LNSP2 for protein product of PHUM596000). The LNSP1 and LNSP2 are 69% identical in deduced amino acid sequence. Both proteins contain the N-terminal sequences that are predicted as signal sequences (colored in blue). Subsequent regions can be divided into three domains each holding characteristic repeat sequences, which are the polyQA sequence (N-terminal domain), the polyGA sequence (middle domain), and the polyQ sequence (C-terminal domain). Conserved residues of Gln (capitalized in red) and Gly (in yellow background) are shown. The protein sequences are translated from our DNA sequencing results.

Figure 5

Temporal and spatial transcription profiles of LNSP1 and LNSP2 in various developmental stages (A) and different female organs (B) of head lice. The ****mark indicates the statistically significant (p < 0.0001) mean value as judged by one-way ANOVA in conjunction with Tukey’s test. Abbreviations in the horizontal axis represent the following: Neo, neonate; 5D-N, 5-day old nymph; 1D-F, 1-day old female; 1D-M, 1-day old male; 5D-F, 5-day old female; 5D-M, 5-day old male; AG, accessory gland, Ov, ovary; AT, alimentary tract. The transcription levels of both LNSP1 and LNSP2 in 5D-N, 1D-M, 5D-M and AT were too low (0.005~0.022) to be seen in the graph.

Figure 6

Adhesive property of LNSP1. (A) Partial LNSP1 was recombinantly expressed in E. coli and was used to glue human hair onto plastic, or stick together laboratory plastic wares. (B) The forces needed to break the polypropylene films attached together using various proteins were measured using a universal testing machine (UTM). The partial LNSP1 showed as much adhesive property as the commercially available fibrin-based glue (Tisseel®) even at a ~500-fold (in grams) less amount (See SI Methods). Chymotrypsin and BSA showed weaker binding than the partial LNSP1. The error bars are for one standard deviation of at least three repeated experiments.