Source: OKLAHOMA STATE UNIVERSITY submitted to NRP
MOLECULAR ASPECTS OF INSECT IMMUNITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0186928
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Entomology And Plant Pathology
Non Technical Summary
This research will continue to elucidate the function and regulation of a serine protease network in the biochemical model insect (Manduca sexta) during innate immune responses. This enzyme system produces phenoloxidase and antimicrobial peptides to entrap and kill pathogens and parasites that cause malaria and other infectious diseases. The acquired knowledge will be useful for disrupting the transmission of human/animal diseases in vector species, protecting beneficial insects, and enhancing the control of agricultural pests and plant pathogens.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21131101040100%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
An extracellular serine protease network coordinates key mechanisms of insect defense against microbial infection. Proteolytic activation of prophenol-oxidase (PPO) generates phenoloxidase that catalyzes the production of reactive compounds to sequester and kill invading pathogens and parasites. Proteolytic processing of spatzle precursor triggers the Toll pathway, which induces the synthesis of antimicrobial peptides and other defense proteins. In insect vectors of human diseases, initiation and regulation of these protease systems may be disrupted by proteins from the intruders. Knowledge of such protein interactions from biochemical model insects such as Manduca sexta will be useful for understanding and manipulating similar systems in arthropod vectors to interfere with disease transmission. We have discovered a branch of the M. sexta protease network that detects Gram-positive bacteria and fungi and mediates PPO activation. In this network, pathogen recognition receptors bind to microbes and initiate protease pathways for PPO and Toll activation. We have identified a new Sushi-domain protease that may participate in a branch of the pathway responsive to Gram-negative bacterial infection. A positive feedback loop in the protease system controls locality and potency of the defense responses. We plan to extend our research on the PPO activation reaction by examining the initiation and regulation of immune protease pathways. The specific aims of this project are: 1) Characterization of the pathway initiation by Gram-negative bacteria; 2) Elucidation of the positive feedback mechanism in the HP system.
Project Methods
To achieve the first objective, we plan to identify plasma proteins that bind to Gram-negative bacteria, characterize a role of M. sexta HP25 in the activation of PPO and Toll pathways, study bacterial binding and autoactivation of proHP25, and elucidate HP25s regulatory functions. To achieve the second objective, we will purify and clone a protease or an adaptor for proHP6 activation, purify proSPH1 activating protease, and explores mechanism for the formation of an active SPH complex.

Progress 10/01/12 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A group of graduate students and colleagues developed skills to perform high-quality gene annotation, evolutionary studies, expression profiling, and structural modeling. These skills are essential to the ongoing and future genome projects. Some students developed expertise in solving protein structure by X-ray crystallography, exploring biochemical problems using mass spectrometry, and analyzing proteomics data qualitatively and quantitatively. These lead to breakthroughs in the functional analysis of the Manduca immune system. How have the results been disseminated to communities of interest?Twenty-one studies have been published in Insect Biochem. Mol. Biol, a prominent journal in the field of entomology (IF: 3.8). All but two of the papers appear in journals with IF > 3.0. The M. sexta proteomic research is published in Mol. Cellular Proteomics, the highest journal in the field of proteomics (IF: 5.9). The PPO8 crystal structure is published in BMC Biology (IF: 7.0). These studies are expected to positively impact the development of insect science. What do you plan to do during the next reporting period to accomplish the goals?With the genome sequence, transcript profiles, and protein level changes available for M. sexta, we are addressing key questions of the insect immunobiochemistry and aimed to make greater progress in the next reporting period using all the techniques available.

Impacts
What was accomplished under these goals? Goal 1: Characterization of the pathway initiation by Gram-negative bacteria We identified in the M. sexta genome 190 putative pattern recognition receptors including PGRPs, βGRPs, LRRPs and CTL-domain proteins, analyzed their structural features, evolutionary relationships, modeled 56 CTL domains to predict their carbohydrate binding capability and specificity, and correlated expression patterns with immune relatedness and transcriptional regulation. We characterized the essential roles of MBP and PGRP1 in recognition of DAP-PG, activation of proHP14, and AMP induction in M. sexta. Goal 2: Elucidation of the positive feedback mechanism in the HP system We isolated a major processing enzyme from M. sexta hemolymph and found it to be PAP3, which activates proPAP3, proPOs, and proSPHs. By activating its precursor, substrate, and cofactor, PAP3 is a key component of the positive feedback mechanism. PAP3 also indirectly activates proHP6, proPAP1, and proHP8 in plasma to stimulate melanization. M. sexta HP1a and HP1b are isoforms, whose genes are specifically expressed in hemocytes in response to developmental or immune signals. The proHP1 is involved in proPO activation by activating proHP6, perhaps. We detected proHP1 in the SDS-stable complexes with a high Mr of 90 kDa. Seven serpins formed SDS complexes with the active proHP1. No proHP6 is activated by the proHP1b mutant cleaved at the designed activation site. Taken together, we proved proHP1 can be activated without being cleaved. M. sexta serpin-9 and -13 are produced in fat body and secreted into plasma to inhibit HPs and affect proPO activation. Serpin-9 uses R366 as P1 to inhibit proteases with different specificities, whereas serpin-13 uses R410, A409, G406, or T404. Serpin-9 forms small amounts of covalent complexes with proHP1*, HP2, and HP6; serpin-13 inhibits proHP1*, HP6 and HP8 to suppress the proPO activation system. Aim 3. Genome, transcriptome, and proteome analyses This aim, not listed in the proposal, evolved as our research moved forward. We published a series of 8 papers on the M. sexta genome, 6 on immunity-related genes, 2 on the genome annotation, and 3 on cuticle and chitin-related proteins. We participated in the genome project of Citrus psyllid and deeply annotated 337 serine protease-related genes in the mosquito A. gambiae. We produced 4 papers on M. sexta whole and immuno-transcriptomes, A. stephensi and H. armigera transcriptomes. We also invested on proteomic analysis of M. sexta and A. gambiae hemolymph samples and published 3 papers. Aim 4. Other collaborative projects We studied acetylcholinesterase expression, functions and inhibition. In three other papers, we characterized microRNAs of M. sexta in terms of developmental, immune, and tissue-specific regulation. We elucidated the crystal structure of A. gambiae PPO8, which yielded crucial insights into its catalytic and activation mechanisms. We solved NMR structure of M. sexta SRP2 and contributed to a protein expression technology.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Schrag, L.G., Cao, X., Alvaro I. Herrera, A.I., Wang, Y., Jiang, H., Prakash, O. (2017) Solution structure and expression profile of an insect cytokine, Manduca sexta stress response peptide-2. Protein Peptide Lett. 24, 3-11
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang, Y., Jiang, H. (2017) Prophenoloxidase activation and antimicrobial peptide expression induced by the recombinant microbe binding protein of Manduca sexta. Insect Biochem. Mol. Biol. 83, 35-43
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Saha, S., Hosmani, P.S., Villalobos-Ayala, K., Miller, S., Shippy. T., Flores, M., Rosendale, A., Cordola, C., Bell, T., Mann, H., DeAvila, G., DeAvila, D., Moore, Z., Buller, K., Ciolkevich, K., Nandyal, S., Mahoney, R., Van Voorhis, J., Dunlevy, M., Farrow, D., Hunter, D., Morgan, T., Shore, K., Guzman, V., Izsak, A., Dixon, D.E., Cridge, A., Cano, L., Cao, X., Jiang, H., Leng, N., Johnson, S., Cantarel, B.L., Richards, S., English, A., Shatter R.G., Childers, C., Chen, M-J., Hunter, W., Cilia, M., Mueller, L.A., Munoz-Torres, M., Nelson, D., Poelchau, M.F., Benoit, J., Wiersma-Koch, H., D'Elia, T., Brown, S.J. (2017) Improved annotation of the insect vector of citrus greening disease: Biocuration by a diverse genomics community. Database (Oxford). 2017, 1-20
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: He, Y., Wang, Y., Yang, F., Jiang, H. (2017) Manduca sexta hemolymph protease-1, activated by an unconventional non-proteolytic mechanism, mediates immune responses. Insect Biochem. Mol. Biol. 84, 23-31
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: He, X., Cao, X., He, Y., Bhattarai, K., Rogers, J., Hartson, S., Jiang, H. (2017) Hemolymph proteins of Anopheles gambiae larvae infected by Escherichia coli. Dev. Comp. Immunol. 74, 110-124
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Steele, K.H., Stone, B.J., Franklin, K., Jesaitis, A., Jiang, H., Webb, B.A., Zhang, X., Geisler, C., Fath-Goodin, A. (2017) Improving the baculovirus expression vector system with vankyrin-enhanced technology. Biotechnol. Progress. 33, 1-12.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Cao, X., Gulati M., Jiang, H. (2017) Serine protease-related proteins in the malaria mosquito, Anopheles gambiae. Insect Biochem. Mol. Biol. 88, 48-62
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Schrag, L.G., Herrera, A.I., Cao, X., Prakash, O., Jiang, H. (2017) Structure and function of stress responsive peptides in insects. In Peptide-based Drug Discovery: Challenges and New Therapeutics (V.P. Srivastava ed), pp. 438-451, Royal Society of Chemistry, London, UK
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Cao, X., Jiang, H. (2017) An analysis of 67 RNA-seq datasets from various tissues at different stages of a model insect, Manduca sexta. BMC Genomics. 18, 796
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: He, Y., Zhao, P, Rayaprolu, S, Wang, Y., Jiang, H. (2017) Serpin-9 and -13 regulate hemolymph proteases during immune responses of Manduca sexta. Insect Biochem. Mol. Biol. 90, 71-81


Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A group of graduate students and colleagues developed skills to perform high-quality gene annotation, evolutionary studies, expression profiling, and structural modeling. These skills are essential to the ongoing and future genome projects. Some students developed expertise in solving protein structure by X-ray crystallography, exploring biochemical problems using mass spectrometry, and analyzing proteomics data qualitatively and quantitatively. These lead to breakthroughs in the functional analysis of the Manduca immune system. How have the results been disseminated to communities of interest?Two studies have been published in Insect Biochemistry and Molecular Biology, a flagship journal in the field of entomology (IF: 3.8). The proteomic research is published in Molecular and Cellular Proteomics, the highest journal in the broad field of proteomics (IF: 5.9). The crystal structure is published in BMC Biology (IF: 7.0). These quality studies are expected to positively impact the insect science. What do you plan to do during the next reporting period to accomplish the goals?With the genome sequence, transcript profiles, and protein level changes available for M. sexta, we are addressing key questions of the insect immunobiochemistry and aimed to make greater progress in the next reporting period using all the techniques available.

Impacts
What was accomplished under these goals? We published four original studies this year. The 1st one on the Manduca sexta proteome revealed 654 proteins (70 up-regulated) in the cell-free hemolymph, their correlation with mRNA level changes, and 66 proteins in the high Mr immune complexes (He et al., 2016). The 2nd on the crystal structure of Anopheles gambiae prophenoloxidase-8 showed a new substrate binding site with E364 acting as the catalytic residue for deprotonation during dihydroxylation and oxidation (Hu et al., 2016). The 3rd on the M. sexta genome is a landmark paper on the model insect representing a large group of agricultural pests (Kanost et al., 2016). The 4th reported a 24-year-long odyssey in search for the function of proHP1 in the PPO activation system of M. sexta (Yang et al., 2016).

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Hu, Y., Wang, Y., Deng, J., Jiang, J. (2016) The structure of a prophenoloxidase (PPO) from Anopheles gambiae provides new insights into the mechanism of PPO activation. BMC Biol. 14, 2.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: He, Y., Cao, X., Zhang, S., Rogers, J., Hartson, S., Jiang, H. (2016) Changes in the plasma proteome of Manduca sexta larvae in relation to the transcriptome variations after an immune challenge: evidence for high molecular weight immune complex formation. Mol. Cell. Proteomics 15, 1176-1187.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Yang, F., Wang, Y., He, Y., Jiang, H. (2016) In search of a function of Manduca sexta hemolymph protease-1 in the innate immune system. Insect Biochem. Mol. Biol. 76, 1-10.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kanost, M.R., Arrese, E.L., Cao, X., Chen, Y-R, Chellapilla, S., Goldsmith, M., Grosse-Wilde, E., Heckel, D.G., Herndon, N., Jiang, H., & Brown, S.J., Scherer, S.E., Richards, S., Blissard, G.W. (2016) Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta. Insect Biochem. Mol. Biol. 76, 118-147.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A group of Ph.D. students and colleagues developed a set of skills to perform high-quality gene annotation, evolutionary studies, expression profiling, and structural modeling. These skills are essential to the ongoing and future genome projects. How have the results been disseminated to communities of interest?All but one of these studies have been published in a special issue of a high-visibility journal Insect Biochemistry and Molecular Biology. Since Manduca sexta is a well-known model insect for biochemical research, these quality studies are expected to positively impact the insect biochemistry and molecular biology. What do you plan to do during the next reporting period to accomplish the goals?Since the genome background is quite clear for Manduca sexta, we plan to focus on proteomic research of insect hemolymph and functional elucidation of individual proteins in the immune system. There are many new questions regarding the insect immunobiochemistry. We plan to address them using all the techniques available.

Impacts
What was accomplished under these goals? We published eight original studies based on the Manduca sexta genome, which revealed major gene families of proteins including pattern recognition proteins (Zhang et al., 2015a); C-type lectins (Rao et al., 2015), non-digestive serine proteases and their homologs (Cao et al., 2015a); signaling pathways (Cao et al., 2015b), antimicrobial proteins (He et al., 2015), cuticular proteins (Dittmer et al., 2015), chitin metabolism enzymes (Tetreau et al., 2015a), and chitin-binding proteins (Tetreau et al., 2015b). The methods we used are gene annotation, phylogenetic analysis, expression profiling, and 3D-structural modeling. In the last of a three-paper series, Zhang et al. (2015b) reported tissue-specific miRNAs in Manduca sexta fat body, hemocytes, and midgut. Cao independently developed a new method to model protein-coding genes in the genome using RNA-Seq data (Cao and Jiang, 2015). In collaboration with Dr. Zhen Zou, we have explored the immunotranscriptome of an agricultural pest, Helicoverpa armigera (Xiong et al., 2015) by RNA-seq analysis and found how its immune system responds to pathogen infections.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Cao, X., He, Y., Hu, Y., Wang, Y., Chen, Y-R., Bryant, B., Clem, R.J., Schwartz, L.M., Blissard, G.W., Jiang, H. (2015) The immune signaling pathways of Manduca sexta. Insect Biochem. Mol. Biol. 62, 64?74.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Rao, X., Cao, X., He, Y., Hu, Y., Zhang, X., Chen, Y., Blissard, G.W., Kanost, M.R., Yu, X-Q., Jiang, H. (2015) Structural features, evolutionary relationships, and transcriptional regulation of C-type lectin-domain proteins in Manduca sexta. Insect Biochem. Mol. Biol. 62, 75?85.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Dittmer, N.T., Tetreau, G., Cao, X., Jiang, H., Wang, P., Kanost, M.R. (2015) Annotation and expression analysis of cuticular proteins from the tobacco hornworm, Manduca sexta. Insect Biochem. Mol. Biol. 62, 100?113.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Cao, X., Jiang, H. (2015) Integrated modeling of protein-coding genes in the Manduca sexta genome using RNA-Seq data from the biochemical model insect. Insect Biochem. Mol. Biol. 62, 2?10.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang, X., Zheng, Y., Cao, X., Ren, R., Yu, X-Q., Jiang, H. (2015) Identification and profiling of Manduca sexta microRNAs and their possible roles in regulating specific transcripts in fat body, hemocytes, and midgut. Insect Biochem. Mol. Biol. 62, 11?22.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Tetreau, G., Cao, X., Chen, Y., Muthukrishnan, S., Jiang, H., Blissard, G.W., Kanost, M.R., Wang, P. (2015) Overview of chitin metabolism enzymes in Manduca sexta: identification, domain organization, phylogenetic analysis and gene expression. Insect Biochem. Mol. Biol. 62, 114?126.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: He, Y., Cao, X., Li, K., Hu, Y., Chen, Y., Blissard, G.W., Kanost, M.R., Jiang, H. (2015) A genome-wide analysis of antimicrobial effector genes and their transcription patterns in Manduca sexta. Insect Biochem. Mol. Biol. 62, 23?37.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang, X., He, Y., Cao, X., Gunaratna, R.T., Chen, Y., Blissard, G.W., Kanost, M.R., Jiang, H. (2015) Phylogenetic analysis and expression profiling of the pattern recognition receptors: insights into molecular recognition of invading pathogens in Manduca sexta. Insect Biochem. Mol. Biol. 62, 38?50.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Cao, X., He, Y., Hu, Y., Zhang, X., Wang, Y., Zou, Z., Chen, Y., Blissard, G.W., Kanost, M.R., Jiang, H. (2015) Sequence conservation, phylogenetic relationships, and expression profiles of nondigestive serine proteases and serine protease homologs in Manduca sexta. Insect Biochem. Mol. Biol. 62, 51?63.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Tetreau, G., Dittmer, N.T., Cao, X., Agrawal, S., Chen, Y., Muthukrishnan, S., Jiang, H., Blissard, G.W., Kanost, M.R., Wang, P. (2015) Analysis of chitin-binding proteins from Manduca sexta provides new insights into evolution of peritrophin A-type chitin-binding domains in insects. Insect Biochem. Mol. Biol. 62, 127?141.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Xiong, G-H, Xing, L-S, Lin, Z., Saha, T.T., Wang, C, Jiang, H., Zou, Z. (2015) High throughput profiling of the cotton bollworm Helicoverpa armigera immunotranscriptome during the fungal and bacterial infections. BMC Genomics, 16, 321.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We have provided training opportunities for graduate students in the laboratory to perform biochemical and molecular biological and develop their professional careers. One Ph.D. student graduated in 2014, who joined the group led by a NAS member. How have the results been disseminated to communities of interest? Through high-caliber scientific journals (5 papers) and meetings (3 posters). What do you plan to do during the next reporting period to accomplish the goals? Publish 6 to 8 original papers in scientific journals and presenting 3 to 5 posters at meetings.

Impacts
What was accomplished under these goals? We continued to publish studies on Manduca sexta as well as other insects in peer-reviewed journals. Using it as a model for insect biochemical and physiological research, we explored the posttranscriptional regulation of immune gene expression by microRNAs (Zhang et al., 2014). We did a semi-quantitative analysis of changes in the plasma peptidome of M. sexta larvae and correlated those with the transcriptome variations upon immune challenge (Zhang et al., 2014). Our biochemical study revealed another positive feedback mechanism involving PAP3, proPAP3, SPHs and proPOs (Wang et al., 2014).

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Chen, K., Liu, C., He, Y., Jiang, H., Lu, Z. (2014) A short-type peptidoglycan recognition protein from the silkworm: expression, characterization and involvement in the prophenoloxidase activation pathway. Dev. Com. Immunol. 45, 1-9.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang, J., Zhang, S., Wang, Y., Xu, W., Zhang, J., Jiang, H., Huang, F. (2014) Modulation of Anopheles stephensi gene expression by nitroquine, an antimalarial drug against Plasmodium yoelii infection in the mosquito. PLoS One, 9, e89473.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang, X., Zheng, Y., Jagadeeswaran, G., Ren, R., Sunkar R., Jiang, H. (2014) Identification of conserved and novel microRNAs in Manduca sexta and their possible roles in the expression regulation of immunity-related genes. Insect Biochem. Mol. Biol. 47, 12-22.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang, S., Cao, X., He, Y., Hartson, S., Jiang, H. (2014) Semi-quantitative analysis of changes in the plasma peptidome of Manduca sexta larvae and their correlation with the transcriptome variations upon immune challenge. Insect Biochem. Mol. Biol. 47, 46-54.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Wang, Y., Lu, Z., Jiang, H. (2014) Manduca sexta proprophenoloxidase activating proteinase-3 (PAP3) stimulates melanization by activating proPAP3, proSPHs, and proPOs. Insect Biochem. Mol. Biol. 50, 82-91.


Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We continue to publish research results in peer-reviewed journals.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhao, P., Wang, Y., Jiang, H. (2013) Characterization of Anopheles gambiae acetylcholinesterase-2 encoded by the orthologous gene (ace-2) from the malaria mosquito. Insect Biochem. Mol. Biol., 43, 260-271
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Gunaratna, R., Jiang, H. (2013) A comprehensive analysis of the Manduca sexta immunotranscriptome. Dev. Com. Immunol., 39, 388-398


Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: MicroRNAs (miRNAs) are involved in translation inhibition or mRNA degradation. Due to its large size, Manduca sexta is used as a model to study insect physiology and biochemistry. While transcriptome studies have enriched our knowledge on structural genes, little is known in this insect about posttranscriptional regulation by miRNAs. We constructed four small RNA libraries from embryos, larvae, pupae and adults, and found 163 conserved and 13 novel miRNAs. We identified precursors of 82 conserved miRNAs, 76 of which had mapped reads in one or more of the libraries. After normalization, we compared numbers of miRNA and miRNA-star reads in these libraries and observed abundance changes during development. Eight miRNA star strands are either more abundant or maintained at similar levels compared to respective mature miRNA strand. Expression profiling of the first miRNA set provided insights to possible involvement in developmental regulation. This study will aid in the annotation of miRNA genes in the genome. M. sexta has greatly contributed to our knowledge on insect immunity. The RNA-Seq approach was implemented in three studies to examine tissue immunotranscriptomes of this species. With the latest and largest focusing on highly regulated process- and tissue-specific genes, we further analyzed the same set of data using BLAST2GO to explore functional aspects of the larval fat body and hemocyte transcriptomes with or without immune challenge. Using immunity-related sequences from other insects, we found 383 homologous contigs and compared them with those discovered based on relative abundance changes. The major overlap of the two lists validated our previous research designed for gene discovery and transcript profiling in organisms lacking sequenced genomes. By concatenating the contigs, we established a repertoire of 232 immunity-related genes and examined their mRNA levels along with attribute classifications and detected prominent differences in nine of the thirty level 2 gene ontology categories. We identified most members of the putative Toll, IMD, MAPK-JNK-p38, and JAK-STAT pathways and small changes in their mRNA levels. Together, these findings set the stage for on-going analysis of the M. sexta immunogenome. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A conserved evolutionary strategy of innate immunity involves serine protease pathways to mediate rapid responses to tissue damage or pathogen invasion. Proteolytically activated PO catalyzes quinone and melanin formation to entrap and kill pathogens. Processed spatzle binds to Toll receptor and induces antimicrobial peptide synthesis. The lepidopteran insect, Manduca sexta, has been established as a model system for studying arthropod immune protease pathways. The acquired knowledge on M. sexta hemolymph protease network would be useful in the future for preventing human diseases transmitted by arthropod vectors.

Publications

  • Lu, Y., Park, Y., Gao, X., Zhang, X., Yao, J., Pang, Y.P., Jiang, H., Zhu, K. Y. (2012) Cholinergic and non-cholinergic functions of two acetylcholinesterase genes revealed by gene-silencing in Tribolium castaneum. Sci. Rep., 2, 288.
  • Gunaratna, R.T., Jiang, H. (2012) A comprehensive analysis of the Manduca sexta immunotrans-criptome. Dev. Com. Immunol., in press.
  • Zhang, X, Zheng, Y., Jagadeeswaran, G., Ren, R., Sunkar, R., Jiang, H. (2012) Identification and developmental profiling of conserved and novel microRNAs in Manduca sexta. Insect Biochem. Mol. Biol., 42, 381-395.


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: We have studied M. sexta microbe binding protein (MBP) which is 61% and 35% identical in sequence to B. mori GNBP and M. sexta bGRP, respectively. Its mRNA and protein levels are up-regulated upon immune challenge. The recombinant MBP associates with intact bacteria and fungi by binding to LTA, LPS, DAP-PG, and laminarin. The binding pattern is influenced by other plasma factors and additional microbial surface molecules. After different amounts of MBP are mixed with larval plasma on ice, a concentration-dependent increase in PO activity occurs in the absence of any microbial elicitor. The activity also increases in the mixture of plasma and a bacterial or fungal cell wall component. The proPO activation becomes more prominent when DAP-PGs, M. luteus Lys-PG, or LTA is included in the mixture of MBP and plasma. Statistic analysis suggests that a synergistic enhancement of proPO activation is caused by an interaction between MBP and the elicitors, but not Lys-PG, LPS, curdlan, or laminarin. Therefore, MBP is a component of the surveillance mechanism and, by working with other recognition molecules and serine proteinases, triggers the M. sexta proPO activation system. Scientists working on non-model organisms are often limited by shortage of information on process-related genes and their expression patterns. We have developed a pyrosequencing-based approach that combines simple mathematical treatment, large-scale gene discovery, expression profiling, and cost effectiveness. It is validated by our investigation of M. sexta transcriptome. We discovered 528 up-regulated contigs and 411 fat body- or hemocyte-specific genes. Instead of relying on a priori knowledge of the genome, we established a sequence database to support future genome annotation, cDNA cloning, and protein identification in this species. PO generates reactive compounds (e.g., DHI), which have a broad-spectrum antibacterial and antifungal activity. DHI and its spontaneous oxidation products are also active against viruses and parasitic wasps. Preincubation of a baculovirus stock with 1.25 mM DHI for 3 h near fully disables recombinant protein production. The LC50's for lambda bacteriophage and eggs of the wasp M. demolitor are 6 and 111 μM, respectively. The toxicity of DHI and related compounds also extends to cells derived from insects that serve as hosts for several of the aforementioned pathogens. Pretreatment of Sf9 cells with 1.0 mM DHI for 4 h results in 97% mortality, and LC50 values are 20 μM in buffer and 132 μM in a culture medium. Symptoms of DHI toxicity in Sf9 cells include DNA polymerization, protein crosslinking, and lysis. Taken together, these data show that proPO activation and DHI production is strongly toxic against various pathogens but can also damage host tissues and cells if not properly controlled. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A conserved evolutionary strategy of innate immunity involves serine protease pathways to mediate rapid responses to tissue damage or pathogen invasion. Proteolytically activated PO catalyzes quinone and melanin formation to entrap and kill pathogens. Processed spatzle binds to Toll receptor and induces antimicrobial peptide synthesis. The lepidopteran insect, Manduca sexta, has been established as a model system for studying arthropod immune protease pathways. The acquired knowledge on M. sexta hemolymph protease network would be useful in the future for preventing human diseases transmitted by arthropod vectors.

Publications

  • Jiang, H., Vilcinskas, A., and Kanost, M.R. (2011) Immunity in lepidopteran insects. In Invertebrate Immunity (K. Soderhall ed), Landes BioScience, in press.
  • Wang, Y., Sumathipala, N., Rayaprolu, S., and Jiang, H. (2011) Recognition of microbial molecular patterns and stimulation of prophenoloxidase activation by a b-1,3-glucanase-related protein in Manduca sexta larval plasma. Insect Biochem. Mol. Biol. 41, 322-331.
  • Zhang, S., Zhang, X., Gunaratna, R., Najar, F., Wang, Y., Roe, B., and Jiang, H. (2011) Pyrosequencing-based expression profiling and identification of differentially regulated genes from Manduca sexta, a lepidopteran model insect that lacks genome sequence. Insect Biochem. Mol. Biol. 41, 733-746.
  • Zhao, P., Lu, Z., Strand, M., and Jiang, H. (2011) Antiviral, antiparasitic, and cytotoxic effects of 5,6-dihydroxyindole (DHI), a reactive compound generated by phenoloxidase during insect immune response. Insect Biochem. Mol. Biol. 41, 645-652.


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: We purified M. sexta peptidoglycan recognition protein-1 (PGRP1) from the baculovirus-insect cell expression system, tested its association with PGs and intact bacteria, and explored its possible link with the prophenoloxidase activation system in larval hemolymph. Sequence comparison suggested that PGRP1 lacks residues for interacting with the carboxyl group of meso-diaminopimelic acid PGs (DAP-PGs). PGRP1 concentration in larval plasma increased in a time-dependent manner after the immune challenge. Purified recombinant PGRP1 specifically bound to soluble DAP-PG of E. coli but not to soluble Lys-type PG of S. aureus. In addition, this recognition protein completely bound to insoluble PGs from M. luteus, B. megaterium and B. subtilis, whereas its association with the bacterial cells was low. After PGRP1 had been added to plasma of naive larvae in the absence of microbial elicitor, there was a concentration-dependent increase in prophenoloxidase activation. Phenoloxidase activity, as usual, increased after the plasma was incubated with PGs or bacterial cells. These increases became more prominent when insoluble M. luteus or B. megaterium PG or soluble E. coli PG and PGRP1 were both present. Statistic analysis suggested a synergistic effect caused by interaction between PGRP1 and these PGs. These results indicated that PGRP1 is a member of the M. sexta prophenoloxidase activation system, which recognizes PGs from certain bacteria and initiates the host defense response. Ticks continue to be a threat to animal and human health, and new and novel control strategies are needed for ticks and tick-borne pathogens. The characterization of the tick-pathogen interface and the tick immune response to microbial infections is fundamental toward the formulation of new control strategies for ticks and the pathogens they transmit. In this study, 454 pyrosequencing, a high-throughput genomic sequencing method, was used to discover tick genes expressed in response to bacterial and fungal infections. ESTs were analyzed from D. variabilis ticks that had been injected with bacteria or fungi and ticks that were naturally infected with the intracellular bacterium, A. marginale. From 5995 contigs, we identified more than 30 genes that are likely to encode for proteins involved in tick immune function. We further analyzed by RT-PCR the expression of 22 of these genes in each of our bacterial or fungal treatment groups and found that seven were up-regulated. Up-regulation of these seven genes was confirmed for bacterial, but not fungal treatment by qPCR. One of these products was novel, encoding a new tick defensin. Our results clearly demonstrate the complexities of the tick immune system and mark new directions for further study and characterization of proteins that modulate microbial infections in the American dog tick. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A conserved evolutionary strategy of innate immunity involves serine protease pathways to mediate rapid responses to tissue damage or pathogen invasion. Proteolytically activated PO catalyzes quinone and melanin formation to entrap and kill pathogens. Processed spatzle binds to Toll receptor and induces antimicrobial peptide synthesis. The lepidopteran insect, Manduca sexta, has been established as a model system for studying arthropod immune protease pathways. The acquired knowledge on M. sexta hemolymph protease network would be useful in the future for preventing human diseases transmitted by arthropod vectors.

Publications

  • Rayaprolu, S., Kanost, M.R., Wang, Y., and Jiang, H. (2010) Functional analysis of four processing products from multiple precursors encoded by a lebocin-related gene from Manduca sexta. Dev. Com. Immunol. 34, 638-647.
  • Jiang, H., Vilcinskas, A., and Kanost, M.R. (2010) Immunity in lepidopteran insects. In Invertebrate Immunity (K. Soderhall ed), Landes BioScience, in press.
  • Sumathipala, N. and Jiang, H. (2010) Involvement of Manduca sexta peptidoglycan recognition protein-1 in the recognition of bacteria and activation of prophenoloxidase system. Insect Biochem. Mol. Biol. 40, 485-495.
  • Jorworski D.C., Zou, Z., Bowen, C.J., Wasala, N.B., Madden, R., Wang, Y., Kocan, K.M., Jiang, H., and Dillwith, J. (2010) Pyrosequencing and characterization of immune responsive genes from the tick Dermacentor variabilis. Insect Mol. Biol. 19, 617-630.


Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: We have solved the crystal structure of Manduca sexta prophenoloxidase (PPO) at 1.97A resolution. It is a heterodimer consisting of two polypeptide chains, PPO1 and PPO2. The active site of each subunit contains a canonical type-3 dinuclear copper center, with each copper ion coordinated with three structurally conserved histidines. The acidic residue Glu395 located at the active site of PPO2 may serve as a general base for deprotonation of monophenolic substrates, which is critical to the o-hydroxylase activity of PO. The structure provides new insights into the mechanism by which type-3 copper proteins differ in their enzymatic activities, albeit sharing a common active center. A drastic change in electrostatic surface induced upon cleavage at Arg51 allows us to propose a model for localized PPO activation. We have elucidated the functions of M. sexta clip-domain proteases HP6 and HP8. HP6 is an apparent ortholog of Drosophila Persephone, whereas HP8 is most similar to spatzle-activating enzymes that activate the Toll pathway. ProHP6 and proHP8 are constitutively expressed in fat body and hemocytes and secreted into plasma, where they are activated by limited proteolysis in response to infection. We have purified recombinant proHP8, proHP6 and mutants of proHP6 and found HP6 activates proPAP1 in vitro to stimulate PPO activation in plasma. HP6 was also determined to activate proHP8. Active HP6 or HP8 injected into larvae induced expression of antimicrobial peptide genes. Therefore, HP6 participates in two pathways: proPAP1 activation, which leads to PPO activation and melanin synthesis, and proHP8 activation, which stimulates a Toll-like pathway in M. sexta. Serpins are a superfamily of proteins, most of which control protease-mediated processes. Determination of the silkworm genome provides us an opportunity to investigate their structure, function, and evolution. There are 34 serpin genes in Bombyx mori, six of which are highly similar in sequence to their M. sexta orthologs that regulate innate immunity. Three alternative exons in serpin1 gene and four in serpin28 encode a variable region including the reactive site loop. There is an increase in the mRNA levels of serpin1, 3, 5, 6, 9, 12, 13, 25, 27, 32 and 34 in induced fat body and hemocytes. The serpin23 transcript level increases in fat body after an immune challenge whereas the serpin4, 28 and 31 mRNA becomes more abundant in hemocytes. The silkworm serpins may play regulatory roles in defense responses. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A conserved evolutionary strategy of innate immunity involves serine protease pathways to mediate rapid responses to tissue damage or pathogen invasion. Proteolytically activated PO catalyzes quinone and melanin formation to entrap and kill pathogens. Processed spatzle binds to Toll receptor and induces antimicrobial peptide synthesis. The lepidopteran insect, Manduca sexta, has been established as a model system for studying arthropod immune protease pathways. The acquired knowledge on M. sexta hemolymph protease network would be useful in the future for preventing human diseases transmitted by arthropod vectors.

Publications

  • Zou, Z., Zhao, P., Weng, H., Mita, K., and Jiang, H. (2009) A comparative analysis of serpin genes in the silkworm genome. Genomics, 93, 367-375.
  • Jiang, H., Liu, S., Zhao, P., and Pope, C. (2009) Recombinant expression and biochemical characterization of the catalytic domain of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae. Insect Biochem. Mol. Biol. 39, 646-653.
  • An, C., Ishibashi, J., Ragan, E.J., Jiang, H., and Kanost, M.R. (2009) Functions of Manduca sexta hemolymph proteinases HP6 and HP8 in two innate immune pathways. J. Biol. Chem. 19716-19726.
  • Ragan, E.J., An, C., Jiang, H., and Kanost, M.R. (2009) Roles of hemolymph proteins in antimicrobial defenses of Manduca sexta. in Insect Infection and Immunity (J. Rolff, S. Reynolds eds), Oxford University Press, Corby, UK, pp. 34-48.
  • Li, Y., Wang, Y., Jiang, H., and Deng, J. (2009) Crystal structure of Manduca sexta prophenoloxidase provides insights into the mechanism of type-3 copper enzymes. Proc. Natl. Acad. Sci. USA. 106, 17001-17005.
  • Zhao, P., Zhu, K.Y., and Jiang, H. (2009) Heterologous expression, purification, and biochemical characterization of a greenbug (Schizaphis graminum) acetylcholinesterase encoded by a paralogous gene (ace-1). J. Biochem. Mol. Toxicol. in press.


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: In Manduca sexta, pathogen recognition triggers a branched serine proteinase cascade which generates active phenoloxidase (PO) in the presence of a proPO-activating proteinase (PAP) and two noncatalytic serine proteinase homologs (SPHs). PO then catalyzes the production of reactive compounds for microbe killing, wound healing, and melanin formation. In this study, we discovered that a minute amount of PAP1 (a final component of the proteinase pathway) caused a remarkable increase in PO activity in plasma from naive larvae, which was significantly higher than that from the same amounts of PAP1, proPO and SPHs incubated in vitro. The enhanced proPO activation concurred with the proteolytic activation of HP6, HP8, PAP1, SPH1, SPH2 and PO precursors. PAP1 cleaved proSPH2 to yield bands with mobility identical to SPH2 generated in vivo. PAP1 partially hydrolyzed proHP6 and proHP8 at a different bond. PAP1 did not directly activate proPAP1. These data suggest a self-reinforcing mechanism built into the proPO activation system. Other plasma proteins are required for cleaving proHP6 and proHP8 at the correct site to strengthen the defense response, perhaps in the early stage of the pathway activation. PO-catalyzed reactions are crucial to the survival of insects after an infection. In M. sexta, active PO is generated from its precursor by a PAP in the presence of noncatalytic SPHs. The PAP and SPHs also require limited proteolysis to become functional. We have investigated the activation of proSPHs by developing a series of vectors for co-expression, secretion, and affinity purification of proSPH1 and 2 from insect cells infected by a baculovirus and by purifying the proSPHs for use as substrates in search for their activators in larval plasma. Proteolysis occurred after the proSPHs had been incubated with hydroxyapatite or gel filtration column fractions. The cleaved proteins were active as a cofactor for proPO activation by PAP, and coexistence of SPH1 and 2 is essential for manifesting the auxiliary effect. To get a broad view of larval hemolymph proteins, particularly those related to immunity, we synthesized and sequenced cDNA fragments from a mixture of M. sexta total RNA samples. Using massively parallel pyrosequencing, we obtained 95,458 ESTs at an average size of 185 bp per read. A majority of these sequences were assembled into 7,231 contigs, 1178 of which had significant similarity with Drosophila genes from various functional groups. Only 606 of the contigs matched known M. sexta cDNA sequences. The remaining 6625 contigs represented newly discovered DNA segments from this well studied biochemical model insect. A search of the dataset using Tribolium castaneum, Drosophila melanogaster, and Bombyx mori immunity-related sequences revealed 424 cDNA contigs with significant similarity. These included 218 previously unknown M. sexta sequences coding for putative defense molecules such as pattern recognition receptors, serine proteinases, serpins, spatzle, Toll-like receptors, intracellular signaling molecules, and antimicrobial proteins. PARTICIPANTS: Haobo Jiang, PI; Yang Wang, Zhen Zou, Zhiqiang Lu, Assoc. & Assist. Researchers; PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A conserved evolutionary strategy of innate immunity involves serine protease pathways to mediate rapid responses to tissue damage or pathogen invasion. A few eliciting molecules, through specific recognition and cascade amplification, lead to the sequential proteolytic activation of a large amount of pathway components within minutes. In insects, the protease cascades for proPO and Toll activation are incompletely understood. PO-catalyzed quinone and melanin formation is a universal mechanism in insects for entrapping and killing pathogens or parasites. Proteolytically processed spatzle binds to the Toll receptor and induces the synthesis of antimicrobial peptides that are active against bacteria, fungi and, in some cases, parasites. The lepidopteran insect, Manduca sexta, has been established as a model system for studying arthropod immune protease pathways. With a sizable collection of molecular probes and purified proteins as well as a great deal of experience in handling hemolymph proteases available, we are at a favorable position to isolate and investigate functional protein complexes formed in vivo during defense responses. The acquired knowledge on M. sexta hemolymph protease network would be useful in the future for preventing human diseases transmitted by arthropod vectors.

Publications

  • Jiang, H. (2008) Insect Science 15, 53-66.
  • Tribolium Genome Sequencing Consortium (2008) The genome of the model beetle and pest Tribolium castaneum. Nature, 452, 949-955.


Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: Upon wounding, a serine proteinase cascade in insect hemolymph leads to prophenoloxidase (proPO) activation and melanization to kill invading microbes. In Manduca sexta, this response is initiated via hemolymph proteinase (HP) 14, a mosaic protein that interacts with fungal β-1,3-glucan and β-1,3-glucan recognition protein-2 to autoactivate. We expressed and characterized M. sexta HP21 precursor, a clip-domain serine proteinase similar to Drosophila Snake. HP14 activated proHP21 by limited proteolysis between Leu152 and Ile153. Active HP21 formed an SDS-stable complex with M. sexta serpin-4, a physiological regulator of the proPO activation system. We determined the P1 site of serpin-4 as Arg355 and, thus, confirmed our prediction that HP21 has trypsin-like specificity. After active HP21 was added to the plasma, there was a major increase in phenoloxidase activity. HP21 cleaved proPO-activating proteinase-2 (PAP-2) precursor after Lys153 and generated an amidase activity which activated proPO in the presence of serine proteinase homolog-1 and -2. We have solved the solution structure of dual clip domains from M. sexta PAP-2. Each domain adopts a new mixed alpha/beta fold (a three-stranded antiparallel beta-sheet flanked by two alpha-helices) and the architecture provides structural information on clip domains from a catalytically active proteinase for the first time. Examination of the structure in conjunction with a multiple sequence alignment of the clip domains from different groups suggests a substrate-binding site, a bacteria-interacting region, and a surface for specific interactions. We have demonstrated antimicrobial effects of reactive compounds produced in PO-catalyzed reactions. After being treated with M. sexta PO and dopamine, E. coli and B. subtilis ceased to grow whereas the growth of P. pastoris was slightly affected. Microscopic analysis showed melanin deposition on cell surface, aggregation of bacteria, and loss of cell mobility. Viability tests revealed major decreases in the bacterial colony counts and, since the decrease remained significant after dispersion of the cell clumps, the reactive intermediates were surmised to have aggregated and killed E. coli and B. subtilis cells. Under the experimental conditions, 60-94% of the Gram-negative bacteria and 52-99% of the Gram-positive bacteria were killed. In the presence of PO, dopamine or 5,6-dihydroxyindole (DHI) exhibited much higher antibacterial activity than L-dopa, N-acetyldopamine or N-beta-alanyldopamine did, suggesting that DHI and its oxidation products were cytotoxic. The antifungal activity of 5,6-DHI was detected using P. pastoris, S. cerevisiae, C. albicans and B. bassiana. PARTICIPANTS: Haobo Jiang, PI; Yang Wang, Senior Research Specialist; Picheng Zhao, GRA;

Impacts
PO catalyzes the formation of quinones which are precursors for melanotic encapsulation, a resistance mechanism against malaria parasites. It is synthesized as proPO, which is activated by proteolytic cleavage at a specific site near its N-terminus. The activation reaction is mediated by a PAP and a protein cofactor. The precursors of PAP and cofactor are also activated by limited proteolysis by a serine proteinase cascade. However, molecular mechanisms that regulate proPO activation remain unclear, and little is known about the pathway initiation. We hypothesize that recognition of microbial polysaccharides causes autoactivation of a hemolymph proteinase. This enzyme then activates one or more proteinase zymogens in a cascade mode and eventually leads to generation of active PAP and cofactor for proPO activation. Specific molecular recognition and serpins ensure that proPO activation occurs as a local reaction against nonself. Our research complements the genetic studies of serine proteinase-related proteins in Drosophila and stimulates similar researches in insect vectors of human diseases.

Publications

  • Wang, Y., Chen, T., Rayaprolu, S., Zou, Z., Xia, Q., Xiang, Z., and Jiang, H. (2007) Dev. Com. Immunol. 31, 1002-1012.
  • Lu, Z. and Jiang, H. (2007) Insect Biochem. Mol. Biol. 37, 478-485.
  • Wang, Y. and Jiang, H. (2007) Insect Biochem. Mol. Biol. 37, 1015-1025.
  • Gorman, M.J., Wang, Y., Jiang, H., and Kanost, M.R. (2007) J. Biol. Chem. 282, 11742-11749.
  • Waterhouse, R.M. et al. (2007) Science. 316 (5832), 1738-1743. Zhao, P., Li, J., Wang, Y. and Jiang, H. (2007) Insect Biochem. Mol. Biol. 37, 952-959.
  • Huang, R., Lu, Z., Dai, H., Vander Velde, D., Prakash, O. and Jiang, H. (2007) Biochemistry 46, 11431-11439.
  • Zou, Z., Evans, J., Lu, Z., Zhao, P., Williams, M., Sumathipala, N., Hetru, C., Hultmark, D. and Jiang, H. (2007) Genome Biol. 8, R177.


Progress 10/01/05 to 09/30/06

Outputs
A serine proteinase pathway in insect hemolymph leads to prophenoloxidase activation, an innate immune response against pathogen infection. We purified and characterized proHP14 from the hemolymph of bacteria-injected Manduca sexta larvae. The zymogen, consisting of a single polypeptide with a molecular mass of 68.5 kDa, is truncated at the amino terminus. It is converted to a two-chain active form in the presence of β-1,3-glucan (a fungal cell wall component) and β-1,3-glucan recognition protein-2. The 45 kDa heavy chain contains four low-density lipoprotein receptor A repeats, one Sushi domain, and one unique cysteine-rich region, whereas the 30 kDa light chain contains a serine proteinase domain, which was labeled by 3H-diisopropyl fluorophosphate. ProHP14 in the plasma binds curdlan, zymosan, yeast, peptidoglycan, and Micrococcus luteus. Addition of autoactivated HP14 elevated phenoloxidase activity level in the larval plasma. Recombinant M. sexta serpin-1I reduced prophenoloxidase activation by inhibiting HP14. These data are consistent with the current model on initiation and regulation of the prophenoloxidase activation cascade upon recognition of pathogen-associated molecular patterns by specific pattern recognition proteins.

Impacts
From our biochemical model insect Manduca sexta, we acquired knowledge on how insect vectors of human diseases defend themselves from microbial infection. Such information is critical for us to genetically manipulate vector species so that they no longer transmit infectious agents to human and other vertebrate hosts.

Publications

  • Wang, Y. and Jiang, H. (2006) J. Biol. Chem. 281, 9271-9278.
  • Wang, Y., Zou, Z. and Jiang, H. (2006) Genomics, 87, 399-409
  • Zou, Z., Lopez, D. Kanost, M., Evans, J. and Jiang, H. (2006) Insect Mol. Biol. 15, 603-614. The Honeybee Genome Consortium (2006) Nature. 443, 931-949.


Progress 10/01/04 to 09/30/05

Outputs
Insect phenoloxidase (PO) participates in melanotic encapsulation and wound healing. It is converted from prophenoloxidase (proPO) by proPO-activating proteinases (PAPs). In an effort to understand the transcriptional regulation of PAP-1, we isolated genomic clones of the gene, determined its nucleotide sequence, and elucidated its exon-intron organization (Zou et al., 2005). Computer analysis revealed immune and hormone responsive elements in the upstream region. Southern blot analysis suggested that the M. sexta genome contains a single copy of PAP-1 gene. Reverse transcription-polymerase chain reaction showed that PAP-1 was constitutively expressed in fat body, trachea, and nerve tissue of the fifth instar larvae. The mRNA levels in hemocytes and fat body markedly increased in response to a bacterial challenge. We also observed tissue-specific and developmental regulation of the gene's transcription. Treating the fat body culture with 20-hydroxyecdysone reduced the PAP-1 mRNA level. These data indicated that the expression of PAP-1 gene is under the dual control of immune and hormonal signals.

Impacts
From our biochemical model insect Manduca sexta, we acquired knowledge on how insect vectors of human diseases defend themselves from microbial infection. Such information is critical for us to genetically manipulate vector species so that they no longer transmit infectious agents to human and other vertebrate hosts.

Publications

  • Zou, Z., Wang, Y., and Jiang, H. (2005) Manduca sexta prophenoloxidase activating proteinase-1 (PAP-1) gene: organization, expression, and regulation by immune and hormonal signals. Insect Biochem. Mol. Biol. 35, 627-636.


Progress 10/01/03 to 09/30/04

Outputs
A serine proteinase cascade in insect hemolymph mediates prophenoloxidase activation, a defense mechanism against pathogen or parasite infection. Little is known about its initiating proteinase or how this enzyme is activated in response to invading microorganisms. We have isolated from the tobacco hornworm, Manduca sexta, a cDNA encoding a modular protein designated hemolymph proteinase 14 (HP14). It contains five low-density lipoprotein receptor class A repeats, a Sushi domain, a unique Cys-rich region, and a proteinase catalytic domain. The HP14 mRNA exists in fat body and hemocytes of the naive larvae, and its level increases significantly at 24 h after a bacterial challenge. We expressed proHP14 with a carboxyl-terminal hexahistidine tag in a baculovirus/insect cell system and detected the recombinant protein in two forms - the 87 kDa protein was primarily intracellular, whereas the 75 kDa form was present in the media. Interaction with peptidoglycan resulted in proteolytic processing of the purified zymogen and generation of an amidase activity. Supplementation of hemolymph with proHP14 greatly enhanced prophenoloxidase activation in response to Micrococcus luteus. These data suggest that proHP14 is a pattern recognition protein that binds to bacteria, autoactivates, and triggers the prophenoloxidase activation system in the hemolymph of M. sexta.

Impacts
We selected a biochemical model insect, Manduca sexta, to study innate immunity. The acquired knowledge will be useful for understanding a similar system in disease vectors and lead to the generation of mosquitoes that are resistant to parasite infection. When released into the environment, the transgenic mosquitoes may replace the susceptible ones and reduce the disease transmission.

Publications

  • No publications reported this period


Progress 10/01/02 to 09/30/03

Outputs
We have isolated an immune-responsive serpin from Manduca sexta. This inhibitor contains a reactive site loop strikingly similar to the proteolytic activation site in prophenoloxidase (proPO). Molecular cloning indicates that it is orthologous to Drosophila serpin 27A, a regulator of melanization and Toll activation. M. sexta serpin-3 is constitutively present in hemolymph and becomes more abundant after a microbial challenge. The recombinant serpin-3 efficiently blocked proPO activation in the hemolymph. It also formed SDS-stable complexes with purified proPO-activating proteinases (PAPs) from the same insect. We observed such complexes in a hemolymph fraction elicited with Micrococcus luteus. Kinetic analysis of PAP-serpin-3 association strongly suggested that serpin-3 could be a physiological regulator of the proPO activation reaction. We have purified a new b-1,3-glucan recognition protein from M. sexta prepupae. This 52 kDa protein, designated bGRP-2, is 57 percent identical in sequence to M. sexta bGRP-1 from larval hemolymph. It differs from bGRP-1 in its absence in the naive larvae before the wandering stage begins. Transcription of the bGRP-2 gene was up-regulated in larvae challenged with yeast or bacteria. bGRP-2 contains a region with sequence similarity to several glucanases but lacks glucanase activity. It aggregates yeasts and bacteria to, perhaps, limit the spread of the invading cells and ensure a localized defense reaction. bGRP-2 binds laminarin and lipoteichoic acid, but not lipopolysaccharide. Laminarin-triggered proPO activation was greatly enhanced in the induced larval hemolymph supplemented with purified bGRP-2.

Impacts
We selected a biochemical model insect, Manduca sexta, to study innate immunity. The acquired knowledge will be useful for understanding a similar system in disease vectors and lead to the generation of mosquitoes that are resistant to parasite infection. When released into the environment, the transgenic mosquitoes may replace the susceptible ones and reduce the disease transmission.

Publications

  • Zhu, Y., Wang, Y., Gorman, M., Jiang, H., and Kanost, M.R. (2003) Manduca sexta serpin-3 regulates prophenoloxidase activation in response to infection by inhibiting prophenoloxidase-activating proteinases. J. Biol. Chem.
  • Jiang, H., Ma, C., Lu, Z., and Kanost, M.R. (2003) $-1,3-glucan recognition protein-2 ($GRP-2) from Manduca sexta: an acute-phase protein that binds $-1,3-glucan and lipoteichoic acid to aggregate fungi and bacteria and stimulate prophenoloxidase activation. Insect Biochem. Mol. Biol.


Progress 10/01/01 to 09/30/02

Outputs
We recently purified and cloned a prophenoloxidase-activating proteinase (PAP-2) from hemolymph of the tobacco hornworm, Manduca sexta. As the terminal component of a putative serine proteinase cascade, this enzyme activates prophenoloxidase (proPO) via limited proteolysis. In order to purify and study the activating proteinase for PAP-2 from this insect, we expressed the zymogen of PAP-2 (proPAP-2) in insect cells infected by a recombinant baculovirus that harbors the cDNA. To facilitate the purification of proPAP-2, we modified a commercial vector (pFastBac1) by inserting a synthetic DNA fragment encoding a hexahistidine sequence, allowing fusion of the affinity tag to the carboxyl terminus of a protein. After Spodoptera frugiperda Sf21 cells were infected by the virus, recombinant proPAP-2 was efficiently secreted into the media at a concentration of 14.8 mg/l under the optimal conditions. After ammonium sulfate precipitation, the proenzyme was purified to near homogeneity by affinity chromatography on nickel-NTA agarose. Western blot analysis indicated that the recombinant proPAP-2 has a mobility slightly lower than that of the zymogen from M. sexta hemolymph. The molecular mass and isoelectric point of proPAP-2 were determined to be 47,573 Da and 6.6, respectively. After the purified proenzyme was added to hemolymph from induced M. sexta larvae, it was rapidly activated by an unknown proteinase in the presence of peptidoglycan.

Impacts
(N/A)

Publications

  • No publications reported this period


Progress 10/01/00 to 09/30/01

Outputs
We produced the zymogen of Manduca sexta prophenoloxidase activating proteinase-1 (PAP-1 precursor) in a bacterial expression system. Using a specific rabbit antiserum against the recombinant protein, we detected a 6- to 8-fold induction of proPAP-1 in hemolymph after bacterial infection. We expressed and characterized native proPAP-1 from baculovirus-infected insect cells. The purified proenzyme, after added to larval hemolymph, was readily activated in the presence of Micrococcus lysodeikticus. Therefore, we are now at the stage to use proPAP-1 as a substrate to modify the purification of its activating enzyme. We have isolated from Manduca hemolymph a new serine proteinase that activates prophenoloxidase (proPO). This enzyme (PAP-2), different from PAP-1, is composed of two clip domains at its amino terminus and a catalytic domain at its carboxyl terminus. Purified PAP-2 cleaved proPO at Arg51 and yield a product which does not have much PO activity. However, in the presence of a mixture of two serine proteinase homologs, active PO was generated at a much higher level and it formed high molecular wight polymers that are covalently linked. Since these proteinase-like molecules also strongly associate with a bacteria-binding lectin in Manduca hemolymph, they may be important for ensuring that the activation of proPO only occurs in the vicinity of invading microorganisms. PAP-2 mRNA was not detected in larval fat body or hemocytes, but became very abundant after the insects were injected with bacterial cells. Since both proPAP-1 and proPAP-2 are present in hemolymph of bacteria-challenged larvae, the proPO-activating system appears to be more complex than previously thought.

Impacts
(N/A)

Publications

  • No publications reported this period


Progress 10/01/99 to 09/30/00

Outputs
This project was new October 2000 and there is no progress to report for this period.

Impacts
(N/A)

Publications

  • No publications reported this period