INTERACTOME ANALYSIS OF PROTEIN KINASES, GERMINATION-RELATED AND HORIZONTALLY TRANSFERRED GENES OF NOSEMA BOMBYCIS USING STRING

Satish L*, Kusuma L, Manthira Moorthy S and Sivaprasad V. Silkworm Breeding and Molecular Biology Laboratory, Central Sericultural Research & Training Institute, Srirampura, Mysore-570 008, Karnataka ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Nosema bombycis infects Bombyx mori upon spore germination utilizing a characteristic mechanism to invade host cell. The uptake of microsporidian spore is either by spore-host interaction or by endocytosis. The molecular mechanism leading to such interactions is not well elucidated. The expansions of N. bombycis genome have acquired many horizontal genes and interact with protein kinases, involved in defense mechanism and cell cycle events. Present study aimed towards understanding these interactions as spore germination being vital process in pebrine infects silkworms through spore endocytosis. Using STRING -the molecular functions of all these proteins and its functional partners in the interactome were analyzed and annotated. Further, the protein-protein interactions network was analyzed to study the functional interaction partners that could decipher the mechanism triggering uptake of spore through endocytosis. A total of 50 proteins including protein kinases, horizontal transfer and germination related genes were studied emphasizing CDC28 activation of VPS34 which inturn mediated the activation of CDC10 leading to spore wall formation. In conclusion, results highlighted the most possible mechanism triggering endocytosis of microsporidia and functional contribution of protein kinases and genes involved in horizontal gene transfer to the N. bombycis germination and survival strategy.

…………………………………………………………………………………………………….... Introduction:-
Microsporidia have attracted much attention as they infect a variety of species ranging from protists to mammals 1 . Almost half of the reported genera of microsporidia use insects as primary hosts, and microsporidian infections usually have chronic and sublethal effects on their hosts. Nosema bombycis, the first named microsporidia species, is the causative agent of devastating pebrine disease in silkworm, Bombyx mori. N. bombycis infects silkworms both vertically (from mother to progenitor eggs) and horizontally (transovarially), damaging gut, malphigian tubules, silk glands and fat bodies causing larval inactivation and retarding the larval development and finally leading to silkworm death 2 . With the appropriate external stimuli trigger or direct contact with host cell, N. bombycis spore rapidly extrudes polar tube from the anterior end in a process called germination. Germination is the most important ISSN: 2320-5407 Int. J. Adv. Res. 5(2), 497-506 498 and foremost step in the infection of silkworm by N. bombycis 3 . The invasion into the host cell is by the mechanism of polar tube extrusion 4 .
Comparative genomics of N. bombycis shows that the genome is astonishingly expanded as compared to the distantly related N. ceranae and this large genome size is due to the proliferation of host-derived transposable elements, horizontally transferred genes (HGT) from prokaryotes, and the production of segmental and tandem duplicates 5 . Based on the review of literature, 55 genes were identified to be involved in horizontal gene transfer, among these, 21 HGT genes had unknown function and 34 had predicted gene functions.
Further, protein kinases that form a large group of enzymes which transfer phosphate group from ATP to a number of proteins are involved in the mechanism of signal transduction leading to the N. bombycis infection 6 . These protein kinases are generally known to be involved in cell cycle events, cell proliferation, development, metabolism, signal transduction and stimulus to external signals. Genomic kinomes of N. bombycis revealed that there are 41 proteinkinases belonging to serine-threonine class 7 . Although there are studies that focused on actions of proteins derived from HGT and also germination related transcriptomics 8 , there are no clear studies that depict the involvement of protein kinases in association to germination. The computational studies of physical and functional interactions prediction generates more robust interactome as the interactions integrate to provide completely annotated information. In view of this, understanding the interactome helps in understanding the biological significance of spore invasion, thereby leading to infection. The present study analyzes the functional interactions of acquired horizontal proteins and N. bombycis genomic protein kinases with highly expressed proteins during germination.

Methodology:-
The present investigation involves analysis of interaction of N. bombycis protein kinases, germination related proteins, horizontal transfer genes and infection mechanism during germination using the STRING software. . Among the fifty proteins, thirteen show specific expression during germination of N. bombycis spore, eighteen proteins acquired through horizontal gene transfer and nineteen protein kinases of N. bombycis. STRING database 9 provides critical assessment and integration of protein-protein interactions, including direct (physical) as well as indirect (functional) associations. All the proteins were queried in the query multiple search box with the interactions restricted to those available for Encephalitozoon Cuniculi as E. cuniculi is the closest microsporidian to N. bombycis as represented by Frankenhuyzen et al., 10 and the sequence information is available. The prediction analysis methods including activation, inhibition, binding, co-expression and gene fusion were utilized. The confidence score >0.9 were selected to obtain interaction network representing >90% confidence in the prediction to establish an interactome network representing the N. bombycis infection in silkworm leading to pebrine disease.

Results and Discussion:-
A total of fifty proteins were analyzed to predict the protein interactions involved in germination of N. bombycis in silkworm (Table 1). Among these, forty proteins showed interactions atleast with one protein, whereas ten proteins did not interact. Fig. 1a represents overall protein-protein interaction network obtained in the present investigation. CDC28 was found to be the most interacting protein followed by Pho85 (ECU08_0230), which are protein kinases. CDC28 showed 19 interactions in the network forming the node 1 and 17 interactions were with other protein kinases and remaining two interactions with proteins expressed during germination. On the other hand, Pho85 showed 18 interactions, which is represented as node 2 ( Table 4) and 16 such protein interactions were other protein kinases and two interactions with proteins expressed during germination. These two major nodes indicate protein kinases form crucial back bone for the interaction of horizontal proteins and N. bombycis protein kinases and could be implicated in eliciting there by causing the infection upon germination of N. bombycis spores. The functional annotation of all the proteins queried for protein-protein interactions utilized in this study is listed in table 1.
Proteins in the proposed network are highly interactive among themselves than for a random set of proteins of similar size that would be expected from the drawn genome. These strong interactions suggest that these proteins are biologically connected to elicit a response upon a specific signal, as a group. However, there was no significant pathway enrichment observed in cellular component and KEGG pathways. N. bombycis HGT, germination and protein kinase gene set includes genes encoding basic cellular functions such as transcription, translation, DNA replication and repair, cell cycle control, protein folding/turnover, intracellular trafficking and key enzymes for glycolysis, pentose phosphate pathway, trehalose metabolism and chitin biosynthesis (Table 1).

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The mode of infection is either through polar tube intrusion or endocytosis and their protein interactors 3,4,11 . In the present study, the potential candidate interactor i. e., VPS34 (vacoular protein sorting 34) represented the major node of 18 protein interactors (Fig. 2) and is known to be involved in sporulation. VPS34 is known to initiate the formation of a forespore membrane at each spindle pole body and extends to form the spore envelope, which further requires binding of CDC10 through the Ptdlns(3) 12 . VPS34 converts phospho-inositol to phosphatidylinositol 3phosphate, the key factor for sporulation and phosphorylates phosphatidylinositol to generate Ptdlns(3)P (Fig. 3). Phosphatidylinositol 3-kinase is also vital for cytoplasm to vacuole transport (Cvt) and autophagy as a part of the autophagy-specific VPS34 PI3-kinase complex I. These proteins are involved in endosome-to-golgi retrograde transport as part of the VPS34 PI3-kinase complex II.
Microsporidia invades host cells in two different ways, the first way of invasion being ejection of spore polar tube and piercing into the host cell in its close proximity. The second mode, host endocytosis of the infective spore but the spore escapes the abjection by endocytic vacuole of the host by discharging its polar tube. However, spore endocytosis mechanism remains unclear and the potential molecular players involved are yet to be elucidated. CDC10 protein or septins are GTPases involved in cytokinesis and spore wall formation 2, 13 .
We hypothesize that major interaction of CDC28, binding and thereby activating CDC10 is crucial by which the microsporidia gets the signal for its invasive entry into the host (Fig. 1b). Endocytosis invasion is known to happen in Encephalitozoon species. The GO biological placement predicts that CDC10 is localised to the membrane of N. bombycis. Septin 7 of O. colligata is also known to be localised in its exospore 12 . Alternatively, a surface septin could also facilitate infection by simply helping to keep the parasite in close proximity to the host cell surface. Further, the present interactome also reveals the binding, catalysis and activation of CDC28 by VPS 34 mediates the activation of CDC10. The activation of VPS34 indeed might be by binding of sugars, anions or small molecules (Fig. 3).
N. bombycis intracellular parasitic lifestyle is designed to have highly reduced metabolism instead of energy investing pathways to synthesize basic biological building blocks (e.g. amino acids, sugars, nucleotides, lipids) and cofactors (e.g. ATP, NAD+, NADP+). The presence of one such enzyme like Mannose-1-phosphate guanyl transferase 2 is the key for carbohydrate metabolism and mannosylation of structural and functional proteins in microsporidians. In Mannosylation of polar tube (PTPs) and spore wall proteins plays very important role in the parasitic lifestyle of E. cuniculi, possibly as virulence factors reported for several fungal pathogens 14 . However, the present interactome reveals that PTP3 does not interact with Mannose-1-phosphate guanyl transferase 2, indicating its involvement in carbohydrate metabolism rather than mannosylation (Fig. 2). This is best evident ( Table 2) with the maximum observed gene count correlated with the functions like phosphorylation and cell cycle processes as far as the biological function was considered. Further, another enzyme, dUTPase is involved in conversion of dUTP to dUMP and pyrophosphate thereby adding on to the ATP stealing mechanism from the host cell. Like many parasites T. hominis has lost the ATP-expensive pathways for the de novo biosynthesis of inosine 59-phosphate and for uridine mono-phosphate, the starting points for the biosynthesis of purines and pyrimidines for DNA and RNA biosynthesis 15 . Probably, N. bombycis might have acquired dUTPase through HGT from bacterium in the predicted manner.
The absence of mitochondria in N. bombycis indicates that they require energy for survival in the host. However, it possess several energy synthesizing pathway enzymes which could compensate for the energy synthesis, which further is achieved by stealing ATP from the host cell. Our analysis also includes such energy synthesizing pathway enzymes viz., glucose-1-phosphate isomerase and transketolase based on transcriptome data of Ma et al., 1 . Glucose-1-phosphate isomerases thus inter converts glucose-6-phosphate to fructose-6-phosphate in glycolysis pathway. This Glucose-6-phosphate can also be utilized by pentose phosphate pathway to synthesize ribulose-6-phosphate and NADPH, which are the key energy molecules that contribute towards the survival of spore in the host cell.
Several studies show the presence of microsporidia hexokinases involvement in glycolytic pathway; but the absence of hexokinase is evident through transcriptome data 8 , furthered by distinctive absence of hexokinase activity in Nosema gryllii, in which activity of several glycolytic enzymes was detected in isolated pathogen cells 16 . Hexokinase catalyzes the first step in glycolysis and the pentose phosphate pathways. Therefore, microsporidia hexokinase activity within host cells could increase host synthesis of building blocks such as nucleotides, amino acids, and lipids, necessary for the rapid growth of parasites. Hexokinases are known to be present in microsporidians like T. hominis, V. culicis and V. corneae 14 . But the absence of hexokinase in our study is 500 compensated by the presence of Glucose-6-phosphate isomerase and transketolase. One of the interesting feature is N. bombycis has acquired phosphoglycerate mutase, which is another important enzyme during glycolysis and pentose phosphate pathways through horizontal gene transfer. In this analysis, there is strong triangular interaction between three enzymes (phosphoglycerate mutase, transketolase and glucose-6-phosphate isomerase) involved in energy synthesis. .
The genes acquired through HGT include enzymes involved in nucleotide synthesis (dUTPase, cytidylate kinase, uridine kinase, thymidine kinase). Further, the pathway analysis based on molecular functions revealed more number of genes participating in nucleotide binding, transferase and kinase activities (Table 3) thus implying its significance leading to elicit the infection upon endocytosis of spore by the host cell. CTP synthetase is the only nucleotide synthesis gene retained in the highly reduced genomes of the microsporidia 14 . The thioredoxin reductase acquired from (bacteria) interacts with thioltransferase, also acquired from bacteria suggesting that these defense proteins are involved in maintaining spore homeostasis and viability. These defense proteins might not interact with any germination expressed proteins. Fine example of role of defense proteins in microsporidia is presence of glutathione reductases and peroxidases, thioredoxin reductases and a superoxide dismutase in T. hominis 17 . Thymidine kinase, the pyrimidine salvage pathway enzyme interacts with phosphoglycerate mutase. Thymidine kinase acquistion as HGT is also found in the apicomplexan Cryptosporidium, from a bacterium 18 .
There is an indication that calcium/calmodulin binding at the spore surface may commence a signaling cascade that causes spore activation 19 . Further, it is of interest to note that E. cuniculi genome encodes five calmodulin-dependent kinases in its minimal set of 32 protein kinases which could potentially participate in such process 7 . The clear concept of activation of germination through signaling pathways however has not been elucidated but, indications of calcium/calmodulin binding at the spore surface may commence signaling cascade cause spore activation. We hypothesize that calmodulin dependent protein kinase (ECU03_0630) interacting with ECU03_1290, ECU08_1620, CDC28, ECU08_0230, MRK1, ECU02_0550, ECU01_1320 and CDC5 (Fig. 1c) could be the stimulant for the host cell to initiate endocytosis (Fig. 3), which further requires experimental validation. The present investigation predicts and emphasizes the possible molecular mechanism of spore uptake through endocytosis and also unravels the protein interactors involved in ATP stealing mechanism and defense mechanism of N. bombycis.