A Novel Spore Wall Protein from Antonospora locustae (Microsporidia: Nosematidae) Contributes to Sporulation

Abstract

Microsporidia are obligate intracellular parasites, existing in a wide variety of animal hosts. Here, we reported AlocSWP2, a novel protein identified from the spore wall of Antonospora locustae (formerly, Nosema locustae, and synonym, Paranosema locustae), containing four cysteines that are conserved among the homologues of several Microspodian pathogens in insects and mammals. AlocSWP2 was detected in the wall of mature spores via indirect immunofluorescence assay. In addition, immunocytochemistry localization experiments showed that the protein was observed in the wall of sporoblasts, sporonts, and meronts during sporulation within the host body, also in the wall of mature spores. AlocSWP2 was not detected in the fat body of infected locust until the 9th day after inoculating spores via RT-PCR experiments. Furthermore, the survival percentage of infected locusts injected with dsRNA of AlocSWP2 on the 15th, 16th, and 17th days after inoculation with microsporidian were significantly higher than those of infected locusts without dsRNA treatment. Conversely, the amount of spores in locusts infected with A. locustae after treated with RNAi AlocSWP2 was significantly lower than those of infected locusts without RNAi of this gene. This novel spore wall protein from A. locustae may be involved in sporulation, thus contributing to host mortality.

Keywords: Locust; RNAi; SWP.

Figure 1

A novel protein, Aloc SWP2, isolated from Antonospora locustae spores. (A) Proteins profile extracted from A. locustae spores in 2 dimensions electrophoresis SDS PAGE. The target protein highlighted (dashed circle). M. protein molecular weight markers, pI, isoelectric point. (B) Amino acid sequence of Aloc SWP2, showing the signal peptide (italicized), and the predicted HBM (box), the asparagine predicted to be N‐glycosylated (underlined) and the potential GPI‐modification (grey shade). (C) Sequence alignment of the amino sequence of Aloc SWP2 with other 14 amino acid sequences of putative spore wall proteins of other species in Microsporidia. Identical and similar residues are highlighted in black and grey, respectively. The conservative cysteine sequences of these Microsporidia marked with an asterisk. Microsporidia and UniProt database ID: A. locustae (Q6E6F1); Encephalitozoon cuniculi (Q8SWG8); Encephalitozoon hellem (I6UB54); Encephalitozoon romaleae (I6ZS97); Enterocytozoon bieneusi (B7XHL8); Encephalitozoon intestinalis (E0S5M3); Edhazardia aedis (J9DKY7); Nosema ceranae (C4VBU8); Nosema bombycis (R0KVG4); Nosema apis (T0KWN0); Vittaforma corneae (L2GPT0); Vavraia culicis (L2GWD6); Trachipleistophora hominis (L7JV52); Spraguea lophii (S7W7E5). (D) Molecular phylogenetic analysis of spore wall protein sequences from Microsporidia. The evolutionary history was inferred via the Maximum Likelihood method based on the JTT matrix‐based model and conducted in MEGA6 (Tamura et al. 2013). The bootstrap consensus tree inferred from 1,000 replicates is taken to represent the evolutionary history of the taxa analyzed.

Figure 2

SDS‐PAGE and Western blot analysis of Aloc SWP2. (A) SDS‐PAGE of purified recombinant Aloc SWP2. (B) SDS‐PAGE of the SDS extract of spore proteins from Antonospora locustae. (C) Western blot of purified recombinant Aloc SWP2, which contains a GST label, using an anti‐Aloc SWP2 antibody. (D) Western blot of extract of endogenous spore proteins using an anti‐Aloc SWP2 antibody. M, protein molecular weight marker.

Figure 3

Localization of Aloc SWP2 by immunofluorescence assay. Light, purified Antonospora locustae were observed via light microscope only. (A) Blue fluorescence signals show samples stained with DAPI, which were observed with a fluorescence microscope; green fluorescence signals show the samples treated with anti‐Aloc SWP2 antibody and FITC‐conjugated goat anti‐rabbit IgG. (B) Negative control. The anti‐Aloc SWP2 antibody was diluted at 10 μg/ml in blocking solution. The secondary antibody was FITC‐conjugated goat anti‐rabbit IgG was diluted 1:64. Bar, 10 μm.

Figure 4

Immunocytochemistry localization of Aloc SWP2 in fat bodies of locusts. (A–D) Negative control, A. locustae‐infected locust fat cells treated with anti‐GST antibody. (E–K) Sections of A. locustae‐infected locust fat cells incubated with anti‐GST‐ Aloc SWP2 antibody. Ex = exospore; En = endospore; Pt = polar tube; Nu = nucleus; Sp = sporont; Sb = sporoblast; M = meront; S = mature spores; F = locust fat cell. Bars in D, H, I, 0.5 μm; Bars in A, C, F, G, J, 1 μm; Bar in B, 2 μm; Bar in E, 5 μm.

Figure 5

Reduction of the mortality of locusts infected by Antonospora locustae after depression of Aloc SWP2 via RNAi. (A) AlocSWP2 gene expression was detected via RT‐PCR in the fat body of locusts after inoculation. LmigActin is a locust actin gene. Lane: 1, 3, …19 locusts on the days after A. locustae inoculation, N is the negative control. (B) qRT‐PCR experimental result of micro‐injection with dsRNA of AlocSWP2 to interfere with the expression of the gene. Reference gene is A. locustae actin. Bar height denotes the mean average of sample‐specific −ΔCt values, and values are plotted as means ± SEM from at least three repeats. Significant difference at the 0.0016 level. (C) The survivals of locusts inoculated with A. locustae spores or non‐inoculated, and those after treatment with RNAi of AlocSWP2. The 3rd instar nymphs of the locust were infected by A. locustae, then treated with or without RNAi of AlocSWP2. The number (n) of in each group treatment group is noted and three independent repeats of each experiment were done. The arrow indicates the time (days) postinoculation when injection was performed. The Kaplan–Meier method in Graphpad Prism 6 was used to analyze survival data. The t‐test was used for significance analysis, the P value of survival proportions of Infected + Aloc SWP2 RNAi group against Infected + GFP control RNAi group by paired one tail t‐test was 0.0278, which was significantly different. The P value of survival proportions of Infected + Aloc SWP2 RNAi group against Infected control group by paired one tail t‐test was 0.0209, which was significantly different. Bars and dotted line height denote the mean average of SEM. (D) Comparisons of A. locustae spores per locust between the treatments (average spore load). *P < 0.05, **P < 0.01.