To facilitate more accurate spore proteomic analysis, the current study focuses on inducing homogeneous sporulation by overexpressing kinA and assesses the effect of synchronized sporulation initiation on spore resistance, structures, the germination behavior at single-spore level and the proteome. The results indicate that, in our set up, the sporulation by overexpressing kinA can generate a spore yield of 70% within 8 h. The procedure increases spore wet heat resistance and thickness of the spore coat and cortex layers, whilst delaying the time to spore phase-darkening and burst after addition of germinant. The proteome analysis reveals that the upregulated proteins in the kinA induced spores, compared to spores without kinA induction, as well as the ‘wildtype’ spores, are mostly involved in spore formation. The downregulated proteins mostly belong to the categories of coping with stress, carbon and nitrogen metabolism, as well as the regulation of sporulation. Thus, while kinA overexpression enhances synchronicity in sporulation initiation, it also has profound effects on the central equilibrium of spore formation and spore germination, through modulation of the spore molecular composition and stress resistance physiology.

BackgroundErythrocyte invasion by merozoites is an essential step in Plasmodium falciparum infection and leads to subsequent disease pathology. Proteins both on the merozoite surface and secreted from the apical organelles (micronemes, rhoptries and dense granules) mediate the invasion of erythrocytes; some of the molecules have been regarded as targets in the development of an anti-malaria vaccine. Recently, a subgroup of rhoptry neck proteins (PfRON2, PfRON4 and PfRON5) associated with the microneme protein apical membrane antigen AMA1 has been described as components of the moving junction complex that assists merozoite invasion into erythrocytes. However, unlike PfRON2, PfRON4 and PfRON5, the latest study suggested that PfRON3 might be located in the rhoptry bulb and participates in a novel PfRON complex (PfRON2, 3 and 4), but does not form a complex with AMA1. Additionally, the full-length PfRON3 protein possesses three transmembrane regions at the N-terminus, which is highly conserved among RON3 orthologues in the genus Plasmodium, Toxoplasma gondii and Eimeria tenella. Overall, these findings suggest that PfRON3 may play an important role in merozoite invasion into erythrocytes.ResultsPfRON3 was primarily expressed during the late trophozoite stage, with a peak in transcription levels at 40 hours post-invasion. The subcellular localization of PfRON3 was confirmed that it is a merozoite rhoptry bulb protein. Additionally, the recombinant form of PfRON3 protein bound to the erythrocyte and was recognized by sera collected from malaria endemic areas in Africa, and anti-PfRON3 antibodies significantly inhibited merozoite invasion into erythrocytes.MethodsThe expression of PfRON3 was analysed via real-time quantitative PCR, and the recombinant PfRON3 proteins were generated with an Escherichia coli expression system. The subcellular localization of PfRON3 was assessed with immunoelectron microscopy and immunofluorescence assay (IFA). The recognition PfRON3 by malaria immune sera was analysed with an enzyme-linked immunosorbent assay (ELISA). Erythrocyte-binding assays were performed using recombinant PfRON3 proteins and invasion inhibition assays were carried out with PfRON3-specific antibodies.ConclusionThis study confirmed that PfRON3 is a rhoptry protein with an erythrocyte-binding property, which is likely associated red blood cell invasion. PfRON3 is a potential vaccine candidate.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-13-490) contains supplementary material, which is available to authorized users.

Toxoplasma gondii is an obligatory intracellular apicomplexan parasite which exploits host cell surface components in cell invasion and intracellular parasitization. Sulfated glycans such as heparin and heparan sulfate have been reported to inhibit cell invasion by T. gondii and other apicomplexan parasites such as Plasmodium falciparum. The aim of this study was to investigate the heparin-binding proteome of T. gondii. The parasite-derived components were affinity-purified on the heparin moiety followed by MS fingerprinting of the proteins. The heparin-binding proteins of T. gondii and P. falciparum were compared based on functionality and affinity to heparin. Among the proteins identified, the invasion-related parasite ligands derived from tachyzoite/merozoite surface and the secretory organelles were prominent. However, the profiles of the proteins were different in terms of affinity to heparin. In T. gondii, the proteins with highest affinity to heparin were the intracellular components with functions of parasite development contrasted to that of P. falciparum, of which the rhoptry-derived proteins were prominently identified. The profiling of the heparin-binding proteins of the two apicomplexan parasites not only explained the mechanism of heparin-mediated host cell invasion inhibition, but also, to a certain extent, revealed that the action of heparin on the parasite extended after endocytosis.

Bacterial endospores (spores) are among the most resistant living forms on earth. Spores of Bacillus subtilis A163 show extremely high resistance to wet heat compared to spores of laboratory strains. In this study, we found that spores of B. subtilis A163 were indeed very wet heat resistant and released dipicolinic acid (DPA) very slowly during heat treatment. We also determined the proteome of vegetative cells and spores of B. subtilis A163 and the differences in these proteomes from those of the laboratory strain PY79, spores of which are much less heat resistant. This proteomic characterization identified 2011 proteins in spores and 1901 proteins in vegetative cells of B. subtilis A163. Surprisingly, spore morphogenic protein SpoVM had no homologs in B. subtilis A163. Comparing protein expression between these two strains uncovered 108 proteins that were differentially present in spores and 93 proteins differentially present in cells. In addition, five of the seven proteins on an operon in strain A163, which is thought to be primarily responsible for this strain’s spores high heat resistance, were also identified. These findings reveal proteomic differences of the two strains exhibiting different resistance to heat and form a basis for further mechanistic analysis of the high heat resistance of B. subtilis A163 spores.

Bacillus subtilis vegetative cells switch to sporulation upon nutrient limitation. To investigate the proteome dynamics during sporulation, high-resolution time-lapse proteomics was performed in a cell population that was induced to sporulate synchronously. Here, we are the first to comprehensively investigate the changeover of sporulation regulatory proteins, coat proteins, and other proteins involved in sporulation and spore biogenesis. Protein co-expression analysis revealed four co-expressed modules (termed blue, brown, green, and yellow). Modules brown and green are upregulated during sporulation and contain proteins associated with sporulation. Module blue is negatively correlated with modules brown and green, containing ribosomal and metabolic proteins. Finally, module yellow shows co-expression with the three other modules. Notably, several proteins not belonging to any of the known transcription regulons were identified as co-expressed with modules brown and green, and might also play roles during sporulation. Finally, levels of some coat proteins, for example morphogenetic coat proteins, decreased late in sporulation.