Spore Surface Proteins Research Articles

Identification of CgeA as a glycoprotein that anchors polysaccharides to the spore surface in Bacillus subtilis.

The Bacillus subtilis spore is composed of a core, containing chromosomal DNA, surrounded by a cortex layer made of peptidoglycan, and a coat composed of concentric proteinaceous layers. A polysaccharide layer is added to the spore surface, and likely anchored to the crust, the coat outermost layer. However, the identity of the coat protein(s) to which the spore polysaccharides (SPS) are attached is uncertain. First, we showed that the crust proteins CotVWXYZ and CgeA were all contained in the peeled SPS layer obtained from a strain missing CotE, the outer coat morphogenetic protein, suggesting that the SPS is indeed bound to at least one of the spore surface proteins. Second, CgeA is known to be located at the most downstream position in the crust assembly pathway. An analysis of truncated variants of CgeA suggested that its N-terminal half is required for localization to the spore surface, while its C-terminal half is necessary for SPS addition. Third, an amino acid substitution strategy revealed that SPS was anchored at threonine 112 (T112), which constitutes a probable O-glycosylation site on CgeA. Our results indicated that CgeA is a glycoprotein required to initiate SPS assembly and serves as an anchor protein linking the crust and SPS layers.

Proteomic Analysis of Spore Surface Proteins and Characteristics of a Novel Spore Wall Protein and Biomarker, EhSWP3, from the Shrimp Microsporidium Enterocytozoon hepatopenaei (EHP).

Enterocytozoon hepatopenaei, a spore-forming and obligate intracellular microsporidium, mainly infects shrimp and results in growth retardation and body length variation, causing huge economic losses to the Asian shrimp aquaculture industry. However, the lack of a full understanding of the surface proteins of spores associated with host infection has hindered the development of technologies for the detection of EHP. In this study, the surface proteins of EHP spores were extracted using the improved SDS method, and 130 proteins were identified via LC-MS/MS analysis. Bioinformatic analysis revealed that these proteins were enriched in biological processes (67), cellular components (62), and molecular functions (71) based on GO terms. KEGG pathway analysis showed that 20 pathways, including the proteasome (eight proteins) and the fatty acid metabolism (15 proteins), were enriched. Among 15 high-abundance surface proteins (HASPs), EhSWP3 was identified as a novel spore wall protein (SWP), and was localized on the endospore of the EHP spores with an indirect immunofluorescence and immunoelectron microscopy assay. Polyclonal antibodies against EhSWP3 showed strong species specificity and high sensitivity to the hepatopancreas of EHP-infected shrimp. As a specific high-abundance protein, EhSWP3 is therefore a promising target for the development of immunoassay tools for EHP detection, and may play a crucial role in the invasion of EHP into the host.

Visualization of fidaxomicin association with the exosporium layer of Clostridioides difficile spores

BackgroundFidaxomicin has novel pharmacologic effects on C. difficile spore formation including outgrowth inhibition and persistent spore attachment. However, the mechanism of fidaxomicin attachment on spores has not undergone rigorous microscopic studies. Materials & methodsFidaxomicin attachment to C. difficile spores of three distinct ribotypes and C. difficile mutant spores with inactivation of exosporium or spore-coat protein-coding genes were visualized using confocal microscopy with a fidaxomicin-bodipy compound (green fluorescence). The pharmacologic effect of the fidaxomicin-bodipy compound was determined. Confocal microscopy experiments included direct effect on C. difficile wild-type and mutant spores, effect of exosporium removal, and direct attachment to a comparator spore forming organism, Bacillus subtilis. ResultsThe fidaxomicin-bodipy compound MIC was 1 mg/L compared to 0.06 mg/L for unlabeled fidaxomicin, a 16-fold increase. Using confocal microscopy, the intracellular localization of fidaxomicin into vegetative C. difficile cells was observed consistent with its RNA polymerase mechanism of action and inhibited spore outgrowth. The fidaxomicin-bodipy compound was visualized outside of the core of C. difficile spores with no co-localization with the membrane staining dye FM4-64. Exosporium removal reduced fidaxomicin-bodipy association with C. difficile spores. Reduced fidaxomicin-bodipy was observed in C. difficile mutant spores for the spore surface proteins CdeC and CotE. ConclusionThis study visualized a direct attachment of fidaxomicin to C. difficile spores that was diminished with mutants of specific exosporium and spore coat proteins. These data provide advanced insight regarding the anti-spore properties of fidaxomicin.

Identification of Native Cross-Links in Bacillus subtilis Spore Coat Proteins

The resistance properties of the bacterial spores are partially due to spore surface proteins, ∼30% of which are said to form an insoluble protein fraction. Previous research has also identified a group of spore coat proteins affected by spore maturation, which exhibit an increased level of interprotein cross-linking. However, the proteins and the types of cross-links involved, previously proposed based on indirect evidence, have yet to be confirmed experimentally. To obtain more insight into the structural basis of the proteinaceous component of the spore coat, we attempted to identify coat cross-links and the proteins involved using new peptide fractionation and bioinformatic methods. Young (day 1) and matured (day 5) Bacillus subtilis spores of wild-type and transglutaminase mutant strains were digested with formic acid and trypsin, and cross-linked peptides were enriched using strong cation exchange chromatography. The enriched cross-linked peptide fractions were subjected to Fourier-transform ion cyclotron resonance tandem mass spectrometry, and the high-quality fragmentation data obtained were analyzed using two specialized software tools, pLink2 and XiSearch, to identify cross-links. This analysis identified specific disulfide bonds between coat proteins CotE–CotE and CotJA–CotJC, obtained evidence of disulfide bonds in the spore crust proteins CotX, CotY, and CotZ, and identified dityrosine and ε-(γ)-glutamyl-lysine cross-linked coat proteins. The findings in this Letter are the first direct biochemical data on protein cross-linking in the spore coat and the first direct evidence of the cross-linked building blocks of the highly ordered and resistant structure called the spore coat.

Nasal Immunization with the C-Terminal Domain of Bcla3 Induced Specific IgG Production and Attenuated Disease Symptoms in Mice Infected with Clostridioides difficile Spores.

Clostridioides difficile is a Gram-positive, spore-forming bacterium that causes a severe intestinal infection. Spores of this pathogen enter in the human body through the oral route, interact with intestinal epithelial cells and persist in the gut. Once germinated, the vegetative cells colonize the intestine and produce toxins that enhance an immune response that perpetuate the disease. Therefore, spores are major players of the infection and ideal targets for new therapies. In this context, spore surface proteins of C. difficile, are potential antigens for the development of vaccines targeting C. difficile spores. Here, we report that the C-terminal domain of the spore surface protein BclA3, BclA3CTD, was identified as an antigenic epitope, over-produced in Escherichia coli and tested as an immunogen in mice. To increase antigen stability and efficiency, BclA3CTD was also exposed on the surface of B. subtilis spores, a mucosal vaccine delivery system. In the experimental conditions used in this study, free BclA3CTD induced antibody production in mice and attenuated some C. difficile infection symptoms after a challenge with the pathogen, while the spore-displayed antigen resulted less effective. Although dose regimen and immunization routes need to be optimized, our results suggest BclA3CTD as a potentially effective antigen to develop a new vaccination strategy targeting C. difficile spores.

Vegetative Cell and Spore Proteomes of Clostridioides difficile Show Finite Differences and Reveal Potential Protein Markers.

Clostridioides difficile-associated infection (CDI) is a health-care-associated infection caused, as the name suggests, by obligate anaerobic pathogen C. difficile and thus mainly transmitted via highly resistant endospores from one person to the other. In vivo, the spores need to germinate into cells prior to establishing an infection. Bile acids and glycine, both available in sufficient amounts inside the human host intestinal tract, serve as efficient germinants for the spores. It is therefore, for better understanding of C. difficile virulence, crucial to study both the cell and spore states with respect to their genetic, metabolic, and proteomic composition. In the present study, mass spectrometric relative protein quantification, based on the 14N/15N peptide isotopic ratios, has led to quantification of over 700 proteins from combined spore and cell samples. The analysis has revealed that the proteome turnover between a vegetative cell and a spore for this organism is moderate. Additionally, specific cell and spore surface proteins, vegetative cell proteins CD1228, CD3301 and spore proteins CD2487, CD2434, and CD0684 are identified as potential protein markers for C. difficile infection.

Identification of the proteoforms of surface localized Rod A of Aspergillus flavus and determination of the mechanism of proteoform generation

Fungal keratitis is a serious, potentially sight-threatening corneal infection that is more prevalent in the tropical parts of the world including India, and A. flavus and Fusarium solani are the predominant etiological agents. The surface of fungal conidia is covered by hydrophobin family proteins, effectively masking the conidial antigens from immune cells. In this study, we report that the outer cell wall layer of A. flavus conidia contain Rod A as well as other hydrophobins, which could be extracted by formic acid. Analysis of these surface proteins by mass spectrometry showed the presence of rodlet forming hydrophobins and other membrane and antigenic proteins. Our analysis revealed that Rod A existed as two proteoforms on the conidial surface. These proteoforms were separated using polyacrylamide gel electrophoresis and the amino acid sequence of these proteoforms was determined by high resolution mass spectrometry. PCR analysis of the mRNA encoding the Rod A showed the retention of intron one, which results in the formation of a truncated proteoform two. This is the first report in which the presence of RodA and its proteoforms and their mechanism of formation has been demonstrated in the corneal pathogenic fungus A. flavus. SignificanceA. flavus is a common fungal pathogen in tropical countries playing a predominant role in causing mycotic keratitis in humans. Surface of fungal conidia is immunologically inert primarily due to the hydrophobin family proteins forming a rodlet layer and masking the conidia from immune cells. In this study we demonstrated the existence two proteoforms of RodA/hydrophobin A and intron retention is shown to be responsible for the formation of one of the proteoforms. In addition, the spore surface proteins of A.flavus corneal isolates and saprophyte are distinctly different, which indicate the spore surface protein profile is ecotype specific. This is the first report showing the presence of two proteoforms of RodA on A.flavus conidial surface and demonstration of the mechanism of formation of the proteoforms.

Survey of Brevibacillus laterosporus insecticidal protein genes and virulence factors.

The pathogenic action of the bacterium Brevibacillus laterosporus against invertebrates involves a toxin-mediated mechanism. Several studies, conducted with specific strains against diverse targets, suggested the implication of different toxins. Recent genome sequencing and annotation of some insecticidal strains revealed several putative virulence factors highly conserved in this species. After determining the pathogenicity of strain UNISS 18 against different Lepidopteran and Dipteran larvae, in this study we have investigated the actual expression of genes encoding for enzymes (i.e., chitinases, proteases), toxins, and other virulence factors, either in vitro and in vivo at the transcriptional level. Selected genes encode for two chitinases, a collagenase-like protease, a GlcNAc-binding protein, two protective antigen proteins, a bacillolysin, a thermophilic serine proteinase, two spore surface proteins, an insecticidal toxin homologous to Cry75Aa. All target genes were well expressed in pure bacterial cultures with significant differences between bacterial growth phases. Their expression level was generally enhanced in the bacterial population developing in the insect body cavity, compared with pure culture. The expression of certain genes increased substantially over time after insect inoculation. These results support a complex mechanism of action leveraging a variety of available virulence factors, and can also explain the ability of this bacterial species to act against diverse invertebrate targets.

Spore surface proteins of Brevibacillus laterosporus are involved in insect pathogenesis

Outer spore envelope proteins of pathogenic bacteria often present specific virulence factors and tools to evade the defence system of their hosts. Brevibacillus laterosporus, a pathogen of invertebrates and an antimicrobial-producing species, is characterised by a unique spore coat and canoe-shaped parasporal body (SC-CSPB) complex surrounding the core spore. In the present study, we identified and characterised major proteins of the SC-CSPB complex of B. laterosporus, and we investigated their entomopathogenic role. Employing a proteomic approach and a B. laterosporus-house fly study model, we found four highly conserved proteins (ExsC, CHRD, CpbA and CpbB) that function as insect virulence factors. CpbA was associated with a significantly higher mortality of flies and greater relative gene expression levels during sporulation, compared to the other SC-CSPB proteins. Taken together, we suggest that spore surface proteins are a part of a complex set of toxins and virulence factors that B. laterosporus employs in its pathogenicity against flies.

Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains

Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75–100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores.

Pre‐infection processes of Botryosphaeriaceae spp.: adhesion of conidia to different substrata

Species of Botryosphaeriaceae are important wound pathogens of grapevines as causal agents of botryosphaeria dieback, but the behaviour of their conidia pre‐infection is unknown and may be important for disease development. Adhesion properties of conidia were investigated for Botryosphaeria dothidea, Neofusicoccum luteum and N. parvum on substrata with different affinities for water. Greatest adhesion on any surface was reached after 5 min for isolates N. luteum MM558, B. dothidea 007 and N. parvum G652 (53·1, 54·0 and 50·6%, respectively) and for N. luteum isolate CC445 after 20 min (61·4%). As conidia adhered well to all artificial substrata, it appeared as if the attachment process was nonspecific. Overall, surface wettability did not play a major role in the adhesion of conidia. Spore surface proteins appeared to play a role in the adhesion process because treatment of conidia of N. luteum MM558 with a protease completely prevented adhesion. Histochemical labelling of conidia and germlings with Coomassie brilliant blue (specific for proteins) was positive for all isolates, with a blue ‘halo’ often seen surrounding conidia or near the germ tube emergence point after incubation times conducive to germination. Alcian blue also stained material surrounding conidia after longer incubation times, which indicated that mucopolysaccharide and protein production may be involved in a second phase of adhesion.

Reinforcement of Bacillus subtilis spores by cross-linking of outer coat proteins during maturation

Resistance characteristics of bacterial endospores towards various environmental stresses such as chemicals and heat are in part attributed to their coat proteins. Heat resistance is developed in a late stage of sporulation and during maturation of released spores. Using our gel-free proteomic approach and LC-FT-ICR-MS/MS analysis we have monitored the efficiency of the tryptic digestion of proteins in the coat during spore maturation over a period of eight days, using metabolically 15N labeled mature spores as reference. The results showed that during spore maturation the loss of digestion efficiency of outer coat and crust proteins synchronized with the increase in heat resistance. This implicates that spore maturation involves chemical cross-linking of outer coat and crust layer proteins leaving the inner coat layer proteins unmodified. It appears that digestion efficiencies of spore surface proteins can be linked to their location within the coat and crust layers. We also attempted to study a possible link between spore maturation and the observed heterogeneity in spore germination.

Metarhizium anisopliae host–pathogen interaction: differential immunoproteomics reveals proteins involved in the infection process of arthropods

Metarhizium anisopliae is an entomopathogenic fungus well characterized for the biocontrol of a wide range of plagues. Its pathogenicity depends on the secretion of hydrolytic enzymes that degrade the host cuticle. To identify proteins involved in the infection process and in host specify, immunoproteomic analysis was performed using antiserum produced against crude extract of M. anisopliae cultured in the presence of Rhipicephalus (Boophilus) microplus and Dysdercus peruvianus cuticles. Spots detected using antisera produced against M. anisopliae cultured in cuticles and spore surface proteins, but not with antiserum against M. anisopliae cultured in glucose, were identified so as to give insights about the infection process. An MS/MS allowed the identification of proteases, like elastase, trypsin, chymotrypsin, carboxypeptidase and subtilisin (Pr1A, Pr1I and PR1J), chitinases, DNase I and proline-rich protein. Chymotrypsin and Pr1I were inferred as host specific, being recognized in D. peruvianus infection only. This research represents an important contribution to the understanding the adaptation mechanisms of M. anisopliae to different hosts.

Expression and Localization of an Hsp70 Protein in the MicrosporidianEncephalitozoon cuniculi

Microsporidia spore surface proteins are an important, under investigated aspect of spore/host cell attachment and infection. For comparison analysis of surface proteins, we required an antibody control specific for an intracellular protein. An endoplasmic reticulum-associated heat shock protein 70 family member (Hsp70; ECU02_0100; “C1”) was chosen for further analysis. DNA encoding the C1 hsp70 was amplified, cloned and used to heterologously express the C1 Hsp70 protein, and specific antiserum was generated. Two-dimensional Western blotting analysis showed that the purified antibodies were monospecific. Immunoelectron microscopy of developing and mature E. cuniculi spores revealed that the protein localized to internal structures and not to the spore surface. In spore adherence inhibition assays, the anti-C1 antibodies did not inhibit spore adherence to host cell surfaces, whereas antibodies to a known surface adhesin (EnP1) did so. In future studies, the antibodies to the ‘C1' Hsp70 will be used to delineate spore surface protein expression.

Conidial surface proteins of Metarhizium anisopliae: Source of activities related with toxic effects, host penetration and pathogenesis

Conidial contact with an arthropod surface is the first step of the fungal penetration and infection process. However, conidia of Metarhizium anisopliae have associated components, like enzymes that could be involved in triggering the penetration process and toxic effects that have not yet been well characterized. Fungi produce many enzymes that also are toxic components found in bacteria and animal venoms and thus may be considered as potential virulence factors. In this work, we report several enzymatic activities from spore surface protein extracts. The major proteolytic activities observed in spore surface proteins (SSP) were Pr1 and Pr2 activities, in that order. According to the zymograms obtained, SSP contain different proteases. SSP contain trehalase, exo- and endo-chitinase activities, and seven different chitinase bands which have been observed in zymograms. Activities involved in protection against reactive oxygen species (ROS) were also detected. Two lipolytic enzymes were also detected in lipase zymograms. Phospholipase C activity, closely related to microbial pathogenesis, was detected for the first time in M. anisopliae conidia. These activities described could be an initial step towards understanding the mechanisms involved in the first stage of M. anisopliae infection process and its toxic effects against arthropod hosts.

Analysis of Bacillus globigii spores by CE

It is imperative in today's world that harmful airborne or solution-based microbes can be detected quickly and efficiently. Bacillus globigii (Bg) spores are used as a simulant for Bacillus anthracis (Ba) due to their similar shape, size, and cellular makeup. The utility of CE to separate and detect low levels of Bg spore concentrations will be evaluated. To differentiate spores from background particulates, several dyes, including fluorescamine, C-10, NN-127, Red-1c, and indocyanine green (ICG), were utilized as noncovalent labels for proteins on the Bg spore surface, as well as for HSA and homoserine standards. On-column labeling, with dye present in the running buffer, was utilized to obtain greater sensitivity and better separation. CE with LIF detection enables interactions between the dye and spore surface proteins to be observed, with enhanced fluorescence occurring upon binding of the dye to surface protein. Resulting electropherograms showed unique fingerprints for each dye with Bg spores. Migration times were under 10 min for all dye-spore complexes, with net mobilities ranging from 3.5x10(-4) to 6.9x10(-4) cm(2) V(-1) s(-1), and calibration curves yielded correlation coefficients of 0.98 or better for four of the dyes studied.

Identification of a Protein Subset of the Anthrax Spore Immunome in Humans Immunized with the Anthrax Vaccine Adsorbed Preparation

We identified spore targets of Anthrax Vaccine Adsorbed (AVA)-induced immunity in humans by screening recombinant clones of a previously generated, limited genomic Bacillus anthracis Sterne (pXO1(+), pXO2(-)) expression library of putative spore surface (spore-associated [SA]) proteins with pooled sera from human adults immunized with AVA (immune sera), the anthrax vaccine currently approved for use by humans in the United States. We identified 69 clones that reacted specifically with pooled immune sera but not with pooled sera obtained from the same individuals prior to immunization. Positive clones expressed proteins previously identified as localized on the anthrax spore surface, proteins highly expressed during spore germination, orthologs of proteins of diverse pathogens under investigation as drug targets, and orthologs of proteins contributing to the virulence of both gram-positive and gram-negative pathogens. Among the reactive clones identified by this immunological screen was one expressing a 15.2-kDa hypothetical protein encoded by a gene with no significant homology to sequences contained in databases. Further studies are required to define the subset of SA proteins identified in this study that contribute to the virulence of this pathogen. We hypothesize that optimal delivery of a subset of SA proteins identified by such studies to the immune system in combination with protective antigen (PA), the principal immunogen in AVA, might facilitate the development of defined, nonreactogenic, more-efficacious PA-based anthrax vaccines. Future studies might also facilitate the identification of SA proteins with potential to serve as targets for drug design, spore inactivation, or spore detection strategies.

An enzyme-linked immunosorbent assay to detect alkali-soluble spore surface antigens of strains of Nosema bombycis (Microspora: Nosematidae)

Spore surface antigens of strains of Nosema bombycis were extracted with alkaline solutions and used in an indirect enzyme-linked immunosorbent assay. Treatment of N. bombycis spores with 0.1 n potassium carbonate or potassium hydroxide solution at 27°C for 30 min was sufficient for the extraction of the antigens. Usually, 10 8 spores of N. bombycis liberated ca. 30 μg spore surface proteins. The indirect enzyme-linked immunosorbent assay detected as little as 60 ng of spore surface proteins (ca. 2000 spore-equivalent antigen). The alkali-soluble spore surface antigens of N. bombycis contained a specific antigen and were stable under storage at −20°C for more than 1 year. The serological assay separated the Nosema isolates pathogenic to the silkworm into three groups.