Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells

Anaplasma marginale is a tick-borne ehrlichial pathogen of cattle for which six major surface proteins (MSPs) have been described. The MSP1 complex, a heterodimer composed of MSP1a and MSP1b, was shown to induce a protective immune response in cattle and both proteins have been identified as putative adhesins for bovine erythrocytes. In this study the role of MSP1a and MSP1b as adhesins for bovine erythrocytes and tick cells was defined. msp1alpha and msp1beta1 genes from the Oklahoma isolate of A. marginale were cloned and expressed in Escherichia coli K-12 under the control of endogenous and tac promoters for both low and high level protein expression. Expression of the recombinant polypeptides was confirmed and localised on the surface of transformed E. coli. The adhesion properties of MSP1a and MSP1b were determined by allowing recombinant E. coli expressing these surface polypetides to react with bovine erythrocytes, Dermacentor variabilis gut cells and cultured tick cells derived from embryonic Ixodes scapularis. Adhesion of the recombinant E. coli to the three cell types was determined using recovery adhesion and microtiter haemagglutination assays, and by light and electron microscopy. MSP1a was shown by all methods tested to be an adhesin for bovine erythrocytes and both native and cultured tick cells. In contrast, recombinant E. coli expressing MSP1b adhered only to bovine erythrocytes and not to tick cells. When low expression vectors were used, single E. coli expressing MSP1a was seen adhered to individual tick cells while reaction of tick cells with the E. coli/MSP1a/high expression vector resulted in adhesion of multiple bacteria per cell. With electron microscopy, fusion of E. coli cell membranes expressing MSP1a or MSP1b with erythrocyte membranes was observed, as well as fusion of tick cell membranes with E. coli membranes expressing MSP1a. These studies demonstrated differential adhesion for MSP1a and MSP1b for which MSP1a is an A. marginale adhesin for both bovine erythrocytes and tick cells while MSP1b is an adhesin only for bovine erythrocytes. The role of the MSP1 complex, therefore, appears to vary among vertebrate and invertebrate hosts.     

Major surface protein 1a effects tick infection and transmission of Anaplasma marginale

Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.     

Sequence Variations in the Boophilus Microplus Bm86 Locus and Implications for Immunoprotection in Cattle Vaccinated with this Antigen

Cattle tick infestations constitute a major problem for the cattle industry in tropical and subtropical regions of the world. Traditional control methods have been only partially successful, hampered by the selection of chemical-resistant tick populations. The Boophilus microplus Bm86 protein was isolated from tick gut epithelial cells and shown to induce a protective response against tick infestations in vaccinated cattle. Vaccine preparations including the recombinant Bm86 are used to control cattle tick infestations in the field as an alternative measure to reduce the losses produced by this ectoparasite. The principle for the immunological control of tick infestations relies on a polyclonal antibody response against the target antigen and, therefore, should be difficult to select for tick-resistant populations. However, sequence variations in the Bm86 locus, among other factors, could affect the effectiveness of Bm86-containing vaccines. In the present study we have addressed this issue, employing data obtained with B. microplus strains from Australia, Mexico, Cuba, Argentina and Venezuela. The results showed a tendency in the inverse correlation between the efficacy of the vaccination with Bm86 and the sequence variations in the Bm86 locus (R² = 0.7). The mutation fixation index in the Bm86 locus was calculated and shown to be between 0.02 and 0.1 amino acids per year. Possible implications of these findings for the immunoprotection of cattle against tick infestations employing the Bm86 antigen are discussed.     

Adaptations of the Tick-Borne Pathogen, Anaplasma marginale, for Survival in Cattle and Ticks

The tick-borne cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) multiplies within membrane-bound inclusions in host cell cytoplasm. Many geographic isolates of A. marginale occur that vary in genotype, antigenic composition, morphology and infectivity for ticks. A tick cell culture system for propagation of A. marginale proved to be a good model for study of tick-pathogen interactions. Six major surface proteins (MSPs) identified on A. marginale from bovine erythrocytes were conserved on A. marginale derived from tick cells. MSP1a and MSP1b were adhesins for bovine erythrocytes, while only MSP1a was found to be an adhesin for tick cells. The tandemly repeated portion of MSP1a was found to be necessary and sufficient for adhesion to both tick cells and bovine erythrocytes. Infectivity of A. marginale isolates for ticks was dependent on the adhesive capacity of the isolate MSP1a, which was found to involve both the adhesive properties and sequence of the repeated peptides. Cattle immunized with A. marginale derived from bovine erythrocytes or tick cells demonstrated a differential antibody response to MSP1a and MSP1b that resulted from the differential expression of these proteins in cattle and ticks cells. MSP2, derived from a multi-gene family, was found to undergo antigenic variation in cattle and ticks and may contribute to establishment of persistent A. marginale infections. MSP1a has been used as a stable genetic marker for geographic isolates because the molecular weight varies due to differing numbers of the tandem repeats. However, phylogenetic studies of A. marginale isolates from North America using MSP1a and MSP4 demonstrated that MSP4 was a good biogeographic marker, while MSP1a varied greatly among and within geographic areas. Infection and development of A. marginale in cattle and tick cells appears to differ and to be mediated by several surface proteins encoded from the small genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium Vibrio anguillarum

Surface display of heterologous peptides and proteins such as receptors, antigens, and enzymes on live bacterial cells is of considerable value for various biotechnological and industrial applications. In this study, a series of novel cell surface display systems were examined by using Vibrio anguillarum outer membrane protein and outer membrane lipoprotein as anchoring motifs. These display systems consist of (i) the signal sequence and first 11 N-terminal amino acids of V. anguillarum outer membrane lipoprotein Wza, or the signal sequence and first 9 N-terminal amino acids of the mature major Escherichia coli lipoprotein Lpp, and (ii) transmembrane domains of V. anguillarum outer membrane proteins Omporf1, OmpU, or Omp26La. In order to assay the translocation efficiency of constructed display systems in bacteria, green fluorescent protein (GFP) was inserted to the systems and the results of GFP surface localization confirmed that four of the six surface display systems could successfully display GFP on the E. coli surface. For assaying its potential application in live bacteria carrier vaccines, an excellent display system Wza-Omporf1 was fused with the major capsid protein (MCP) of large yellow croaker iridovirus and introduced into attenuated V. anguillarum strain MVAV6203, and subsequent analysis of MCP surface localization proved that the novel display system Wza-Omporf1 could function as a strong tool in V. anguillarum carrier vaccine development.     

Major surface protein 1a effects tick infection and transmission of Anaplasma marginale.

Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.     

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.     

Functional cell surface display and controlled secretion of diverse Agarolytic enzymes by Escherichia coli with a novel ligation-independent cloning vector based on the autotransporter YfaL

Autotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) of Escherichia coli for the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes from Saccharophagus degradans 2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 10(4) molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growing E. coli.     

Cloning and modeling of CD8 beta in the amphibian ambystoma Mexicanum. Evolutionary conserved structures for interactions with major histocompatibility complex (MHC) class I molecules

Bovine anaplasmosis is a rickettsial disease of world-wide economic importance caused by Anaplasma marginale. Several major surface proteins with conserved gene sequences have been examined as potential candidates for vaccines and/or diagnostic assays. Major surface protein 1 (MSP1) is composed of polypeptides MSP1a and MSP1b. MSP1a is expressed from the single copy gene msp1 alpha and MSP1b is expressed by members of the msp1 beta multigene family. In order to determine if the msp1 genes are conserved, primers specific for msp1 alpha, msp1 beta(1), and msp1 beta(2) genes were synthesized and used to amplify msp1 sequences of A. marginale from tick cell cultures, from cattle during acute and chronic infections and from salivary glands of Dermacentor variabilis. Protein sequences of MSP1a, MSP1b(1) and MSP1b(2) were conserved during the life cycle of the parasite. No amino acid changes were observed in MSP1a. However, small variations were observed in the MSP1b(1) and MSP1b(2) protein sequences, which could be attributed to recombination, selection for sub-populations of A. marginale in the vertebrate host and/or PCR errors. Several isolate-specific sequences were also observed. Based on the information obtained in this study, the MSP1 protein appears to be fairly well conserved and a potential vaccine candidate.     

Development of PLEX, a plasmid-based expression system for production of heterologous gene products by the gram-positive bacteria Streptococcus gordonii

While Escherichia coli expression systems have been widely utilized for the production of heterologous proteins, these systems have limitations with regard to the production of particular protein products, including poor expression, expression of insoluble proteins into inclusion bodies, and/or expression of a truncated product. Using the surface protein expression (SPEX) system, chromosomally integrated heterologous genes are expressed and secreted into media by the naturally competent gram-positive organism Streptococcus gordonii. After E. coli turned out to be an inappropriate expression system to produce sufficient quantities of intact product, we successfully utilized SPEX to produce the heterologous antigen BH4XCRR that is designed from sequences homologous to the S. pyogenes M-protein C-repeat region. To further enhance production of this product by S. gordonii, we sought to develop a novel system for the production and secretion of heterologous proteins. We observed that under various growth conditions, S. gordonii secreted high levels of a 172 kDa protein, which was identified by N-terminal sequence analysis as the glucosyltransferase GTF. Here we report on the development of a plasmid-based expression system, designated as PLEX, which we used to enhance production of BH4XCRR by S. gordonii. A region from the S. gordonii chromosome that contains the positive regulatory gene rgg, putative gtfG promoter, and gtfG secretion-signal sequence was cloned into the E. coli/Streptococcus shuttle plasmid pVA838. Additionally, the bh4xcrr structural gene was cloned into the same plasmid downstream and in-frame with rgg and gtfG. This plasmid construct was transformed into S. gordonii and BH4XCRR was detected in culture supernatants from transformants at greater concentrations than in supernatants from a SPEX strain expressing the same product. BH4XCRR was easily purified from culture supernatant using a scalable two-step purification process involving hydrophobic-interaction and gel-filtration chromatography.     

Electrospun micro- and nanofiber tubes for functional nervous regeneration in sciatic nerve transections

Background

Rhipicephalus (Boophilus) spp. ticks economically impact on cattle production in Africa and other tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The R. microplus Bm86 protective antigen has been produced by recombinant DNA technology and shown to protect cattle against tick infestations.

Results

In this study, the genes for Bm86 (R. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) were cloned and characterized from African or Asian tick strains and the recombinant proteins were secreted and purified from P. pastoris. The secretion of recombinant Bm86 ortholog proteins in P. pastoris allowed for a simple purification process rendering a final product with high recovery (35–42%) and purity (80–85%) and likely to result in a more reproducible conformation closely resembling the native protein. Rabbit immunization experiments with recombinant proteins showed immune cross-reactivity between Bm86 ortholog proteins.

Conclusion

These experiments support the development and testing of vaccines containing recombinant Bm86, Ba86 and Bd86 secreted in P. pastoris for the control of tick infestations in Africa.     

Immunity against Boophilus annulatus induced by the Bm86 (Tick-GARD) vaccine

Friesian cattle were immunized with two inoculations of anti-tick Bm86 (Tick-GARD) vaccine and were challenged 30 or 90 d later with Boophilus annulatus larvae derived from 1.2 g of eggs. No nymphs or adult ticks were found on the immunized cattle during four weeks after challenge. Repeated infestations (2 to 4) with larvae on three other calves during a period of 160 and 390 d after the immunization did not result in development of nymphal and adult stages. In control, non-immunized cattle infested with corresponding batches of larvae 1380 to 4653 replete adult female ticks were collected. Larvae issued from Babesia bovis-infected female ticks transmitted the infection to Bm86-immunized cattle, but the progeny of B. bigemina-infected females did not. Since B. bigemina is transmitted exclusively by nymphal stages of Bo. annulatus these results support the observation that immunity induced by Bm86 affects the larval stage of this tick.     

Decorating microbes: surface display of proteins on Escherichia coli

Bacterial surface display entails the presentation of recombinant proteins or peptides on the surface of bacterial cells. Escherichia coli is the most frequently used bacterial host for surface display and, as such, a variety of E. coli display systems have been described that primarily promote the surface exposure of peptides and small proteins. By contrast, display systems based on autotransporter proteins (ATs) and ice nucleation protein (INP) are excellent systems for the display of large and complex proteins, and are therefore of considerable biotechnological relevance. Here, we review recent advances in AT and INP-mediated display and their biotechnological applications. Additionally, we discuss several promising alternative display methods, as well as novel bacterial host organisms.     

Cell surface presentation of recombinant (poly-) peptides including functional T-cell epitopes by the AIDA autotransporter system

For the efficient surface presentation and release of virulence factors especially pathogenic Gram-negative bacteria have developed several distinct secretion mechanisms. An increasing number of pathogens in various species employs a mechanism denoted the 'autotransporter' pathway. This pathway is characterised by an outer membrane translocator module representing the C-terminal domain of the transported protein itself. An intriguing potential application of such systems involves the transport and surface expression of recombinant proteins or peptides, like e.g. the presentation of antigens for the generation of live oral vectors as vaccine carriers. Here we report on the incorporation of heterologous (poly-) peptides in permissive sites of the translocator module of the adhesin-involved-in-diffuse-adherence (AIDA) autotransporter system. We demonstrate the presentation of the B subunit of the heat labile enterotoxin of Escherichia coli (LTB) as well as of functional T-cell epitopes of Yersinia enterocolitica heat-shock protein 60 (Y-hsp60) on the surface of E. coli.     

Development of enzyme linked immunosorbent assays to measure Bm86 antigen of Boophilus microplus (cattle tick) and to detect anti-Bm86 antibodies in serum samples

The immunization of cattle with the Boophilus microplus Bm86 antigen has been successful for the control of cattle tick infestations. To monitor the Bm86 production process and to measure the anti-Bm86 antibody titers in vaccinated cattle, mAb-based ELISA were developed and validated. The development of both immunological methods is essential to obtain a product with high quality and immunogenic properties and to monitor the immunological protection induced in vaccinated cattle against B. microplus.     

Heterologous expression, purification and characterisation of the extracellular domain of trypanosome invariant surface glycoprotein ISG75

The invariant surface glycoprotein ISG75 is a transmembrane glycoprotein occurring on the surface of the bloodstream-form Trypanozoon. This study describes the expression and purification of the N-terminal extracellular domain of ISG75, a novel target for development of diagnostic tests for trypanosomosis. To facilitate disulfide formation in the cytoplasm, a 1287-bp cDNA fragment encoding ISG75 from Trypanosoma brucei gambiense was expressed in a thioredoxin reductase, glutathione oxidoreductase double mutant Escherichia coli strain. An accessory plasmid pRIL, providing the argI, ileY, and leuW tRNAs, was necessary for efficient heterologous translation of the ISG75 mRNA. The recombinant double-tagged (streptavidine and histidine) ISG75 was purified by two-step affinity chromatography. Addition of L-glutamic acid and L-arginine in the buffer solutions was crucial to stabilise the protein during purification. The purified soluble protein was characterised by circular dichroism spectroscopy, reverse-phase high pressure liquid chromatography and mass spectrometry. It has an alpha-helical folded conformation, is homogeneous and pure (99%). Furthermore, sera of Trypanosoma brucei-infected animals specifically recognise this recombinant ISG75; and rabbit antiserum raised against the recombinant ISG75 detects all species of the Trypanozoon subgenus in parasite preparations.     

Production and biochemical characterization of the recombinant Boophilus microplus Bm95 antigen from Pichia pastoris

The new antigen Bm95 from the cattle tick Boophilus microplus was recently isolated, cloned and expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein has shown to induce protection in cattle against infestations of B. microplus under controlled and production conditions. In this paper we report the production and large-scale purification of the Bm95 protein, following a simple and cost-effective process. The antigen was obtained highly aggregated, forming particles ranging from 26 to 30 nm and with purity higher than 80%. The process yield was 0.55 g of pure Bm95 protein per liter of culture. The 98% of the primary structure of the recombinant protein was verified by mass spectrometry. Three amino acid changes in comparison with the sequence deduced from cDNA were detected by LC-MS/MS. The antigen was also obtained N-glycosylated, as previously reported for heterologous protein expression in P. pastoris.     

Cell surface display of hepatitis B virus surface antigen by using Pseudomonas syringae ice nucleation protein

A new system designed for cell surface display of recombinant proteins on Escherichia coli was evaluated for expression of eukaryotic viral antigens. The major surface antigen of hepatitis B virus (HBsAg) was fused to the ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. Western blotting, immunofluorescence microscopy, whole-cell ELISA, and ice nucleation activity assay confirmed expression of recombinant proteins on the surface of Escherichia coli. This study indicated that INP-based cell surface display can be used for epitope mapping and recombinant bacteria expressing hepatitis viral antigens may be used for developing live vaccines.     

Functional genomic studies of tick cells in response to infection with the cattle pathogen, Anaplasma marginale

The coevolution of ticks and the pathogens that they transmit has ensured their mutual survival. In these studies, we used a functional genomics approach to characterize tick genes regulated in response to Anaplasma marginale infection. Differentially regulated genes/proteins were identified by suppression-subtractive hybridization and differential in-gel electrophoresis analyses of cultured IDE8 tick cells infected with A. marginale. Nine of 17 of these genes were confirmed by real-time RT-PCR to be differentially regulated in ticks and/or IDE8 tick cells in response to A. marginale infection. RNA interference was used for functional studies. Six genes, which encode putative selenoprotein W2a, hematopoietic stem/progenitor cells protein-like, proteasome 26S subunit, ferritin, GST, and subolesin control, were found to affect A. marginale infection in IDE8 tick cells. Four genes, which encode putative GST, salivary selenoprotein M, vATPase, and ubiquitin, affected A. marginale infection in different sites of development in ticks. The results of these studies demonstrated that a molecular mechanism occurs by which tick cell gene expression mediates the A. marginale developmental cycle and trafficking through ticks.     

A partially structured region of a largely unstructured protein, Plasmodium falciparum merozoite surface protein 2 (MSP2), forms amyloid-like fibrils.

Merozoite surface protein 2 (MSP2) from the human malaria parasite Plasmodium falciparum is expressed as a GPI-anchored protein on the merozoite surface. It has been implicated in the process of erythrocyte invasion and is a leading vaccine candidate. MSP2 is an intrinsically unstructured protein (IUP), and recombinant MSP2 forms amyloid-like fibrils upon storage. We have examined synthetic peptides corresponding to sequences in the conserved N-terminal region of MSP2 for the presence of local structure and the ability to form fibrils related to those formed by full-length MSP2. In a 25-residue peptide corresponding to the entire N-terminal region of mature MSP2, structures calculated from NMR data show the presence of nascent helical and turn-like structures. An 8-residue peptide from the central region of the N-terminal domain (residues 8-15) also formed a turn-like structure. Both peptides formed fibrils that were similar but not identical to the amyloid-like fibrils formed by full-length MSP2. Notably, the fibrils formed by the peptides bound both Congo Red and Thioflavin T, whereas the fibrils formed by full-length MSP2 bound only Congo Red. The propensity of peptides from the N-terminal conserved region of MSP2 to form amyloid-like fibrils makes it likely that this region contributes to fibril formation by the full-length protein. Thus, in contrast to the more common pathway of amyloid formation by structured proteins, which proceeds via partially unfolded intermediates that then undergo beta-aggregation, MSP2 is an example of a largely unstructured protein with at least one small structured region that has an important role in fibril formation.