Author Correction: Hidden mycota of pine needles: Molecular signatures from PCR-DGGE and Ribosomal DNA phylogenetic characterization of novel phylotypes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hidden mycota of pine needles: Molecular signatures from PCR-DGGE and Ribosomal DNA phylogenetic characterization of novel phylotypes.

Previous studies for enumerating fungal communities on pine needles relied entirely on phenotypic diversity (microscopy) or identification based on DNA sequence data from those taxa recovered via cultural studies. To bypass limitations of the culturing methods and provide a more realistic diversity estimate, we employed and assessed a PCR-DGGE based method coupled with rDNA phylogenetic sequence analyses to characterize fungal taxa associated with pine needles. Fresh (living) and decayed needles from three hosts of the Pinaceae (Keteleeria fortunei, Pinus elliottii and P. massoniana) were examined. Morphological studies reveal that the most abundant species associated with decayed needles were Cladosporium cladosporioides and an unidentified Trichoderma species followed by Gliocephalotrichum sp., Gliocladium sp., Lophodermium pinastri, Paecilomyces varioti, Phaeostalagmus cyclosporus and a Phoma sp, which are commonly occurring fungi. Community genomic data from freshly collected and decayed pine needles recovered 40 operational taxonomic units, which appear to be mostly undetected members of the natural fungal consortium. Sequence analyses revealed a number of phylotypes or "species" that were not recovered using traditional morphological and cultural approaches previously used. Phylogenetic data from partial 18S rDNA sequence data reveal that most phylotypes represent potential novel phylogenetic fungal lineages with affinities to the Dothideomycetes, Leotiomycetes, Lecanoromycetes and Sordariomycetes and were not identical to previously known endophytes or saprobes. Although the major ecological roles of these phylotypes in pine needles are still enigmatic, this study provides new insights in hidden fungal diversity that mycologists are possibly ignoring given the discrepancies associated with available methods. To what extent do previously recovered identified species (either as saprobes or endophytes) from morphological or culturing studies act as pioneer decomposers or constitute an integral part of endophytic community warrants further investigation.

Direct activation of dendritic cells by the malaria parasite, Plasmodium chabaudi chabaudi.

A primary infection of mice with Plasmodium chabaudi chabaudi (AS) is characterized by a rapid and marked inflammatory response. Typically, IL-12, TNF-alpha and IFN-gamma are produced in the spleen, and are transiently present in plasma. The cells involved in this early response are unknown. Here we show that dendritic cells derived from GM-CSF-stimulated mouse bone marrow cultures produce TNF-alpha within 30 min of exposure to P.c.chabaudi schizonts. IL-6, IL-12p40 and p70 follow this. The production of these cytokines was not dependent on the presence of T cells or NK cells and did not require CD40. Incubation of dendritic cells with P.c.chabaudi schizonts also resulted in up-regulation of MHC class II, CD40 and CD86 but not CD80. In contrast to some strains of the human parasite, P. falciparum, P.c. chabaudi (AS) did not inhibit the up-regulation of MHC class II, CD86 or CD40 induced by LPS. Therefore, the erythrocytic stages of P.c.chabaudi are able to activate dendritic cells directly. The consequences of such an interaction could be rapid activation of TH1 cells and induction of immunity, and in the event of a large response also induction of TNF-alpha associated pathology.

Different regions of the malaria merozoite surface protein 1 of Plasmodium chabaudi elicit distinct T-cell and antibody isotype responses.

In this study we have investigated the antibody and CD4 T-cell responses to the well-characterized malaria vaccine candidate MSP-1 during the course of a primary Plasmodium chabaudi chabaudi (AS) infection. Specific antibody responses can be detected within the first week of infection, and CD4 T cells can be detected after 3 weeks of infection. The magnitude of the CD4 T-cell response elicited during a primary infection depended upon the region of MSP-1. In general, the highest precursor frequencies were obtained when a recombinant MSP-1 fragment corresponding to amino acids 900 to 1507 was used as the antigen in vitro. By contrast, proliferative and cytokine responses against amino acids 1508 to 1766 containing the C-terminal 21-kDa region of the molecule were low. The characteristic interleukin 4 (IL-4) switch that occurs in the CD4 T-cell population after an acute blood stage P. c. chabaudi infection was only consistently observed in the response to the amino acid 900 to 1507 MSP1 fragment. A lower frequency of IL-4-producing cells was seen in response to other regions. Although the magnitudes of the immunoglobulin G antibody responses to the different regions of MSP-1 were similar, the isotype composition of each response was distinct, and there was no obvious relationship with the type of T helper cells generated. Interestingly, a relatively high antibody response to the C-terminal region of MSP-1 was observed, suggesting that T-cell epitopes outside of this region may provide the necessary cognate help for specific antibody production.

Low CD4(+) T cell responses to the C-terminal region of the malaria merozoite surface protein-1 may be attributed to processing within distinct MHC class II pathways.

The C-terminal fragment of merozoite surface protein-1 (MSP-1) of the mouse malaria parasite Plasmodium chabaudi chabaudi (AS) stimulates a weak CD4 T cell response when compared to the response to a more structurally simple region of the molecule. The tertiary structure of the C-terminal region of MSP-1 is maintained by five disulfide bonds. A peptide from this region could only be processed and loaded onto newly synthesized MHC class II molecules, whereas a peptide from the structurally simple region was available for loading onto recycling MHC class II. CD4(+) T cell hybridomas took longer to recognize an epitope derived from the disulfide-bonded region whether native parasite or recombinant MSP-1 antigen was used. Reduction of disulfide bonds in the C-terminal region subsequently allowed peptides to be loaded onto recycling MHC class II and greatly enhanced the rapidity of the T cell response. These data demonstrate that differential processing occurs intramolecularly in MSP-1, which may be responsible for the observed weak CD4 T cell responses against this region. The consequences of this in vivo may be that limited T cell help is available for protective antibody production which has important implications for designing vaccines based on MSP-1.