The antibody response to Plasmodium falciparum Merozoite Surface Protein 4: comparative assessment of specificity and growth inhibitory antibody activity to infection-acquired and immunization-induced epitopes.

BACKGROUND: Malaria remains a global public health challenge. It is widely believed that an effective vaccine against malaria will need to incorporate multiple antigens from the various stages of the parasite's complex life cycle. Plasmodium falciparum Merozoite Surface Protein 4 (MSP4) is a vaccine candidate that has been selected for development for inclusion in an asexual stage subunit vaccine against malaria. METHODS: Nine monoclonal antibodies (Mabs) were produced against Escherichia coli-expressed recombinant MSP4 protein and characterized. These Mabs were used to develop an MSP4-specific competition ELISA to test the binding specificity of antibodies present in sera from naturally P. falciparum-infected individuals from a malaria endemic region of Vietnam. The Mabs were also tested for their capacity to induce P. falciparum growth inhibition in vitro and compared against polyclonal rabbit serum raised against recombinant MSP4. RESULTS: All Mabs reacted with native parasite protein and collectively recognized at least six epitopes. Four of these Mabs recognize reduction-sensitive epitopes within the epidermal growth factor-like domain found near the C-terminus of MSP4. These sera were shown to contain antibodies capable of inhibiting the binding of the six Mabs indicating infection-acquired responses to the six different epitopes of MSP4. All of the six epitopes were readily recognized by human immune sera. Competition ELISA titres varied from 20 to 640, reflecting heterogeneity in the intensity of the humoral response against the protein among different individuals. The IgG responses during acute and convalescent phases of infection were higher to epitopes in the central region than to other parts of MSP4. Immunization with full length MSP4 in Freund's adjuvant induced rabbit polyclonal antisera able to inhibit parasite growth in vitro in a manner proportionate to the antibody titre. By contrast, polyclonal antisera raised to individual recombinant fragments rMSP4A, rMSP4B, rMSP4C and rMSP4D gave negligible inhibition. Similarly, murine Mabs alone or in combination did not inhibit parasite growth. CONCLUSIONS: The panel of MSP4-specific Mabs produced were found to recognize six distinct epitopes that are also targeted by human antibodies during natural malaria infection. Antibodies directed to more than three epitope regions spread across MSP4 are likely to be required for P. falciparum growth inhibition in vitro.

Mutations in pimE restore lipoarabinomannan synthesis and growth in a Mycobacterium smegmatis lpqW mutant.

Lipoarabinomannans (LAMs) and phosphatidylinositol mannosides (PIMs) are abundant glycolipids in the cell walls of all corynebacteria and mycobacteria, including the devastating human pathogen Mycobacterium tuberculosis. We have recently shown that M. smegmatis mutants of the lipoprotein-encoding lpqW gene have a profound defect in LAM biosynthesis. When these mutants are cultured in complex medium, spontaneous bypass mutants consistently evolve in which LAM biosynthesis is restored at the expense of polar PIM synthesis. Here we show that restoration of LAM biosynthesis in the lpqW mutant results from secondary mutations in the pimE gene. PimE is a mannosyltransferase involved in converting AcPIM4, a proposed branch point intermediate in the PIM and LAM biosynthetic pathways, to more polar PIMs. Mutations in pimE arose due to insertion of the mobile genetic element ISMsm1 and independent point mutations that were clustered in predicted extracytoplasmic loops of this polytopic membrane protein. Our findings provide the first strong evidence that LpqW is required to channel intermediates such as AcPIM4 into LAM synthesis and that loss of PimE function results in the accumulation of AcPIM4, bypassing the need for LpqW. These data highlight new mechanisms regulating the biosynthetic pathways of these essential cell wall components.

Plasmodium falciparum Pf34, a novel GPI-anchored rhoptry protein found in detergent-resistant microdomains.

Apicomplexan parasites are characterised by the presence of specialised organelles, such as rhoptries, located at the apical end of invasive forms that play an important role in invasion of the host cell and formation of the parasitophorous vacuole. In this study, we have characterised a novel Plasmodium falciparum rhoptry protein, Pf34, encoded by a single exon gene located on chromosome 4 and expressed as a 34kDa protein in mature asexual stage parasites. Pf34 is expressed later in the life cycle than the previously described rhoptry protein, Rhoptry Associated Membrane Antigen (RAMA). Orthologues of Pf34 are present in other Plasmodium species and a potential orthologue has also been identified in Toxoplasma gondii. Indirect immunofluorescence assays show that Pf34 is located at the merozoite apex and localises to the rhoptry neck. Pf34, previously demonstrated to be glycosyl-phosphatidyl-inositol (GPI)-anchored [Gilson, P.R., Nebl, T., Vukcevic, D., Moritz, R.L., Sargeant, T., Speed, T.P., Schofield, L., Crabb, B.S. (2006) Identification and stoichiometry of GPI-anchored membrane proteins of the human malaria parasite Plasmodium falciparum. Mol. Cell. Proteomics 5, 1286-1299.], is associated with parasite-derived detergent-resistant microdomains (DRMs). Pf34 is carried into the newly invaded ring, consistent with a role for Pf34 in the formation of the parasitophorous vacuole. Pf34 is exposed to the human immune system during infection and is recognised by human immune sera collected from residents of malaria endemic areas of Vietnam and Papua New Guinea.

Identification of a novel protein with a role in lipoarabinomannan biosynthesis in mycobacteria.

All species of Mycobacteria synthesize distinctive cell walls that are rich in phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). PIM glycolipids, having 2-4 mannose residues, can either be channeled into polar PIM species (with 6 Man residues) or hypermannosylated to form LM and LAM. In this study, we have identified a Mycobacterium smegmatis gene, termed lpqW, that is required for the conversion of PIMs to LAM and is highly conserved in all mycobacteria. A transposon mutant, Myco481, containing an insertion near the 3' end of lpqW exhibited altered colony morphology on complex agar medium. This mutant was unstable and was consistently overgrown by a second mutant, represented by Myco481.1, that had normal growth and colony characteristics. Biochemical analysis and metabolic labeling studies showed that Myco481 synthesized the complete spectrum of apolar and polar PIMs but was unable to make LAM. LAM biosynthesis was restored to near wild type levels in Myco481.1. However, this mutant was unable to synthesize the major polar PIM (AcPIM6) and accumulated a smaller intermediate, AcPIM4. Targeted disruption of the lpqW gene and complementation of the initial Myco481 mutant with the wild type gene confirmed that the phenotype of this mutant was due to loss of LpqW. These studies suggest that LpqW has a role in regulating the flux of early PIM intermediates into polar PIM or LAM biosynthesis. They also suggest that AcPIM4 is the likely branch point intermediate in polar PIM and LAM biosynthesis.

Selectively impaired CD8+ but not CD4+ T cell cycle arrest during priming as a consequence of dendritic cell interaction with plasmodium-infected red cells.

Individuals living in malaria-endemic areas show generally low T cell responses to malaria Ags. In this study, we show murine dendritic cell (DC) interaction with parasitized erythrocytes (pRBC) arrested their maturation, resulting in impaired ability to stimulate naive, but not recall T cell responses in vitro and in vivo. Moreover, within the naive T cell population, pRBC-treated DC were selectively deficient in priming CD8(+) but not CD4(+) T cells. Indeed, DC that had taken up pRBC were shown for the first time to efficiently prime CD4(+) T cell responses to a known protective merozoite Ag, MSP4/5. In contrast, impaired priming resulted in decreases in both proliferation and cytokine production by CD8(+) T cells. Deficient priming was observed to both a model and a Plasmodium berghei-specific CD8(+) T cell epitope. The mechanisms underlying the inability of parasite-treated DC to prime CD8(+) T cells were explored. pRBC treatment of DC from wild-type C57BL/6, but not from IL-10 knockout animals, suppressed DC-mediated T cell priming across a Transwell, suggesting active IL-10-dependent suppression. CD8(+) T cells were arrested at the G(0) stage of the cell cycle after two cell divisions post-Ag stimulation. The proliferation arrest was partially reversible by the addition of IL-2 or IL-7 to responder cultures. These results suggest that in malaria-endemic areas, priming of CD8(+) T cell responses may be more difficult to induce via vaccination than the priming of CD4(+) T cells. Moreover, pathogens may selectively target the CD8(+) T cell arm of protective immunity for immune evasion.

Inactivation of mshB, a key gene in the mycothiol biosynthesis pathway in Mycobacterium smegmatis.

The mshB gene encoding N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis. Disruption of mshB in Mycobacterium smegmatis resulted in decreased production of mycothiol (5-10 % of the parent strain mc(2)155) but did not abolish mycothiol synthesis completely. Complementation of the MshB(-) mutants with the mshB gene resulted in increased mycothiol production towards the exponential and stationary phases of the bacterial growth cycle. These results suggest that another enzyme is capable of mycothiol biosynthesis by providing N-acetylglucosaminylinositol deacetylation activity in the absence of MshB. One of the candidate enzymes capable of carrying out such reactions is the MshB orthologue mycothiol amide hydrolase, MCA. However, epichromosomal expression of mca in the MshB(-) mutants did not restore mycothiol levels to the level of the parent strain. Unlike other mutants, which have little or no detectable levels of mycothiol, the MshB(-) mutant did not exhibit increased resistance to isoniazid. However, the MshB(-) mutant was resistant to ethionamide. Phenotypic analysis of other mutants lacking mycothiol revealed that MshA(-) mutants also exhibit ethionamide resistance but that a MshC(-)mutant was sensitive to ethionamide, suggesting that mycothiol or its early intermediates influence ethionamide activation.