Hypervariability within the Rifin, Stevor and Pfmc-2TM superfamilies in Plasmodium falciparum
Catherine Lavazec
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Sohini Sanyal
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Thomas J. Templeton
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Department of Microbiology and Immunology, Weill Cornell Medical College and the Program in Immunology and Microbial Pathogenesis, Weill Graduate School of Medical Sciences of Cornell University
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New York, NY 10021, USA
The human malaria parasite, Plasmodium falciparum, possesses a broad repertoire of proteins that are proposed to be trafficked to the erythrocyte cytoplasm or surface, based upon the presence within these proteins of a Pexel/VTS erythrocytetrafficking motif. This catalog includes large families of predicted 2 transmembrane (2TM) proteins, including the Rifin, Stevor and Pfmc-2TM superfamilies, of which each possesses a region of extensive sequence diversity across paralogs and between isolates that is confined to a proposed surface-exposed loop on the infected erythrocyte. Here we express epitope-tagged versions of the 2TM proteins in transgenic NF54 parasites and present evidence that the Stevor and Pfmc-2TM families are exported to the erythrocyte membrane, thus supporting the hypothesis that host immune pressure drives antigenic diversity within the loop. An examination of multiple P.falciparum isolates demonstrates that the hypervariable loop within Stevor and Pfmc-2TM proteins possesses sequence diversity across isolate boundaries. The Pfmc-2TM genes are encoded within large amplified loci that share profound nucleotide identity, which in turn highlight the divergences observed within the hypervariable loop. The majority of Pexel/VTS proteins are organized together within sub-telomeric genome neighborhoods, and a mechanism must therefore exist to differentially generate sequence diversity within select genes, as well as within highly defined regions within these genes.
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The bloodstream stages of the human malaria parasite,
Plasmodium falciparum, reside enveloped by a parasitophorous
vacuole within mature erythrocytes. As the parasite develops
within this niche, it extensively modifies the erythrocyte
surface and cytoplasm in order to display parasite-encoded
receptors on the erythrocyte surface as well as to establish
erythrocyte surface solute channels that are involved in
nutrient uptake and elimination of metabolic waste products.
The requisite extra-parasitic, intra-erythrocytic trafficking
network is of parasite origin and invention, because the
erythrocyte itself is devoid of trafficking machinery that can be
co-opted for parasitic means. Little is known regarding the
molecular mechanisms of Plasmodium extra-parasitic
trafficking, but it likely involves components that are either
soluble within the erythrocyte cytoplasm or integral within
the parasitophorous vacuolar membrane; the Maurers clefts
that dot the erythrocyte cytoplasm; or possibly small vesicles
budding from either of these membrane surfaces [(17),
reviewed in (8,9)]. Our understanding of protein targeting
to the erythrocyte cytoplasm was recently given a revelatory
boost by the identification of a trafficking motif that appears
to be widely, but not inclusively, present in parasite-encoded,
signal peptide-containing proteins that are trafficked to the
erythrocyte cytoplasm or surface (1012). The motif is termed
Pexel [Plasmodium export element, (12)] or VTS [vacuolar
transport signal, (10)] and has a simple core consensus
sequence, RxLxQ/E. All Pexel/VTS-containing proteins
identified to date possess a two exon gene structure in
which the signal peptide, oftentimes recessed from the start
methionine, is encoded on the short N-terminal exon, whereas
the Pexel/VTS motif is encoded on the second exon near
the splice junction site [reviewed in (13,14)].
Using the above descriptive features of
Pexel/VTSencoding genes heuristically it is possible to compile a
catalog of P.falciparum proteins that are predicted to be trafficked
to the erythrocyte. This catalog most notably includes all
proteins encoded by the 2 transmembrane (2TM)-containing
members of the stevor, rifin and Pfmc-2TM gene families;
the knob-associated proteins KAHRP and MESA; the DnaJ
domain-containing RESA family; the superfamily of a-helical
helical PHIST domain proteins (9) that contains over 70
members, including the RESA-like proteins; as well as a
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
family of 20 serine/threonine protein kinases of the R45
family that are predicted to be trafficked to the erythrocyte
cytoplasm (8,15). In addition, as much as 25% of the total
Pexel/VTS catalog is represented by unique proteins. We
have determined such a catalog [see also the recent reviews,
(9,16)] by combining the catalogs compiled by Marti et al.
(12) and Hiller et al. (10); and additionally performing
exhaustive BLAST screening of Plasmodium sp. genome
sequence databases and conducting extensive genome
walking on the P.falciparum genome sequence. The resulting
catalog contains over 300 genes that, although daunting in it (...truncated)