Host sequences in Plasmodium falciparum and Plasmodium vivax genomic DNA: horizontal transfer or contamination artifact?

Genome sequencing projects have led to new insights into the evolutionary impact of horizontal gene transfer between species [1]. The processes mediating such transfer in eukaryotes could, hypothetically, involve incorporation of host cell DNA into intracellular parasites, viruses and symbionts. Indeed, recent observations show that the Plasmodium falciparum genome may be frequently exposed to host sequences, as intra-erythrocytic malaria parasites spontaneously take up and express DNA from host red blood cells that can contain residual nuclear material [2]. We were therefore intrigued by the presence of Alu elements in reported Plasmodium vivax and P. falciparum DNA sequences [3,4] as well as in several entries in databases from P. vivax and P. falciparum genome projects (GenBank accession numbers AA550283, AF010561; University of Florida Gene Sequence Tag Project http://parasite.vetmed.u£.edu/, clone numbers UFL_211PvG06, UFL_214PvC04, UFL_217PvA08, UFL_212PvE06). LINE (L1) sequences have also been identi¢ed in some of these databases (University of Florida clone numbers UFL_202PvC10, UFL_211PvE06, UFL_214PvH07, UFL_213PvB11). Both Alu and LINE sequences are abundant interspersed repetitive DNA elements that are characteristic of primate genomes. Since parasite DNA extraction methods are not guaranteed to remove host DNA completely, and it has been shown that host-like sequences in schistosome parasite sequence databases can arise from contamination artifacts [5], we performed experiments to reassess the origin of these Alu and LINE elements. The reported Alu sequence from P. vivax [3] was identi¢ed in an expression library that had been constructed with DNA from the blood of an infected patient. This sequence was proposed to be integrated within a gene, Pv-Alu, encoding a P. vivax antigen. To test this proposal, we performed PCR using oligonucleotide primers speci¢c to the reported sequence to amplify across the junction of the Alu element and the putative parasite gene. These reactions used pure human leukocyte DNA and P. vivax DNA prepared from a monkey infection as templates. Results of the ampli¢cations showed a product from human but not parasite DNA (Fig. 1A). Direct sequencing con¢rmed the product ampli¢ed from human DNA to be identical to the reported sequence [3]. We also used the Pv-Alu sequence and eight other randomly chosen Alu and L1 DNA fragments identi¢ed from the University of Florida P. vivax Gene Sequence Tag Project to probe P. vivax and P. falciparum chromosomal DNAs separated by pulsed ¢eld gradient electrophoresis (PFGE). All nine clones hybridized to the same high MW band in the compression region of the P. vivax lanes that varied in intensity from preparation to preparation, relative to the intensities of the lower chromosome bands. (Fig. 1B and data not shown). In previous work this band was assumed to be parasite in origin and was described as P. vivax chromosome XIV [6]. However, in the light of these results, this assignment appears to be erroneous. We also note that if the repetitive host sequences were indeed true integrants in the parasite genome, they would likely be found at random chromosomal locations. The band to which the sequences hybridize is instead evidently a `pseudo-chromosome', composed of sheared host material which migrates within the compression region under the PFGE conditions used to separate P. vivax chromosomes. In addition, no sequences of con¢rmed P. vivax origin have been found to hybridize to this band ([7] and data not shown). None of the Alu or L1 elements hybridized to gels of separated P. falciparum chromosomes, which are from in vitro cultures containing erythrocytes cleared of leukocytes and platelets before use (Fig. 1B). Southern blots of restricted P. falciparum genomic DNA were also probed for the presence of Alu sequences. Under low stringency conditions (3USSPE at 503C), these probings did not detect bands that would suggest the presence of Alulike sequences in the P. falciparum genome (data not shown). PCR ampli¢cation experiments also failed to con¢rm other Alu elements [4] that have appeared in P. falciparum genome project databases (data not shown). The above experiments indicate that the human repeat elements cited in the literature and as DNA entries in public sequence databases are most likely contaminants of parasite DNA preparations by host DNA. While this ¢nding does not preclude the possibility that other sequences may have been acquired by horizontal transfer in the evolutionary lineage of Plasmodium, caution should be exercised to ensure that such sequences are in fact integrants in the parasite genome. As increasing amounts of data from protozoan genome sequencing projects become available, a more thorough assessment of the issue of horizontal gene transfer in the evolutionary lineage of Plasmodium, raised in [7], should be possible.