Many studies have generated convincing evidence of the unique antigenic and binding properties of P. falciparum
iEs collected from the placenta. Previous analysis of the parasite variant surface antigens has pointed to PfEMP1 variants as the major target of antigenic variation and mediator of iE adhesion properties. Our prior studies have shown that VAR2CSA PfEMP1 accounts for CSA binding by iEs originating from the placenta 
. The present study has identified differences in gene expression compared to in vitro
3D7 parasite reference, using the whole-genome microarray analysis of P. falciparum
parasites directly collected from the placenta. First, this analysis confirmed var2csa
(represented by 16 oligonucleotides) as the only var
gene clearly over-expressed in all placental parasite isolates. This however does not exclude possible over-expression of other var
genes, which were not represented on the microarray due to sequence diversity. Second, along side with the var2csa
, a significant number of genes were over-expressed by placental parasites, including genes predicted to encode exported proteins and thus possibly contributing to parasite placental tropism. For this whole-genome analysis of in vivo
transcription of placental parasites, stringent selection criteria were used i.e. differential expression in at least 2 out of 3 pooled samples after stringent Bonferroni p value adjustment. A common reference containing all parasite erythrocytic stages with an emphasis on schizonts and trophozoites, forms predominantly found in placental isolates, was used. Although there are clearly defined morphologic differences between the schizont, merozoite, and ring stages, biological processes may be shared in contiguous stages. Using a common reference was preferred to three separate controls attempting to match the composition of each pool, as such matching on morphological criteria is very difficult on late stages, if not impossible. This allowed identification of 38 and 46 genes over- or under-expressed in placental parasites respectively.
Of special interest is gene PFI1785w that was highly and specifically over-expressed in placental parasites. The transcription of PFI1785w displayed a clearly defined profile, similar to that of var2csa
, suggesting a specific association with placental parasites. However, no evidence of its relation to the CSA binding phenotype was seen in parasite lines biologically selected for in vitro
CSA binding. Several other genes were also over-expressed in placental parasites, but not in in vitro
selected CSA binding parasites. This suggests either that these genes are selectively over-expressed during pregnancy but are not directly involved in CSA binding, or that conditions required for in vivo
binding to proteoglycans expressed by the placenta and in vitro
binding to CSA differ. A recent microarray study on individual parasites by Francis et al. 
also observed high transcription of PFI1785w by placental parasites, and the expression of this particular PHIST gene at the protein level was described by Fried et al. 
in membrane extracts from placental parasites. Although CSA binding property represents the major specific phenotype of placental parasites that can be reproduced in laboratory-adapted parasite lines in vitro
, some characteristics of parasites causing PAM might require in vivo
conditions for expression. We hypothesise that this could be the case for PFI1785w expression.
The presence of a stop codon (...KKP-stop-KKPHP...) within the 3D7 PFI1785w explains why the gene was miss-annotated as an atypical three–exon PHIST gene and suggests that the sequence downstream the stop codon is not necessary for 3D7 survival in vitro. However in other strains such as those causing PAM the entire protein structure may be needed for placental tropism of the parasite.
The recombinant protein (NP561) corresponding to part of the protein encoded by this gene was more highly recognized by serum samples from women living in malaria-endemic areas as compared to serum from men. The relation to gender was less strong than that of VAR2CSA, raising the question of this gene's exact role in the mechanisms governing P. falciparum
placental tropism. However, the importance of this relation was similar to the one observed with recombinant PFD1140w in the Francis et al. study 
, and the presence of significant levels of antibodies to VAR2CSA has been reported among some men and children 
. Whether antibodies to PFI1785w express functional activity against PAM parasites will be addressed in future studies. Differential acquisition of these antibodies between men and pregnant women should also be confirmed in a dedicated seroepidemiological survey.
PFI1785w is a single copy gene that encodes a small protein likely exported to the red blood cell as judged by its predicted signal sequence and export motif. The gene is found conserved (>98%) at the nucleotide level in all 4 species of Plasmodium spp infecting humans as well as in the Plasmodium reichenowi genome, suggesting that the product of this gene plays an important role for the survival of Plasmodium, but argues against a direct role in antigenic variation on the surface of the infected red blood cell. No evidence has been presented that the PHIST proteins in general are surface antigens, and none of them have TM domains. In contrast to PfEMP1 and RIFIN proteins, they exhibit no amino acid diversity between isolates; for example, comparison of PFI1785w alleles between 3D7 and HB3 (Broad Institute browser), the Ghanaian isolate (Sanger browser) or placental isolates from our study shows essentially 100% sequence conservation.
A recent study identified a ~22 KDa protein as being involved in the binding of iEs to CSA 
. In the present study, high transcription of a number of genes encoding low molecular weight proteins with predictive characteristics for red blood cell trafficking was specifically observed in placental isolates. Notable examples include, along with PFI1785w (44 KDa corrected here), PFA0700c (12.83 KDa), PF14_0757 (24.95 KDa), and PFB0105c (35.3 KDa). In our study, PF14_0757 with its theoretical molecular weight of 24.95 KDa is the closest in size to this potential protein, but its transcription was not shown to significantly differ between placental and non-placental parasites. Among the 5 genes found by Francis et al. 
to be specifically co-transcribed with var2csa
by placental parasites when analyzing individual isolates, four were also identified in our study (PFI1785w, PFL0050c, PFD1140w, PFB0115w). However only PFI1785w met our own criteria of interesting genes (high expression in at least 2 out of the 3 test pools). PFD1140w, the main candidate gene characterized in the Francis et al. study 
was also detected here, but only in the pool with domination of trophozoites. This apparent discrepancy suggests a stage-dependent expression of the PFD1140w gene. Proteins from 2 (PFI1785w and PFA700c) genes with interesting scores in our study were also detected by Fried et al 
. For 3 other genes (PFB0115w, PF14_0016, PF14_0260) for which proteins were detected in the same study, differential expression was only recorded in 1 of the 3 test pools. With PF14_0016 and PF14_0260 rather showing PAM under-expression in our study.
Our results suggest that many sub-telomeric genes in P. falciparum
genome play a role in host-parasite interactions. As sub-telomeric genes, PHIST appears dissimilar to any known structure, making it difficult to assign possible functions to members of this family. A published microarray study of field isolates has shown that most genes from PHISTb and PHISTc families are over-expressed in P. falciparum
parasites directly collected from infected children compared to 3D7 
. As shown in table S2
, in the present study, 4 genes belonging to the three PHIST families, i.e. PF14_0757, PFA110w, PFI1785w and PFB105c were found over-expressed in PAM parasites compared to 3D7. Thus, it is all the more interesting that proteomic data support consistent presence of proteins belonging to PHIST family in iE membrane preparations 
These findings suggest that other parasite proteins beside VAR2CSA may contribute to the pathogenesis of PAM. Prospective clinical studies are needed to estimate the importance of those new proteins in the mechanism controlling acquisition of distinct binding and serological phenotypes by P. falciparum and thus, identification of critical targets for intervention.