From In Vivo to In Vitro: Dynamic Analysis of Plasmodium falciparum var Gene Expression Patterns of Patient Isolates during Adaptation to Culture

Abstract

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family, plays a crucial role in disease virulence through its involvement in binding to various host cellular receptors during infection. Growing evidence suggests that differential expression of the various var subgroups may be involved in parasite virulence. To further explore this issue, we have collected isolates from symptomatic patients in south China-Myanmar border, and characterized their sequence diversity and transcription profiles over time of var gene family, and cytoadherence properties from the time of their initial collection and extending through a two month period of adaptation to culture. Initially, we established a highly diverse, DBLα (4 cysteines) subtype-enriched, but unique local repertoire of var-DBL1α sequences by cDNA cloning and sequencing. Next we observed a rapid transcriptional decline of upsA- and upsB-subtype var genes at ring stage through qRT-PCR assays, and a switching event from initial ICAM-I binding to the CD36-binding activity during the first week of adaptive cultivation in vitro. Moreover, predominant transcription of upsA var genes was observed to be correlated with those isolates that showed a higher parasitemia at the time of collection and the ICAM-1-binding phenotype in culture. Taken together, these data indicate that the initial stage of adaptive process in vitro significantly influences the transcription of virulence-related var subtypes and expression of PfEMP1 variants. Further, the specific upregulation of the upsA var genes is likely linked to the rapid propagation of the parasite during natural infection due to the A-type PfEMP1 variant-mediated growth advantages.

The antigenic switching network of Plasmodium falciparum and its implications for the immuno-epidemiology of malaria

“…Previous studies reported that UpsA var genes from patient isolates were down-regulated faster than other var genes during adaptation to culture (Peters et al, 2007; Zhang et al, 2011). The HB3 repertoire contains seven UpsA genes, all but one of which are subtelomeric.…”

“…Several studies have noted that var genes located near the centres of chromosomes tend to be more highly transcribed in vitro than those in subtelomeric location (Frank et al, 2007; Peters et al, 2007; Enderes et al, 2011; Zhang et al, 2011; Fastman et al, 2012), and it has been suggested that this might be due to low deactivation rates (Frank et al, 2007). In this study, we found that high transcription levels are confined to a subset of central genes that encode only four PfEMP1 binding domains (termed Type 1 var genes [Gardner et al, 2002]) and, importantly, are the result of high activation biases rather than low off-rates.…”

“…In this setting, Horrocks et al (Horrocks et al, 2004) found that the rates at which var genes activate or deactivate are non-random, gene-specific and highly dissimilar. It has furthermore been suggested that var genes occupying subtelomeric loci tend to switch off faster than those positioned in central chromosomal regions (Frank et al, 2007), offering a possible explanation for the rapid decline in the transcription of subtelomeric genes in parasites during culture adaptation (Peters et al, 2007; Zhang et al, 2011). More recently, Recker et al (2011) proposed that var gene transcriptional change involves a highly structured pathway that has evolved in response to a trade-off between the within-host and the between-host level fitness.…”

Silence, activate, poise and switch! Mechanisms of antigenic variation in P lasmodium falciparum

“…Two recently published var expression studies from malaria patients, however, support the idea that host factors contribute to var gene transcriptional control. Unlike in vitro cultured parasites, ups A-type var genes were frequently expressed in P. falciparum-infected patients (Bachmann et al, 2011;Zhang et al, 2011b). Subsequent cultivation resulted in random expression of many var genes mostly of the ups B and C-type.…”

Antígenos variantes de superfície de hemácias infectadas por Plasmodium falciparum na Amazônia brasileira: aderência a receptores do endotélio vascular (CD36 e ICAM-1) e reconhecimento por anticorpos.

“…Porém, não está claro se a capacidade de aderir a mais de um receptor se deve à expressão de um gene var que codifica uma PfEMP1 "poliaderente" ou se é devido à co-expressão de genes var (Joergensen et al, 2010). (Frankland et al, 2007 Adicionalmente, a literatura também relata alterações no padrão de adesão de isolados de campo (Gray et al, 2003;Zhang et al, 2011). Experimentos em fluxo com isolados, provenientes de pacientes que foram a óbito devido à malária cerebral, que aderiam bem a ICAM-1 estaticamente, não se mostraram eficientes em aderir a ICAM-1 em fluxo, e o inverso também foi observado, o isolado que se ligava menos a ICAM-1 estaticamente, obteve uma adesão em fluxo mais eficiente (Gray et al 2003).…”