In order to gain a better understanding of the sequence of molecular events in hosts that succumb to or recover from a malaria infection, global gene expression profiling on high-density oligonucleotide microarrays was exploited to assess the function of differentially regulated genes in the context of the kinetics of transcription and to describe the biological processes that are regulated by such genes. It is reasonable to hypothesize that clusters of up- or down-regulated genes would show patterns of expression that mirrored the course of parasitemia and that the magnitude of transcriptional activity would vary as a function of disease progression or resolution. Factors that may influence such transcriptional changes include both the molecular interactions that regulate gene expression and the dynamic changes in cellular composition that occur in the spleens of animals during the course of infection. Using multiple biological replicates at specific intervals during 17XNL infection, a large number of statistically significant genes were identified, such as heat shock protein genes, whose expression correlated directly with the pattern of ascending and descending parasitemia.
Biological and molecular processes linking sets of up-regulated genes to gene ontology-annotated terms were discovered from gene clusters identified through hierarchical clustering and self-organizing maps. The overrepresented GO terms assigned to sets of differentially expressed genes during specific phases of infection suggested that the functions of coexpressed genes may also be coregulated. The networks of cis
-regulatory elements and transcription factors that regulate induction or silencing of gene expression during malaria infection play critical but as-yet-undefined roles in coordinating the activation of gene expression and may help explain why transcriptional activation continues after the host successfully clears the infection. The coordination of gene expression with gene regulation is tightly controlled and influences the expression of many genes involved in the pathological manifestations of disease. In particular, the differential expression of transcription factors and their target genes in regulating erythropoiesis illustrates how expression profiling differentiates between groups of animals that die (24
; this paper) or recover from infection and associates that outcome with the host's response to severe malaria anemia. Such transcription factors and cis
-regulatory elements, singly or in molecular complexes, regulate red cell proliferation and expression of erythrocyte genes for band 4.2, uroporphyrinogen III synthase, transferrin receptor 2, glycophorin A, and hemoglobin (3
As erythropoiesis is not limited to the spleen, future investigations should include genome-wide expression profiling of tissues such as the bone marrow and liver, in addition to correlating the expression patterns of regulatory proteins (transcription factors) with their target genes to improve our understanding of the molecular interactions that may predict prognostic signatures for severe malarial anemia in young children.
Physiologic adaptations to malaria infection have indicated that lactic acidosis resulting from the conversion of pyruvate to lactate is implicated in the pathogenesis of human P. falciparum
and lethal murine P. berghei
infection and is a prognostic indicator of severe disease (8
). Sexton et al. have recently reported on the up-regulation of nine glycolytic pathway genes in the spleens of mice infected with P. berghei
that favor the conversion of pyruvate to lactate and the down-regulation of counterregulatory genes which shunt or divert products from this pathway (24
). Other reports have shown that acidosis correlates with increased parasitemia, progression of disease, and increased glycolytic activity (7
). In the study presented here, mice infected with lethal 17XL P. yoelii
parasites showed increased gene expression of all the enzymes present in the glycolytic pathway throughout infection. Conversely, in the nonlethal infection, transient increases in gene expression were followed by a dramatic reversal in gene transcription in the interval of time when the parasitemia rose from 5% to 25%. Whether the divergence in patterns of expression between lethal and nonlethal infections reflects the cause or effect of mechanisms that influence death or survival is unknown. However, the observation that the reversal in transcriptional activation of glycolytic pathway genes in nonlethal infection precedes the peak of maximal parasite density suggests that expression patterns may have prognostic significance as predictors of clinical outcome.
Signature patterns of gene expression in immune response genes that distinguished 17XNL from 17XL infection were associated primarily with B-cell proliferation and immunoglobulin production. Antibody responses and plasma cell production in lethal P. berghei
and P. yoelii
infection are suppressed (21
), while nonlethal P. chabaudi
and P. yoelii
infections induce a large expansion of plasma cells within the red pulp of the spleen (1
). The suppression of gene expression of CXCL13
, a chemokine important for B-cell homing to germinal centers (14
), observed here coincided with the suppression observed for other B-cell proliferation genes, such as Lsp1
, necessary for cytoskeletal rearrangements and lymphocyte motility and proliferation (4
), while genes involved in plasma cell differentiation were induced. Critically, the relative contribution of antibody-mediated immune mechanisms that appear to control the 17XNL infection studied here in an immunologically intact animal may mask or impede the transcription of genes that are crucial for resolving infections primarily by cell-mediated immune mechanisms (22
). In addition, care must be taken not to overinterpret the differences in B-cell and plasma cell gene expression in animals infected with a lethal compared to a nonlethal infection, since the rate of 17XL parasite replication may outstrip the host's ability to elicit a timely and effectual immunologic response. The immune response in animals infected with the lethal strain of P. yoelii
may in fact be normal (as shown by the early induction of gene ontology functional groups associated with immune response and cytokine and chemokine signaling pathways), but these animals die prior to the acquisition of antiparasite antibodies.
In conclusion, expression profiling is an extraordinarily useful tool to explore the molecular features of the host's response to infection and can provide insights into transcriptional regulatory mechanisms that influence both the pathogenesis of disease and the host's recovery from infection. While immune responses in human P. falciparum and P. vivax malaria may share many similar features of the global gene expression program observed in murine malaria, important differences in expression profiles in humans infected clinically or experimentally with malaria will depend heavily on the type of tissue (peripheral blood, bone marrow, spleen, or brain) and the stage of infection (early asymptomatic versus clinical malaria) that is studied.