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MICROBIOLOGY COMMENT |
Division of Biological Science, University of California at San Diego, La Jolla, CA 92093-0116, USA
Correspondence
Milton H. Saier, Jr
(msaier{at}ucsd.edu)
Malaria is a global health threat placing approximately 40 % of the world's population at risk. Nearly one million deaths can be attributed to malaria every year with an additional 300 million acute illnesses resulting annually. About 90 % of these cases occur in the tropical and subtropical regions of the world (World Health Organization, http://www.who.int). While mosquitoes transmit malaria, the disease is caused by protozoan parasites of the genus Plasmodium. Virulent Plasmodium species include Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium falciparum. Most cases of malaria are caused by P. falciparum and P. vivax, with P. falciparum inducing the most severe symptoms. Traditionally, chloroquine and other quinoline-based drugs have been used for the prophylaxis and treatment of malaria; however, Plasmodium resistance to chloroquine has been increasingly problematic. Since chloroquine is the most efficacious low-cost drug available for treatment of malaria, chloroquine resistance has been exceptionally detrimental in third-world countries where the spread of malaria is rampant.
Point mutations in a 13 exon gene, crt, in Asian, African and South American chloroquine-resistant P. falciparum lines have implicated its product, a putative transmembrane protein, PfCRT, as the primary culprit behind chloroquine resistance (Fidock et al., 2000
). It is believed that mutations in PfCRT affect either drug binding to haematin through alteration of pH levels in the digestive vacuole or changes in chloroquine flux (Fidock et al., 2000). Evidence supporting the hypothesis that PfCRT could be a membrane transport protein includes two key factors. First, resistance to chloroquine appears to involve translocation away from the target area rather than alteration of the drug's chemical structure, and, second, PfCRT is predicted to have 10 membrane-spanning 
-helices, a characteristic of many transporters.
After four iterations of searching the non-redundant NCBI database with PSI-BLAST using PfCRT as the query sequence, a putative nucleotide sugar transporter (NST) in the drug/metabolite transporter (DMT) superfamily (TC : 2.A.7) was uncovered (Table 1) (Altschul et al., 1997; Jack et al., 2001). Comparison of the putative NST against TC-DB (Saier, 2000; Tran et al., 2003) using BLAST showed strong sequence similarity with a UDP-N-acetylglucosamine (UDP-GlcNAc) transporter (TC : 2.A.7.15.1). The hypothetical NST, PfCRT, and many proteins of the DMT superfamily are predicted to have 10 transmembrane spanners (TMSs) (Table 1); the UDP-GlcNAc transporters as well as many other DMT superfamily members have been shown to have arisen by duplication of a primordial 5 TMS element (Jack et al., 2001).
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) uncovered significant sequence similarity between the first five TMSs of the PfCRT protein and the first five TMSs of the putative NST. With homologues of PfCRT and homologues of the UDP-GlcNAc transporter taken from the PSI-BLAST search, we constructed a multiple alignment, revealing residues conserved primarily in the hydrophobic TMSs. Most conserved residues are glycines and hydrophobic residues, but some conserved polar residues were also identified (Fig. 1). The multiple sequence alignment was then applied to HMMER to create a Hidden Markov model (HMM) (Eddy, 1998). Searching the non-redundant NCBI protein database with the HMM resulted in the retrieval of both PfCRT homologues and UDP-GlcNAc homologues. No extraneous sequence artefacts were found with this search, signifying the specificity of the HMMER motif. The consensus sequence emitted by this HMM showed 27 well-conserved residues.
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). Additionally, we employed the PRSS program with 1000 shuffles (Table 2) (Pearson, 2000). After computing pairwise alignments between several pairs of proteins included in the multiple sequence alignment, we found significant sequence similarity between a PfCRT homologue in Arabidopsis thaliana and a DMT homologue in Homo sapiens with a Z-score exceeding 9 standard deviations (Saier, 1994). The strength of the sequence similarity between these two proteins strongly implies a common evolutionary link between PfCRT and the DMT superfamily. Since all functionally characterized members of the DMT superfamily are transporters (Tran et al., 2003), and since most families of transport proteins lack proteins that function in any capacity other than transport (Saier, 2003), we conclude that transport is the probable function of PfCRT.
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