Sequence polymorphism in the beta-tubulin gene reveals heterogeneous and variable population structures in Cryptosporidium parvum

Abstract

Restriction fragment length polymorphism (RFLP) analysis of isolates of Cryptosporidium parvum has revealed two subgroups, termed H and C. The limited resolution of the RFLP method precludes an in-depth study of the genetic structure of C. parvum populations. Published C. parvum restriction polymorphisms lie within protein-coding regions known to be more homogeneous than noncoding sequences. To better assess the degrees of heterogeneity between and within C. parvum isolates, sequence polymorphism in the beta-tubulin intron, the only C. parvum intron described to date, was investigated. In contrast to the two genotypes distinguished by multilocus RFLP, several alleles were detected by sequence and RFLP analysis of the beta-tubulin intron and adjacent exon 2. Isolates carrying different beta-tubulin alleles were found. Significantly, one of the beta-tubulin alleles present in two geographically unrelated isolates combined features of C- and H-type isolates, suggesting that it might have arisen from a recombination event. A comparison of multiple samples of a calf-propagated laboratory isolate showed that the ratio of different beta-tubulin alleles fluctuated during serial passage.

FIG. 1

(A) Restriction maps of four β-tubulin alleles of C. parvum. The fragment sizes are indicated for each allele. The approximate locations of the Tsp509I sites (vertical lines), the intron (black box), and the T repeat (hatched box) were derived from multiple, aligned sequences. The PCR fragment is 478 bp long in GCH1. The codes for the isolates from which these sequences were amplified are shown at the left. Arrows show the approximate locations of the PCR primers used in this study. btub2 is located 60 bp downstream of btub4 and, together with primer btub5, amplifies a 538-bp fragment. The location of the restriction site defining the Tsp509I⁺ and Tsp509I⁻ alleles is marked (*). The dashed line indicates the approximate range of the sequence included in the alignment shown in Fig. 2. (B) Tsp509I restriction profiles of isolates shown in panel A. GCH1 lanes − and + designate unrestricted and restricted btub5-btub4 PCR products, respectively. The arrow indicates the 99-bp fragment mentioned in the text. Lane M, 100-bp DNA ladder. The sizes (in base pairs) of three bands are shown at the right.

FIG. 2

Sequence alignment of β-tubulin sequences from 14 C. parvum isolates. The alignment was generated with the PileUp program (Wisconsin Sequence Analysis Package). Isolate codes are shown on the left. The number following the hyphen designates the plasmid number. The multilocus RFLP genotype (15) is indicated leftmost. Only the polymorphic positions are shown. Changes from the sequence under GenBank accession no. Y12615 are shaded. A 6-bp duplication in Y12615 presumed to be artifactual was omitted (see text). Selected positions are numbered below the sequences according to accession no. Y12615. The intron-exon boundary is indicated with a vertical line. The arrowhead at position 399 indicates the 5′-most nucleotide of a polymorphic Tsp509I site in exon 2. Clones icp-1, tamu-2, tamu-5, 0541L-5, 0541L-6, 0541L-8, 2066K-5, and 0583K-8 were sequenced on both strands. All others were sequenced on one strand. A sequence ambiguity in 0541L-9 is indicated in lowercase.

FIG. 3

Variable β-tubulin restriction profiles in GCH1 oocysts from three calf passages. A 538-bp fragment was PCR amplified with primers btub5 and btub2 from three samples of GCH1 oocyst DNA collected from calf passages at the dates shown (lanes 2 to 4). The difference in restriction profiles was caused by variable ratios of β-tubulin alleles with or without a Tsp509I site at position 399. The sizes of the uncut PCR product (lane 1) and the relevant restriction fragments are shown (in base pairs) at the left. The length of the uncut PCR product (538 bp) and digests (408 bp or 309 and 99 bp) is not additive due to the presence of several unresolved small fragments totalling 129 to 133 bp (Fig. 1A). The positions of 600- and 100-bp size markers are indicated on the right.