Myosins ofBabesia bovis: Molecular characterisation, erythrocyte invasion, and phylogeny

Abstract

Using degenerate primers, three putative myosin sequences were amplified from Australian isolates of Babesa bovis and confirmed as myosins (termed Bbmyo-A, Bbmyo-B, and Bbmyo-C) from in vitro cultures of the W strain of B. bovis. Comprehensive analysis of 15 apicomplexan myosins suggests that members of Class XIV be defined as those with greater than 35% myosin head sequence identity and that these be further subclassed into groups bearing above 50-60% identity. Bbmyo-A protein bears a strong similarity with other apicomplexan myosin-A type proteins (subclass XIVa), the Bbmyo-B myosin head protein sequence exhibits low identity (35-39%) with all members of Class XIV, and 5'-sequence of Bbmyo-C shows strong identity (60%) with P. falciparum myosin-C protein. Domain analysis revealed five divergent IQ domains within the neck of Pfmyo-C, and a myosin-N terminal domain as well as a classical IQ sequence unusually located within the head converter domain of Bbmyo-B. A cross-reacting antibody directed against P. falciparum myosin-A (Pfmyo-A) revealed a zone of approximately 85 kDa in immunoblots prepared with B. bovis total protein, and immunofluorescence inferred stage-specific myosin-A expression since only 25% of infected erythrocytes with mostly paired B. bovis were immuno-positive. Multiplication of B. bovis in in vitro culture was inhibited by myosin- and actin-binding drugs at concentrations lower than those that inhibit P. falciparum. This study identifies and classifies three myosin genes and an actin gene in B. bovis, and provides the first evidence for the participation of an actomyosin-based motor in erythrocyte invasion in this species of apicomplexan parasite.

New insights into myosin evolution and classification

“…Class XIV had previously been described as exclusively comprising apicomplexan myosins and was divided into two subclasses (25,26). In contrast, our analyses (Fig.…”

“…2 and Table 2). That this robust association had not been recognized previously (4,19,25,26), with the ciliate myosins recently having been assigned to a separate class (19), is most likely because of the inclusion of rogue sequences, small dataset size, and the use of full-length myosin sequences in previous analyses. Based on our strong statistical support for this extended class XIV and on independent corroborating evidence (see below), we now include these 12 ciliate myosins in class XIV as subclass XIVd (Fig.…”

“…Researchers commonly have named new apicomplexan myosins in the order of discovery (MyoA, MyoB, etc. ), often independently for each species (22)(23)(24)(25)(26)(27)(28). This has led to the confusing situation where nonorthologous apicomplexan sequences were designated with the same letter, e.g., PfMyoC (PfM-C) and TgMyoC of Plasmodium falciparum and T. gondii, respectively (25).…”

“…2), and thus represent the first examples of ATS1-like domains associated with myosin tails. Class XIVc myosins also feature six or more IQ motifs and include the previously unclassified BbMyoH (Table 4) (25,26).…”

Myosin genes in Tetrahymena

“…In the apicomplexa, a group of parasitic protists that includes Babesia, Gregarina, Plasmodium, and Toxoplasma, 17 myosins have been identified; all but one cluster in class XIV, and all have been identified only in apicomplexa [Chaparro-Olaya et al, 2005;Heintzelman and Schwartzman, 1997;Heintzelman, 2004;Hettmann et al, 2000;Lew et al, 2002]. In the ciliophora, Whether the myosins in Tetrahymena are limited to this organism will have to await further identification of myosins in other protists.…”