Molecular and phylogenetic characterization of syntaxin genes from parasitic protozoa

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Abstract

Vesicular transport is an integral process in eukaryotic cells and the syntaxins, a member of the SNARE protein superfamily, are a critical piece of the vesicular transport machinery. We have obtained syntaxin homologues from diverse protozoan parasites (including Entamoeba, Giardia, Trichomonas and Trypanosoma), determined the paralogue affinity of the homologues by molecular phylogenetics and compared functionally critical amino acid sites identified in other syntaxins. Surprisingly, three sequences deviate at the signature glutamine residue position, conserved in all previously identified syntaxin homologues. It is known that, despite conserved structure and function of both the syntaxins and the proteins of the regulatory SM superfamily, the various syntaxin paralogues bind their respective SM partners at different regions of the syntaxin molecule. These sites of interactions have been identified down to the individual residues. The pattern of conservation at these residues, in our evolutionarily diverse sampling of syntaxin paralogues, is therefore used to gain further insight into the interaction of these proteins. Phylogenetic analysis confirms and extends previous conclusions that the syntaxin families are present in diverse eukaryotes and that the syntaxin sub-families diverged early in eukaryotic evolution. This result is expanded with the inclusion of new homologues for previously sampled taxa, newly sampled taxa, and newly sampled syntaxin sub-families. Because of their integral role in membrane trafficking, the syntaxin genes represent a valuable potential molecular marker for the experimental study of the endomembrane system of disease-causing protists.

Introduction

The membrane-trafficking system of eukaryotes enables critical functions such as secretion and endocytosis [1]. An important component of this membrane-trafficking machinery is the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs). These proteins have been implicated in a variety of processes including vesicle tethering [2], docking [3], fusion [4] as well as specificity of vesicular transport in the eukaryotic cell [5]. SNARE proteins are characterized by the presence of, at least, one copy of a homologous coiled-coil forming motif, entitled the SNARE motif [6]. There are four general classes of SNAREs (Qa, Qb, Qc and R) based on the presence of a signature glutamine (Q) or arginine (R) residue at a critical amino acid position in the SNARE motif, with the Q-SNAREs further classified based on the role that each one plays in the formation of the SNARE complex [1]. R-SNAREs, present on the membrane of an incoming transport vesicle, interact with those embedded in the membrane of the target organelle (Q-SNAREs) to form a complex containing four copies of the SNARE motif [1]. It is thought that this combinatorial nature of the SNARE complex provides the specificity of transport [5].

Syntaxins, the Qa class of SNAREs, comprise a coherent protein family based on primary and secondary structure [7]. In addition to the homologous SNARE motif, syntaxins have three N-terminal helices, interspaced with linker regions of variable size. These helices, denoted Ha, Hb and Hc, have been assigned a regulatory role in the fusion process [8], as has the linker region between the helices and the SNARE motif [9]. Syntaxins generally possess a C-terminal, membrane-spanning domain [10]. The syntaxin family itself contains several paralogous sub-families [7]. Each is involved in a specific step in the transport pathway, or is associated with a specific intracellular location, as summarized in Table 1. This specificity makes them attractive potential markers both for identifying cryptic organelles and for use in the study of membrane-trafficking processes.

While many studies have confirmed the conserved presence and function of the vesicular transport system in eukaryotes, a number of recent studies have uncovered differences in endomembrane system organization and function of molecular components among evolutionarily distant taxa [11], [12], [13]. This emphasizes the importance of a broad comparative approach in obtaining a general understanding of the eukaryotic endomembrane system. Much of the information regarding syntaxin function and diversity has been obtained from animal and fungal models, as well as a few studies in plants and slime molds. Our previous molecular phylogenetic examination of syntaxins from diverse eukaryotic taxa provided an evolutionary framework of the protein family’s history and established the wide spread presence of syntaxin homologues in eukaryotes [14].

As both secretion and endocytosis are incorporated into the cellular mechanism of pathogenesis in several protozoa, molecular sequence data of vesicular transport machinery from parasitic protists may provide targets for cell biological study of their endomembrane systems thus providing specific insight into the biology underpinning their pathogenesis. We chose to examine syntaxin genes from five taxa including the agricultural parasite Phytophthora, and the human parasites Entamoeba, Trypanosoma, Giardia and Trichomonas.

Organisms in the genus Phytophthora are pathogens of non-cereal crops, most famously as the cause of the Irish potato famine of the 1840s [15]. While traditionally grouped with parasitic fungi due to their hyphal growth patterns, Phytophthora is, in fact, much more closely related to brown algae and diatoms as a member of the stramenopile clade [16], [17]. Studies of Phytophthora’s pathogenic mechanism have shown secretion to play a major role with delivery of adhesins allowing for the infection of plant material [18].

A member of the Amoebozoa clade [19], Entamoeba histolytica is the third largest cause of parasitic death, responsible for up to 111,000 fatalities per year [20]. Cysteine proteinases, either on the amoeba surface, or secreted into the medium are very important for the organism in both infection and immune system evasion [20].

The remaining protozoan parasites studied here have been evolutionarily assigned to a large eukaryotic supergroup called the Excavates, based on a combination of morphological and molecular data [21]. Kinetoplastid flagellates of the genus Trypanosoma cause sleeping sickness in Africa (T. brucei) and Chagas disease in South America (T. cruzi) [22]. Immune system evasion mechanisms in T. brucei include cell surface receptor recycling and immunoglobin degradation via the endocytic pathway [23]. In the mammalian bloodstream form of the trypanosomatid, the rate of endocytosis is upregulated 10-fold [23]. Secretion plays a major role in the infectious process of Giardia intestinalis, one of the world’s leading agents of human enteric disease [24]. Cell wall proteins are exported to form the resistant Giardia cysts crucial for transfer of the pathogen between hosts. Variable surface proteins are also routed via a secretory pathway and are displayed at the cell surface in a mechanism of antigenic variation involved in host immune system evasion [24]. Our final organism of study, the parabasalid flagellate Trichomonas vaginalis, is the causative agent of the most prevalent, non-viral, sexually transmitted disease in the world [25]. Characterized by its prominent Golgi apparatus, Trichomonas secretes cysteine proteinases to evade the host immune response [25]. The endocytic uptake of iron-binding proteins has also been shown to be correlated with pathogenicity of the organism [26].

Here we present nine novel syntaxin gene sequences from these diverse parasitic protozoa. The sequences are characterized at a primary sequence and molecular phylogenetic level to further examine the evolution of the syntaxin gene family, the conservation of previously identified functional sites within the protein and to yield insight into the biology of membrane trafficking in the parasites themselves.

Section snippets

Syntaxin homologue identification

Putative syntaxin genes were identified using a reciprocal BLAST method to search various genomic databases. BLAST analysis, performed at the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/), used default settings. A cut-off value of 0.05 was used when selecting potential homologues due to the short length of the protein and due to their low conservation. Each retrieved sequence was reciprocally used as a query back to the “non-redundant” database. Only sequences that retrieved the

Identification and characterization of syntaxin homologues

Partial putative syntaxin sequences from the chosen parasitic protozoa were identified using the BLAST algorithm to search various genomic databases. After obtaining the polished, and in all cases but the T. bru-syn16 and T. cru-syn7 genes (Table 2), full length sequence of the ORF, BLASTp analysis was used to confirm the validity of the putative identification. The results of these analyses are summarized in Table 2. The E. histolytica syntaxin 5 sequence produced a Drosophila melanogaster

Evolution of syntaxin sub-families

Previous work on the evolution of the syntaxin family in eukaryotes showed that five paralogous syntaxin sub-families (syntaxin PM, syntaxin 5, 6, 16, and the endosomal syntaxins) arose by a series of gene duplications early on in the history of eukaryotes [14]. The eight novel syntaxin protein sequences from parasitic protozoa determined here have extended these results in a number of ways. Deductions regarding the antiquity of the syntaxin gene family are based on the premise that, if at

Conclusion

Examining the syntaxin genes from a wide range of parasitic protozoa has yielded insight into the evolution of the gene family, as well as provided some important comparative data for crucial amino acid positions. This comparative data, in turn, may well allow for a more generalisable understanding of syntaxin function in vesicular fusion. The gene sequence of syntaxin homologues also provide an avenue of experimentation for the better understanding of how the endomembrane system of these

Acknowledgements

We would like to thank A. Roger, S. Hadjuk, P. Hoffman for providing genomic DNA and the Phytophthora Genome Initiative, Daniel O. Sanchez, and the Protist EST project (John Logsdon, Mark Ragan, Andew Roger, Robert Hirt and T. Martin Embley) for generously sending clones to be analyzed. Thanks also to the Giardia, Entamoeba and Trypanosoma genome initiatives for making their data publicly available. We would also like to thank R. Hirt, M. Field and H. Lujan for critical reading of this

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    Note: Nucleotide sequence data reported in this paper are available in the GenBank, EMBL and DDBJ databases under the accession numbers AY344235–AY344243.

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