Identification and analysis of putative homologues of mechanosensitive channels in pathogenic protozoa

Abstract

Mechanosensitive channels play important roles in the physiology of many organisms, and their dysfunction can affect cell survival. This suggests that they might be therapeutic targets in pathogenic organisms. Pathogenic protozoa lead to diseases such as malaria, dysentery, leishmaniasis and trypanosomiasis that are responsible for millions of deaths each year worldwide. We analyzed the genomes of pathogenic protozoa and show the existence within them of genes encoding putative homologues of mechanosensitive channels. Entamoeba histolytica, Leishmania spp., Trypanosoma cruzi and Trichomonas vaginalis have genes encoding homologues of Piezo channels, while most pathogenic protozoa have genes encoding homologues of mechanosensitive small-conductance (MscS) and K(+)-dependent (MscK) channels. In contrast, all parasites examined lack genes encoding mechanosensitive large-conductance (MscL), mini-conductance (MscM) and degenerin/epithelial Na(+) (DEG/ENaC) channels. Multiple sequence alignments of evolutionarily distant protozoan, amoeban, plant, insect and vertebrate Piezo channel subunits define an absolutely conserved motif that may be involved in channel conductance or gating. MscS channels are not present in humans, and the sequences of protozoan and human homologues of Piezo channels differ substantially. This suggests the possibility for specific targeting of mechanosensitive channels of pathogens by therapeutic drugs.

Figure 1. Homologues of Piezo channel subunits in pathogenic protozoa.

Phylogram showing the relationship between homologues of Piezo channel subunits (see Methods: based on 63 high-confidence positions from a multiple sequence alignment; gamma shape parameter 1.974; proportion of invariant sites 0.085). Homologues from different groups of organisms are indicated, along with the two phylogenetically distinct groups of homologues in trypanosomatid parasites. Branch length scale bar (amino acid substitutions per site) and branch support values >0.5 are shown. Protozoan homologues with incomplete sequences (see Table 1 ) are not shown.

Figure 2. Conserved domains in homologues of Piezo channel subunits.

Locations of predicted transmembrane domains (TMDs) and conserved motifs in the homologues of Piezo channel subunits from humans and pathogenic protozoa. Horizontal lines depict the length of each protein, while predicted TMDs are indicated by blue bars. The positions of TMDs along the length of each protein are shown to scale. A conserved arrangement of five TMDs near the C-terminal end of each protein is indicated by red shading. This region contains the conserved domain pfam12166 (Conserved Domains Database, NCBI). The conserved PFEW motif is indicated by a magenta triangle. Protozoan homologues with incomplete sequences (see Table 1 ) are not shown. Topologies were drawn using MyDomains (Swiss Institute of Bioinformatics; http://prosite.expasy.org/mydomains). Abbreviations are as follows: h, human; Eh, E. histolytica; Lm, L. major; Li, L. infantum; Lb, L. braziliensis; Tc, T.cruzi; Tv, T. vaginalis.

Figure 3. Conserved residues in homologues of Piezo channel subunits.

(A) Alignment of human Piezo1 with a homologue in L. infantum. Predicted TMDs are underlined. Asterisks below the alignment indicate residues conserved in human Piezo1 and the homologue shown from L. infantum. Colons indicate residues with highly similar properties. Magenta triangles indicate residues, including the PFEW motif, that are conserved absolutely in predicted homologues of Piezo in all organisms examined (Figure S1). An open circle above the alignment indicates a residue in human Piezo1 that is mutated in familial xerocytosis and alters channel gating . An open triangle above the alignment indicates a position at which the introduction of a stop codon alters gating . (B) Multiple sequence alignment of predicted Piezo homologues from protozoa, humans, mouse, Dictyostelium discoideum, Drosophila melanogaster, Danio rerio, Arabidopsis thaliana and Oryza sativa. The absolutely conserved PFEW motif is indicated and residues comprising this motif in each protein are shaded. Asterisks indicate absolutely conserved residues, while colons indicate residues with highly similar properties. Protozoan homologues with incomplete sequences (see Table 1 ) are not shown.

Figure 4. Homologues of MscS and MscK channel subunits in pathogenic protozoa.

Phylogram showing the relationship between bacterial and protozoan homologues of MscS and MscK channel subunits (see Methods: based on 113 high-confidence positions from a multiple sequence alignment; gamma shape parameter 3.83; proportion of invariant sites 0). Homologues from different groups of organisms are indicated. Branch length scale bar (amino acid substitutions per site) and branch support values >0.5 are shown.

Figure 5. The transmembrane regions of protozoan and bacterial MscS homologues have similar sequences.

Multiple sequence alignment of MscS homologues from Escherichia coli and trypanosomatid parasites. The TMD2 and TMD3 regions of MscS are indicated by bars above the alignment, and the predicted TMDs of individual proteins are underlined. Asterisks below the alignment indicate positions that have a single fully conserved residue, while colons below the alignment indicate positions that have residues with highly similar properties. Black triangles above the alignment indicate two glycine residues in MscS (G113 and G121) that may form gating hinges . Red triangles above the alignment indicate two leucine residues in MscS (L105 and L109) that may occlude the pore in the closed state . Open circles above the alignment indicate residues in MscS or the related MscK, which when mutated lead to a gain-of-function phenotype .