Actin-interacting and flagellar proteins in Leishmania spp.: Bioinformatics predictions to functional assignments in phagosome formation

Abstract

Several motile processes are responsible for the movement of proteins into and within the flagellar membrane, but little is known about the process by which specific proteins (either actin-associated or not) are targeted to protozoan flagellar membranes. Actin is a major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin-interacting proteins (AIPs) during both processes of motility and host cell entry might be key events for successful infection. For a better understanding the eukaryotic flagellar dynamics, we have surveyed genomes, transcriptomes and proteomes of pathogenic Leishmania spp. to identify pertinent genes/proteins and to build in silico models to properly address their putative roles in trypanosomatid virulence. In a search for AIPs involved in flagellar activities, we applied computational biology and proteomic tools to infer from the biological meaning of coronins and Arp2/3, two important elements in phagosome formation after parasite phagocytosis by macrophages. Results presented here provide the first report of Leishmania coronin and Arp2/3 as flagellar proteins that also might be involved in phagosome formation through actin polymerization within the flagellar environment. This is an issue worthy of further in vitro examination that remains now as a direct, positive bioinformatics-derived inference to be presented.

Keywords: Leishmania; actin-interacting proteins (AIPs); coronin and Arp2/3; flagellar proteins; phagosome.

Figure 1

Bioinformatics methods employed in this work, shown as a briefly simplified scheme of tools to illustrate a stepwise process in biological information technology.

Figure 2

Sequence-structural alignments of four Leishmania coronins. a), b), c) and d): 3D models of L. major (LmjF23.1165/CAC44941.1), L. infantum (LinJ23.1360), L. donovani (AAY56362.1) and L. braziliensis (LbrM23.1230), respectively, illustrating the putative actin-binding region (N-terminus). Left-panel: schematic diagram of corresponding regions of other coronin family proteins. The first 30 amino acids are shown to illustrate residues conserved across members of the coronin family (Trypanosoma brucei, Cryptosporidium parvum, Toxoplasma gondii, Plasmodium falciparum, Mus musculus and Bos taurus).

Figure 3

3D visualization of Arp2/3 complex modeling, including possible binding site to a coronin WDR domain at central (blue) region and multiple alignment of the amino acid sequences of Arp2/3 complex proteins from different organisms. A) PDB solved structure of Bos taurus Arp2/3 complex (1K8K) used as a template for building comparative models of Leishmania Arps with sequence from B) L. braziliensis (LbrM02.0360); C) L. infantum (LinJ02.0520) and D) L. major (XP_822258.1). *N-terminus; **C-terminus. E) Multiple alignment of the amino acid sequences of Arp2/3 complex proteins. Accession numbers are: T. cruzi (XP_810627.1), T. brucei (XP_951567.1), D. rerio (NP_991100.1), H. sapiens (NP_005709.1), S. cerevisiae (NP_012912.1), L. infantum (LinJ02.0520), L. major (XP_822258.1) and L. braziliensis (LbrM02.0360). Residues identical or similar are shaded dark or light gray, respectively. Dashes indicate gaps introduced for optimal alignment.

Figure 4

Two dimensional gel electrophoresis of Leishmania amazonensis flagellar fraction. Proteomic analysis after visualization by silver staining. Imaging covering narrow range IPG strips of pH 3-10 - observed in the gel top. The standard of molecular mass range used is 14400 Da to 97000 Da; -1 is a software configuration parameter. Six (06) putative spots for coronin are circled and indicated by a red arrow; four (04) Arp2/3 putative spots are circled and pointed by a yellow arrow. The molecular weight (in KDa) and the corresponding isoeletric point of each protein are shown in white.

Figure 5

Schematic illustration of Leishmania spp. proteins that are seemingly homologous (by sequence alignments) to the proteins of the virtual phagosome depicted on the article by Garin et al., 2001. Selected proteins were used in the present study in an attempt to suggest their potential interaction with phagosomes. Assigned localization to the lumen, the membrane, or the cytoplasmic aspect of the phagosome was the same indicated by Garin et al. (2001).