Regulation of Flagellar Motility in Giardia lamblia and Trichomonas vaginalis

Abstract

Protozoan pathogens are responsible for infections that are highly prevalent, especially in developing and under-developed countries. Flagellar motility is exhibited by a variety of organisms ranging from bacteria to certain cell types in mammals. In the context of pathogens, flagellar motility plays a significant role in the establishment of infection in the host. Unlike bacterial motility, where the mechanism and its contribution to pathogenesis is well studied, flagellar motility in protozoan parasites is an ill-explored field. Understanding the regulation of motility in these organisms would highly enhance our understanding of pathogenesis in these organisms and may possibly open new avenues of interventions. All eukaryotic flagella are made of microtubules and driven by dynein motor proteins. However, every organism is unique in terms of its flagellar waveform, beat frequency and its general motility pattern. In the current study, using biophysical and biochemical approach we studied the motility characteristics of Giardia lamblia and Trichomonas vaginalis, both flagellated protozoan pathogens which establish infection in the small intestine and the urogenital tract respectively. We have addressed the question of “how flagellar motility may be differentially regulated in an organelle as structurally conserved as the flagella”. We used a biophysical approach to first characterize the patterns of motility in both the organisms by combining microscopy and imaging with a high-speed camera to understand the movements of the cells to bring out the differences in terms of speed and beat frequency of the flagella. Our results demonstrate that despite overall conservation in flagellar structure, the pattern of tubulin post-translational modifications within the flagella are diverse and may contribute to variations in their patterns of motility. In this study, we have examined the tubulin post-translational modification in the protozoan parasites Giardia lamblia and Trichomonas vaginalis using global, untargeted mass spectrometry. Using cellular fractions generated by density gradient centrifugation we enriched flagellar fractions from the cell homogenate and examined the presence of novel post translational modifications by western blotting using specific antibodies as well as by mass spectrometry. Our results highlight organism specific 'tubulin code'. For example, we find that tubulin monoglycylation is a modification localized to the flagella present in G.lamblia but not so evident in T.vaginalis. We also showed the presence of glutamylated tubulin in both G.lamblia and T.vaginalis. Using MS/MS, we were also able to identify the previously unknown sites of monoglycylation in beta tubulin in G.lamblia as well as mono and tri glutamylation in T.vaginalis. Using isolated flagella, we also characterized the flagellar proteome in G.lamblia and T.vaginalis and identified 468 proteins in G.lamblia and 380 proteins in T.vaginalis flagella. The flagellar proteomes reveal unique mechanisms supporting energy generation required for flagellar motility in these organisms. Altogether, the flagellar proteomes as well as the sites of tubulin PTMs in these organisms, our study reveals potential mechanisms in regulating flagellar motilities in these neglected protozoan parasites.