Wheeler, R. | bioRxiv Science

bioRxiv2019

Crippling the bacterial cell wall molecular machinery

Lytic transglycosylases (LT) are redundant enzymes that play a critical role in peptidoglycan (PG) recycling and metabolism. LT(s) role in cell wall-modifying complexes and usefulness as antimicrobial drug targets remain elusive. We determined at high-resolution a structure of the membrane-bound homolog of the soluble LT from Neisseria species with a disordered active site helix (alpha helix 30). Alpha helix 30 is crucial for binding PG during catalysis1. Here we show using an alpha helix 30 deletion strain that LT (LtgA) determines the integrity of the cell wall, participates in cell division and separation, and can be manipulated to impair the fitness of the human pathogen Neisseria meningitidis during infection. Characterization of ltgA helix deleted strain interactome identified glycan chain remodeling enzymes whose function appear to be modulated by LTs. Targeting LTs can disrupt the PG machinery, which is fatal for the bacterium, a new approach for antibiotic development.

biochemistry↗

bioRxiv2019

High-speed multifocal plane fluorescence microscopy for three-dimensional visualisation of beating flagella

Analysis of flagellum beating in three dimensions is important for understanding how cells can undergo complex flagellum-driven motility and the ability to use fluorescence microscopy for such three-dimensional analysis would be extremely powerful. Trypanosoma and Leishmania are unicellular parasites which undergo complex cell movements in three dimensions as they swim and would particularly benefit from such an analysis. Here, high-speed multifocal plane fluorescence microscopy, a technique in which a light path multi-splitter is used to visualise 4 focal planes simultaneously, was used to reconstruct the flagellum beating of Trypanosoma brucei and Leishmania mexicana in three dimensions. It was possible to use either an organic fluorescent stain or a genetically-encoded fluorescence fusion protein to visualise flagellum and cell movement in three dimensions at a 200 Hz frame rate. This high-speed multifocal plane fluorescence microscopy approach was used to address two open questions regarding Trypanosoma and Leishmania swimming: To quantify the planarity of the L. mexicana flagellum beat and analyse the nature of flagellum beating during T. brucei tumbling.

cell biology↗