BIC: Journal Articles

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    ItemOpen Access
    Biomechanical responses of encysted zoospores of the oomycete Achlya bisexualis to hyperosmotic stress are consistent with an ability to turgor regulate
    (2022) Lacalendola N; Tayagui A; Ting M; Malmstrom J; Willmott GR; Garrill A; Nock, Volker
    Zoospores are motile, asexual reproductive propagules that enable oomycete pathogens to locate and infect new host tissue. While motile, they have no cell wall and maintain tonicity with their external media using water expulsion vacuoles. Once they locate host tissue, they encyst and form a cell wall, enabling the generation of turgor pressure that will provide the driving force for germination and invasion of the host. It is not currently known how these spores respond to the osmotic stresses that might arise due to different environments on and around their hosts that have different osmotic strengths. We have made microaspiration (MA) measurements on >800 encysted zoospores and atomic force microscopy (AFM) measurements on 12 encysted zoospores to determine their mechanical properties and how these change after hyperosmotic stress. Two types of encysted zoospores (Type A and Type B) were produced from the oomycete Achlya bisexualis, that differed in their morphology and response. With a small hyperosmotic stress (using 0.1 and 0.2 M sorbitol to give media osmolality changes of 155.4 and 295.6 mOsmol/kg), Type A zoospores initially became stiffer, with an increase in the Young’s modulus (E) over 30 mins from 0.16 MPa to 0.25 and 0.22 MPa respectively. E then returned to its original value after 120 min. With a greater osmotic stress (using 0.3, 0.4 and 0.5 M sorbitol to give media osmolality changes of 438.2, 587.2 and 787.6 mOsmol/kg) the reverse occurred, with an initial decrease in E over 30 – 60 mins to values of 0.1, 0.08 and 0.09 MPa respectively, before recovery to the original value after 120 min. In 0.5 M sorbitol this recovery was only observed with AFM, but not with MA. Type B zoospores, which may be primary/secondary spores about to release secondary/tertiary spores, or else spores that were damaged during encystment, initially stiffened in response to the lower hyperosmotic stresses with a slight increase in E (from 0.077 to 0.1 MPa after 15 min (with both 0.1 and 0.2 M sorbitol) before recovering to the original value after 60 min. These spores showed no change in response to the higher osmotic stresses. The responses of the Type A spores are consistent with rapid changes in cell wall thickness and a turgor regulation mechanism. Turgor regulation is further supported by microscopic observations of the Type A spores showing protoplast retraction from the cell wall followed by deplasmolysis, coupled with measurements of spore volume. As far as we are aware this is the first demonstration of turgor regulation, not just in encysted zoospores, but in oomycetes in general.
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    ItemOpen Access
    Three sites and you are out: Ternary synergistic allostery controls aromatic amino acid biosynthesis in Mycobacterium tuberculosis.
    (University of Canterbury. Biomolecular Interaction Centre, 2013) Blackmore, N.J.; Reichau, S.; Jiao, W.; Hutton, R.D.; Baker, E.N.; Jameson, G.B.; Parker, E.J.
    3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step in the shikimate pathway, the pathway responsible for the biosynthesis of the aromatic amino acids Trp, Phe, and Tyr. Unlike many other organisms that produce up to three isozymes, each feedback-regulated by one of the aromatic amino acid pathway end products, Mycobacterium tuberculosis expresses a single DAH7PS enzyme that can be controlled by combinations of aromatic amino acids. This study shows that the synergistic inhibition of this enzyme by a combination of Trp and Phe can be significantly augmented by the addition of Tyr.We used X-ray crystallography, mutagenesis, and isothermal titration calorimetry studies to show that DAH7PS from M. tuberculosis possesses a Tyr-selective site in addition to the Trp and Phe sites, revealing an unusual and highly sophisticated network of three synergistic allosteric sites on one enzyme. This ternary inhibitory response, by a combination of all three aromatic amino acids, allows a tunable response of the protein to changing metabolic demands.