Browsing by Author "Woodward, Jeremy"

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    Cryo-electron microscopy of HPV16 pseudovirions reveal changes in capsid conformation upon furin cleavage
    (2021) Marx, Melissa Lauren; Schafer, Georgia; Woodward, Jeremy
    Persistent infection by oncogenic human papillomavirus (HPV) is the primary cause of cervical cancer, a leading cause of cancer deaths in women worldwide. There are no treatments for HPV infection, and although prophylactic vaccines are effective and safe, they are HPV type specific, provide little therapeutic benefit and developing countries often have limited access to these. Therefore, additional measures against HPV infection are urgently needed. Preventing HPV entry into host cells is an attractive option for therapeutic intervention. The HPV capsid is icosahedral and consists of two proteins, L1 and L2, which participate in entry and infection of host cells. During entry, the virus capsid attaches to the cell surface via binding to heparan sulphate proteoglycans (HSPGs). Cleavage of L2 by a host protease, furin, is necessary for infection and is thought to facilitate a conformational change in the virus capsid. Furin cleavage may affect the ability of HPV to bind to sulphated glycoproteins and a HSPG substitute, heparin. Understanding these proposed structural changes may aid in the development of therapeutics targeting virus entry. Here, we directly visualize the conformation changes to HPV16 pseudovirions (HPV16 PsVs) resulting from cleavage of L2 by exogenous furin using cryoelectron microscopy (cryo-EM). At 5 Å resolution, we observed that furin-cleaved HPV16 PsVs capsids display widespread changes in the arrangement of capsomeres relative to uncleaved control virions. This structural change is relevant because heparin has previously been observed to bind to the HPV16 capsid in the canyon surrounding the capsomere at the five-fold icosahedral symmetry axis, but not in other canyons between capsomeres, related by pseudo-symmetry. This suggests that differences in the relative orientations of the surrounding capsomeres to each other either prevent or allow heparin binding. We observed a narrowing of the putative heparin binding site by 0.4 Å after furin cleavage and propose that this change may be responsible for the transfer of HPV from cell-surface HSPGs to the unknown entry receptor(s) by a yet unidentified mechanism.
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    Real-time investigation of tuberculosis transmission: developing the Respiratory Aerosol Sampling Chamber (RASC)
    (Public Library of Science, 2016) Wood, Robin; Morrow, Carl; III, Clifton E Barry; Bryden, Wayne A; Call, Charles J; Hickey, Anthony J; Rodes, Charles E; Scriba, Thomas J; Blackburn, Jonathan; Issarow, Chacha; Mulder, Nicola; Woodward, Jeremy; Moosa, Atica; Singh, Vinayak; Mizrahi, Valerie; Warner, Digby F
    Knowledge of the airborne nature of respiratory disease transmission owes much to the pioneering experiments of Wells and Riley over half a century ago. However, the mechanical, physiological, and immunopathological processes which drive the production of infectious aerosols by a diseased host remain poorly understood. Similarly, very little is known about the specific physiological, metabolic and morphological adaptations which enable pathogens such as Mycobacterium tuberculosis ( Mtb ) to exit the infected host, survive exposure to the external environment during airborne carriage, and adopt a form that is able to enter the respiratory tract of a new host, avoiding innate immune and physical defenses to establish a nascent infection. As a first step towards addressing these fundamental knowledge gaps which are central to any efforts to interrupt disease transmission, we developed and characterized a small personal clean room comprising an array of sampling devices which enable isolation and representative sampling of airborne particles and organic matter from tuberculosis (TB) patients. The complete unit, termed the Respiratory Aerosol Sampling Chamber (RASC), is instrumented to provide real-time information about the particulate output of a single patient, and to capture samples via a suite of particulate impingers, impactors and filters. Applying the RASC in a clinical setting, we demonstrate that a combination of molecular and microbiological assays, as well as imaging by fluorescence and scanning electron microscopy, can be applied to investigate the identity, viability, and morphology of isolated aerosolized particles. Importantly, from a preliminary panel of active TB patients, we observed the real-time production of large numbers of airborne particles including Mtb , as confirmed by microbiological culture and polymerase chain reaction (PCR) genotyping. Moreover, direct imaging of captured samples revealed the presence of multiple rod-like Mtb organisms whose physical dimensions suggested the capacity for travel deep into the alveolar spaces of the human lung.
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    The 2.7 resolution cryo-EM reconstruction of Mycobacterium tuberculosis encapsulin nanocompartment containing DyP peroxidase
    (2024) Willmore, Rhys; Woodward, Jeremy
    Mycobacterium tuberculosis has evolved many persistence factors in response to the host generated immune response as a means of survival. One such immune response generated by humans is the use of reactive oxygen species (ROS) such as H2O2 to cause damage to M. tuberculosis. Encapsulin (from here on referred to as Enc) nanocompartments and the cargo proteins within have been implicated as persistence factors and decreased viability of cells has been shown when they are knocked out. Previous research has found that dye-decolorizing peroxidase (DyP) is encapsulated by these Enc nanocompartments, as has been shown in Mycobacterium smegmatis. This is done by way of a C-terminal targeting peptide, with Enc and DyP also being part of the same operon in the genome of M. tuberculosis. However, not much is understood about the structure and function of this system. Both encapsulin and DyP were expressed and purified recombinantly in E. coli. Cryo- EM particle processing and EM map reconstruction was carried in an attempt to generate a density map of encapsulated DyP along with model building. Native expression and purification were carried out followed by negative-stain EM on this sample.Successful recombinant expression and purification of DyP and Enc was achieved, with a high-resolution 2.7 Å cryo-EM structure of the Enc nanocompartment obtained, but no encapsulated DyP was visualized. A model of both the Enc monomer and multimer was built, with comparisons to M. smegmatis in charge around the fivefold pore showing differences. The purification of Enc from M. tuberculosis was successful and a negative stain reconstruction of the nanocompartment was obtained. 2D classes showed what could have been DyP but it did not show up in any 3D models. Ferritin was shown to only be outside of the nanocompartment. The high-resolution map showed high similarity to other T=1 Enc nanocompartments. The multimer model built called into question the exact function of this nanocompartment as the charge distribution differed from closely related M. smegmatis. When these charge changes are correlated to findings of catalytic analysis done in the same lab, it indicates that more research is needed to understand the exact function of this system in M. tuberculosis. The 2D classes showed that DyP is the only cargo protein that appears to be present in M. tuberculosis nanocompartments, with ferritin being identified outside the nanocompartment.