Carreno, D. | Search bioRxiv

bioRxiv2023

On-grid purification of electron microscopy samples via a 3D-printed flow-cell

While recent advances in cryo-EM, coupled with single particle analysis, have the potential to allow structure determination in a near-native state from vanishingly few individual particles, this vision has yet to be realised in practise. Requirements for particle numbers that currently far exceed the theoretical lower limits, challenges with the practicalities of achieving high concentrations for difficult-to-produce samples, and inadequate sample-dependent imaging conditions, all result in significant bottlenecks preventing routine structure determination using cryo-EM. Therefore, considerable efforts are being made to circumvent these bottlenecks by developing affinity purification of samples on-grid; at once obviating the need to produce large amounts of protein, as well as more directly controlling the variable, and sample-dependent, process of grid preparation. In this proof-of-concept study, we demonstrate a further practical step towards this paradigm, developing a 3D-printable flow-cell device to allow on-grid affinity purification from raw inputs such as whole cell lysates, using graphene oxide-based affinity grids. Our flow-cell device can be interfaced directly with routinely-used laboratory equipment such as liquid chromatographs, or peristaltic pumps, fitted with standard chromatographic (1/16") connectors, and can be used to allow binding of samples to affinity grids in a controlled environment prior to the extensive washing required to remove impurities. Furthermore, by designing a device which can be 3D printed and coupled to routinely used laboratory equipment, we hope to increase the accessibility of the techniques presented herein to researchers working towards single-particle macromolecular structures.

biophysics↗

bioRxiv2022

Diurnal differences in intracellular replication within splenic macrophages correlates with the outcome of pneumococcal infection.

Circadian rhythms affect the progression and severity of bacterial infections including those caused by Streptococcus pneumoniae, but the mechanisms responsible for this phenomenon remain largely elusive. Following advances in our understanding of the role of replication of S. pneumoniae within a specific subset of splenic macrophages, we sought to investigate events within the spleen that correlate with differential outcomes of invasive pneumococcal infection. Utilising murine invasive pneumococcal disease (IPD) models, here we report that infection during the murine active phase (zeitgeber time; 15h after start of light cycle, 3h after start of dark cycle) resulted in significantly faster onset of moderate septicaemia compared to rest phase (zeitgeber time 3; 3h after start of light cycle) infection. These findings correlated with significantly higher pneumococcal burden within the spleen of active phase-infected mice at early time points compared to rest phase-infected mice. Whole-section confocal microscopy analysis of these spleens revealed that the number of pneumococci is significantly higher exclusively within marginal zone metallophilic macrophages (MMMs), known to allow intracellular pneumococcal replication as a prerequisite step to the onset of septicaemia. Pneumococcal clusters within MMMs were more abundant and increased in size in active phase-infected mice compared to those in rest phase-infected mice which decreased in size over time and were present in a lower percentage of MMMs. This phenomenon preceded significantly higher levels of bacteraemia alongside serum IL-6 and TNF- concentrations in active phase-infected mice following re-seeding of pneumococci into the blood. In summary, these data link the difference in susceptibility to invasive pneumococcal infection to variation in the ability of MMMs to successfully control and digest phagocytosed bacteria. Author summaryCircadian rhythms are present within the majority of multicellular organisms and influence almost all aspects of our physiology. As such, circadian rhythm disorders have been shown to result in an increased susceptibility to certain diseases. The effects of host circadian rhythm have been also mirrored in rodent studies, with the outcome of Streptococcus pneumoniae infection being dependent on the time of challenge. Whilst studies into the functional effects of circadian rhythm on the host immune system are present, knowledge of how these contribute to the control of invasive S. pneumoniae infection are lacking, especially considering the recent breakthrough in understanding the stages of pneumococcal pathogenesis. We show here that mice infected with S. pneumoniae during their active phase developed septicaemia quicker than those infected during their rest phase. We demonstrate that this is likely due to increased replication of pneumococci specifically within a subset of splenic macrophages, which subsequently results in increased numbers of pneumococci in the blood and higher levels of pro-inflammatory cytokines which result in septicaemia. These data provide novel insights into how circadian rhythm influences the immune functionality of the spleen, and how the regulation of function of one macrophage subtype can significantly alter the course of infection.

microbiology↗