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Guerrero-Ferreira, R. C.

Publications and source records attributed to Guerrero-Ferreira, R. C..

4 recordsLinked to original sources

Title: Multimodal profiling reveals Mycobacterium tuberculosis restricts lung mitochondrial immunometabolism to promote pathogenesis

Early events in the lung that shape protective immune responses to M. tuberculosis (Mtb) infection are not well understood but are critical for developing better vaccines and immunomodulatory therapies for tuberculosis. Here, we used high-dimensional flow cytometry, single-cell transcriptomics, and untargeted metabolomics to define the early lung immune environment that precedes the development of protective versus pathogenic outcomes following aerosolized Mtb infection of mice. We show that Mtb induced sustained glycolysis in the lung while restricting oxidative phosphorylation (OXPHOS) and impairing mitochondria, in part through the Mtb serine protease Hip1, leading to low energy output and suboptimal macrophage-T cell interactions that promoted pathogenic immunity. However, robust induction of mitochondrial OXPHOS, amino acid metabolism, and fatty acid oxidation in the early lung resulted in high ATP output and enhanced innate-T cell signaling networks that stimulated protective immune responses. Moreover, we identified a novel mitochondrial immunometabolic lung signature associated with protective outcomes to Mtb infection in animal models and humans. Our studies identify induction of mitochondrial dysfunction as a mechanism employed by Mtb to manipulate lung immunometabolism to its benefit and reveal that maintenance of intact mitochondrial metabolism in the early lung is pivotal for generating protective outcomes to Mtb infection.

immunology↗

Pulsed-electron illumination does not reduce beam damage for imaging biological macromolecules

Radiation damage remains a fundamental limitation in cryo-electron microscopy (cryo-EM), constraining the total electron dose that can be used and thus limiting high-resolution imaging of biological specimens. Recent studies have proposed that temporally structured or pulsed electron beams could reduce radiation damage by allowing time for energy dissipation between individual electron interactions. To evaluate this hypothesis, we conducted a systematic investigation using a radiofrequency (RF) driven 300 kV Titan Krios microscope equipped with cold field emission gun (c-FEG) to generate highly regular pulsed electron beams for specimens under cryogenic conditions. We compared radiation damage in three representative samples: paraffin 2D crystals, bacteriorhodopsin (purple membrane) 2D crystals, and plunge-frozen tobacco mosaic virus (TMV) in vitreous ice, under both pulsed and conventional (= random) illumination, while keeping all other imaging conditions constant. Radiation damage was quantified by tracking the decay of computed diffraction intensities to determine the critical dose (Ne). We observed no statistically significant difference in critical dose between pulsed and random illumination across all 3 samples. Our findings provide a critical reference point for future development and evaluation of temporally modulated electron sources in cryo-EM instrumentation.

molecular biology↗

Low-dose cryo-electron ptychography of proteins at sub-nanometer resolution

Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, cryo-EM and cryo-electron tomography are limited by the low signal-to-noise ratio (SNR) of recorded images, making detection of smaller particles challenging. For dose-resilient samples often studied in the physical sciences, electron ptychography - a coherent diffractive imaging technique using 4D scanning transmission electron microscopy (4D-STEM) - has recently demonstrated excellent SNR and resolution down to tens of picometers for thin specimens imaged at room temperature. Here we applied 4D-STEM and ptychographic data analysis to frozen hydrated proteins, reaching sub-nanometer resolution 3D reconstructions. We employed low-dose cryo-EM with an aberration-corrected, convergent electron beam to collect 4D-STEM data for our reconstructions. The high frame rate of the electron detector allowed us to record large datasets of electron diffraction patterns with substantial overlaps between the interaction volumes of adjacent scan positions, from which the scattering potentials of the samples were iteratively reconstructed. The reconstructed micrographs show strong SNR enabling the reconstruction of the structure of apoferritin protein at up to 5.8 [A] resolution. We also show structural analysis of the Phi92 capsid and sheath, tobacco mosaic virus, and bacteriorhodopsin at slightly lower resolutions.

biophysics↗

Flagellar structures from the bacterium Caulobacter crescentus and implications for phage {Phi}CbK predation of multi-flagellin bacteria

Caulobacter crescentus is a gram-negative alpha-proteobacterium that commonly lives in oligotrophic fresh and salt-water environments. C. crescentus is a host to many bacteriophages, including {phi}CbK and {phi}CbK-like bacteriophages, which first adsorb to cells by interaction with the bacterial flagellum. It is commonly thought that the six paralogs of the flagellin gene present in C. crescentus are important for bacteriophage evasion. Here, we show that deletion of specific flagellins in C. crescentus can indeed attenuate {phi}CbK adsorption efficiency, although no single deletion completely ablates {phi}CbK adsorption. Thus, bacteriophage {phi}CbK likely recognizes a common motif amongst the six known flagellins in C. crescentus with varying degrees of efficiency. Interestingly, we observe that most deletion strains still generate flagellar filaments, with the exception of a strain that contains only the most divergent flagellin, FljJ, or a strain that contains only FljN and FljO. To visualize the surface residues that are likely recognized by {phi}CbK, we determined two high-resolution structures of the FljK filament, with and without an amino acid substitution that induces straightening of the filament. We observe post-translational modifications on conserved surface threonine residues of FljK that are likely O-linked glycans. The possibility of interplay between these modifications and {phi}CbK adsorption is discussed. We also determined the structure of a filament composed of a heterogeneous mixture of FljK and FljL, the final resolution of which was limited to approximately 4.6 [A]. Altogether, this work builds a platform for future investigation of how phage {phi}CbK infects C. crescentus at the molecular level.

microbiology↗