Label-free imaging of nuclear membrane for analysis of nuclear import of viral complexes.

HIV-1 enters the nucleus of non-dividing cells through the nuclear pore complex where it integrates into the host genome. The mechanism of HIV-1 nuclear import remains poorly understood. A powerful means to investigate the docking of HIV-1 at the nuclear pore and nuclear import of viral complexes is through single virus tracking in live cells. This approach necessitates fluorescence labeling of HIV-1 particles and the nuclear envelope, which may be challenging, especially in the context of primary cells. Here, we leveraged a deep neural network model for label-free visualization of the nuclear envelope using transmitted light microscopy. A training image set of cells with fluorescently labeled nuclear Lamin B1 (ground truth), along with the corresponding transmitted light images, was acquired and used to train our model to predict the morphology of the nuclear envelope in fixed cells. This protocol yielded accurate predictions of the nuclear membrane and was used in conjunction with virus infection to examine the nuclear entry of fluorescently labeled HIV-1 complexes. Analyses of HIV-1 nuclear import as a function of virus input yielded identical numbers of fluorescent viral complexes per nucleus using the ground truth and predicted nuclear membrane images. We also demonstrate the utility of predicting the nuclear envelope based on transmitted light images for multicolor fluorescence microscopy of infected cells. Importantly, we show that our model can be adapted to predict the nuclear membrane of live cells imaged at 37 °C, making this approach compatible with single virus tracking. Collectively, these findings demonstrate the utility of deep learning approaches for label-free imaging of cellular structures during early stages of virus infection.

Rho factor mediates flagellum and toxin phase variation and impacts virulence in Clostridioides difficile.

The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the "flagellar switch" upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained single-nucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5' leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile.

Discovery and Characterization of FMN-Binding ß-Glucuronidases in the Human Gut Microbiome.

The human gut microbiota encodes ß-glucuronidases (GUSs) that play key roles in health and disease via the metabolism of glucuronate-containing carbohydrates and drugs. Hundreds of putative bacterial GUS enzymes have been identified by metagenomic analysis of the human gut microbiome, but less than 10% have characterized structures and functions. Here we describe a set of unique gut microbial GUS enzymes that bind flavin mononucleotide (FMN). First, we show using mass spectrometry, isothermal titration calorimetry, and x-ray crystallography that a purified GUS from the gut commensal microbe Faecalibacterium prausnitzii binds to FMN on a surface groove located 30 Šaway from the active site. Second, utilizing structural and functional data from this FMN-binding GUS, we analyzed the 279 unique GUS sequences from the Human Microbiome Project database and identified 14 putative FMN-binding GUSs. We characterized four of these hits and solved the structure of two, the GUSs from Ruminococcus gnavus and Roseburia hominis, which confirmed that these are FMN binders. Third, binding and kinetic analysis of the FMN-binding site mutants of these five GUSs show that they utilize a conserved site to bind FMN that is not essential for GUS activity, but can affect KM. Lastly, a comprehensive structural review of the PDB reveals that the FMN-binding site employed by these enzymes is unlike any structurally characterized FMN binders to date. These findings reveal the first instance of an FMN-binding glycoside hydrolase and suggest a potential link between FMN and carbohydrate metabolism in the human gut microbiota.

Three structurally and functionally distinct ß-glucuronidases from the human gut microbe Bacteroides uniformis.

The glycoside hydrolases encoded by the human gut microbiome play an integral role in processing a variety of exogenous and endogenous glycoconjugates. Here we present three structurally and functionally distinct ß-glucuronidase (GUS) glycoside hydrolases from a single human gut commensal microbe, Bacteroides uniformis We show using nine crystal structures, biochemical, and biophysical data that whereas these three proteins share similar overall folds, they exhibit different structural features that create three structurally and functionally unique enzyme active sites. Notably, quaternary structure plays an important role in creating distinct active site features that are hard to predict via structural modeling methods. The enzymes display differential processing capabilities toward glucuronic acid-containing polysaccharides and SN-38-glucuronide, a metabolite of the cancer drug irinotecan. We also demonstrate that GUS-specific and nonselective inhibitors exhibit varying potencies toward each enzyme. Together, these data highlight the diversity of GUS enzymes within a single Bacteroides gut commensal and advance our understanding of how structural details impact the specific roles microbial enzymes play in processing drug-glucuronide and glycan substrates.