RavN is a member of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases.

The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway.

25-Hydroxycholesterol Production by the Cholesterol-25-Hydroxylase Interferon-Stimulated Gene Restricts Mammalian Reovirus Infection.

Following the initial detection of viral infection, innate immune responses trigger the induction of numerous interferon-stimulated genes (ISGs) to inhibit virus replication and dissemination. One such ISG encodes cholesterol-25-hydroxylase (CH25H), an enzyme that catalyzes the oxidation of cholesterol to form a soluble product, 25-hydroxycholesterol (25HC). Recent studies have found that CH25H is broadly antiviral; it inhibits infection by several viruses. For enveloped viruses, 25HC inhibits membrane fusion, likely by altering membrane characteristics such as hydrophobicity or cholesterol aggregation. However, the mechanisms by which 25HC restricts infection of nonenveloped viruses are unknown. We examined whether 25HC restricts infection by mammalian reovirus. Treatment with 25HC restricted infection by reovirus prototype strains type 1 Lang and type 3 Dearing. In contrast to reovirus virions, 25HC did not restrict infection by reovirus infectious subvirion particles (ISVPs), which can penetrate either directly at the cell surface or in early endosomal membranes. Treatment with 25HC altered trafficking of reovirus particles to late endosomes and delayed the kinetics of reovirus uncoating. These results suggest that 25HC inhibits the efficiency of cellular entry of reovirus virions, which may require specific endosomal membrane dynamics for efficient membrane penetration.IMPORTANCE The innate immune system is crucial for effective responses to viral infection. Type I interferons, central components of innate immunity, induce expression of hundreds of ISGs; however, the mechanisms of action of these antiviral proteins are not well understood. CH25H, encoded by an ISG, represents a significant constituent of these cellular antiviral strategies, as its metabolic product, 25HC, can act in both an autocrine and a paracrine fashion to protect cells from infection and has been shown to limit viral infection in animal models. Further investigation into the mechanism of action of 25HC may inform novel antiviral therapies and influence the use of mammalian reovirus in clinical trials as an oncolytic agent.

Host Cell-catalyzed S-Palmitoylation Mediates Golgi Targeting of the Legionella Ubiquitin Ligase GobX.

The facultative intracellular pathogen Legionella pneumophila, the causative agent of Legionnaires disease, infects and replicates within human alveolar macrophages. L. pneumophila delivers almost 300 effector proteins into the besieged host cell that alter signaling cascades and create conditions that favor intracellular bacterial survival. In order for the effectors to accomplish their intracellular mission, their activity needs to be specifically directed toward the correct host cell protein or target organelle. Here, we show that the L. pneumophila effector GobX possesses E3 ubiquitin ligase activity that is mediated by a central region homologous to mammalian U-box domains. Furthermore, we demonstrate that GobX exploits host cell S-palmitoylation to specifically localize to Golgi membranes. The hydrophobic palmitate moiety is covalently attached to a cysteine residue at position 175, which is part of an amphipathic α-helix within the C-terminal region of GobX. Site-directed mutagenesis of cysteine 175 or residues on the hydrophobic face of the amphipathic helix strongly attenuated palmitoylation and Golgi localization of GobX. Together, our study provides evidence that the L. pneumophila effector GobX exploits two post-translational modification pathways of host cells, ubiquitination and S-palmitoylation.

Second Time's the Charm? Assessing the Sensitivity and Yield of Inpatient Diagnostic Algorithms for Pulmonary Tuberculosis in a Low-Prevalence Setting.

Background: For persons with suspected pulmonary tuberculosis, the guidelines of the Centers for Disease Control and Prevention recommend collecting 3 respiratory specimens 8 to 24 hours apart for acid-fast bacilli (AFB) smear and culture, in addition to 1 nucleic acid amplification test (NAAT). However, data supporting this approach are limited. Our objective was to estimate the performance of 1, 2, or 3 AFB smears with or without NAATs to detect pulmonary tuberculosis in a low-prevalence setting. Methods: We conducted a retrospective study of hospitalized persons at 8 Massachusetts acute care facilities who underwent mycobacterial culture on 1 or more respiratory specimens between July 2016 and December 2022. We evaluated percentage positivity and yield on serial AFB smears and NAATs among people with growth of Mycobacterium tuberculosis on mycobacterial cultures. Results: Among 104 participants with culture-confirmed pulmonary tuberculosis, the first AFB smear was positive in 41 cases (39%). A second AFB smear was positive in 11 (22%) of the 49 cases in which it was performed. No third AFB smears were positive following 2 initial negative smears. Of 52 smear-negative cases, 36 had a NAAT performed, leading to 23 additional diagnoses. Overall sensitivity to detect tuberculosis prior to culture positivity was higher in any strategy involving 1 or 2 NAATs (74%-79%), even without AFB smears, as compared with 3 smears alone (60%). Conclusions: Tuberculosis diagnostic testing with 2 AFB smears offered the same yield as 3 AFB smears while potentially reducing laboratory burden and duration of airborne infection isolation. Use of 1 or 2 NAATs increased sensitivity to detect culture-positive pulmonary tuberculosis when added to AFB smear-based diagnostic testing alone.

Impact of Enlarged Nonhypermetabolic Lymph Nodes on Outcomes After Stereotactic Body Radiotherapy for Early-Stage Non-Small-Cell Lung Cancer.

BACKGROUND: Up to 15% of patients undergoing positron emission tomography (PET)/computed tomography (CT) before stereotactic body radiotherapy (SBRT) harbor occult nodal disease. In the absence of invasive mediastinal staging, the clinical significance of enlarged nonhypermetabolic lymph nodes (LNs) remains unclear. We performed what is to our knowledge the first study to address whether enlarged nonhypermetabolic LNs were associated with higher post-SBRT failure rates. PATIENTS AND METHODS: Two academic centers assessed 157 consecutive patients treated with SBRT for cT1-2aN0M0 non-small-cell lung cancer who underwent PET/CT without pathologic nodal staging. The cutoff of an enlarged node was ≥ 1.0 cm, although a 7 mm threshold was also evaluated. Local recurrence-free survival (RFS), regional RFS, distant metastasis-free survival, RFS, and overall survival (OS) were calculated by Kaplan-Meier methodology. Multivariate Cox modeling addressed factors associated with RFS and OS. RESULTS: There were 120 patients (76%) with LNs < 1 cm and 37 (24%) with nodes ≥ 1 cm. Most patients had peripheral and/or T1 tumors. Median follow-up was 25.5 months. There were no differences between cohorts in actuarial local RFS, regional RFS, distant metastasis-free survival, RFS, or OS (P > .05 for all). Thirteen percent of patients experienced any nodal relapse, 15% of which occurred in the same station as that of the largest pre-SBRT LN. Stratification by largest LN location in an N1 versus N2 station showed no differences in RFS or OS (P > .05 for both). A 7 mm cutoff also showed no differences in outcomes (P > .05 for all). LN size was not correlated with RFS/OS on multivariable analysis (P > .05 for both). CONCLUSION: The presence of enlarged nonhypermetabolic LNs on PET/CT is not associated with increased post-SBRT failure rates.