Coxiella co-opts the Glutathione Peroxidase 4 to protect the host cell from oxidative stress-induced cell death.

The causative agent of human Q fever, Coxiella burnetii, is highly adapted to infect alveolar macrophages by inhibiting a range of host responses to infection. Despite the clinical and biological importance of this pathogen, the challenges related to genetic manipulation of both C. burnetii and macrophages have limited our knowledge of the mechanisms by which C. burnetii subverts macrophages functions. Here, we used the related bacterium Legionella pneumophila to perform a comprehensive screen of C. burnetii effectors that interfere with innate immune responses and host death using the greater wax moth Galleria mellonella and mouse bone marrow-derived macrophages. We identified MceF (Mitochondrial Coxiella effector protein F), a C. burnetii effector protein that localizes to mitochondria and contributes to host cell survival. MceF was shown to enhance mitochondrial function, delay membrane damage, and decrease mitochondrial ROS production induced by rotenone. Mechanistically, MceF recruits the host antioxidant protein Glutathione Peroxidase 4 (GPX4) to the mitochondria. The protective functions of MceF were absent in primary macrophages lacking GPX4, while overexpression of MceF in human cells protected against oxidative stress-induced cell death. C. burnetii lacking MceF was replication competent in mammalian cells but induced higher mortality in G. mellonella, indicating that MceF modulates the host response to infection. This study reveals an important C. burnetii strategy to subvert macrophage cell death and host immunity and demonstrates that modulation of the host antioxidant system is a viable strategy to promote the success of intracellular bacteria.

Eat, prey, love: Pathogen-mediated subversion of lysosomal biology.

The mammalian lysosome is classically considered the 'garbage can' of the cell, contributing to clearance of infection through its primary function as a degradative organelle. Intracellular pathogens have evolved several strategies to evade contact with this harsh environment through subversion of endolysosomal trafficking or escape into the cytosol. Pathogens can also manipulate pathways that lead to lysosomal biogenesis or alter the abundance or activity of lysosomal content. This pathogen-driven subversion of lysosomal biology is highly dynamic and depends on a range of factors, including cell type, stage of infection, intracellular niche and pathogen load. The growing body of literature in this field highlights the nuanced and complex relationship between intracellular pathogens and the host lysosome, which is critical for our understanding of infection biology.

Validation of a non-oncogene encoded vulnerability to exportin 1 inhibition in pediatric renal tumors.

BACKGROUND: Malignant rhabdoid tumors (MRTs) and Wilms' tumors (WTs) are rare and aggressive renal tumors of infants and young children comprising ∼5% of all pediatric cancers. MRTs are among the most genomically stable cancers, and although WTs are genomically heterogeneous, both generally lack therapeutically targetable genetic mutations. METHODS: Comparative protein activity analysis of MRTs (n = 68) and WTs (n = 132) across TCGA and TARGET cohorts, using metaVIPER, revealed elevated exportin 1 (XPO1) inferred activity. In vitro studies were performed on a panel of MRT and WT cell lines to evaluate effects on proliferation and cell-cycle progression following treatment with the selective XPO1 inhibitor selinexor. In vivo anti-tumor activity was assessed in patient-derived xenograft (PDX) models of MRTs and WTs. FINDINGS: metaVIPER analysis identified markedly aberrant activation of XPO1 in MRTs and WTs compared with other tumor types. All MRT and most WT cell lines demonstrated baseline, aberrant XPO1 activity with in vitro sensitivity to selinexor via cell-cycle arrest and induction of apoptosis. In vivo, XPO1 inhibitors significantly abrogated tumor growth in PDX models, inducing effective disease control with sustained treatment. Corroborating human relevance, we present a case report of a child with multiply relapsed WTs with prolonged disease control on selinexor. CONCLUSIONS: We report on a novel systems-biology-based comparative framework to identify non-genetically encoded vulnerabilities in genomically quiescent pediatric cancers. These results have provided preclinical rationale for investigation of XPO1 inhibitors in an upcoming investigator-initiated clinical trial of selinexor in children with MRTs and WTs and offer opportunities for exploration of inferred XPO1 activity as a potential predictive biomarker for response. FUNDING: This work was funded by CureSearch for Children's Cancer, Alan B. Slifka Foundation, NIH (U01 CA217858, S10 OD012351, and S10 OD021764), Michael's Miracle Cure, Hyundai Hope on Wheels, Cannonball Kids Cancer, Conquer Cancer the ASCO Foundation, Cycle for Survival, Paulie Strong Foundation, and the Grayson Fund.

What Are We Missing by Using Hydrophilic Enrichment? Improving Bacterial Glycoproteome Coverage Using Total Proteome and FAIMS Analyses.

Hydrophilic interaction liquid chromatography (HILIC) glycopeptide enrichment is an indispensable tool for the high-throughput characterization of glycoproteomes. Despite its utility, HILIC enrichment is associated with a number of shortcomings, including requiring large amounts of starting materials, potentially introducing chemical artifacts such as formylation when high concentrations of formic acid are used, and biasing/undersampling specific classes of glycopeptides. Here, we investigate HILIC enrichment-independent approaches for the study of bacterial glycoproteomes. Using three Burkholderia species (Burkholderia cenocepacia, Burkholderia Dolosa, and Burkholderia ubonensis), we demonstrate that short aliphatic O-linked glycopeptides are typically absent from HILIC enrichments, yet are readily identified in whole proteome samples. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS) fractionation, we show that at high compensation voltages (CVs), short aliphatic glycopeptides can be enriched from complex samples, providing an alternative means to identify glycopeptide recalcitrant to hydrophilic-based enrichment. Combining whole proteome and FAIMS analyses, we show that the observable glycoproteome of these Burkholderia species is at least 25% larger than what was initially thought. Excitingly, the ability to enrich glycopeptides using FAIMS appears generally applicable, with the N-linked glycopeptides of Campylobacter fetus subsp. fetus also being enrichable at high FAIMS CVs. Taken together, these results demonstrate that FAIMS provides an alternative means to access glycopeptides and is a valuable tool for glycoproteomic analysis.

Evidence-based path to newborn screening for Duchenne muscular dystrophy.

OBJECTIVE: Creatine kinase (CK) levels are increased on dried blood spots in newborns related to the birthing process. As a marker for newborn screening, CK in Duchenne muscular dystrophy (DMD) results in false-positive testing. In this report, we introduce a 2-tier system using the dried blood spot to first assess CK with follow-up DMD gene testing. METHODS: A fluorometric assay based upon the enzymatic transphosphorylation of adenosine diphosphate to adenosine triphosphate was used to measure CK activity. Preliminary studies established a population-based range of CK in newborns using 30,547 deidentified anonymous dried blood spot samples. Mutation analysis used genomic DNA extracted from the dried blood spot followed by whole genome amplification with assessment of single-/multiexon deletions/duplications in the DMD gene using multiplex ligation-dependent probe amplification. RESULTS: DMD gene mutations (all exonic deletions) were found in 6 of 37,649 newborn male subjects, all of whom had CK levels>2,000U/l. In 3 newborns with CK>2,000U/l in whom DMD gene abnormalities were not found, we identified limb-girdle muscular dystrophy gene mutations affecting DYSF, SGCB, and FKRP. INTERPRETATION: A 2-tier system of analysis for newborn screening for DMD has been established. This path for newborn screening fits our health care system, minimizes false-positive testing, and uses predetermined levels of CK on dried blood spots to predict DMD gene mutations.