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Objective: To develop a rapid functional assay to validate variants of uncertain significance (VUS) in the MEFV gene. Methods: Overactivity of the pyrin inflammasome pathway and ASC speck oligomerisation in response to stimulation with low concentrations of Clostridium difficile toxin A was directly visualised by immunofluorescence microscopy. A semi‐automated algorithm was developed to count cells and ASC specks. Results: The semi‐automated ASC speck assay is able to discriminate between healthy controls and patients with familial Mediterranean fever (FMF) and pyrin inflammasome overactivity with high sensitivity. It is also able to discriminate pyrin inflammasome overactivity from other autoinflammatory disease controls with high specificity. Conclusion: The semi‐automated ASC speck assay may be a useful test to functionally validate VUS in the MEFV gene and screen for pyrin inflammasome overactivity. A semi‐automated ASC speck assay using machine learning is able to discriminate between healthy controls and patients with familial Mediterranean fever (FMF) with high sensitivity. It is also able to discriminate FMF from other autoinflammatory diseases with high specificity.

In the arms race between a pathogen and the host, the defense mechanisms of the host cell, including the ubiquitin system, are often counteracted by bacteria. Simkania negevensis (Sne), an obligate intracellular Chlamydia-like bacterium connected with respiratory diseases, possesses numerous deubiquitinases, but not much is known about its other ubiquitin-modifying enzymes. Sne infects a wide range of hosts, developing inside a tubular vacuole in close contact with the host endoplasmic reticulum (ER) and mitochondria. Our study describes an uncharacterized Sne ubiquitin E3 RING-ligase (SNE_A12920 or SneRING), which primarily generates K63- and K11-linked ubiquitin chains and preferentially interacts with UbcH5b and UBE2T E2 enzymes. SneRING is expressed upon infection of various human cell lines, as well as amoebae. We show that a portion of the expressed SneRING co-localizes with mitochondria and ER and that the SneRING interactome includes mitochondrial and ER proteins involved in organelle morphology and stress response. Our work offers an initial characterization of a bacterial RING ligase potentially involved in the host cell remodeling to accommodate the unique intracellular lifestyle of Sne. Author summaryUbiquitination is a protein modification system that regulates protein degradation, localization, or interactions. As such, ubiquitination has many important functions in cell signalling, and its dysregulation can lead to cancer and neurodegenerative diseases. Bacteria that live and develop inside human or other eukaryotic cells, such as Chlamydia, often modulate the ubiquitination system to ensure their own survival. Simkania negevensis is a Chlamydia-like bacterium connected to respiratory diseases in humans. We have discovered a novel enzyme expressed by these bacteria that can ubiquitinate other proteins and thus potentially modify host cell processes that would otherwise hinder infection. In this work, we explore the function of this enzyme and determine its possible cellular localization, as well as some of the proteins it interacts with. Our study provides new insights into how bacterial pathogens adapt to and manipulate host cells using one of the major cell function regulatory systems.

Staphylococcus aureus is a notorious human pathogen that thrives in macrophages. It resides in mature phagolysosomes, where a subset of the bacteria eventually begin to proliferate. How S. aureus acquires essential nutrients, such as amino acids, for growth in this niche is poorly understood. Using a long-term primary human macrophage infection model, we show that branched-chain amino acid (BCAA) uptake mediated by the major transporter BrnQ1 is required by S. aureus for intracellular replication in macrophages and we provide mechanistic insight into the role of BCAAs in the success of intracellular S. aureus. Loss of BrnQ1 function renders intracellular S. aureus non-replicative and non-cytotoxic. The defective intracellular growth of S. aureus brnQ1 mutants can be rescued by supplementation with BCAAs or by overexpression of the BCAA transporters BrnQ1 or BcaP. Inactivation of the CodY repressor rescues the ability of S. aureus brnQ1 mutants to proliferate intracellularly independent of endogenous BCAA synthesis but dependent on BcaP expression. Non-replicating brnQ1 mutants in primary human macrophages become metabolically quiescent and display aberrant gene expression marked by failure to respond to intraphagosomal iron starvation. The bacteria remain, however, viable for an inordinate length of time. This dormant, yet viable bacterial state is distinct from classical persisters and small colony variants. Author summaryStaphylococcus aureus is a prominent human pathogen causing acute and chronic disease. It is facultatively intracellular and can reside within many host cell types, including professional phagocytes such as macrophages. The intracellular state contributes to dissemination, recurrence and infection chronicity. Chronic and relapsing infections are often associated with so-called persister phenotypes. Growth arrest and metabolic quiescence, accompanied by antibiotic tolerance, are hallmarks of persistence in bacteria. Antibiotic pressure is a major factor in triggering intracellular persistence. The small colony variant (SCV), an extensively studied form of S. aureus persister, can arise in the absence of antibiotic pressure and exhibits very distinctive phenotypic characteristics.Here, we describe a different growth-arrested state of S. aureus, which conforms to the definition of a non-antibiotic-driven form of intracellular dormancy, triggered by branched-chain amino acid starvation in macrophages. We show that loss of function of the major branched-chain amino acid transporter BrnQ1 renders intracellular S. aureus non-replicative and metabolically quiescent for an inordinate period of time. Upon stochastic exit from infected macrophages, brnQ1 mutants retain full virulence. This dormancy differs from classical persistence or SCVs and uncovers an underestimated role for BCAA uptake in the success of intracellular S. aureus.