Search

Septins are cytoskeletal proteins implicated in numerous cellular processes including cytokinesis and morphogenesis. In the case of infection by Shigella flexneri, septins assemble into cage-like structures that entrap cytosolic bacteria targeted by autophagy. The interplay between septin cage entrapment and bacterial autophagy is poorly understood. We used a correlative light and cryo-soft X-ray tomography (cryo-SXT) pipeline to study septin cage entrapment of Shigella in its near-native state. Septin cages could be identified as X-ray dense structures, indicating they contain host cell proteins and lipids consistent with their autophagy links. Airyscan confocal microscopy of Shigella-septin cages showed that septins and lysine 63 (K63)-linked ubiquitin chains are present in separate bacterial microdomains, suggesting they are recruited separately. Finally, Cryo-SXT and live-cell imaging revealed an interaction between septins and microtubule-associated protein light chain 3B (LC3B)-positive membranes during autophagy of Shigella. Collectively our data present a new model for how septin-caged Shigella are targeted to autophagy.

Many cellular processes are regulated by ubiquitin-mediated proteasomal degradation. Pathogens can regulate eukaryotic proteolysis through the delivery of proteins with de-ubiquitinating (DUB) activities. The obligate intracellular pathogen Chlamydia trachomatis secretes Cdu1 (ChlaDUB1), a dual deubiquitinase and Lys-acetyltransferase, that promotes Golgi remodeling and survival of infected host cells presumably by regulating the ubiquitination of host and bacterial proteins. Here, we determined that Cdu1’s acetylase but not its DUB activity is important to protect Cdu1 from ubiquitin-mediated degradation. We further identified three C. trachomatis proteins on the pathogen-containing vacuole (InaC, IpaM, and CTL0480) that required Cdu1‘s acetylase activity for protection from degradation and determined that Cdu1 and these Cdu1-protected proteins are required for optimal egress of Chlamydia from host cells. These findings highlight a non-canonical mechanism of pathogen-mediated protection of virulence factors from degradation after their delivery into host cells and the coordinated regulation of secreted effector proteins.

Protein UFMylation downstream of the E1 enzyme UBA5 plays essential roles in development and endoplasmic reticulum stress. Variants in the UBA5 gene are associated with developmental and epileptic encephalopathy 44 (DEE44), an autosomal recessive disorder characterized by early-onset encephalopathy, movement abnormalities, global developmental delay, intellectual disability, and seizures. DEE44 is caused by at least 12 different missense variants described as loss of function (LoF), but the relationships between genotypes and molecular or clinical phenotypes remain to be established. We developed a humanized UBA5 fly model and biochemical activity assays in order to describe in vivo and in vitro genotype–phenotype relationships across the UBA5 allelic series. In vivo, we observed a broad spectrum of phenotypes in viability, developmental timing, lifespan, locomotor activity, and bang sensitivity. A range of functional effects was also observed in vitro across comprehensive biochemical assays for protein stability, ATP binding, UFM1 activation, and UFM1 transthiolation. Importantly, there is a strong correlation between in vivo and in vitro phenotypes, establishing a classification of LoF variants into mild, intermediate, and severe allelic strengths. By systemically evaluating UBA5 variants across in vivo and in vitro platforms, this study provides a foundation for more basic and translational UBA5 research, as well as a basis for evaluating current and future individuals afflicted with this rare disease.