SummaryCaspase-1 activation by inflammasome signaling scaffolds initiates inflammation and antimicrobial responses. Caspase-1 proteolytically converts newly induced pro-interleukin 1 beta (IL-1β) into its mature form and directs its secretion, triggering pyroptosis and release of non-substrate alarmins such as interleukin 1 alpha (IL-1α) and HMGB1. While some caspase-1 substrates involved in these events are known, the identities and roles of non-proteolytic targets remain unknown. Here, we use unbiased proteomics to show that the UBE2L3 ubiquitin conjugase is an indirect target of caspase-1. Caspase-1, but not caspase-4, controls pyroptosis- and ubiquitin-independent proteasomal degradation of UBE2L3 upon canonical and non-canonical inflammasome activation by sterile danger signals and bacterial infection. Mechanistically, UBE2L3 acts post-translationally to promote K48-ubiquitylation and turnover of pro-IL-1β and dampen mature-IL-1β production. UBE2L3 depletion increases pro-IL-1β levels and mature-IL-1β secretion by inflammasomes. These findings regarding UBE2L3 as a molecular rheostat have implications for IL-1-driven pathology in hereditary fever syndromes and in autoinflammatory conditions associated with UBE2L3 polymorphisms.

The Escherichia coli eukaryote-like serine/threonine kinase, encoded by yeaG, is expressed in response to diverse stresses, including nitrogen (N) starvation. A role for yeaG in bacterial stress response is unknown. Here we reveal for the first time that wild-type E. coli displays metabolic heterogeneity following sustained periods of N starvation, with the metabolically active population displaying compromised viability. In contrast, such heterogeneity in metabolic activity is not observed in an E. coli ∆yeaG mutant, which continues to exist as a single and metabolically active population and thus displays an overall compromised ability to survive sustained periods of N starvation. The mechanism by which yeaG acts, involves the transcriptional repression of two toxin/antitoxin modules, mqsR/mqsA and dinJ/yafQ. This, consequently, has a positive effect on the expression of rpoS, the master regulator of the general bacterial stress response. Overall, results indicate that yeaG is required to fully execute the rpoS-dependent gene expression program to allow E. coli to adapt to sustained N starvation and unravels a novel facet to the regulatory basis that underpins adaptive response to N stress.

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Highlights d Streptococcus pneumoniae infection induces dephosphorylation of histone H3 d The bacterial factors PLY and SpxB contribute to H3S10 dephosphorylation d Host PP1 mediates H3S10 dephosphorylation and is important for intracellular infection d PP1 is activated upon S. pneumoniae infection through T320 dephosphorylation

The NAD-dependent deacetylase Sirtuin-2 (SIRT2) functions in diverse cellular processes including the cell cycle, metabolism, and has important roles in tumorigenesis and bacterial infection. SIRT2 predominantly resides in the cytoplasm but can also function in the nucleus. Consequently, SIRT2 localisation and its interacting partners may greatly impact its function and need to be defined more clearly. In this study we used mass spectrometry to determine the interactomes of SIRT2 in whole cells and in specific cellular fractions; cytoplasm, nucleus and chromatin. Using this approach, we identified novel interacting partners of SIRT2. These included a number of proteins that function in nuclear import. We show that multiple importins interact with and contribute to the basal nuclear shuttling of SIRT2 and that one of these, IPO7 is required for SIRT2 mediated H3K18 deacetylation in response to bacterial infection. Furthermore, we reveal that the unstructured C-terminus of SIRT2 negatively regulates importin-binding and nuclear transport. This study demonstrates that SIRT2 is actively transported into the nucleus via a process regulated by its C-terminus and provides a resource of SIRT2 interacting partners.The Sirtuin family of NAD-dependent deacetylases consists of 7 members (SIRT1-7) which play key protective roles in age-related diseases and act as metabolic-stress response regulators 1 . Despite sharing conserved NAD-binding and catalytic domains Sirtuins have diverse roles across multiple subcellular compartments. SIRT1, 6 and 7 are primarily nuclear proteins; SIRT3, 4 and 5 localise to the mitochondria and SIRT2 is the only Sirtuin which predominantly resides in the cytoplasm 1 . These differences are in large part due to the distinct N-and/ or C-terminal extensions of different Sirtuins 2 . These regions can regulate substrate binding; catalytic activity; contain specialised domains such as nuclear-localisation signals (NLSs), nuclear-export signals (NESs) and mitochondrial-targeting sequences (MTSs) which control subcellular localisation; and serve as platforms for the addition of post-translational modifications (PTMs) which adjust Sirtuin function 3-5 .SIRT2 has been studied primarily for its roles within the cytoplasmic milieu where it was first identified as a tubulin deacetylase 6 . It has since been demonstrated to have regulatory roles during oxidative stress and inflammatory responses via the direct deacetylation of FOXO3 and NF-κB respectively, as well as multiple pathways relating to glucose and lipid metabolism 7 . The functionality of SIRT2 is extended further by its capacity to localise to different cellular compartments including the ER-Golgi intermediate compartment (ERGIC) 8 , mitochondria 9,10 and notably the nucleus and chromatin [11][12][13] . Despite having a predominantly cytoplasmic localisation, SIRT2 is in fact continuously shuttled between the cytosol and the nuclear compartment 11 . During various physiological conditions, such as mitosis 6,14 and Listeria monocytogenes infection ...

For many intracellular bacterial pathogens manipulating host cell survival is essential for maintaining their replicative niche, and is a common strategy used to promote infection. The bacterial pathogen Listeria monocytogenes is well known to hijack host machinery for its own benefit, such as targeting the host histone H3 for modification by SIRT2. However, by what means this modification benefits infection, as well as the molecular players involved, were unknown. Here we show that SIRT2 activity supports Listeria intracellular survival by maintaining genome integrity and host cell viability. This protective effect is dependent on H3K18 deacetylation, which safeguards the host genome by counteracting infection-induced DNA damage. Mechanistically, infection causes SIRT2 to interact with the nucleic acid binding protein TDP-43 and localise to genomic R-loops, where H3K18 deacetylation occurs. This work highlights novel functions of TDP-43 and R-loops during bacterial infection and identifies the mechanism through which L. monocytogenes co-opts SIRT2 to allow efficient infection.

During infection, the foodborne bacterial pathogen Listeria monocytogenes dynamically influences the gene expression profile of host cells. Infection-induced transcriptional changes are a typical feature of the host-response to bacteria and contribute to the activation of protective genes such as inflammatory cytokines. However, by using specialized virulence factors, bacterial pathogens can target signaling pathways, transcription factors, and epigenetic mechanisms to alter host gene expression, thereby reprogramming the response to infection. Therefore, the transcriptional profile that is established in the host is delicately balanced between antibacterial responses and pathogenesis, where any change in host gene expression might significantly influence the outcome of infection. In this review, we discuss the known transcriptional and epigenetic processes that are engaged during Listeria monocytogenes infection, the virulence factors that can remodel them, and the impact these processes have on the outcome of infection.

26Pathogenic bacteria can alter host gene expression through post-translational 27 modifications of histones. We show for the first time that a natural colonizer, Streptococcus 28 pneumoniae, also induces specific histone modifications, including robust dephosphorylation of 29 histone H3 on serine 10, during infection of respiratory epithelial cells. Two bacterial factors are 30 important for the induction of this modification: the bacterial toxin PLY, a pore-forming toxin, and 31 the pyruvate oxidase SpxB, an enzyme responsible for H 2 O 2 production. The combined effects 32 of PLY and H 2 O 2 lead to host signaling which culminates in H3S10 dephosphorylation, mediated 33 by the host cell phosphatase PP1. Strikingly, S. pneumoniae infection induces 34 dephosphorylation and associated activation of PP1 catalytic activity. Colonization of cells, 35 which lacked active PP1, resulted in the impairment of intracellular S. pneumoniae survival. 36Interestingly, PP1 activation mediating H3S10 dephosphorylation is not restricted to S. 37 pneumoniae and appears to be a general epigenomic mechanism favoring intracellular survival. 38