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Ubiquitination of proteins, like phosphorylation and acetylation, is an important regulatory aspect influencing numerous and various cell processes, such as immune response signaling and autophagy. The study of ubiquitination has become essential to learning about host–pathogen interactions, and a better understanding of the detailed mechanisms through which pathogens affect ubiquitination processes in host cell will contribute to vaccine development and effective treatment of diseases. Pathogenic bacteria (e.g., Salmonella enterica, Legionella pneumophila and Shigella flexneri) encode many effector proteins, such as deubiquitinating enzymes (DUBs), targeting the host ubiquitin machinery and thus disrupting pertinent ubiquitin-dependent anti-bacterial response. We focus here upon the host ubiquitination system as an integral unit, its interconnection with the regulation of inflammation and autophagy, and primarily while examining pathogens manipulating the host ubiquitination system. Many bacterial effector proteins have already been described as being translocated into the host cell, where they directly regulate host defense processes. Due to their importance in pathogenic bacteria progression within the host, they are regarded as virulence factors essential for bacterial evasion. However, in some cases (e.g., Francisella tularensis) the host ubiquitination system is influenced by bacterial infection, although the responsible bacterial effectors are still unknown.

Francisella tularensis influences several host molecular/signaling pathways during infection. Ubiquitination and deubiquitination are among the most important regulatory mechanisms and respectively occur through attachment or removal of the ubiquitin molecule. The process is necessary not only to mark molecules for degradation, but also, for example, to the activation of signaling pathways leading to pro-inflammatory host response. Many intracellular pathogens, including Francisella tularensis , have evolved mechanisms of modifying such host immune responses to escape degradation. Here, we describe that F. tularensis interferes with the host’s ubiquitination system. We show increased total activity of deubiquitinating enzymes (DUBs) in human macrophages after infection, while confirm reduced enzymatic activities of two specific DUBs (USP10 and UCH-L5), and demonstrate increased activity of USP25. We further reveal the enrichment of these three enzymes in exosomes derived from F. tularensis -infected cells. The obtained results show the regulatory effect on ubiquitination mechanism in macrophages during F. tularensis infection.

Ubiquitination is an important protein modification that regulates various essential cellular processes, including the functions of innate immune cells. Deubiquitinases are enzymes responsible for removing ubiquitin modification from substrates, and the regulation of deubiquitinases in macrophages during infection with Typhimurium and remains unknown. To identify deubiquitinases regulated in human macrophages during bacterial infection, an activity-based proteomics screen was conducted. The effects of pharmacological inhibition of the identified deubiquitinase, USP8, were examined, including its impact on bacterial survival within macrophages and its role in autophagy regulation during infection. Several deubiquiitnases were differentially regulated in infected macrophages. One of the deubiquitinases identified was USP8, which was downregulated upon infection. Inhibition of USP8 was associated with a decrease in bacterial survival within macrophages, and it was found to play a distinct role in regulating autophagy during infection. The inhibition of USP8 led to the downregulation of the p62 autophagy adaptor. The findings of this study suggest a novel role of USP8 in regulating autophagy flux, which restricts intracellular bacteria, particularly during infection.

Zika virus is considered a major global threat to human kind. Here, we present a crystal structure of one of its essential enzymes, the methyltransferase, with the inhibitor sinefungin. This structure, together with previously solved structures with bound substrates, will provide the information needed for rational inhibitor design. Based on the structural data we suggest the modification of the adenine moiety of sinefungin to increase selectivity and to covalently link it to a GTP analogue, to increase the affinity of the synthesized compounds.

Human APOBEC3A is a single-stranded DNA cytidine deaminase that restricts viral pathogens and endogenous retrotransposons, and has a role in the innate immune response. Furthermore, its potential to act as a genomic DNA mutator has implications for a role in carcinogenesis. A deeper understanding of APOBEC3A’s deaminase and nucleic acid-binding properties, which is central to its biological activities, has been limited by the lack of structural information. Here we report the nuclear magnetic resonance solution structure of APOBEC3A and show that the critical interface for interaction with single-stranded DNA substrates includes residues extending beyond the catalytic centre. Importantly, by monitoring deaminase activity in real time, we find that A3A displays similar catalytic activity on APOBEC3A-specific TT C A- or A3G-specific CC C A-containing substrates, involving key determinants immediately 5′ of the reactive C. Our results afford novel mechanistic insights into APOBEC3A-mediated deamination and provide the structural basis for further molecular studies. The cytidine deaminase APOBEC3A has potent antiviral activity, degrading foreign DNA, and inhibiting viral replication, retrotransposition and reverse transcription. Byeon et al . present the solution structure of APOBEC3A, and reveal insights into its substrate specificity.

Background The genome of Pseudomonas aeruginosa contains at least three genes encoding eukaryotic-type Ser/Thr protein kinases, one of which, ppkA , has been implicated in P. aeruginosa virulence. Together with the adjacent pppA phosphatase gene, they belong to the type VI secretion system (H1-T6SS) locus, which is important for bacterial pathogenesis. To determine the biological function of this protein pair, we prepared a pppA-ppkA double mutant and characterised its phenotype and transcriptomic profiles. Results Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pyoverdine. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the plant model of infection. Whole-genome transcriptome analysis revealed that pppA-ppkA deletion affects the expression of oxidative stress-responsive genes, stationary phase σ-factor RpoS-regulated genes, and quorum-sensing regulons. The transcriptome of the pppA-ppkA mutant was also analysed under conditions of oxidative stress and showed an impaired response to the stress, manifested by a weaker induction of stress adaptation genes as well as the genes of the SOS regulon. In addition, expression of either RpoS-regulated genes or quorum-sensing-dependent genes was also affected. Complementation analysis confirmed that the transcription levels of the differentially expressed genes were specifically restored when the pppA and ppkA genes were expressed ectopically. Conclusions Our results suggest that in addition to its crucial role in controlling the activity of P. aeruginosa H1-T6SS at the post-translational level, the PppA-PpkA pair also affects the transcription of stress-responsive genes. Based on these data, it is likely that the reduced virulence of the mutant strain results from an impaired ability to survive in the host due to the limited response to stress conditions.

Francisella tularensis is the causative agent of tularemia, a zoonotic disease named after the city of Tulare, California. Symptoms include sudden fever, chills, fatigue, and swollen lymph nodes, among others, and without treatment it is very serious or even fatal. In addition, F. tularensis is considered a potential bioterrorism threat due to its high infectivity and lethality. Ribosomes are key targets for many classes of antibiotics. In this study, we examined the F. tularensis ribosome and determined its structure at 2.8Å resolution using cryo-electron microscopy. Notably, we observed the stress-induced ribosome-associated inhibitor A (RaiA) protein bound to the ribosome. RaiA functions as a molecular hibernation factor, inhibiting bacterial translation in response to stress or nutrient deprivation. This mechanism parallels that described in the model organism Escherichia coli and in several pathogenic bacteria, such as Staphylococcus aureus. Furthermore, we solved structures of the antibiotics chloramphenicol and gentamicin bound to the F. tularensis ribosome. Collectively, these results provide structural insights that highlight previously unexplored opportunities for therapeutic intervention.

Abasic (Ap) sites arise frequently in genomic DNA and can form interstrand crosslinks (Ap-ICLs) that block DNA replication and threaten genome stability. The DNA glycosylase NEIL3 is required for replication-coupled repair of Ap-ICLs, yet its catalytic mechanism has remained unclear, as biochemical studies report lyase-dependent strand cleavage whereas cellular systems indicate incision-free unhooking. Here, we show that the catalytic outcome of NEIL3 is determined by the N-terminal processing of its NEI domain. Using biochemically and structurally defined NEI variants, we demonstrate that a native-like processed form (V2M), in which valine 2 is replaced by an initiating methionine, efficiently unhooks Ap-ICLs by releasing the crosslinked strand without generating toxic DNA strand breaks, and without β- or δ-elimination. In contrast, an unprocessed form (M1) exhibits elevated Ap-lyase activity and generates strand breaks. Time-resolved Schiff-base trapping in the presence of a reducing agent reveals distinct high-molecular-weight intermediates during Ap-ICL unhooking. A crystal structure of NEIL3 bound to native-like substrate in form of a single-stranded DNA identifies features underlying its preference for fork-like substrates. Together, these findings reconcile previously conflicting models of NEIL3 function and define a mechanistic framework for replication-coupled repair of endogenous crosslinks, Ap-ICL, preserving fork integrity.Competing Interest StatementThe authors have declared no competing interest.Funder Information DeclaredCzech Science Foundation, 24-12306SNew Technologies for Translational Research in Pharmaceutical Sciences, New Technologies for Translational Research in Pharmaceutical SciencesCzech Academy of Sciences, Institute of Organic Chemistry and Biochemistry, RVO: 61388963

Listeria monocytogenes is a deadly foodborne pathogen that primarily affects pregnant women, infants, and the elderly. The vast array of environments that this organism can survive in contributes to its virulence and nuisance in the food industry. This chapter will focus upon -omics-based approaches that are being utilized to determine how this microbe is able to overcome such a wide variety of environments. Additionally, this chapter will also analyze strain-to-strain variations of this bacterial species and how one strain does not fit all scenarios.