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Summary Due to their role in many diseases, enzymes of the ubiquitin system have recently become interesting drug targets. Despite efforts, primary screenings of compound libraries targeting E2 enzymes and E3 ligases have been strongly limited by the lack of robust and fast high-throughput assays. Here we report a label-free high-throughput screening assay for ubiquitin E2 conjugating enzymes and E3 ligases based on MALDI-TOF mass spectrometry. The MALDI-TOF E2/E3 assay allows testing E2 enzymes and E3 ligases for their ubiquitin transfer activity, identifying E2/E3 active pairs, inhibitor potency and specificity and screening compound libraries in vitro without chemical or fluorescent probes. We demonstrate that the MALDI-TOF E2/E3 assay is a universal tool for drug discovery screening in the ubiquitin pathway as it is suitable for working with all E3 ligase families and requires a reduced amount of reagents, compared with standard biochemical assays., Graphical Abstract, Highlights • We have developed a high-throughput MALDI-TOF assay for E2/E3 enzymes • It allows screening compound libraries without chemical or fluorescent probes • We tested the screen on three disease-relevant E3 ligases: MDM2, ITCH, and HOIP • We performed a proof-of-concept high-throughput screen against 1,430 compounds, Due to their role in many diseases, there is a need to identify drug candidates for enzymes of the ubiquitin system in robust and high-throughput assays. Here we report as label-free high-throughput screening assay for ubiquitin E2 conjugating enzymes and E3 ligases based on MALDI-TOF mass spectrometry.

The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently (‘specialised’) or non-covalently (‘cargo’ effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a ‘core’ T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the machinery with differential effector specificity and efficiency of target cell delivery., Author Summary Pathogenic bacteria use a biological nanomachine called the ‘Type VI secretion system’ (T6SS) to fire toxic proteins into other cells. These target cells can be rival bacterial cells, allowing the T6SS-wielding bacteria to outcompete other pathogens or harmless bacteria, or cells of a human, animal or plant, to help the bacteria cause an infection. The T6SS can deliver multiple different toxins, known as ‘effectors’, into target cells. This work aimed to learn more about how the machinery is able to recruit effectors and fire them from the bacterial cell into the target cell. To do this, we used a T6SS in Serratia marcescens, a pathogen causing hospital-acquired infections, which kills competitor bacteria very efficiently and is a good model for other T6SSs. We found that recently-identified components of the T6SS, called PAAR proteins, are an essential part of the machinery and also that several different versions of the machinery can form. These versions are able to fire different subsets of effectors and to deliver effectors into target cells with different efficiency. Our results help us to better understand how the T6SS machinery is able to deploy so many different toxins and kill competitor bacterial cells so effectively.

Streptococcus agalactiae , also known as group B Streptococcus (GBS), is a primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30 to 76% of the cases of neonatal meningitis. In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low-, and non-biofilm-forming strains, and to facilitate interpretation of data. This protocol was used to screen the biofilm-forming abilities of 366 GBS clinical isolates from pregnant women and from neonatal infections of different serotypes in relation to medium composition and pH. The results identified a subset of isolates of serotypes III and V that formed strong biofilms under acidic conditions. Importantly, the best biofilm formers belonged to serotype III hypervirulent clone ST-17. Moreover, the abilities of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm initiation and contribute to biofilm structural stability.