Evolution of paralogous multicomponent systems for site-specific O-sialylation of flagellin in Gram-negative and Gram-positive bacteria.

Many bacteria glycosylate flagellin on serine or threonine residues using pseudaminic acid (Pse) or other sialic acid-like donor sugars. Successful reconstitution of Pse-dependent sialylation by the conserved Maf-type flagellin glycosyltransferase (fGT) may require (a) missing component(s). Here, we characterize both Maf paralogs in the Gram-negative bacterium Shewanella oneidensis MR-1 and reconstitute Pse-dependent glycosylation in heterologous hosts. Remarkably, we uncovered distinct acceptor determinants and target specificities for each Maf. Whereas Maf-1 uses its C-terminal tetratricopeptide repeat (TPR) domain to confer flagellin acceptor and O-glycosylation specificity, Maf-2 requires the newly identified conserved specificity factor, glycosylation factor for Maf (GlfM), to form a ternary complex with flagellin. GlfM orthologs are co-encoded with Maf-2 in Gram-negative and Gram-positive bacteria and require an invariant aspartate in their four-helix bundle to function with Maf-2. Thus, convergent fGT evolution underlies distinct flagellin-binding modes in tripartite versus bipartite systems and, consequently, distinct O-glycosylation preferences of acceptor serine residues with Pse.

Predicting cytogenetic risk in multiple myeloma using conventional whole-body MRI, spinal dynamic contrast-enhanced MRI, and spinal diffusion-weighted imaging.

OBJECTIVES: Cytogenetic abnormalities are predictors of poor prognosis in multiple myeloma (MM). This paper aims to build and validate a multiparametric conventional and functional whole-body MRI-based prediction model for cytogenetic risk classification in newly diagnosed MM. METHODS: Patients with newly diagnosed MM who underwent multiparametric conventional whole-body MRI, spinal dynamic contrast-enhanced (DCE-)MRI, spinal diffusion-weighted MRI (DWI) and had genetic analysis were retrospectively included (2011-2020/Ghent University Hospital/Belgium). Patients were stratified into standard versus intermediate/high cytogenetic risk groups. After segmentation, 303 MRI features were extracted. Univariate and model-based methods were evaluated for feature and model selection. Testing was performed using receiver operating characteristic (ROC) and precision-recall curves. Models comparing the performance for genetic risk classification of the entire MRI protocol and of all MRI sequences separately were evaluated, including all features. Four final models, including only the top three most predictive features, were evaluated. RESULTS: Thirty-one patients were enrolled (mean age 66 ± 7 years, 15 men, 13 intermediate-/high-risk genetics). None of the univariate models and none of the models with all features included achieved good performance. The best performing model with only the three most predictive features and including all MRI sequences reached a ROC-area-under-the-curve of 0.80 and precision-recall-area-under-the-curve of 0.79. The highest statistical performance was reached when all three MRI sequences were combined (conventional whole-body MRI + DCE-MRI + DWI). Conventional MRI always outperformed the other sequences. DCE-MRI always outperformed DWI, except for specificity. CONCLUSIONS: A multiparametric MRI-based model has a better performance in the noninvasive prediction of high-risk cytogenetics in newly diagnosed MM than conventional MRI alone. CRITICAL RELEVANCE STATEMENT: An elaborate multiparametric MRI-based model performs better than conventional MRI alone for the noninvasive prediction of high-risk cytogenetics in newly diagnosed multiple myeloma; this opens opportunities to assess genetic heterogeneity thus overcoming sampling bias. KEY POINTS: • Standard genetic techniques in multiple myeloma patients suffer from sampling bias due to tumoral heterogeneity. • Multiparametric MRI noninvasively predicts genetic risk in multiple myeloma. • Combined conventional anatomical MRI, DCE-MRI, and DWI had the highest statistical performance to predict genetic risk. • Conventional MRI alone always outperformed DCE-MRI and DWI separately to predict genetic risk. DCE-MRI alone always outperformed DWI separately, except for the parameter specificity to predict genetic risk. • This multiparametric MRI-based genetic risk prediction model opens opportunities to noninvasively assess genetic heterogeneity thereby overcoming sampling bias in predicting genetic risk in multiple myeloma.

Extracellular transfer of a conserved polymerization factor for multi-flagellin filament assembly in Caulobacter.

Unidirectional growth of filamentous protein assemblies including the bacterial flagellum relies on dedicated polymerization factors (PFs). The molecular determinants and structural transitions imposed by PFs on multi-subunit assembly are poorly understood. Here, we unveil FlaY from the polarized α-proteobacterium Caulobacter crescentus as a defining member of an alternative class of specialized flagellin PFs. Unlike the paradigmatic FliD capping protein, FlaY relies on a funnel-like ß-propeller fold for flagellin polymerization. FlaY binds flagellin and is secreted by the flagellar secretion apparatus, yet it can also promote flagellin polymerization exogenously when donated from flagellin-deficient cells, serving as a transferable, extracellular public good. While the surge in FlaY abundance precedes bulk flagellin synthesis, FlaY-independent filament assembly is enhanced by mutation of a conserved region in multiple flagellin paralogs. We suggest that FlaYs are (multi-)flagellin PFs that evolved convergently to FliDs yet appropriated the versatile ß-propeller fold implicated in human diseases for chaperone-assisted filament assembly.

Auto-immune hemolytic anemia and hemophagocytic lymphohistiocytosis as immune-related adverse event in patients with metastatic melanoma and concurrent chronic lymphocytic leukemia: a case series and literature review.

Auto-immune hemolytic anemia (AIHA) and hemophagocytic lymphohistiocytosis (HLH) are both rare immune-related adverse events (irAEs) following treatment with immune checkpoint inhibitors. Consensus treatment guidelines are currently lacking. Patients with a solid malignancy and a concurrent lymphoproliferative disorder, such as chronic lymphocytic leukemia (CLL), might be more prone to develop hematological irAEs. We report the case history of two patients, diagnosed with CLL, who during treatment for metastatic melanoma with nivolumab, a PD-1 immune checkpoint blocking mAb, developed AIHA and HLH in combination with AIHA. Furthermore, we provide a review of the literature on published cases of immune-related AIHA and HLH and their correlation with CLL.

Stereoisomer-specific reprogramming of a bacterial flagellin sialyltransferase.

Glycosylation of surface structures diversifies cells chemically and physically. Nucleotide-activated sialic acids commonly serve as glycosyl donors, particularly pseudaminic acid (Pse) and its stereoisomer legionaminic acid (Leg), which decorate eubacterial and archaeal surface layers or protein appendages. FlmG, a recently identified protein sialyltransferase, O-glycosylates flagellins, the subunits of the flagellar filament. We show that flagellin glycosylation and motility in Caulobacter crescentus and Brevundimonas subvibrioides is conferred by functionally insulated Pse and Leg biosynthesis pathways, respectively, and by specialized FlmG orthologs. We established a genetic glyco-profiling platform for the classification of Pse or Leg biosynthesis pathways, discovered a signature determinant of eubacterial and archaeal Leg biosynthesis, and validated it by reconstitution experiments in a heterologous host. Finally, by rewiring FlmG glycosylation using chimeras, we defined two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that either donates Leg or Pse and a specialized flagellin-binding domain that identifies the acceptor.

Selinexor, Bortezomib and Dexamethasone: An Effective Salvage Regimen for Heavily Pretreated Myeloma Patients.

Purpose: Multiple myeloma (MM) patients with triple- and penta-refractory disease have a poor survival and limited treatment options. Selinexor, in combination with bortezomib and dexamethasone, demonstrated clinical activity in the STOMP study as well as in the BOSTON study in previously treated patients with disease refractory to a proteasome inhibitor (PI). Patients and Methods: Here, we report a real-world case series of 7 heavily pretreated MM patients who had been extensively pretreated with bortezomib and had disease refractory to PIs, including carfilzomib; who were administered a starting dose of 100 mg of selinexor, 20-40 mg dexamethasone and 1.3 mg/m2 of bortezomib, each once weekly. The majority of these patients (6 patients, 86.0%) had penta-refractory disease, with 5 patients (71.4%) having disease refractory to bortezomib and carfilzomib, and all 7 patients having pomalidomide refractory disease. The median number of prior lines of therapy was 8 (range 4-12). Results: The seven patients in this case series received selinexor for a median of 5 cycles (range 1-10). Four patients (57.1%) had a dose reduction of selinexor. Five patients (71.4%) had a response, of which 2 (29.0%) had a very good partial response (VGPR) and 3 (43.0%) had a partial response (PR). One patient (14.3%) had stable disease (SD) and 1 (14.3%) had progressive disease (PD). There were no new safety signals. Conclusion: The selinexor, bortezomib, and dexamethasone triplet combination demonstrates activity in PI-resistant MM and patients with heavily pretreated MM with refractory disease and after multiple lines of therapy.

Specificity and modularity of flagellin nonulosonic acid glycosyltransferases.

Many bacterial flagella are specifically O-glycosylated with nonulosonic acids, including the sialic acid derivatives, pseudaminic acid or legionaminic acid. Unlike protein glycosyltransferases that are extracytoplasmic, flagellin glycosyltransferases (fGTs) act cytoplasmically with unknown donor or acceptor specificities. The recent reconstitution of fGT-based glycosylation in heterologous hosts enables analyses underpinning such specificity.

Oxygen response and tolerance mechanisms in Clostridioides difficile.

While the gut is typically thought of as anoxic, there are two intersecting and decreasing oxygen gradients that are observed in the gut: oxygen decreases from the small to the large intestine and from the intestinal epithelium toward the colon lumen. Gut oxygen levels also increase following antibiotic induced-dysbiosis. While dysbiosis favors growth of Clostridioides difficile, the oxygen increase also causes stress to this anaerobic enteropathogen. To circumvent oxygen threat, C. difficile has developed efficient strategies: sporulation, biofilm formation, the rerouting of central metabolism and the production of oxygen detoxification enzymes. Especially, reverse rubrerythrins and flavodiiron proteins involved in oxygen reduction are crucial in C. difficile ability to tolerate and survive the oxygen concentrations encountered in the gastrointestinal tract. Two regulators, σB and PerR, play pivotal role in the mastering of these adaptive responses by controlling the various systems that protect cells from oxidative damages.

Responses of Clostridia to oxygen: from detoxification to adaptive strategies.

Clostridia comprise bacteria of environmental, biotechnological and medical interest and many commensals of the gut microbiota. Because of their strictly anaerobic lifestyle, oxygen is a major stress for Clostridia. However, recent data showed that these bacteria can cope with O2 better than expected for obligate anaerobes through their ability to scavenge, detoxify and consume O2 . Upon O2 exposure, Clostridia redirect their central metabolism onto pathways less O2 -sensitive and induce the expression of genes encoding enzymes involved in O2 -reduction and in the repair of oxidized damaged molecules. While Faecalibacterium prausnitzii efficiently consumes O2 through a specific extracellular electron shuttling system requiring riboflavin, enzymes such as rubrerythrins and flavodiiron proteins with NAD(P)H-dependent O2 - and/or H2 O2 -reductase activities are usually encoded in other Clostridia. These two classes of enzymes play indeed a pivotal role in O2 tolerance in Clostridioides difficile and Clostridium acetobutylicum. Two main signalling pathways triggering O2 -induced responses have been described so far in Clostridia. PerR acts as a key regulator of the O2 - and/or reactive oxygen species-defence machinery while in C. difficile, σB , the sigma factor of the general stress response also plays a crucial role in O2 tolerance by controlling the expression of genes involved in O2 scavenging and repair systems.

Immunomodulators in newly diagnosed multiple myeloma: current and future concepts.

Introduction: Impressive therapeutic progress is being made in the management of multiple myeloma (MM). his progress is related to the introduction of several new classes of therapeutic agents including proteasome inhibitors, immunomodulatory drugs (IMiDs) and monoclonal antibodies (MoAbs).Areas covered: In this manuscript, the role of the IMiDs thalidomide and lenalidomide in the management of newly diagnosed MM is discussed. The mode of action of IMiDs and their role in the management of newly diagnosed MM patients is highlighted. In addition, clinical data on how MoAbs such as the anti-CD38 antibody daratumumab can further increase the efficacy of IMiD-based first-line anti-myeloma regimens are provided. A database search in PubMed was carried out.Expert Opinion: Immunomodulation has become an indispensable part of successful anti-myeloma regimens both at relapse and at diagnosis. The combination of lenalidomide plus dexamethasone with an anti-CD38 MoAb such as daratumumab and a proteasome inhibitor such as bortezomib is currently one of the most potent first-line treatment regimens for MM. A better understanding on how IMiDs synergize with existing and new anti-myeloma treatments can further improve the outcome for patients. Optimal first-line therapy will continue to benefit the long-term outcome of a growing population of young and elderly MM patients.

Secretion Relieves Translational Co-repression by a Specialized Flagellin Paralog.

How cellular checkpoints couple the orderly assembly of macromolecular machines with cell-cycle progression is poorly understood. The alpha-proteobacterium Caulobacter crescentus assembles a single polar flagellum during each cell cycle. We discovered that the expression of multiple flagellin transcripts is licensed by a translational checkpoint responsive to a dual input signal: a secretion-competent hook-basal-body (HBB) structure and a surge in the FlaF secretion chaperone during cytokinesis, instructed by the cell-cycle program. We find that the unorthodox FljJ flagellin, one of the six flagellin paralogs, acts as a checkpoint linchpin, binding both FlaF and the FlbT translational regulator. FljJ recruits FlbT to inhibit translation at the 5' untranslated region in other flagellin transcripts before HBB assembly. Once FlaF is synthesized and stabilized, it directs FljJ secretion through the HBB, thereby separating FlbT from its co-activator and relieving translational inhibition. The FlbT/FlaF pair is wide spread and its functional properties are conserved in alpha-proteobacteria, including pathogens.

Specificity in glycosylation of multiple flagellins by the modular and cell cycle regulated glycosyltransferase FlmG.

How specificity is programmed into post-translational modification of proteins by glycosylation is poorly understood, especially for O-linked glycosylation systems. Here we reconstitute and dissect the substrate specificity underpinning the cytoplasmic O-glycosylation pathway that modifies all six flagellins, five structural and one regulatory paralog, in Caulobacter crescentus, a monopolarly flagellated alpha-proteobacterium. We characterize the biosynthetic pathway for the sialic acid-like sugar pseudaminic acid and show its requirement for flagellation, flagellin modification and efficient export. The cognate NeuB enzyme that condenses phosphoenolpyruvate with a hexose into pseudaminic acid is functionally interchangeable with other pseudaminic acid synthases. The previously unknown and cell cycle-regulated FlmG protein, a defining member of a new class of cytoplasmic O-glycosyltransferases, is required and sufficient for flagellin modification. The substrate specificity of FlmG is conferred by its N-terminal flagellin-binding domain. FlmG accumulates before the FlaF secretion chaperone, potentially timing flagellin modification, export, and assembly during the cell division cycle.

Transcriptomic and Phenotypic Analysis of a spoIIE Mutant in Clostridium beijerinckii.

SpoIIE is a phosphatase involved in the activation of the first sigma factor of the forespore, σ F , during sporulation. A ΔspoIIE mutant of Clostridium beijerinckii NCIMB 8052, previously generated by CRISPR-Cas9, did not sporulate but still produced granulose and solvents. Microscopy analysis also showed that the cells of the ΔspoIIE mutant are elongated with the presence of multiple septa. This observation suggests that in C. beijerinckii, SpoIIE is necessary for the completion of the sporulation process, as seen in Bacillus and Clostridium acetobutylicum. Moreover, when grown in reactors, the spoIIE mutant produced higher levels of solvents than the wild type strain. The impact of the spoIIE inactivation on gene transcription was assessed by comparative transcriptome analysis at three time points (4 h, 11 h and 23 h). Approximately 5% of the genes were differentially expressed in the mutant compared to the wild type strain at all time points. Out of those only 12% were known sporulation genes. As expected, the genes belonging to the regulon of the sporulation specific transcription factors (σ F , σ E , σ G , σ K ) were strongly down-regulated in the mutant. Inactivation of spoIIE also caused differential expression of genes involved in various cell processes at each time point. Moreover, at 23 h, genes involved in butanol formation and tolerance, as well as in cell motility, were up-regulated in the mutant. In contrast, several genes involved in cell wall composition, oxidative stress and amino acid transport were down-regulated. These results indicate an intricate interdependence of sporulation and stationary phase cellular events in C. beijerinckii.

How the Anaerobic Enteropathogen Clostridioides difficile Tolerates Low O2 Tensions.

Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2 O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon.IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile This enteropathogen has developed efficient strategies to detoxify O2 In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.

The σB signalling activation pathway in the enteropathogen Clostridioides difficile.

Clostridium difficile is the main cause of antibiotic-associated diarrhoea. Inside the gut, C. difficile must adapt to the stresses it copes with, by inducing protection, detoxification and repair systems that belong to the general stress response involving σB . Following stresses, σB activation requires a PP2C phosphatase to dephosphorylate the anti-anti-sigma factor RsbV that allows its interaction with the anti-sigma factor RsbW and the release of σB . In this work, we studied the signalling pathway responsible for the activation of σB in C. difficile. Contrary to other firmicutes, the expression of sigB in C. difficile is constitutive and not autoregulated. We confirmed the partner switching mechanism that involved RsbV, RsbW and σB . We also showed that CD2685, renamed RsbZ, and its phosphatase activity are required for RsbV dephosphorylation triggering σB activation. While CD0007 and CD0008, whose genes belong to the sigB operon, are not involved in σB activity, depletion of the essential iron-sulphur flavoprotein, CD2684, whose gene forms an operon with rsbZ, prevents σB activation. Finally, we observed that σB is heterogeneously active in a subpopulation of C. difficile cells from the exponential phase, likely leading to a 'bet-hedging' strategy allowing a better chance for the cells to survive adverse conditions.

Discovery of new type I toxin-antitoxin systems adjacent to CRISPR arrays in Clostridium difficile.

Clostridium difficile, a major human enteropathogen, must cope with foreign DNA invaders and multiple stress factors inside the host. We have recently provided an experimental evidence of defensive function of the C. difficile CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system important for its survival within phage-rich gut communities. Here, we describe the identification of type I toxin-antitoxin (TA) systems with the first functional antisense RNAs in this pathogen. Through the analysis of deep-sequencing data, we demonstrate the general co-localization with CRISPR arrays for the majority of sequenced C. difficile strains. We provide a detailed characterization of the overlapping convergent transcripts for three selected TA pairs. The toxic nature of small membrane proteins is demonstrated by the growth arrest induced by their overexpression. The co-expression of antisense RNA acting as an antitoxin prevented this growth defect. Co-regulation of CRISPR-Cas and type I TA genes by the general stress response Sigma B and biofilm-related factors further suggests a possible link between these systems with a role in recurrent C. difficile infections. Our results provide the first description of genomic links between CRISPR and type I TA systems within defense islands in line with recently emerged concept of functional coupling of immunity and cell dormancy systems in prokaryotes.

Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes.

Unraveling the mechanism of action and molecular target of small molecules remains a major challenge in drug discovery. While many cancer drugs target genetic vulnerabilities, loss-of-function screens fail to identify essential genes in drug mechanism of action. Here, we report CRISPRres, a CRISPR-Cas-based genetic screening approach to rapidly derive and identify drug resistance mutations in essential genes. It exploits the local genetic variation created by CRISPR-Cas-induced non-homologous end-joining (NHEJ) repair to generate a wide variety of functional in-frame mutations. Using large sgRNA tiling libraries and known drug-target pairs, we validate it as a target identification approach. We apply CRISPRres to the anticancer agent KPT-9274 and identify nicotinamide phosphoribosyltransferase (NAMPT) as its main target. These results present a powerful and simple genetic approach to create many protein variants that, in combination with positive selection, can be applied to reveal the cellular target of small-molecule inhibitors.

The alternative sigma factor σB plays a crucial role in adaptive strategies of Clostridium difficile during gut infection.

Clostridium difficile is a major cause of diarrhoea associated with antibiotherapy. Exposed to stresses in the gut, C. difficile can survive by inducing protection, detoxification and repair systems. In several firmicutes, most of these systems are controlled by the general stress response involving σB . In this work, we studied the role of σB in the physiopathology of C. difficile. We showed that the survival of the sigB mutant during the stationary phase was reduced. Using a transcriptome analysis, we showed that σB controls the expression of ∼25% of genes including genes involved in sporulation, metabolism, cell surface biogenesis and the management of stresses. By contrast, σB does not control toxin gene expression. In agreement with the up-regulation of sporulation genes, the sporulation efficiency is higher in the sigB mutant than in the wild-type strain. sigB inactivation also led to increased sensitivity to acidification, cationic antimicrobial peptides, nitric oxide and ROS. In addition, we showed for the first time that σB also plays a crucial role in oxygen tolerance in this strict anaerobe. Finally, we demonstrated that the fitness of colonisation by the sigB mutant is greatly affected in a dixenic mouse model of colonisation when compared to the wild-type strain.