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 β-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 β-propeller fold implicated in human diseases for chaperone-assisted filament assembly.
Display omitted
•The FlaY β-propeller protein promotes multi-flagellin filament assembly in Caulobacter•Extracellular donation of FlaY allows flagellar filament assembly in recipient cells•Mutations in the flagellin D0 domain facilitate FlaY-independent filament assembly
Kint and Viollier show that the β-propeller protein FlaY promotes multi-flagellin assembly of the flagellar filament in Caulobacter crescentus. FlaY binds flagellin and is secreted by the flagellar secretion apparatus, but it can also promote flagellin polymerization when donated extracellularly. Mutations in the flagellin D0 domain enable FlaY-independent flagellin polymerization.
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.
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
, 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.
The strict anaerobe Clostridium difficile is the most common cause of nosocomial diarrhea, and the oxygen-resistant spores that it forms have a central role in the infectious cycle. The late stages ...of sporulation require the mother cell regulatory protein σK. In Bacillus subtilis, the onset of σK activity requires both excision of a prophage-like element (skinBs) inserted in the sigK gene and proteolytical removal of an inhibitory pro-sequence. Importantly, the rearrangement is restricted to the mother cell because the skinBs recombinase is produced specifically in this cell. In C. difficile, σK lacks a pro-sequence but a skinCd element is present. The product of the skinCd gene CD1231 shares similarity with large serine recombinases. We show that CD1231 is necessary for sporulation and skinCd excision. However, contrary to B. subtilis, expression of CD1231 is observed in vegetative cells and in both sporangial compartments. Nevertheless, we show that skinCd excision is under the control of mother cell regulatory proteins σE and SpoIIID. We then demonstrate that σE and SpoIIID control the expression of the skinCd gene CD1234, and that this gene is required for sporulation and skinCd excision. CD1231 and CD1234 appear to interact and both proteins are required for skinCd excision while only CD1231 is necessary for skinCd integration. Thus, CD1234 is a recombination directionality factor that delays and restricts skinCd excision to the terminal mother cell. Finally, while the skinCd element is not essential for sporulation, deletion of skinCd results in premature activity of σK and in spores with altered surface layers. Thus, skinCd excision is a key element controlling the onset of σK activity and the fidelity of spore development.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Graphical Abstract
SpoIIE is a phosphatase involved in the activation of the first sigma factor of the forespore, σ
, during sporulation. A Δ
mutant of
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 Δ
mutant are elongated with the presence of multiple septa. This observation suggests that in
, SpoIIE is necessary for the completion of the sporulation process, as seen in
and
. Moreover, when grown in reactors, the
mutant produced higher levels of solvents than the wild type strain. The impact of the
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
, σ
, σ
, σ
) were strongly down-regulated in the mutant. Inactivation of
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
.
•Low oxygen tensions are present throughout the gastrointestinal tract.•Although Clostridioides difficile is considered as obligate anaerobe, this bacterium can tolerate low oxygen ...tensions.•Clostridioides difficile oxygen tolerance involves O2-reductases that scavenge and detoxify oxygen.•ơB and PerR are key actors of the adaptive response to oxygen.
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.
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.
Display omitted
•Shewanella Maf-1 and Maf-2 are flagellin sialyltransferase paralogs•Maf-1 and Maf-2 differ in serine-target-site selectivity and flagellin binding•Maf-2 function requires GlfM, a co-encoded and conserved four-helix-bundle protein•Invariant aspartate 77 of GlfM promotes Maf-2 function and flagellin binding
Unay et al. show that Shewanella oneidensis MR-1 encodes two flagellin sialyltransferases with distinct target-serine selectivity and flagellin-binding modes. Whereas Maf-1 activity can be reconstituted with a suitable sialic acid donor and flagellin acceptor alone, Maf-2 depends on a conserved, cis-encoded specificity factor, GlfM.
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.
PDF
