The structural integrity of the Gram-negative cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. ...coli upon addition of antibacterial compounds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compound screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes.
Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by pathogenic variants in TCF4, leading to intellectual disability, specific morphological features, and autonomic nervous system ...dysfunction. Epigenetic dysregulation has been implicated in PTHS, prompting the investigation of a DNA methylation (DNAm) "episignature" specific to PTHS for diagnostic purposes and variant reclassification and functional insights into the molecular pathophysiology of this disorder. A cohort of 67 individuals with genetically confirmed PTHS and three individuals with intellectual disability and a variant of uncertain significance (VUS) in TCF4 were studied. The DNAm episignature was developed with an Infinium Methylation EPIC BeadChip array analysis using peripheral blood cells. Support vector machine (SVM) modeling and clustering methods were employed to generate a DNAm classifier for PTHS. Validation was extended to an additional cohort of 11 individuals with PTHS. The episignature was assessed in relation to other neurodevelopmental disorders and its specificity was examined. A specific DNAm episignature for PTHS was established. The classifier exhibited high sensitivity for TCF4 haploinsufficiency and missense variants in the basic-helix-loop-helix domain. Notably, seven individuals with TCF4 variants exhibited negative episignatures, suggesting complexities related to mosaicism, genetic factors, and environmental influences. The episignature displayed degrees of overlap with other related disorders and biological pathways. This study defines a DNAm episignature for TCF4-related PTHS, enabling improved diagnostic accuracy and VUS reclassification. The finding that some cases scored negatively underscores the potential for multiple or nested episignatures and emphasizes the need for continued investigation to enhance specificity and coverage across PTHS-related variants.
Unveiling a DNA methylation signature specific to Pitt-Hopkins syndrome (PTHS), this study enhances diagnostic accuracy and variant reclassification. The signature shows high sensitivity for TCF4 variants, with complexities like mosaicism emphasized. This finding underscores the need for continued research to enhance specificity and coverage across PTHS-related variants.
The self-sufficient autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a ...secreted passenger domain and a β-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein.
Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a β-helical core structure (β-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated β-stem appeared unstable after translocation, demonstrating the importance of an intact β-stem. By interrupting the cleavage site between passenger and β-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development.
We developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria.
Chung-Jansen syndrome is a neurodevelopmental disorder characterized by intellectual disability, behavioral problems, obesity and dysmorphic features. It is caused by pathogenic variants in the
PHIP
...gene that encodes for the Pleckstrin homology domain-interacting protein, which is part of an epigenetic modifier protein complex. Therefore, we hypothesized that
PHIP
haploinsufficiency may impact genome-wide DNA methylation (DNAm). We assessed the DNAm profiles of affected individuals with pathogenic and likely pathogenic
PHIP
variants with Infinium Methylation EPIC arrays and report a specific and sensitive DNAm episignature biomarker for Chung–Jansen syndrome. In addition, we observed similarities between the methylation profile of Chung–Jansen syndrome and that of functionally related and clinically partially overlapping genetic disorders, White–Kernohan syndrome (caused by variants in
DDB1
gene) and Börjeson–Forssman–Lehmann syndrome (caused by variants in
PHF6
gene). Based on these observations we also proceeded to develop a common episignature biomarker for these disorders. These newly defined episignatures can be used as part of a multiclass episignature classifier for screening of affected individuals with rare disorders and interpretation of genetic variants of unknown clinical significance, and provide further insights into the common molecular pathophysiology of the clinically-related Chung–Jansen, Börjeson–Forssman–Lehmann and White–Kernohan syndromes.
The Autotransporter pathway, ubiquitous in Gram-negative bacteria, allows the efficient secretion of large passenger proteins via a relatively simple mechanism. Capitalizing on its crystal structure, ...we have engineered the Escherichia coli autotransporter Hemoglobin protease (Hbp) into a versatile platform for secretion and surface display of multiple heterologous proteins in one carrier molecule.
As proof-of-concept, we demonstrate efficient secretion and high-density display of the sizeable Mycobacterium tuberculosis antigens ESAT6, Ag85B and Rv2660c in E. coli simultaneously. Furthermore, we show stable multivalent display of these antigens in an attenuated Salmonella Typhimurium strain upon chromosomal integration. To emphasize the versatility of the Hbp platform, we also demonstrate efficient expression of multiple sizeable antigenic fragments from Chlamydia trachomatis and the influenza A virus at the Salmonella cell surface.
The successful efficient cell surface display of multiple antigens from various pathogenic organisms highlights the potential of Hbp as a universal platform for the development of multivalent recombinant bacterial vector vaccines.
Mutations in the gene coding for Sequestosome 1 (SQSTM1) have been genetically associated with amyotrophic lateral sclerosis (ALS) and Paget disease of bone. In the present study, we analyzed the
...SQSTM1
coding sequence for mutations in an extended cohort of 1,808 patients with frontotemporal lobar degeneration (FTLD), ascertained within the European Early-Onset Dementia consortium. As control dataset, we sequenced 1,625 European control individuals and analyzed whole-exome sequence data of 2,274 German individuals (total
n
= 3,899). Association of rare
SQSTM1
mutations was calculated in a meta-analysis of 4,332 FTLD and 10,240 control alleles. We identified 25 coding variants in FTLD patients of which 10 have not been described. Fifteen mutations were absent in the control individuals (carrier frequency <0.00026) whilst the others were rare in both patients and control individuals. When pooling all variants with a minor allele frequency <0.01, an overall frequency of 3.2 % was calculated in patients. Rare variant association analysis between patients and controls showed no difference over the whole protein, but suggested that rare mutations clustering in the UBA domain of
SQSTM1
may influence disease susceptibility by doubling the risk for FTLD (RR = 2.18 95 % CI 1.24–3.85; corrected
p
value = 0.042). Detailed histopathology demonstrated that mutations in
SQSTM1
associate with widespread neuronal and glial phospho-TDP-43 pathology. With this study, we provide further evidence for a putative role of rare mutations in
SQSTM1
in the genetic etiology of FTLD and showed that, comparable to other FTLD/ALS genes,
SQSTM1
mutations are associated with TDP-43 pathology.
Antibodies recognizing plasminogen, a key component of the fibrinolytic system, associate with venous thrombotic events in PR3-ANCA vasculitis. Here, we investigated the prevalence and function of ...anti-plasminogen antibodies in independent UK and Dutch cohorts of patients with ANCA-associated vasculitis (AAV). We screened Ig isolated from patients (AAV-IgG) and healthy controls by ELISA. Eighteen of 74 (24%) UK and 10/38 (26%) Dutch patients with AAV had anti-plasminogen antibodies compared with 0/50 and 1/61 (2%) of controls. We detected anti-plasminogen antibodies in both PR3-ANCA- and MPO-ANCA-positive patients. In addition, we identified anti-tissue plasminogen activator (tPA) antibodies in 13/74 (18%) patients, and these antibodies were more common among patients with anti-plasminogen antibodies (P = 0.011). Eighteen of 74 AAV-IgG (but no control IgG) retarded fibrinolysis in vitro, and this associated with anti-plasminogen and/or anti-tPA antibody positivity. Only 4/18 AAV-IgG retarded fibrinolysis without harboring these antibodies; dual-positive samples retarded fibrinolysis to the greatest extent. Patients with anti-plasminogen antibodies had significantly higher percentages of glomeruli with fibrinoid necrosis (P < 0.05) and cellular crescents (P < 0.001) and had more severely reduced renal function than patients without these antibodies. In conclusion, anti-plasminogen and anti-tPA antibodies occur in AAV and associate with functional inhibition of fibrinolysis in vitro. Seropositivity for anti-plasminogen antibodies correlates with hallmark renal histologic lesions and reduced renal function. Conceivably, therapies that enhance fibrinolysis might benefit a subset of AAV patients.
Coalition formation is often analysed in an almost non-cooperative way, as a two-stage game that consists of a first stage comprising membership actions and a second stage with physical actions, such ...as the provision of a public good. We formalised this widely used approach for the case where actions are simultaneous in each stage. Herein, we give special attention to the case of a symmetric physical game. Various theoretical results, in particular, for cartel games, are provided. As they are crucial, recent results on the uniqueness of coalitional equilibria of Cournot-like physical games are reconsidered. Various concrete examples are included. Finally, we discuss research strategies to obtain results about equilibrium coalition structures with abstract physical games in terms of qualitative properties of their primitives.
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