Shiga toxin-producing
(STEC) and the STEC subgroup enterohemorrhagic
cause intestinal infections with symptoms ranging from watery diarrhea to hemolytic-uremic syndrome (HUS). A key tool for the ...epidemiological differentiation of STEC is serotyping. The serotype in combination with the main virulence determinants gives important insight into the virulence potential of a strain. However, a large fraction of STEC strains found in human disease, including strains causing HUS, belongs to less frequently detected STEC serovars or their O/H antigens are unknown or even untypeable. Recent implementation of whole-genome sequence (WGS) analysis, in principle, allows the deduction of serovar and virulence gene information. Therefore, here we compared classical serovar and PCR-based virulence marker detection with WGS-based methods for 232 STEC strains, focusing on less frequently detected STEC serovars and nontypeable strains. We found that the results of WGS-based extraction showed a very high degree of overlap with those of the more classical methods. Specifically, the rate of concordance was 97% for O antigens (OAGs) and 99% for H antigens (HAGs) of typeable strains and >99% for
,
, or
for all strains. Ninety-eight percent of nontypeable OAGs and 100% of nontypeable HAGs were defined by WGS analysis. In addition, the novel methods enabled a more complete analysis of strains causing severe clinical symptoms and the description of four novel STEC OAG loci. In conclusion, WGS is a promising tool for gaining serovar and virulence gene information, especially from a public health perspective.
Legionella pneumophila, the causative agent of a life‐threatening pneumonia, intracellularly replicates in a specialized compartment in lung macrophages, the Legionella‐containing vacuole (LCV). ...Secreted proteins of the pathogen govern important steps in the intracellular life cycle including bacterial egress. Among these is the type II secreted PlaA which, together with PlaC and PlaD, belongs to the GDSL phospholipase family found in L. pneumophila. PlaA shows lysophospholipase A (LPLA) activity which increases after secretion and subsequent processing by the zinc metalloproteinase ProA within a disulfide loop. Activity of PlaA contributes to the destabilization of the LCV in the absence of the type IVB‐secreted effector SdhA. We here present the 3D structure of PlaA which shows a typical α/β‐hydrolase fold and reveals that the uncleaved disulfide loop forms a lid structure covering the catalytic triad S30/D278/H282. This leads to reduction of substrate access before activation; however, the catalytic site gets more accessible when the disulfide loop is processed. After structural modeling, a similar activation process is suggested for the GDSL hydrolase PlaC, but not for PlaD. Furthermore, the size of the PlaA substrate‐binding site indicated preference toward phospholipids comprising ~16 carbon fatty acid residues which was verified by lipid hydrolysis, suggesting a molecular ruler mechanism. Indeed, mutational analysis changed the substrate profile with respect to fatty acid chain length. In conclusion, our analysis revealed the structural basis for the regulated activation and substrate preference of PlaA.
The 3D structure of PlaA shows a typical α/β‐hydrolase fold and reveals that a disulfide loop forms a lid structure covering the catalytic triad S30/D278/H282. After disulfide loop processing by ProA, access of lipid substrates to the catalytic site is facilitated and thus leads to an increase of LPLA activity. The size of the substrate‐binding site indicated preference toward phospholipids comprising ~16 carbon fatty acid residues suggesting regulation by a ruler‐like mechanism.
L. pneumophila, an important facultative intracellular bacterium, infects the human lung and environmental protozoa. At least fifteen phospholipases A (PLA) are encoded in its genome. Three of which, ...namely PlaA, PlaC, and PlaD, belong to the GDSL lipase family abundant in bacteria and higher plants. PlaA is a lysophospholipase A (LPLA) that destabilizes the phagosomal membrane in absence of a protective factor. PlaC shows PLA and glycerophospholipid: cholesterol acyltransferase (GCAT) activities which are activated by zinc metalloproteinase ProA via cleavage of a disulphide loop. In this work, we compared GDSL enzyme activities, their secretion, and activation of PlaA. We found that PlaA majorly contributed to LPLA, PlaC to PLA, and both substrate-dependently to GCAT activity. Western blotting revealed that PlaA and PlaC are type II-secreted and both processed by ProA. Interestingly, ProA steeply increased LPLA but diminished GCAT activity of PlaA. Deletion of 20 amino acids within a predicted disulfide loop of PlaA had the same effect. In summary, we propose a model by which ProA processes PlaA via disulfide loop cleavage leading to a steep increase in LPLA activity. Our results help to further characterize the L. pneumophila GDSL hydrolases, particularly PlaA, an enzyme acting in the Legionella-containing phagosome.
•State of the art on secreted phospholipases of L. pneumophila.•Phospholipases secreted via type II, type IVB and unknown secretion systems.•Localization and growth-phase dependent abundance of ...phospholipases.•Classification, activities and activation of L. pneumophila phospholipases.•Role of L. pneumophila phospholipases in infection.
Legionella pneumophila is an intracellular pathogen and the main causative agent of Legionnaires’ disease, a potentially fatal pneumonia. The bacteria infect both mammalian cells and environmental hosts, such as amoeba. Inside host cells, the bacteria withstand the multifaceted defenses of the phagocyte and replicate within a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). For establishment and maintenance of the infection, L. pneumophila secretes many proteins including effector proteins by means of different secretion systems and outer membrane vesicles. Among these are a large variety of lipolytic enzymes which possess phospholipase/lysophospholipase and/or glycerophospholipid:cholesterol acyltransferase activities. Secreted lipolytic activities may contribute to bacterial virulence, for example via modification of eukaryotic membranes, such as the LCV. In this review, we describe the secretion systems of L. pneumophila, introduce the classification of phospholipases, and summarize the state of the art on secreted L. pneumophila phospholipases. We especially highlight those enzymes secreted via the type II secretion system Lsp, via the type IVB secretion system Dot/Icm, via outer membrane vesicles, and such where the mode of secretion has not yet been defined. We also give an overview on the complexity of their activities, activation mechanisms, localization, growth-phase dependent abundance, and their role in infection.
Provider: - Institution: - Data provided by Europeana Collections- Kiel, Univ., Diss., 2010- All metadata published by Europeana are available free of restriction under the Creative Commons CC0 1.0 ...Universal Public Domain Dedication. However, Europeana requests that you actively acknowledge and give attribution to all metadata sources including Europeana
Provider: - Institution: - Data provided by Europeana Collections- Regensburg, Universität Regensburg, Diss., 2012- All metadata published by Europeana are available free of restriction under the ...Creative Commons CC0 1.0 Universal Public Domain Dedication. However, Europeana requests that you actively acknowledge and give attribution to all metadata sources including Europeana
Chronic mucocutaneous candidiasis disease (CMCD) may be caused by autosomal dominant (AD) IL-17F deficiency or autosomal recessive (AR) IL-17RA deficiency. Here, using whole-exome sequencing, we ...identified heterozygous germline mutations in STAT1 in 47 patients from 20 kindreds with AD CMCD. Previously described heterozygous STAT1 mutant alleles are loss-of-function and cause AD predisposition to mycobacterial disease caused by impaired STAT1-dependent cellular responses to IFN-γ. Other loss-of-function STAT1 alleles cause AR predisposition to intracellular bacterial and viral diseases, caused by impaired STAT1-dependent responses to IFN-α/β, IFN-γ, IFN-λ, and IL-27. In contrast, the 12 AD CMCD-inducing STAT1 mutant alleles described here are gain-of-function and increase STAT1-dependent cellular responses to these cytokines, and to cytokines that predominantly activate STAT3, such as IL-6 and IL-21. All of these mutations affect the coiled-coil domain and impair the nuclear dephosphorylation of activated STAT1, accounting for their gain-of-function and dominance. Stronger cellular responses to the STAT1-dependent IL-17 inhibitors IFN-α/β, IFN-γ, and IL-27, and stronger STAT1 activation in response to the STAT3-dependent IL-17 inducers IL-6 and IL-21, hinder the development of T cells producing IL-17A, IL-17F, and IL-22. Gain-of-function STAT1 alleles therefore cause AD CMCD by impairing IL-17 immunity.
Muscle formation is a coordinated process driven by extensive gene expression changes where single cells fuse together to form multinucleated muscle fibers. Newly synthesized mRNAs are then regulated ...by RNA binding proteins (RBPs), affecting post-transcriptional transcript metabolism. Here, we determined how large-scale gene expression changes affect the catalog of RBPs by studying proliferating and differentiated muscle cells in healthy and dystrophic conditions. Transcriptomic analysis showed that the expression of more than 7000 genes was affected during myogenesis. We identified 769 RBPs, of which 294 were muscle-specific and 49 were uniquely shared with cardiomyocytes. A subset of 32 RBPs (half of which were muscle-specific) was found to be preferentially associated with target mRNAs in either myoblasts (MBs) or myotubes (MTs). A large proportion of catalytic proteins were bound to mRNAs even though they lack classical RNA binding domains. Finally, we showed how the identification of cell-specific RBPs enabled the identification of biomarkers that can separate healthy individuals from dystrophic patients. Our data show how interactome data can shed light on new basic RNA biology as well as provide cell-specific data that can be used for diagnostic purposes.
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•Benchmarking the E. coli S30 lysate core proteome.•Classification of proteome subsets relevant for cell-free expression.•Tuning S30 cell-free lysate production for improved protein ...quality.•Quantitative proteome analysis of SOS response induced S30 lysates.
Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a “black box”. Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures.
This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as a platform for the generation of customized cell-free systems and product analysis.
Biallelic pathogenic variants in SZT2 result in a neurodevelopmental disorder with shared features, including early-onset epilepsy, developmental delay, macrocephaly, and corpus callosum ...abnormalities. SZT2 is as a critical scaffolding protein in the amino acid sensing arm of the mTORC1 signalling pathway. Due to its large size (3432 amino acids), lack of crystal structure, and absence of functional domains, it is difficult to determine the pathogenicity of SZT2 missense and in-frame deletions, but these variants are increasingly detected and reported by clinical genetic testing in individuals with epilepsy. To exemplify this latter point, here we describe a cohort of 12 individuals with biallelic SZT2 variants and phenotypic overlap with SZT2-related neurodevelopmental disorders. However, the majority of individuals carried one or more SZT2 variants of uncertain significance (VUS), highlighting the need for functional characterization to determine, which, if any, of these VUS were pathogenic. Thus, we developed a novel individualized platform to identify SZT2 loss-of-function variants in the context of mTORC1 signalling and reclassify VUS. Using this platform, we identified a recurrent in-frame deletion (SZT2 p.Val1984del) which was determined to be a loss-of-function variant and therefore likely pathogenic. Haplotype analysis revealed that this single in-frame deletion is a founder variant in those of Ashkenazi Jewish ancestry. Moreover, this approach allowed us to tentatively reclassify all of the VUS in our cohort of 12 individuals, identifying five individuals with biallelic pathogenic or likely pathogenic variants. Clinical features of these five individuals consisted of early-onset seizures (median 24 months), focal seizures, developmental delay and macrocephaly similar to previous reports. However, we also show a widening of the phenotypic spectrum, as none of the five individuals had corpus callosum abnormalities, in contrast to previous reports. Overall, we present a rapid assay to resolve VUS in SZT2, identify a founder variant in individuals of Ashkenazi Jewish ancestry, and demonstrate that corpus callosum abnormalities is not a hallmark feature of this condition. Our approach is widely applicable to other mTORopathies including the most common causes of the focal genetic epilepsies, DEPDC5, TSC1/2, MTOR and NPRL2/3.
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