Edvard Wigren

The EAL‐like protein STM1697 regulates virulence phenotypes, motility and biofilm formation in Salmonella typhimurium

The ubiquitous second messenger c‐di‐GMP regulates the switching of bacterial lifestyles from motility to sessility and acute to chronic virulence to adjust bacterial fitness to altered environmental conditions. Conventionally, EAL proteins being c‐di‐GMP phosphodiesterases promote motility and acute virulence phenotypes such as invasion into epithelial cells and inhibit biofilm formation. We report here that in contradiction, the EAL‐like protein STM1697 of Salmonella typhimurium suppresses motility, invasion into HT‐29 epithelial cell line and secretion of the type three secretion system 1 effector protein SipA, whereas it promotes rdar biofilm formation and CsgD expression. STM1697 can, however, functionally replace the EAL‐like protein STM1344 and vice versa, whereby both proteins neither degrade nor bind c‐di‐GMP. Like STM1344, STM1697 suppresses the transcription of class 2 and class 3 flagella regulon genes by binding to FlhD, a component of the master regulator of the flagella regulon FlhD4C2 and act additively under numerous conditions. Interestingly, the interaction interface of STM1697 with FlhD2 is distinct from its paralogue STM1344. We predict that the stand alone EAL domain proteins STM1697 and STM1344 belong to a subclass of EAL domain proteins in S. typhimurium, which are all involved in motility, biofilm and virulence regulation through interaction with proteins that regulate flagella function.

New Insights Into Multiple Coagulation Factor Deficiency from the Solution Structure of Human Mcfd2.

Human MCFD2 (multiple coagulation factor deficiency 2) is a 16-kDa protein known to participate in transport of the glycosylated human coagulation factors V and VIII along the secretory pathway. Mutations in MCFD2 or in its binding partner, the membrane-bound transporter ERGIC (endoplasmic reticulum-Golgi intermediate compartment)-53, cause a mild form of inherited hemophilia known as combined deficiency of factors V and VIII (F5F8D). While ERGIC-53 is known to be a lectin-type mannose binding protein, the role of MCFD2 in the secretory pathway is comparatively unclear. MCFD2 has been shown to bind both ERGIC-53 and the blood coagulation factors, but little is known about the binding sites or the true function of the protein. In order to facilitate understanding of the function of MCFD2 and the mechanism by which mutations in the protein cause F5F8D, we have determined the structure of human MCFD2 in solution by NMR. Our results show the folding of MCFD2 to be dependent on availability of calcium ions. The protein, which is disordered in the apo state, folds upon binding of Ca(2+) to the two EF-hand motifs of its C-terminus, while retaining some localized disorder in the N-terminus. NMR studies on two disease-causing mutant variants of MCFD2 show both to be predominantly disordered, even in the presence of calcium ions. These results provide an explanation for the previously observed calcium dependence of the MCFD2-ERGIC-53 interaction and, furthermore, clarify the means by which mutations in this protein result in inefficient secretion of blood coagulation factors V and VIII.

Structural Characterization of Carbohydrate Binding by LMAN1 Protein Provides New Insight into the Endoplasmic Reticulum Export of Factors V (FV) and VIII (FVIII).

Background: LMAN1 is an important mammalian cargo receptor for endoplasmic reticulum (ER)-to-Golgi trafficking. Results: Crystal structures pinpoint critical residues on LMAN1 for mannose binding, which is sensitive to Ca2+ concentration. Conclusion: Changes in Ca2+ concentration can cause ligand release without disrupting the LMAN1·MCFD2 receptor complex. Significance: The results shed new light on how LMAN1 binds and releases its cargo in the ER-to-Golgi transport pathway. LMAN1 (ERGIC-53) is a key mammalian cargo receptor responsible for the export of a subset of glycoproteins from the endoplasmic reticulum. Together with its soluble coreceptor MCFD2, LMAN1 transports coagulation factors V (FV) and VIII (FVIII). Mutations in LMAN1 or MCFD2 cause the genetic bleeding disorder combined deficiency of FV and FVIII (F5F8D). The LMAN1 carbohydrate recognition domain (CRD) binds to both glycoprotein cargo and MCFD2 in a Ca2+-dependent manner. To understand the biochemical basis and regulation of LMAN1 binding to glycoprotein cargo, we solved crystal structures of the LMAN1-CRD bound to Man-α-1,2-Man, the terminal carbohydrate moiety of high mannose glycans. Our structural data, combined with mutagenesis and in vitro binding assays, define the central mannose-binding site on LMAN1 and pinpoint histidine 178 and glycines 251/252 as critical residues for FV/FVIII binding. We also show that mannobiose binding is relatively independent of pH in the range relevant for endoplasmic reticulum-to-Golgi traffic, but is sensitive to lowered Ca2+ concentrations. The distinct LMAN1/MCFD2 interaction is maintained at these lowered Ca2+ concentrations. Our results suggest that compartmental changes in Ca2+ concentration regulate glycoprotein cargo binding and release from the LMAN1·MCFD2 complex in the early secretory pathway.

Crystal structure of the LMAN1‐CRD/MCFD2 transport receptor complex provides insight into combined deficiency of factor V and factor VIII

MINT‐7557086: lman1 (uniprotkb:P49257) and mcfd2 (uniprotkb:Q8NI22) bind (MI:0407) by X‐ray crystallography (MI:0114)

Protein homeostasis — more than resisting a hot bath

Maintenance of protein homeostasis is essential for survival of all organisms. In bacteria, the protein quality control system has a broad physiological impact beyond heat shock resistance, being involved in virulence, antibiotic resistance, as well as protection against environmental stresses. Its contribution to rejuvenation and growth arrest suggests interference with protein quality control to be a novel antimicrobial strategy. Remarkably, a protein quality control module originating from environmental strains has been found to be horizontally transferred to predominant clonal groups of bacteria providing exquisite thermotolerance to recently emerged global pathogens suggesting that novel features related to protein homeostasis contribute to the transition to new environments.

A novel protein quality control mechanism contributes to heat shock resistance of worldwide-distributed Pseudomonas aeruginosa clone C strains

Pseudomonas aeruginosa is a highly successful nosocomial pathogen capable of causing a wide variety of infections with clone C strains most prevalent worldwide. In this study, we initially characterize a molecular mechanism of survival unique to clone C strains. We identified a P. aeruginosa clone C-specific genomic island (PACGI-1) that contains the highly expressed small heat shock protein sHsp20c, the founding member of a novel subclass of class B bacterial small heat shock proteins. sHsp20c and adjacent gene products are involved in resistance against heat shock. Heat stable sHsp20c is unconventionally expressed in stationary phase in a wide temperature range from 20 to 42°C. Purified sHsp20c has characteristic features of small heat shock protein class B as it is monodisperse, forms sphere-like 24-meric oligomers and exhibits significant chaperone activity. As the P. aeruginosa clone C population is significantly more heat shock resistant than genetically unrelated P. aeruginosa strains without sHsp20c, the horizontally acquired shsp20c operon might contribute to the survival of worldwide-distributed clone C strains.

First report of molecular diagnosis of Tunisian hemophiliacs A: Identification of 8 novel causative mutations

AbstractIntroductionHemophilia A is an X linked recessive hemorrhagic disorder caused by mutations in the F8 gene that lead to qualitative and/or quantitative deficiencies of coagulation factor VIII (FVIII). Molecular diagnosis of hemophilia A is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. Molecular studies of these mutations are essential in order to reinforce our understanding of their pathogenic effect responsible for the disorder.AimIn this study we have performed molecular analysis of 28 Tunisian hemophilia A patients and analyzed the F8 mutation spectrum.MethodsWe screened the presence of intron 22 and intron 1 inversion in severe hemophilia A patients by southern blotting and polymerase chain reaction (PCR). Detection of point mutations was performed by dHPLC/sequencing of the coding F8 gene region. We predict the potential functional consequences of novel missense mutations with bioinformatics approaches and mapping of their spatial positions on the available FVIII 3D structure.ResultsWe identified 23 different mutations in 28 Tunisian hemophilia A patients belonging to 22 unrelated families. The identified mutations included 5 intron 22 inversions, 7 insertions, 4 deletions and 7 substitutions. In total 18 point mutations were identified, of which 9 are located in exon 14, the most mutated exonic sequence in the F8 gene. Among the 23 mutations, 8 are novel and not deposited in the HAMSTeRS database nor described in recently published articles.ConclusionThe mutation spectrum of Tunisian hemophilia A patients is heterogeneous with the presence of some characteristic features.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1693269827490715

Finally! The structural secrets of a HD-GYP phosphodiesterase revealed.

The major sessility‐motility lifestyle change and additional fundamental aspects of bacterial physiology, behaviour and morphology are regulated by the secondary messenger cyclic di‐GMP (c‐di‐GMP). Although the c‐di‐GMP metabolizing enzymes and many receptors have been readily characterized upon discovery, the HD‐GYP domain c‐di‐GMP phosphodiesterase family remained underinvestigated. In this issue of Molecular Microbiology, Bellini et al. provide an important step towards functional and structural characterization of the previously neglected HD‐GYP domain family by resolving the crystal structure of PmGH, a catalytically active family member from the thermophilic bacterium Persephonella marina. The crystal structure revealed a novel tri‐nuclear catalytic iron centre involved in c‐di‐GMP binding and catalysis and provides the structural basis to subsequently characterize in detail the catalytic mechanism of hydrolysis of c‐di‐GMP to GMP by HD‐GYP domains.

Analysis of newly detected mutations in the MCFD2 gene giving rise to combined deficiency of coagulation factors V and VIII.

Summary.  Combined deficiency of coagulation factor V (FV) and factor VIII (FVIII) (F5F8D) is a rare autosomal recessive disorder characterized by mild‐to‐moderate bleeding and reduction in FV and FVIII levels in plasma. F5F8D is caused by mutations in one of two different genes, LMAN1 and MCFD2, which encode proteins that form a complex involved in the transport of FV and FVIII from the endoplasmic reticulum to the Golgi apparatus. Here, we report the identification of a novel mutation Asp89Asn in the MCFD2 gene in a Tunisian patient. In the encoded protein, this mutation causes substitution of a negatively charged aspartate, involved in several structurally important interactions, to an uncharged asparagine. To elucidate the structural effect of this mutation, we performed circular dichroism (CD) analysis of secondary structure and stability. In addition, CD analysis was performed on two missense mutations found in previously reported F5F8D patients. Our results show that all analysed mutant variants give rise to destabilized proteins and highlight the importance of a structurally intact and functional MCFD2 for the efficient secretion of coagulation factors V and VIII.