The Vi Capsular Polysaccharide Enables Salmonella enterica Serovar Typhi to Evade Microbe-Guided Neutrophil Chemotaxis

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.     

PRO40 Is a Scaffold Protein of the Cell Wall Integrity Pathway,Linking the MAP Kinase Module to the Upstream Activator Protein Kinase C

Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.     

Cathepsin E generates a sumoylated intracellular fragment of the cell adhesion molecule L1 to promote neuronal and Schwann cell migration as well as myelination

The cell adhesion molecule L1 regulates cellular responses in the developing and adult nervous system. Here, we show that stimulation of cultured mouse cerebellar neurons by a function‐triggering L1 antibody leads to cathepsin E‐mediated generation of a sumoylated 30 kDa L1 fragment (L1‐30) and to import of L1‐30 into the nucleus. Mutation of the sumoylation site at K₁₁₇₂ or the cathepsin E cleavage site at E₁₁₆₇ abolishes generation of L1‐30, while mutation of the nuclear localization signal at K₁₁₄₇ prevents nuclear import of L1‐30. Moreover, the aspartyl protease inhibitor pepstatin impairs the generation of L1‐30 and inhibits L1‐induced migration of cerebellar neurons and Schwann cells as well as L1‐dependent in vitro myelination on axons of dorsal root ganglion neurons by Schwann cells. L1‐stimulated migration of HEK293 cells expressing L1 with mutated cathepsin E cleavage site is diminished in comparison to migration of cells expressing non‐mutated L1. In addition, L1‐stimulated migration of HEK293 cells expressing non‐mutated L1 is also abolished upon knock‐down of cathepsin E expression and enhanced by over‐expression of cathepsin E. The findings of the present study indicate that generation and nuclear import of L1‐30 regulate neuronal and Schwann cell migration as well as myelination.

     

Complex Regional Pain Syndrome Type I Affects Brain Structure in Prefrontal and Motor Cortex

The complex regional pain syndrome (CRPS) is a rare but debilitating pain disorder that mostly occurs after injuries to the upper limb. A number of studies indicated altered brain function in CRPS, whereas possible influences on brain structure remain poorly investigated.We acquired structural magnetic resonance imaging data from CRPS type I patients and applied voxel-by-voxel statistics to compare white and gray matter brain segments of CRPS patients with matched controls. Patients and controls were statistically compared in two different ways: First, we applied a 2-sample ttest to compare whole brain white and gray matter structure between patients and controls. Second, we aimed to assess structural alterations specifically of the primary somatosensory (S1) and motor cortex (M1) contralateral to the CRPS affected side. To this end, MRI scans of patients with left-sided CRPS (and matched controls) were horizontally flipped before preprocessing and region-of-interest-based group comparison. The unpaired ttest of the “non-flipped” data revealed that CRPS patients presented increased gray matter density in the dorsomedial prefrontal cortex. The same test applied to the “flipped” data showed further increases in gray matter density, not in the S1, but in the M1 contralateral to the CRPS-affected limb which were inversely related to decreased white matter density of the internal capsule within the ipsilateral brain hemisphere. The gray-white matter interaction between motor cortex and internal capsule suggests compensatory mechanisms within the central motor system possibly due to motor dysfunction. Altered gray matter structure in dorsomedial prefrontal cortex may occur in response to emotional processes such as pain-related suffering or elevated analgesic top-down control.     

Construction of a 42 base pair double stranded DNA microcircle.

A procedure for the construction of double stranded DNA microcircles is described that overcomes the natural limits of established circularization procedures. Starting with two synthetic oligonucleotides which are able to form dumbbell shaped structures, two subsequent ligation reactions yield a microcircle of double stranded DNA of 42 base pairs. This is by far the smallest circle of double stranded DNA yet described. These microcircles can be constructed in quantities required for high resolution structural analyses such as X-ray crystallography and NMR spectroscopy.     

Probing FhuA'-'PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K12

Summary The FhuA protein (formerly TonA) is located in the outer membrane of Escherichia coli K12. Fusions between fhuA and phoA genes were constructed. They determined proteins containing a truncated but still active alkaline phosphatase of constant size and a variable FhuA portion which ranged from 11%–90% of the mature FhuA protein. The fusion sites were nearly randomly distributed along the FhuA protein. The FhuA segments directed the secretion of the truncated alkaline phosphatase across the cytoplasmic membrane. The fusion proteins were proteolytically degraded up to the size of alkaline phosphatase and no longer reacted with anti-FhuA antibodies. The fusion proteins were more stable in lon and pep mutants lacking cytoplasmic protease and peptidases, respectively. The larger fusion proteins above a molecular weight of 64000 dalton were predominantly found in the outer membrane fraction. They were degraded by trypsin when cells were converted to spheroplasts so that trypsin gained access to the periplasm. In contrast, FhuA protein in the outer membrane was largely resistant to trypsin. It is concluded that the larger FhuA-PhoA fusion proteins were associated with, but not properly integrated into, the outer membrane.     

New Gromov-Inspired Metrics on Phylogenetic Tree Space

We present a new class of metrics for unrooted phylogenetic X-trees inspired by the Gromov–Hausdorff distance for (compact) metric spaces. These metrics can be efficiently computed by linear or quadratic programming. They are robust under NNI operations, too. The local behaviour of the metrics shows that they are different from any previously introduced metrics. The performance of the metrics is briefly analysed on random weighted and unweighted trees as well as random caterpillars.     

Living on the edge: substrate competition explains loss of robustness in mitochondrial fatty-acid oxidation disorders

Background

Defects in genes involved in mitochondrial fatty-acid oxidation (mFAO) reduce the ability of patients to cope with metabolic challenges. mFAO enzymes accept multiple substrates of different chain length, leading to molecular competition among the substrates. Here, we combined computational modeling with quantitative mouse and patient data to investigate whether substrate competition affects pathway robustness in mFAO disorders.

Results

First, we used comprehensive biochemical analyses of wild-type mice and mice deficient for medium-chain acyl-CoA dehydrogenase (MCAD) to parameterize a detailed computational model of mFAO. Model simulations predicted that MCAD deficiency would have no effect on the pathway flux at low concentrations of the mFAO substrate palmitoyl-CoA. However, high concentrations of palmitoyl-CoA would induce a decline in flux and an accumulation of intermediate metabolites. We proved computationally that the predicted overload behavior was due to substrate competition in the pathway. Second, to study the clinical relevance of this mechanism, we used patients’ metabolite profiles and generated a humanized version of the computational model. While molecular competition did not affect the plasma metabolite profiles during MCAD deficiency, it was a key factor in explaining the characteristic acylcarnitine profiles of multiple acyl-CoA dehydrogenase deficient patients. The patient-specific computational models allowed us to predict the severity of the disease phenotype, providing a proof of principle for the systems medicine approach.

Conclusion

We conclude that substrate competition is at the basis of the physiology seen in patients with mFAO disorders, a finding that may explain why these patients run a risk of a life-threatening metabolic catastrophe.