The Listeria protein internalin B mimics hepatocyte growth factor-induced receptor trafficking

Increased hepatocyte growth factor receptor (HGFR) signaling correlates closely with neoplastic invasion and metastatic potential of many human cancers. Hepatocyte growth factor receptor signaling is initiated by binding the physiological ligand HGF or the internalin B (InlB) protein of Listeria monocytogenes. Subsequent degradation of endocytosed HGFR terminates receptor signaling. Previously reported discrepancies in InlB and HGF-induced HGFR signaling could reflect differences in receptor internalization and degradation in response to these distinct ligands. We report that soluble InlB and HGF are mechanistically equivalent in triggering clathrin-dependent endocytosis and lysosomal degradation of HGFR. After internalization, InlB and HGF colocalize with Rab5, EEA1 and the transferrin receptor in classical early endosomes. Hepatocyte growth factor receptor internalization was prevented by overexpression of dominant negative mutants of dynamin 1 and epidermal growth factor phosphorylation substrate 15, but not caveolin 1, the GTPase Arf6 or the cholesterol-chelating drug Nystatin. Thus, HGFR internalization is principally clathrin-mediated and is not regulated by clathrin- independent pathways. Phosphatidylinositol 3-kinase signaling and HGF-regulated tyrosine kinase substrate were not required for ligand-triggered internalization of HGFR but were essential for subsequent lysosomal degradation. Thus, soluble InlB and HGF induce HGFR endocytosis and degradation by indistinguishable mechanisms, suggesting that InlB may be exploited to regulate pathogenic HGFR signaling.     

InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase

The Listeria monocytogenes surface protein InlB promotes bacterial entry into mammalian cells. Here, we identify a cellular surface receptor required for InlB-mediated entry. Treatment of mammalian cells with InlB protein or infection with L. monocytogenes induces rapid tyrosine phosphorylation of Met, a receptor tyrosine kinase (RTK) for which the only known ligand is Hepatocyte Growth Factor (HGF). Like HGF, InlB binds to the extracellular domain of Met and induces "scattering" of epithelial cells. Experiments with Met-positive and Met-deficient cell lines demonstrate that Met is required for InlB-dependent entry of L. monocytogenes. InlB is a novel Met agonist that induces bacterial entry through exploitation of a host RTK pathway.     

Evidence for subsites in the galectins involved in sugar binding at the nonreducing end of the central galactose of oligosaccharide ligands: sequence analysis, homology modeling and mutagenesis studies of hamster galectin-3

A model of the carbohydrate recognition domain CRD, residues 111-245, of hamster galectin-3 has been made using homology modeling and dynamics minimization methods. The model is based on the known x-ray structures of bovine galectin-1 and human galectin-2. The oligosaccharides NeuNAc-alpha2,3-Gal-beta1,4-Glc and GalNAc-alpha1, 3- [Fuc-alpha1,2]-Gal-beta1,4-Glc, known to be specific high-affinity ligands for galectin-3, as well as lactose recognized by all galectins were docked in the galectin-3 CRD model structure and a minimized binding conformation found in each case. These studies indicate a putative extended carbohydrate-binding subsite in the hamster galectin- 3 involving Arg139, Glu230, and Ser232 for NeuNAc-alpha2,3-; Arg139 and Glu160 for fucose-alpha1,2-; and Arg139 and Ile141 for GalNAc-alpha1,3- substituents on the primary galactose. Each of these positions is variable within the whole galectin family. Two of these residues, Arg139 and Ser232, were selected for mutagenesis to probe their importance in this newly identified putative subsite. Residue 139 adopts main-chain dihedral angles characteristic of an isolated bridge structural feature, while residue 232 is the C-terminal residue of beta- strand-11, and is followed immediately by an inverse gamma-turn. A systematic series of mutant proteins have been prepared to represent the residue variation present in the aligned sequences of galectins-1, - 2, and -3. Minimized docked models were generated for each mutant in complex with NeuNAc-alpha2,3-Gal-beta1,4-Glc, GalNAc-alpha1, 3-[Fuc- alpha1,2]-Gal-beta1,4- Glc, and Gal-beta1,4-Glc. Correlation of the computed protein-carbohydrate interaction energies for each lectin- oligosaccharide pair with the experimentally determined binding affinities for fetuin and asialofetuin or the relative potencies of lactose and sialyllactose in inhibiting binding to asiolofetuin is consistent with the postulated key importance of Arg139 in recognition of the extended sialylated ligand.      

Interactions among mutations that cause altered timing of gene expression during sporulation in Bacillus subtilis.

The ski4::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but that are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ). ski4::Tn917lac caused a small defect in sporulation, but in combination with a null mutation in kinA, it caused a much more severe defect. The insertion mutation was in an 87-amino-acid open reading frame (orf87 bofA) that controls the activation of a sigma factor, sigma K, at intermediate times during sporulation. The ski4 mutation caused the premature expression of cotA, a gene controlled by sigma K. An independent mutation that causes the premature activation of sigma K also caused a synthetic (synergistic) sporulation phenotype in combination with a null mutation in kinA, indicating that the defect was due to altered timing of gene expression directed by sigma K. Expression of ski4 was shown to be controlled by the sporulation-specific sigma factor sigma E.     

Host endoplasmic reticulum COPII proteins control cell‐to‐cell spread of the bacterial pathogen Listeria monocytogenes

Listeria monocytogenes is a food‐borne pathogen that uses actin‐dependent motility to spread between human cells. Cell‐to‐cell spread involves the formation by motile bacteria of plasma membrane‐derived structures termed ‘protrusions’. In cultured enterocytes, the secreted Listeria protein InlC promotes protrusion formation by binding and inhibiting the human scaffolding protein Tuba. Here we demonstrate that protrusions are controlled by human COPII components that direct trafficking from the endoplasmic reticulum. Co‐precipitation experiments indicated that the COPII proteins Sec31A and Sec13 interact directly with a Src homology 3 domain in Tuba. This interaction was antagonized by InlC. Depletion of Sec31A or Sec13 restored normal protrusion formation to a Listeria mutant lacking inlC, without affecting spread of wild‐type bacteria. Genetic impairment of the COPII component Sar1 or treatment of cells with brefeldin A affected protrusions similarly to Sec31A or Sec13 depletion. These findings indicated that InlC relieves a host‐mediated restriction of Listeria spread otherwise imposed by COPII. Inhibition of Sec31A, Sec13 or Sar1 or brefeldin A treatment also perturbed the structure of cell–cell junctions. Collectively, these findings demonstrate an important role for COPII in controlling Listeria spread. We propose that COPII may act by delivering host proteins that generate tension at cell junctions.     

Lung function,symptoms and inflammation during exacerbations of non-cystic fibrosis bronchiectasis: a prospective observational cohort study

Background

Exacerbations of non-cystic fibrosis bronchiectasis cause significant morbidity but there are few detailed data on their clinical course and associated physiological changes. The biology of an exacerbation has not been previously described.The purpose of this study was to describe changes in lung function, symptoms, health status and inflammation during the development and recovery from community-treated exacerbations.

Methods

This was a prospective observational cohort study of 32 outpatients with non-cystic fibrosis bronchiectasis conducted between August 2010 and August 2012. Patients completed a symptom diary card and measured their peak expiratory flow rate (PEFR) daily. Exacerbations were defined as oral antibiotic treatment taken for a worsening of respiratory symptoms. Symptoms and peak flow at exacerbation were analysed, and further measurements including the COPD Assessment Test (CAT) and inflammatory markers were also compared to baseline values.

Results

At baseline, health status was significantly related to lung function, prognostic severity and systemic inflammation. 51 exacerbations occurred in 22 patients. Exacerbation symptoms began a median (interquartile range) of 4 (2, 7) days before treatment started and the median exacerbation duration was 16 (10, 29) days. 16% had not recovered by 35 days. At exacerbation, mean PEFR dropped by 10.6% (95% confidence interval 6.9-14.2, p < 0.001) and mean CAT score increased by 6.3 units (3.6-9.1, p = 0.001), median symptom count by 4 (2.25, 6, p < 0.001), and mean CRP by 9.0mg/L (2.3-15.8, p = 0.011). Exacerbations where PEFR fell by ≥10% were longer with more symptoms at onset.

Conclusion

Exacerbations of non-CF bronchiectasis are inflammatory events, with worsened symptoms, lung function and health status, and a prolonged recovery period. Symptom diary cards, PEFR and CAT scores are responsive to changes at exacerbation and may be useful tools for their detection and monitoring.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0167-9) contains supplementary material, which is available to authorized users.     

Possible role for the essential GTP-binding protein Obg in regulating the initiation of sporulation in Bacillus subtilis.

We fused obg, encoding an essential GTP-binding protein in Bacillus subtilis, to the LacI-repressible, IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter Pspac. Depletion of Obg, following removal of IPTG, caused a defect in sporulation and in expression of sporulation genes that are activated by Spo0A approximately P. These defects were significantly relieved by a mutation in spo0A (rvtA11) that bypasses the normal phosphorylation pathway, indicating that Obg might normally be required, either directly or indirectly, to stimulate activity of the phosphorelay that activates Spo0A.     

On the origins of esterases

Comparisons among the primary sequences of five cloned eukaryotic esterases reveal two distinct lineages, neither bearing any significant overall sequence similarity to the functionally related serine protease multigene family. We have not eliminated the possibility that the esterases may have residual conformational similarities to the serine proteases. However, our profile analysis and analyses of the predicted conformations of the esterases reveal little similarity to the serine proteases. Four of the esterase proteins share 27%-53% overall sequence similarity and evidence of a catalytic mechanism involving the same Arg- Asp-Ser or His-Asp-Ser charge relay. We propose that these four esterases, three of them cholinesterases, form part of a multigene family essentially separate from the serine proteases.