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The Group A streptococcus Interleukin-8 Protease spyCEP Promotes Bacterial Intracellular survival by Evasion of Autophagy

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Infectious Microbes & Diseases 2022年第3期4卷 116-123页

作者： RenéBergmann Giuseppe Gulotta Federica Andreoni Tomoko sumitomo shigetada Kawabata Annelies s.Zinkernagel Gursharan s.Chhatwal Victor Nizet Manfred Rohde satoshi Uchiyama Central Unit for Microscopy(ZEIM) Helmholtz Centre for Infection ResearchBraunschweigGermany Division of Infectious Diseases and Hospital Epidemiology University Hospital ZurichZurichSwitzerland Department of Oral and Molecular Microbiology Osaka University Graduate School of DentistrySuita-OsakaJapan Division of Host-Microbe systems and Therapeutics Department of PediatricsUniversity of CaliforniaSan DiegoLa JollaCalifornia

Autophagy serves an innate immune function in defending the host against invading bacteria,including group A streptococcus(GAs).Autophagy is regulated by numerous host proteins,including the endogenous negative regulator calpain,a cytosolic protease.Globally disseminated serotypeM1T1 GAs strains associated with high invasive disease potential express numerous virulence factors and resist autophagic clearance.Upon in vitro infection of human epithelial cell lines with representative wild-type GAs M1T1 strain 5448(M1.5448),we observed increased calpain activation linked to a specific GAs virulence factor,the interleukin-8 protease spyCEP.Calpain activation inhibited autophagy and decreased capture of cytosolic GAs in autophagosomes.In contrast,the serotype M6 GAs strain JRs4(M6.JRs4),which is highly susceptible to host autophagy-mediated killing,expresses low levels of spyCEP and does not activate calpain.Overexpression of spyCEP in M6.JRs4 stimulated calpain activation,inhibited autophagy,and significantly decreased bacterial capture in autophagosomes.These paired loss-and gain-of-function studies reveal a novel role for the bacterial protease spyCEP in enabling GAs M1 evasion of autophagy and host innate immune clearance.

关键词： group A streptococcus spyCEP virulence factor epithelial cells intracellular survival autophagy xenophagy calpain

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Multimodal imaging of experimental choroidal neovascularization

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International Journal of Ophthalmology(English edition) 2022年第6期15卷 886-893页

作者： Ioanna Tsioti Xuan Liu Petra schwarzer Martin s.zinkernagel Despina Kokona Department of Ophthalmology InselspitalBern University Hospitaland Department of BioMedical ResearchUniversity of BernBern 3010Switzerland Department of Ophthalmology the First Affiliated Hospital of Xi’an Jiaotong UniversityXi’an 710061Shaanxi ProvinceChina

AIM:To compare choroidal neovascularization(CNV)lesion measurements obtained by in vivo imaging modalities,with whole mount histological preparations stained with isolectin Gs-IB4,using a murine laser-induced CNV model.METHODs:B6 N.Cg-Tg(Csf1 r-EGFP)1 Hume/J heterozygous adult mice were subjected to laser-induced CNV and were monitored by fluorescein angiography(FA),multicolor(MC)fundus imaging and optical coherence tomography angiography(OCTA)at day 14 after CNV induction.Choroidalretinal pigment epithelium(RPE)whole mounts were prepared at the end of the experiment and were stained with isolectin Gs-IB4.CNV areas were measured in all different imaging modalities at day 14 after CNV from three independent raters and were compared to choroidal-RPE whole mounts.Intraclass correlation coefficient(ICC)type 2(2-way random model)and its 95%confidence intervals(CI)were calculated to measure the correlation between different raters’measurements.spearman’s rank correlation coefficient(spearman’s r)was calculated for the comparison between FA,MC and OCTA data and histology data.REsULTs:FA(early and late)and MC correlates well with the CNV measurements ex vivo with FA having slightly better correlation than MC(FA early spearman’s r=0.7642,FA late spearman’s r=0.7097,and MC spearman’s r=0.7418),while the interobser ver reliability was good for both techniques(FA early ICC=0.976,FA late ICC=0.964,and MC ICC=0.846).In contrast,OCTA showed a poor correlation with ex vivo measurements(spearman’s r=0.05716)and high variability between different raters(ICC=0.603).CONCLUsION:This study suggests that FA and MC imaging could be used for the evaluation of CNV areas in vivo while caution must be taken and comparison studies should be performed when OCTA is employed as a CNV monitoring tool in small rodents.

关键词： choroidal neovascularization in vivo imaging fluorescein angiography multicolor fundus imaging optical coherence tomography angiography

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selection Vector for Direct Cloning of Proof Reading Polymerase Chain Reaction Products Based on the Lethal <i>ccdB</i>Gene in <i>Escherichia coli </i>

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Advances in Microbiology 2013年第1期3卷 14-20页

作者： Pascal Weibel Miriam Ender Jerzy Madon annelies s. zinkernagel Reto A. schuepbach Clinic of Infectious Diseases and Hospital Hygiene University Hospital Zurich University of Zurich Zurich Switzerland surgical Intensive Care Medicine University Hospital Zurich University of Zurich Zurich Switzerland

Introducing PCR products into plasmids vectors is key for molecular techniques. Ideally cloning vectors are easy to construct, modify and propagate, neither require advanced techniques nor special equipment or reagents and efficiently incorporate PCR products at close to zero empty vector background. We provide an easy to engineer self-made cloning vector, neither requiring sophisticated tools or techniques nor advanced cloning knowledge. Through recombination we obtained the pUC18ccdB vector, carrying the ccdB suicide gene within the pUC18 backbone. When smaI cleaved (within the ccdB) vector was T4 ligated with small (0.2 kbp) and intermediate (1.3 to 2.2 kbp) blunt end PCR-products and transformed into E. coli, the amount of clones with incorporated PCR product was comparable to commercial PCR-cloning kits and at a close to zero PCR product negative background. In conclusion we present a simple, versatile and cheap approach to an efficient “home made” PCR-cloning vector that allows integration of crude blunt end PCR products at close to zero background.

关键词： PCR-Cloning Vector Blunt End ccdB Toxic Gene

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