Charakterisierung einer Hüllprotein-vermittelten Restriktion von HIV-2 in humanen Zellen

Die Erforschung von Restriktionen der HIV-Infektion spielt eine wichtige Rolle für die Aufklärung und das Verständnis von Virus-Wirt-Interaktionen und liefert neue Ansätze für die Entwicklung antiviraler Therapien. In der vorliegenden Arbeit wurde die Restriktion eines HIV-2 Isolates in humanen Zellen analysiert. Das Primärisolat prCBL23 erfährt im Vergleich zum T-Zell-adaptierten Isolat CBL23 eine Restriktion der Infektion in einigen humanen Zelllinien und primären Zellen. Die restriktiven Zellen exprimieren dabei die für die HIV-Infektion notwendigen Rezeptoren CD4 und CXCR4 und teilen sich aktiv. Zunächst wurde eine virale Determinante der Restriktion charakterisiert, dann die Phase des Replikationszyklus bestimmt, in der sich die Restriktion manifestiert, und anschließend Versuche zur Aufklärung des Mechanismus der Restriktion durchgeführt. In Infektionsstudien mit pseudotypisierten HIV-2-Partikeln konnte gezeigt werden, dass das Hüllprotein von prCBL23 die virale Determinante der Restriktion in GHOST/X4-Zellen ist. Es vermittelt im Vergleich zum Hüllprotein von CBL23 eine 60fache Restriktion der Infektion. Durch Mutationsanalyse der Hüllproteine prCBL23-Env und CBL23-Env konnte gezeigt werden, dass die Aminosäure an Position 74 im aminoterminalen Bereich von prCBL23-Env die für die Restriktion kritische Determinante ist. Die Aminosäure an Position 111 und die Verkürzung der zytoplasmatischen Domäne von prCBL23 spielen nur eine untergeordnete Rolle. Durch die Quantifizierung der Hüllproteine in den pseudotypisierten Partikeln konnte ausgeschlossen werden, dass die Restriktion durch einen schlechten Einbau des Hüllproteins in das virale Partikel vermittelt wird. Mit Hilfe des BlaM-Assays konnte gezeigt werden, dass die restringierten prCBL23-Env-Partikel mit ähnlicher Effizienz mit GHOST/X4-Zellen fusionieren können wie die nicht-restringierten CBL23-Partikel. Dies bedeutet, dass die Restriktion erst nach der Fusion innerhalb der Zelle stattfindet. Eine Analyse der viralen DNA in GHOST/X4-Zellen durch quantitative PCR zeigte weiterhin, dass sich die Restriktion bereits vor oder während der reversen Transkription manifestiert. Dies wirft die Frage auf, wie das Hüllprotein Prozesse beeinflussen kann, die nach der Fusion innerhalb der Zelle stattfinden. In Infektionsstudien mit gemischten Partikeln konnte beobachtet werden, dass bereits wenig CBL23-Env in prCBL23-Partikeln die Restriktion drastisch reduziert. Andererseits konnten keine Hinweise gefunden werden, dass die Aktivierung von Signalkaskaden eine Rolle für die Vermeidung der Restriktion spielt. Dies könnte bedeuten, dass die Restriktion nur vermieden werden kann, wenn CBL23-Env die Fusion selbst vermittelt. Eine Hypothese für den der Restriktion zugrunde liegenden Mechanismus wäre, dass durch die kritische Aminosäureposition 74 die Interaktion des Hüllproteins mit zellulären Oberflächenmolekülen beeinflusst wird, so dass die beiden Hüllproteine, möglicherweise unter Ausnutzung spezieller Mikrodomänen in der Zellmembran, das virale Kapsid auf verschiedenen Routen in die Zelle schleusen, die unterschiedlich günstige Bedingungen für die Replikation bieten. So könnten prCBL23-Env-Partikel in einem zellulären Kompartiment festsitzen, das keine günstigen Bedingungen bietet, oder die Degradation fördert. Die weiterführende Aufklärung des Mechanismus der Restriktion kann maßgeblich zu einem detaillierten Verständnis von Post-Fusions-Ereignissen wie Uncoating, Zusammenlagerung des Präintegrationskomplexes und Start der reversen Transkriptase beitragen, die bislang nur unzureichend erforscht sind

Local Persistence of Novel MRSA Lineage after Hospital Ward Outbreak, Cambridge, UK, 2011–2013

To the Editor: Previously, we reported the use of whole-genome sequencing to investigate a putative methicillin-resistant Staphylococcus aureus (MRSA) outbreak in 2011 in the special care baby unit (SCBU) at the Cambridge University Hospitals National Health Service Foundation Trust (CUH) in the United Kingdom (1). The report identified 26 related cases of infection with or asymptomatic carriage of MRSA and showed that transmission occurred within the SCBU, between mothers on a postnatal ward, and in the community; the outbreak apparently resolved at the end of 2011. The outbreak strain, sequence type (ST) 2371, was of a novel multilocus ST related to the dominant hospital-associated lineage in the UK (ST22, EMRSA-15), but unlike most ST22 strains, this strain was Panton-Valentine leucocidin–positive (2). Since then, ST2371 has been identified as a prevalent communityassociated MRSA clone in Southern India, and sporadic isolates have also been detected by whole-genome sequencing of MRSA in Denmark (3–5)The study was supported by grants from the UKCRC Translational Infection Research (TIR) Initiative, and the Medical Research Council (Grant Number G1000803) with contributions to the Grant from the Biotechnology and Biological Sciences Research Council, the National Institute for Health Research on behalf of the Department of Health, and the Chief Scientist Office of the Scottish Government Health Directorate (to Prof. Peacock); by a Healthcare Infection Society Major Research Grant; and by Wellcome Trust grant number 098051 awarded to the Wellcome Trust Sanger Institute

The thrombophilic network of autoantibodies in celiac disease

BACKGROUND: Celiac disease is a life-long autoimmune condition, affecting genetically susceptible individuals that may present with thromboembolic phenomena. This thrombophilia represents a puzzle with multiple constituents: hyperhomocysteinemia, B12 and\or folate deficiency, methylenetetrahydrofolate reductase mutations, and protein C and S deficiency due to vitamin K deficiency. However, the well known thrombogenic factors, antiphosphatidylserine/prothrombin and antiprothrombin have never been explored in celiac disease. METHODS: The serum autoantibody levels were determined in 248 individuals, classified into three groups. Group 1 comprised 70 children with definitive celiac disease (age: 7.04 ±4.3 years, male to female ratio 1.06) and group 2 comprised 88 normal children (age: 6.7 ±4.17 years, male to female ratio 0.87), representing controls. The pediatric populations were compared to group 3, which included 90 adults who were family members (parents) of group 1 (age: 34.6 ±11.35 years, male to female ratio 1.2). Antibodies were checked by enzyme-linked immunosorbent assay. RESULTS: Mean optical density levels of serum antiphosphatidylserine/prothrombin immunoglobulin G antibodies were 32.4 ±19.4, 3.6 ±2.5 and 16.1 ±15.8 absorbance units in groups 1, 2 and 3 respectively (P <0.0001), with 45.7%, 0% and 7.8% of groups 1, 2 and 3 respectively positive for the antibody (P <0.01). Mean optical density levels of serum antiphosphatidylserine/prothrombin immunoglobulin M antibodies were 14.2 ±8.7, 6.7 ±6.4 and 12.4 ±15.5 absorbance units in groups 1, 2 and 3 respectively (P <0.0001), with 7.1%, 3.4% and 9.9% of groups 1, 2 and 3 positive for the antibody. Mean optical density levels of serum antiprothrombin and antiphospholipid immunoglobulin G antibodies were higher in groups 1 and 3 compared with 2 (P <0.005) and in groups 1 and 2 compared with 3 (P <0.01), respectively. Groups 1, 2 and 3 were positive for antiphospholipid immunoglobulin G antibodies (groups 1 and 2 compared with 3) . Celiac disease sera harbor a higher antiprothrombin immunoglobulin G level compared with controls. CONCLUSIONS: It is suggested that the intestinal injury, endothelial dysfunction, platelet abnormality and enhanced apoptosis recently described in celiac disease are at the origin of the increased exposure of phospholipids or new epitopes representing autoantigens. Those autoantibodies might play a pathogenic role in the thrombophilia associated with celiac disease and represent markers for potential anticoagulant preventive therapy

Comparison of bacterial genome assembly software for MinION data and their applicability to medical microbiology.

Translating the Oxford Nanopore MinION sequencing technology into medical microbiology requires on-going analysis that keeps pace with technological improvements to the instrument and release of associated analysis software. Here, we use a multidrug-resistant Enterobacter kobei isolate as a model organism to compare open source software for the assembly of genome data, and relate this to the time taken to generate actionable information. Three software tools (PBcR, Canu and miniasm) were used to assemble MinION data and a fourth (SPAdes) was used to combine MinION and Illumina data to produce a hybrid assembly. All four had a similar number of contigs and were more contiguous than the assembly using Illumina data alone, with SPAdes producing a single chromosomal contig. Evaluation of the four assemblies to represent the genome structure revealed a single large inversion in the SPAdes assembly, which also incorrectly integrated a plasmid into the chromosomal contig. Almost 50 %, 80 % and 90 % of MinION pass reads were generated in the first 6, 9 and 12 h, respectively. Using data from the first 6 h alone led to a less accurate, fragmented assembly, but data from the first 9 or 12 h generated similar assemblies to that from 48 h sequencing. Assemblies were generated in 2 h using Canu, indicating that going from isolate to assembled data is possible in less than 48 h. MinION data identified that genes responsible for resistance were carried by two plasmids encoding resistance to carbapenem and to sulphonamides, rifampicin and aminoglycosides, respectively.Health Innovation Challenge Fund (WT098600, HICF-T5-342) (Department of Health, Wellcome Trust)This is the final version of the article. It first appeared from the Microbiology Society via http://dx.doi.org/10.1099/mgen.0.00008

Use of whole-genus genome sequence data to develop a multilocus sequence typing tool that accurately identifies Yersinia isolates to the species and subspecies levels

The genus Yersinia is a large and diverse bacterial genus consisting of human-pathogenic species, a fish-pathogenic species, and a large number of environmental species. Recently, the phylogenetic and population structure of the entire genus was elucidated through the genome sequence data of 241 strains encompassing every known species in the genus. Here we report the mining of this enormous data set to create a multilocus sequence typing-based scheme that can identify Yersinia strains to the species level to a level of resolution equal to that for whole-genome sequencing. Our assay is designed to be able to accurately subtype the important human-pathogenic species Yersinia enterocolitica to whole-genome resolution levels. We also report the validation of the scheme on 386 strains from reference laboratory collections across Europe. We propose that the scheme is an important molecular typing system to allow accurate and reproducible identification of Yersinia isolates to the species level, a process often inconsistent in nonspecialist laboratories. Additionally, our assay is the most phylogenetically informative typing scheme available for Y. enterocolitica

Two TRPV1 receptor antagonists are effective in two different experimental models of migraine

Background The capsaicin and heat responsive ion channel TRPV1 is expressed on trigeminal nociceptive neurons and has been implicated in the pathophysiology of migraine attacks. Here we investigate the efficacy of two TRPV1 channel antagonists in blocking trigeminal activation using two in vivo models of migraine. Methods Male Sprague–Dawley rats were used to study the effects of the TRPV1 antagonists JNJ-38893777 and JNJ-17203212 on trigeminal activation. Expression of the immediate early gene c-fos was measured following intracisternal application of inflammatory soup. In a second model, CGRP release into the external jugular vein was determined following injection of capsaicin into the carotid artery. Results Inflammatory up-regulation of c-fos in the trigeminal brain stem complex was dose-dependently and significantly reduced by both TRPV1 antagonists. Capsaicin-induced CGRP release was attenuated by JNJ-38893777 only in higher dosage. JNJ-17203212 was effective in all doses and fully abolished CGRP release in a time and dose-dependent manner. Conclusion Our results describe two TRPV1 antagonists that are effective in two in vivo models of migraine. These results suggest that TRPV1 may play a role in the pathophysiological mechanisms, which are relevant to migraine

Limited Multidrug Resistance Efflux Pump Overexpression among Multidrug-Resistant Escherichia coli Strains of ST131

Gram-negative bacteria partly rely on efflux pumps to facilitate growth under stressful conditions and to increase resistance to a wide variety of commonly used drugs. In recent years E. coli ST131 has emerged as a major cause of extraintestinal infection frequently exhibiting an MDR phenotype. The contribution of efflux to MDR in emerging E. coli MDR clones however, is not well studied. We characterized strains from an international collection of clinical MDR-E. coli isolates by MIC testing with and without the addition of the AcrAB-TolC efflux inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). MIC data for 6 antimicrobial agents and their reversion by NMP were analyzed by Principal Component Analysis (PCA). PCA revealed a group of 17/34 MDR-E. coli exhibiting increased susceptibility to treatment with NMP suggesting an enhanced contribution of efflux pumps to antimicrobial resistance in these strains (termed "enhanced efflux phenotype" [EEP]). Only 1/17 EEP strains versus 12/17 non-EEP MDR strains belonged to the ST131 clonal group. Whole-genome sequencing revealed marked differences in efflux-related genes between EEP and control strains, with the majority of notable amino-acid substitutions occurring in AcrR, MarR and SoxR. qRT-PCR of multiple efflux-related genes showed significant overexpression of the AcrAB-TolC-system in EEP strains, whereas in the remaining strains we found enhanced expression of alternative efflux proteins. We conclude that a proportion of MDR E. coli exhibit an EEP, which is linked to an overexpression of the AcrAB-TolC-efflux-pump and a distinct array of genomic variations. Members of ST131, although highly successful, are less likely to exhibit the EEP

Taking hospital pathogen surveillance to the next level

High-throughput bacterial genomic sequencing and subsequent analyses can produce large volumes of high-quality data rapidly. Advances in sequencing technology, with commensurate developments in bioinformatics, have increased the speed and efficiency with which it is possible to apply genomics to outbreak analysis and broader public health surveillance. This approach has been focused on targeted pathogenic taxa, such as Mycobacteria, and diseases corresponding to different modes of transmission, including food-and-water-borne diseases (FWDs) and sexually transmitted infections (STIs). In addition, major healthcare-associated pathogens such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and carbapenemase-producing Klebsiella pneumoniae are the focus of research projects and initiatives to understand transmission dynamics and temporal trends on both local and global scales. Here, we discuss current and future public health priorities relating to genome-based surveillance of major healthcare-associated pathogens. We highlight the specific challenges for the surveillance of healthcare-associated infections (HAIs), and how recent technical advances might be deployed most effectively to mitigate the increasing public health burden they cause

Whole-genome sequencing reveals transmission of vancomycin-resistant Enterococcus faecium in a healthcare network.

BACKGROUND: Bacterial whole-genome sequencing (WGS) has the potential to identify reservoirs of multidrug-resistant organisms and transmission of these pathogens across healthcare networks. We used WGS to define transmission of vancomycin-resistant enterococci (VRE) within a long-term care facility (LTCF), and between this and an acute hospital in the United Kingdom (UK). METHODS: A longitudinal prospective observational study of faecal VRE carriage was conducted in a LTCF in Cambridge, UK. Stool samples were collected at recruitment, and then repeatedly until the end of the study period, discharge or death. Selective culture media were used to isolate VRE, which were subsequently sequenced and analysed. We also analysed the genomes of 45 Enterococcus faecium bloodstream isolates collected at Cambridge University Hospitals NHS Foundation Trust (CUH). RESULTS: Forty-five residents were recruited during a 6-month period in 2014, and 693 stools were collected at a frequency of at least 1 week apart. Fifty-one stool samples from 3/45 participants (7 %) were positive for vancomycin-resistant E. faecium. Two residents carried multiple VRE lineages, and one carried a single VRE lineage. Genome analyses based on single nucleotide polymorphisms (SNPs) in the core genome indicated that VRE carried by each of the three residents were unrelated. Participants had extensive contact with the local healthcare network. We found that VRE genomes from LTCF residents and hospital-associated bloodstream infection were interspersed throughout the phylogenetic tree, with several instances of closely related VRE strains from the two settings. CONCLUSIONS: A proportion of LTCF residents are long-term carriers of VRE. Evidence for genetic relatedness between these and VRE associated with bloodstream infection in a nearby acute NHS Trust indicate a shared bacterial population.We gratefully acknowledge the contribution of the staff at the LTCF in sample collection, and thank the patients who agreed to participate. We thank Kirsty Ambridge and Angela Kidney for technical assistance. We are grateful for assistance from the library construction, sequencing and core informatics teams at the Wellcome Trust Sanger Institute. This publication presents independent research supported by the Health Innovation Challenge Fund (WT098600, HICF-T5-342), a parallel funding partnership between the Department of Health and Wellcome Trust. The views expressed in this publication are those of the author(s) and not necessarily those of the Department of Health or Wellcome Trust. MET is a Clinician Scientist Fellow supported by the Academy of Medical Sciences, The Health Foundation and the NIHR Cambridge Biomedical Research Centre.This is the final version of the article. It was first available from BioMed Central via http://dx.doi.org/10.1186/s13073-015-0259-

Systematic Surveillance Detects Multiple Silent Introductions and Household Transmission of Methicillin-Resistant Staphylococcus aureus USA300 in the East of England.

BACKGROUND: The spread of USA300 methicillin-resistant Staphylococcus aureus (MRSA) across the United States resulted in an epidemic of infections. In Europe, only sporadic cases or small clusters of USA300 infections are described, and its prevalence in England is unknown. We conducted prospective surveillance for USA300 in the east of England. METHODS: We undertook a 12-month prospective observational cohort study of all individuals with MRSA isolated from community and hospital samples submitted to a microbiology laboratory. At least 1 MRSA isolate from each individual underwent whole-genome sequencing. USA300 was identified on the basis of sequence analysis, and phylogenetic comparisons were made between these and USA300 genomes from the United States. RESULTS: Between April 2012 and April 2013, we sequenced 2283 MRSA isolates (detected during carriage screening and in clinical samples) from 1465 individuals. USA300 was isolated from 24 cases (1.6%). Ten cases (42%) had skin and soft tissue infection, and 2 cases had invasive disease. Phylogenetic analyses identified multiple introductions and household transmission of USA300. CONCLUSIONS: Use of a diagnostic laboratory as a sentinel for surveillance has identified repeated introductions of USA300 in eastern England in 2012-2013, with evidence for limited transmission. Our results show how systematic surveillance could provide an early warning of strain emergence and dissemination.This work was supported by grants from the UK Clinical Research Collaboration Translational Infection Research Initiative, and the Medical Research Council (Grant Number G1000803) with contributions to the Grant from the Biotechnology and Biological Sciences Research Council, the National Institute for Health Research on behalf of the Department of Health, and the Chief Scientist Office of the Scottish Government Health Directorate (to Prof. Peacock); by a Healthcare Infection Society Major Research Grant (to Prof. Peacock), and by Wellcome Trust grant number 098051 awarded to the Wellcome Trust Sanger Institute. MST is a Wellcome Trust Clinical PhD Fellow. MET is a Clinician Scientist Fellow, supported by the Academy of Medical Sciences and the Health Foundation, and by the National Institute for Health Research Cambridge Biomedical Research Centre.This is the final version of the article. It first appeared from Oxford University Press via https://doi.org/10.1093/infdis/jiw16