Institut für Hygiene und Mikrobiologie
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- Broad Institute, USA (1)
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- Department of Hematology and Oncology, Sana Hospital Hof, Hof, Germany (1)
- Department of Laboratory Medicine and Medicine Huddinge, Karolinska Institutet and University Hospital, Stockholm, Sweden (1)
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- Krankenhaushygiene und Antimicrobial Stewardship (1)
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- 2016 FGR 0053 (1)
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- 847507 (1)
Background:
Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence.
Results:
Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region.
Conclusions:
Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis.
Introduction: Although there has been a worldwide emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA), little is known about the molecular epidemiology of MRSA in Tanzania.
Methodology: In this study, we characterized MRSA strains isolated from clinical specimens at the Bugando Medical Centre, Tanzania, between January and December 2008. Of 160 S. aureus isolates from 600 clinical specimens, 24 (15%) were found to be MRSA. Besides molecular screening for the Panton Valentine leukocidin (PVL) genes by PCR, MRSA strains were further characterized by Multi-Locus Sequence Typing (MLST) and spa typing.
Results: Despite considerable genetic diversity, the spa types t690 (29.1%) and t7231 (41.6%), as well as the sequence types (ST) 88 (54.2%) and 1797 (29.1%), were dominant among clinical isolates. The PVL genes were detected in 4 isolates; of these, 3 were found in ST 88 and one in ST1820. Resistance to erythromycin, clindamicin, gentamicin, tetracycline and co-trimoxazole was found in 45.8%, 62.5%, 41.6%, 45.8% and 50% of the strains, respectively.
Conclusion: We present the first thorough typing of MRSA at a Tanzanian hospital. Despite considerable genetic diversity, ST88 was dominant among clinical isolates at the Bugando Medical Centre. Active and standardized surveillance of nosocomial MRSA infection should be conducted in the future to analyse the infection and transmission rates and implement effective control measures.
Einfluss des Komplementsystems und der neuartigen Meningokokken-Vakzine 4CMenB auf cnl-Meningokokken
(2016)
In dieser Arbeit wurden verschiedene Vakzine-relevante Oberflächenantigene von cnl-Meningokokken typisiert und die Interaktion von cnl-Meningokokken mit dem Komplementsystem, v.a. mit dessen Hauptregulatoren fH und C4bp, analysiert. Mit den gewonnenen Daten sollten Schlussfolgerungen bzgl. der erwarteten Wirkung von 4CMenB, einem 2013 in Deutschland eingeführten und auf Meningokokken der Serogruppe B abzielenden Impfstoff, auf cnl-Meningokokken gezogen werden. Des Weiteren sollte die Interaktion der natürlicherweise unbekapselten cnl-Meningokokken, die als apathogen und möglicherweise günstig für die Entwicklung einer natürlichen Immunität eingeschätzt werden, untersucht werden.
Eine Auswahl von cnl-Meningokokken-Stämmen, die die genetische Variabilität dieser Bakterienpopulation abbilden, wurde mittels PCR (porA, porB, fetA, opc, fHbp, nhba und nadA) oder Western Blot-Analyse (Opc) typisiert. Hierbei konnte eine deutliche Assoziation einzelner Allele zu klonalen Komplexen gezeigt werden. Allerdings lässt die Analyse bezweifeln, dass cnl-Meningokokken durch Bexsero-induzierte Antikörper erkannt werden,
da ihr Antigenmuster stark von den Vakzineantigenen abweicht. Unklarheit herrscht lediglich bzgl. des Antigens NhbA.
In der Folge wurde die fH- und C4bp-Bindung bei cnl-Meningokokken mittels Durchflusszytometrie untersucht. Es konnte beobachtet werden, dass im Vergleich zu fH bzw. C4bp bindenden Kontrollstämmen die Bindung der Hauptregulatoren des Komplementsystems an cnl-Meningokokken sehr gering ist. Weiterhin konnte gezeigt werden, dass cnl-Meningokokken eine sehr geringe Serumresistenz in vitro haben, was ebenfalls für eine schwache Akquirierung der Komplementregulatoren spricht. Dieser Befund unterstreicht die apathogene Natur der Bakterien. Er zeigt aber auch, dass mit herkömmlichen Methoden wie dem Serumbakterizidietest, der bei bekapselten Stämmen angewendet wird, funktionelle Aussagen bzgl. der Wirkung bakterizider Antikörper, die durch Impfstoffe auf Proteinbasis induziert werden, nur schwer zu tätigen sein werden. Sehr geringe Komplementmengen müssten eingesetzt werden oder alternative Verfahren wie die Opsonophagozytose Anwendung finden.
Background
Early developmental patterns of flatworms are extremely diverse and difficult to compare between distant groups. In parasitic flatworms, such as tapeworms, this is confounded by highly derived life cycles involving indirect development, and even the true orientation of the tapeworm antero-posterior (AP) axis has been a matter of controversy. In planarians, and metazoans generally, the AP axis is specified by the canonical Wnt pathway, and we hypothesized that it could also underpin axial formation during larval metamorphosis in tapeworms.
Results
By comparative gene expression analysis of Wnt components and conserved AP markers in the tapeworms Echinococcus multilocularis and Hymenolepis microstoma, we found remarkable similarities between the early stages of larval metamorphosis in tapeworms and late embryonic and adult development in planarians. We demonstrate posterior expression of specific Wnt factors during larval metamorphosis and show that scolex formation is preceded by localized expression of Wnt inhibitors. In the highly derived larval form of E. multilocularis, which proliferates asexually within the mammalian host, we found ubiquitous expression of posterior Wnt factors combined with localized expression of Wnt inhibitors that correlates with the asexual budding of scoleces. As in planarians, muscle cells are shown to be a source of secreted Wnt ligands, providing an explanation for the retention of a muscle layer in the immotile E. multilocularis larva.
Conclusions
The strong conservation of gene expression between larval metamorphosis in tapeworms and late embryonic development in planarians suggests, for the first time, a homologous developmental period across this diverse phylum. We postulate these to represent the phylotypic stages of these flatworm groups. Our results support the classical notion that the scolex is the true anterior end of tapeworms. Furthermore, the up-regulation of Wnt inhibitors during the specification of multiple anterior poles suggests a mechanism for the unique asexual reproduction of E. multilocularis larvae.
Neisseria meningitidis is a commensal bacterium which sometimes causes serious disease in humans. Recent studies in numerous human pathogenic bacteria have shown that the stringent response contributes to bacterial virulence. Therefore, this study analyzed the regulation of the stringent response in meningococci and in particular of RelA as well as its contribution to ex vivo fitness in a strain- and condition- dependent manner by using the carriage strain α522 and the hyperinvasive strain MC58 in different in vitro and ex vivo conditions.
Growth experiments revealed that both wild-type strains were almost indistinguishable in their ex vivo phenotypes. However, quantitative real time PCR (qRT-PCR) found differences in the gene expression of relA between both strains. Furthermore, in contrast to the MC58 RelA mutant strain α522 deficient in RelA was unable to survive in human whole blood, although both strains showed the same ex vivo phenotypes in saliva and cerebrospinal fluid. Moreover, strain α522 was depended on a short non-coding AT-rich repeat element (ATRrelA) in the promoter region of relA to survive in human blood. Furthermore, cell culture experiments with human epithelial cells revealed that in both strains the deletion of relA resulted in a significantly decreased invasion rate while not significantly affecting adhesion. In order to better understand the conditional lethality of the relA deletion, computational and experimental analyses were carried out to unravel differences in amino acid biosynthetic pathways between both strains. Whereas strain MC58 is able to synthesize all 20 amino acids, strain α522 has an auxotrophy for cysteine and glutamine. In addition, the in vitro growth experiments found that RelA is required for growth in the absence of external amino acids in both strains. Furthermore, the mutant strain MC58 harboring an ATRrelA in its relA promoter region showed improved growth in minimal medium supplemented with L-cysteine and/or L-glutamine compared to the wild-type strain. Contrary, in strain α522 no differences between the wild-type and the ATRrelA deletion mutant were observed.
Together this indicates that ATRrelA interferes with the complex regulatory interplay between the stringent response pathway and L-cysteine as well as L-glutamine metabolism. It further suggests that meningococcal virulence is linked to relA in a strain- and condition- depended manner. In conclusion, this work highlighted the role of the stringent response and of non-coding regulatory elements for bacterial virulence and indicates that virulence might be related to the way how meningococci accomplish growth within the host environments.
Die Interaktion mit Gehirnendothelzellen stellt ein zentraler Schritt in der Infektionspathogenese von Neisseria meningitidis dar. In dieser Promotionsarbeit konnte gezeigt werden, dass die Infektion von menschlichen Gehirnendothelzellen mit N. meningitidis zu einer transienten Aktivierung der sauren Sphingomyelinase (ASM) gefolgt von einer vermehrten Ceramidproduktion führt. Als Antwort auf die Infektion mit N. meningitidis kommt es zu einer vermehrten Präsentation der ASM und von Ceramiden an der äusseren Seite der Plasmamembran und zu einer Ausbildung von großen Ceramid-reichen Membran-Domänen, welche mit cortical plaque assoziierten Proteinen kolokalisieren. Bei dieser N. meningitids vermittelten Aktivierung der ASM spielt das bakterielle Aussenmembranprotein Opc sowie die Aktivierung der Phosphatidylcholin-spezifische Phospholipase C über die Interaktion von Opc mit Heparansulfat-Proteoglykane eine entscheidende Rolle. Die pharmakologische oder genetische Inhibition der ASM Funktion führt zu einer geringeren Invasivität der Meningokokken ohne dabei die Adhärenz zu beeinflussen. Im Einklang mit diesen Ergebnissen steht die Beobachtung, dass die geringere Invasivität von ausgewählten Isolaten des ST-11/ST-8 Komplex in menschlichen Gehirnendothelzellen direkt mit ihrer eingeschränkter Fähigkeit korreliert, die ASM zu aktivieren bzw. eine Ceramidproduktion zu induzieren. Schlussfolgernd ist die ASM Aktivierung und eine nachfolgende Ceramidproduktion essenziell für die Internalisierung von Opc-exprimierende Meningokokken in Gehirnendothelzellen und bietet einen Erklärungsansatz für die unterschiedliche Invasivität von verschiedenen N. meningitidis Stämmen.
Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.
Alveolar and cystic echinococcosis, caused by Echinococcus multilocularis and Echinococcus
granulosus respectively, are severe zoonotic diseases with limited treatment
options. The sole curative treatment is the surgical removal of the complete parasite
material. Due to late diagnosis, chemotherapeutic treatment often is the only treatment
option. Treatment is based on benzimidazoles, which merely act parasitostatic
and often display strong side effects. Therefore, new therapeutic drugs are urgently
needed.
Evolutionarily conserved signalling pathways are known to be involved in hostparasite
cross-communication, parasite development and survival. Moreover, they
represent potential targets for chemotherapeutic drugs. In this context the roles of
the serotonin- and cAMP-signalling pathways in Echinococcus were studied.
Genes encoding serotonin receptors, a serotonin transporter and enzymes involved in
serotonin biosynthesis could be identified in the E. multilocularis and E. granulosus
genomes indicating that these parasites are capable of synthesizing and perceiving
serotonin signals. Also the influence of exogenous serotonin on parasite development
was studied. Serotonin significantly increased metacestode vesicle formation
from primary cells and re-differentiation of protoscoleces. Inhibition of serotonin
transport with citalopram significantly reduced metacestode vesicle formation from
primary cells and caused death of protoscoleces and metacestodes. Furthermore, it
could be shown that serotonin increased phosphorylation of protein kinase A substrates.
Taken together, these results show that serotonin and serotonin transport
are essential for Echinococcus development and survival. Consequently, components
of the serotonin pathway represent potential drug targets.
In this work the cAMP-signalling pathway was researched with focus on G-protein
coupled receptors and adenylate cyclases. 76 G-protein coupled receptors, including
members of all major families were identified in the E. multilocularis genome.
Four genes homologous to adenylate cyclase IX were identified in the E. multilocularis
genome and three in the E. granulosus genome. While glucagon caused
no significant effects, the adenylate cyclase activator forskolin and the adenylate
cyclase inhibitor 2’, 5’ didesoxyadenosine influenced metacestode vesicle formation
from primary cells, re-differentiation of protoscoleces and survival of metacestodes.
It was further shown that forskolin increases phosphorylation of protein kinase A
substrates, indicating that forskolin activates the cAMP-pathway also in cestodes.
These results indicate that the cAMP signalling pathway plays an important role in
Echinococcus development and survival.
To complement this work, the influence of different media and additives on E. granulosus protoscoleces was investigated. Anaerobic conditions and the presence of FBS
prolonged protoscolex survival while different media influenced protoscolex activation
and development.
Taken together, this work provided important insights into developmental processes
in Echinococcus and potential drug targets for echinococcosis chemotherapy.
Background: Alveolar echinococcosis, caused by Echinococcus multilocularis larvae, is a chronic disease associated with considerable modulation of the host immune response. Dendritic cells (DC) are key effectors in shaping the immune response and among the first cells encountered by the parasite during an infection. Although it is assumed that E. multilocularis, by excretory/secretory (E/S)-products, specifically affects DC to deviate immune responses, little information is available on the molecular nature of respective E/S-products and their mode of action. Methodology/Principal Findings: We established cultivation systems for exposing DC to live material from early (oncosphere), chronic (metacestode) and late (protoscolex) infectious stages. When co-incubated with Echinococcus primary cells, representing the invading oncosphere, or metacestode vesicles, a significant proportion of DC underwent apoptosis and the surviving DC failed to mature. In contrast, DC exposed to protoscoleces upregulated maturation markers and did not undergo apoptosis. After pre-incubation with primary cells and metacestode vesicles, DC showed a strongly impaired ability to be activated by the TLR ligand LPS, which was not observed in DC pre-treated with protoscolex E/S-products. While none of the larvae induced the secretion of pro-inflammatory IL-12p70, the production of immunosuppressive IL-10 was elevated in response to primary cell E/S-products. Finally, upon incubation with DC and naive T-cells, E/S-products from metacestode vesicles led to a significant expansion of Foxp3+ T cells in vitro. Conclusions: This is the first report on the induction of apoptosis in DC by cestode E/S-products. Our data indicate that the early infective stage of E. multilocularis is a strong inducer of tolerance in DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. The induction of CD4+CD25+Foxp3+ T cells through metacestode E/S-products suggests that these cells fulfill an important role for parasite persistence during chronic echinococcosis.