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Rabies virus (RABV) is an ancient, highly neurotropic rhabdovirus that causes lethal encephalitis. Most RABV pathogenesis determinants have been identified with laboratory-adapted or attenuated RABVs, but details of natural RABV pathogenesis and attenuation mechanisms are still poorly understood. To provide a deeper insight in the cellular mechanism of pathogenies of field RABV, this work was performed to assess virus strain specific differences in intra-neuronal virus transport, to identify cell culture adaptive mutations in recombinant field viruses and to explore shRNA-expressing RABVs as research tools for targeted host manipulation in infected cells.
Comparison of chimeric RABVs with glycoprotein (G) ecto-domains of different lyssaviruses, together with field RABVs from dog and fox in dorsal root ganglion (DRG) neurons revealed no detectable differences in the axonal accumulation of the viruses. This indicates that previously described G-dependent transport of newly formed RABV in axons can occur both in laboratory-adapted and field RABV. Moreover, partial overlap of nucleoprotein (N) and G protein particles in field virus infected DRG axons supported the hypothesis of the “separate model” for anterograde RABV transport.
Serial passages of recombinant dog and fox field clones in different cell lines led to the identification of general (D266N) and cell line specific (K444N) adaptive mutations in the G ecto-domain of both viruses. In BHK cells, synergistic effects of D226N, K444N and A417T on field dog virus G protein surface localization led to the loss of endoplasmic reticulum (ER) retention of G and increased virus titers in the supernatant, indicating that limited virus release by ER retention is a major bottleneck in cell culture adaptation. In addition, selection of mutations within the C-terminus of the RABV phosphoprotein (P) (R293H and R293C in fox and dog viruses, respectively) led to the hypothesis of altered binding affinities to nucleoprotein and RNP complexes. Identification of the above mentioned amino acid substitutions together with alterations in a suboptimal transcription stop signal in the P/M gene border indicated that adaptation to cell culture replication occurs on both levels, RNA transcription/replication and virus release.
To evaluate the possibility of an expression of a functional microRNA-adapted short-hairpin RNAs (miR-shRNA) expressing RABV, recombinant RABVs encoding miR-shRNAs against cellular Dynein Light Chain 1 (DYNLL1) and Acidic Nuclear Phosphoprotein 32 family member B (ANP32B) were generated. In spite of cytoplasmic transcription of the respective mRNAs, downregulation of DYNLL1 and ANP32B mRNA and respective protein levels in infected cells revealed correct processing to functional shRNAs. Specific downregulation of the cellular genes at 2, 3 and 4 days post infection further demonstrated feasibility of the approach in standard cell lines. However, it remained open whether miR-shRNA expressing RABV can be used to study neuro-infection in vivo. Since first attempts in primary rat neuron cultures failed, it has to be clarified in further experiments whether this strategy can be used in mature, non-dividing neurons or whether breakdown of the nucleus in the course of cell division is a requirement for the processing of cytoplasmically expressed miR-RNA by nuclear RNases.
By providing novel insights in axonal RABV transport and cell culture adaptive mutations this work extends the current understanding of RABV pathogenesis in natural and non-natural cell environments. Moreover, it provides a basis for further pathogenicity studies in which the impact of cell culture adaptation through increased virus release on RABV virulence can be investigated. With successful expression of functional miR-shRNAs from RABV vectors, this work also provides a tool for RABV gene targeting in infected cell lines and thus may contribute to the further investigation of RABV-host-cell-interactions.
Streptococcus pneumoniae is a commensal of the human upper respiratory tract and
the etiological agent of several life-threatening diseases. This pathogen is the model bacterium
for natural competence. Furthermore, the pneumococci played an important role in the
identification of DNA as the main molecule involved in bacterial transformation. As a result,
studies on the pneumococcal genome provided an initial overview of the genetic potential of
this pathogen. The pneumococcus is a highly versatile bacterium possessing a high rate of
uptake and recombination of exogenous DNA from neighboring bacteria. As such, a significant
diversity in the genome content among the different pneumococcal strains has been reported.
The capsular polysaccharide, an important pneumococcal virulence factor, is the best example
on the pneumococcal diversity. There are over 98 serotypes characterized to date presenting
differences in their capsule (cps) locus. Additional to the cps locus, the pneumococcus also
presents 13 genomic islets annotated as regions of diversity (RD) encoded in the auxiliary
genome. Remarkably, 8 of the pneumococcal RD studied so far have been associated with
virulence. Furthermore, the ongoing sequencing of over 4000 pneumococcal genomes have
shed light on the conservation level of well-known pneumococcal virulence factors.
Interestingly, important pneumococcal virulence determinants show variations in the gene and
protein sequence among the different strains. Prototypes are for example the pneumococcal
surface protein C (PspC) and pneumococcal adherence and virulence factor B (PavB).
Conversely, gene regulation in S. pneumoniae is carried out by highly conserved and genome-
wide distributed transcriptional factors. Overall, the pneumococci interplays with its
environment with 4 major regulatory systems: quorum sensing (QS), stand-alone
transcriptional regulators, small RNAs (sRNAs) and two-component regulatory systems (TCS).
Some of these systems are multifaceted and share more than one feature. Furthermore, there
is crosstalk among the different systems, requiring the activation of a signaling cascade to
function properly.
A comprehensive analysis of the distribution and conservation of pneumococcal
virulence factors and TCS was obtained in this study. The results are summarized as a
simplified variome in which 25 pneumococcal strains with a complete sequenced genome were
analyzed. Interestingly, the genes encoding the glycolytic protein enolase and the toxin
pneumolysin were the most conserved virulence determinants. Additionally, the high level of
conservation was confirmed for the pneumococcal TCS regulators, especially for WalKR,
CiaRH and TCS08.
The main focus of this study was on the regulatory functions of pneumococcal TCS.
With this in mind, an extensive and detailed systematic review of the 13 pneumococcal TCS
and its orphan RR was undertaken. For this purpose, every pneumococcal TCS was analyzed
for its reported functional and structural information along with its contribution to the main
pathophysiology of the pneumococci. In brief, S. pneumoniae can utilize its TCS for the
regulation of important cellular processes and the sensing of detectable signals in the
environment. Additionally, the role of TCS in pneumococcal processes and signal sensing can
be divided further. In the first place, pneumococcal TCS regulate competence and fratricide,
the production of bacteriocins and host-pathogen interaction processes, while the detectable
signals include cell-wall perturbations, environmental stress, and nutrients. As a conclusion
from this section, it is possible to analyze the pneumococcal TCS in a comprehensive manner.
There is a complex network among the different pneumococcal regulators and the TCS play
an important role. Moreover, these systems are highly conserved and essential for the proper
functioning of the pneumococcus as a pathogen.
Following up on pneumococcal TCS, this study focused especially on the TCS08.
Interestingly, the pneumococcal TCS08 has been previously associated with the regulation of the cellobiose metabolism. Furthermore, this system has also been reported to regulate the
expression of genes encoded in the RD4 (Pilus-1). Remarkably, the pneumococcal TCS08
was shown to be highly homologous to the SaeRS system of Staphylococcus aureus. Initially,
mutant strains lacking a single (Δrr08 or Δhk08) or both components (Δtcs08) of the TCS08
were generated in pneumococcal D39 and TIGR4 strains. Transcriptomics and functional
assays showed a downregulation of the PI-1 in the absence of the complete tcs08, while PavB
presented an upregulation in the Δhk08 knockout. Moreover, an important number of genes
coding for intermediary metabolism proteins were also found to be differentially expressed by
microarray analysis. As such, the TIGR4Δhk08 strain presented a downregulation for the
cellobiose operon (cel). In contrast, an upregulation was reported for the fatty acid biosynthesis
(fab) and arginine catabolism (arc) operons. Conversely, a decrease in gene expression was
seen in the TIGR4Δrr08 strain for the arc operon. Finally, in vivo murine pneumonia and sepsis
models highlighted an involvement of TCS08 in pneumococcal virulence. Remarkably, the
different TCS08 mutants presented a strain dependent effect on their virulence severity. The
TIGR4Δrr08, and all TCS08 mutants in D39 showed a decrease in virulence in the pneumonia
model, with no changes in sepsis. Conversely, the absence of HK08 in TIGR4 presented a
highly virulent phenotype in both pneumonia and sepsis models. To sum up, the pneumococcal
TCS08 influenced the expression of genes involved in fitness and colonization. Specifically,
those coding for the adhesins PavB and PI-1 and fitness proteins from the cel, arc and fab
operons. Remarkably, the highest changes in expression were observed in the strains lacking
the HK08. Additionally, TCS08 has a strain dependent impact on pneumococcal virulence as
showed by murine pneumonia and sepsis models when comparing the effects in D39 and
TIGR4.
Mast cells reside on and near the cerebral vasculature, the predominant site of pneumococcal entry into the central nervous system (CNS). Although mast cells have been reported to be crucial in protecting from systemic bacterial infections, their role in bacterial infections of the CNS remained elusive. Here, we assessed the role of mast cells in pneumococcal infection in vitro and in vivo. In introductory experiments using mouse bone marrow-derived mast cells (BMMC), we found that (i) BMMC degranulate and release selected cytokines upon exposure to Streptococcus pneumoniae, (ii) the response of BMMC varies between different pneumococcal serotypes and (iii) is dependent on pneumolysin. Intriguingly though, apart from a slight enhancement of cerebrospinal fluid (CSF) pleocytosis, neither two different mast cell-deficient Kit mutant mouse strains (WBB6F1-KitW/Wv and C57BL/6 KitW-sh/W-sh mice) nor pharmacologic mast cell stabilization with cromoglycate had any significant impact on the disease phenotype of experimental pneumococcal meningitis. The incomplete reversal of the enhanced CSF pleocytosis by local mast cell engraftment suggests that this phenomenon is caused by other c-Kit mutation-related mechanisms than mast cell deficiency. In conclusion, our study suggests that mast cells can be activated by S. pneumoniae in vitro. However, mast cells do not play a significant role as sentinels of pneumococcal CSF invasion and initiators of innate immunity in vivo.
Intranasal Vaccination With Lipoproteins Confers Protection Against Pneumococcal Colonisation
(2018)
Streptococcus pneumoniae is endowed with a variety of surface-exposed proteins representing putative vaccine candidates. Lipoproteins are covalently anchored to the cell membrane and highly conserved among pneumococcal serotypes. Here, we evaluated these lipoproteins for their immunogenicity and protective potential against pneumococcal colonisation. A multiplex-based immunoproteomics approach revealed the immunogenicity of selected lipoproteins. High antibody titres were measured in sera from mice immunised with the lipoproteins MetQ, PnrA, PsaA, and DacB. An analysis of convalescent patient sera confirmed the immunogenicity of these lipoproteins. Examining the surface localisation and accessibility of the lipoproteins using flow cytometry indicated that PnrA and DacB were highly abundant on the surface of the bacteria. Mice were immunised intranasally with PnrA, DacB, and MetQ using cholera toxin subunit B (CTB) as an adjuvant, followed by an intranasal challenge with S. pneumoniae D39. PnrA protected the mice from pneumococcal colonisation. For the immunisation with DacB and MetQ, a trend in reducing the bacterial load could be observed, although this effect was not statistically significant. The reduction in bacterial colonisation was correlated with the increased production of antigen-specific IL-17A in the nasal cavity. Immunisation induced high systemic IgG levels with a predominance for the IgG1 isotype, except for DacB, where IgG levels were substantially lower compared to MetQ and PnrA. Our results indicate that lipoproteins are interesting targets for future vaccine strategies as they are highly conserved, abundant, and immunogenic.