Characterizing Uncultured Bacteria Michael Wagner Division of Microbial Ecology University of...
-
Upload
timothy-crawford -
Category
Documents
-
view
223 -
download
2
Transcript of Characterizing Uncultured Bacteria Michael Wagner Division of Microbial Ecology University of...
Characterizing Uncultured Bacteria
Michael WagnerDivision of Microbial Ecology
www.microbial-ecology.net
University of Vienna
Vienna Roundtable Pathogenomics
• April 4
• 16 Scientists, 4 members of the bmbwk, 1 member of the FWF
• Introduction: ERA-NET
• Overview presentation by each scientist
• Identification of three major topics which are suggested for the call
ERA-NET Partner Austria:Key topic I: Host-Pathogen Interaction
Focus on obligate and facultative intracellular bacteriaand on pathogenic fungi
Decker, Horn, Müller, Kuchler
Toll-like receptors (TLR) and Interferons (IFN) in Host-Pathogen
Interaction
• TLR recognize bacteria and trigger an antibacterial and inflammatory response.
• Stimulation of several TLR causes the synthesis of type I IFN in infected cells.
• Antimicrobial gene expression results from genes directly stimulated by TLR-derived signals, by IFN signaling, or both.
Interferons and TLR together determine the gene expression signature of pathogen-infected cells.
Open Questions
• What are the signaling molecules (signalosomes) linking the interferon genes to extra- or intracellular nonviral pathogens (similarities and differences between TLR-mediated and intracellular recognition)?
• Is there a causal relationship between the degree, timing, intensity of an IFN response and the effect of IFN immune responses to nonviral pathogenes?
• Can pathogen virulence be linked to IFN synthesis (positively or negatively)?
Parachlamydia acanthamoebae
outgroup
CandidatusRhabdochlamydia porcellionis
Fritschea eriococciWaddlia chondrophila 2032
Waddlia chondrophila WSU851044
Waddlia sp. RLUH1
Chlamydia suis
Chlamydia trachomatisChlamydia muridarum
Chlamydophila pneumoniae
Chlamydophila abortusChlamydophila psittaci
Chlamydophila caviaeChlamydophila felisChlamydophila pecorum
Endosymbiont of Acanthamoeba sp. Berg17Parachlamydia sp. Hall's coccus
sp. PL9
Endosymbiont von Acanthamoeba sp. UWC22Endosymbiont von Acanthamoeba sp. TUME1
Neochlamydia hartmannellae
Endosymbiont von Acanthamoeba sp. UWE1
Endosymbiont KcontProtochlamydia amoebophila UWE25
Endosymbiont von Acanthamoeba sp. SS1Endosymbiont von Acanthamoeba sp. TUMSJ61
0.10outgroup
Simkania negevensis Z
Fritschea eriococciFritschea bemisiae Waddlia chondrophila 2032
Waddlia sp. RLUH1
Chlamydia suis
Chlamydia trachomatisChlamydia muridarum
Chlamydophila pneumoniae
Chlamydophila abortusChlamydophila psittaci
Chlamydophila caviaeChlamydophila felisChlamydophila pecorum
Bn9
Parachlamydia
Endosymbiont von Acanthamoeba sp. UWC22Endosymbiont von Acanthamoeba sp. TUME1
Neochlamydia hartmannellae
Endosymbiont von Acanthamoeba sp. UWE1
Endosymbiont KcontProtochlamydia amoebophila UWE25
Endosymbiont von Acanthamoeba sp. SS1Endosymbiont von Acanthamoeba sp. TUMSJ61
0.10
Model System: Environmental chlamydiae which thrive in
protozoa, salmon, fruit bats etc.
Protochlamydia amoebophila UWE25
Comparative genome analysis of Protochlamydia amoebophila UWE25 –
Inferring the evolutionary history of chlamydiae
Horn et al. 2004. Science 304: 728
Environmental Chlamydia are ATP, NTP and NAD+ parasites – a unique adaptation
to intracellular life
ADP
ADP
NAD+
NADP+
GTP
UTP
ATP
H+
ATP
ADP
NTT2
NT
T1 N
TT4
ADP
ADP
CTP
H+
ATP
ATP
NAD+
GTP
UTPATP
Energie Redox-CoenzymeDNA/RNA Synthese
Wirtszelle
Inklusion
Symbiont
ADP
ADP
NAD+
NADP+
GTP
UTP
ATP
H+
ATP
ADP
NTT2
NT
T1 N
TT4
ADP
ADP
CTP
H+
ATP
ATP
NAD+
GTP
UTPATP
energy electron carrierDNA/RNA synthesis
host
inclusion
symbiont
ADP
ADP
NAD+
NADP+
GTP
UTP
ATP
H+
ATP
ADP
NTT2
NT
T1 N
TT4
ADP
ADP
CTP
H+
ATP
ATP
NAD+
GTP
UTPATP
Energie Redox-CoenzymeDNA/RNA Synthese
Wirtszelle
Inklusion
Symbiont
ADP
ADP
NAD+
NADP+
GTP
UTP
ATP
H+
ATP
ADP
NTT2
NT
T1 N
TT4
ADP
ADP
CTP
H+
ATP
ATP
NAD+
GTP
UTPATP
energy electron carrierDNA/RNA synthesis
host
inclusion
symbiont
Haferkamp et al. 2004. Nature 432: 622
ERA-NET Partner Austria. Key topic II:Regulatory Networks of Microbial Virulence
Bläsi, Charpentier, Kuchler,Rosengarten
Virulence Genome of Candida glabrataVirulence Genome of Candida glabrata
C.g. 2nd most frequent C.g. 2nd most frequent human fungal pathogen - haploidhuman fungal pathogen - haploid
Genome sequenced - Genome sequenced - closest in evolution to closest in evolution to S. cerevisiaeS. cerevisiae
Virulence of Virulence of C. glabrata - C. glabrata - KNOCK-OUT KNOCK-OUT ~ 6500 Genes~ 6500 Genes
Bar-Codes (Up-Down) Bar-Codes (Up-Down) of each of each C.g.C.g. knock-out as in knock-out as in S.c.S.c.
Test & correlate Test & correlate pathogenicity of all knockouts in vivo & in vitropathogenicity of all knockouts in vivo & in vitro
Pathogenicity Pathogenicity genom/proteom of a fungal pathogengenom/proteom of a fungal pathogen
• Cell-density dependent regulatory systems (quorum sensing/auto-inducing molecules) controlling virulence in Salmonella typhimurium, Pseudomonas aeruginosa and Streptococcus pyogenes
ERA-NET Partner Austria. Key topic III:Metagenomics and postgenomicsof bacterial communities colonizing humans
Focus on oral cavity, skin, and gut system
Daims, Superti-Furga, Wagner
BAC ends linked to ANAMMOX rRNA
three different BAC libraries
• 200,000 shot gun reads• 12,000 BAC end sequences
• 32 BAC full sequences• 4.3 Mb in 4 contigs
Metagenomics –Reconstructing the ANAMMOX genome
substratum
surface
NH4+
NO2-
NO2- NH4
+
N2
substratum
surface
NH4+
NO2-
NO2- NH4
+
N2
shotgun library
Key topic III:Metagenomics and postgenomicsof bacterial communities colonizing humans
Key topic II: Regulatory Networks ofMicrobial Virulence
ERA-NET Partner Austria:
Key topic I: Host-Pathogen Interaction
Antimicrobial Action of Interferons
• Type II IFN (IFN-) activates macrophages and enhances immunity to predominantly nonviral pathogens, particularly when intracellular.
• Type I IFN (>10 genes) mediate antiviral innate immunity. It is unclear why their synthesis is an obligatory response to many or even most nonviral pathogens.
• The immunological effect of type I IFN can be benefitial or detrimental for the host, depending on the pathogen and parameters of infection. The underlying causes are not known.
Approach• Proteomics to identify the signaling molecules
(signalosomes) linking the interferon genes and inflammatory genes to extra- or intracellular nonviral pathogens.
• Gene expression patterns in mice with defined mutations in IFN synthesis or response after infection infection with pathogens stimulating different types of immune responses. Link between gene expression and protective or deterimental response.
• Infection of wildtype and mutant mice with microbial mutants representing different degrees of virulence. Virulence enhanced or reduced by defects in IFN system?
Chlamydiae are …
… among the most important bacterial pathogens of humans
Chlamydia suis
Chlamydia trachomatisChlamydia muridarum
Chlamydophila pneumoniae
Chlamydophila abortusChlamydophila psittaci
Chlamydophila caviaeChlamydophila felisChlamydophila pecorum
Chlamydia suis
Chlamydia trachomatisChlamydia muridarum
Chlamydophila pneumoniae
Chlamydophila abortusChlamydophila psittaci
Chlamydophila caviaeChlamydophila felisChlamydophila pecorum
… a phylogenetically well separated group of closely related bacteria
… intracellular bacteria with a unique biphasic developmental cycle
M.E. Ward (www.chlamydiae.com)