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)