Characterizing Uncultured Bacteria Michael Wagner Division of Microbial Ecology 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 bacteria and 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)?

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 Science 304: 728

Environmental Chlamydia are ATP, NTP and NAD + parasites – a unique adaptation to intracellular life Haferkamp et al Nature 432: 622

ERA-NET Partner Austria. Key topic II: Regulatory Networks of Microbial Virulence Bläsi, Charpentier, Kuchler, Rosengarten

Virulence Genome of Candida glabrata  C.g. 2nd most frequent human fungal pathogen - haploid  Genome sequenced - closest in evolution to S. cerevisiae  Virulence of C. glabrata - KNOCK-OUT ~ 6500 Genes  Bar-Codes (Up-Down) of each C.g. knock-out as in S.c.  Test & correlate pathogenicity of all knockouts in vivo & in vitro  Pathogenicity genom/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 postgenomics of 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 shotgun library

Key topic III: Metagenomics and postgenomics of bacterial communities colonizing humans Key topic II: Regulatory Networks of Microbial 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 trachomatis Chlamydia muridarum Chlamydophila pneumoniae Chlamydophila abortus Chlamydophila psittaci Chlamydophila caviae Chlamydophila felis Chlamydophila pecorum Chlamydia suis Chlamydia trachomatis Chlamydia muridarum Chlamydophila pneumoniae Chlamydophila abortus Chlamydophila psittaci Chlamydophila caviae Chlamydophila felis Chlamydophila pecorum … a phylogenetically well separated group of closely related bacteria … intracellular bacteria with a unique biphasic developmental cycle M.E. Ward (