Research Report 2010 2011 - Helmholtz-Zentrum für ...

124 SCIENTIFIC REPORTS | PoFII Independent Research
03 Structural Analysis of the Innate Immune System
PROJECT LEADER | Prof. Dr. Wolf-Dieter Schubert | Former Research Group Molecular Host-Pathogen
Interactions | Department of Biotechnology, University of the Western Cape, Cape Town, South Africa |
wschubert@uwc.ac.za
PROJECT MEMBERS | Nils Kuklik | Clive Mketsu | Mujaahida Mohammed | Edukondalu Mullapudi | Lilia Polle |
Donné Simpson | Jason Stark
In the Research Group “Molecular Host-Pathogen Interactions”
(MHPI) we previously focussed on the molecular
details of human defence mechanisms against invading
pathogens (innate immunity) as well as the corresponding
molecular strategies of pathogens in infecting humans.
Novel Listerial Virulence Factors InlJ and Auto The bacterium
Listeria monocytogenes infects humans following the
consumption of contaminated food. To breach the intestinal
barrier and for all steps of the infection process Listeria has
evolved a dedicated set of highly specific protein factors that
it produces and secretes in a highly coordinated manner. To
understand how their physical structure relates to their physiological
function, we have undertaken to investigate their
three-dimensional structure at the atomic level of resolution.
“Auto” The cell wall of Gram-positive bacteria is a complex
envelope that helps cells to maintain their shape and to
counteract the turgor pressure of their cytosol. It consists of
long, linear chains of alternating sugar residues (N-acetylglucosamine
and N-acetylmuramic acid) crosslinked by short
peptides. Cell growth, cell division and many other processes
crucially depend on the constant remodelling of the cell wall.
Enzymes involved in this process are generally referred to as
autolysins.
Studies revealed that only one of the autolysins is involved
in the pathogenesis of Listeria monocytogenes. This autolysin
named “Auto” is essential for listerial entry into numerous
eukaryotic cell lines.
Left: Crystal structure of autolysin Auto of L. monocytogenes
is depicted in cartoon style and with transparent surface.
The active site of the pro-enzyme is blocked by an N-terminal
α-helix (red). Right: Crystal structure of InlJ (right) with enlarged
view of the cysteine ladder (left): reduced cysteines align
in the hydrophobic core (Sulphur - yellow). Graphics: HZI
Auto is an N-acetylglucosaminidase, meaning that it cleaves
the sugar backbone of the cell wall exposing the reducing
end of N-acetylglucosamine. Solving the crystal structure
of Auto we observed that it is structurally related to lytic
transglycosylases and some lysozymes despite catalysing an
unrelated chemical reaction. By mutating residues potentially
involved in catalysis, we identified the glutamic acid residues
Glu122 and Glu156 as being essential for the enzymatic
reaction. Fascinatingly, we found that Auto, as originally produced,
is autoinhibited by an N-terminal α-helix. Its specific
N-terminal cleavage by an as yet unidentified protease following
an extracellular signal may, therefore, allow its rapid
and coordinated activation. In addition, Auto has a highly
acidic pH optimum making it essentially inactive at a pH of 7.
Overall we interpret our data to indicate that Auto participates
in liberating the pathogen from the partially acidified
phagolysosome. By increasing the porosity of the cell wall it
would help to coordinate the release of other virulence factors
such as listeriolysin. Their activity in turn would result
in the breaching of the phagolysosomal membrane, raising
the pH to physiological levels and de-activating Auto.
InlJ The internalin family of proteins constitutes a group
of virulence factors of Listeria monocytogenes. The founding
member, Internalin or InlA, is central to the forced uptake
of the bacterium into epithelial cells of the human small intestine.
Only recently this family was found to possess an additional,
previously unrecognized member, InlJ. This protein
has been shown to be crucial to bacterial adhesion to host
cells. InlJ is distinct from other family members in that its 16
LRR units mostly comprise 21 rather than the standard 22
residues and by one LRR-defining hydrophobic residue being
replaced with a conserved cysteine. Solving the crystal structure
of this protein we could show that the cysteines line up
to form a unique cysteine ladder in consecutive repeats. InlJ
thus presents a very rare case of an extracellular protein
(oxidizing environment) bearing a large number of reduced
cysteine residues. This implies that the cysteines must serve
to significantly stabilize the structure, offsetting the danger
of its unfolding followed by cysteine oxidation.
Bröcker, M.J., Schomburg, S., Heinz, D.W., Jahn, D., Schubert, W.-D., & Moser, J (2010)
Crystal structure of the nitrogenase-like dark operative protochlorophyllide oxidoreductase
catalytic complex (ChlN/ChlB)2. Journal of Biological Chemistry 285, 27336-27345.
Heinemann, I.U., Schulz, C., Schubert, W.-D., Heinz, D.W., Wang, Y.G., Kobayashi, Y.,
Awa, Y., Wachi, M., Jahn, D., & Jahn, M. (2010) Structure of the heme biosynthetic
Pseudomonas aeruginosa porphobilinogen synthase in complex with the antibiotic
alaremycin. Antimicrobial Agents and Chemotherapy 54, 267-272.
Bublitz, M., Polle, L., Holland, C., Heinz, D.W., Nimtz, M., & Schubert, W.-D. (2009)
Structural basis for autoinhibition and activation of Auto, a virulence-associated peptidoglycan
hydrolase of L. monocytogenes. Molecular Microbiology 71, 1509–1522.