download report - Istituto Pasteur

P a r t i c i p a n t s :
Alessandro Giuffrè, CNR researcher; Elena Forte, researcher;
Daniela Mastronicola, Francesca Maria Scandurra, postdoc
fellows; Fabrizio Testa, PhD student.
C o l l a b o r a t i o n s :
Instituto de Tecnologia Química e Biológica, Universidade Nova de
Lisboa, Portugal (Prof. Miguel Teixeira); Dipartimento di Scienze
Biomediche, Università di Sassari (Prof. Pier Luigi Fiori); Istituto
Nazionale per le Malattie Infettive “L. Spallanzani”, Roma (Dr.
Leopoldo Paolo Pucillo); Dipartimento di Scienze Biochimiche,
Sapienza-Università di Roma (Prof. Maurizio Brunori).
Report of Activity
During infection, pathogenic microorganisms are
exposed to the toxic effects exerted by NO, O 2 and
their reactive species, a condition currently referred
to as “oxidative/nitrosative stress”. Reactive oxygen
and nitrogen species play a key role in the
human immune response against microbial infection;
hence, it is not surprising that pathogens are
able to cope with these harmful species. A number
of wide spread severe human diseases, such as giardiasis,
amoebiasis and trichomoniasis, are caused by
anaerobic protozoa, whose infection peculiarly often
evolves into chronic inflammatory states. These
protists are somehow able to resist to the host
NO/O 2 -mediated immune response, but the molecular
mechanisms at the basis of their resistance are
largely unknown yet. Notably, genes coding for
flavodiiron proteins (FDP), have been recently identified
in the genome of several human pathogenic
anaerobic protozoa, including Giardia intestinalis,
Trichomonas vaginalis and Entamoeba histolytica.
FDPs are typical prokaryotic enzymes that are
endowed with O 2 - and/or NO-reductase activity,
and thus believed to be implicated in the defense of
anaerobes against oxidative and nitrosative stress.
The current project aims at exploiting the role of
Pathogenetic mechanisms of microbially associated diseases - AREA 2
Nitric oxide detoxification in pathogenic protozoa: role of
flavodiiron proteins
Principal investigator: Paolo Sarti
Professor of Chemistry and Biochemistry
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel: (+39) 06 49910944; Fax: (+39) 06 4440062
Paolo.sarti@uniroma1.it
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these enzymes in pathogenic protozoa, particularly
in G. intestinalis.
The flavodiiron protein from the human
parasite Giardia intestinalis
The FDP from G. intestinalis has been expressed in E.
coli, purified and characterized from a structural and
functional standpoint. This is the first eukaryotic
member of the flavodiiron proteins family to have
been investigated. The crystallographic structure of
the enzyme, solved at 1.9 Å resolution, shows that
the FDP is a dimer of homodimers, where each
monomer encompasses a flavodoxin-like domain, that
binds a FMN moiety, and a β-lactamase-like domain
with a non-heme diiron site. The enzyme maintains a
tetrameric structure also in solution. Monomers are
arranged in a head-to-tail configuration, so that the
redox cofactors bound to opposing monomers are at
a close distance. Compared to bacterial FDPs, the
enzyme from Giardia shows remarkable overall similarities
and a highly conserved structure at the level
of the redox cofactors.
The functional properties of the G. intestinalis FDP
have been investigated both by time-resolved spectrophotometry
and by amperometry using Clarktype
electrodes selective for O2 and NO.
Experiments carried out with a stopped-flow instrument
show that the enzyme in the fully reduced state
reacts with O2 rapidly (ms) and with high affinity,
whereas the reaction with NO is much slower. In
agreement with these results, NO- and O2-ampero metric measurements led us to conclude that the G.
intestinalis FDP is an efficient O2-scavenging enzyme
forming H2O as the reaction product, whereas it is a
poor NO-reductase. O2 is scavenged at high rate
(> 40 s-1 , at room temperature) and with high affinity
(KM ≤ 2 µM). Remarkably, these results may be
relevant in terms of microbial physiology because,
although Giardia intestinalis is a highly O2-suscepti ble anaerobic parasite, it preferentially colonizes a
fairly aerobic tract of the human intestine, i.e., the