Bariloche protein symposium argentine society for biochemistry and molecular biology

67
BIOCELL, 27 (Suppl. I), 2003
MI-P30.
ROLE OF QUORUM SENSING MECHANISM ON THE
RESPONSE OF Pseudomonas aeruginosa TO UVA
Fernández Rubén O.
1
; Poncelas Silvina
2
; Nikel Pablo
2
 and Pizarro
Ramón A.
1
1
CNEA; 
2
UNSAM, San Martín, Bs. As., Argentina. E-mail:
[e-mail]
Our previous results demonstrated a high UVA sensitivity of P.
aeruginosa. Together with reproductive cell death, alterations of
several membrane associated functions were informed. In the
present work the participation of Quorum Sensing (QS)  in  P.
aeruginosa UVA response is shown. This cell-cell communication
mechanism is involved in the regulation of transcription of around
600 genes including catalase and superoxide dismutase (SOD).
When  P. aeruginosa cells were harvested at exponential phase
strains carrying rhlI mutation were more sensitive than the wild
type strain or lasI mutant, indicating a specific involvement of
QS system II. A linear correlation has been found between survival
fraction and catalase residual specific activity, irrespective from
which strain was analysed. This suggests a possible participation
of this enzyme in the resistance to UVA. SOD activity was not
modified by UVA treatment and the levels of SOD were similar
for wild type and QS mutants. Our previous reports also informed
that a short nutritional stress before irradiation protects P.
aeruginosa from the UVA lethal effect. This protection is dependent
on protein synthesis during starvation. In the present study it has
been found that the mentioned protection was independent on QS
system. In conclusion this study shows the participation of QS II
and possibly catalase activity in the modification of the UVA lethal
effect, and the independence of the radioprotection induced by
nutritional stress from QS.
MI-P31.
A 20.9 kDa NOVEL PUTATIVE LIPOPROTEIN FROM
Mycobacterium paratuberculosis IS STRONGLY ANTIGENIC
Gioffré A, Bigi F, Alito A, Caimi K, Klepp L, Santángelo MP,
Zumárraga M, Moretta R, Meikle V, Paolicchi F, Cataldi A,
Romano MI.
E-mail: [e-mail]
Mycobacterium avium subsp. paratuberculosis (MPTB) is an
important cause of ruminant animal disease such as
paratuberculosis.  The sequence of MPTB genome has recently
been finished however a large proportion of the putative genes
still have no identified functions. To identify a novel potential
diagnostic antigen molecule we analyzed an expression library
with serum of animals naturally infected with the bacteria or
immunized mice. We identified an ORF of 611 pb that codes a
hypothetical protein of 20872 MW. The sequence has 99% of
identity with the corresponding ORF from de genome of M.avium
subsp.  avium (BLAST search). The presence of the gene was
evaluated by Southern Blot and PCR in other species of
mycobacterium genera. The results demonstrate that this sequence
was only presents in these species and as well in M. plhei. Database
of protein families and domains searching (PROSITE) revealed
that the protein could have prokaryotic membrane lipoprotein lipid
attachment site. The localisation to the bacterial membrane is
hypothesized. The ORF was cloned an expressed in pRSET vector,
and a recombinant peptide of 32.7kDa was obtained and purified.
Mice were immunized to obtain policlonal sera to perform cellular
localization. Humoral response was evaluated in sera and milk
serum samples from different animals (bovine, ovine) by
immunoblot, demonstrating that this protein is strongly recognized.
MI-P32.
FERREDOXIN FROM Trypanosoma cruzi AND  ITS
POTENTIAL ROLE IN BIOREDUCTION OF
ANTIPROTOZOAL COMPOUNDS
Girardini, J.E.; Manarin, R.; Serra E.C.
Instituto de Biología Molecular y Celular de Rosario. IBR.
CONICET. E-mail: [e-mail]
Ferredoxins (Frd) act as low potential electron carriers. This
proteins can be classified according to their structure and the type
and number of Fe-S clusters that contain. Vertebrate type
ferredoxins contain one [2Fe2S] cluster and they are present in a
wide variety of organisms, including prokariotes. Its biological
function is poorly understood. In mammals, they are involved in
the biosynthesis of steroids in mitochondria. However, the presence
of this protein, and its reducing partner ferredoxin-NADP(H)
oxidoreductase (FNR), in organisms incapable of sinthetizing
steroids suggests that they may perform another function. They
were implicated in and Fe-S cluster assembly in yeasts, in the
metabolization of xenobiotics in prokaryotes and in induction of
apoptosis in human cell lines.  The observation that mammalian
Frd and FNR can reduce in vitro compounds with antiprotozoal
activity prompted us to search for this kind of enzymes in T. cruzi.
We have previously identified sequences that code for FNR
(TcFNR) and Frd (TcFrd). In the present work recombinant TcFrd
was expressed fused to thioredoxin from Escherichia coli. The
[2fe-2S] cluster was characterized by UV-visible spectroscopy and
circular dichroism. The expression of the enzyme in T cruzi was
analyzed by northern blot and western blot. We were able to
confirm the ability of the recombinant enzyme to act as electron
carrier. This results make TcFrd an interest candidate for the
bioactivation of trypanocidal compounds.
MI-P33.
ANTI-Trypanosoma cruzi ACTIVITY OF GREEN TEA
(Camellia sinensis)  CATECHINS
María C. Güida, Mónica I. Esteva, Mirtha M. Flawiá, Héctor N.
Torres, Cristina Paveto.
Instituto de Investigaciones en Ingeniería Genética y Biología
Molecular y Facultad de Ciencias Exactas y Naturales (INGEBI-
CONICET-UBA), Buenos Aires, Argentina. E-mail:
[e-mail]
Flavan-3-ols compounds known as catechins posses a strong
trypanocidal activity being the most active gallocatechin gallate
(GCg) and epigallocatechin gallate (EGCg). Both compounds were
assayed against three different Trypanosoma cruzi stages: IC
50
values lower than 1 pM were obtained against blood-stream
trypomastigote form; Vero cell culture infection by amastigote was
50% inhibited at  approximately 100 nM without visible host cell
damage; and growth of epimastigote in liquid cultures was 50%
at 150 µM. Trypomastigotes morphological alteration and normal
erythrocytes stages from 1 nM EGCg treated Balb/c mice blood at
15 and 60 min were confirmed by electronic microscopy. In
toxicological assays no hepatic damage was observed under
histological techniques in mice treated with 100 µM EGCg but
reversible alteration with 400 µM EGCg were detected. Catechins
mode of action in the parasite cell are under studies. This results
allows us to suggest that these compounds could be used to sterilize
blood and continuous working on the possibility to use they on the
Chagas’ disease chemotherapy.

---