Bariloche protein symposium argentine society for biochemistry and molecular biology

76
BIOCELL, 27 (Suppl. I), 2003
MI-P66.
COMPETITIVE REVERSE TRANSCRIPTION-
POLYMERASE CHAIN REACTION ASSAYS FOR
QUANTIFICATION OF HEPATITIS G VIRUS (HGV) RNA
STRANDS
Ruiz, Vanesa; Mathet, Verónica L. and Oubiña, José R.
Depto. Microbiología Facultad de Medicina. UBA, Argentina.
E-mail: [e-mail]
Objective: To design and develop quantitative PCR-based assays
for detection of either positive (genomic) or negative (replicating)
HGV RNA strands. Material and methods: A 329 bp c-DNA
fragment from the 5' UTR of HGV genome was cloned in a vector
where a 67 bp deletion was introduced. Clones harboring both
insert orientations -corresponding to [+] and [-] strands- were
selected by sequencing. Linearized plasmids served as templates
for T7 RNA polymerase activity. Fully purified competitive RNA
copies were quantified by UV absorbance. HGV [+] RNA strand
was quantified in serum samples (n= 5) by competitive RT-PCR
(cRT-PCR). Each RNA sample was mixed with different amounts
of competitor RNA and RT-PCR amplified with the highly specific
Tth enzyme. PCR products were separated by  gel electrophoresis.
Digital images were analyzed by Scion Image (NIH) software.
Results: The sensitivity of RT-PCR was 10
3 
and 10
4
 molecules for
[-] and [+] competitor RNAs, respectively. This method has  a
discrimination of at least 5 log
10
 between both RNA strands. Titers
obtained by cRT-PCR correlated exactly with those obtained by a
limiting dilution method. Negative HGV strand proved
undetectable in serum samples. Conclusion: The availability of
an in-house made HGV [+] and [-] strand quantification method
will shed some light on future studies concerning its poorly
understood pathogenesis.
MI-P67.
CELL WALL MODIFICATIONS BY OSMOTIC STRESS IN
Lb. casei ATCC 393
Mariana Piuri, Carmen Sanchez-Rivas and Sandra M. Ruzal.
Depto. Química Biológica, FCEN-UBA. E-mail:
[e-mail]
Lb. casei was more sensitive to mutanolysine (MtLz) and
antibiotics with target in the cell wall when grown in high salt
medium (MRS+1M NaCl, condition N). EM showed that in N
cell were 60% bigger than those grown in the control condition
(MRS, C) and cell wall was detached from the cytoplasmic
membrane. Purified cell wall of both conditions obtained by SDS-
PronaseE treatment also showed the differential sensitivity to
MtLz. Therefore differences observed would be related to structural
modifications. In particular a decreased in peptidoglycan (PG)
cross-linking was found by FACE analysis and DNF-derivatization.
Treatment with 10% TCA or 0.1N HCl that enabled extraction of
wall associated polymers such as Teichoic acid (WTA) showed
that C condition produce 8 fold greater WTA than N. Alterations
in PG could be attributed to the biosynthetic process dependant of
the Penicillin-binding proteins (PBP). PBP are associated to the
membrane with is also altered by osmotic stress. Membranes
isolated from both conditions were developed with Biocillin that
specifically binds PBP. Saturation assays with increasing Pen G
concentrations showed a modified pattern between both conditions.
Three of the 9 PBP here first described, were fully saturated in N
at lower Pen G concentrations, and related to a different
functionality in vivo. Taken all together these results showed that
growth in high salt modified the cell wall (PG and WTA) and its
related functions (PBP).
MI-P68.
USE OF Brucella abortus AS A  VECTOR  FOR THE
EXPRESSION OF HETEROLOGOUS ANTIGENS
Sabio y García, Julia; Carrica, Mariela; Farber, Marisa; Bigi,
Fabiana; Campos, Eleonora; Cravero, Silvio and Rossetti,
Osvaldo.
Inst. de Biotecnología. CICVYA-INTA, Buenos Aires, Argentina.
E-mail: [e-mail]
Brucella abortus S19 and RB51 are attenuated strains used as
live vaccines to control bovine brucellosis. Due to the strong
cellular and humoral immune response that they elicit, they  are
particular attractive vectors for the delivery of heterologous
antigens. The objective of the present study is to express antigens
of veterinary pathogens that require for their control the same type
of  immune response elicited by Brucella. We have cloned esat-6
and  cfp-10  of  Mycobacteium bovis,  rap-1  of  Bavesia bovis and
msp-1a  of Anaplasma marginale into the replicative plasmid
pBBR1MCS and evaluated their expression in Brucella abortus
S19 and RB51. We have also cloned esat-6,  cfp-10 and msp-1a
under the control of bp26 promoter and in frame with its signal
peptide in order to express the heterologous protein in the
periplasmic space. Immune response against the heterologous
antigens and against Brucella is at present being evaluated. We
expect to obtain valuable information about the use of Brucella
abortus as a vector for the expression of heterologous antigens.
MI-P69.
SIMULTANEOUS EXPRESSION OF VAG AND VRG GENES
DOES NOT ALTER Bordetella bronchiseptica –HOST
INTERACTION
Fernández J
1,2
, Sisti F
1
, Fingermann M
1
, Rodriguez ME
2
, and
Hozbor D.
1,2
1
Instituto de Bioquímica y Biología Molecular. 
2
Centro de
Investigación y Desarrollo en Fermentaciones Industriales. Fac.
Cs. Exactas. UNLP. 47 y 115. La Plata.
In  Bordetella the expression of the main virulence factors is
regulated by bvgAS locus, a two-component transduction system.
This locus enables Bordetella to alternate between two distinct
phenotypic states. The virulent state (Bvg
+
) arise when BvgAS
induces the expression of genes that encode for most of the
virulence factors (vag genes), while at the same time represses
other genes called vir repressed genes (vrg genes).  By contrast,
the avirulent state (Bvg
-
) is characterized by the expression of vrg
genes and occurs when BvgAS activity is suppressed. Although
the role of vag genes in the infection process is undisputed, the
involvement, if any, of vrg genes in virulence as well as the
molecular mechanism of their repression remain to be determined.
Only in B. pertussis the involvement of a repressor gene called
bvgR was recently reported. Therefore, we investigated whether
bvgR has a role in B. bronchiseptica. Using PCR, sequencing
methods, and Southern blot we could determine the presence of
bvgR in B. bronchiseptica. On this basis we constructed a defective
mutant by site-specific insertional mutagenesis. This mutant
(Bb:bvgR) expresses at the same time vag and vrg genes when
BvgAS system is activated. We could observe in vitro that both
adhesion and intracellular survival in epithelial human cells were
not affected. In vivo assays using intranasal Balb/C mice infection,
showed colonization kinetics of Bb:bvgR comparable to the
parental strain. Our results showed that the vrg gene products
together with the already known vag gene products do not interfere
in the infectious cycle.

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