Brucellosis 2003 proceedings - PHIDIAS

Poster Session
adaptation to starvation. In pathogenic or symbiotic bacteria like Legionella and
Rhizobium, a single protein called Rsh is involved in the adaptation to the
intracellular environement.
In order to understand the role of rsh in the virulence of Brucella, we created a
strain of B. melitensis 16M deleted for rsh using a non polar cassette called aphA4. It
is expected that rsh deletion will have pleiotropic effects, as demonstrated in E. coli.
In agreement with this, we observed that the rsh mutant presents a particular
morphology: after growth in a rich medium, the rsh mutant is two to three-fold bigger
than the wild-type strain when observed with electron microscopy. This phenotype
was very similar to the one described for the double relA spoT mutant of E. coli. The
non polar rsh mutant fail to replicate in ovine macrophages and HeLa cells. The
virulence of the rsh mutant in a murine model is currently under investigation.
Western blot analysis revealed that some virB proteins are less abundant in the rsh
mutant compared to the wild-type strain when they are grown until mid-exponential
phase in rich medium. These data suggest that Rsh may indirectly regulate the
expression of the virB operon.
KOHLER, S., FOULONGNE, V., OUAHRANI-BETTACHE, S., BOURG, G., TEYSSIER, J.,
RAMUZ, M., AND LIAUTARD, J-P. (2002). Proc Natl Acad Sci USA 99(24):15711-6.
KIM, S., WATARAI, M., KONDO, Y., ERDENEBAATAR, J., MAKINO, S, AND SHIRAHATA, T.
(2003). Infect Immun 71(6): 3020-3027.
80- LIPOPOLYSACCHARIDE MODIFICATIONS IN Brucella abortus MUTATED IN
THE TWO-COMPONENT REGULATORY SYSTEM BvrR/BvrS.
L. Manterola 1 , I. Moriyón 1 , E. Moreno 2 , K. Brandenburg 3 , and I. López-Goñi 1 . (1) Departamento de
Microbiología, Universidad de Navarra, Pamplona, Spain. (2) PIET, Escuela de Medicina Veterinaria,
Universidad Nacional, Costa Rica. (3) Forschungszentrum Borstel, Borstel, Germany.
The BvrR/BvrS two-component sensory-regulatory system is essential for
penetration and intracellular survival of B. abortus. BvrR/BvrS mutants display
increased surface hydrophobicity, sensitivity to polycationic bactericidal peptides and
adherence to epithelial cells and macrophages. This set of properties is suggestive of
envelope changes and, in fact, we have shown that these mutants do not express
Omp3a (Omp25) and Omp3b (Omp22) (Guzmán-Verri et al. Proc. Natl. Acad. Sci. U.
S. A. 99:12375-12380). To test whether other envelope molecules are changed in
BvrR/BvrS mutants, several envelope fractions were analyzed. No quantitative
differences were found in periplasmic cyclic 1,2-β-glucans, lipopolysaccharide (LPS)
or native hapten polysaccharide content, or in free lipid profiles. However, chimeric
bvrS mutant cells bearing the parental LPS showed increased polymyxin resistance
and, conversely, chimeric parental cells bearing the LPS of the bvrS mutant
displayed increased sensitivity and, by electron microscopy, the envelope
morphological alterations associated with the peptide action. Increased binding by
the LPS of bvrR and bvrS mutants was observed by fluorimetry with dansylated
polymyxin, and polymyxin had a stronger acyl-chain fluidifying effect on the LPS of
the mutants. No differences in the degree polymerization or 13 C-NMR spectra of the
O-polysaccharides were observed, an increase of underacylated forms in the lipid A
of the bvrR and bvrS mutants was observed by high-performance thin layer
chromatography. Although further research is necessary to ascertain whether this
lipid A change represents a true regulation by BvrR/BvrS or simply results from a
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Brucellosis 2003 International Research Conference