Brucellosis 2003 proceedings - PHIDIAS

Poster Session
pleiotropic effect caused by the absence of Omp3a, and Omp3b, lipid A alterations
could contribute to explain the lack of virulence of the BvrS/BvrR mutants.
81- EXPRESSION OF Brucella abortus omp3a AND omp3b UNDER DIFFERENT
GROWTH CONDITIONS AND EFFECT OF omp3a OR omp3b DELETION ON
OUTER MEMBRANE PROPERTIES AND VIRULENCE.
L. Manterola 1 , C. Guzmán-Verri 2 , M. J. Grilló 3 , M. J. de Miguel 3 , I. Moriyón 1 , E. Chaves-Olarte 2 , E.
Moreno 2 , 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) Unidad de
Sanidad Animal, Servicio de Investigación Agroalimentaria, Gobierno de Aragón, Zaragoza, Spain.
The two-component regulatory system BvrR/BvrS of Brucella abortus is
essential for intracellular penetration and survival within mammalian cells. We have
previously shown that this system controls the expression of B. abortus Omp3a
(Omp25) and Omp3b (Omp22) (Guzmán-Verri et al. Proc. Natl. Acad. Sci. U. S. A.
99:12375-12380). Thus, we explored the kinetics of Omp3a and Omp3b expression
and their role in B. abortus virulence. The level of expression of omp3a and omp3b
promoters in response to different growth conditions was measured by the β-
galactosidase assay in chromosomal omp3a::lacZ and omp3b::lacZ transcriptional
fusions. The level of omp3a transcription increased when cells growth in rich medium
(TSB or BHI) were incubated for 6 h. in minimum medium. On the contrary, the level
of omp3b transcription increased when cells growth in minimum medium were
incubated in rich medium.
In contrast to BvrS/BvrR mutants, the sensitivity of these Omps mutants to
sodium dodecyl sulphate, Sarkosyl and polymyxin B were not significantly different
from that exhibit by the virulent strain. Similarly, the adherence, internalization and
replication of both Omp mutants in non-professional phagocytes HeLa cells and
RAW 264.7 macrophages, assayed by double immunofluorescence and direct
bacterial counts, were not significantly different from that of the virulent parental
strain. Whereas a large number of bvrS mutant bacteria were found extracellularly in
HeLa cell cultures, omp3a and omp3b mutants were seldom found extracellularly.
Finally, the survival of both Omp mutants in BALB/c mice was similar to that of the
wild type strain. These results demonstrate that the previously described attenuation
of BvrR/BvrS mutants is not due only to down-regulation of Omp3a and Omp3b and
indicate that additional factors are involved in the attenuation.
82- CLONING, EXPRESSION AND PURIFICATION OF B. suis OUTER
MEMBRANE PROTEINS.
X. Ding 1 , C. Paranavitana 1 , M. Izadjoo 2 and D. Hoover 1 . (1) Department of Bacterial Diseases, Walter
Read Army Institute of Reseach, Silver Spring, MD, USA. (2) Armed Forces Institute of Pathology,
Washington, DC, USA.
Brucella, an aerobic, nonsporeforming, nonmotile Gram-negative
coccobacillus, is a NIH/CDC category B bioterror threat agent that causes
incapacitating human illness. Medical defense against the bioterror threat posed by
Brucella would be strengthened by development of a human vaccine and improved
diagnostic tests. Central to advancement of these goals is discovery of bacterial
Brucellosis 2003 International Research Conference
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