Brucellosis 2003 proceedings - PHIDIAS

Poster Session
BMEI2000 is flanked by: (1), a putative transcription terminator for the gene situated
immediately upstream coding for the (putative) chaperon protein DnaJ; and (2) a
ORF situated about 150 bp downstream with homology to genes involved in
pyrimidine metabolism, suggesting that BMEI2000 may be an independent
transcriptional unit. Two possible translation starting codons were identified in the
sequence but only the second is preceded by a putative ribosome binding site. The
corresponding protein contained an amino acid stretch (VLEFGPGTGV) which
corresponds to the consensus amino acid sequence described in methyltransferases.
To ascertain its function, BMEI2000 was cloned in the expression vector pET21a and
introduced in E. coli BL21(DE3). Cells induced with IPTG synthesized a new protein
of the expected molecular weight. Research is in progress to ascertain whether
expression of Brucella PmtA in E. coli results in a change in its phospholipid profile,
as a first step to determine the role of phosphatidyl-choline in the unusual properties
of Brucella cell envelopes.
118- EXPRESSION OF THE ExoS PROTEIN FROM Sinorhizobium meliloti
COMPLEMENTS BIOLOGICAL PROPERTIES OF A Brucella abortus MUTANT
LACKING THE BvrS PROTEIN.
Esteban Chaves-Olarte 1,2 , Caterina Guzmán-Verri 1 and Edgardo Moreno 1 . (1) Programa de
Investigación en Enfermedades Tropicales (PIET), Escuela de Medicina Veterinaria, Universidad
Nacional, Aptdo 304-3000 Heredia, Costa Rica. (2) Centro de Investigación en Enfermedades
Tropicales, Facultad de Microbiología, Costa Rica.
Brucella abortus is an intracellular pathogen that relies upon unconventional
virulence factors to infect hosts. Of these factors, a two component regulatory system
has proven to be essential for virulence since mutants in either the sensor (BvrS) or
the regulator (BvrR) have lost the ability to penetrate to and survive within epithelial
and phagocytic cell lines. In this work we have evaluated the degree of
complementation provided by the sensor (ExoS) of an analogous two component
system in the related plant pathogen Sinorhizobium meliloti. A Brucella abortus strain
lacking BvrS (B. abortus 2.13) was transformed with a cosmid containing ExoS and a
tetracycline resistance cassette which allows selection. Isolation of the cosmid from
resistant strains confirmed successfull transformation. The complemented strain
(2.13-ExoS) was tested for its ability to penetrate and survive within phagocytic and
non-phagocytic cell lines. 2.13-ExoS recuperated the ability to invade non-phagocytic
HeLa cells as determined by Colony Forming Units counting and
Immunofluorescence. Mirroring the wildtype strain (2308), 2.13-ExoS was able to
reach the endoplasmic reticulum avoiding lisosomes and replicated successfully
within this compartment. In the same manner, 2.13-ExoS invaded the phagocytic cell
line RAW 264.7 and followed a similar replication kinetic than strain 2308. Altogether
these results imply that the BvrR-BvrS system from Brucella abortus is functionally
interchangeable with the ExoS system from Sinorhizobium meliloti. The implications
of these observations for the pathophysiology of brucellosis is discussed.
Brucellosis 2003 International Research Conference
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