4 research outputs found
Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis
Molecular studies of bacterial virulence are enhanced by expression of
recombinant DNA during infection to allow complementation of mutants and
expression of reporter proteins in vivo. For highly pathogenic
bacteria, such as Yersinia pestis, these studies are currently
limited because deliberate introduction of antibiotic resistance is restricted
to those few which are not human treatment options. In this work, we report the
development of alternatives to antibiotics as tools for host-pathogen research
during Yersinia pestis infections focusing on the
diaminopimelic acid (DAP) pathway, a requirement for cell wall synthesis in
eubacteria. We generated a mutation in the dapA-nlpB(dapX)
operon of Yersinia pestis KIM D27 and CO92 which eliminated the
expression of both genes. The resulting strains were auxotrophic for
diaminopimelic acid and this phenotype was complemented in
trans by expressing dapA in single and multi-copy.
In vivo, we found that plasmids derived from the p15a
replicon were cured without selection, while selection for DAP enhanced
stability without detectable loss of any of the three resident virulence
plasmids. The dapAX mutation rendered Y.
pestis avirulent in mouse models of bubonic and septicemic plague
which could be complemented when dapAX was inserted in single
or multi-copy, restoring development of disease that was indistinguishable from
the wild type parent strain. We further identified a high level, constitutive
promoter in Y. pestis that could be used to drive expression of
fluorescent reporters in dapAX strains that had minimal impact
to virulence in mouse models while enabling sensitive detection of bacteria
during infection. Thus, diaminopimelic acid selection for single or multi-copy
genetic systems in Yersinia pestis offers an improved
alternative to antibiotics for in vivo studies that causes
minimal disruption to virulence