82 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
Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice
Background: Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phageresistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. Methodology/Principal Findings: The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD 50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. Conclusions/Significance: We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophag
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