Discovery of chromosomal factors contributing to the virulence of Yersinia pseudotuberculosis, including Fis, a regulator of resistance to reactive oxygen species.
Abstract: All three
pathogenic Yersinia species share a conserved virulence plasmid that encodes a Type 3
Secretion System (T3SS) and its effectors. This system contributes to growth and
virulence in mammalian infection by injecting effector proteins into innate immune cells
to dismantle their bactericidal functions. However, in the absence of this plasmid, the
enteric pathogen Yersinia pseud... read moreotuberculosis (Yptb) retains the ability to colonize and
grow within host organs. To uncover chromosomal factors that contribute to pathogenicity
of Yptb in the absence of the virulence plasmid, we utilized a high-throughput,
transposon screen (TnSeq). More than 30 genes, including many that were previously
uncharacterized, were identified. Next, I developed a deep sequencing-based approach to
validate and further characterize the phenotype of 18 of those genes in both WT and
plasmid-deficient Yptb strains by infecting mixed pools of in-frame knockouts into
immunocompetent mice. Fourteen mutants in the plasmid-deficient strain and 13 in the WT
strain were attenuated for growth in livers, indicating that most of these factors were
essential, even in the presence of the virulence plasmid. The growth of these mutants
was assessed in immunocompromised mice to determine which of these factors disarm host
defenses produced by innate immune cells. Mutants containing deletions of the dusB-fis
operon, which encodes the nucleoid-associated protein Fis, were restored for growth in
mice lacking neutrophils and inflammatory monocytes, two of the major cell types
responsible for controlling Yersinia infection. This operon was not important for
secretion or translocation of T3SS effectors, but protected Yptb from oxidative stress
by regulating the transcription of several ROS-detoxifying genes. Strikingly, this
protection was critical for virulence, as mice unable to produce ROS were more sensitive
to infection by ΔdusB-fis than WT mice. Notably, this is the first report of the
requirement for Fis during Yersinia infection and also highlights a novel mechanism by
which Yptb defends against ROS in mammalian
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Joan Mecsas.
Committee: Andrew Camilli, Carol Kumamoto, and Ralph Isberg.
Keyword: Microbiology.read less