High-Throughput Identification and Characterization of Klebsiella pneumoniae Virulence Determinants in the Lungs
Klebsiella pneumoniae (Kp) is a gram-negative bacterial pathogen that causes a range of
infections, including pneumonias, and has become an increasing concern due to the rise
and spread of antibiotic resistant and hypervirulent strains. Despite the importance of
this pathogen, its virulence determinants remain largely understudied. Therefore, to
identify novel Kp virulence determina... read morents needed to cause pneumonia, a high-throughput
screen was performed in the lungs of WT mice using TnSeq with an arrayed library of more
than 13,000 Kp transposon insertion mutants. Furthermore, Kp is classically an
opportunistic pathogen that typically infects the immunosuppressed, such as those with
conditions that result in defective neutrophil responses like diabetes and malignancies,
and there remains much left to be discovered about Kp's interactions with the immune
response of which neutrophils have been shown to play a strong role in the lungs.
Therefore, the genes needed for Kp to infect neutropenic lungs were also assessed. A
comparison of the two studies reveals genes Kp needs to protect against neutrophils, as
well as genes that Kp requires in both immunocompetent and immunosuppressed hosts. The
latter could encode factors that may potentially serve as therapeutic targets. Using
this method, mutants with transposons in 166 genes were significantly less fit infecting
lungs of WT mice. Of these, 51 genes were still significantly less fit in neutropenic
mice, 52 were able to colonize neutropenic mice, and the differences in the fitness of
the remainder in the absence versus the presence of neutrophils was modest. In vitro
screens using a mini-library of Kp insertion mutants of genes with fitness defects in
the initial screen identified a number of putative functions for a subset of these
genes, such as in growth under different conditions, exopolysaccharide production,
resistance to ROS and RNS, and de novo synthesis of amino acids and metabolites. Further
in vivo studies using single strain and mini-library competition lung infections
confirmed roles in Kp virulence in the lungs for dedA, dsbA, gntR, VK055_RS17445,
VK055_26085, ycgE, yhjH, aroA, gltB, leuA, leuB, metA, nadB, serA, serB, trpE, and tyrA,
all of which were assigned putative functions using the above-described in vitro assays.
When aroA was assessed as a potential therapeutic target against Kp, treatment with the
AroA-specific inhibitor glyphosate was found to significantly decrease Kp growth in
vitro and bacterial loads in the lungs in vivo. Importantly, resistance to glyphosate
occurred only rarely when assessing spontaneous resistance in the lab strain Kp ATCC
43816 and in clinical isolates, including ESBL and KPC expressing strains. Overall, this
study provided evidence for dedA, dsbA, gntR, VK055_RS17445, VK055_RS26085, ycgE, yhjH,
aroA, gltB, leuA, leuB, metA, nadB, serA, serB, trpE and tyrA as novel Kp virulence
factors in the lungs, uncovered putative functional roles in growth and protection
against the neutrophil response for these factors, and identified aroA as a gene with
strong therapeutic potential as a target of
Thesis (Ph.D.)--Tufts University, 2017.
Submitted to the Dept. of Immunology.
Advisor: Joan Mecsas.
Committee: Henry Wortis, John Leong, Alexander Poltorak, and Celeste Thorpe.
Keywords: Microbiology, and Immunology.read less