Mycobacterial growth adaptations to the host environment.
Logsdon, Michelle.
2019
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Asymmetric
elongation and division in mycobacteria give rise to increased variability in cell size
compared to model bacteria such as E. coli and B. subtilis. Their innate asymmetry
creates unequal birth sizes and growth rates for daughter cells with each division,
generating phenotypically heterogeneous populations that may serve as a bet-hedging
mechanism under host stress. Cell size ... read moreheterogeneity increases during infection in
clinical strains of Mycobacterium tuberculosis (Mtb), but its role in infection
progression has yet to be determined. Here, we used time-lapse microscopy and
fluorescent reporters of DNA replication and polar elongation to examine the
coordination of growth, division, and chromosome dynamics at a single-cell level in
Mycobacterium smegmatis (M. smegmatis), Mycobacterium bovis Bacillus Calmette-Guerin
(BCG), and Mtb. Using measurements and stochastic modeling of mycobacterial cell size
and cell-cycle timing in both slow and fast growth conditions, we found that
well-studied models of bacterial cell-size control are insufficient to explain
mycobacterial growth and division patterns. Instead, we showed that mycobacterial cell
size control is regulated by an unprecedented mechanism involving parallel adders (i.e.,
constant growth increments) that start at replication initiation and control both cell
division and replication initiation in the subsequent generation. Additionally, when
initiation of DNA replication is inhibited through depletion or mutation of essential
DNA replication protein DciA, cell size control is abrogated. These molecular
perturbations indicate a mechanistic basis for our initiation dependent model of cell
size control. We sought to define changes in Mtb growth patterns during macrophage
infection and understand how growth behaviors contribute to survival within host
macrophages. Cell-to-cell variability in mycobacterial size, cell cycle, and growth
properties are likely maintained and contribute to survival during infection, but Mtb
size, growth, and cell cycle patterns within a host macrophage have never been measured
with the single cell techniques required to capture population heterogeneity.
Fluorescent D-amino acid pulse labelling and 3D image analysis were utilized to evaluate
single cell Mtb growth properties in laboratory strains and clinical isolates during
macrophage infection. Mtb laboratory strains have lost their size responsiveness to
macrophage infection, however clinical isolates increase in length after 4 days
infection. Additionally, pH stress alone is sufficient to induce cell size plasticity in
both Mtb lab strains and clinical isolate. Mtb growth and size properties shift to adapt
to infection and likely have a role in infection establishment and persistence.
Understanding how cellular processes allow mycobacteria to adapt to the intracellular
host environment could provide novel targets to reduce Mtb virulence and treatment
time.
Thesis (Ph.D.)--Tufts University, 2019.
Submitted to the Dept. of Molecular Microbiology.
Advisors: Bree Aldridge, and Ralph Isberg.
Committee: Andrew Camilli, and Joan Mecsas.
Keyword: Microbiology.read less - ID:
- fj236f83n
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