Type IV P ilus retractive forces during microcolony development K B Eckenrode 12 I Spielman 1 K Alzurqa 1 CA Weber ID: 653846
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Neisseria gonorrhoeae segregate cells lacking Type IV Pilusretractive forces during microcolony development
K. B. Eckenrode1,2, I. Spielman1, K. Alzurqa1, C.A, Weber3, W. Poenisch3, V. Zaburdaev3, N. Biais1,2 1Brooklyn College, Brooklyn, NY, 2CUNY Graduate Center, NY, NY, 3Max-Planck-Institute for the Physics of Complex Systems, Dresden, Germany
Neisseria gonorrhoeae (Ng), a human pathogen, colonize urogenital epithelial cells, which produces a threatening infection. During colonization, Neisseria gonorrhea's extracellular protein “sticky” filament named type IV pilus (Tfp) attract gonococci together to assemble microcolonies. The dynamics of microcolony formation are still unclear. Specifically, we are interested in the role of forces at single cell resolution during microcolony formation. Tfp is sufficient to produce loose microcolonies in Neisseria gonorrhoeae; however, to assemble dense microcolonies an ATPase motor named pilT is required. We aim to investigate a heterogeneous mixture of bacteria: 50% with pilT and 50% without pilT. How will a heterogeneous population mix to form an microcolony aggregate? We see spatiotemporal arrangements during bacterial microcolony development that segregates cells lacking retractive force along the perimeter of the colony. This work will advance our knowledge of microcolony architecture and the initial stages of Neisseria gonorrhoeae infection.
PILUS AS A FORCE MODEL
CELLS WITHOUT FORCE ARE OUTCASTS
Pilus dynamics have proven to be a robust model for studying how mechanical force influences microorganisms. When pilus retract, they can produce dense
microcolonies
(A). When pilus cannot retract, the colony morphology is much looser (B) than the WT strain. In previous work, WT pilus bundles were measured to pull at forces greater than 200pN for short (seconds) to long (hours) periods of time (C, D) (Biais, 2008).
FUTURE DIRECTIONS
Eukaryotes produce varieties of heterogeneous populations during development. By creating spatiotemporal patterns, animals can create complex and evolving body systems. We are interestedin creating prokaryotic heterogeneouspopulations by altering forces from Tfp. This model will allow us to investigate the role of colony development and survival inbacterial heterogeneous populations.
CELLS ACTIVELY DIFFUSE IN MICROCOLONY
MOTIVATION
TYPE IV PILUS DYNAMICS
www.mechano-micro-biology.org
BIAIS LAB WEBSITE
To study how single cells move within a
microcolony
, we tracked live fluorescent cells within
microcolonies
to measure
the total distance travelled over
short time intervals (
schematic above
). Our model simulation shows cells diffuse in the
microcolony
because velocity (
µ
m
2
/ s) decreases as
distance (
µ
m) increases, which is representative of diffusion.
Type IV pilus (
Tfp
) dynamics play a central role in prokaryotic survival and pathogenesis.
Tfp
filament construction relies on a set of conserved proteins to orchestrate polymerization and
depolymerization
of monomers that create an active appendage. The figure above is a simplified version of
Tfp
dynamics; therefore, many key proteins are not displayed for clarity.
A C
B D
Planktonic cells
Microcolony
Movement of 2
fluorescent cells in colony
v
D (Diffusion coefficient)
We mixed 50% WT (fluorescently labeled
tdTomato
) and 50%
∆
pilT
fluorescently labeled
GFP)
planktonic
cells, and after 3 hours we observed the above
microcolonies
. Our results indicate that cells without
pilT
ATPase motor proteins are segregated to the outside perimeter of the
microcolony
. Our model simulation (A) and our microscopy images (B) below. Captured with Nikon Eclipse
Ti
at 60X magnification.