Date of Degree

9-2019

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Nicolas Biais

Committee Members

Theodore Muth

Lars Dietrich

Peter Lipke

Luis Quadri

Subject Categories

Bacteriology | Biophysics | Molecular Biology

Keywords

Biofilm, Gene expression, Type IV Pilus

Abstract

Many bacteria rely on the dynamics of their extracellular appendages to perform important tasks, like motility and biofilm formation. Interestingly, these dynamics have been linked to physiological responses in some pathogenic bacteria; therefore, it is important to understand more about the role of physical forces in bacteria. I used the causative agent of the human disease gonorrhea, Neisseria gonorrhoeae, as a model system to study the role of physical force on early biofilm formation. The advantage of this system is that cell-cell interactions are controlled by extracellular filaments called type IV pili (tfp). Tfp is composed of monomers that give bacteria the ability to produce a dynamic filament undergoing cycles of elongations and retractions, and thus to exert forces on their surroundings.

Through experiments and modeling, I demonstrated that pilus interactions produce motility gradients in microcolonies potentially establishing a force gradient across the microcolonies. I was interested in testing the biological implications of those motility and force gradients, so I utilized an established genetic mutant, ∆pilT, which lacks the pilus retraction motor pilT (Merz, So, and Sheetz 2000). A ∆pilT mutant allowed us to measure physiological response in cells that do not produce retractive force from its pilus. I measured the level of gene expression of seven pilus-related genes in two backgrounds: WT and a pilus retraction-deficient mutant, ∆pilT. I found that some WT microcolonies express pilus-related genes in a heterogeneous fashion, while others are homogeneous. Spatiotemporal patterns in the microcolony are modified in a ∆pilT background. The presence or absence of retraction forces between bacteria have a profound impact on bacterial physiology: the WT and ∆pilT background do not survive in a classical static biofilm assay at the same rate. Together these results point toward a fundamental role for intracellular forces in shaping bacteria physiology.

The work of biologists has been dominated by a biochemical perspective. Although biochemical processes, like metabolism and information transfer, are certainly essential in all hierarchical levels of life, there is growing evidence that physical forces may provide an alternate physiological mechanism. The introduction in Chapter 1 provides context for understanding the role of force pattern formation in multicellular structures, in the hopes to extend this line of thinking to microbial communities.

The development of microbial communities relies on self-assembly of single cells. The development of Neisseria gonorrhoeae cellular aggregates rely exclusively on type IV pili interactions (Taktikos et al. 2015a). In Chapter 2 is a transcription of the publication where I explore the dynamics of the microcolonies (W. Pönisch et al. 2018a). We found that cells have differential motility depending where in the microcolony cells are located. Differential motility is a result of fewer pili-pili interactions on the perimeter of the microcolony, and more pili-pili interactions closer to the center. Therefore, due to frequency of pili-pili interactions, a gradient of motility produces heterogeneous behavior in the microcolony.

To investigate whether heterogenous behavior is extended beyond motility, I investigated whether there is a connection between retraction force and the physiology of microcolonies. In Chapter 3 I used a quantitative approach to analyze seven pilus-related genes using fluorescent reporters. Using fluorescence and confocal microscopy, I quantified fluorescence intensity within space and time in microcolonies. Here, I provide evidence that physical intracellular cues in a three-dimensional bacterial aggregate provide context for spatial organization, since spatiotemporal patterning and survival in ∆pilT background are compromised in comparison to WT microcolonies. This suggests the important role PilT retraction force plays in regulating spatiotemporal patterning during early biofilm development.

Lastly, in Chapter 4 I characterized some physical features of microcolonies. I measured the formation size and survival rates of microcolonies when exposed to a range of osmotic pressures. These experiments were motivated by my interest in understanding the native context of developing microcolonies. Microcolonies inhabit the viscous mucosal membranes of epithelial cells; therefore, I measured one aspect of the environmental effects of microcolony when exposed to similar osmotic pressure created by mucus. I also measured the plasticity of WT and ∆pilT microcolonies through squeezing microplate experiments.

The overall aim of this work is to understand the role of physical force on microbial development. I largely focused on role of tfp forces on Neisseria gonorrhoeae microcolony formation. Characterizing gene expression in microcolonies provided key evidence for spatiotemporal heterogeneity in developing WT microcolonies. Heterogeneity was minimized without pilus retraction forces, which suggests that retraction forces play a role in the early development of biofilm formation.

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