Defense Date

4-26-2022

Document Type

Thesis

Degree Type

Master of Science

Degree Name

Biological Sciences

First Advisor

Robert P. Smith, Ph.D.

Second Advisor

Omar T. Eldakar, Ph.D.

Third Advisor

Travis J.A. Craddock, Ph.D.

Keywords

autoinducer, biofilm, gene expression, pathogenicity, shear force

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen associated with severe acute and chronic illnesses. Current antibiotic-based approaches fail to effectively treat P. aeruginosa infections due to the effectiveness and robustness of the quorum sensing signaling system (QS). Pathogenic bacteria, such as P. aeruginosa, employ this population density-dependent communication mechanism to confer antimicrobial resistance, propagate infection, and coordinate the expression of virulence factors, through the production and detection of autoinducing signaling molecules (AI). As such, there is a growing interest in developing novel non-antibiotic-based techniques to attenuate the pathogenicity of P. aeruginosa by disrupting the functionality of its QS system. Previous studies suggest that periodic disturbances to the biofilm structures of bacteria performing QS alter the distribution of bacteria and autoinducer, thus attenuating the expression of its QS-regulated genes; but this has yet to be explored in P. aeruginosa. Accordingly, we examined the effect of periodic spatial disturbances on the expression of QS-regulated virulence genes in P. aeruginosa. We found that periodically disturbing biofilms composed of P. aeruginosa at various frequencies and amplitudes alter the distribution of bacteria in the biofilm and surrounding planktonic states, with higher frequencies and amplitudes greatly increasing the distribution of cells into the planktonic state. Moreover, qRT-PCR analysis suggests periodic disturbances decrease the expression of QS-transcription regulators and effector virulence factors across all major QS systems in planktonic state cells compared to an undisturbed control (0/hr). Finally, the range of disturbance frequencies that resulted in a reduction in the expression of QS-transcription regulators was dependent on the amplitude of the disturbance; at an intermediate amplitude of 0.3mm, a reduction in the expression of these critical genes occurred over a broader range of shaking frequencies relative to smaller or larger amplitudes. This suggests that an optimal combination of physical and high shear forces favors this reduction at intermediate disturbance amplitudes. Comparative gene expression analysis of wildtype PA14 and QS knockout strains DLasR, DRhlR, and DLasR/DRhlR suggests that periodic disturbances alter QS stability and function in P. aeruginosa. Overall, these results establish that disrupting the spatial structure of P. aeruginosa biofilms has an effect on QS-regulated gene expression and suggest that modulating shaking frequency and amplitude of disturbance could lead to novel non-antibiotic-based approaches to mitigate pathogenesis, cooperation, and virulence of P. aeruginosa.

ORCID ID

https://orcid.org/0000-0003-4535-0138

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