Defense Date

4-27-2021

Document Type

Thesis

Degree Type

Master of Science

Degree Name

Biological Sciences

First Advisor

Robert P. Smith, Ph.D.

Second Advisor

Omar Tonsi Eldakar, Ph.D.

Third Advisor

Travis J.A. Craddock, Ph.D.

Keywords

Staphylococcus aureus, virulence, disturbance, biofilm

Abstract

Staphylococcus aureus is an opportunistic pathogen that can cause a variety of acute and chronic illnesses. The severity of these illnesses such as sepsis, necrotizing pneumonia, and toxic shock syndrome is measured through the virulence that S. aureus inflicts on its host. Methicillin-resistant S. aureus (MRSA) is commonly associated with secondary infections and is challenging to treat given the limited selection of antibiotics that are effective against it. Accordingly, novel approaches to reduce S. aureus pathogenicity are required. S. aureus regulates pathogenesis through a cell-to-cell communication system referred to as quorum sensing. Effective communication determines the production of two broad classes of virulence factors: surface attachment proteins and exotoxins. To initiate quorum sensing, S. aureus secretes a diffusible autoinducer, AIP, which is shared amongst members of the population. As AIP diffuses away from the cell that produces it, the relative positioning of AIP and bacteria determines the effective concentration that each bacterium senses. Interestingly, a previous study has suggested that periodically disturbing the spatial structure of bacterial populations participating in quorum sensing can decouple the positions of bacteria, respective autoinducers, and thus affect quorum sensing. However, this finding has yet to be extended to S. aureus. Based on these previous findings, we hypothesized that the expression of quorum sensing regulated virulence factors in S. aureus can be perturbed through the application of periodic spatial disturbance. To test this hypothesis, we used a microplate reader assay that disturbs the structure of biofilms and RT-PCR analysis to measure gene expression. We found that increasing the frequency of periodic disturbance increases exotoxin, surface attachment protein, and internal regulator expression in nutrient limited environments. In contrast, we found that increasing the frequency of periodic spatial disturbance significantly decreases the expression of all surface attachment proteins in nutrient rich environments compared to the undisturbed control (0/hr). Moreover, the expression of several exotoxin genes remained relatively unchanged with increasing disturbance frequency. Interestingly, when altering amplitude, another determinant of shear force, we found a biphasic relationship between shaking amplitude and expression of quorum sensing regulated genes. For a high disturbance frequency, an intermediate shaking amplitude (0.3 mm) was able to attenuate the expression of both surface attachment and exotoxin genes. This coincided with a decrease in surface attachment protein expression at the same intermediate point. Our results highlight the ability of periodic spatial disturbance to affect quorum sensing regulated behaviors and may very well represent a strategy to reduce pathogenesis and infection.

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