Thermoregulation of PrpL, a Virulence Factor from a Dangerous Pathogen Open Access

Abstract

Pseudomonas aeruginosa is a dangerous pathogen that has been identified as a “critical priority” by the World Health Organization. Its virulence factors make the bacterium adaptable and help it to promote infection in a variety of environments. One of its virulence factors is Pvds-regulated endoprotease, lysyl class (PrpL), a protease that degrades several important host defenses and increases the severity of corneal and lung infections in mammals. The regulation of prpL expression is complex and not fully understood. The quorum sensing transcriptional activator, LasR, has been identified in some studies to activate prpL expression at later growth phases. The protease is also uniquely expressed more at room temperature (25°C) compared to human body temperature (37°C). We hypothesized that prpL would be thermoregulated by a transcriptional regulator, and that thermoregulation of prpL would cause it to be more important to the severity of infections at 25°C than at 37°C. By using a reporter plasmid of the prpL promoter transcribing green fluorescence protein (GFP), we were able to assess prpL promoter activity in different strains and with different mutations. We found that LasR is necessary for prpL thermoregulation. Deleterious mutations to a potential site of LasR binding on the prpL promoter inhibited transcriptional activity. Truncating the prpL promoter on the reporter plasmid led to a diverse set of phenotypes, which suggests more complexity to thermoregulation. To understand how prpL is thermoregulated in clinical isolates, we identified clinical isolates with single nucleotide polymorphisms in regulators of prpL. We assessed prpL promoter activity in these clinical isolates with the reporter plasmid. We found that prpL is thermoregulated in diverse lineages of P. aeruginosa and that lasR mutant clinical isolates show no promoter activity. Experiments to determine the impact of thermoregulation on the relative severity of infections by using the model organism, Galleria mellonella, found temperature dependent virulence but no significant effect of PrpL on virulence. These studies found that LasR directly regulates prpL and identified the complexity of prpL thermoregulation. More research is needed to create a complete model of thermoregulation and understand its role in infections.

Table of Contents

Introduction..........................................................................1

Methodology............................................................................5

           Table 1. Strains used in this study......................................5

           Table 2. Plasmids used in this study......................................6

           Table 3. Primers used in this study......................................7

Results....................................................................12

           Figure 1. prpL thermoregulation is dependent on lasR......................................14

Figure 2. Mutations to the las-box lead to novel thermoregulatory phenotypes......................................17

Figure 3. Truncations of the prpL promoter cause unique thermoregulatory phenotypes......................................19

Figure 4. Phylogenetic tree of clinical and environmental Pseudomonas aeruginosa isolates......................................22

Figure 5. Transcriptional activity of diverse clinical isolates......................................24

Table 4. Median survival and CFUs from Galleria mellonella survival assays......................................24

Figure 6. Galleria mellonella survival is temperature dependent but not strain dependent......................................26

Discussion....................................................................28

Refrences....................................................................32

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Microbiology
Keyword	LasR PrpL Virulence Temperature Thermoregulation Pseudomonas aeruginosa Galleria mellonella
Committee Chair / Thesis Advisor	Joanna B. Goldberg, Emory University
Committee Members	Skye Comstra, Emory University Levi Morran, Emory University

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