AT THE INTERFACE OF THE HUMAN HOST AND BACTERIA: STRESS SIGNALING VIA CYCLIC AMP AND CALCIUM Öffentlichkeit

Abstract

Outer membrane vesicles (OMVs) play a critical role in bacterial-host interactions, influencing immune signaling and cellular responses. These OMVs influence host responses to bacterial infections, thereby regulating immunity and pathogenicity. This study investigates the differences in OMV production and host signaling activation between the laboratory-adapted strain Pseudomonas aeruginosa PAO1 and two clinically derived isolates. PAO1 and clinical isolates were cultured under controlled conditions, and OMVs were isolated using size-exclusion chromatography. Nanoparticle tracking analysis (NTA) quantified OMV production, while a bioluminescence assay measured cAMP and calcium signaling activation in a THP-1 monocyte cell line through CREB and NFAT reporter systems.

PAO1 exhibited a higher OMV yield per cell but showed a decreasing trend in production over time, whereas clinical isolates maintained relatively stable OMV output. Bioluminescence assays demonstrated that PAO1 OMVs elicited a consistent, dose-dependent activation of CREB and NFAT, aligning with expected cAMP and calcium stimulation. In contrast, the clinical isolates displayed more variable activation patterns, with fluctuations in NFAT and CREB responses.

These findings highlight strain-specific differences in OMV biogenesis and signaling capacity. The observed variation in cellular activation suggests that clinical isolates may have evolved

distinct OMV-mediated strategies to modulate host responses. Further investigations into these mechanisms will provide insight into the evolutionary pressures shaping human immune

responses to bacterial pathogens.

Table of Contents

Table of Contents

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

1.1 Host and environment ...........................................................................................................................1

1.1.1 Signal transduction ............................................................................................................................ 1

1.1.2 Role of secondary messengers in biological systems .............................................................................. 1

1.1.3 cAMP and Ca²⁺ regulation of membrane channels ................................................................................. 3

1.2 Host-pathogen interactions .................................................................................................................... 4

1.2.1 Pathogen-associated molecular patterns and damage-associated molecular patterns ............................... 4

1.2.2 Extracellular vesicles and outer membrane vesicles ................................................................................ 5

1.2.3 The effect of pathogens on cAMP and Ca²⁺ levels ................................................................................... 6

1.2.4 Altered cAMP and Ca²⁺ levels impair immune function ........................................................................... 6

1.3 Significance of research project ................................................................................................................ 7

1.3.1 Pathogenic bacteria (Pseudomonas) ...................................................................................................... 7

1.3.2 CREB- and NFAT-responsive human monocyte reporter cell lines ............................................................. 8

1.3.3 Project: investigating the effects of P. aeruginosa OMVs on immune cell signaling. .................................... 8

2. Methods .................................................................................................................................................. 10

2.1 Bacterial culture ..................................................................................................................................... 10

2.2 OMV isolation ........................................................................................................................................ 10

2.3 Nanoparticle tracking analysis (NTA) ....................................................................................................... 11

2.4 THP-1 cell culture, OMV exposure and luminescence assay ....................................................................... 11

3. Results ..................................................................................................................................................... 13

3.1 Growth rate of the PAO1 lab-adapted strain of P. aeruginosa ..................................................................... 13

3.3 Cystic Fibrosis P. aeruginosa clinical isolate 2 (CF2) ................................................................................... 20

3.4 Comparative analysis between P. aeruginosa strains .................................................................................. 22

4. Discussion ............................................................................................................................................... 26

5. Anthropological implications ................................................................................................................... 28

6. References ............................................................................................................................................... 29

About this Honors Thesis

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School	Emory College
Department	Anthropology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Anthropology, Medical and Forensic Biology, Microbiology
Stichwort	bacteria stress signaling
Committee Chair / Thesis Advisor	Tirouvanziam, Rabindra, Emory University
Committee Members	Lindo, John, Emory University Comstra, Skye, Emory University

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