1. Development of Synthetic Cystic Fibrosis Growth
Medium for Pseudomonas Iron Regulation Studies
Katherine Markin, Angela Nguyen,
Alexandria Reinhart, Dr. Oglesby-Sherrouse
2. Introduction/Background
• Cystic fibrosis (CF) is a hereditary disorder that
predisposes patients to chronic lung infections.
• Pseudomonas aeruginosa colonizes the lungs of CF
patients and causes life-long infections.
• Iron is critical for P. aeruginosa pathogenesis.
Goal:
To analyze P. aeruginosa’s ability to maintain iron
homeostasis in a synthetic CF sputum medium
3. PrrF
holo
FUR
holo
FUR
High Iron
Fur represses PrrF, siderophore production and PQS
Low Iron
No Fur repression
apo
FUR apo
FUR
PrrF
F
e
Iron
containing
proteins
Iron
uptake
Steal iron from
other bacteria
PQS
PQS
Iron and Heme Regulation of PrrF and PrrH
sRNA in P. aeruginosa
7. Conclusion
• The synthetic CF sputum medium appears to
induce more iron stress than other media
previously used to study P. aeruginosa.
• Next step would be to repeat studies at
different growth times and analyze other iron-
related phenotypes.
8. References:
1.Nguyen AT, Jones JW, Ruge MA, Kane MA, and Oglesby-Sherrouse AG. (2015). Iron
depletion enhances production of antimicrobials by Pseudomonas aeruginosa. Journal
of Bacteriology 197(14):2265-75.
2.Oglesby-Sherrouse AG, Vasil ML. (2010) Characterization of a heme-regulated non-
coding RNA encoded by the prrF locus of Pseudomonas aeruginosa. PLoS
One 5(4):e9930.
3.Oglesby AG, Farrow JM 3rd, Lee JH, Tomaras AP, Greenberg EP, Pesci EC, Vasil ML.
(2008) The influence of iron on Pseudomonas aeruginosa physiology: a regulatory link
between iron and quorum sensing. Journal of Biological Chemistry 283(23):15558-67.
4.Palmer, K. L., Mashburn, L. M., Singh, P. K., & Whiteley, M. (2005). Cystic Fibrosis
Sputum Supports Growth and Cues Key Aspects of Pseudomonas aeruginosa Physiology.
Journal of Bacteriology, 187(15), 5267–5277. doi:10.1128/JB.187.15.5267-5277.2005
5.Mashburn, L. M., Jett, A. M., Akins, D. R., & Whiteley, M. (2005). Staphylococcus
aureus Serves as an Iron Source for Pseudomonas aeruginosa during In Vivo Coculture.
Journal of Bacteriology, 187(2), 554–566. doi:10.1128/JB.187.2.554-566.2005
Acknowledgements:
I would like to thank the Oglesby-Sherrouse Lab members for their help. This work was
supported by funding from the University of Maryland School of Pharmacy.
Editor's Notes
Pseudomonas highly resistant to multiple antibiotics
Long term goals: using this information to target heme regulatory and uptake mechanisms for therapeutic purposes
Pyovirdine gives yellow pigment (protein)
Pyrocyane – toxin in blue
Iron is a major factor in virulence b/c free iron is very limited and often tightly bound to proteins like hemoglobin
Bacteria utilize siderophores (large and expensive molecules) to help get iron from the environment
CAS assays utilize a dye called chrome azurol that complexes tightly with free iron to turn blue when siderophores remove iron from the environment the color changes to yellow
To quantitatively assess the amount of siderophores for each iron interval, I compared the OD of the culture before and after the CAS assay
More siderophores were released (more iron starved) in 0, 1, and 5 uM concentrations of iron than 50, 100, and 200 uM iron. Most media in the lab only use 100 uM which doesn’t appear to completely turn off iron acquisition. We could look at this using a range of iron concentrations over different time periods.
For wildtype P. aeruginosa, prrF and prrH are repressed in high iron/high heme environments.
PrrF1 and 2 located in tandem on genome which allows expression for 3rd sRNA
PrrF = iron regulation (regulated by iron)
PrrH = heme homeostasis (regulated by heme abundant in CF lung during infection but don’t know how heme regulates)
AQ (alkyl quinalones) produced and found in CF lung
Likely contribute to Pseudomonas’s competitive advantage in CF lung
PrrFs required for PQS expression in low iron conditions
PQS is the chemical language used by bacteria to turn on group behaviors
When bacteria are in small numbers, they secrete a hormone-like molecule that drifts around in the environment and no response occurs. When more bacteria are present, more molecules are secreted and are recognized by a specific membrane protein on the bacteria. The binding tells the bacteria that there are many more of its kind around and causes a group response.
In addition, PQS molecules help Pseudomonas get and compete for iron. Iron represses PQS production but we don’t know exactly how that happens.
This data indicates that with no iron present, pseudomonas is not making as much PQS. This could be due to the fact that it is so iron starved that it is putting all its energy into making siderophores to acquire iron rather than making signaling molecules or may not have grown to the same level. The spots are lighter for the 18 hour culture which could be contributed to the fact that by 18 hours the cells are dying, or they don’t need to compete against other bacteria in the monoculture and don’t need to secrete any more.
Using this media for future testing could give more realistic and meaningful results
Long term goals: using this information to target heme regulatory and uptake mechanisms for therapeutic purposes
