Original Article: Chen, A., Dolben, E., Okegbe, C., Harty, C., Golub, Y., Thao, S., . . . Hogan, D. (2014). Candida albicans Ethanol Stimulates Pseudomonas aeruginosa WspR-Controlled Biofilm Formation as Part of a Cyclic Relationship Involving Phenazines. PLoS Pathog PLoS Pathogens.
Post By: Liana Ghiron and Maddie Brudos
Pseudomonas aeruginosa and Cystic Fibrosis
From Batman and Robin to Bonnie and Clyde, we’ve seen teamwork always makes things easier. Even in the microbial world researchers have found cooperative interactions between bacteria. In the case of infections, P. aeruginosa and Candida albicans seem to be partners in crime.
P. aeruginosa is a gram-negative bacterium that is responsible for various diseases in plants and animals. It is an incredibly versatile organism, retrieving its energy sources from a wide array of organic materials. Because it can have many sources of energy, P. aeruginosa is especially infectious in tissue of immune-compromised patients. The most prevalent symptoms of these infections are sepsis and inflammation. Furthermore, P. aeruginosa can cause chronic infections in the lungs of patients with cystic fibrosis.
Cystic fibrosis is a genetic disease, in which a buildup of mucous in the lungs clogs airways. P. aeruginosa can cause chronic lung infections in patients with cystic fibrosis. Persistence of the infection can be attributed to the formation of a P. aeruginosa biofilm, a congregation of bacteria embedded in a matrix. Biofilms can become resistant to antibiotics and phagocytosis of the immune system, causing chronic infections.
Curiously, patients with CF seem to deteriorate more quickly when they have an infection of both C. albicans and P. aeruginosa. The thick mucus in the lungs of CF patients appears to be a good medium for bacteria to grow on. Researchers, Chen et al., deduced that the combination of the two bacteria must engage in some sort of interaction to make it harder for infected patients to recover. In their discoveries they found that P. aeruginosa more quickly creates a biofilm on the lungs causing worsening of the patients conditions when the bacteria is in the presence of C. albicans.
Biofilm growth: A microbial interaction
P. aeruginosa was found to have a positive relationship with another microorganism, C. albicans. P. aeruginosa produces phenazines which, in low levels, cause decreased cellular respiration in C. albicans. This in turn causes the C. albicans to produce higher concentrations of ethanol. Specifically, the ethanol production can increase 3 to 5 times more than usual production. The ethanol produced had two main effects on P. aeruginosa. First, C. albicans seemed to be engaged in a feedback pathway with P. aeruginosa where the ethanol production subsequently effected the levels of, and type of, phenazines produced. Secondly, ethanol contributed to higher levels of c-di-GMP. This c-di-GMP was seen to be involved with activation of WspR and WspA. The Wsp system was found to be necessary in the cascade pathway to cause the ethanol production of C. albicans to have any effect on P. aeruginosa. The activation of the c-di-GMP, through WspA/R, then activated pel polysaccharides, which contributed positively to biofilm growth and development. It is suspected that the growth occurs due to decreased swarming abilities and flagellar motility by the bacteria, as well as increased matrix production. (Figure above displays the pathway)
In other tests run, the researchers found that each of the steps of the signal cascade, particularly in the Wsp system, were important to the interaction between P. aeruginosa and C. albicans. For example, tests were run using WspR mutants deleted for that gene. In these mutants, the missing WspR gene inhibited the effects of the increased ethanol production by the C. albicans. Shown in figure 2, we can see that without the WspR to activate the pel polysaccharides, the biofilm creation was no different than the tests run on the P. aeruginosa without ethanol.
Another finding during the trials was the observation of green crystal formations on the plates that contained the ethanol. Tests were run to figure out why these formations occurred, because they were consistent with formations from PCN, a toxin secreted by P. aeruginosa. Through these tests they found elevated numbers (22.4 and 5.8 times more) of PCN and PCA (the precursor to phenazines including PCN). Through this they deduced that perhaps ethanol had a direct effect on the PCA. More tests were run using PCA mutants, unable to create PCA, on ethanol containing plates. The results showed that ethanol did have an effect on aiding production of PCA, but perhaps is more pertinent in the determination of types of phenazines produced. Even more tests were run studying the effects of ethanol production on a different type of phenazine, 5MPCA. Through these tests they concurred that the increase of production was still minimal, but the real effect had to do with specialization of the type of phenazines created. In addition, the ethanol production is furthered by the release of the specialized phenazines, creating a positive feedback system. The ethanol that is continually produced also increases biofilm production through the other pathway described above.
But how can this research become useful in hospitals?
It is important to note that the article proposes that research about interactions between bacteria could suggest indirect treatment for fungal and bacterial infections. For example, treating one bacterial infection may have an effect on stopping the spread of many other bacteria. In fact, at the end of the study I wondered whether current treatment was used to treat the P. aeruginosa or both the P. aeruginosa and the C. albicans together. The article specifies that the infection of P. aeruginosa is worse due to the growth of a biofilm. How does P. aeruginosa act if it is swarming? Is the treatment different for different strains of the bacteria? If the researchers were to run further tests, it would be productive to start testing drugs that might affect different pathways of bacteria interaction. For example, would a drug that acts on the Wsp pathway be more or less effective than one that destroys the pel polysaccharides? I also wondered if there was a way to prevent the P. aeruginosa from creating the phenazines that were needed for the feedback loop that caused the C. albicans to produce more ethanol. In addition it mentioned in the article that too much phenazine production would lead to death of C. albicans, so I wondered whether there was a way to control both the growth of C. albicans and P. aeruginosa by adding higher concentrations of phenazines or different types to stop the growth of either bacteria.
Even though this study focuses on CF patients I still wanted to know whether this interaction was common in patients with other infections or conditions. Is this interaction specific to worsening patients with CF only? Or does it mainly affect CF patients? When doing research on P. aeruginosa, patients with CF seemed to come up in the search, so I wonder why, or if, this strain of bacteria seems to be more common in these patients.
Fun facts
Curious to see the motility of a Pseudomonas aeruginosa biofilm under the microscope?
Visit https://www.youtube.com/watch?v=xJIvtp44Kb8 for you viewing pleasure!
Journal Source
Chen, A., Dolben, E., Okegbe, C., Harty, C., Golub, Y., Thao, S., . . . Hogan, D. (2014). Candida albicans Ethanol Stimulates Pseudomonas aeruginosa WspR-Controlled Biofilm Formation as Part of a Cyclic Relationship Involving Phenazines. PLoS Pathog PLoS Pathogens.
Other Sources
"Cystic Fibrosis." Genetics Home Reference. U.S. National Library of Medicine, Aug. 2012. Web. 17 Dec. 2015.
Fujitani, Shigeki, Kathryn S. Moffett, and Victor L. Yu. "Pseudomonas Aeruginosa." Infectious Disease and Antimicrobial Agents. Web. 29 Nov. 2015.
Image Sources
1. https://s-media-cache-ak0.pinimg.com/236x/ae/fb/db/aefbdbfd5482394660bcced5ae1f70be.jpg
2. https://img0.etsystatic.com/000/0/5369791/il_fullxfull.146359624.jpg
3.http://www.dentalproductshopper.com/documents/31920/0/biofilm.jpg/7e8b88c8-19ad-49c4-b1e5-8fc5acbf3de5?t=1427491271764
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