Airway mucus is part of inborn immunity that facilitates the elimination of inhaled microorganisms. Antibacterial mechanisms such as lysozymes and defensins, which are contained in airway mucus, are key components of this inborn immunity (Gerson et al. 2000). While this defense mechanisms remains prevalent in cystic fibrosis patients it appears to be compromised in that it allows the growth of Pseudomonas aeruginosa in airway mucus.
The fact that people with cystic fibrosis are equally exposed to pathogens as any healthy person it remains a question as to why Pseudomonas aeruginosa has been successful at inhabiting and multiplying in the lungs of cystic fibrosis patients. A recent study, A novel siderophore system is essential for the growth of Pseudomonas aeruginosa in airway mucus, published in Nature investigates this uncertainty. Using ex vivo airway mucus secretion treatments and mouse models, the researchers study the interaction between airway mucus and the Pseudomonas aeruginosa.
The following paper details and reviews the approach, methods and finding into why Pseudomonas aeruginosa, unlike other bacterial species, is capable of proliferating in airway mucus of patients with cystic fibrosis. Pseudomonas aeruginosa is a gram-negative bacteria very commonly carried around by healthy individuals without any symptoms. This bacteria can cause minor infections like swimmer’s ear. But for people who are ill and have weak immune system, this bacteria can cause deadly infection to many parts of the body. The infection is hard to treat because Pseudomonas aeruginosa can resist many antibiotics.
Pseudomonas aeruginosa is spread easily in hospitals by health care professionals and uncleansed medical equipment. This serious infection can cause pneumonia in the lungs and can cause septic shock if released into the blood stream. Symptoms include high fever, chills, confusion, and shock (CDC 2014). However, cystic fibrosis patients the persistent existence of Pseudomonas aeruginosa can be deadly. In search for why Pseudomonas aeruginosa has the capability to flourish in the airway mucus, the researchers examined how airway mucus secretions affected various bacterial species.
This was done by evaluating the number of viable cells before and after incubation with airway mucus secretion. Seven different bacterial strains were incubated with airway mucus secretion cultures that were prepared from normal human tracheal epithelial cells. Figure 1C in the above referenced research paper shows the growth index of the various bacterial strains. After sixteen hours of incubation with airway mucus all bacteria species were not viable with the exception of Pseudomonas aeruginosa. In fact the growth index of the PAO1, the prototype of Pseudomonas aeruginosa, actually increased.
Pseudomonas aeruginosa demonstrates that it has the ability to grow when incubated with normal airway mucus secretion. Since the Pseudomonas aeruginosa does response differently to airway mucus than other bacterial species, the study began to search for mechanisms by which Pseudomonas aeruginosa is resistant to airway mucus. The method used to detect increased expression of genes was microarray analysis. The thirty-five genes listed in table one, of the above referenced study, were upregulated in the wild type strain PAO1 when incubated with airway mucus.
The table included genes that encode siderophore molecules used for iron sequestration called pyoverdine and pyochelin. Also, genes that make up the operon PA4834-PA4837 were highly expressed in response to airway mucus. Next, they disrupted the genes upregulated in the wild type PAO1 strain when in contact with airway mucus. This was done using transposon insertion mutants grown in airway mucus and then their growth index were measured. All of the genes in the PA4834-PA4837 operon when mutant grew to wild type levels in lysogeny media (Figure 2C).
But in airway mucus, mutants varied in degree of growth. PA4834 mutant was susceptible to treatment by airway mucus. It should be noted that restoration of mutant PA4834 strain ability to grow in airway mucus was observed when inserted with a plasmid containing the wild type PA4834 gene (Figure 2A). Thus, the PA4834 gene encodes a protein sufficient and necessary for infection. The researchers inoculated wild type PAO1 strain, Staphylcoccus aereus, and mutant PA4837 strain with normal human tracheal epithelial cells to investigate bacterial colonization in airway mucus.
Figure 2B shows a scanning electron microscope of the infected epithelial cells. PAO1 was shown to invade the human tracheal epithelial cells. In contrast only a few cells of the mutant PA4834 strain and Staphlycoccus aureus remained viable in human tracheal epithelial cells (Figure 2B). Thus, demonstrating the ability of the wild type PAO1 strain to resistant airway mucus secreted by normal human tracheal epithelial cells. Also, demonstrating the need for PA4834 gene to carry out the mechanism to invade.
Previous results showed PA4834 gene was found to be highly expressed when treated with airway mucus. Also, previous results showed PA4834 gene when mutant failed to resist airway mucus. All of this information leads to the fact that Pseudomonas aeruginosa is activated when in contact with airway mucus iron limiting environment. Signaling the PA4834 gene to carry out the process of up taking iron. In figure 3A when mutant PA4834 strain treated with airway mucus was supplemented with ferric iron it was able to survive. Also to be noted, the wild type strain PAO1 was comparable in growth.
Then, the researchers wanted to compare the growth of the PAO1 strain and mutant PA4834 strain in iron depleted conditions. PAO1 strain grew significantly better than the mutant PA4834 strain. Iron supplementation was able to rescue the mutant PA4834 suggesting that the PA4834 encodes an iron sequestration molecule. Inductively coupled plasma mass spectrometry (ICP-MS) measures intracellular iron concentration in bacteria. In nutrient rich broth, PAOI strain and PA4834 mutant strain had similar intracellular iron concentrations (Fig. C).
However, a dramatic difference was seem when treated with airway mucus. PAO1 strain had increased intracellular iron concentration in airway mucus while PA4834 mutant strain had decrease in intracellular iron concentration (Fig. 3C). This result shows PAO1 strains ability to actively uptake iron. Also, the researchers used a green fluorescent heavy metal indicator to detect intracellular iron. The PAO1 strain in airway mucus had increased in green fluorescent color when compared to lysogeny broth incubation.
