Researchers at the Ohio State University Wexner Medical Center Division of Infectious Diseases Department of Internal Medicine at Columbus, Ohio have found that respiratory failure caused by chronic lung infection with Pseudomonas aeruginosa bacteria is a common cause of death in patients with cystic fibrosis (CF). The study, published in the journal PLOS Pathogens, demonstrates that an antimicrobial peptide produced by human immune cells can promote mutations in the bacterium that make it more lethal.
CF is genetic disease that is more common in individuals of European descent, and the most common fatal genetic disease affecting North American children and young adults. There is currently no cure.
A multi-system disorder, CF causes a variety of symptoms that may include:
• Persistent cough with productive thick mucous
• Wheezing and shortness of breath
• Frequent chest infections, which may include pneumonia
• Bowel disturbances, such as intestinal obstruction or frequent, oily stools
• Weight loss or failure to gain weight despite possible increased appetite
• Salty tasting sweat
• Infertility (men) and decreased fertility (women)
However, CF mainly affects the digestive system and lungs. The degree of disease involvement varies from person to person, but the persistence and ongoing infection in the lungs, with consequent destruction of tissues and loss of lung function, eventually causes death in most people who have cystic fibrosis. Typical complications caused by cystic fibrosis include difficulty digesting fats and proteins; vitamin deficiencies due to loss of pancreatic enzymes; and progressive loss of lung function.
The open access PLOS Pathogens paper entitled “Cationic Antimicrobial Peptides Promote Microbial Mutagenesis and Pathoadaptation in Chronic Infections” (April 24, 2014 PLoS Pathog 10(4): e1004083. doi:10.1371
journal.ppat.1004083) is co-authored by Dominique H. Limoli, Andrea B. Rockel, Kurtis M. Host, Anuvrat Jha, Benjamin T. Kopp, Thomas Hollis, and corresponding author Daniel Wozniak, Ph.D.
Dr. Wozniak, Professor of Internal Medicine Microbiology and Microbial Interface Biology with the Ohio State University Wexner Medical Center Division of Infectious Diseases Department of Internal Medicine and his colleagues studied a process called “mucoid conversion,” which involves mutations in Pseudomonas that produce a sticky sugar coating of the bacteria which makes them more resistant to various treatments. They note that the process is fairly well understood, and involves mutation of a particular Pseudomonas gene called mucA.
Searching for factors of the human host that facilitate mucA mutation, the scientists found that specific immune system cells called polymorphonucleocytes (or neutrophils), which are present in large numbers in lung cells of patients with CF, can trigger Pseudomonas mucoid conversion, and that a specific antimicrobial factor produced by these cells called LL-37 plays a key role.
At high doses, LL-37 can kill bacteria by poking large holes into their cell walls. However, at lower concentrations (which seem to mimic the situation in the lungs of CF patients), the scientists found that some LL-37 molecules can enter the bacterial cells without killing them. Once inside, LL-37 appears to be able to directly interact with and alter the bacterial DNA, leading to mutation of the mucA gene. The resulting mucoid conversion makes the sugar-coated bacteria then resistant to higher doses of LL-37, including doses that would readily kill the “naked” Pseudomonas bacteria prior to mucoid conversion.
The scientists go on to show that LL-37 can induce mutations besides those in mucA in both Pseudomonas and E. coli, showing that its function as a mutagen is neither restricted to a particular gene nor a particular pathogen.
The coauthors explain in the study abstract that acquisition of adaptive mutations is essential for microbial persistence during chronic infections — particularly so during chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients. Thus far, they note that mutagenesis has been attributed to the generation of reactive species by polymorphonucleocytes (PMN) and antibiotic treatment. However, their current studies of mutagenesis leading to P. aeruginosa mucoid conversion have revealed a potential new mutagen. The researchers’ findings confirm the current view that reactive oxygen species can promote mucoidy in vitro, but revealed PMNs are proficient at inducing mucoid conversion in the absence of an oxidative burst.
That observation led to the discovery that cationic antimicrobial peptides can be mutagenic and promote mucoidy. Of specific interest was the human cathelicidin LL-37, canonically known to disrupt bacterial membranes leading to cell death. An alternative role was revealed at sub-inhibitory concentrations, where LL-37 was found to induce mutations within the mucA gene encoding a negative regulator of mucoidy and to promote rifampin resistance in both P. aeruginosa and Escherichia coli. The mechanism of mutagenesis was found to be dependent upon sub-inhibitory concentrations of LL-37 entering the bacterial cytosol and binding to DNA. LL-37/DNA interactions then promote translesion DNA synthesis by the polymerase DinB, whose error-prone replication potentiates the mutations. A model of LL-37 bound to DNA was generated, which reveals amino termini-helices of dimerized LL-37 bind the major groove of DNA, with numerous DNA contacts made by LL-37 basic residues. This demonstrates a mutagenic role for antimicrobials previously thought to be insusceptible to resistance by mutation, highlighting a need to further investigate their role in evolution and pathoadaptation in chronic infections.
The coauthors summarize that antimicrobial peptides (AMPs) are produced by the mammalian immune system to fight invading pathogens, with their best understood function interaction with the membranes of microbes, thereby disrupting and killing cells. However, they note that the amount of AMP available during chronic bacterial infections may not be sufficient to kill pathogens (sub-inhibitory), and in their study, they found that at sub-inhibitory levels, AMPs promote mutations in bacterial DNA, a function not previously attributed to them. In particular, they found that in the bacteria Pseudomonas aeruginosa, one AMP called LL-37 can promote mutations, which enable the bacteria to overproduce a protective sugar coating, a process called mucoid conversion. P. aeruginosa mucoid conversion is a major risk factor for those suffering from cystic fibrosis (CF). The researchers found that LL-37 is able to produce these mutations by penetrating the bacterial cell and binding to the bacterial DNA. This DNA binding disrupts normal DNA replication and allows mutations to occur. Furthermore, they observed LL-37 induced mutagenesis in processes apart from mucoid conversion, in both P. aeruginosa and E. coli, suggesting that AMP-induced mutagenesis may be important for a broad range of chronic diseases and pathogens.
Taken together, the researchers conclude that an antimicrobial substance can, at low dose, function as a mutagen that makes bacteria more dangerous, and given that antimicrobial peptides similar to LL-37 are being discussed as promising leads for the development of new antibiotics, the scientists say their data “reinforce how important it is to consider the impact of current and novel treatments and the host immune response on evolution of microbial communities during chronic infections.”
Dr. Wozniak’s main research interests are in microbial pathogenesis and gene regulation, and the major goal of the Wozniak laboratory is to understand the molecular biology and pathogenesis of the bacterium Pseudomonas aeruginosa. This soil and water organism is a common, yet serious opportunistic pathogen. Additionally, P. aeruginosa causes severe pulmonary infections in patients with cystic fibrosis. Failure to control colonization with P. aeruginosa in CF patients is now the major cause of pulmonary debilitation in this group. The lab’s research has centered on genes involved in the regulation of several P. aeruginosa virulence factors. Molecular, biochemical, and genetic techniques are used address these issues.
The Wozniak lab team is currently investigating the biosynthesis and genetic regulation of two polysaccharides called alginate and Psl, which are critical factors in biofilm formation and thus pathogenesis of P. aeruginosa. Biofilms, which are defined as communities of microorganisms that are attached to a surface, play a critical role in infectious diseases. Because of their innate resistance to antibiotics, phagocytic cells, and other biocides, biofilms are difficult, if not impossible, to eradicate. Since the matrix contributes considerably to the highly resistant nature of the biofilm, it is anticipated that this work will lead to agents that could disrupt the matrix and be of significant therapeutic value patients colonized with P. aeruginosa. In this regard, we have ongoing collaborations with scientists to develop a conjugate vaccine for use in CF patients and to develop small molecule inhibitors of bacterial biofilms.
Among infectious diseases, respiratory infections are the leading cause of morbidity and a primary cause of death for children. At OSU, Dr. Wozniak is part of a team of investigators that study the host-pathogen interface in persistent airway infections. The long-term goals of their laboratory are to deepen our understanding of the interplay between host immunity and bacterial biofilms and to develop therapies to prevent persistence of infectious childhood agents by eliminating initial infection or progression to the biofilm mode of growth.
Ohio State University Wexner Medical Center Division of Infectious Diseases
Ohio State University Wexner Medical Center Division of Infectious Diseases