The Dodging Biofilms of P. aeruginosa in Cystic Fibrosis

S U Vandhana, School of Biosciences and Technology, VIT-Vellore

Introduction

Cystic fibrosis (CF) is a notable example of a chronic disease whose manifestations are visible as dysfunctional, hyperinflammatory immune response with chronic pulmonary infections creating a huge catastrophe to the airways. This leads to substantial morbidity accompanied by shortened life span. Being an opportunistic pathogen, Pseudomonas aeruginosa has a profound impact on the lungs of CF patients by decelerating pulmonary function. It is also known to exhibit a demarcating resistance to antibiotics, and the innate immunity of the host. This is made possible by the expression of certain virulence factors (such as exopolysaccharides and antioxidants) as well as through adaptive immunity acquired by the bacterium during chronic infections. 

Understanding Cystic Fibrosis

While analyzing at the genetic level, Cystic Fibrosis is the result of a mutation in a gene that encodes for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). CFTR is a transport channel of chloride and bicarbonate ions that aim at maintaining osmotic balance across the epithelial surfaces in our body. The inheritance pattern of CFTR mutations is autosomal recessive. A rough estimation of 2,000 disease-causing mutations has been discovered to the present date, paving way for further categorization of the mutations into six classes. However, the most common mutation is the deletion of Phenylalanine at the 508th position. This is observed in almost two-thirds of the patients suffering from Cystic Fibrosis. CFTR dysfunction causes deregulation of the transport of chloride and bicarbonate ions across epithelial cells. This leads to multiple organ dysfunction and the major clinical manifestations include increased chloride content of the sweat (major diagnostic test-sweat chloride test), meconium ileus, distal intestinal obstruction, pancreatic problems, growth-related disorders, infertility in males along with chronic and recurrent pulmonary infections. Long-term manifestations also include diabetes and mood disorders like depression and anxiety. 

Patients suffering from Cystic fibrosis exhibit permanent changes to the architecture of the lungs within the first five years of life. CF patients also start displaying air trapping, mucus obstruction, and bronchiectasis (enduring enlargement of the airways) which are revealed in the reports from Computed Tomography (CT scans). Other significant manifestations of the disease include elevated inflammatory markers and the presence of polymicrobial populations such as Staphylococcus aureus, Pseudomonas aeruginosa, etc. 

P. aeruginosa as key players

Pseudomonas aeruginosa is a gram-negative aerobic or facultative anaerobic bacterium commonly found in soil and aquatic environments. It is an oxidase-positive and a non-lactose fermenting bacterium. Through genomic analysis, it is also proved that the bacterium exhibits a greater capacity for horizontal gene transfer through transformation, transduction, and conjugation. The bacterium also exhibits three forms of motility: flagellum-dependent swimming, flagellum- and pilus-dependent swarming, and pilus-dependent twitching. The bacterium has been described by Gessard in 1882, to play a key role in various acute and chronic infections by functioning as an opportunistic pathogen. The bacterium is also proposed to be the causative organism of several diseases like bacteremia or endocarditis, skin, and soft tissue infections, urinary tract infections, gastrointestinal infections, meningitis, ocular infections, ear infections, ventilator-associated pneumonia, etc. Recent studies have revealed that this bacterium poses a greater threat by manifesting diseases through multi-drug resistant strains, making treatment options problematic. Some of the virulence factors expressed by the bacterium include lipases, proteases, rhamnolipids, pyocyanin, quorum sensing molecules, catalases, exopolysaccharides, etc. Studies have also proven that patients with inherited immune disorders such as Leukocyte Adhesion Disease (LAD) and specific granule deficiency (both of which contribute to decreased neutrophil functions) are highly susceptible to infections by P. aeruginosa. 

The chronic manifestation of the pulmonary infection by P. aeruginosa in patients with cystic fibrosis is characterized by downregulation of some virulence factors, increased biofilm formation, and upregulation of exopolysaccharide expression. Among these, biofilm formation is gaining widespread importance, which is characterized by a sessile, multicellular lifestyle as opposed to planktonic characteristics. Bacterial communities are seen as microcolonies or aggregation in intimate contact with airway epithelial cells surrounded by neutrophils in biofilms. These biofilms also exhibit a notable resistance to antibiotics as well as innate immune effectors. Resistance to antibiotics has laid importance on the use of combination therapy to conquer the heterogeneous bacterial population of the biofilms. Additionally, P. aeruginosa biofilms exhibit reduced complement activation compared to planktonic cultures. The interaction of neutrophils with these biofilms results in their immobilization of the upper layers of the microcolonies, thereby leading to the inability to clear the bacterial infection. The accumulation of neutrophils also results in NET formation and their components (including DNA) are unable to trap or kill the bacteria. Instead, they get embedded into the extracellular polymeric substances (EPS) of the biofilms themselves. This creates a complex mixture of substances present in the biofilms, thereby creating a greater challenge to therapeutic options. 

The biofilm formation by P. aeruginosa enables the bacterium to sustain harsh conditions while establishing chronic infection. The EPS matrix constitutes 90% of the biofilm, which is produced by bacteria that accounts for less than 10% of the biofilm biomass. The EPS matrix consists of polysaccharides, proteins, extracellular DNA, and lipids. The matrix allows a synergistic functioning between the microbes as a community by maintaining close contact via intercellular communication pathways and sharing of group resources. These biofilms are also regulated by a property known as Quorum Sensing (QS) and small RNAs. Quorum sensing allows bacteria to coordinate transcription of genes and group activity in response to population density. P. aeruginosa displays two primary interconnected quorum sensing systems namely the Las system and Rhl system that depend on Acyl-Homoserine Lactone (AHL) signaling molecules. Nevertheless, two additional QS systems do not involve acyl-homoserine molecules. These include the Pseudomonas quinolone signal system and the integrated quorum-sensing system. Quorum sensing in biofilms is the major driving force for the formation of essential biofilm components such as extracellular DNA.

A Ray of Hope in Therapeutics

Modern-day advancements in molecular and analytical techniques have been an eye-opener for devising many therapeutic options for Cystic Fibrosis. Some of the therapeutic strategies include:

1. Biofilm adhesion inhibition by the engineering of artificial devices. These are designed to be more resistant to bacterial attachment and the development of biofilms.
2. Inhibition of Quorum sensing. This is made possible by prohibiting bacterial quorum sensing signaling.
3. Quorum quenching by enzymatic degradation of AHL quorum sensing molecules (lactonases, acylases).
4. Augmentation of host response by enzymatic inactivation of AHL molecules along with the trigger of innate immune effectors (anti-biofilm response) in the host.
5. Inhibition of c-di-GMP signaling. This will reduce the formation of biofilms and promote dispersal favoring the planktonic state of bacteria. 
6. Production of peptides and molecules with anti-biofilm activity. These compounds have direct anti-bacterial activity as well as a considerate activity against extracellular polymeric substances (EPS).

Recent years have witnessed tremendous growth in the understanding of biofilms from P. aeruginosa. Novel therapies and strategies are being developed in full swing to combat the multi-drug resistant strains of the bacterium in the biofilms. Strategies that are presently under investigation include direct blocking of biofilm attachment and development and promotion of their dispersal and degradation, thereby maintaining the planktonic state. It is noteworthy that targeting bacteria with antibiotics in their planktonic state is far easier compared to that of biofilms. Biofilms not only induce a higher rate of off-target effects of antibiotics but also the increased dose of combinational antibiotic therapy leaves the patients at a greater risk for adverse side effects. There have also been several cases of relapse of infections due to biofilm formation with the major reason attributed to a heterogeneous population of bacteria in the biofilms (aerobes and anaerobes, etc.). 

Future-prospects

In recent years, scientists have been ardently working to devise an effective treatment against biofilms and their efforts have paved the way for a better understanding of these phenomena. There have been several hypotheses and theses. Many are at several stages of clinical trials and there have also been many treatment options in current use. As a concluding note, it is wise to remember that no treatment option is the final step in the complete eradication of these infections. A regular update of techniques and understanding is required to tackle any further disastrous manifestations of any bacterial disease.

Also read: CRISPR-Cas9 as an advance tool for eliminating HIV/AIDS

Sources:

1. Malhotra, S., Hayes, D., Jr, & Wozniak, D. J. (2019). Cystic Fibrosis and Pseudomonas aeruginosa: the Host-Microbe Interface. Clinical microbiology reviews, 32(3), e00138-18. https://doi.org/10.1128/CMR.00138-18
2. Maurice, N. M., Bedi, B., & Sadikot, R. T. (2018). Pseudomonas aeruginosa Biofilms: Host Response and Clinical Implications in Lung Infections. American journal of respiratory cell and molecular biology, 58(4), 428–439. https://doi.org/10.1165/rcmb.2017-0321TR

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