cystic fibrosis caused by bacteria :: Article CreatorA Lung Pathogen's Dilemma: Infect Or Resist Antibiotics?
Imagine trying to settle into a new home while constantly being attacked. That's what the bacterium Pseudomonas aeruginosa faces when it infects the lungs, and it can't both spread and protect itself from antibiotics at the same time. Nonetheless, it's one of the top culprits in hospital-acquired infections and it's notorious for causing long-lasting, antibiotic-resistant infections, causing damage especially in people with lung diseases like cystic fibrosis, COPD, or bronchiectasis.
To survive tough conditions, P. Aeruginosa forms colonies known as "biofilms" -- clusters of bacteria encased in a self-produced matrix that provides them with significant advantages, including protection from antibiotics.
But biofilms come at a cost: the clustered bacteria also lose the ability to move around, find nutrients, and spread effectively. For P. Aeruginosa infecting a lung, this poses a dilemma: should it spread across the lung's surface or bunker down to resist incoming antibiotics? Achieving the right balance can mean life and death for the pathogen -- and disrupting it can mean life or death for patients.
New research by the group of Alexandre Persat at EPFL's Global Health Institute has now uncovered how P. Aeruginosa manages the trade-off between colonizing and surviving during infection by switching between biofilm formation for antibiotic protection and a more mobile, "planktonic" state to spread and access nutrients, depending on the environmental pressures they face. The study is published in Nature Microbiology.
Mimicking natural infection environments to observe the bacteria
To better understand P. Aeruginosa's behavior, the researchers grew the bacteria on mucus-covered tissue models that mimic human lungs. These tissue models, known as "organoids," are at the cutting-edge of bioengineering.
"We then used a high throughput screening technique called transposon-insertion sequencing (Tn-seq), combined with metabolic modeling and live imaging, to study how P. Aeruginosa adapts to colonize the mucosal surface of the lung and tolerate antibiotic treatment," says Lucas Meirelles, who led the study.
Thanks to the Tn-seq technique, the scientists identified which genes were important for the bacterium's survival under different conditions: those which contributed to fitness during mucosal colonization and those which helped the bacteria tolerate antibiotics.
The scientists also used computational modeling to simulate how the bacteria metabolize nutrients in the lung environment, which helped pinpoint the exact metabolic pathways P. Aeruginosa relies on during infection.
Finding the right balance
The study found that P. Aeruginosa adapts to the lung's mucus by relying on sugars and lactate, nutrients that are abundant in infected lungs. However, to survive on the mucus, the bacterium also needs to synthesize essential but less available nutrients, like amino acids. This self-sufficiency, or "metabolic independence," helps the bacterium thrive in the early stages of lung infection.
What Persat's team uncovered is the mechanism behind this dilemma. They found that biofilm formation imposes a "metabolic burden," meaning that producing the sticky matrix that holds the biofilm together consumes resources, slowing down the bacteria's ability to spread. In experiments, bacteria that couldn't form biofilms spread more efficiently but were left vulnerable to antibiotics. This new insight into the metabolic costs of biofilm formation explains how the bacterium balances growth and antibiotic tolerance.
The study highlights the delicate balancing act that Pseudomonas aeruginosa must perform during infections. While the bacteria need to colonize the lung effectively, their best survival strategy -- forming biofilms -- limits their access to nutrients and, therefore, their ability to spread. However, once antibiotics are introduced, biofilm formation becomes advantageous, protecting the bacteria from being wiped out.
Exploring new paths
The discovery opens the door for the exploration of new treatment strategies: if we can find a way to disrupt the bacteria's ability to form biofilms without giving them more room to spread, it could make them more vulnerable to existing treatments. And therapies that target the bacteria's metabolic pathways may also prove to be effective at weakening Pseudomonas infections.
More broadly, the scientists believe that studying pathogens like P. Aeruginosa in infection models that replicate the physiology of human tissues is crucial for combating antibiotic resistance.
"Antibiotic resistance is set to become one of the most serious healthcare challenges of this century, and P. Aeruginosa is a major contributor to this issue," says Meirelles. "By using tissue-engineering to replicate the airway environment in the lab, we aim to better understand the physiology of this pathogen. Our hope is that this will uncover previously unknown targets to help us combat these infections and address antibiotic resistance."
Structural Biology Analysis Of A Pseudomonas Bacterial Virus Reveals A Genome Ejection Motor
The viruses that infect bacteria are the most abundant biological entities on the planet. For example, a recent simple study of 92 showerheads and 36 toothbrushes from American bathrooms found more than 600 types of bacterial viruses, commonly called bacteriophages or phages. A teaspoon of coastal seawater has about 50 million phages.
While largely unnoticed, phages do not harm humans. On the contrary, these viruses are gaining increasing popularity as biomedicines to eradicate pathogenic bacteria, especially those associated with antibiotic-resistant infections.
In a study published in the journal Nature Communications, Gino Cingolani, Ph.D., of the University of Alabama at Birmingham, and Federica Briani, Ph.D., of the Università degli Studi di Milano, Milan, Italy, have described the full molecular structure of the phage DEV. DEV infects and lyses Pseudomonas aeruginosa bacteria, an opportunistic pathogen in cystic fibrosis and other diseases. DEV is part of an experimental phage cocktail developed to eradicate P. Aeruginosa infection in pre-clinical studies.
A peculiar feature of DEV is the presence of a 3,398-amino acid virion-associated RNA polymerase inside the capsid expelled into the bacterium upon infection. Unexpectedly, Cingolani and Briani's study revealed the virion-associated RNA polymerase is part of a genome ejection motor that pulls the DNA of the phage out of its head after the phage has attached to the surface of a Pseudomonas bacteria using its tail fibers and has penetrated the cell's outer and inner membranes using its tail tube.
"We posit that the design principles of the DEV ejection apparatus are conserved in all Schitoviridae phages," Cingolani said. "As of October 2024, over 220 Schitoviridae genomes have been sequenced and are available in the public database. As these genomes are largely unannotated and many open-reading frames have unknown functions, our work paves the way for the facile identification of structural components when a new Schitoviridae phage is discovered."
The Schitoviridae family of phages "represents some of biology's most understudied bacterial viruses, increasingly utilized in phage therapy," Cingolani said. "We are using structural biology to decipher the building blocks and map gene products. This is vital when the amino acid sequence evolves too rapidly for conventional phylogenetic analysis."
The researchers used cryo-electron microscopy localized reconstruction, biochemical methods and genetic knockouts to describe the complete molecular architecture of DEV, whose DNA genome has 91 open-reading frames that include the giant virion-associated RNA polymerase. "This vRNAP is part of a three-gene operon conserved all Schitoviridae genomes we analyzed," Cingolani said. "We propose these three proteins are ejected into the host to form a genome ejection motor spanning the cell envelope."
The structure of DEV and many other phages resembles a minuscule version of Neil Armstrong's 1969 lunar lander, with a large head, or capsid, that contains the genome and leg-like fibers supporting the phage as it lands on the surface of bacteria, preparing to infect the living bacterial cell.
The researchers determined structures of all the protein capsid factors and tail components in DEV involved in host attachment. Through genetic experiments, they showed that the DEV long tail fibers were essential for infection of P. Aeruginosa but were not needed to infect P. Aeruginosa mutants whose surface lipopolysaccharide lacked the O-antigen. In general, viruses attach to different cell surface molecules as the first step of infection.
While this study provides several still images of the phage structure, the researchers do not completely understand the movie of DEV infection. They envision three steps in that infection process.
In step one, as a single DEV phage drifts in isolation, its flexible long tail fibers fluctuate to improve the chance of touching a Pseudomonas lipopolysaccharide surface molecule. After the first touch, all five fibers attach to tether the phage perpendicularly close to the bacterial outer surface.
In step two, the short tail fiber, which also acts as a tail plug, touches a secondary receptor on the Pseudomonas and a mechanical signal releases the tail plug.
Up to this point, the three proteins called gp73, gp72 and gp71 have been stored inside the phage head near its tail, with shapes that will dramatically change when they exit the phage head. In step three, when the plug is gone, the three proteins are expelled out of the head and into the bacterial cell envelope. The lead protein, gp73, refolds its shape to form an outer membrane pore with a hollow center. Below that, gp72 refolds into a hollow tube that spans the Pseudomonas periplasm, the space between the bacteria's outer membrane and its inner membrane. Finally, gp71 crosses the inner membrane and refolds into a large RNA polymerase motor in the bacterial cytoplasm that pulls the phage DNA through the hollow gp73 and gp72 channels and into the Pseudomonas cell.
Cingolani, a professor in the Department of Biochemistry and Molecular Genetics, recently came to UAB to head the new Center for Integrative Structural Biology, approved by the University of Alabama System Board of Trustees this summer. The center will help UAB researchers study the three-dimensional structures of biological macromolecules, such as proteins and nucleic acids, to decipher their function and mechanisms of action.
Integrative structural biology seeks to visualize a complete movie of how macromolecules function, using multiple methods to view molecular structures and how they interact with each other. The primary focus of the UAB Center for Integrative Structural Biology will be studying biological problems related to infection, inflammation, immunity, cancer and neurodegeneration.
Haemophilus Influenzae In Adults With Cystic Fibrosis
Photo Credit: Kateryna Kon
The following is a summary of "Clinical epidemiology and impact of Haemophilus influenzae airway infections in adults with cystic fibrosis," published in the October 2024 issue of Infectious Disease by Weyant et al.
Haemophilus influenzae is prevalent in the airways of persons with cystic fibrosis (pwCF), and its association with pulmonary exacerbations (PEx), yields a decline in lung function.
Researchers conducted a retrospective study to examine the prevalence, natural history, and clinical impact of H. Influenzae in adults pwCF.
They reviewed all adults pwCF who had positive sputum cultures for H influenzae between 2002 and 2016. Persistently individuals infected (≥2 samples with the same pulsotype and > 50% sputum culture-positive for H. Influenzae each year) were matched 1:2 with controls without H. Influenzae. Demographic and clinical data, baseline and during PEx, were collected at each H. Influenzae-positive visit. Pulsed-field gel electrophoresis (PFGE) was used annually on biobank isolates to assess genetic relatedness.
The results showed that over the study period, 30% (n = 70/240) of adults pwCF were culture-positive for H. Influenzae, with 54% (n = 38) testing positive multiple times and 17% (n = 12) having a persistent infection, PFGE on 137 isolates revealed 94 unique pulsotypes; 1.5% (n = 2) were serotype f, while the remaining 98.5% were non-typeable. H. Influenzae isolation increased the risk of PEx (RR = 1.61 [1.14–2.27], P = 0.006), though this association disappeared for those who only produced sputum during PEx and annual lung function decline showed no difference.
Investigators concluded that H. Influenzae infection was common but transient among adult pwCF and was not associated with acute PEx decline.
Source: bmcinfectdis.Biomedcentral.Com/articles/10.1186/s12879-024-10050-7
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