Expression of a peptide called RpoN* in antibiotic-resistant Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients made the bacteria more susceptible to antibiotics and reduced virulence in a roundworm infection model, a study has found.
The study, “Blocking RpoN reduces virulence of Pseudomonas aeruginosa isolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain,” was published in the journal Nature Scientific Reports.
Pseudomonas aeruginosa is an opportunistic bacterial strain, and the most common pathogen found in CF patients. The ability of P. aeruginosa to produce biofilm (layers of bacteria sticking to surfaces and other bacteria) can protect the bacteria from antibiotics and the host’s immune response.
RNA polymerase, nitrogen-limitation N (RpoN) is a sigma factor — a protein needed to start bacterial DNA transcription (the process of copying a gene’s DNA sequence to make an RNA molecule for protein production) — and regulates bacterial motility, biofilm formation, and other virulent (pathogen-linked harmful) factors. RpoN is also linked to antibiotic resistance.
Researchers developed a so-called “molecular roadblock” in the form of a peptide (RpoN*), which can block DNA transcription controlled by RpoN and other factors.
Although previous studies showed that RpoN* expression reduced the virulence of laboratory strains of P. aeruginosa, its effect is not known in clinical isolates of the bacteria. The difference is critical, as clinical isolates from patients may encompass different genetic and observable backgrounds.
The researchers evaluated the effect of RpoN* expression on 12 different P. aeruginosa strains isolated from patients with CF, especially regarding virulence and antibiotic susceptibility. Four laboratory bacterial strains were used as controls.
The features of the bacterial strains were either tested in culture plates (in vitro), or using live models (in vivo).
Researchers first tested the different strains’ motility of flagella (a structure used by bacteria to move) and pili (surface structures used to transfer genetic material between bacteria), and formation of biofilms.
Assay results showed that four of the strains were motile based on flagella movement, and that most strains had motile pili and produced moderate biofilms.
The team next assessed the pathogenic (disease-causing) effect of the bacterial strains in a P. aeruginosa-C. elegans infection model by looking at the survival of C. elegans — a transparent roundworm commonly used in research. They found that the pathogenic effect varied among the different strains.
To further assess changes in motility and biofilm formation, the researchers induced RpoN* expression in four of the 12 isolates.
They found that expression of RpoN* significantly reduced flagellar motility in all four isolates. When assessing pili motility, they found it was significantly lower in three of the four isolates. Additionally, RpoN* expression significantly reduced biofilm formation in two of the isolates.
These results indicated that RpoN* reduced the virulence caused by P. aeruginosa isolates derived from CF patients.
Finally, the researchers investigated the protective features of RpoN* expression in P. aeruginosa-C. elegans infection models. While the control isolate without RpoN* expression killed about 80% of C. elegans, RpoN* expression resulted in significant survival of the roundworm.
These results suggested that RpoN* expression reduced the pathogenic effect of the bacteria in vivo.
Following the protective feature of RpoN* expression on C. elegans survival, the researchers also found that expression of the molecular roadblock resulted in more susceptibility to several antibiotics — RpoN* increased the bacterial sensitivity to several of the tested antibiotics by two to four times compared with controls not expressing the peptide.
The antibiotics with improved susceptibility included cefotaxime, cefepime, ceftazidime, piperacillin, and imipenem.
Although RpoN* is currently only a tool to study antimicrobial development, similar peptides could be used to reduce bacterial pathogenesis, according to the researchers.
“Finding a small molecule or stapled-peptide that works in the same … manner as RpoN* would be an effective, clinically relevant strategy to combat P. aeruginosa virulence and antibiotic resistance,” they concluded.
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