Identification and characterisation of genes involved in Pseudomonas aeruginosa L-form biogenesis

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𝘗𝘴𝘦𝘶𝘥𝘰𝘮𝘰𝘯𝘢𝘴 𝘢𝘦𝘳𝘶𝘨𝘪𝘯𝘰𝘴𝘢 is an opportunistic pathogen and a common cause of nosocomial infections. It is resistant to most antimicrobial agents including many of the last resort antibiotics. It is able to survive high concentrations of β-lactam antibiotics by undergoing a rapid and reversible 𝘦𝘯 𝘮𝘢𝘴𝘴𝘦 transition from normal rod-shaped cells to viable, cell wall-deficient (CWD) cells. The aims of this study were to examine the proliferation mechanisms of meropenem induced 𝘗. 𝘢𝘦𝘳𝘶𝘨𝘪𝘯𝘰𝘴𝘢 CWD cells, to identify and characterise the genes involved in reversible 𝘗. 𝘢𝘦𝘳𝘶𝘨𝘪𝘯𝘰𝘴𝘢 L-form lifestyle and to investigate the effectiveness of synthetic derivatives of LL-37 peptide on meropenem induced L-forms and bacillary cells. Here I demonstrated that CWD 𝘗. 𝘢𝘦𝘳𝘶𝘨𝘪𝘯𝘰𝘴𝘢 not only survives but proliferates in high concentrations of β-lactam antibiotics (meropenem) via mechanisms consistent with those described for the proliferation of L-form bacteria. Remarkably, upon transfer to media without antibiotic, the progeny of these L-form cells rapidly revert back to bacillary cells that have elevated resistance to the antibiotic. These findings indicate that the ability to transition to unstable L-forms enables 𝘗. 𝘢𝘦𝘳𝘶𝘨𝘪𝘯𝘰𝘴𝘢 to survive, proliferate and adapt to β-lactam antibiotics. To investigate the molecular mechanisms underlying β-lactam tolerance in 𝘗. 𝘢𝘦𝘳𝘶𝘨𝘪𝘯𝘰𝘴𝘢, a comprehensive nonredundant mutant library was screened for mutants that are unable to survive or recover from meropenem treatment through reversible transition to the spherical cell morphotype. Using this approach we identified a total of 76 genes whose inactivation caused clear meropenem tolerance defects arising from specific deficiencies in spherical cell formation, survival or transition back to the bacillary form after treatment. The majority of these genes involved in lipid, biotin, and flagella synthesis. The genes identified in this study not only provide important insights into the biology underlying β-lactam tolerance in 𝘗. 𝘢𝘦𝘳𝘶𝘨𝘪𝘯𝘰𝘴𝘢, but may also inform future drug development efforts to selectively target the spherical cell morphotype. In an attempt to explore new active antimicrobial agents against 𝘗. 𝘢𝘦𝘳𝘶𝘨𝘪𝘯𝘰𝘴𝘢, I have investigated the effectiveness of six LL-37 derivatives on meropenem induced L-forms and bacillary cells of 𝘗. 𝘢𝘦𝘳𝘶𝘨𝘪𝘯𝘰𝘴𝘢. L-form cells were found to be highly susceptible to killing by LL-37 derivatives. Combinations of meropenem with all tested LL-37 derivatives also exhibited a significant activity against bacillary cells. Remarkably, the LL-37 derivatives Bob5 and Bob6 were extremely efficient in killing 100% of bacillary cells within the first two hours of treatment. Synthetic LL-37 analogues show great promise as effective antimicrobial agents against 𝘗. 𝘢𝘦𝘳𝘶𝘨𝘪𝘯𝘰𝘴𝘢.
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