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RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells
Published on: June 4, 2016
A novel promoter trap identifies Listeria monocytogenes promoters expressed at a low pH within the macrophage
Helena M Stack1, Cormac G M Gahan, Colin Hill
1Department of Microbiology and Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland.
Abstract:
The Gram-positive pathogen Listeria monocytogenes encounters acid environments in low-pH foods, during passage through the stomach and within the macrophage phagosome during systemic infection. A novel promoter-trap system termed pGAD-HLY was developed, based on a plasmid containing a promoterless copy of gadB (required for survival at low pH) and hly (whose product facilitates escape from the macrophage phagosome) to identify loci that are induced under different stress conditions in vitro as well as identifying in vivo inducible promoters expressed during intracellular infection. This system facilitated the identification of 11 acid-inducible genes in L. monocytogenes. Transcriptional analysis and acid tolerance response assays confirmed the low-pH induction of these loci, validating this promoter-trap system. Macrophage assays revealed the phagosomal induction of three clones, corresponding to lmo0095, lmo2565 and lmo2371, with two of these clones (lmo0095 and lmo2565) also being induced during murine infection. However, virulence studies did not show any significant difference between strains carrying insertional mutations in these genes and the wild type strain. Although the loci that were identified by this screening procedure do not appear to be central to listerial pathogenesis, it is evident from studies that they contribute to the 'fitness' of this pathogen in adverse acid conditions.
Insights
A new promoter-trap system identified 11 acid-inducible genes in Listeria monocytogenes. While not central to pathogenesis, these genes enhance bacterial fitness in acidic environments.
Area of Science:
- Microbiology
- Pathogen Biology
- Bacterial Genetics
Background:
- Listeria monocytogenes, a Gram-positive pathogen, faces acid stress in foods, the stomach, and macrophage phagosomes.
- Understanding acid-inducible genes is crucial for controlling L. monocytogenes infections.
Purpose of the Study:
- To develop a novel promoter-trap system (pGAD-HLY) for identifying stress- and in vivo-inducible genes in L. monocytogenes.
- To characterize genes induced by low pH and during intracellular infection.
Main Methods:
- Development of the pGAD-HLY promoter-trap system using gadB and hly genes.
- Screening for acid-inducible and phagosome-inducible loci in L. monocytogenes.
- Transcriptional analysis, acid tolerance assays, and macrophage infection models.
Main Results:
- The pGAD-HLY system identified 11 acid-inducible genes.
- Three genes (lmo0095, lmo2565, lmo2371) were induced in phagosomes; two (lmo0095, lmo2565) were also induced during murine infection.
- Mutations in these genes did not significantly affect virulence but contributed to bacterial fitness in acid.
Conclusions:
- The pGAD-HLY system is effective for identifying stress-inducible genes in L. monocytogenes.
- Identified genes contribute to bacterial fitness under acid stress, though not essential for pathogenesis.
- Further research into these fitness factors could offer novel control strategies.
