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Bacillus subtilis expressing a haemolysin gene from Listeria monocytogenes can grow in mammalian cells
J Bielecki1, P Youngman, P Connelly
1Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6076.
Abstract:
Intracellular parasites can be classified into those that reside within a host vacuole and those which grow directly in the host cytoplasm. Members of the latter group, which includes Rickettsia, Shigellae, Trypanosoma cruzi, and Listeria monocytogenes, possess haemolytic activity associated with the ability to enter the host cytoplasm. Therefore mutants of L. monocytogenes lacking a pore-forming haemolysin, listeriolysin O, do not escape from the endosomal compartment and consequently fail to become established in the cytoplasm. To examine the role of listeriolysin O, we cloned the structural gene for the L. monocytogenes haemolysin, hlyA, into an asporogenic mutant of Bacillus subtilis under the control of an IPTG-inducible promoter. After being internalized by the macrophage-like cell line J774, haemolytic B. subtilis disrupted the phagosomal membrane and grew rapidly within the macrophage cytoplasm. These results show that a single gene product is sufficient to convert a common soil bacterium into a parasite that can grow in the cytoplasm of a mammalian cell.
Insights
A single bacterial gene, listeriolysin O, enables bacteria to escape host cell vacuoles and grow in the cytoplasm. This finding demonstrates how a common soil bacterium can become an intracellular parasite.
Area of Science:
- Microbiology
- Cell Biology
- Genetics
Background:
- Intracellular parasites are categorized by their location within host cells: either in vacuoles or the cytoplasm.
- Cytoplasmic-dwelling parasites like Listeria monocytogenes possess hemolytic activity, crucial for escaping the endosomal compartment.
- Mutants of L. monocytogenes lacking listeriolysin O (LLO) are impaired in cytoplasmic invasion.
Purpose of the Study:
- To investigate the role of listeriolysin O (LLO) in bacterial intracellular parasitism.
- To determine if LLO alone is sufficient for a bacterium to invade and replicate within the host cell cytoplasm.
Main Methods:
- The listeriolysin O gene (hlyA) from L. monocytogenes was cloned into Bacillus subtilis.
- The gene was placed under an IPTG-inducible promoter in an asporogenic B. subtilis strain.
- The modified B. subtilis was internalized by J774 macrophage-like cells to observe its behavior.
Main Results:
- Haemolytic B. subtilis, expressing LLO, successfully disrupted the phagosomal membrane after internalization by macrophages.
- These bacteria exhibited rapid growth within the cytoplasm of the host cells.
- The study confirmed that LLO facilitates escape from the endosome into the host cytoplasm.
Conclusions:
- A single gene product, listeriolysin O, is sufficient to confer the ability of intracellular parasitism.
- This research demonstrates the potential for converting a non-pathogenic bacterium into a cytoplasmic parasite by introducing a single virulence factor.
- The findings highlight the critical role of pore-forming haemolysins in bacterial invasion and intracellular survival strategies.