Revertant Listeria monocytogenes and conditions for their persistence as intracellular L-forms

Abstract

L-forms are cell wall deficient forms of bacteria that are able to survive and multiply without a rigid cell wall. Since their discovery in 1935, numerous case studies have reported an observation or isolation of such bacterial L-forms in diseased patients or animals. They have largely been associated with in chronic or recurrent diseases, where L-forms are believed to represent a strategy that allows the bacteria to escape from eradication by the host immune system and/or antibiotic therapy in the clinical setting. However, hitherto no study has definitively established the role of the L-form state in diseases nor was the occurrence of L-forms undoubtedly demonstrated and quantified. This is in particular true for the hypothesised relevance of L-forms for relapsing infectious diseases. Several cases of recurrent listeriosis have been reported so far. Sub-typing analyses suggested that the identical Listeria monocytogenes strain persisted in the patient during these cases. In addition, Listeria L-forms have been reported to be isolated from the cerebrospinal fluid of a patient and from sheep diagnosed with listeriosis. Therefore, Listeria monocytogenes L-forms are assumed to be induced, or selected, by the antibiotic therapy and to be responsible for the persistence of the pathogen although antibiotic treatment. After termination of the treatment, L-forms are hypothesised to revert to walled state by rebuilding their cell wall. However, the triggers for this reversion back to a walled state is still not known. With the reestablishment of the cell wall, the bacteria regain their virulence properties that confer the bacteria the possibility to restart the infection process. The aim of this thesis is to investigate whether antibiotic treatment might promote the development of L-forms inside previously infected non-professional phagocytes and whether the occurring L-forms can survive and persist inside the host cells. To be able to achieve this major goal, the first step was to generate transient L-form strains, called revertant strains, that have the capability to switch back and forth between the L-form and the walled state. By adapting previously established protocols it was possible to generate revertant strains that were not impaired in growth or pathogenicity compared to their wild-type strains. Thus, the revertant strains turned out to be suited for the investigation of the development of intracellular L-forms after infection and antibiotic treatment of host cells in cell culture experiments. Confocal laser scanning microscopy analysis of cell cultures infected with L. monocytogenes revertant strains expressing Green Fluorescent Protein demonstrated the development of intracellular L. monocytogenes L-forms after an antibiotic treatment with Ampicillin. The presence and identification of L. monocytogenes L-forms was further confirmed by fluorescence in situ hybridisation using oligonucleotide probes specific for Listeria genus which allowed visualisation of intracellular L-forms in situ. In order to quantify the intracellular conversion of rods to L-form, cell cultures showing intracellular L-forms were lysed and plated on suitable media. However, the results obtained with this method were not conclusive, as growth of L-forms or reverted walled bacteria from the cell culture lysate could not be observed. Validation of the viability of L-forms occurring inside the host cells was provided by extraction of L- forms using a micro-manipulator and subsequent growth of the extracted L-forms in vitro. In conclusion, the results from this study demonstrated the development of Listeria monocytogenes L- forms inside infected eukaryotic host cells by an antibiotic treatment of the cell cultures with Ampicillin. In addition, it was proved that Listeria L-forms can survive and persist intracellularly for a couple of weeks in eukaryotic host cells. These are important findings that provide evidence for he potential role of L-forms of Listeria monocytogenes in recurrent listeriosis.