Auxotrophic mutants may be defective for intracellular growth however none seem to have been isolated, a point mentioned by Sun et al. A study by Marquis et al (1993) investigated whether there was a difference in intracellular growth in auxotrophic mutants. A culture of L. monocytogenes was mutagenised using Tn917-LTV3 and auxotrophic mutants were selected for by their lack of growth on a defined minimal medium, Welshimer’s medium.
These mutants were used to infect L2 cells, and LD50 in mice was established. Most of the mutants grew ‘normally’ and were fully virulent in mice. Those requiring three aromatic amino acids (phenylalanine, tryptophan and tyrosine) or adenine were less virulent. This study concluded that the cytoplasm of the host must be a rich nutrient source that L. monocytogenes has been able to take advantage of because of its requirement to grow intracellularly.
Most of the studies mentioned above point to the importance of LLO in intracellular growth in that LLO mutants fail to escape the phagolysosome. The investigation of subsequent steps in LLO mutants has been prevented because of this fact, therefore LLO may have other roles in cell-to-cell spread. This theory was tested by Gedde et al (2000) when the authors found that LLO binds to LLO mutant bacteria. The addition of bound LLO to these mutants (also being defective for two PLCs) rendered the bacteria able to escape from the phagolysosome, form a pseudopod and infect the neighbouring cell. Further cell-to-cell spread does not take place because the bacteria cannot escape from the double membrane that forms. Thus it can be said that LLO and PLCs are not required for cell-to-cell spread, but are for escaping mebraned-vacuoles.
All of the above studies are relevant to this project as they either describe methods that shall be used (Sun et al and Camilli et al), provide information on mutants that I may or may not find (Sun et al, Barry et al and Gedde et al), or state other methods of mutagenesis (Hodgson and Autret). The aims of this project are to mutagenise a culture of L. monocytogenes using ultra violet light and nitrosoguanidine and subject it to in vivo ampicillin enrichment, thus aiming to isolate intracellular mutants caused by point mutations. Due to time limitations, the methods stated in an investigation very similar to this project previously carried out by a summer student, Goddard (2001), are closely followed. Other aspects described in the above mentioned studies will also be incorporated.
A culture of L. monocytogenes was subjected to UV and NTG mutagenesis that brought about approximately 99.9% killing of the total cell population. This percentage was chosen because doses that are too high will not yield enough viable cells, and lower doses will not damage DNA sufficiently. An approximate assay to see if sufficient mutagenesis had occurred was carried out in which mutagenised cells were spread onto agar plates containing the antibiotic rifampicin. Rifampicin was chosen because resistance is generated by a simple point mutation in the target RNA polymerase and thus is likely to occur in UV and NTG mutagenesis.
The penicillin enrichment procedure described in Camilli et al was used in this project but using ampicillin instead, only because it was more readily available than penicillin. To establish an efficient ampicillin enrichment method, an initial enrichment of hly- cells from hly+ cells was carried out. The exact same method was then used to enrich for intracellular mutants from UV and NTG mutagenised cells.
Characterisation of intracellular mutants involved infecting the mouse fibroblast cell line, L2, and looking for abnormal plaques, as carried out by Sun et al and Marquis et al. Growth rates were also investigated, as generation of small plaques may just be due to mutations in essential genes, such as RNA polymerase, which would slow the growth, and thus decrease the normal plaque size.
MATERIALS AND METHODS
Bacterial strains and growth conditions. L. monocytogenes DP-L184 was the initial strain used in this study and the one from which mutants were derived. This strain is hemolysin positive (hly+). Another strain, DP-L476, was used in a practice ampicillin enrichment because it is hemolysin negative (hly-). The bacteria were grown in brain heart infusion (BHI) agar and broth, and blood plates consisted of Luria-Bertani agar and 5% sheep’s blood. Cultures were incubated overnight at 37oC in a roller-drum for those grown in broth, or just in an oven for that grown on agar.
Tissue culture cells and growth medium. The L2 cell line was used and maintained in Dubelco modified Eagle medium (DMEM) supplemented with 5% fetal calf serum (FCS), glutamine and gentamycin at a concentration of 1.125 ?g/ml.
Ultra violet light mutagenesis. This method was adapted from a second year mutagenesis experiment. First a 99.9% kill curve was estimated. A fresh culture of DP-L184 was washed and resuspended in 5 ml TM buffer (pH 7.5) in a glass petri dish. This inorganic buffer is used because broth absorbs radiation. The bacteria were held 15 cm away from the UV lamp and the dish was continually swirled whilst being exposed to short-wave UV.
The dish was exposed for 60 seconds with 300 ?l samples being withdrawn every 5 seconds. From these samples, 100??l was used to create a dilution series from 100 to 10-7, of which 50 ?l was spread onto BHI agar plates. A further 100 ?l from the time samples was used to inoculate BHI broth and grown overnight for any mutations to be expressed. Colonies from agar plates were counted, cell numbers per ml were calculated and the UV time sample with 99.9% killing was estimated to be 30 seconds. This value was later changed to 35 seconds for reasons explained in the discussion section.
Rifampicin resistance. Bacteria expressing UV mutations from all time samples were grown on plates containing rifampicin. To determine an effective rifampicin concentration to use in the agar plates, the minimum inhibitory concentration (MIC) was estimated. Thus, four 3 mm wells were cut out of a BHI agar plate with DP-L184 spread on to it. Varying concentrations of rifampicin were added to the wells. The plates were incubated and the zones of inhibition were measured. The appropriate concentration of rifampicin was added to BHI agar, which was 10?g/ml. 100 ?l from each of the mutation-expressing samples was spread onto the rifampicin BHI plates. After incubation, colonies were counted and compared with the results from the kill curve.
Nitrosoguanidine mutagenesis. 5 ml of an overnight culture of DP-L184 were washed twice with sodium citrate buffer (0.1M pH 7) and resuspended in 5 ml sodium citrate buffer with 400 ?g/ml NTG. This concentration was found to produce 99.4% killing in Goddard’s investigation. The bacteria were incubated in the rota-deck at 37oC for one hour. The cells were then washed twice with, and resuspended in, 5 ml warm BHI broth. From this, 100 ?l samples were used to (i) inoculate 10 ml BHI broth and; (ii) spread onto 100 ?g/ml rifampicin plates.
Ampicillin enrichment of hemolysin negative L. monocytogenes. 1 ml DP-L184 and 0.1 ml DP-L476, both grown in BHI broth, were added to 8.9 ml BHI broth and mixed. The bacteria were washed and resuspended in 5 ml PBS. A 200 ml flask of L2 cells was infected with the bacteria and incubated at 37oC for 30 minutes. The remaining supernatant was aspirated, the L2 cells were washed in 5ml PBS, then 10 ml DMEM with FCS and gentamycin was added and the cells were incubated for a further 30 minutes at 37oC.
This media was removed and 10 ml DMEM with FCS, gentamycin and ampicillin (at a concentration of 100 ?l/ml). The cells were incubated for 24 hours then washed with 5 ml PBS. L2 cells were hypotonically lysed by adding 10 ml sterile distilled water (SDW). The cells were extracted to a universal tube, centrifuged and resuspended in 1 ml PBS. A dilution series using these cells was set up (100 to 10-4) and 100 ?l were spread onto blood agar plates. Plates were incubated overnight at 37oC and numbers of hly+ and hly- were counted.
Enrichment of UV and NTG mutagenised L. monocytogenes. The bacteria exposed to 35 seconds UV light and NTG mutagenised bacteria that had grown in BHI broth were washed and resuspended in 5 ml PBS. The enrichment method used is exactly the same as that for hly- L. monocytogenes described previously. Any colonies that grew on the blood plates were examined, and those that might be mutants were picked, streaked onto separate blood and BHI agar plates and used to inoculate BHI broth.
Characterisation of intracellular mutants. The colonies that were picked following the enrichment process, as well as 9 colonies isolated previously by Goddard, were used to (i) establish whether the colonies isolated were in fact mutant strains by infecting L2 cells and examining any resultant plaques. To do this the colonies that were grown in BHI broth were washed and resuspended in 4 ml PBS.
They were added to a 60 mm petri dish of L2 cells and 4 ml DMEM with FCS was added. The petri dishes were incubated overnight at 37oC. Any plaques were examined for abnormalities. And to (ii) measure the growth rates of the putative mutants. 1 ml mutant L. monocytogenes was used to inoculate 10 ml BHI broth in a 25 ml side arm flask. A spectrophotometer with a wavelength of 540 nm was used to measure the absorbance of the cultures. A control culture of L. monocytogenes DP-L184 that had not been mutagenised was also used.
RESULTS
Ultra violet light kill curve. This method had to be repeated three times because of unsuitable killing the first two times. This could have been due to errors in the method, such as biased dilutions, cells sticking to eppindorfs in the dilution series and the fact that, during the first two attempts, the samples were not kept in the dark, thus the cells may have had a chance to repair any UV damage. The final kill curve can be seen in graph 1. The time at which 99.9% killing has occurred was worked out to be 30 seconds. The raw data for these results and the calculation of the 99.9% killing can be seen in Appendix I. Graph 1 shows a clear negative correlation between the number of cells per ml and exposure to UV light.
After 15 seconds the average number of cells per ml oscillates, but overall a decrease is seen. Rifampicin resistance in UV mutagenised cells. The number of mutagenised colonies that grew on 10 ?g/ml rifampicin BHI agar plates from each time sample can be seen in Table 1. There is an overall positive correlation between length of exposure to UV and number of colonies that have acquired resistance to rifampicin between 5 and 50 seconds.