Another method of mutagenesis is signature-tagged transposon mutagenesis (STM). In this method a transposon flanked by two unique oligopeptides is inserted randomly into the bacterial genome. The culture is pooled with half being used to infect a murine model. Bacteria are recovered from the animal, DNA is extracted, the unique oligopeptides are tagged and undergo PCR amplification. These then hybridise with the other half of the pooled mutants. Bacteria that are avirulent are unable to grow within the animal and thus cannot hybridise. These bacteria are easily identified.
Whilst this is an in vivo method that aims to identify novel virulence genes, it is possible that novel intracellular mutants may be found. Indeed an investigation using this method by Autret et al (2001) may have found bacteria that are defective for intracellular growth because of changes to cell wall components. However, whilst these mutants showed reduced growth in vitro, the growth in vivo was not as severely reduced.
As stated previously, intracellular mutants are being investigated for use as vaccines against virulent L. monocytogenes. Intracellular mutants, such as those identified by Sun et al, have been used to generate immunity against fully virulent L. monocytogenes in mice, as demonstrated in the study by Barry et al (1992). Fifty percent lethal dose assays (LD50) were carried out on mice using different classes of intracellular mutants. The mice were then challenged with fully virulent L. monocytogenes and relative levels of immune protection were calculated. This investigation found that some LLO production is required for immunity to be generated, mutants that did not produce LLO provided no protection. In other words, the bacteria must be able to escape the phagolysosome into the host cytoplasm for immunity to be induced. Other factors that were required for the induction of immunity are compromised phospholipase activity, intracellular growth and cell-to-cell spread. The fact that not all intracellular mutants induce immunity may tie in with the finding by Autret et al, which is usually the case with in vitro studies, in that characteristics exhibited by cells in vitro may not be the same as those exhibited in vivo.
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 μg/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 μl samples being withdrawn every 5 seconds. From these samples, 100 μl was used to create a dilution series from 100 to 10-7, of which 50 μl was spread onto BHI agar plates. A further 100 μl 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 10μg/ml. 100 μl 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 μg/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 μl samples were used to (i) inoculate 10 ml BHI broth and; (ii) spread onto 100 μg/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 μl/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 μl 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 μg/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.
The number of colonies that grew on the rifampicin plates between 20 and 60 seconds oscillates.
Rifampicin resistance in NTG mutagenised cells. The number of colonies that grew on this plate was 50.
Enrichment of hly- L. monocytogenes. The initial concentration of hly- compared to hly+ was 10%. After enrichment the concentration of hly- colonies rose to 69% (raw data and calculations can be seen in Appendix II).
Enrichment of intracellular mutants. A large number of colonies grew on the blood agar plates after enrichment, about 200 in all. Roughly equal numbers of colonies survived the enrichment process from each method of mutagenesis. They were all hly+ with normal hemolytic patterns. Two from each method of mutagenesis were randomly picked.
Characterisation of intracellular mutants. (i) I was unable to establish whether the colonies picked were in fact intracellular mutants because I was unable to successfully infect a culture of L2 cells. This was because the L2 cells did not adhere properly to the petri dish and there was insufficient time to repeat the experiment. (ii) I established the growth curves of the putative mutants as well as the previously isolated mutants, regardless of whether it was established they were intracellular mutants or not. The generation times of the mutants can be seen in table 2 (raw data can be seen in Appendix III). The colonies I picked are numbered 1 to 4, those previously isolated are numbered 5 to 13. Generation times that are significantly different to that of non-mutagenised DP-L184 have been highlighted.
DISCUSSION
Whilst there was insufficient time to establish whether the colonies picked from the enrichment were intracellular mutants, the results show that efficient mutagenesis of a culture of L. monocytogenes took place.
UV mutagenesis. The negative correlation seen in graph 1 is supported by the positive correlation in table 1, as it shows that the number of mutations increases with exposure to UV. The 99.9% killing time was altered from 30 to 35 seconds after seeing that there was growth on the rifampicin plates at 35 seconds and not at 30. This suggested that a more efficient mutagenesis had occurred at 35 seconds.
The oscillations seen in both results may be due to experimental error, or the natural error generated when calculating averages, or it may be due to the fact that the dose is so high, the length of exposure is incidental and the variation in the number of viable cells is negligible. This becomes apparent between 50 and 60 seconds in graph 1, where the difference is only 1.83 x 104 cells per ml (compared with a difference of 1.61588 x 108 cells per ml between 5 and 20 seconds), and at 55 and 60 seconds in table 1 where there is virtually no growth on the rifampicin plates.
NTG mutagenesis. NTG is a more powerful mutagen than UV. This is demonstrated by the concentration of rifampicin in the BHI agar plates being increased 10 times more than that used in the UV rifampicin plates, as colony numbers on the plate containing 10 μg/ml were too many too count. This increase in colony numbers compared to UV is probably due to the different ways in which these mutagens cause mutations. Exposure to UV light induces the formation of pyrimidine dimers in DNA. This structure increases the probability of DNA polymerase inserting an incorrect nucleotide when the cell comes to replicate. The mutations generated tend to be point mutations. NTG is a powerful bifunctional alkylating agent that can induce point mutations in non-replicating DNA.
Enrichment of hly- L. monocytogenes. The results demonstrate that an efficient enrichment took place. This means that when enriching for intracellular mutants, 69% of them should be genuine intracellular mutants and not just cells that have survived the enriching process.
Enrichment of intracellular mutants. As no one method of mutagenesis yielded more intracellular mutants than the other, it can be suggested that even though the methods of mutation are different, the resulting mutations are similar. However this may not be the case; although the number of intracellular mutants may be the same, the numbers within each type of mutation may be different (e.g. UV may yield more mutants with poor phospholipase activity than NTG).
Characterisation of intracellular mutants. I can only comment on the growth curves of the colonies isolated and not on the type of plaque they generated. However, some of the generation times of the putative mutants are very interesting. Mutant 1 has a significantly slower generation time than the non-mutagenised strain, with it being 88.2 minutes. As I hypothesised previously, this may mean that a mutation has occurred in an essential gene, such as RNA polymerase, causing this bacterium to grow at a much slower rate. This would probably mean that the plaques generated would be very small.
Mutants 4, 8 and 10 have incredibly fast generation times (43.8, 39.6 and 33.6 minutes respectively) and it is difficult to believe that they are this quick. Maybe these genes were mutated so that they are more efficient at metabolising nutrients. It is also difficult to see how these mutants can be intracellular mutants as well, unless the bacteria have been mutated such that they are more efficient at growing in broth than they are at growing in tissue cells. It is thus impossible to predict the size of the plaque that these mutants would generate. However, these putative intracellular mutants may just be cells that were able to escape the enrichment process. If this is the case, it is possible that the accelerated generation times are an incidental consequence.
The other mutants that have generation times similar to the non-mutagenised strain obviously show that no mutations have occurred in genes essential for growth. The mutations in these mutants may have occurred in genes previously mentioned, such as the phospholipase genes. I would like to have carried out an assay for phospholipase activity on these mutants, but the strain DP-L184 is a derivative of 10403 Mack, which does not easily show phospholipase activity in this assay.
These generation times may not be the values that they are because of errors in the readings by the spectrophotometer. The growth curves were measured in side arm flasks which, when placed in the spectrophotometer, meant that the lid could not be closed properly over the samples. As a result light was able to filter in and may have disrupted the true readings of the samples.
I did not isolate any hly- mutants, but this may not be that surprising. In this project 200 colonies survived the enriching process, the studies that I reviewed previously screened at least 10,000 colonies. Also, this project was specifically looking at point mutations, which may not be enough to knock out genes such as the hlyA gene.
It is difficult to assess where the mutations in these putative mutants occurred if there is no selectable phenotype, a problem encountered by many of the authors in the studies previously reviewed. It is especially difficult in this project because there are no selectable markers flanking the mutations, as is the case when using transposon mutagenesis. Similarly, some of the mutations may occur in pleiotropic genes or regulatory genes. With mutations in such genes, a defect in intracellular growth may only be due to consequence. In these mutants more than one phenotype would be knocked out. Again, mutations in genes such as these would be indistinguishable from the ones defective for just intracellular growth unless the other phenotypes affected were easily selectable. However, once the genome of L. monocytogenes is sequenced identification of mutations in such genes may become easier.
Experiments to be carried out in the future. After completing this project there are a number of experiments that I would either repeat or continue, had I the time to do so. Firstly I would repeat the enrichment for intracellular mutants so as I could screen more colonies. I would then, of course, repeat the infection of L2 cells with putative mutants to see if there were any abnormal plaque sizes. It may be interesting to see if there were any growth differences in the putative mutants when grown on different cell lines, such as the macrophagelike cell line J774, or the human epithelial cell line Henle 407 (as used by Camilli et al, Gedde et al, Hodgson, Marquis et al and Sun et al). It was reported by Portnoy et al (1988) that LLO was required for growth in murine macrophages and fibroblasts but not in human epithelial cell lines. It would be interesting to see if there were any more differences.
Once there was confirmation that the colonies picked were intracellular mutants I would repeat the growth curve experiments, but this time either using cuvettes instead of the side-arm flasks, or using a machine that could measure the number of cells, such as ‘Cellfacs’. Although both these methods are not without their inaccuracies themselves, they might be more accurate than the method that was used in this project.
I would then examine a variety of virulent properties of L. monocytogenes. I would look at cell-to-cell spread in the intracellular mutants as Sun et al did. These authors found that some mutants were able to successfully replicate in the initial tissue cell, but then was either (i) unable to establish infection in a neighbouring cell (i.e. not able to form pseudopodia); (ii) once in the neighbouring cell, was unable to escape from the double-membrane vacuole or; (iii) was able to escape from the double-membrane vacuole but unable to form pseudopodia in this second cell.
Finally, I would examine mutants for actin polymerisation, especially ones that were unable to form pseupdopodia, as the association between L. monocytogenes and actin polymerisation in the cell is essential for the bacterium to be able to progress to the cells edge.
