Listeria monocytogenes is a facultative intracellular pathogen. This project aimed to produce strains that were defective for intracellular growth by mutagenising a culture with ultra violet light and nitrosoguanidine. Intracellular mutants were selected for using a method of ampicillin enrichment. Approximately 200 colonies survived the enrichment process but only four colonies were characterised.
These putative mutants, as well as nine putative mutants isolated from a previous experiment, were used to infect a culture of the mouse fibroblast cell line, L2, to look for mutants that generate small plaques. Unfortunately this infection was unsuccessful, but the generation times of the putative mutants were calculated. One mutant had a slow generation time of 88.2 minutes, three had fast generation times of 43.8, 39.6 and 33.6 minutes. The rest had relatively normal generation times of 60 minutes.
Listeria monocytogenes is a facultatively intracellular, gram positive, food-borne pathogen. Infection with L. monocytogenes can cause listeriosis, a relatively rare disease, which can be fatal to the fetus in pregnant women, or can present it self as meningitis in the immunocompromised. L. monocytogenes is widespread in the environment and transmission to man can be via infected animals (such as sheep, cattle and pigs) or through ingestion of contaminated foods, especially unpasteurized milk or soft cheese as L. monocytogenes is capable of replication at temperatures as low as 3oC.
When ingested L. monocytogenes is internalised within gut epithelial cells or macrophages into a host vacuole, referred to as a phagolysosome. The bacteria escapes from this phagolysosome via, in part, production of listeriolysin O (LLO). LLO is a member of the pore-forming protein family, cytolysin. It can lyse red blood cells, and thus its production can be detected on blood plates. Once in the cytoplasm of the host cell the bacteria replicates and concomitantly becomes covered in host actin filaments.
When the actin polymerises at the base of the actin ‘tail’ the bacteria is propelled to the edge of the host cell. Once at the cells’ edge the bacteria protrude from the surface forming pseudopods. These are in turn recognised by neighbouring cells and the bacterium is internalised. The bacterium this time is internalised into a double membraned vacuole. These membranes are lysed by another pore-forming protein, phospholipase C (PLC), and the cycle begins again (adapted from Cossart and Lecuit, 1998). This cycle can be seen in figure 1.
Figure 1. Morphological stages in the entry, growth, movement, and spread of L. monocytogenes from one cell to another. Taken from Sun et al (1990) As described, the intracellular growth of L. monocytogenes requires a number of virulence factors and loss of one or more of these factors can render the bacteria incapable of establishing an infection. These mutants are of particular interest because of their potential use as vaccines against virulent L. monocytogenes. Various studies have investigated virulence factors involved in intracellular replication and cell-to-cell spread by mutagenising L. monocytogenes and assaying them in tissue cultures. L. monocytogenes grows rapidly in a variety of tissue cultures derived from macrophages and lymphoid cells, forming zones of tissue cell lysis, or plaques. Most intracellular mutants can be characterised by the formation of abnormally small plaques, or in rare cases, no plaques at all.
Most studies have mutagenised L. monocytogenes with transposons, one of the first ones being that by Sun et al (1990). The transposon Tn917-LTV3 was inserted randomly into the L. monocytogenes genome constructing a library of mutants that were used to infect the macrophagelike cell line J774. Small plaque mutants were identified and various tests were carried out to determine which genes had been affected. These tests included an assay for phospholipase activity, an assay for intracellular growth, cell-to-cell spread, flourescence labelling of F-actin and mapping sites of transposon insertion.
Ten classes of mutants were identified as a result of these tests, of which most were defective in either LLO production, cytoplasmic induction of actin polymerisation and/or phospholipase activity. At the time when this study was conducted only a small region of the L. monocytogenes genome had been sequenced, therefore it was difficult to map the transposon insertion sites outside of this region. Incidentally, the sequenced region included the LLO gene (hlyA) and four of the ten classes of mutants found in this study had mutations mapping to this region.
A similar study in which intracellular mutants were selected for directly is that by Camilli et al (1989). Intracellular mutants (derived from the insertion of Tn916) were selected using penicillin enrichment. The theory is that penicillin enters the tissue cells and kills any bacteria that are able to replicate. Thus, when the tissue cells are lysed, the only viable bacteria are intracellular mutants. Eight mutants were isolated in this study, all of which were hly-, or had reduced hemolytic activity. However, three of the eight mutations were point mutations (i.e. not due to the transposon) and the genes mutated were unable to be identified.
A pioneering study by Hodgson (2000) reported 34 bacteriophage, specific to L. monocytogenes, that were capable of generalised transduction. Thus, point mutations may be transposon-tagged and genes with non-selectable phenotypes may be isolated. Transduction using these phage can show whether a transposon insertion has or has not caused a mutant phenotype – an experiment that would have been very useful in Camilli’s study.
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.