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Complementation of Listeria seeligeri with the plcA-prfA genes from L. monocytogenes activates transcription of seeligerolysin and leads to bacterial escape from the phagosome of infected mammalian cells

Iddya Karunasagar , Robert Lampidis , Werner Goebel , Jürgen Kreft
DOI: http://dx.doi.org/10.1111/j.1574-6968.1997.tb10209.x 303-310 First published online: 1 January 1997

Abstract

Infection experiments have shown that the avirulent species Listeria seeligeri invaded the enterocyte-like cell line Caco-2 with low efficiency but was unable to escape from the phagosome. Introduction of the listeriolysin gene (hly) from L. monocytogenes into L. seeligeri via a recombinant plasmid did not change these characteristics. No measurable transcription of this gene or of the structurally intact chromosomal seeligerolysin gene (lso) was detected. Transformation with a plasmid carrying the bicistronically transcribed plcA-prfA genes from L. monocytogenes resulted in the efficient expression of the plasmid-encoded transcription activator PrfA, a readily detectable synthesis of seeligerolysin and the escape of the bacteria from the phagosome of infected mammalian cells, followed by intracytoplasmic multiplication.

Keywords
  • Intracellular bacteria
  • Listeria seeligeri
  • Listeriolysin
  • Transcription activator

1 Introduction

Within the bacterial genus Listeria the three species L. monocytogenes, L. ivanovii and L. seeligeri are hemolytic. The former two species are known as pathogens whereas L. seeligeri is considered non-pathogenic; in one case a strain of this species could be isolated from the cerebrospinal fluid of an adult suffering from meningitis [1]. L. monocytogenes and L. ivanovii are capable of invading non-professional phagocytic cells, e.g. the enterocyte-like cell line Caco-2, by induced phagocytosis whereas L. seeligeri has been reported to be non-invasive in this system [2].

Following entry into a eukaryotic cell, L. monocytogenes is capable of escaping from the phagosomal vesicle mainly by the action of a cholesterol-binding (thiol-activated) cytolysin, listeriolysin O [24], encoded by the hly gene. DNA sequences with homology to hly have been detected in L. ivanovii and L. seeligeri and we have previously reported the complete nucleotide sequences for these hly homologues [5]. In the case of L. monocytogenes and L. ivanovii, following entry into the cytoplasm, the bacteria replicate, exhibit intracellular motility and cell-to-cell spread. This infection process requires the coordinate expression of a number of genes (plcA, hly/ilo, mpl, actA, plcB) which constitute a chromosomal virulence gene cluster [6]. Its transcription is positively regulated by the product of prfA, the first gene in the cluster [7]. DNA hybridization experiments by Gouin et al. [8] indicated that DNA sequences homologous not only to the listeriolysin gene but rather to all genes of the L. monocytogenes virulence gene cluster are present in L. seeligeri. It remained unknown, however, if the observed avirulence of this species is caused by structural defects in genes of the virulence cluster or by a block in their expression.

2 Materials and methods

2.1 Bacterial strains, growth conditions and plasmids

L. seeligeri SLCC3954 and L. monocytogenes Sv1/2a EGD (provided by S.H.E. Kaufmann, Ulm) were the wild-type strains used in this study; they were grown in brain heart infusion (BHI) broth at 37°C. The complementation plasmids contained different virulence genes from L. monocytogenes EGD: pERL3 50-1 and pERL3 51-1 carried a functional prfA or hly gene, respectively [7]; pERL3 50-2 carried prfA plus plcA in a bicistronic arrangement and pERL3 50-4 contained hly followed by a divergently transcribed sequence of a non-functional plcA* (with an internal 300 bp deletion) and an intact prfA gene; both plasmids were kindly provided by Dr. T. Chakraborty. L. seeligeri was transformed with these plasmids by electroporation [9].

2.2 Hemolytic activity

Supernatants from bacterial cultures grown in charcoal-treated proteose peptone broth for 10 h at 37°C were assayed by a microplate technique [10], one minimal hemolysin unit was defined as the reciprocal of the highest dilution of supernatant at which hemolysis could be detected.

2.3 Extraction of RNA and transcription analysis

RNA extraction from Listeria strains and transcription analysis by Northern blotting was performed as previously described [11]. Gene- and species-specific DNA probes were generated by PCR amplification of appropriately selected chromosomal regions and random-primed radiolabelled using a commercial kit.

2.4 Mammalian cell culture and infection

The enterocyte-like human colon carcinoma cell line Caco2 (ECACC 86010202) and the mouse macrophage-like cell line J774 (ATCC TIB 67) were cultured in appropriately supplemented MEM or RPMI medium, respectively. Bacterial invasion and intracellular multiplication were studied essentially according to Gaillard et al. [2] with minor modifications, as previously described [12]. A multiplicity of infection of 1 bacterium per cell was used in these experiments, the initial uptake period prior to the addition of 10 μg ml−1 gentamicin was 1 h. Infected cells were lysed by the addition of distilled water and viable bacterial counts, defined as colony forming units (cfu), were determined from serial dilutions plated on BHI agar.

2.5 Electron microscopy

J774 cells were seeded out on cover slips and infected (see above), then fixed and stained as previously described [12]. Photographs from ultrathin sections were taken with a Zeiss EM 900 electron microscope at 80 kV.

3 Results

3.1 Phenotypic expression of virulence genes

L. seeligeri wild-type (WT) and strains transformed with the different recombinant plasmids were streaked on BHI agar plates supplemented with blood (5%, v/v) or phosphatidylinositol (0.2%, w/v), respectively, and tested for their hemolytic or phosphatidylinositol-specific phospholipase C (PI-PLC) activity (Fig. 1). The strongly hemolytic and phospholipolytic L. ivanovii was used as a positive control. L. seeligeri with pERL3 50-1 (prfA) or pERL3 51-1 (hly) showed no activity on all test plates, as did the wild-type strain (not shown). The strains with pERL3 50-2 (bicistronic plcA-prfA) or pERL3 50-4 (partially deleted plcA*+intact prfA and hly) clearly exhibited PI-PLC or hemolytic activity, respectively. In addition, the hemolytic activity of all the strains was determined in a more sensitive and quantitative microtiter assay (Table 1).

1

Phenotype of the L. seeligeri strains used in this study. A: BHI agar with 0.2% (w/v) phosphatitylinositol. PI-PLC activity was visualized by the formation of a faint halo of precipitated diacylglycerol. B: BHI agar with 5% (v/v) human blood. L. seeligeri with (a) pERL3 50-1 (prfA), (b). pERL3 50-4 (plcA*-prfA+hly), (c) pERL3 50-2 (plcA-prfA), (d) pERL3 51-1 (hly). (e) L. ivanovii ATCC19119 as a positive control.

View this table:
1

Genotype and phenotype of the L. seeligeri strains used

3.2 Transcription analysis

L. seeligeri WT, the different transformed strains and L. monocytogenes EGD as a control were grown in BHI and then transferred into MEM for 30 min in order to induce PrfA-dependent transcription [11]. Total RNA preparations were electrophoresed on formaldehyde-containing agarose gels and the RNA was visualized with ethidium bromide in order to check for equal concentrations and integrity (not shown). RNA blots from gels run in parallel were hybridized with radioactively labelled gene- and species-specific DNA probes. The autoradiograph in Fig. 2B shows that in the transformed L. seeligeri strains the plasmid-encoded prfA gene from L. monocytogenes is strongly transcribed into transcripts of 0.9 kb (monocistronic prfA) and 2.1 kb (bicistronic plcA-prfA), respectively, depending on the plasmid construct. In the strain with pERL3 50-2 large amounts of the monocistronic 1.0-kb plcA transcript were also made (Fig. 2C). If L. seeligeri was complemented with hly from L. monocytogenes only (pERL3 51-1), no transcription of this gene was detectable (Fig. 2D, lane f). In the strain with pERL3 50-4 (bicistronic plcA*-prfA plus hly, with a small deletion in plcA) a very strong transcription of hly was observed (Fig. 2D, lane e). Most interestingly, only in L. seeligeri with the bicistronic plcA-prfA construct pERL3 50-2 was transcription from the chromosomal lso gene clearly activated (Fig. 2E). The absence of unspecific hybridization signals with the lso probe demonstrated that the RNA preparations were free of contaminating chromosomal DNA.

2

Promoter sequence of lso and transcription analysis by Northern blots. A: Nucleotide sequence of the L. seeligeri chromosome ustream of the lso gene. +1 denotes the transcription start, as determined by primer extension experiments (not shown) and corresponds to position −132 from the lso start codon. B–E: Total RNA from Listeria strains grown in BHI and then tranferred into MEM for 30 min was electrophoresed on formaldehyde-agarose gels, blotted onto nylon membranes and hybrdized to radiolabelled gene-specific DNA probes (indicated in parentheses). (a) L. seeligeri WT, (b) L. monocytogenes EGD, (c) L. seeligeri pERL3 50-1, (d) L. seeligeri pERL3 50-2, (e) L. seeligeri pERL3 50-4, (f) L. seeligeri pERL3 51-1. Transcript sizes are indicated in kb.

3.3 Invasion and multiplication in enterocyte-like Caco-2 cells

L. seeligeri WT invaded Caco-2 cells very poorly, only 0.3% of the bacteria used for infection were found intracellularly at 1 h post infection (p.i.) compared to 4% with L. monocytogenes EGD and 45% with L. monocytogenes NCTC 7973 (not shown). Complementation of L. seeligeri with any of the plasmids had no detectable effect on the initial invasion rate for this cell line (Fig. 3A). After infection, L. seeligeri WT and L. seeligeri pERL3 51-1 (hly) showed a very slight increase in numbers at 8 h p.i. while the number of intracellular bacteria remained constant in the case of L. seeligeri pERL3 50-1 (prfA) (Fig. 3A). The differences between these strains were not significant. In contrast, the strains with pERL3 50-4 (plcA*-prfA+hly) or pERL3 50-2 (plcA-prfA) showed a slight increase at 4 h and a significant increase at 8 h p.i.

3

Intracellular multiplication of L. seeligeri wild-type and transformed strains in Caco-2 enterocyte-like cells (A) or J774 macrophage-like cells (B). (△) Wild-type, (+) pERL3 50-1, (◻) pERL3 51-1, (×) pERL3 50-2, (*) pERL3 50-4. The graph shows a typical result from three independent experiments.

Caco-2 cells infected with these multiplication-proficient L. seeligeri strains were stained for F-actin with FITC-phalloidin as previously described [12]. No F-actin surrounding the intracellular bacteria could be detected (results not shown).

3.4 Intracellular multiplication in the macrophage-like cell line J774

All L. seeligeri strains were readily taken up by the phagocytic cell line J774. The differences in intracellular bacterial number observed after the initial uptake period (1 h) may have been caused by slightly differing inocula or/and strain-specific differences in uptake and survival. Following ingestion, the bacterial numbers showed a constant decrease over the time in the case of L. seeligeri WT, L. seeligeri pERL3 50-1 and pERL3 51-1 (Fig. 3B). With L. seeligeri pERL3 50-4 the number of intracellular bacteria increased considerably until 4 h p.i. and then slightly declined until 8 h p.i., while L. seeligeri pERL3 50-2 showed a significant and constant increase in bacterial counts during the test period (8 h).

3.5 Electron microscopy of intracellular L. seeligeri

As mentioned above, invasion of Caco-2 cells by L. seeligeri was very inefficient, therefore the intracellular fate of these bacteria was analyzed electron-microscopically only in J774 macrophages. At 2 h after uptake, L. seeligeri WT and the strains with pERL3 51-1 (hly) or pERL3 50-1 (prfA) were found exclusively within the phagocytic vacuole, seeming morphologically still intact (Fig. 4a,c,e). The majority of L. seeligeri carrying pERL3 50-4 (plcA*-prfA+hly) was also still inside a vacuole (Fig. 4g), however, a significant amount of the bacteria was already found free in the cytoplasm (Fig. 4h). At 4 h (and 8 h, not shown) p.i., the former three strains were still in the vacuole, showing signs of more or less severe degradation (Fig. 4b,d,f). At 2 h p.i. several discrete, closely associated phagosomes could be observed in some cases (Fig. 4c), later on larger vesicles with more than one bacterial cell were found (Fig. 4d). This suggests that later in the infection process primary phagosomes may have fused. At 4 h p.i. most of the bacteria with pERL3 50-4 were found free in the cytoplasm (not shown). In the case of L. seeligeri pERL3-502 (plcA-prfA), already at 2 h p.i. some of the infecting bacteria were found free in the cytoplasm (Fig. 4j), until 8 h p.i. the cytoplasmic proportion reached almost 80% (Fig. 4k). However, one could also find large vesicles containing multiple bacteria, some of which were clearly dividing (Fig. 4l).

4

Transmission electron micrographs of L. seeligeri WT and transformed strains at different times after infection (p.i.) of J774 macrophages. L. seeligeri WT at 2 h p.i. (a) and 4 h p.i. (b); pERL3 51-1 at 2 h (c) and 4 h (d); pERL3 50-1 at 2 h (e) and 4 h (f); pERL3 50-4 at 2 h (g,h); pERL3 50-2 at 2 h (i,j) and 8 h (k,l). The bar represents 2 μm.

4 Discussion

It has previously been shown that L. seeligeri synthesizes and secretes, yet in minor amounts, a hemolysin which belongs to the group of cholesterol-binding (thiol-activated) cytolysins [5, 13] and which has been termed seeligerolysin (LSO). Our published DNA sequence of the lso gene revealed an 80% identity of the deduced amino acid sequence of seeligerolysin to listeriolysin O from L. monocytogenes[5]. Upstream of the lso gene all the transcription signals known to precede the listeriolysin (hly) gene of L. monocytogenes are present: an 18-bp palindrome containing the canonical ‘PrfA box’ which in L. monocytogenes is required for the transcriptional activation of hly and other virulence genes by the PrfA protein (review in [14]), and a consensus −10 promoter region. The latter, however, is somewhat shifted towards the ribosome binding site, when compared to the other Listeria species. From the presence of these expression signals we concluded that the very low expression of lso has mainly to be attributed to insufficient amounts of the PrfA homologue or inactivity of this protein in L. seeligeri. Work on the prfA gene from L. seeligeri is in progress but not yet finished (Lampidis et al., in preparation) and there are no data available on its activity. Therefore we tested L. seeligeri strains complemented with the hly, prfA and plcA genes from L. monocytogenes, present in different combinations on the multi-copy vector plasmid pERL3, for their hemolytic activity and their intracellular behavior after infection of suitable mammalian cell lines. Table 1 summarizes the results from these experiments. This, together with the results from transcription analyses, led us to the following conclusions.

(i) In L. seeligeri WT transcription of the lso gene was below the limits of detection. The same was true for the related hly gene from L. monocytogenes when introduced into L. seeligeri on a recombinant plasmid without the homologous prfA gene. The slight increase in hemolytic activity of the latter strain can be attributed to a very weak transcription, undetectable in our experiments, from a PrfA-independent promoter [15]. These very weak hemolytic activities obviously were not sufficient to mediate escape of these strains from the phagosome. (ii) Introduction of an intact prfA gene from L. monocytogenes on a plasmid construct which allowed for a strong synthesis of the monocistronic prfA transcript could not activate lso transcription and hence hemolytic activity. This was different from previous results obtained with a prfA deletion mutant of L. monocytogenes[7], but is in line with results from others obtained with wild-type L. monocytogenes. It has been reported there that enhanced synthesis of the monocistronic prfA mRNA only did not necessarily result in a concomitant transcriptional activation of PrfA-dependent genes [11, 16]. This has partially been attributed to the negative autoregulation of prfA, in addition it seems possible that the monocistronic prfA mRNA is poorly translated. (iii) Transformation of L. seeligeri with plasmids containing both plcA and prfA in the normal tandem array (pERL3 50-4 and pERL3 50-2) led to the synthesis of large amounts of a bicistronic plcA-prfA transcript. This, however, resulted in a readily measurable transcriptional activation of the chromosomal lso gene by PrfA only if no intact hly gene was present on the same plasmid, i.e. in the case of pERL3 50-2. A possible, yet not proven explanation is that the PrfA protein might be titrated if an intact and strongly as well as divergently transcribed hly gene is present in cis, i.e. on the transforming plasmid. This question, however, needs further clarification.

In any case, the dramatic increase in hemolytic activity and the efficient phagosomal escape of L. seeligeri (pERL3 50-4) only resulted from the expression of the heterologous plasmid-encoded hly gene. (iv) The considerable increase in hemolytic activity following synthesis of the bicistronic plcA-prfA mRNA (from pERL3 50-2) in the absence of the hly gene has to be attributed to the readily detectable transcription from the chromosomal lso gene. This also led to the capacity of this strain to escape efficiently from the phagosome of both enterocyte-like and macrophage-like cells. For L. monocytogenes it has been demonstrated that in Caco-2 cells listeriolysin is absolutely required for phagosomal escape [3], PI-PLC has an enhancing effect only. Therefore the PI-PLC activity of the plcA-complemented L. seeligeri strain (pERL3 50-2) cannot alone be responsible for the escape-proficient phenotype.

To conclude, our results clearly show that the efficient expression of the functional transcription activator protein PrfA from L. monocytogenes in L. seeligeri led to a relatively strong expression of the chromosomally encoded listeriolysin (seeligerolysin, lso) gene. Furthermore, listeriolysin-mediated escape of such strains from the phagosome of infected enterocyte- or macrophage-like cells enabled the avirulent L. seeligeri to multiply in the cytoplasm of the host cell. This, however, does not mean that the sole introduction of a fully functional prfA gene converted L. seeligeri into an intracellular pathogen. It has clearly been demonstrated that additional properties, in particular actin-based motility which had not been observed in the case of the L. seeligeri strains studied here, are required for Listeria virulence.

Acknowledgements

We thank Drs. T. Chakraborty and M. Leimeister-Wächter for kindly providing the plasmids used in this study. A. Knopp is thanked for expert technical assistance and C. Gehrig for her skilful help in the electron-microscopic work. I.K. is grateful to the Alexander von Humboldt Foundation for the award of a fellowship. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 165) and the Fonds der Chemischen Industrie.

Footnotes

  • 1University of Agricultural Sciences, College of Fisheries, P.O. Box 527, Mangalore-575 002, Karnataka, India.

References

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