Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA
Correspondence
Anthony T. Maurelli
amaurelli{at}usuhs.mil
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ABSTRACT |
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INTRODUCTION |
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DNA methylation is a mechanism by which bacteria regulate gene expression and control cellular functions such as DNA replication, mismatch repair, and segregation of chromosomal DNA (Marinus, 1996). Enzymes known as DNA-methyltransferases mediate methylation, which occurs at the C-5 or N-4 positions of cytosine and at the N-6 position of adenine. DNA-methyltransferases are found in many genera of enteric bacteria, but DNA methylation is not essential for viability of Escherichia coli or Salmonella (García-del Portillo et al., 1999
; Low et al., 2001
).
Recently, DNA adenine methylase (Dam), which methylates the N-6 of adenine in GATC sequences, has been reported to play a key role in regulation of virulence of several bacterial pathogens. Dam Salmonella typhimurium display reduced M-cell cytotoxicity and reduced invasion in tissue culture (García-del Portillo et al., 1999). In an animal model, these mutants colonize Peyer's patches but fail to invade enterocytes and fail to cause disease (Heithoff et al., 1999
). It has also been reported that Dam is required for expression of virulence genes in Yersinia pseudotuberculosis and Vibrio cholerae. In these organisms, Dam activity is essential for viability. However, overexpression of dam inhibits colonization of V. cholerae in the suckling mouse model and strongly attenuates virulence of Y. pseudotuberculosis in a murine model (Julio et al., 2001
). Heithoff et al. (1999
, 2001)
showed that dam mutants of Salmonella provide effective immunity when used as live attenuated vaccines in the mouse model. These authors also proposed that because of the high conservation of DNA adenine methylases among pathogenic bacteria, these proteins are potential targets for the development of vaccines and antimicrobial agents.
In light of these reports, it has been suggested that Dam is likely to be important for pathogenesis of other bacterial pathogens as well as those just described (Low et al., 2001). In this study, we tested this hypothesis by constructing a dam mutant of Shigella flexneri 2a and examining the virulence properties of the mutant. We report that, contrary to what has been observed in Salmonella, a dam mutant of Sh. flexneri retains the ability to invade, grow intracellularly and spread intercellularly, and is only minimally altered in its virulence phenotypes.
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METHODS |
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Secretion of invasion plasmid antigens (Ipas).
Secretion of IpaB and IpaC from 2457T and 2457T/dam was examined as previously described (Bahrani et al., 1997).
Virulence assays.
Invasion assays with 2457T and 2457T/dam were performed using a gentamicin protection assay with semiconfluent monolayers of L2 mouse fibroblasts, as described before (Hromockyj & Maurelli, 1989). Plaque assays were performed using confluent L2 monolayers, as described by Oaks et al., (1985)
. The Sereny test was used as an in vivo virulence assay (Hartman et al., 1991
).
Bacterial growth in liquid culture.
Strains 2457T, 2457T/dam, BS103 and BS103/dam were cultured overnight with shaking at 30 °C, subcultured 1 : 100 into fresh TSB, and then incubated at 30 °C or 37 °C (for 2457T and 2457T/dam), or at 37 °C (for BS103 and BS103/dam). Samples were taken every hour and plated to determine viable cell numbers.
Measurements of intracellular bacterial growth.
Assays for growth of intracellular bacteria were carried out as previously described, with some modifications (Schuch et al., 1999). Briefly, strain 2457T and 2457T/dam were grown to earlymid log phase in TSB. Bacteria were washed with PBS and suspended in Dulbecco's minimal essential medium (DMEM) to an OD600 of 0·72. One millilitre of this bacterial suspension (containing about 68x108 c.f.u.) was applied to each monolayer of semiconfluent L2 cells in 35 mm tissue culture dishes. After 10 min centrifugation at 1000 g and a 30 min invasion period at 37 °C in CO2, infected monolayers were washed with PBS and further incubated in DMEM containing 50 µg gentamicin ml1. For measurement of intracellular growth, t=0 was set at the beginning of the incubation period after centrifugation. At the indicated intervals, cell monolayers (in triplicate) were washed to remove extracellular bacteria and gentamicin, lysed with 0·5 % Triton X-100 in H2O to release intracellular bacteria, and the recovered bacteria were plated on Congo red agar for counting.
Spontaneous mutation rate.
To determine the mutation rate in dam mutants of Shigella, spontaneous mutation to rifampicin resistance was examined, as described previously (Bale et al., 1979). Briefly, a few hundred cells from each strain were inoculated into 5 ml LB and the cells were cultured overnight to saturation at 37 °C with shaking. Bacteria from each culture were plated on LB agar, with or without 100 µg rifampicin ml1. After incubation overnight at 37 °C, the plates were scored for rifampicin-resistant colonies and the frequency of spontaneous mutation calculated.
Statistical analysis.
Values are reported as mean±standard error of the mean (SEM) in tables. All results were analysed using Student's t-test.
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RESULTS |
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Secretion of Ipa proteins
The mutant strain 2457T/dam was first tested for the ability to secrete invasion effectors via the Shigella type III secretion system. This phenotype is tightly regulated by growth temperature and is essential for invasion and intracellular growth. 2457T/dam synthesized as much IpaBC as the parent 2457T, and secreted similar amounts of these effector proteins into the growth medium, as detected by Western blotting (data not shown).
Virulence assays
Next, 2457T/dam was tested for the ability to invade tissue culture cells and to spread from cell to cell (as measured in the plaque assay). No significant difference in the efficiency of invasion was detected between 2457T and 2457T/dam (Table 1, P=0·1) and only a modest difference in plaque formation ratio was found (Table 1
, P=0·04). Plaques formed by 2457T/dam tended to be smaller in size compared to 2457T (Fig. 1
). In the Sereny test, 2457T/dam produced keratoconjunctivitis in all three of the animals tested, although fully developed keratoconjunctivitis was delayed in two of the animals (Table 1
).
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DISCUSSION |
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In this study, we constructed a dam mutant of Sh. flexneri 2a strain 2457T. The mutant 2457T/dam was slightly attenuated in virulence in tissue culture invasion assays, plaque assays and, in vivo, in the guinea pig Sereny test. However, 2457T/dam formed smaller plaques in the plaque assay, and gave a delayed positive reaction in some of the animals in the Sereny test, compared to 2457T. Growth in TSB was also slower for the dam mutant, and in invaded cells some 2457T/dam bacteria were filamentous. Genes involved in stress and the SOS response are expressed at higher levels in dam mutants of E. coli (Marinus, 1996; Oshima et al., 2002
), and the filamentous shape of some dam mutant Sh. flexneri in invaded cells suggested an increased SOS response in the Sh. flexneri dam mutant.
We were rather surprised that the efficiency of invasion of the Sh. flexneri dam mutant was indistinguishable from that of the wild-type parent, in spite of its high mutability and slower growth. In contrast, a Sal. typhimurium dam mutant is non-invasive in tissue culture (García-del Portillo et al., 1999). The Sh. flexneri dam mutant was also virulent in an animal model, while a Sal. typhimurium dam mutant is completely attenuated in an animal model (Heithoff et al., 2001
). It has been proposed that since motility is decreased in a dam mutant of E. coli (Oshima et al., 2002
), this phenotype might be responsible for the lack of invasiveness by the mutant of Sal. typhimurium. Motility is important for invasiveness of Salmonella typhi (Liu et al., 1988
), and increases the rate of invasion of Sal. typhimurium (Khoramian-Falsafi et al., 1990
; Moens & Vanderleyden 1996
), but it is not important for invasion of Shigella, which is non-motile. The different behaviour of the dam mutants underscores the fact that the fundamental mechanism of pathogenesis of Shigella differs from that of Salmonella.
DNA adenine methylase in bacteria regulates a variety of functions, including chromosome replication, transcription, and DNA repair (Marinus, 1996). Spontaneous mismatch mutations that occur in rapidly growing bacteria are normally repaired by the dam-directed mismatch repair system. Thus, one phenotype of dam mutants is that they display an increased rate of mutation. We found that 2457T/dam had a mutation rate about 1000-fold higher compared to the wild-type parent. This result is in stark contrast with the spontaneous mutation rate of a dam mutant of E. coli, which was reported to be only 20- to 80-fold higher than wild-type (Bale et al., 1979
). The molecular basis for this increased mutation rate remains to be elucidated.
These results suggest that a general anti-bacterial pathogen vaccine strategy based on dam mutants needs to be re-evaluated. Although inactivation or overexpression of dam has a strong attenuating effect on pathogens such as Salmonella, Yersinia and V. cholerae (Heithoff et al., 2001; Julio et al., 2001
), a mutation in dam does not significantly alter virulence in Sh. flexneri. Although the Sh. flexneri dam mutant produced a positive reaction in guinea pigs in the Sereny test, we cannot exclude the possibility that the mutant might be attenuated in a human host. However, the increased rate of spontaneous mutation we observed in the Sh. flexneri dam mutant would not be a desirable trait for a live vaccine strain.
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ACKNOWLEDGEMENTS |
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The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defence or the Uniformed Services University of the Health Sciences.
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REFERENCES |
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Bale, A., d'Alarcao M. & Marinus, M. G. (1979). Characterization of DNA adenine methylation mutants of Escherichia coli K12. Mutat Res 59, 157165.[Medline]
Formal, S. B., Dammin, G. J., Labrec, E. H. & Schneider, H. (1958). Experimental Shigella infections: characteristics of a fatal infection produced in guinea pigs. J Bacteriol 75, 604610.[Medline]
García-del Portillo, F., Pucciarelli, M. G. & Casadesus, J. (1999). DNA adenine methylase mutants of Salmonella typhimurium show defects in protein secretion, cell invasion, and M cell cytotoxicity. Proc Natl Acad Sci U S A 96, 1157811583.
Glickman, B. W. (1979). Spontaneous mutagenesis in Escherichia coli strains lacking 6-methyladenine residues in their DNA: an altered mutational spectrum in dam mutants. Mutat Res 61, 153162.[Medline]
Hartman, A. B., Powell, C. J., Shultz, C. L., Oaks, E. V. & Eckels, K. H. (1991). Small-animal model to measure efficacy and immunogenicity of Shigella vaccine strains. Infect Immun 59, 40754083.[Medline]
Heithoff, D. M., Sinsheimer, R. L., Low, D. A. & Mahan, M. J. (1999). An essential role for DNA adenine methylation in bacterial virulence. Science 284, 967970.
Heithoff, D. M., Enioutina, E. Y., Daynes, R. A., Sinsheimer, R. L., Low, D. A. & Mahan, M. J. (2001). Salmonella DNA adenine methylase mutants confer cross-protective immunity. Infect Immun 69, 67256730.
Hromockyj, A. E. & Maurelli, A. T. (1989). Identification of Shigella invasion genes by isolation of temperature-regulated inv : : lacZ operon fusions. Infect Immun 57, 29632970.[Medline]
Julio, S. M., Heithoff, D. M., Provenzano, D., Klose, K. E., Sinsheimer, R. L., Low, D. A. & Mahan, M. J. (2001). DNA adenine methylase is essential for viability and plays a role in the pathogenesis of Yersinia pseudotuberculosis and Vibrio cholerae. Infect Immun 69, 76107615.
Khoramian-Falsafi, T., Harayama, S., Kutsukake, K. & Pechère, J. C. (1990). Effect of motility and chemotaxis on the invasion of Salmonella typhimurium into HeLa cells. Microb Pathog 9, 4753.[Medline]
Liu, S. L., Ezaki, T., Miura, H., Matsui, K. & Yabuuchi, E. (1988). Intact motility as a Salmonella typhi invasion related factor. Infect Immun 56, 19671973.[Medline]
Low, D. A., Weyand, N. J. & Mahan, M. J. (2001). Roles of DNA adenine methylation in regulating bacterial gene expression and virulence. Infect Immun 69, 71977204.
Marinus, M. G. (1996). Methylation of DNA. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, vol. 1, pp. 782791,. Edited by F. C. Neidhardt et al., Washington, DC: American Society for Microbiology.
Marinus, M. G., Carraway, M., Frey, A. Z., Brown, L. & Arraj, J. A. (1983). Insertion mutations in the dam gene of Escherichia coli K-12. Mol Gen Genet 192, 288289.[Medline]
Maurelli, A. T., Blackmon, B. & Curtiss R, III. (1984). Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid. Infect Immun 43, 397401.[Medline]
Moens, S. & Vanderleyden, J. (1996). Functions of bacterial flagella. Crit Rev Microbiol 22, 67100.[Medline]
Oaks, E. V., Wingfield, M. E. & Formal, S. B. (1985). Plaque formation by virulent Shigella flexneri. Infect Immun 48, 124129.[Medline]
Oshima, T., Wada, C., Kawagoe, Y., Ara, T., Maeda, M., Masuda, Y., Hiraga, S. & Mori, H. (2002). Genome-wide analysis of deoxyadenosine methyltransferase-mediated control of gene expression in Escherichia coli. Mol Microbiol 45, 673695.[CrossRef][Medline]
Parsot, C. & Sansonetti, P. J. (1996). Invasion and the pathogenesis of Shigella infections. Curr Top Microbiol Immunol 209, 2542.[Medline]
Sansonetti, P. J., Kopecko, D. J. & Formal, S. B. (1982). Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect Immun 35, 852856.[Medline]
Schuch, R., Sandlin, R. C. & Maurelli, A. T. (1999). A system for identifying post-invasion functions of invasion genes: requirements for Mxi-Spa type III secretion pathway of Shigella flexneri in intercellular dissemination. Mol Microbiol 34, 675689.[CrossRef][Medline]
Received 23 September 2003;
revised 17 December 2003;
accepted 22 December 2003.
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