University of Manchester, School of Biological Science, 1.800, Stopford Building, Manchester M13 9PT, UK1
Author for correspondence: Nicola J. High. Tel: +44 161 275 5749. Fax: +44 161 275 5656. e-mail: nicky.high{at}man.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: Haemophilus influenzae type b, phase variation, invasive disease, lic genes
Abbreviations: CSF, cerebrospinal fluid; i.n., intranasal; i.p., intraperitoneal
a Present address: Unilever Research, Port Sunlight, Quarry Road East, Wirral L63 3JW, UK.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analysis of the genome sequence of H. influenzae strain Rd has identified a further nine loci which contain multiple repeats of a tetrameric sequence other than 5'-CAAT-3' (Hood et al., 1996 ). Tandem repeats of 5'-GCAA-3' have also been found in genes involved LPS biosynthesis (Jarosik & Hansen, 1994
). All of these genes encode candidate virulence genes, indicating that H. influenzae has a massive potential for variation during the course of infection which may contribute to its success as a pathogen. The significance of the phase variation of each of these genes in the pathogenesis of invasive disease is unknown. In the case of LPS, phase variation is thought to enable H. influenzae to evade antigen-specific host immune defences by promoting variation in cell surface composition. The ability to vary the expression of multiple virulence determinants may optimize the virulence potential of H. influenzae by enabling the expression of the most appropriate phenotypic attributes for a given environment, or stage in pathogenesis. This may be of particular significance in the development of invasive disease which, in the case of meningitis, requires translocation of H. influenzae from the nasopharynx to the central nervous system. This sequence of events involves the colonization of several distinct environments and the interaction of the organism with a variety of different host cells.
In this paper we investigate the role of the phase variation of lic1A, lic2A and lic3A in colonization of the major compartments colonized by H. influenzae during the development of invasive disease. This was achieved by monitoring the number of 5'-CAAT-3' repeats present in each gene from organisms isolated from the nasopharynx, bloodstream and cerebrospinal fluid (CSF) of infant rats infected with H. influenzae strain Eagan. Organisms colonizing each of these environments were shown to express different combinations of lic genes. Phase variation of these genes may therefore play an important role in facilitating the survival and persistence of H. influenzae in the diverse environments encountered during the development of invasive disease.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The infant rat model of H. influenzae invasive disease.
Natural litters of 5-d-old SpragueDawley rats were reduced to 12 rats at birth and randomized. Infant rats were challenged with H. influenzae strain Eagan by intranasal (i.n.) inoculation so that the expression status of lic genes during colonization of the nasopharynx could be established (Moxon et al., 1974 ). Although invasive disease occurs following i.n. challenge the level of bacteraemia generated is often low and does not always lead to meningitis. A separate group of animals were therefore challenged intraperitoneally (i.p.) to enable efficient isolation of organisms from the bloodstream and CSF (Smith et al., 1973
). For both i.n. and i.p. challenges the inocula were prepared and quantified as described by Moxon et al. (1974)
and Smith et al. (1973)
, and adjusted to the desired con-centration. After 48 h approximately 20 µl blood was taken from a tail vein of which 5 µl was plated onto supplemented BHI agar to detect and quantify bacteraemia. CSF was also removed from each animal by cisterna magna puncture and 10% of the volume obtained plated onto supplemented BHI agar. Bacteria colonizing the nasopharynx were harvested by washing one nostril with 50 µl PBS and then withdrawing approximately 20 µl from the opposite nostril. Two microlitres of each sample was cultured as described above, in the presence of bacitracin (1 mg ml-1).
PCR amplification of the 5'-CAAT-3' regions of lic1A, lic2A and lic3A.
Blood, CSF and nasal washings were boiled for 5 min. Whole-cell debris was removed by centrifugation and the supernatant decanted to a fresh tube. Samples were then treated with Hybaid pre-amplification reagent, according to the manufacturers recommendations, to remove compounds which might inhibit the PCR. The PCR was carried out in a buffer containing 500 mM KCl, 100 mM Tris/HCl, pH 8, 0·1% (w/v) gelatin and 25 mM MgCl2. [-32P]dCTP [10 µCi (0·37 MBq)] was added to the dNTP mixture to increase the sensitivity of the PCR. The 5'-CAAT-3' region from lic1A was amplified using 5'-GGAATGGAATGCTGATGAAG-3' and 5'-TCATAAGATTCAGAGCCT-3' or, in the case of nasal lavage samples, 5'-TCTTTCAGCTAACCGAGC-3'. The 5'-CAAT-3' region from lic2A was amplified using 5'-GCAACTGAACGTCGCAAACAT-3' and 5'-CACACACTTATTCCATAATAAG-3'. The primers used to amplify the 5'-CAAT-3' from lic3A were 5'-CGGAGATAGTACAACTGATA-3' and 5'-AACTTGTTCCATTACCTGCA-3'. The reaction mixture was incubated at 94 °C for 5 min and then 45 cycles of PCR were carried out, each cycle consisting of 94 °C for 1 min, 57 °C for 1 min and 72 °C for 1 min. The resultant PCR products were analysed by electrophoresis through a 6% polyacrylamide gel. The size of each product and hence the number of 5'-CAAT-3' repeats was determined by comparison with an M13 sequencing ladder. To confirm the predicted number of 5'-CAAT-3' repeats a number of samples from each PCR set was sequenced directly as previously described (High et al., 1993
). Autoradiographs were scanned using a Phoretix densitometer and the captured images analysed using Phoretix ID gel analysis software. Bands were identified and densitometry measurements obtained after an automatic background subtraction had been performed.
Statistical analysis.
Densitometry measurements obtained for each set of PCR products were compared statistically. PCR products derived from lic1A, which comprised two bands, were compared using a MannWhitney non-parametric test. PCR products derived from lic2A and lic3A, which comprised three bands, were compared using the KruskalWallis non-parametric test. In both cases P values <0·05 were considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Phase variation of lic3A in H. influenzae colonizing the nasopharynx, bloodstream and CSF of infant rats
Amplification of the 5'-CAAT-3' repeats in lic3A from organisms present in nasal lavage samples generated a predominant PCR product consistent with the presence of 28 repeat units in this gene (Fig. 3a, lanes 18; Fig. 3b
). This number of 5'-CAAT-3' repeats is permissive for the expression of lic3A (Maskell et al., 1992
). PCR products corresponding to 29 and 27 repeat units were also detectable in one sample (Fig. 3
, lane 3), but were present at low levels. In the inoculum used to challenge these animals the majority of organisms contained 27 copies of 5'-CAAT-3', which is non-permissive for expression of lic3A, although a subpopulation containing 28 copies was also present. Colonization of the nasopharynx by H. influenzae therefore appears to involve selection of organisms which have the ability to express lic3A. In contrast, in the bloodstream and CSF the predominant population of organisms were unable to express lic3A. Three PCR products were amplified from blood and CSF samples (Fig. 3a
and b
). The relative distribution of these products was identical to that of organisms in the original inoculum. The most intense PCR product was consistent with the presence of 27 copies of 5'-CAAT-3' in lic3A, which does not permit translation of the ORF. Subpopulations of organisms, containing 28 and 26 copies of 5'-CAAT-3', both of which permit expression of lic3A, were also detected at low levels.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Evidence of a selection pressure influencing the distribution of lic genotypes during nasopharyngeal colonization was most apparent in the case of lic3A. During the 48 h period following i.n. challenge the predominant lic3A genotype changed, relative to the inoculum, resulting in a marked reduction in the proportion of organisms unable to express lic3A. This observation suggests that expression of this gene is important for nasopharyngeal colonization, although its precise role has yet to be determined. The distribution of lic1A genotypes also altered following i.n. challenge. However, this did not result in colonization by a predominant population since equal proportions of organisms which either expressed or did not express lic1A were detected. This even distribution of genotypes would suggest that during the first 48 h the expression status of lic1A is not important for nasopharyngeal colonization. In a study by Weiser et al. (1998) , a gradual increase in the proportion of organisms able to express lic1A was shown to occur in the nasopharynx over a period of 10 d. If the distribution of lic1A genotypes had been monitored over a longer period, a gradual increase in the proportion of organisms expressing this gene might therefore have been expected to occur. In contrast to lic1A and lic3A, the distribution of lic2A genotypes in the nasopharynx was indistinguishable from that of the original inoculum. No conclusive evidence suggesting that organisms expressing this gene were selected in the nasopharynx was therefore obtained.
Following i.p. challenge, differences were observed in the distribution of 5'-CAAT-3' repeats in blood and CSF samples, relative to that in the original inoculum. The numbers of 5'-CAAT-3' repeats also varied between paired blood and CSF samples, indicating that phase variation of lic2A had occurred in vivo. Despite phase variation organisms expressing lic2A always formed the predominant population suggesting that this phenotype was important in the persistence of H. influenzae in the bloodstream and CSF. This notion is consistent with previous observations that show that H. influenzae mutants which lack a functional copy of lic2A have reduced virulence in the infant rat model (Cope et al., 1991 ). No evidence was obtained, however, that phase variation followed by selection of specific lic1A and lic3A genotypes had occurred. The distribution of genotypes in organisms present in the bloodstream and CSF was indistinguishable from that of broth grown organisms used as the inoculum. If a specific genotype was essential for survival at these sites, the relative proportion of organisms with this attribute would be expected to increase. This was not the case for either lic1A or lic3A. Expression of these genes may therefore be neither an advantage nor a disadvantage in these environments.
In summary, the phase variation of lic genes has been monitored in vivo using an infant rat model of H. influenzae invasive disease. We have demonstrated that organisms colonizing the nasopharynx express a different combination of lic genes to those colonizing the bloodstream and CSF. Phase variation of these genes may therefore play an important role in the development of invasive disease by H. influenzae.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson, P., Flesher, A., Shaw, S., Harding, L. & Smith, D. H. (1980). Phenotypic and genetic variation in the susceptibility to Haemophilus influenzae type b to antibodies to somatic antigens. J Clin Invest 65, 885-891.[Medline]
Cope, L. D., Jogev, R., Mertsola, J., Latimer, J. L., Hanson, M. S., McCracken, G. H. & Hansen, E. J. (1991). Molecular cloning of a gene involved in lipooligosaccharide biosynthesis and virulence expression by Haemophilus influenzae type b. Mol Microbiol 5, 1113-1124.[Medline]
High, N. J., Deadman, M. E. & Moxon, E. R. (1993). The role of a repetitive DNA motif (5'-CAAT-3') in the variable expression of the Haemophilus influenzae lipopolysaccharide epitope Gal(14)ßGal. Mol Microbiol 9, 1275-1282.[Medline]
Hood, D. W., Deadman, M. E., Jennings, M. P., Bisercic, M., Fleischmann, R. D., Venter, J. C. & Moxon, E. R. (1996). DNA repeats identify novel virulence genes in Haemophilus influenzae. Proc Natl Acad Sci USA 93, 11121-11125.
Jarosik, G. P. & Hansen, E. J. (1994). Identification of a new locus involved in the expression of Haemophilus influenzae type b lipooligosaccharide. Infect Immun 62, 4861-4867.[Abstract]
Kimura, A. & Hansen, E. J. (1986). Antigenic and phenotypic variants of Haemophilus influenzae type b lipopolysaccharide and their relationship to virulence. Infect Immun 51, 60-79.[Medline]
Levinson, G. & Gutman, G. A. (1987). High frequencies of short frameshifts in poly-CA/TG tandem repeats borne by bacteriophage M13 in Escherichia coli K-12. Nucleic Acids Res 15, 5323-5338.[Abstract]
Maskell, D. J., Szabo, M. J., Butler, P. D., Williams, A. E. & Moxon, E. R. (1992). Molecular analysis of a complex locus from Haemophilus influenzae involved in phase-variable lipopolysaccharide biosynthesis. Mol Microbiol 5, 1013-1022.
Moxon, E. R. (1985). Haemophilus influenzae. In Principles and Practice of Infectious Disease, pp. 1274-1279. Edited by G. Mandrell, R. Douglas & J. Bennet. New York: Wiley.
Moxon, E. R., Smith, A. L., Averill, D. R. & Smith, D. H. (1974). Haemophilus influenzae meningitis in infant rats after intranasal inoculation. J Infect Dis 129, 154-162.[Medline]
Smith, A. L., Smith, D. H., Averill, D. R. & Moxon, E. R. (1973). Production of Haemophilus influenzae type B meningitis in infant rats by intraperitoneal inoculation. Infect Immun 8, 278-290.[Medline]
Weiser, J. N., Love, J. & Moxon, E. R. (1989). The molecular mechanism of phase-variation in Haemophilus influenzae lipopolysaccharide. Cell 59, 657-665.[Medline]
Weiser, J. N., Maskell, D. J., Butler, P. D. & Moxon, E. R. (1990). Characterisation of repetitive sequences controlling phase variation of Haemophilus influenzae lipopolysaccharide. J Bacteriol 172, 3304-3309.[Medline]
Weiser, J. N., Shchepetov, M. & Chong, S. T. (1997). Decoration of lipopolysaccharide with phosphorylcholine: a phase-variable characteristic of Haemophilus influenzae. Infect Immun 65, 943-950.[Abstract]
Weiser, J. N., Pan, N., McGowan, K. L., Musher, D., Martin, A. & Richards, J. (1998). Phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae contributes to persistence in the respiratory tract and sensitivity to serum killing mediated by C-reactive protein. J Exp Med 187, 631-640.
Whittle, H. C. & Greenwood, B. M. (1977). Cerebrospinal fluid immunoglobulins and complement in meningococcal meningitis. J Clin Pathol 31, 213-216.[Abstract]
Zwahlen, A., Nydegger, U. E., Vaudaux, P., Lambert, P.-H. & Waldvogel, F. A. (1982). Complement-mediated opsonic activity in normal and infected human cerebrospinal fluid: early response during bacterial meningitis. J Infect Dis 145, 635-646.[Medline]
Received 14 December 1998;
revised 30 June 1999;
accepted 8 July 1999.