当前位置: 首页 > 医学版 > 期刊论文 > 基础医学 > 感染与免疫杂志 > 2005年 > 第10期 > 正文
编号:11254267
Digalactoside Expression in the Lipopolysaccharide of Haemophilus influenzae and Its Role in Intravascular Survival
     Molecular Infectious Diseases Group, University of Oxford Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom

    Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, KIA 0R6Canada

    ABSTRACT

    Digalactoside (gal-1-4 gal) structures of the lipopolysaccharide (LPS) of Haemophilus influenzae are implicated in virulence. A confounding factor is that tetranucleotide repeats within the lic2A, lgtC, and lex2 genes mediate phase-variable expression of the digalactosides. By deleting these repeats, we constructed recombinant strains of RM153 constitutively expressing either one or two LPS digalactosides. Expression of two digalactosides, rather than one, was associated with increased virulence of H. influenzae in vivo.

    TEXT

    Lipopolysaccharide (LPS) is one of the major virulence determinants of the human pathogen Haemophilus influenzae. The number of hexoses and phosphate groups replacing the triheptose (HepI to HepIII) backbone (Fig. 1) is variable within any strain owing to high-frequency translational switching (phase variation) of LPS genes containing repeat tracts. Phase-variable LPS genes include lic2A and lgtC, which are involved in the assembly of gal-1-4 gal into the oligosaccharide extensions from the conserved triheptose backbone (7, 10), and lex2, which is involved in completion of the HepI-attached diglucoside acceptor for this digalactoside (Fig. 1) (4). The investigation of the association between digalactoside expression on H. influenzae LPS and virulence has relied on the monoclonal antibody (MAb) 4C4 for detecting the expression of the digalactoside (1, 2, 12, 13). The findings from these studies have been difficult to interpret because of the confounding factor of phase variation and the different numbers and locations of the digalactoside on oligosaccharide extensions in different strains (7, 17, 18).

    Recently, it has been shown that MAb 4C4 binds digalactosides that are part of the extensions from both HepI and HepII of the triheptose LPS backbone (Fig. 1) (4, 5). A type b clinical strain, RM153, used in previous virulence studies (9, 16), typically generates LPS molecules containing only four hexose sugars (17), depending on whether lic2A, lgtC, or lex2 is out of frame (4, 7, 10). This is in contrast to the related strain RM7004, which has up to nine hexose sugars in its LPS due to these three loci being predominantly in frame (Fig. 1). Knowing the LPS structure, the genes required for digalactoside assembly, and the fact that tetranucleotide repeats mediate phase variation, we constructed recombinant strains of RM153 in which variable expression of digalactosides was eliminated by removing the repeat tracts located within each of these genes. Briefly, two isogenic strains were constructed by transformation (6) using appropriate chromosomal DNA or plasmid constructs in which the repeats had been deleted for lic2A (8), lex2 (5), or lgtC. All three genes were constitutively expressed in the first strain, RM153lic2A+tlgtC+lex2+k, to facilitate expression of two digalactosides, while in the second strain, RM153lic2A+tlgtC+lex2–k, lex2 was mutated such that only a single digalactoside in the extension from HepII was expressed (Fig. 1).

    The strains were constructed as follows. An in-frame deletion of the repeat tract of lic2A in RM153 was generated by transformation with chromosomal DNA of a derivative of RM7004 in which the 5'-CAAT-3' repeats had been deleted (8). Our construct differed from that of High and coworkers (8) only in that the kanR antibiotic resistance cassette was replaced with tetR. Into this strain (designated RM153lic2A+t), we introduced an in-frame deletion of the repeats in lex2A by transformation with pBlex25'-GCAA-3'k (4) and selection for kanamycin resistance. The resultant strain, RM153lic2A+lex2+k, was then transformed with pUClgtC5'-GACA-3'lacZ, in which lacZ is fused to the 5' end of lgtC (Fig. 2C). The disrupted lgtC gene of a lacZ-positive transformant of RM153lic2A+lex2+k was rescued by transformation with pUClgtC5'-GACA-3', which carries an in-frame deletion of the repeat tract of lgtC (Fig. 2B). Briefly, pUClgtC5'-GACA-3' and pUClgtC5'-GACA-3'lacZ were created as follows. A primer (LGTC3268R) was designed to include BglII and XhoI sites followed by sequence complementary to sequence upstream of and including the initiation codon of lgtC (Fig. 2A). Another primer, LGTCRPS1 (5'-TCGAGATCTACGGACTGTCAGTCAGACAATG-3'), was designed to include a BglII site followed by sequence immediately downstream of the repeat tract (Fig. 2A). Plasmid pBSHI, incorporating the region encompassing lgtC of RM153 (Fig. 2A), was used as a source of DNA. The region upstream of lgtC was amplified using primers LGTC3268R (5'-TGACTGACAGTCCGTCCGTCAGATCTCGAGACGCGTTCATGAAATTATCTCTGATT-3') and 6024J (5'-TCGTAAGGAATAAGCGTG-3'), and the downstream region was amplified using primers LGTCRPS1 and T7 (Fig. 2A) (23). These fragments were cloned separately and then fused using the BglII site and other appropriate cloning sites in the vector plasmids to generate pUClgtC5'-GACA-3' (Fig. 2B). Plasmid pUClgtC5'-GACA-3'lacZ was created by replacing the XhoI-HindIII fragment of pUClgtC5'-GACA-3' with a XhoI-BamHI fragment of pGZMCS (3), incorporating lacZ without an initiation codon. Transformants constitutively expressing all three loci were selected by their ability to react with MAb 4C4 and designated RM153lic2A+tlgtC+lex2+k.

    To obtain organisms expressing one digalactoside only, lex2 was disrupted in a strain in which lic2A and lgtC expression was constitutive. This strain was constructed as follows. RM153lic2A+t was transformed with a construct carrying lgtC disrupted by kanR (10), and then lgtC expression was restored by transformation with pUClgtC5'-GACA-3' (Fig. 2B). Transformants were selected for their restored ability to react with MAb 4C4. Finally, lex2 was disrupted by transformation with pDL2 (4), and transformants were selected by growth on kanamycin. The resultant strain was designated RM153lic2A+tlgtC+lex2–k. To confirm that the loci lacked repeats and were maintained constitutively in frame in these strains, the repeat region was amplified by PCR and sequenced using appropriate primers. Note that a plus in a strain designation indicates that the relevant gene lacks repeats and is therefore constitutively expressed. A minus in a strain designation indicates a disrupted gene that is not expressed. The designations "t" and "k" indicate that selection for these genes was dependent upon tetracycline or kanamycin antibiotic resistance cassettes, respectively.

    The magnitude of bacteremia for organisms expressing two digalactosides, compared to that for organisms expressing one digalactoside, was investigated in infant rats to assess the role of gal-1-4 gal expression in intravascular survival in an in vivo model.

    Prior to mixed infection of infant rats, isogenic streptomycin-resistant (Strr) mutants were obtained for RM153lic2A+tlgtC+lex2+k and RM153lic2A+tlgtC+lex2–k. Use of these mutants permitted discrimination between these strains following challenge of the rats with a mixed inoculum by plating blood cultures from infected animals onto medium with or without streptomycin. The strains were transformed with chromosomal DNA of a spontaneous Strr clone of RM153. The resultant Strr isogenic strains showed no alteration in their LPS compared to that of their progenitors and maintained the deletions in the repeat tracts. Each of the four recombinant strains showed no difference in growth rate (data not shown).

    Twenty-eight 5-day-old Sprague-Dawley rats were each given mixed infections by the intraperitoneal route (9): 15 rats were inoculated with approximately 150 CFU of RM153lic2A+tlgtC+lex2+k and 150 CFU of Strr RM153lic2A+tlgtC+lex2–k, while 13 received 150 CFU of each strain in which the antibiotic resistance marker was switched so that the former strain carrying the Strr marker was now streptomycin sensitive. Forty-eight hours after inoculation, significantly higher numbers of bacteria expressing two digalactosides than of the single-digalactoside-expressing strain were recovered from tail vein blood from infant rats, as determined by the nonparametric Mann-Whitney U test (P value, 0.0287) (Table 1). The paired data in Table 1 were also used to derive a competition ratio for bacteria expressing two digalactosides versus one digalactoside (ratio determined by dividing the number of CFU of strain RM153lic2A+tlgtC+lex2+k by that of strain RM153lic2A+tlgtC+lex2–k). The average ratio was higher when the single-digalactoside-expressing strain was streptomycin resistant, although this antibiotic resistance mutation could be associated with a small fitness deficit. However, as the average ratio was greater than 1 in both groups of animals, this result provides further evidence that increased digalactoside expression leads to enhanced virulence.

    In order to substantiate the data from the animal studies, we investigated the LPS expression patterns of the two isogenic test strains used to infect rats. These strains had been cultured in 1% galactose, which was previously shown to encourage the incorporation of galactose into the LPS (16).

    First, the reactivity of colonies of the test strains was investigated using MAb 4C4 (21). RM153lic2A+tlgtC+lex2–k showed the reactive (R) phenotype only, suggesting the expression of a single digalactoside, while RM153lic2A+tlgtC+lex2+k demonstrated the strongly reacting (S) phenotype only, indicative of the expression of two digalactosides (Fig. 3A) as documented for RM7004 (4).

    Second, duplicate sodium dodecyl sulfate-polyacrylamide gels were run with whole-cell lysates (14, 22). One gel was stained with silver (20), while the other was blotted onto a membrane and reacted with MAb 4C4 (24). The predominant band of LPS from RM153lic2A+tlgtC+lex2–k showed migration equivalent to that expected for glycoforms comprising six hexose sugars and showed staining, albeit weak, with MAb 4C4, thus confirming the presence of a single digalactoside (Fig. 3). Strain RM153lic2A+tlgtC+lex2+k showed additional bands; each incremental band size is considered to represent an additional hexose (9). A band corresponding to the presence of nine hexose sugars and reactive with MAb 4C4 was detected for RM153lic2A+tlgtC+lex2+k, as for RM7004, which typically contains each of the three loci in frame and expresses two digalactoside-containing oligosaccharides (Fig. 3) (18).

    Finally, the presence of nine hexose sugars in RM153lic2A+tlgtC+lex2+k was confirmed by electrospray-ionization mass spectrometry (Table 2) (15). Compositional sugar analysis (15) indicated a 5:4 ratio of galactose to glucose, analogous to the fully extended glycoform of RM7004.

    In conclusion, variants capable of expressing two rather than one digalactoside were more virulent in vivo. These data provide strong indications of the importance of the gal-1-4 gal digalactoside structure of LPS in virulence, but some caveats must be considered in interpreting these results. First, the relevance of findings from an infant rat model for humans is of course open to question, especially since host cells in the rat express digalactosides in which the galactose linkage is 1-3 (19). In contrast, humans express an 1-4 digalactoside identical to that found on the LPS of H. influenzae. Another important potentially confounding factor is the extent of LPS sialylation. We cannot exclude the possibility that the differences in virulence observed could be attributed, at least in part, to differences in sialylated LPS glycoforms (11), or indeed other unrecognized but relevant and subtle differences in LPS structure that are independent of digalactoside expression.

    ACKNOWLEDGMENTS

    This work was funded by the Medical Research Council, United Kingdom.

    MAb 4C4 was kindly provided by E. J. Hansen (University of Texas). We thank Adele Martin for LPS purification and O deacylation and Don Krajcarski for electrospray-ionization mass spectrometry.

    Present address: Centre for Molecular Microbiology and Infection, Level 3, Flowers Building, Imperial College, London SW7 2AZ, United Kingdom. Phone: 0207 5943094. Fax: 0207 5943095. E-mail: r.griffin@imperial.ac.uk.

    REFERENCES

    1. Cope, L. D., R. Yogev, J. Mertsola, J. C. Argyle, G. H. McCracken, Jr., and E. J. Hansen. 1990. Effect of mutations in lipooligosaccharide biosynthesis genes on virulence of Haemophilus influenzae type b. Infect. Immun. 58:2343-2351.

    2. Cope, L. D., R. Yogev, J. Mertsola, J. L. Latimer, M. S. Hanson, G. H. McCracken, Jr., and E. J. Hansen. 1991. Molecular cloning of a gene involved in lipooligosaccharide biosynthesis and virulence expression by Haemophilus influenzae type b. Mol. Microbiol. 5:1113-1124.

    3. De Bolle, X., C. D. Bayliss, D. Field, T. van de Ven, N. J. Saunders, D. W. Hood, and E. R. Moxon. 2000. The length of a tetranucleotide repeat tract in Haemophilus influenzae determines the phase variation rate of a gene with homology to type III DNA methyltransferases. Mol. Microbiol. 35:211-222.

    4. Griffin, R., A. D. Cox, K. Makepeace, J. C. Richards, E. R. Moxon, and D. W. Hood. 2003. The role of lex2 in lipopolysaccharide biosynthesis in Haemophilus influenzae strains RM7004 and RM153. Microbiology 149:3165-3175.

    5. Griffin, R., A. D. Cox, K. Makepeace, J. C. Richards, E. R. Moxon, and D. W. Hood. 2005. Elucidation and role in serum resistance of the monoclonal antibody 5G8-reactive, virulence-associated lipopolysaccharide epitope of Haemophilus influenzae and its role in bacterial resistance to complement-mediated killing. Infect. Immun. 73:2213-2221.

    6. Herriott, R. M., E. Y. Meyer, and M. Vogt. 1970. Defined nongrowth media for stage II development of competence in Haemophilus influenzae. J. Bacteriol. 101:517-524.

    7. High, N. J., M. E. Deadman, and E. R. Moxon. 1993. The role of a repetitive DNA motif (5'-CAAT-3') in the variable expression of the Haemophilus influenzae lipopolysaccharide epitope alpha Gal(1-4)beta Gal. Mol. Microbiol. 9:1275-1282.

    8. High, N. J., M. P. Jennings, and E. R. Moxon. 1996. Tandem repeats of the tetramer 5'-CAAT-3' present in lic2A are required for phase variation but not lipopolysaccharide biosynthesis in Haemophilus influenzae. Mol. Microbiol. 20:165-174.

    9. Hood, D. W., M. E. Deadman, T. Allen, H. Masoud, A. Martin, J. R. Brisson, R. Fleischmann, J. C. Venter, J. C. Richards, and E. R. Moxon. 1996. Use of the complete genome sequence information of Haemophilus influenzae strain Rd to investigate lipopolysaccharide biosynthesis. Mol. Microbiol. 22:951-965.

    10. Hood, D. W., M. E. Deadman, M. P. Jennings, M. Bisercic, R. D. Fleischmann, J. C. Venter, and E. R. Moxon. 1996. DNA repeats identify novel virulence genes in Haemophilus influenzae. Proc. Natl. Acad. Sci. USA 93:11121-11125.

    11. Hood, D. W., G. Randle, A. D. Cox, K. Makepeace, J. Li, E. K. H. Schweda, J. C. Richards, and E. R. Moxon. 2004. Biosynthesis of cryptic lipopolysaccharide glycoforms in Haemophilus influenzae involves a mechanism similar to that required for O-antigen synthesis. J. Bacteriol. 186:7429-7439.

    12. Kimura, A., and E. J. Hansen. 1986. Antigenic and phenotypic variations of Haemophilus influenzae type b lipopolysaccharide and their relationship to virulence. Infect. Immun. 51:69-79.

    13. Kimura, A., C. C. Patrick, E. E. Miller, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen. 1987. Haemophilus influenzae type b lipooligosaccharide: stability of expression and association with virulence. Infect. Immun. 55:1979-1986.

    14. Lesse, A. J., A. A. Campagnari, W. E. Bittner, and M. A. Apicella. 1990. Increased resolution of lipopolysaccharides and lipooligosaccharides utilising tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. J. Immunol. Methods 126:109-117.

    15. Lysenko, E., J. C. Richards, A. D. Cox, A. Stewart, A. Martin, M. Kapoor, and J. N. Weiser. 2000. The position of phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae affects binding and sensitivity to C-reactive protein mediated killing. Mol. Microbiol. 35:234-245.

    16. Maskell, D. J., M. J. Szabo, M. E. Deadman, and E. R. Moxon. 1992. The gal locus from Haemophilus influenzae: cloning, sequencing and the use of gal mutants to study lipopolysaccharide. Mol. Microbiol. 6:3051-3063.

    17. Masoud, H., E. R. Moxon, A. Martin, D. Krajcarski, and J. C. Richards. 1997. Structure of the variable and conserved lipopolysaccharide oligosaccharide epitopes expressed by Haemophilus influenzae serotype b strain Eagan. Biochemistry 36:2091-2103.

    18. Masoud, H., A. Martin, P. Thibault, E. R. Moxon, and J. C. Richards. 2003. Structure of extended lipopolysaccharide glycoforms containing two globotriose units in the Haemophilus influenzae serotype b strain, RM7004. Biochemistry 42:4463-4475.

    19. Naiki, M., and D. M. Marcus. 1975. An immunochemical study of the human blood group P1, P, and PK glycosphingolipid antigens. Biochemistry 14:4837-4841.

    20. Roche, R. J., N. J. High, and E. R. Moxon. 1994. Phase variation of Haemophilus influenzae lipopolysaccharide: characterisation of lipopolysaccharide from individual colonies. FEMS Microbiol. Lett. 120:279-284.

    21. Roche, R. J., and E. R. Moxon. 1995. Phenotypic variation in Haemophilus influenzae: the interrelationship of colony opacity, capsule and lipopolysaccharide. Microb. Pathog. 18:129-140.

    22. Serino, L., and M. Virji. 2000. Phosphorylcholine decoration of lipopolysaccharide differentiates commensal Neisseriae from pathogenic strains: identification of licA-type genes in commensal Neisseriae. Mol. Microbiol. 35:1550-1559.

    23. Short, J. M., J. M. Fernandez, J. A. Sorge, and W. D. Huse. 1988. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 16:7583-7600.

    24. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354.(Ruth Griffin, Chris D. Ba)