Chromosomal location of Hif cap locus. Sequence analysis of the DNA adjacent to the Hif cap locus within cosmid pTS66 identified six additional ORFs, five highly homologous to previously identified ORFs from H. influenzae Rd (GenBank accession no. NC_ 000907): merT, sapA, HI1048, HI1637, and an incomplete copy of dmsA. An additional gene, sodC, not found in the Rd genome, is located immediately adjacent to bexA and the 5′ junction of the Hif cap locus. The layout of the flanking sequences with respect to the Hif cap locus is shown in Fig. 1. HI1048 and HI1637 are hypothetical proteins (GenBank accession no. AAC22710 and AAC23282, respectively); dmsA ( HI1047) encodes an anaerobic dimethyl sulfoxide reductase, chain A protein (GenBank accession no. AAC22706); merT ( HI1049) encodes a mercuric ion transport protein (GenBank accession no. AAC22707); sapA ( HI1638) encodes a peptide ABC transporter periplasmic protein (GenBank accession no. AAC23285); and sodC (GenBank accession no. M84012) encodes a copper-zinc superoxide dismutase (Cu,Zn-SOD). Unlike the DNA flanking the Hib cap locus, which is contiguous in the H. influenzae Rd genome, the DNA outside of the Hif cap locus is found in two widely separated sections of the Rd genome. dmsA ( HI1047), HI1048, and merT ( HI1049) are found in section 100 of the Rd genome, while HI1637 and sapA ( HI1638) are in sections 151 and 152, respectively ( sodC is not present in the Rd genome). Sections 100 and 151 to 152 are separated by 589,419 bp in Rd (GenBank accession no. NC_000907) ( 6). This finding was not necessarily unexpected. Rd, formerly an encapsulated H. influenzae serotype d strain, is a division I H. influenzae strain, and all H. influenzae serotype f strains belong to division II. Divisions I and II have been demonstrated to be widely separated phylogenetic divisions ( 30). Furthermore, sodC is present only in division II and not present in division I strains with the exception of serotype e, which shares features of both divisions I and II ( 15, 22). To determine if the layout of the Hif cap locus and flanking sequences found on the cosmid clones was identical in the type f chromosome, PCR analysis of the end junctions of the Hif cap locus was performed using primers specific to region I, sodC, merT, region III, and HI1637 (Table 1) and chromosomal DNA from Hif strain 700222. The same layout of genes was observed in Hif strain 700222 and the sequenced cosmid pTS66 (data not shown). We looked further at a collection of 69 Hif strains isolated from patients with invasive H. influenzae disease. Isolates were confirmed to be H. influenzae serotype f strains by serologic and molecular methods ( 5). All 69 Hif isolates were examined by one-colony PCR ( 32) using primer pairs as described above and were found to have the Hif cap locus in the identical location as that of Hif 700222 and associated with the same flanking genes (data not shown). This suggests that the chromosomal location of the Hif cap locus is highly conserved and is as presented in Fig. 1. It has been suggested that all division I H. influenzae strains have the same chromosomal cap locus location, between direct repeats of IS 1016 ( 34). It is possible that all cap loci in H. influenzae division II strains are located between sodC and HI1637. Previously, we noted that H. influenzae serotype a strain ATCC 9006 and H. influenzae serotype e strain 8142 did not have the same cap locus chromosomal location shared by most division I H. influenzae strains ( 34). Although most Hia belong to division I, some have been classified as division II. Hie, while classified as division I, has the greatest genetic distance from all other division I H. influenzae ( 29) and is the only division I H. influenzae reported to have sodC ( 22). We also examined the end junctions of Hia strain 9006 and Hie strain 8142 using primers specific to region I, sodC, merT, region III, and HI1637 (Table 1). Both the Hia and the Hie strains had the same PCR profile as the Hif strains, suggesting the identical chromosomal location of the cap loci in these strains (Fig. 3). Further studies will be necessary to determine whether the cap locus location is uniformly conserved among all H. influenzae division II strains (all Hif, and a minority of Hia and Hib) and division I Hie strains. | FIG. 3.PCR analysis of the end junctions and outside DNA of the Hif cap locus and H. influenzae serotypes a and e. Lanes 1 and 14, 1-kb standard marker (Promega Corp.). Hif 700222 chromosomal DNA was amplified with primer pairs capfMerT and SodC 3970-2951 (lane (more ...) |
The location of sodC adjacent to bexA and the capsule locus of H. influenzae NCTC8468, a division II type b strain, have been previously reported ( 15). Hif sodC is 98.4% identical to the Hib sodC described in detail by Kroll et al. ( 15) and also located adjacent to bexA. The identical palindromic pairs that match the 11-bp Haemophilus DNA uptake motif seen 19 nucleotides downstream from the stop codon of Hib sodC (GenBank accession no. M84012) ( 17) are found at the same location downstream from the termination codon in the Hif sodC. The sodC gene encodes for Cu,Zn-SOD, a metallo-enzyme catalyzing the conversion of superoxide radicals into hydrogen peroxide and oxygen ( 9, 12). Cu,Zn-SOD has been found in the periplasm of a wide range of gram-negative commensal and pathogenic bacteria, including Haemophilus species ( 22), and its role in virulence has been suggested for pathogenic bacteria ( 23). Unlike Fe-SODs and Mn-SODs, which are located in the cytosol and have a primary role of minimizing the effects from anaerobic respiration, Cu,Zn-SODs are exported from the cytosol to the periplasm or beyond. Since superoxide generated within the cytosol cannot cross the cytoplasmic membrane, this suggests that Cu,Zn-SODs provide protection from extracellular superoxide production by phagocytic host cells ( 23). The possession of a polysaccharide capsule greatly enhances the virulence of H. influenzae and its ability to cause invasive disease. Although the Hib conjugate vaccine has been highly successful in dramatically reducing Hib invasive disease, occurrence of invasive disease due to other capsule serotypes as well as nontypeable H. influenzae has persisted and may be increasing in some populations. The apparent increase of Hif disease in the post-Hib vaccine era suggests the need for further characterization of H. influenzae serotype f. We report the genetic analysis of the Hif cap locus and its surrounding DNA. Future studies to better understand the functional activities of the capsule-specific genes fcs1, fcs2, and fcs3 are warranted. |
REFERENCES 1. Adlakha, A., S. H. Yale, R. Patel, R. S. Edson, H. J. Schultz, and A. W. Stanson. 1994. Haemophilus influenzae serotype f: an unusual cause of a mycotic aneurysm in an adult. Mayo Clin. Proc. 69:467-468. [PubMed]. 2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. [PubMed]. 3. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. [PubMed]. 4. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1989. Current protocols in molecular biology. John Wiley & Sons, Inc., New York, N.Y. 5. Falla, T. J., D. W. M. Crook, L. N. Brophy, D. Maskell, J. S. Kroll, and E. R. Moxon. 1994. PCR for capsular typing of Haemophilus influenzae. J. Clin. Microbiol. 32:2382-2386. [PubMed]. 6. Fleischmann, R. D., M. D. Adams, O. White, R. A. Clayton, E. F. Kirkness, A. R. Kerlavage, C. J. Bult, J. F. Tomb, B. A. Dougherty, J. M. Merrick, K. McKenney, G. Sutton, W. FitzHugh, C. Fields, J. D. Gocayne, J. Scott, R. Shirley, L.-I. Liu, A. Glodek, J. M. Kelley, J. F. Weidman, C. A. Phillips, T. Spriggs, E. Hedblom, M. D. Cotton, T. R. Utterback, M. C. Hanna, D. T. Nguyen, D. M. Saudek, R. C. Brandon, L. D. Fine, J. L. Fritchman, J. L. Fuhrmann, N. S. M. Geoghagen, C. L. Gnehm, L. A. McDonald, K. V. Small, C. M. Fraser, H. O. Smith, and J. C. Venter. 1995. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496-512. [PubMed]. 7. Follens, A., M. Veiga-da-Cunha, R. Merckx, E. van Schaftingen, and J. van Eldere. 1999. acs1 of Haemophilus influenzae type a capsulation locus region II encodes a bifunctional ribulose 5-phosphate reductase-CDP-ribitol pyrophosphorylase. J. Bacteriol. 181:2001-2007. [PubMed]. 8. Frayha, H. H., A. K. Kalloghlian, and M. M. deMoor. 1996. Endocarditis due to Haemophilus influenzae serotype f. Clin. Infect. Dis. 23:401-402. [PubMed]. 9. Fridovich, I. 1986. Superoxide dismutases. Adv. Enzymol. Rel. Areas Mol. Biol. 58:61-97. 10. Frosch, M., and A. Muller. 1993. Phospholipid substitution of capsular polysaccharides and mechanisms of capsule formation in Neisseria meningitidis. Mol. Microbiol. 8:483-493. [PubMed]. 11. Geer, L. Y., M. Domrachev, D. J. Lipman, and S. H. Bryant. 2002. CDART: protein homology by domain architecture. Genome Res. 12:1619-1623. [PubMed]. 12. Hassan, H. M. 1989. Microbial superoxide dismutases. Adv. Genet. 26:65-97. [PubMed]. 13. Hoiseth, S. K., C. J. Connelly, and E. R. Moxon. 1985. Genetics of spontaneous, high-frequency loss of b capsule expression in Haemophilus influenzae. Infect. Immun. 49:389-395. [PubMed]. 14. Hoiseth, S. K., E. R. Moxon, and R. P. Silver. 1986. Genes involved in Haemophilus influenzae type b capsule expression are part of an 18-kilobase tandem duplication. Proc. Natl. Acad. Sci. USA 83:1106-1110. [PubMed]. 15. Kroll, J. S., P. R. Langford, and B. M. Loynds. 1991. Copper-zinc superoxide dismutase of Haemophilus influenzae and H. parainfluenzae. J. Bacteriol. 173:7449-7457. [PubMed]. 16. Kroll, J. S., B. Loynds, L. N. Brophy, and E. R. Moxon. 1990. The bex locus in encapsulated Haemophilus influenzae: a chromosomal region involved in capsule polysaccharide export. Mol. Microbiol. 4:1853-1862. [PubMed]. 17. Kroll, J. S., B. M. Loynds, and P. R. Langford. 1992. Palindromic Haemophilus DNA uptake sequences in presumed transcriptional terminators from H. influenzae and H. parainfluenzae. Gene 114:151-152. [PubMed]. 18. Kroll, J. S., B. M. Loynds, and E. R. Moxon. 1991. The Haemophilus influenzae capsulation gene cluster: a compound transposon. Mol. Microbiol. 5:1549-1560. [PubMed]. 19. Kroll, J. S., and E. R. Moxon. 1988. Capsulation and gene copy number at the cap locus of Haemophilus influenzae type b. J. Bacteriol. 170:859-864. [PubMed]. 20. Kroll, J. S., S. Zamze, B. Loynds, and E. R. Moxon. 1989. Common organization of chromosomal loci for production of different capsular polysaccharides in Haemophilus influenzae. J. Bacteriol. 171:3343-3347. [PubMed]. 21. Kunst, F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, V. Azevedo, M. G. Bertero, P. Bessieres, A. Bolotin, S. Borchert, R. Borriss, L. Boursier, A. Brans, M. Braun, S. C. Brignell, S. Bron, S. Brouillet, C. V. Bruschi, B. Caldwell, V. Capuano, N. M. Carter, S. K. Choi, J. J. Codani, I. F. Connerton, A. Danchin, et al. 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390:249-256. [PubMed]. 22. Langford, P. R., B. M. Loynds, and J. S. Kroll. 1992. Copper-zinc superoxide dismutase in Haemophilus species. J. Gen. Microbiol. 138:517-522. [PubMed]. 23. Langford, P. R., B. J. Sheehan, T. Shaikh, and J. S. Kroll. 2002. Active copper- and zinc-containing superoxide dismutase in the cryptic genospecies of Haemophilus causing urogenital and neonatal infections discriminates them from Haemophilus influenzae sensu stricto. J. Clin. Microbiol. 40:268-270. [PubMed]. 24. Leaves, N. I., T. J. Falla, and D. W. M. Crook. 1995. The elucidation of novel capsular genotypes of Haemophilus influenzae type b with the polymerase chain reaction. J. Med. Microbiol. 43:120-124. [PubMed]. 25. Lo, R. Y., L. J. McKerral, T. L. Hills, and M. Kostrzynska. 2001. Analysis of the capsule biosynthetic locus of Mannheimia ( Pasteurella) haemolytica A1 and proposal of a nomenclature system. Infect. Immun. 69:4458-4464. [PubMed]. 26. Marchler-Bauer, A., J. B. Anderson, C. DeWeese-Scott, N. D. Fedorova, L. Y. Geer, S. He, D. I. Hurwitz, J. D. Jackson, A. R. Jacobs, C. J. Lanczycki, C. A. Liebert, C. Liu, T. Madej, G. H. Marchler, R. Mazumder, A. N. Nikolskaya, A. R. Panchenko, B. S. Rao, B. A. Shoemaker, V. Simonyan, J. S. Song, P. A. Thiessen, S. Vasudevan, Y. Wang, R. A. Yamashita, J. J. Yin, and S. H. Bryant. 2003. CDD: a curated Entrez database of conserved domain alignments. Nucleic Acids Res. 31:383-387. [PubMed]. 27. Marchler-Bauer, A., A. R. Panchenko, B. A. Shoemaker, P. A. Thiessen, L. Y. Geer, and S. H. Bryant. 2002. CDD: a database of conserved domain alignments with links to domain three-dimensional structure. Nucleic Acids Res. 30:281-283. [PubMed]. 28. Moxon, E. R., and J. S. Kroll. 1990. The role of bacterial polysaccharide capsules as virulence factors. Curr. Top. Microbiol. Immunol. 150:65-85. [PubMed]. 29. Musser, J. M., J. S. Kroll, D. M. Granoff, E. R. Moxon, B. R. Brodeur, J. Campos, H. Dabernat, W. Frederiksen, J. Hamel, G. Hammond, E. A. Hoiby, K. E. Jonsdottir, M. Kabeer, I. Kallings., W. N. Khan, M. Kilian, K. Knowles, H. J. Koornhof, B. Law, K. I. Li, J. Montgomery, P. E. Pattison, J.-C. Piffaretti, A. K. Takala, M. L. Thong, R. A. Wall, J. I. Ward, and R. K. Selander. 1990. Global genetic structure and molecular epidemiology of encapsulated Haemophilus influenzae. Rev. Infect. Dis. 12:75-111. [PubMed]. 30. Musser, J. M., J. S. Kroll, E. R. Moxon, and R. K. Selander. 1988. Evolutionary genetics of the encapsulated strains of Haemophilus influenzae. Proc. Natl. Acad. Sci. USA 85:7758-7762. [PubMed]. 31. Pittman, M. 1931. Variation and type specificity in the bacterial species Haemophilus influenzae. J. Exp. Med. 53:471-492. 32. Read, T. D., S. W. Satola, J. A. Opdyke, and M. M. Farley. 1998. Copy number of pilus gene clusters in Haemophilus influenzae and variation in the hifE pilin gene. Infect. Immun. 66:1622-1631. [PubMed]. 33. Sambrook, J., E. F. Fritsh, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 34. Satola, S. W., P. L. Schirmer, and M. M. Farley. 2003. Complete sequence of the cap locus of Haemophilus influenzae serotype b and nonencapsulated b capsule-negative variants. Infect. Immun. 71:3639-3644. [PubMed]. 35. Slater, L. N., J. Guarnaccia, S. Makintubee, and G. R. Istre. 1990. Bacteremic disease due to Haemophilus influenzae capsular type f in adults: report of five cases and review. Rev. Infect. Dis. 12:628-635. [PubMed]. 36. Swartley, J. S., L. J. Liu, Y. K. Miller, L. E. Martin, S. Edupuganti, and D. S. Stephens. 1998. Characterization of the gene cassette required for biosynthesis of the (α1→6)-linked N-acetyl-d-mannosamine-1-phosphate capsule of serogroup A Neisseria meningitidis. J. Bacteriol. 180:1533-1539. [PubMed]. 37. Tatusov, R. L., E. V. Koonin, and D. J. Lipman. 1997. A genomic perspective on protein families. Science 278:631-637. [PubMed]. 38. Tatusov, R. L., D. A. Natale, I. V. Garkavtsev, T. A. Tatusova, U. T. Shankavaram, B. S. Rao, B. Kiryutin, M. Y. Galperin, N. D. Fedorova, and E. V. Koonin. 2001. The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Res. 29:22-28. [PubMed]. 39. Trollfors, B., B. Claesson, T. Lagergard, and T. Sandberg. 1984. Incidence, predisposing factors and manifestations of invasive Haemophilus influenzae infections in adults. Eur. J. Clin. Microbiol. 3:180-184. [PubMed]. 39a. Tzeng, Y.-L., C. Noble, and D. S. Stephens. 2003. Genetic basics for biosynthesis of the (α1->4)-linked N-acetyl-d-glucosamine 1-phosphate capsule of Neisseria meningitidis serogroup X. Infect. Immun. 71:6712-6720. [PubMed]. 40. Urwin, G., J. A. Krohn, K. Deaver-Robinson, J. D. Wenger, M. M. Farley, et al. 1996. Invasive disease due to Haemophilus influenzae serotype f: clinical and epidemiologic characteristics in the H. influenzae serotype b vaccine era. Clin. Infect. Dis. 22:1069-1076. [PubMed]. 41. van Eldere, J., L. Brophy, B. Loynds, P. Celis, I. Hancock, S. Carman, J. S. Kroll, and E. R. Moxon. 1995. Region II of the Haemophilus influenzae type b capsulation locus is involved in serotype-specific polysaccharide synthesis. Mol. Microbiol. 15:107-118. [PubMed]. 42. van Kranenburg, R., I. I. van Swam, J. D. Marugg, M. Kleerebezem, and W. M. de Vos. 1999. Exopolysaccharide biosynthesis in Lactococcus lactis NIZO B40: functional analysis of the glycosyltransferase genes involved in synthesis of the polysaccharide backbone. J. Bacteriol. 181:338-340. [PubMed]. 43. Wallace, R. J., Jr., D. M. Musher, E. J. Septimus, J. E. McGowan, Jr., F. J. Quinones, K. Wiss, P. H. Vance, and P. A. Trier. 1981. Haemophilus influenzae infections in adults: characterization of strains by serotypes, biotypes, and beta-lactamase production. J. Infect. Dis. 144:101-106. [PubMed]. 44. Wenger, J. D., R. Pierce, K. Deaver, R. Franklin, G. Bosley, N. Pigott, and C. V. Broome. 1992. Invasive Haemophilus influenzae disease: a population-based evaluation of the role of capsular polysaccharide serotype. J. Infect. Dis. 165:(Suppl. 1) :S34-S35. [PubMed]. 45. Zacharisen, M. C., S. K. Watters, and J. Edwards. 2003. Rapidly fatal Haemophilus influenzae serotype f sepsis in a healthy child. J. Infect. 46:194-196. [PubMed]. |