1887

Abstract

To identify transcripts that are expressed during human infection, we used selective capture of transcribed sequences (SCOTS) with RNA isolated from pustules obtained from three volunteers infected with , and with RNA isolated from broth-grown bacteria used to infect volunteers. With SCOTS, competitive hybridization of tissue-derived and broth-derived sequences identifies genes that may be preferentially expressed . Among the three tissue specimens, we identified 531 genes expressed . Southern blot analysis of 60 genes from each tissue showed that 87 % of the identified genes hybridized better with cDNA derived from tissue specimens than with cDNA derived from broth-grown bacteria. RT-PCR on nine additional pustules confirmed expression of 10 of 11 selected genes in other volunteers. Of the 531 genes, 139 were identified in at least two volunteers. These 139 genes fell into several functional categories, including biosynthesis and metabolism, regulation, and cellular processes, such as transcription, translation, cell division, DNA replication and repair, and transport. Detection of genes involved in anaerobic and aerobic respiration indicated that likely encounters both microenvironments within the pustule. Other genes detected suggest an increase in DNA damage and stress . Genes involved in virulence in other bacterial pathogens and 32 genes encoding hypothetical proteins were identified, and may represent novel virulence factors. We identified three genes, , and , known to be required for virulence in humans. This is the first study to broadly define transcripts expressed by in humans.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/013953-0
2008-04-01
2019-08-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/4/1152.html?itemId=/content/journal/micro/10.1099/mic.0.2007/013953-0&mimeType=html&fmt=ahah

References

  1. Al-Tawfiq, J. A., Thornton, A. C., Katz, B. P., Fortney, K. R., Todd, K. D., Hood, A. F. & Spinola, S. M. ( 1998; ). Standardization of the experimental model of Haemophilus ducreyi infection in human subjects. J Infect Dis 178, 1684–1687.[CrossRef]
    [Google Scholar]
  2. Al-Tawfiq, J. A., Fortney, K. R., Katz, B. P., Elkins, C. & Spinola, S. M. ( 2000; ). An isogenic hemoglobin receptor-deficient mutant of Haemophilus ducreyi is attenuated in the human model of experimental infection. J Infect Dis 181, 1049–1054.[CrossRef]
    [Google Scholar]
  3. Baltes, N. & Gerlach, G. F. ( 2004; ). Identification of genes transcribed by Actinobacillus pleuropneumoniae in necrotic porcine lung tissue by using selective capture of transcribed sequences. Infect Immun 72, 6711–6716.[CrossRef]
    [Google Scholar]
  4. Bauer, M. E. & Spinola, S. M. ( 2000; ). Localization of Haemophilus ducreyi at the pustular stage of disease in the human model of infection. Infect Immun 68, 2309–2314.[CrossRef]
    [Google Scholar]
  5. Bauer, M. E., Goheen, M. P., Townsend, C. A. & Spinola, S. M. ( 2001; ). Haemophilus ducreyi associates with phagocytes, collagen, and fibrin and remains extracellular throughout infection of human volunteers. Infect Immun 69, 2549–2557.[CrossRef]
    [Google Scholar]
  6. Bauer, M. E., Townsend, C. A., Ronald, A. R. & Spinola, S. M. ( 2006; ). Localization of Haemophilus ducreyi in naturally acquired chancroidal ulcers. Microbes Infect 8, 2465–2468.[CrossRef]
    [Google Scholar]
  7. Bong, C. T. H., Throm, R. E., Fortney, K. R., Katz, B. P., Hood, A. F., Elkins, C. & Spinola, S. M. ( 2001; ). DsrA-deficient mutant of Haemophilus ducreyi is impaired in its ability to infect human volunteers. Infect Immun 69, 1488–1491.[CrossRef]
    [Google Scholar]
  8. Bong, C. T. H., Bauer, M. E. & Spinola, S. M. ( 2002a; ). Haemophilus ducreyi: clinical features, epidemiology, and prospects for disease control. Microbes Infect 4, 1141–1148.[CrossRef]
    [Google Scholar]
  9. Bong, C. T. H., Harezlak, J., Katz, B. P. & Spinola, S. M. ( 2002b; ). Men are more susceptible to pustule formation than women in the experimental model of Haemophilus ducreyi infection. Sex Transm Dis 29, 114–118.[CrossRef]
    [Google Scholar]
  10. Camilli, A. & Mekalanos, J. J. ( 1995; ). Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection. Mol Microbiol 18, 671–683.[CrossRef]
    [Google Scholar]
  11. Cano, D. A., Pucciarelli, M. G., García-del Portillo, F. & Casadesús, J. ( 2002; ). Role of the RecBCD recombination pathway in Salmonella virulence. J Bacteriol 184, 592–595.[CrossRef]
    [Google Scholar]
  12. Chiang, S. L., Mekalanos, J. J. & Holden, D. W. ( 1999; ). In vivo genetic analysis of bacterial virulence. Annu Rev Microbiol 53, 129–154.[CrossRef]
    [Google Scholar]
  13. Cunin, R., Glansdorff, N., Piérard, A. & Stalon, V. ( 1986; ). Biosynthesis and metabolism of arginine in bacteria. Microbiol Rev 50, 314–352.
    [Google Scholar]
  14. Daigle, F., Graham, J. E. & Curtiss, R., III ( 2001; ). Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences (SCOTS). Mol Microbiol 41, 1211–1222.
    [Google Scholar]
  15. Daigle, F., Hou, J. Y. & Clark-Curtiss, J. E. ( 2002; ). Microbial gene expression elucidated by selective capture of transcribed sequences (SCOTS). Methods Enzymol 358, 108–122.
    [Google Scholar]
  16. Dozois, C. M., Daigle, F. & Curtiss, R., III ( 2003; ). Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain. Proc Natl Acad Sci U S A 100, 247–252.[CrossRef]
    [Google Scholar]
  17. Faucher, S. P., Porwollik, S., Dozois, C. M., McClelland, M. & Daigle, F. ( 2006; ). Transcriptome of Salmonella enterica serovar Typhi within macrophages revealed through the selective capture of transcribed sequences. Proc Natl Acad Sci U S A 103, 1906–1911.[CrossRef]
    [Google Scholar]
  18. Fortney, K. R., Young, R. S., Bauer, M. E., Katz, B. P., Hood, A. F., Munson, R. S., Jr & Spinola, S. M. ( 2000; ). Expression of peptidoglycan-associated lipoprotein is required for virulence in the human model of Haemophilus ducreyi infection. Infect Immun 68, 6441–6448.[CrossRef]
    [Google Scholar]
  19. Froussard, P. ( 1992; ). A random-PCR method (rPCR) to construct whole cDNA library from low amounts of RNA. Nucleic Acids Res 20, 2900 [CrossRef]
    [Google Scholar]
  20. Fulcher, R. A., Cole, L. E., Janowicz, D. M., Toffer, K. L., Fortney, K. R., Katz, B. P., Orndorff, P. E., Spinola, S. M. & Kawula, T. H. ( 2006; ). Expression of Haemophilus ducreyi collagen binding outer membrane protein NcaA is required for virulence in swine and human challenge models of chancroid. Infect Immun 74, 2651–2658.[CrossRef]
    [Google Scholar]
  21. Gourse, R. L., Gaal, T., Bartlett, M. S., Appleman, J. A. & Ross, W. ( 1996; ). rRNA transcription and growth rate-dependent regulation of ribosome synthesis in Escherichia coli. Annu Rev Microbiol 50, 645–677.[CrossRef]
    [Google Scholar]
  22. Graham, J. E. & Clark-Curtiss, J. E. ( 1999; ). Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc Natl Acad Sci U S A 96, 11554–11559.[CrossRef]
    [Google Scholar]
  23. Graham, J. E., Peek, R. M., Jr, Krishna, U. & Cover, T. L. ( 2002; ). Global analysis of Helicobacter pylori gene expression in human gastric mucosa. Gastroenterology 123, 1637–1648.[CrossRef]
    [Google Scholar]
  24. Grass, S., Buscher, A. Z., Swords, W. E., Apicella, M. A., Barenkamp, S. J., Ozchlewski, N. & St. Geme, J. W., III ( 2003; ). The Haemophilus influenzae HMW1 adhesin is glycosylated in a process that requires HMW1C and phosphoglucomutase, an enzyme involved in lipooligosaccharide biosynthesis. Mol Microbiol 48, 737–751.[CrossRef]
    [Google Scholar]
  25. Haydel, S. E., Benjamin, W. H., Jr, Dunlap, N. E. & Clark-Curtiss, J. E. ( 2002; ). Expression, autoregulation, and DNA binding properties of the Mycobacterium tuberculosis TrcR response regulator. J Bacteriol 184, 2192–2203.[CrossRef]
    [Google Scholar]
  26. Hays, R. C. & Mandell, G. L. ( 1974; ). pO2, pH, and redox potential of experimental abscesses. Proc Soc Exp Biol Med 147, 29–30.[CrossRef]
    [Google Scholar]
  27. Hou, J. Y., Graham, J. E. & Clark-Curtiss, J. E. ( 2002; ). Mycobacterium avium genes expressed during growth in human macrophages detected by selective capture of transcribed sequences (SCOTS). Infect Immun 70, 3714–3726.[CrossRef]
    [Google Scholar]
  28. Janowicz, D. M., Fortney, K. R., Katz, B. P., Latimer, J. L., Deng, K., Hansen, E. J. & Spinola, S. M. ( 2004; ). Expression of the LspA1 and LspA2 proteins by Haemophilus ducreyi is required for virulence in human volunteers. Infect Immun 72, 4528–4533.[CrossRef]
    [Google Scholar]
  29. Janowicz, D. M., Leduc, I., Fortney, K. R., Katz, B. P., Elkins, C. & Spinola, S. M. ( 2006a; ). A DltA mutant of Haemophilus ducreyi is partially attenuated in its ability to cause pustules in human volunteers. Infect Immun 74, 1394–1397.[CrossRef]
    [Google Scholar]
  30. Janowicz, D. M., Luke, N. R., Fortney, K. R., Katz, B. P., Campagnari, A. A. & Spinola, S. M. ( 2006b; ). Expression of OmpP2A and OmpP2B is not required for pustule formation by Haemophilus ducreyi in human volunteers. Microb Pathog 40, 110–115.[CrossRef]
    [Google Scholar]
  31. Jennings, M. P. & Beacham, I. R. ( 1990; ). Analysis of the Escherichia coli gene encoding l-asparaginase II, ansB, and its regulation by cyclic AMP receptor. J Bacteriol 172, 1491–1498.
    [Google Scholar]
  32. Liu, S., Graham, J. E., Bigelow, L., Morse, P. D., II & Wilkinson, B. J. ( 2002; ). Identification of Listeria monocytogenes genes expressed in response to growth at low temperature. Appl Environ Microbiol 68, 1697–1705.[CrossRef]
    [Google Scholar]
  33. Mahan, M. J., Slauch, J. M. & Mekalanos, J. J. ( 1993; ). Selection of bacterial virulence genes that are specifically induced in host tissues. Science 259, 686–688.[CrossRef]
    [Google Scholar]
  34. Mahan, M. J., Heithoff, D. M., Sinsheimer, R. L. & Low, D. A. ( 2000; ). Assessment of bacterial pathogenesis by analysis of gene expression in the host. Annu Rev Genet 34, 139–164.[CrossRef]
    [Google Scholar]
  35. Morrow, B. J., Graham, J. E. & Curtiss, R., III ( 1999; ). Genomic subtractive hybridization and selective capture of transcribed sequences identify a novel Salmonella typhimurium fimbrial operon and putative transcriptional regulator that are absent from the Salmonella typhi genome. Infect Immun 67, 5106–5116.
    [Google Scholar]
  36. Munson, R. S., Jr, Harrison, A., Gillaspy, A., Ray, W. C., Carson, M., Armbruster, D., Gipson, J., Gipson, M., Johnson, L. & other authors ( 2004; ). Partial analysis of the genomes of two nontypeable Haemophilus influenzae otitis media isolates. Infect Immun 72, 3002–3010.[CrossRef]
    [Google Scholar]
  37. Nika, J. R., Latimer, J. L., Ward, C. K., Blick, R. J., Wagner, N. J., Cope, L. D., Mahairas, G. G., Munson, R. S., Jr & Hansen, E. J. ( 2002; ). Haemophilus ducreyi requires the flp gene cluster for microcolony formation in vitro. Infect Immun 70, 2965–2975.[CrossRef]
    [Google Scholar]
  38. Palmer, K. L., Schnizlein-Bick, C. T., Orazi, A., John, K., Chen, C.-Y., Hood, A. F. & Spinola, S. M. ( 1998; ). The immune response to Haemophilus ducreyi resembles a delayed-type hypersensitivity reaction throughout experimental infection of human subjects. J Infect Dis 178, 1688–1697.[CrossRef]
    [Google Scholar]
  39. Post, D. M., Mungur, R., Gibson, B. W. & Munson, R. S., Jr ( 2005; ). Identification of a novel sialic acid transporter in Haemophilus ducreyi. Infect Immun 73, 6727–6735.[CrossRef]
    [Google Scholar]
  40. Rediers, H., Rainey, P. B., Vanderleyden, J. & De Mot, R. ( 2005; ). Unraveling the secret lives of bacteria: use of in vivo expression technology and differential fluorescence induction promoter traps as tools for exploring niche-specific gene expression. Microbiol Mol Biol Rev 69, 217–261.[CrossRef]
    [Google Scholar]
  41. Sawers, G. & Bock, A. ( 1988; ). Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12. J Bacteriol 170, 5330
    [Google Scholar]
  42. Shelburne, S. A. & Musser, J. M. ( 2004; ). Virulence gene expression in vivo. Curr Opin Microbiol 7, 283–289.[CrossRef]
    [Google Scholar]
  43. Spinola, S. M., Wild, L. M., Apicella, M. A., Gaspari, A. A. & Campagnari, A. A. ( 1994; ). Experimental human infection with Haemophilus ducreyi. J Infect Dis 169, 1146–1150.[CrossRef]
    [Google Scholar]
  44. Spinola, S. M., Orazi, A., Arno, J. N., Fortney, K., Kotylo, P., Chen, C.-Y., Campagnari, A. A. & Hood, A. F. ( 1996; ). Haemophilus ducreyi elicits a cutaneous infiltrate of CD4 cells during experimental human infection. J Infect Dis 173, 394–402.[CrossRef]
    [Google Scholar]
  45. Spinola, S. M., Bauer, M. E. & Munson, R. S., Jr ( 2002; ). Immunopathogenesis of Haemophilus ducreyi infection (chancroid). Infect Immun 70, 1667–1676.[CrossRef]
    [Google Scholar]
  46. Spinola, S. M., Bong, C. T. H., Faber, A. L., Fortney, K. R., Bennett, S. L., Townsend, C. A., Zwickl, B. E., Billings, S. D., Humphreys, T. L. & other authors ( 2003a; ). Differences in host susceptibility to disease progression in the human challenge model of Haemophilus ducreyi infection. Infect Immun 71, 6658–6663.[CrossRef]
    [Google Scholar]
  47. Spinola, S. M., Fortney, K. R., Katz, B. P., Latimer, J. L., Mock, J. R., Vakevainen, M. & Hansen, E. J. ( 2003b; ). Haemophilus ducreyi requires an intact flp gene cluster for virulence in humans. Infect Immun 71, 7178–7182.[CrossRef]
    [Google Scholar]
  48. St. Geme, J. W., III & Grass, S. ( 1998; ). Secretion of the Haemophilus influenzae HMW1 and HMW2 adhesins involves a periplasmic intermediate and requires the HMWB and HMWC proteins. Mol Microbiol 27, 617–630.[CrossRef]
    [Google Scholar]
  49. Steen, R. ( 2001; ). On eradicating chancroid. Bull World Health Organ 79, 818–826.
    [Google Scholar]
  50. Stevens, M. K., Latimer, J. L., Lumbley, S. R., Ward, C. K., Cope, L. D., Lagergård, T. & Hansen, E. J. ( 1999; ). Characterization of a Haemophilus ducreyi mutant deficient in expression of cytolethal distending toxin. Infect Immun 67, 3900–3908.
    [Google Scholar]
  51. Throm, R. E. & Spinola, S. M. ( 2001; ). Transcription of candidate virulence genes of Haemophilus ducreyi during infection of human volunteers. Infect Immun 69, 1483–1487.[CrossRef]
    [Google Scholar]
  52. Throm, R. E., Al-Tawfiq, J. A., Fortney, K. R., Katz, B. P., Hood, A. F., Hansen, E. J. & Spinola, S. M. ( 2000; ). Evaluation of an isogenic MOMP-deficient mutant in the human model of Haemophilus ducreyi infection. Infect Immun 68, 2602–2607.[CrossRef]
    [Google Scholar]
  53. Vakevainen, M., Greenberg, S. & Hansen, E. J. ( 2003; ). Inhibition of phagocytosis by Haemophilus ducreyi requires expression of the LspA1 and LspA2 proteins. Infect Immun 71, 5994–6003.[CrossRef]
    [Google Scholar]
  54. Ward, C. K., Lumbley, S. R., Latimer, J. L., Cope, L. D. & Hansen, E. J. ( 1998; ). Haemophilus ducreyi secretes a filamentous hemagglutinin-like protein. J Bacteriol 180, 6013–6022.
    [Google Scholar]
  55. Young, R. S., Fortney, K. R., Gelfanova, V., Phillips, C. L., Katz, B. P., Hood, A. F., Latimer, J. L., Munson, R. S., Jr, Hansen, E. J. & Spinola, S. M. ( 2001; ). Expression of cytolethal distending toxin and hemolysin are not required for pustule formation by Haemophilus ducreyi in human volunteers. Infect Immun 69, 1938–1942.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/013953-0
Loading
/content/journal/micro/10.1099/mic.0.2007/013953-0
Loading

Data & Media loading...

SCOTS procedure. [PDF file](74 KB)

PDF

[PDF file](13 KB)

PDF

[PDF file](140 KB)

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error