1887

Microbiology Society logo

Volume 150, Issue 4

The MspA porin promotes growth and increases antibiotic susceptibility of both BCG and Free

Abstract

Porins mediate the diffusion of hydrophilic solutes across the outer membrane of mycobacteria, but the efficiency of this pathway is very low compared to Gram-negative bacteria. To examine the importance of porins in slow-growing mycobacteria, the major porin MspA of was expressed in and . Approximately 20 and 35 MspA molecules per μm cell wall were observed in and BCG, respectively, by electron microscopy and quantitative immunoblot experiments. Surface accessibility of MspA in was demonstrated by flow cytometry. Glucose uptake was twofold faster, indicating that the outer membrane permeability of BCG to small and hydrophilic solutes was increased by MspA. This significantly accelerated the growth of BCG, identifying very slow nutrient uptake as one of the determinants of slow growth in mycobacteria. The susceptibility of both BCG and to zwitterionic -lactam antibiotics was substantially enhanced by MspA, decreasing the minimal inhibitory concentration up to 16-fold. Furthermore, became significantly more susceptible to isoniazid, ethambutol and streptomycin. Fluorescence with the nucleic acid binding dye SYTO 9 was 10-fold increased upon expression of . These results indicated that MspA not only enhanced the efficiency of the porin pathway, but also that of pathways mediating access to large and/or hydrophobic agents. This study provides the first experimental evidence that porins are important for drug susceptibility of .

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): OM, outer membrane , OPOE, n-octylpolyethylene oxide and TB, tuberculosis
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26902-0
2004-04-01
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/4/mic1500853.html?itemId=/content/journal/micro/10.1099/mic.0.26902-0&mimeType=html&fmt=ahah

References

  1. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidmann J. G., Smith J. A., Struhl K. 1987 Current Protocols in Molecular Biology New York: Wiley;
  2. Bardou F., Raynaud C., Ramos C., Laneelle M. A., Laneelle G. 1998; Mechanism of isoniazid uptake in Mycobacterium tuberculosis. Microbiology 144:2539–2544 [CrossRef]
     [Google Scholar]
  3. Bercovier H., Kafri O., Sela S. 1986; Mycobacteria possess a surprisingly small number of ribosomal RNA genes in relation to the size of their genome. Biochem Biophys Res Commun 136:1136–1141 [CrossRef]
     [Google Scholar]
  4. Brennan P. J., Nikaido H. 1995; The envelope of mycobacteria. Annu Rev Biochem 64:29–63 [CrossRef]
     [Google Scholar]
  5. Chambers H. F., Moreau D., Yajko D. 7 other authors 1995; Can penicillins and other beta-lactam antibiotics be used to treat tuberculosis?. Antimicrob Agents Chemother 39:2620–2624 [CrossRef]
     [Google Scholar]
  6. Delcour A. H. 2003; Solute uptake through general porins. Front Biosci 8:D1055–1071 [CrossRef]
     [Google Scholar]
  7. Draper P. 1998; The outer parts of the mycobacterial envelope as permeability barriers. Front Biosci 3:1253–1261
     [Google Scholar]
  8. Engelhardt H., Heinz C., Niederweis M. 2002; A tetrameric porin limits the cell wall permeability of Mycobacterium smegmatis. J Biol Chem 277:37567–37572 [CrossRef]
     [Google Scholar]
  9. Ferenci T. 1999; Regulation by nutrient limitation. Curr Opin Microbiol 2:208–213 [CrossRef]
     [Google Scholar]
  10. Förtsch D., Röllinghoff M., Stenger S. 2000; IL-10 converts human dendritic cells into macrophage-like cells with increased antibacterial activity against virulent Mycobacterium tuberculosis. J Immunol 165:978–987 [CrossRef]
     [Google Scholar]
  11. Gaora P. O. 1998; Expression of genes in mycobacteria. Methods Mol Biol 101:261–273
     [Google Scholar]
  12. Harshey R. M., Ramakrishnan T. 1977; Rate of ribonucleic acid chain growth in Mycobacterium tuberculosis H37Rv. J Bacteriol 129:616–622
     [Google Scholar]
  13. Heinz C., Niederweis M. 2000; Selective extraction and purification of a mycobacterial outer membrane protein. Anal Biochem 285:113–120 [CrossRef]
     [Google Scholar]
  14. Hiriyanna K. T., Ramakrishnan T. 1986; Deoxyribonucleic acid replication time in Mycobacterium tuberculosis H37Rv. Arch Microbiol 144:105–109 [CrossRef]
     [Google Scholar]
  15. Jackson M., Raynaud C., Laneelle M. A., Guilhot C., Laurent-Winter C., Ensergueix D., Gicquel B., Daffé M. 1999; Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope. Mol Microbiol 31:1573–1587 [CrossRef]
     [Google Scholar]
  16. Jarlier V., Nikaido H. 1990; Permeability barrier to hydrophilic solutes in Mycobacterium chelonei. J Bacteriol 172:1418–1423
     [Google Scholar]
  17. Jarlier V., Nikaido H. 1994; Mycobacterial cell wall: structure and role in natural resistance to antibiotics. FEMS Microbiol Lett 123:11–18 [CrossRef]
     [Google Scholar]
  18. Joux F., Lebaron P. 2000; Use of fluorescent probes to assess physiological functions of bacteria at single-cell level. Microbes Infect 2:1523–1535 [CrossRef]
     [Google Scholar]
  19. Kaps I., Ehrt S., Seeber S., Schnappinger D., Martin C., Riley L. W., Niederweis M. 2001; Energy transfer between fluorescent proteins using a co-expression system in Mycobacterium smegmatis. Gene 278:115–124 [CrossRef]
     [Google Scholar]
  20. Kartmann B., Stenger S., Niederweis M. 1999; Porins in the cell wall of Mycobacterium tuberculosis. J Bacteriol 181:6543–6546 (correction in J Bacteriol 181 7650)
    [Google Scholar]
  21. Kremer L., Besra G. S. 2002a; Re-emergence of tuberculosis: strategies and treatment. Expert Opin Investig Drugs 11:153–157 [CrossRef]
     [Google Scholar]
  22. Kremer L. S., Besra G. S. 2002b; Current status and future development of antitubercular chemotherapy. Expert Opin Investig Drugs 11:1033–1049 [CrossRef]
     [Google Scholar]
  23. Lambert P. A. 2002; Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria. J Appl Microbiol 92 Suppl:46S–54S [CrossRef]
     [Google Scholar]
  24. Liu J., Barry C. E. 1996; Mycolic acid structure determines the fluidity of the mycobacterial cell wall. J Biol Chem 271:29545–29551 [CrossRef]
     [Google Scholar]
  25. Liu J., Rosenberg E. Y., Nikaido H. 1995; Fluidity of the lipid domain of cell wall from Mycobacterium chelonae. Proc Natl Acad Sci U S A 92:11254–11258 [CrossRef]
     [Google Scholar]
  26. Matsumoto S., Furugen M., Yukitake H., Yamada T. 2000; The gene encoding mycobacterial DNA-binding protein I (MDPI) transformed rapidly growing bacteria to slowly growing bacteria. FEMS Microbiol Lett 182:297–301 [CrossRef]
     [Google Scholar]
  27. Niederweis M. 2003; Mycobacterial porins – new channel proteins in unique outer membranes. Mol Microbiol 49:1167–1177 [CrossRef]
     [Google Scholar]
  28. Niederweis M., Ehrt S., Heinz C., Klöcker U., Karosi S., Swiderek K. M., Riley L. W., Benz R. 1999; Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis. Mol Microbiol 33:933–945 [CrossRef]
     [Google Scholar]
  29. Nikaido H. 1986; Transport through the outer membrane of bacteria. Methods Enzymol 125:265–278
     [Google Scholar]
  30. Nikaido H., Jarlier V. 1991; Permeability of the mycobacterial cell wall. Res Microbiol 142:437–443 [CrossRef]
     [Google Scholar]
  31. Nikaido H., Rosenberg E. Y. 1983; Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins. J Bacteriol 153:241–252
     [Google Scholar]
  32. Nikaido H., Rosenberg E. Y., Foulds J. 1983; Porin channels in Escherichia coli: studies with beta-lactams in intact cells. J Bacteriol 153:232–240
     [Google Scholar]
  33. Nikaido H., Kim S. H., Rosenberg E. Y. 1993; Physical organization of lipids in the cell wall of Mycobacterium chelonae. Mol Microbiol 8:1025–1030 [CrossRef]
     [Google Scholar]
  34. Novo D. J., Perlmutter N. G., Hunt R. H., Shapiro H. M. 2000; Multiparameter flow cytometric analysis of antibiotic effects on membrane potential, membrane permeability, and bacterial counts of Staphylococcus aureus and Micrococcus luteus. Antimicrob Agents Chemother 44:827–834 [CrossRef]
     [Google Scholar]
  35. Pratt L. A., Hsing W., Gibson K. E., Silhavy T. J. 1996; From acids to osmZ: multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli. Mol Microbiol 20:911–917 [CrossRef]
     [Google Scholar]
  36. Rastogi N., Legrand E., Sola C. 2001; The mycobacteria: an introduction to nomenclature and pathogenesis. Rev Sci Tech 20:21–54
     [Google Scholar]
  37. Raynaud C., Laneelle M. A., Senaratne R. H., Draper P., Laneelle G., Daffé M. 1999; Mechanisms of pyrazinamide resistance in mycobacteria: importance of lack of uptake in addition to lack of pyrazinamidase activity. Microbiology 145:1359–1367 [CrossRef]
     [Google Scholar]
  38. Raynaud C., Papavinasasundaram K. G., Speight R. A., Springer B., Sander P., Böttger E. C., Colston M. J., Draper P. 2002; The functions of OmpATb, a pore-forming protein of Mycobacterium tuberculosis. Mol Microbiol 46:191–201 [CrossRef]
     [Google Scholar]
  39. Scholz O., Thiel A., Hillen W., Niederweis M. 2000; Quantitative analysis of gene expression with an improved green fluorescent protein. Eur J Biochem 267:1565–1570 [CrossRef]
     [Google Scholar]
  40. Senaratne R. H., Mobasheri H., Papavinasasundaram K. G., Jenner P., Lea E. J., Draper P. 1998; Expression of a gene for a porin-like protein of the OmpA family from Mycobacterium tuberculosis H37Rv. J Bacteriol 180:3541–3547
     [Google Scholar]
  41. Stahl C., Kubetzko S., Kaps I., Seeber S., Engelhardt H., Niederweis M. 2001; MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis. Mol Microbiol 40:451–464 [CrossRef]
     [Google Scholar]
  42. Stover C. K., de la Cruz V. F., Fuerst T. R. 11 other authors 1991; New use of BCG for recombinant vaccines. Nature 351:456–460 [CrossRef]
     [Google Scholar]
  43. Takayama K., Kilburn J. O. 1989; Inhibition of synthesis of arabinogalactan by ethambutol in Mycobacterium smegmatis. Antimicrob Agents Chemother 33:1493–1499 [CrossRef]
     [Google Scholar]
  44. Trias J., Benz R. 1994; Permeability of the cell wall of Mycobacterium smegmatis. Mol Microbiol 14:283–290 [CrossRef]
     [Google Scholar]
  45. Winder F. G., Collins P. B. 1970; Inhibition by isoniazid of synthesis of mycolic acids in Mycobacterium tuberculosis. J Gen Microbiol 63:41–48 [CrossRef]
     [Google Scholar]
  46. Yuan Y., Zhu Y., Crane D. D., Barry C. E. 1998; The effect of oxygenated mycolic acid composition on cell wall function and macrophage growth in Mycobacterium tuberculosis. Mol Microbiol 29:1449–1458 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26902-0
Loading
The MspA porin promotes growth and increases antibiotic susceptibility of both Mycobacterium bovis BCG and Mycobacterium tuberculosis
Microbiology 150, 853 (2004); https://doi.org/10.1099/mic.0.26902-0
/content/journal/micro/10.1099/mic.0.26902-0
/content/journal/micro/10.1099/mic.0.26902-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

IMAGE

Most cited Most Cited RSS feed

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