Previous Article | Next Article 
Infection and Immunity, June 2003, p. 3020-3027, Vol. 71, No. 6
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.6.3020-3027.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Isolation and Characterization of Mini-Tn5Km2 Insertion Mutants of Brucella abortus Deficient in Internalization and Intracellular Growth in HeLa Cells
Suk Kim, Masahisa Watarai,* Yuki Kondo, Janchivdorj Erdenebaatar, Sou-ichi Makino, and Toshikazu Shirahata
Department of Applied Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
Received 9 January 2003/
Returned for modification 11 February 2003/
Accepted 28 February 2003
Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. To identify genes related to internalization and multiplication in host cells, Brucella abortus was mutagenized by mini-Tn5Km2 transposon that carryied the kanamycin resistance gene, 4,400 mutants were screened, and HeLa cells were infected with each mutant. Twenty-three intracellular-growth-defective mutants were screened and were characterized for internalization and intracellular growth. From these results, we divided the mutants into the following three groups: class I, no internalization and intracellular growth within HeLa cells; class II, an internalization similar to that of the wild type but with no intracellular growth; and class III, internalization twice as high as the wild type but with no intracellular growth. Sequence analysis of DNA flanking the site of transposon showed various insertion sites of bacterial genes that are virulence-associated genes, including virB genes, an ion transporter system, and biosynthesis- and metabolism-associated genes. These internalization and intracellular-growth-defective mutants in HeLa cells also showed defective intracellular growth in macrophages. These results suggest that the virulence-associated genes isolated here contributed to the intracellular growth of both nonprofessional and professional phagocytes.
* Corresponding author. Mailing address: Department of Applied Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro-shi, Hokkaido 080-8555, Japan. Phone: 81-155-49-5387. Fax: 81-155-49-5386. E-mail: watarai{at}obihiro.ac.jp.
Editor: D. L. Burns
Infection and Immunity, June 2003, p. 3020-3027, Vol. 71, No. 6
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.6.3020-3027.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
This article has been cited by other articles:
-
den Hartigh, A. B., Rolan, H. G., de Jong, M. F., Tsolis, R. M.
(2008). VirB3 to VirB6 and VirB8 to VirB11, but Not VirB7, Are Essential for Mediating Persistence of Brucella in the Reticuloendothelial System. J. Bacteriol.
190: 4427-4436
[Abstract]
[Full Text]
-
Rolan, H. G., den Hartigh, A. B., Kahl-McDonagh, M., Ficht, T., Adams, L. G., Tsolis, R. M.
(2008). VirB12 Is a Serological Marker of Brucella Infection in Experimental and Natural Hosts. CVI
15: 208-214
[Abstract]
[Full Text]
-
Ammendola, S., Pasquali, P., Pistoia, C., Petrucci, P., Petrarca, P., Rotilio, G., Battistoni, A.
(2007). High-Affinity Zn2+ Uptake System ZnuABC Is Required for Bacterial Zinc Homeostasis in Intracellular Environments and Contributes to the Virulence of Salmonella enterica. Infect. Immun.
75: 5867-5876
[Abstract]
[Full Text]
-
Rolan, H. G., Tsolis, R. M.
(2007). Mice Lacking Components of Adaptive Immunity Show Increased Brucella abortus virB Mutant Colonization. Infect. Immun.
75: 2965-2973
[Abstract]
[Full Text]
-
Haine, V., Sinon, A., Van Steen, F., Rousseau, S., Dozot, M., Lestrate, P., Lambert, C., Letesson, J.-J., De Bolle, X.
(2005). Systematic Targeted Mutagenesis of Brucella melitensis 16M Reveals a Major Role for GntR Regulators in the Control of Virulence. Infect. Immun.
73: 5578-5586
[Abstract]
[Full Text]
-
Sun, Y.-H., Rolan, H. G., den Hartigh, A. B., Sondervan, D., Tsolis, R. M.
(2005). Brucella abortus VirB12 Is Expressed during Infection but Is Not an Essential Component of the Type IV Secretion System. Infect. Immun.
73: 6048-6054
[Abstract]
[Full Text]
-
Kim, S., Watanabe, K., Suzuki, H., Watarai, M.
(2005). Roles of Brucella abortus SpoT in morphological differentiation and intramacrophagic replication. Microbiology
151: 1607-1617
[Abstract]
[Full Text]
-
Danese, I., Haine, V., Delrue, R.-M., Tibor, A., Lestrate, P., Stevaux, O., Mertens, P., Paquet, J.-Y., Godfroid, J., De Bolle, X., Letesson, J.-J.
(2004). The Ton System, an ABC Transporter, and a Universally Conserved GTPase Are Involved in Iron Utilization by Brucella melitensis 16M. Infect. Immun.
72: 5783-5790
[Abstract]
[Full Text]
-
den Hartigh, A. B., Sun, Y.-H., Sondervan, D., Heuvelmans, N., Reinders, M. O., Ficht, T. A., Tsolis, R. M.
(2004). Differential Requirements for VirB1 and VirB2 during Brucella abortus Infection. Infect. Immun.
72: 5143-5149
[Abstract]
[Full Text]
Copyright © 2003 by the American Society for Microbiology. All rights reserved.