Tài liệu Báo cáo khoa học: The Mycobacterium tuberculosis membrane protein Rv2560 ) biochemical and

The Mycobacterium tuberculosis membrane protein

Rv2560 ) biochemical and functional studies

David F. Plaza1

, Hernando Curtidor2,4, Manuel A. Patarroyo1,4, Julie A. Chapeton-Montes1

,

Claudia Reyes3

, Jose Barreto3 and Manuel E. Patarroyo1,5

1 Molecular Biology Department, Fundacion Instituto de Inmunologia de Colombia, Bogota, Colombia

2 Receptor–Ligand Department, Fundacion Instituto de Inmunologia de Colombia, Bogota, Colombia

3 Chemical Synthesis Department, Fundacion Instituto de Inmunologia de Colombia, Bogota, Colombia

4 Universidad del Rosario, Bogota, Colombia

5 Universidad Nacional de Colombia, Bogota, Colombia

Tuberculosis (TB) is a major public health problem

throughout the world, affecting almost nine million

people [1] and causing more than three million deaths

per year. An increasing incidence of TB, related to the

high risk of developing the disease in immuno￾suppressed individuals and the increasing proportion

of Mycobacterium tuberculosis drug-resistant strains,

has contributed to this problem [2,3]. This makes the

Keywords

high-activity binding peptide; invasion

inhibition; Mycobacterium tuberculosis –

host cell interaction; Rv2560 membrane

protein

Correspondence

M. E. Patarroyo, Carrera 50 # 26-00,

Bogota, Colombia

Fax: +57 (1) 4815269

Tel: +57 (1) 4815219 or +57 (1) 3244672

Ext 125

E-mail: [email protected]

(Received 31 July 2007, revised 11 October

2007, accepted 17 October 2007)

doi:10.1111/j.1742-4658.2007.06153.x

The characterization of membrane proteins having no identified function in

Mycobacterium tuberculosis is important for a better understanding of the

biology of this pathogen. In this work, the biological activity of the

Rv2560 protein was characterized and evaluated. Primers used in PCR and

RT-PCR assays revealed that the gene encoding protein Rv2560 is present

in M. tuberculosis complex strains, but transcribed in only some of them.

Sera obtained from rabbits inoculated with polymer peptides from this pro￾tein recognized a 33 kDa band in the M. tuberculosis lysate and a mem￾brane fraction corresponding to the predicted molecular mass (33.1 kDa)

of this protein. Immunoelectron microscopy analysis found this protein on

the mycobacterial membrane. Sixteen peptides covering its entire length

were chemically synthesized and tested for their ability to bind to A549

and U937 cells. Peptide 11024 (121VVALSDRATTAYTNTSGVSS140)

showed high specific binding to both cell types (dissociation constants of

380 and 800 nm, respectively, and positive receptor–ligand interaction

cooperativity), whereas peptide 11033 (284LIGIPVAALIHVYTYRKLS

GG304) displayed high binding activity to A549 cells only. Cross-linking

assays showed the specific binding of peptide 11024 to a 54 kDa membrane

protein on U937. Invasion inhibition assays, in the presence of shared

high-activity binding peptide identified for U937 and A549 cells, presented

maximum inhibition percentages of 50.53% and 58.27%, respectively. Our

work highlights the relevance of the Rv2560 protein in the M. tuberculosis

invasion process of monocytes and epithelial cells, and represents a funda￾mental step in the rational selection of new antigens to be included as

components in a multiepitope, subunit-based, chemically synthesized, anti￾tuberculosis vaccine.

Abbreviations

GRAVY, grand average of hydropathicity; HABP, high-activity binding peptide; SPf66, synthetic Plasmodium falciparum 66; SSP, simple

sequence protein; TB, tuberculosis.

6352 FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS