Tuberculosis remains the major cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. The molecular mechanisms of infection and persistence have not been completely elucidated for this pathogen. Studies involving nucleoid-associated proteins (NAPs), which have been related to the control and influence of virulence genes in pathogenic bacteria, can help unveil the virulence process of M. tuberculosis. Here, we describe the initial characterization of an ORF for an M. tuberculosis putative NAP. The Rv3852 gene was cloned and expressed, and its product purified to homogeneity. A qualitative protein-DNA binding assay was carried out by gelretardation and the protein affinity for specific DNA sequences was assessed quantitatively by surface plasmon resonance (SPR). A stoichiometry of 10 molecules of monomeric protein per molecule of DNA was determined. The monophasic apparent dissociation rate constant values increased to a saturable level as a function of protein concentration, yielding two limiting values for the molecular recognition of proU2 DNA. A protein-DNA binding mechanism is proposed. In addition, functional complementation studies with an Escherichia coli hns mutant reinforce the likelihood that the Rv3852 protein represents a novel NAP in M. tuberculosis.
INTRODUCTIONTuberculosis (TB) is one of the major causes of death worldwide caused by a single infectious agent, Mycobacterium tuberculosis. TB resurgence in the late 1980s was caused by a combination of several factors, such as HIV co-infection, increased poverty in urban areas and emergence of M. tuberculosis multidrug-resistant strains (MDR-TB) (Raviglione, 2003). According to the 2008 Global TB Control Report of the World Health Organization (WHO, 2008), there were approximately 9.2 million new TB cases in 2006, of which 0.5 million were MDR-TB. Moreover, the emergence of extensively drugresistant (XDR) TB cases (CDC, 2007), defined as cases in persons with TB whose isolates are MDR-TB as well as resistant to any one of the fluoroquinolone drugs and to at least one of the three injectable second-line drugs, amikacin, kanamycin or capreomycin, and their global distribution (Dorman & Chaisson, 2007), raise the prospect of virtually incurable TB worldwide. To compound the problem, it has been estimated that of 9.27 million incident TB cases in 2007, 1.37 million (15 %) were HIV-positive (WHO, 2009).M. tuberculosis has been considered the world's most successful pathogen. It is able to resist macrophage killing and persist in body tissues, thereby establishing a latent infection which can be reactivated when the host immune system wanes (Gomez & McKinney, 2004;Hingley-Wilson et al., 2003). The mechanism by which M. tuberculosis establishes latency and persistence is largely unknown and efforts have been made to address this and other issues, such as virulence (Andersen, 2007;Gandotra et al., 2007;Saunders & Britton, 2007;Schnappinger et al., 2006). Nucleoid-associated proteins (NAPs), also known as histone-like proteins, are a divers...