Identification of Rv3852 as a nucleoid-associated protein in Mycobacterium tuberculosis

Abstract

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...

Regulation of Lipid Biosynthesis, Sliding Motility, and Biofilm Formation by a Membrane-Anchored Nucleoid-Associated Protein of Mycobacterium tuberculosis

“…The protein bound to DNA in a sequence-independent manner, and with increasing protein concentrations, the DNA-protein complexes were increased. At higher concentrations (75 to 100 nM) of the protein, the DNA-protein complexes failed to enter the gel, suggesting the formation of higher-order oligomeric DNA-protein structures, as reported previously (28). These structures presumably formed due to the cooperative binding of the protein on DNA.…”

Virulence Regulator EspR of Mycobacterium tuberculosis Is a Nucleoid-Associated Protein

“…Together, this suggests that EspR and Lsr2 may control gene expression, including that of many cell wall functions, in a divergent manner with EspR possibly replacing Lsr2 at certain sites and vice-versa. It is striking that in all sequenced mycobacterial genomes espR is very close to the hns gene encoding another NAP [33] and this may also indicate functional interplay. These hypotheses can be tested experimentally by using the corresponding antibodies to perform ChIP-Seq experiments on Mtb strains at different stages in the growth cycle to localize their binding sites.…”

Rv3852 (H-NS) of Mycobacterium tuberculosis Is Not Involved in Nucleoid Compaction and Virulence Regulation

“…Since Rv3852 had been hypothesized to play a role as a NAP (19), thus contributing to shaping the chromosome, the MICs of drugs that have an effect on the topology of DNA were measured (28). We used the fluoroquinolones moxifloxacin and novobiocin targeting the GyrA and GyrB subunits of DNA gyrase, respectively, for this purpose together with rifampin, inhibiting RNA synthesis, as a control.…”

“…Curiously, Werlang and colleagues reported that M. tuberculosis Rv3852 does not complement hns mutations in E. coli (19), whereas Lsr2, another NAP, does (20) and is therefore a functional homologue of E. coli H-NS. Ectopic expression of M. tuberculosis rv3852 in M. smegmatis led to a less-compacted nucleoid, altered biofilm formation, and decreased sliding motility (21).…”