Mycobacterium tuberculosis universal stress protein Rv2623 regulates bacillary growth by ATP-Binding: requirement for establishing chronic persistent infection.

Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

Structure of the ternary complex of phosphomevalonate kinase: the enzyme and its family.

The galacto-, homoserine-, mevalonate-, phosphomevalonate-kinase (GHMP) superfamily encompases a wide-range of protein function. Three members of the family (mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase) comprise the mevalonate pathway found in S. pneumoniae and other organisms. We have determined the 1.9 A crystal structure of phosphomevalonate kinase (PMK) from S. pneumoniae in complex with phosphomevalonate and AMPPNP.Mg(2+). Comparison of the apo and ternary PMK structures suggests that ligand binding reverses the side-chain orientations of two antiparallel lysines residues (100 and 101) with the result that Lys101 is switched into a position in which its ammonium ion is in direct contact with the beta,gamma-bridging atom of the nucleotide, where it is expected to stabilize both the ground and transition states of the reaction. Analysis of all available GHMP kinase ternary complex structures reveals that while their C(alpha)-scaffolds are highly conserved, their substrates bind in one of two conformations, which appear to be either reactive or nonreactive. The active site of PMK seems spacious enough to accommodate interconversion of the reactive and nonreactive conformers. A substantial fraction of the PMK active site is occupied by ordered water, which clusters near the charged regions of the substrate. Notably, a water pentamer that interacts extensively with the reactive groups of both substrates was discovered at the active site.

Crystal structure of the Streptococcus pneumoniae mevalonate kinase in complex with diphosphomevalonate.

Streptococcus pneumoniae, a ubiquitous gram-positive pathogen with an alarming, steadily evolving resistance to frontline antimicrobials, poses a severe global health threat both in the community and in the clinic. The recent discovery that diphosphomevalonate (DPM), an essential intermediate in the isoprenoid biosynthetic pathway, potently and allosterically inhibits S. pneumoniae mevalonate kinase (SpMK) without affecting the human isozyme established a new target and lead compound for antimicrobial design. Here we present the crystal structure of the first S. pneumoniae mevalonate kinase, at a resolution of 2.5 A and in complex with DPM.Mg(2+) in the active-site cleft. Structural comparison of SpMK with other members of the GHMP kinase family reveals that DPM functions as a partial bisubstrate analog (mevalonate linked to the pyrophosphoryl moiety of ATP) in that it elicits a ternary-complexlike form of the enzyme, except for localized disordering in a region that would otherwise interact with the missing portion of the nucleotide. Features of the SpMK-binding pockets are discussed in the context of established mechanistic findings and inherited human diseases linked to MK deficiency.

Antibodies against immunodominant antigens of Mycobacterium tuberculosis in subjects with suspected tuberculosis in the United States compared by HIV status.

The immunodominance of Mycobacterium tuberculosis proteins malate synthase (MS) and MPT51 has been demonstrated in case-control studies with patients from countries in which tuberculosis (TB) is endemic. The value of these antigens for the serodiagnosis of TB now is evaluated in a cross-sectional study of pulmonary TB suspects in the United States diagnosed to have TB, HIV-associated TB, or other respiratory diseases (ORD). Serum antibody reactivity to recombinant purified MS and MPT51 was determined by enzyme-linked immunosorbent assays (ELISAs) of samples from TB suspects and well-characterized control groups. TB suspects were diagnosed with TB (n = 87; 49% sputum microscopy negative, 20% HIV(+)) or ORD (n = 63; 58% HIV(+)). Antibody reactivity to MS and MPT51 was significantly higher in U.S. HIV(+)/TB samples than in HIV(-)/TB samples (P < 0.001), and it was significantly higher in both TB groups than in control groups with latent TB infection (P < 0.001). Antibody reactivity to both antigens was higher in U.S. HIV(+)/TB samples than in HIV(+)/ORD samples (P = 0.052 for MS, P = 0.001 for MPT51) but not significantly different between HIV(-)/TB and HIV(-)/ORD. Among U.S. HIV(+) TB suspects, a positive anti-MPT51 antibody response was strongly and significantly associated with TB (odds ratio, 11.0; 95% confidence interval, 2.3 to 51.2; P = 0.002). These findings have implications for the adjunctive use of TB serodiagnosis with these antigens in HIV(+) subjects.

The structure of the carboxyltransferase component of acetyl-coA carboxylase reveals a zinc-binding motif unique to the bacterial enzyme.

Acetyl-coA carboxylase (ACC) is a central metabolic enzyme that catalyzes the committed step in fatty acid biosynthesis: biotin-dependent conversion of acetyl-coA to malonyl-coA. The bacterial carboxyltransferase (CT) subunit of ACC is a target for the design of novel therapeutics that combat severe, hospital-acquired infections resistant to the established classes of frontline antimicrobials. Here, we present the structures of the bacterial CT subunits from two prevalent nosocomial pathogens, Staphylococcus aureus and Escherichia coli, at a resolution of 2.0 and 3.0 A, respectively. Both structures reveal a small, independent zinc-binding domain that lacks a complement in the primary sequence or structure of the eukaryotic homologue.

Crystal structure of the ancient, Fe-S scaffold IscA reveals a novel protein fold.

IscA belongs to an ancient family of proteins responsible for iron-sulfur cluster assembly in essential metabolic pathways preserved throughout evolution. We report here the 2.3 A resolution crystal structure of Escherichia coli IscA, a novel fold in which mixed beta-sheets form a compact alpha-beta sandwich domain. In contrast to the highly mobile secondary structural elements within the bacterial Fe-S scaffold protein IscU, a protein which is thought to have a similar function, the great majority of the amino acids that are conserved in IscA homologues are located in elements that constitute a well-ordered fold. However, the 10-residue C-terminal tail segment that contains two invariant cysteines critical for the Fe-S-binding function of a cyanobacterial (Synechocystis PCC) IscA homologue is not ordered in our structure. In addition, the crystal packing reveals a helical assembly that is constructed from two possible tetrameric oligomers of IscA.