Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosis universal 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-Å-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.

Approximately one-third of the human population is latently infected with Mycobacterium tuberculosis, comprising a critical reservoir for disease reactivation. Despite the importance of latency in maintaining M. tuberculosis in the human population, little is known about the mycobacterial factors that regulate persistence and reactivation. Previous in vitro studies have implicated a family of five related M. tuberculosis proteins, called resuscitation promoting factors (Rpfs), in regulating mycobacterial growth. We studied the in vivo role of M. tuberculosis rpf genes in an established mouse model of M. tuberculosis persistence and reactivation. After an aerosol infection with the M. tuberculosis Erdman wild type (Erdman) or single-deletion rpf mutants to establish chronic infections in mice, reactivation was induced by administration of the nitric oxide (NO) synthase inhibitor aminoguanidine. Of the five rpf deletion mutants tested, one (⌬Rv1009) exhibited a delayed reactivation phenotype, manifested by delayed postreactivation growth kinetics and prolonged median survival times among infected animals. Immunophenotypic analysis suggested differences in pulmonary B-cell responses between Erdman-and ⌬Rv1009-infected mice at advanced stages of reactivation. Analysis of rpf gene expression in the lungs of Erdman-infected mice revealed that relative expression of four of the five rpf-like genes was diminished at late times following reactivation, when bacterial numbers had increased substantially, suggesting that rpf gene expression may be regulated in a growth phase-dependent manner. To our knowledge, ⌬Rv1009 is the first M. tuberculosis mutant to have a specific defect in reactivation without accompanying growth defects in vitro or during acute infection in vivo.Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an extraordinarily successful human pathogen. Based on surveys of tuberculin reactivity, it is estimated that up to one-third of the world's population, or 2 billion individuals, is infected with the bacillus. The vast majority of these individuals are asymptomatic latent carriers who exhibit no signs of disease and are noncontagious. However, these latently infected persons represent a critically important reservoir for disease reactivation. For immunocompetent individuals, the risk of reactivation is estimated to be 2 to 23% over their lifetimes; however, for persons coinfected with human immunodeficiency virus, the risk is a considerably higher 10% per year (13,35). It is crucial that this reservoir of latent infection be addressed if attempts to control the TB epidemic are to succeed.Although numerous host factors responsible for limiting M. tuberculosis growth have been identified (for a review, see reference 11), much less is known regarding the mycobacterial factors which contribute to persistence, and perhaps even less is known about the mycobacterial signals governing reactivation from a "persistent" or "dormant" state. The resuscitationpromoting factor (Rpf) of Micrococcus l...

We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (ΔRv2623) displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs), while the ΔRv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ΔRv2623 is hypervirulent, while ΔRv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ΔRv2623 and ΔRv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv1747 might function as a transporter of PIMs. Because these glycolipids are major mycobacterial cell envelope components that can impact on the immune response, our findings raise the possibility that Rv2623 may regulate bacterial growth, virulence, and entry into persistence, at least in part, by modulating the levels of bacillary PIM expression, perhaps through negatively regulating the Rv1747-dependent export of the immunomodulatory PIMs to alter host-pathogen interaction, thereby influencing the fate of M. tuberculosis in vivo.

A novel design total knee arthroplasty (TKA) system has been introduced to improve patient outcomes and increase longevity. However, we have encountered a high rate of debonding of tibial implant-cement interface. In addition, multiple reports have been filed in Manufacturer and User Facility Device Experience database (MAUDE) with the same mechanism of failure. Therefore, we evaluated: clinical, radiographic, and intraoperative findings of patients who received this system and required a revision surgery, and findings from MAUDE database compiled to this date. We reviewed three hospital databases for patients who had revision TKA for tibial loosening at the implant-cement interface. This yielded 15 cases with a mean age of 61 years (range, 47-84). All patients received a novel knee system at another institution. Radiographic analysis was performed by treating orthopaedist. The MAUDE database was reviewed for reports of aseptic failure. Patients presented with pain on weight bearing, effusion, and decreased range of motion (ROM) within 2 years after surgery. Radiographic evaluation demonstrated loosening of the tibial components in 2 of 15 knees. This included cruciate retaining, posterior stabilized, fixed bearing, and rotating platform bearing designs. Intraoperative findings demonstrated gross loosening of the tibial component at the implant-cement interface. Femoral and patellar components were well fixed. There were 21 reports of tibial loosening at the implant-cement interface in MAUDE database in the past 2 months alone. Numerous other tibial failures were reported; however, the mechanisms of failures were not specified. Tibial component loosening is a rare complication of cemented TKA at short-term follow-up. Several possible reasons include increased constraint, reduced cement pockets, and reduced keel rotational stabilizers. The tibial component, which has greater torsional loads, has lower surface roughness than femoral component. We believe that this complication is