The mechanisms utilized by Mycobacterium tuberculosis to establish, maintain, or reactivate from latent infection in the host are largely unknown but likely include genes that mediate adaptation to conditions encountered during persistence. Previously, a two-component signal transduction system, mprAB, was found to be required in M. tuberculosis for establishment and maintenance of persistent infection in a tissue-and stage-specific fashion. To begin to characterize the role of this system in M. tuberculosis physiology and virulence, a functional analysis of the mprA and mprB gene products was initiated. Here, evidence is presented demonstrating that sensor kinase MprB and response regulator MprA function as an intact signal-transducing pair in vitro and in vivo. Sensor kinase MprB can be autophosphorylated, can donate phosphate to MprA, and can act as a phospho-MprA phosphatase in vitro. Correspondingly, response regulator MprA can accept phosphate from MprB or from small phosphodonors including acetyl phosphate. Mutagenesis of residues His249 in MprB and Asp48 in MprA abolished the ability of these proteins to be phosphorylated in vitro. Introduction of these alleles into Mycobacterium bovis BCG attenuated virulence in macrophages in vivo. Together, these results support a role for the mprAB two-component system in M. tuberculosis physiology and pathogenesis. Characterization of two-component signal transduction systems will enhance our understanding of processes regulated by M. tuberculosis during acute and/or persistent infection in the host.Persistent Mycobacterium tuberculosis infections afflict nearly 2 billion people worldwide (31). The combination of M. tuberculosis reactivation in these individuals and subsequent primary infection of immunocompromised hosts unable to control the infection accounts for more than 2.4 million deaths annually due to tuberculosis (34). This disease is the world's leading cause of death due to infection by a single bacterial agent (24). While the majority of individuals latently infected with M. tuberculosis do not undergo reactivation of disease, factors that suppress the host's immune system, including infection with human immunodeficiency virus, steroid therapy, malnutrition, and age, can increase the risk of reactivation tuberculosis to 10% per annum (44). While antitubercular therapeutics are effective in treating individuals with acute primary or reactivated tuberculosis infections, these antibiotics are largely ineffective against M. tuberculosis in the latent stages of infection (40), a poorly understood disease state characterized by little or no bacterial proliferation and the lack of overt disease symptoms. With the continuing increase in human immunodeficiency virus infection in many parts of the world, the emergence of multidrug-resistant isolates of M. tuberculosis (35), and the lack of antitubercular drugs exhibiting activity against organisms during latent infection, new therapeutics targeting persistent bacilli or preventing reactivation are needed.The abili...

The hemA gene codes for one of two synthases in Rhodobacter sphaeroides 2.4.1 which catalyze the formation of 5-aminolevulinic acid. We have examined the role of PrrA, a DNA binding protein that is associated with the metabolic switch between aerobic growth and anoxygenic photosynthetic growth, in hemA expression and found that hemA transcription is directly activated by PrrA. Using electrophoretic mobility shift assays and DNase I protection assays, we have mapped two binding sites for PrrA within the hemA upstream sequences, each of which contains an identical 9-bp motif. Using lacZ transcription reporter plasmids in wild-type strain 2.4.1 and PrrA ؊ mutant strain PRRA2, we showed that PrrA was required for maximal expression. We also found that the relative impacts of altering DNA sequences within the two binding sites are different depending on whether cells are growing aerobically or anaerobically. This reveals a greater level of complexity associated with PrrA-mediated regulation of transcription than has been heretofore described. Our findings are of particular importance with respect to those genes regulated by PrrA having more than one upstream binding site. In the case of the hemA gene, we discuss possibilities as to how these new insights can be accommodated within the context of what has already been established for hemA transcription regulation in R. sphaeroides.

In the facultatively phototrophic proteobacterium Rhodobacter sphaeroides, formation of the photosynthetic apparatus is oxygen dependent. When oxygen tension decreases, the response regulator PrrA of the global two-component PrrBA system is believed to directly activate transcription of the puf, puh, and puc operons, encoding structural proteins of the photosynthetic complexes, and to indirectly upregulate the photopigment biosynthesis genes bch and crt. Decreased oxygen also results in inactivation of the photosynthesis-specific repressor PpsR, bringing about derepression of the puc, bch, and crt operons. We uncovered a hierarchical relationship between these two regulatory systems, earlier thought to function independently. We also more accurately assessed the spectrum of gene targets of the PrrBA system. First, expression of the appA gene, encoding the PpsR antirepressor, is PrrA dependent, which establishes one level of hierarchical dominance of the PrrBA system over AppA-PpsR. Second, restoration of the appA transcript to the wild-type level is insufficient for rescuing phototrophic growth impairment of the prrA mutant, whereas inactivation of ppsR is sufficient. This suggests that in addition to controlling appA transcription, PrrA affects the activity of the AppA-PpsR system via an as yet unidentified mechanism(s). Third, PrrA directly activates several bch and crt genes, traditionally considered to be the PpsR targets. Therefore, in R. sphaeroides, the global PrrBA system regulates photosynthesis gene expression (i) by rigorous control over the photosynthesis-specific AppA-PpsR regulatory system and (ii) by extensive direct transcription activation of genes encoding structural proteins of photosynthetic complexes as well as genes encoding photopigment biosynthesis enzymes.In the facultatively phototrophic alphaproteobacterium Rhodobacter sphaeroides, photosynthesis (PS) operates under anoxic conditions. A decrease in oxygen tension triggers significant upregulation of PS gene transcription (35,38). The photosynthetic apparatus is comprised of the reaction center, encoded by the puh and puf operons, and two light-harvesting complexes, encoded by the puf and puc operons. Enzymes involved in the biosynthesis of photosynthetic pigments, i.e., bacteriochlorophyll a and carotenoids, are encoded by the bch and crt genes, respectively. Most PS-specific genes are located in the R. sphaeroides PS gene cluster, whereas the puc operons are located separately (4, 49). Three major regulatory systems control oxygen-dependent transcription of PS genes. One of these is composed of the antirepressor AppA (15, 17) and the repressor PpsR (7, 36) and is primarily responsible for the regulation of PS genes (30). Two other systems are global regulatory systems, i.e., the redox-responsive two-component system PrrBA (2, 47) and the anaerobic activator FnrL (48).

The American Academy of Pediatrics recommends screening newborns ≥35 weeks' gestation with total serum bilirubin (TSB) or transcutaneous bilirubin (TcB) to detect hyperbilirubinemia. Retrospective studies show TcB measurements strongly correlate with TSB; however, few prospective trials document this relationship. Furthermore, Dräger's newest TcB instrument, JM-105, remains unstudied in the United States. We measure TcB on foreheads and sternums of newborns using JM-105 and Bilichek devices within 30 minutes of TSB measurement. We find best overall TcB/TSB correlation with JM-105 on the sternum (mean TcB-TSB difference: -0.21 ± 1.15 mg/dL). Correlations between paired measurements for TcB on the sternum using JM-105 were 0.93 for all TSB levels (n = 178), 0.82 for TSB > 10 (n = 19), 0.69 for TSB > 12 (n = 11), and 0.52 for TSB > 15 (n = 6). TcB accuracy via JM-105 on the sternum significantly differed among races ( P < .001). For 5% of paired measurements, TcB with JM-105 on the sternum underestimated TSB by ≥2 mg/dL, and for <1% by ≥3 mg/dL.

ObjectivesTo describe critical features of the Ethiopian Pediatric Society (EPS) Quality Improvement (QI) Initiative and to present formative research on mentor models.SettingGeneral and referral hospitals in the Addis Ababa area of Ethiopia.ParticipantsEighteen hospitals selected for proximity to the EPS headquarters, prior participation in a recent newborn care training cascade and minimal experience with QI.InterventionsEducation in QI in a 2-hour workshop setting followed by implementation of a facility-based QI project with the support of virtual mentorship or in-person mentorship.Primary and secondary outcome measuresPrimary outcome—QI progress, measured using an adapted Institute for Healthcare Improvement Scale; secondary outcome—contextual factors affecting QI success as measured by the Model for Understanding Success in Quality.ResultsThe dose and nature of mentoring encounters differed based on a virtual versus in-person mentoring approach. All QI teams conducted at least one large-scale change. Education of staff was the most common change implemented in both groups. We did not identify contextual factors that predicted greater QI progress.ConclusionsThe EPS QI Initiative demonstrates that education in QI paired with external mentorship can support implementation of QI in low-resource settings. This pragmatic approach to facility-based QI may be a scalable strategy for improving newborn care and outcomes. Further research is needed on the most appropriate instruments for measuring contextual factors in low/middle-income country settings.