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Background: We have previously identified Mycobacterium tuberculosis PknD to be an important virulence factor required for the pathogenesis of central nervous system (CNS) tuberculosis (TB). Specifically, PknD mediates bacillary invasion of the blood-brain barrier, which can be neutralized by specific antisera, suggesting its potential role as a therapeutic target against TB meningitis. Methodology/Principal Findings: We utilized an aerosol challenge guinea pig model of CNS TB and compared the protective efficacy of recombinant M. tuberculosis PknD subunit protein with that of M. bovis BCG against bacillary dissemination to the brain. BCG vaccination limited the pulmonary bacillary burden after aerosol challenge with virulent M. tuberculosis in guinea pigs and also reduced bacillary dissemination to the brain (P = 0.01). PknD vaccination also offered significant protection against bacterial dissemination to the brain, which was no different from BCG (P>0.24), even though PknD vaccinated animals had almost 100-fold higher pulmonary bacterial burdens. Higher levels of PknD-specific IgG were noted in animals immunized with PknD, but not in BCG-vaccinated or control animals. Furthermore, pre-incubation of M. tuberculosis with sera from PknD-vaccinated animals, but not with sera from BCG-vaccinated or control animals, significantly reduced bacterial invasion in a human blood-brain barrier model (P<0.01). Conclusion: Current recommendations for administering BCG at birth are based on protection gained against severe disease, such as TB meningitis, during infancy. We demonstrate that vaccination with recombinant M. tuberculosis PknD subunit offers a novel strategy to protect against TB meningitis, which is equivalent to BCG in a guinea pig model. Moreover, since BCG lacks the PknD sensor, BCG could also be boosted to develop a more effective vaccine against TB meningitis, a devastating disease that disproportionately affects young children. [ABSTRACT FROM AUTHOR]

Hyperglycemia, the diagnostic feature of diabetes also occurs in non-diabetics associated with chronic inflammation and systemic insulin resistance. Since the increased risk of active TB in diabetics has been linked to the severity and duration of hyperglycemia, we investigated what effect diet-induced hyperglycemia had on the severity of Mycobacterium tuberculosis (Mtb) infection in non-diabetic guinea pigs. Post-prandial hyperglycemia was induced in guinea pigs on normal chow by feeding a 40% sucrose solution daily or water as a carrier control. Sucrose feeding was initiated on the day of aerosol exposure to the H37Rv strain of Mtb and continued for 30 or 60 days of infection. Despite more severe hyperglycemia in sucrose-fed animals on day 30, there was no significant difference in lung bacterial or lesion burden until day 60. However the higher spleen and lymph node bacterial and lesion burden at day 30 indicated earlier and more severe extrapulmonary TB in sucrose-fed animals. In both sucrose- and water-fed animals, serum free fatty acids, important mediators of insulin resistance, were increased by day 30 and remained elevated until day 60 of infection. Hyperglycemia mediated by Mtb infection resulted in accumulation of advanced glycation end products (AGEs) in lung granulomas, which was exacerbated by sucrose feeding. However, tissue and serum AGEs were elevated in both sucrose and water-fed guinea pigs by day 60. These data indicate that Mtb infection alone induces insulin resistance and chronic hyperglycemia, which is exacerbated by sucrose feeding. Moreover, Mtb infection alone resulted in the accumulation tissue and serum AGEs, which are also central to the pathogenesis of diabetes and diabetic complications. The exacerbation of insulin resistance and hyperglycemia by Mtb infection alone may explain why TB is more severe in diabetics with poorly controlled hyperglycemia compared to non- diabetics and patients with properly controlled blood glucose levels. [ABSTRACT FROM AUTHOR]

Low-income countries with high Tuberculosis burden have few reference laboratories able to perform TB culture. In 2006, the Zanzibar National TB Control Programme planned to decentralize TB diagnostics. The Italian Cooperation Agency with the scientific support of the "L. Spallanzani" National Institute for Infectious Diseases sustained the project through the implementation of a TB reference laboratory in a low-income country with a high prevalence of TB. The implementation steps were: 1) TB laboratory design according to the WHO standards; 2) laboratory equipment and reagent supplies for microscopy, cultures, and identification; 3) on-the-job training of the local staff; 4) web- and telemedicine-based supervision. From April 2007 to December 2010, 921 sputum samples were received from 40 peripheral laboratories: 120 TB cases were diagnosed. Of all the smear-positive cases, 74.2% were culture- positive. During the year 2010, the smear positive to culture positive rate increased up to 100%. In March 20, 2010 the Ministry of Health and Social Welfare of Zanzibar officially recognized the Public Health Laboratory- Ivo de Carneri as the National TB Reference Laboratory for the Zanzibar Archipelago. An advanced TB laboratory can represent a low cost solution to strengthen the TB diagnosis, to provide capacity building and mid-term sustainability. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, presumably utilizes fatty acids as a major carbon source during infection within the host. Metabolism of even-chain-length fatty acids yields acetyl-CoA, whereas metabolism of oddchain- length fatty acids additionally yields propionyl-CoA. Utilization of these compounds by tubercle bacilli requires functional glyoxylate and methylcitrate cycles, respectively. Enzymes involved in both pathways are essential for M. tuberculosis viability and persistence during growth on fatty acids. However, little is known about regulatory factors responsible for adjusting the expression of genes encoding these enzymes to particular growth conditions. Here, we characterized the novel role of PrpR as a transcription factor that is directly involved in regulating genes encoding the key enzymes of methylcitrate (methylcitrate dehydratase [PrpD] and methylcitrate synthase [PrpC]) and glyoxylate (isocitrate lyase [Icl1]) cycles. Using cell- free systems and intact cells, we demonstrated an interaction of PrpR protein with prpDC and icl1 promoter regions and identified a consensus sequence recognized by PrpR. Moreover, we showed that an M. tuberculosis prpR-deletion strain exhibits impaired growth in vitro on propionate as the sole carbon source. Real-time quantitative reverse transcription-polymerase chain reaction confirmed that PrpR acts as a transcriptional activator of prpDC and icl1 genes when propionate is the main carbon source. Similar results were also obtained for a non-pathogenic Mycobacterium smegmatis strain. Additionally, we found that ramB, a prpR paralog that controls the glyoxylate cycle, is negatively regulated by PrpR. Our data demonstrate that PrpR is essential for the utilization of odd- chain-length fatty acids by tubercle bacilli. Since PrpR also acts as a ramB repressor, our findings suggest that it plays a key role in regulating expression of enzymes involved in both glyoxylate and methylcitrate pathways. [ABSTRACT FROM AUTHOR]

Background: The Mycobacterium tuberculosis PhoP/PhoR two-component signal transduction system controls the expression of about 2% of the genome and plays a major role in pathogenicity. However, its regulon has not been well characterized. Methodology/Principal Findings: The binding site of PhoP transcription regulator was identified in the upstream regions of msl3, pks2, lipF and fadD21 genes, by using gene fusions, electrophoretic mobility shift assays and DNase I footprinting experiments. A consensus sequence for PhoP binding was deduced. It consists of two direct repeats, DR1/DR2, associated with a third repeat, DR3, important in some cases for PhoP binding to DR1/DR2 but located at a variable distance from these direct repeats. DR1/DR2 and DR3 consensus sequences were used to screen the whole-genome sequence for other putative binding sites potentially corresponding to genes directly regulated by PhoP. The identified 87 genes, encoding transcription regulators, and proteins involved in secondary metabolites biosynthesis, transport and catabolism are proposed to belong to the PhoP regulon. Conclusions/Significance: A consensus sequence derived from the analysis of PhoP binding to four gene promoter regions is proposed. We show for the first time the involvement of a third direct repeat motif in this binding reaction. The consensus sequence was instrumented to study the global regulation mediated by PhoP in M. tuberculosis. This analysis leads to the identification of several genes that are potentially regulated by this key player. [ABSTRACT FROM AUTHOR]