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Kurthkoti et al. 2017. The Capacity of Mycobacterium tuberculosis To Survive Iron Starvation Might Enable It To Persist in Iron-Deprived Microenvironments of Human Granulomas.

One-third of the world population may harbor persistent M. tuberculosis, causing an asymptomatic infection that is refractory to treatment and can reactivate to become potentially lethal tuberculosis disease. However, little is known about the factors that trigger and maintain M. tuberculosis persistence in infected individuals. Iron is an essential nutrient for M. tuberculosis growth. In this study, we show, first, that in human granulomas the immune defense creates microenvironments in which M. tuberculosis likely experiences drastic Fe deprivation and, second, that Fe-starved M. tuberculosis is capable of long-term persistence without growth. Together, these observations suggest that Fe deprivation in the lung might trigger a state of persistence in M. tuberculosis and promote chronic TB. We also identified vulnerabilities of iron-restricted persistent M. tuberculosis,which can be exploited for the design of new antitubercular therapies.

Kurthkoti K, Amin H, Marakalala MJ, Ghanny S, Subbian S, Sakatos A, Livny J, Fortune SM, Berney M, Rodriguez GM (2017) The Capacity of Mycobacterium tuberculosis To Survive Iron Starvation Might Enable It To Persist in Iron-Deprived Microenvironments of Human Granulomas. MBio 8. PMI: 28811344

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Arrigucci et al. 2017. FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry.

This Nature Method manuscript describes a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which is called FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ~30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described.

Arrigucci R, Bushkin Y, Radford F, Lakehal K, Vir P, Pine R, Martin D, Sugarman J, Zhao Y, Yap GS, Lardizabal AA, Tyagi S, Gennaro ML (2017) FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry. Nat Protoc 12: 1245-1260. PMI: 28518171

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Vinnard C et al. 2017. Long-term Mortality of Patients With Tuberculous Meningitis in New York City: A Cohort Study.

Tuberculosis meningitis (TBM) is the most devastating clinical presentation of infection with M. tuberculosis; delayed initiation of effective anti-tuberculosis (TB) therapy is associated with poor treatment outcomes. Our objective was to determine the relationship between drug resistance and 10-year mortality among TBM patients. We conducted a retrospective cohort study of patients with culture-confirmed TBM reported to the TB registry in New York City, NY (US) between January 1, 1992 and December 31, 2001. Date of death was ascertained by matching the TB registry with death certificate data for 1992 through 2012 from the New York Office of Vital Statistics. Among TBM patients without rifampin-resistant isolates, isoniazid resistance was associated with mortality after the first 60 days of treatment when controlling for age and HIV infection (adjusted HR 2.77, 95% CI: 1.28-6.01). We observed rapid early mortality in patients with rifampin-resistant isolates, and an independent association between isoniazid-resistant isolates and death after the first 60 days of therapy. These findings support the continued evaluation of rapid diagnostic techniques and the empiric addition of second-line drugs for patients with clinically suspected drug-resistant TBM.

Vinnard C, King L, Munsiff S, Crossa A, Iwata K, Pasipanodya J, Proops D, Ahuja S (2016) Long-term Mortality of Patients With Tuberculous Meningitis in New York City: A Cohort Study. Clin Infect Dis. PMI: 27927856

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Guerrini V et al. 2016. Experimental Evolution of Mycobacterium tuberculosis in Human Macrophages Results in Low-Frequency Mutations Not Associated with Selective Advantage

Isolates of the human pathogen Mycobacterium tuberculosis recovered from clinical samples exhibit genetic heterogeneity. Such variation may result from the stressful environment encountered by the pathogen inside the macrophage, which is the host cell tubercle bacilli parasitize. To study the evolution of the M. tuberculosis genome during growth inside macrophages, we developed a model of intracellular culture in which bacteria were serially passaged in macrophage-like THP-1 cells for about 80 bacterial generations. Genome sequencing of single bacterial colonies isolated before and after the infection cycles revealed that M. tuberculosis developed mutations at a rate of about 5.7 × 10-9 / bp/ generation, consistent with mutation rates calculated during in vivo infection. Analysis of mutant growth in macrophages and in mice showed that the mutations identified after the cyclic infection conferred no advantage to the mutants relative to wild-type. Furthermore, activity testing of the recombinant protein harboring one of these mutations showed that the presence of the mutation did not affect the enzymatic activity. The serial infection protocol developed in this work to study M. tuberculosis genome microevolution can be applied to exposure to stressors to determine their effect on genome remodeling during intra-macrophage growth.

Guerrini V, Subbian S, Santucci P, Canaan S, Gennaro ML, Pozzi G (2016) Experimental Evolution of Mycobacterium tuberculosis in Human Macrophages Results in Low-Frequency Mutations Not Associated with Selective Advantage. PLoS One 11: e0167989. PMI: 27959952

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Manganelli R and Gennaro ML 2016. Protecting from Envelope Stress: Variations on the Phage-Shock-Protein Theme

During envelope stress, critical inner-membrane functions are preserved by the phage-shock-protein (Psp) system, a stress response that emerged from work with Escherichia coli and other Gram-negative bacteria. Reciprocal regulatory interactions and multiple effector functions are well documented in these organisms. Searches for the Psp system across phyla reveal conservation of only one protein, PspA. However, examination of Firmicutes and Actinobacteria reveals that PspA orthologs associate with non-orthologous regulatory and effector proteins retaining functions similar to those in Gram-negative counterparts. Conservation across phyla emphasizes the long-standing importance of the Psp system in prokaryotes, while inter- and intra-phyla variations within the system indicate adaptation to different cell envelope structures, bacterial lifestyles, and/or bacterial morphogenetic strategies.

Manganelli R, Gennaro ML (2016) Protecting from Envelope Stress: Variations on the Phage-Shock-Protein Theme. Trends Microbiol. PMI: 27865622

Subbian et al. 2016. Pharmacologic Inhibition of Host Phosphodiesterase-4 Improves Isoniazid-Mediated Clearance of Mycobacterium tuberculosis

The lengthy duration of multidrug therapy needed to cure tuberculosis (TB) poses significant challenges for global control of the disease. Moreover, chronic inflammation associated with TB leads to pulmonary damage that can remain even after successful cure. Thus, there is a great need for the development of effective shorter drug regimens to improve clinical outcome and strengthen TB control. Host-directed therapy (HDT) is emerging as a novel adjunctive strategy to enhance the efficacy and shorten the duration of TB treatment. Previously, we showed that the administration of CC-3052, a phosphodiesterase-4 inhibitor (PDE4i), reduced the host inflammatory response during Mycobacterium tuberculosis (Mtb) infection and improved the antimicrobial efficacy of isoniazid (INH) in both the mouse and rabbit models. In the present study, we evaluated the pharmacokinetics and explored the mechanism underlying the efficacy of a more potent PDE4i, CC-11050, as adjunct to INH treatment in a mouse model of pulmonary Mtb infection. Genome-wide lung transcriptome analysis confirmed the dampening of inflammation and associated network genes that we previously reported with CC-3052. Consistent with the reduction in inflammation, a significant improvement in Mtb control and pathology was observed in the lungs of mice treated with CC-11050 plus INH, compared to INH alone. This important confirmatory study will be used to help design upcoming human clinical trials with CC-11050 as an HDT for TB treatment.

Subbian S, Koo MS, Tsenova L, Khetani V, Zeldis JB, Fallows D, Kaplan G (2016) Pharmacologic Inhibition of Host Phosphodiesterase-4 Improves Isoniazid-Mediated Clearance of Mycobacterium tuberculosis. Front Immunol 7: 238. PMI: 27379099

Shi et al. 2016. Immunometabolism in Tuberculosis

Immunometabolism, the study of the relationship between bioenergetic pathways and specific functions of immune cells, has recently gained increasing appreciation. In response to infection, activation of the host innate and adaptive immune cells is accompanied by a switch in the bioenergetic pathway from oxidative phosphorylation to glycolysis, a metabolic remodeling known as the Warburg effect, which is required for the production of antimicrobial and pro-inflammatory effector molecules. In this review, we summarize the current understanding of the Warburg effect and discuss its association with the expression of host immune responses in tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb). We also discuss potential mechanisms underlying the Warburg effect with a focus on the expression and regulation of hypoxia-inducible factor 1 alpha (HIF-1alpha), the regulatory subunit of HIF-1, a major transcription regulator involved in cellular stress adaptation processes, including energy metabolism and antimicrobial responses. We also propose a novel hypothesis that Mtb perturbs the Warburg effect of immune cells to facilitate its survival and persistence in the host. A better understanding of the dynamics of metabolic states of immune cells and their specific functions during TB pathogenesis can lead to the development of immunotherapies capable of promoting Mtb clearance and reducing Mtb persistence and the emergence of drug resistant strains.

Shi L, Eugenin E, Subbian S (2016) Immunometabolism in Tuberculosis. Front Immunol 7: 150. PMI: 27148269

Subbian et al. 2016. Adjunctive Phosphodiesterase-4 Inhibitor Therapy Improves Antibiotic Response to Pulmonary Tuberculosis in a Rabbit Model

OBJECTIVES: Adjunctive host-directed therapy is emerging as a new potential approach to improve the outcome of conventional antimicrobial treatment for tuberculosis (TB). We tested the ability of a phosphodiesterase-4 inhibitor (PDE4i) CC-11050, co-administered with the first-line anti-TB drug isoniazid (INH), to accelerate bacillary killing and reduce chronic inflammation in the lungs of rabbits with experimental Mycobacterium tuberculosis (Mtb) infection. METHODS: A rabbit model of pulmonary TB that recapitulates the pathologic manifestations seen in humans was used. Rabbits were infected with virulent Mtb by aerosol exposure and treated for eight weeks with INH with or without CC-11050, starting at four weeks post infection. The effect of CC-11050 treatment on disease severity, pathology, bacillary load, T cell proliferation and global lung transcriptome profiles were analyzed. RESULTS: Significant improvement in bacillary clearance and reduced lung pathology and fibrosis were noted in the rabbits treated for eight weeks with INH + CC-11050, compared to those treated with INH or CC-11050 only. In addition, expression of host genes associated with tissue remodeling, tumor necrosis factor alpha (TNF-alpha) regulation, macrophage activation and lung inflammation networks was dampened in CC-11050-treated, compared to the untreated rabbits. CONCLUSIONS: Adjunctive CC-11050 therapy significantly improves the response of rabbits with experimental pulmonary TB to INH treatment. We propose that CC-11050 may be a promising candidate for host directed therapy of patients with pulmonary TB, reducing the duration and improving clinical outcome of antibiotic treatment.

Subbian S, Tsenova L, Holloway J, Peixoto B, O'Brien P, Dartois V, Khetani V, Zeldis JB, Kaplan G (2016) Adjunctive Phosphodiesterase-4 Inhibitor Therapy Improves Antibiotic Response to Pulmonary Tuberculosis in a Rabbit Model. EBioMedicine 4: 104-114. PMI: 26981575

Vargas et al. 2016. Multiplex real-time PCR assays that measure the abundance of extremely rare mutations associated with cancer

The paper describes the use of “SuperSelective” primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time multiplex PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies), thereby providing information that can be used to modify therapy for individual patients, prolonging (and improving the quality of) life. These relatively rapid, low-cost assays are carried out on standard spectrofluorometric thermal cyclers that are available in hospitals and clinical diagnostic laboratories throughout the world. In addition to selectively amplifying the mutations of interest, these assays simultaneously amplify a section of a normal gene that serves as a reference for the amount of DNA present in the sample. Since these are real-time assays, the number of thermal cycles that it takes to generate a pre-selected number of amplified copies (the threshold value) is inversely linearly proportional to the logarithm of the initial number of target molecules present in the sample. The results are then looked at in three ways (i) the generation of amplified copies of the reference gene indicates that the assay worked properly; (ii) the threshold value obtained for the reference gene must occur sufficiently early to confirm that there was enough DNA in the sample to include the rare mutant fragments of interest; and (iii) the difference between the threshold value of each mutant (if it is present in the sample) and the threshold value of the reference gene provides a direct measure of the abundance of each mutant compared to the abundance of the reference gene (without needing to predetermine the amount of DNA in the sample). These assays are extraordinarily specific (essentially amplifying the mutant fragments, while ignoring the abundant related wild-type fragments, even though the only difference between the mutant and the wild-type is as small as a single-nucleotide polymorphism); and these assays are extraordinarily sensitive, potentially able to distinguish even a single mutant fragment in a blood sample, without generating false-negative results.

Structure of a SuperSelective primer for the detection of BRAF V600E mutant sequences in the presence of abundant BRAF wild-type sequences, and a demonstration of its use in monoplex real-time PCR assays. (A) SuperSelective primer BRAF V600E 24-14/14-5:1:1 contains a long 5'-anchor sequence that binds strongly to template strands, a short 3'-foot sequence that includes an interrogating nucleotide that is perfectly complementary to the corresponding nucleotide in a mutant template (but mismatches the corresponding nucleotide in a wild-type template), and a bridge sequence that links the anchor sequence to the foot sequence, and that is chosen to not be complementary to the corresponding intervening sequence in the template strand, thereby forming a single-stranded bubble that separates the function of the anchor from the function of the foot. (B) Real-time PCR assays employing SuperSelective primer BRAF V600E 24-14/14-5:1:1. Six reactions initiated with 1,000,000 BRAF wild-type templates plus different quantities of mutant templates (10, 100, 1,000, 10,000, 100,000, and 1,000,000) are plotted in blue; a reaction initiated with only 1,000,000 wild-type templates is plotted with a dotted orange line; and a control reaction containing no template DNA is plotted in red. (C) The threshold cycle measured for each reaction that contained mutant templates is plotted as a function of the logarithm of the number of mutant templates initially present in each reaction. The dotted orange line indicates the threshold cycle of the reaction containing only wild-type templates.

Vargas DY, Kramer FR, Tyagi S, Marras SAE (2016) Multiplex real-time PCR assays that measure the abundance of extremely rare mutations associated with cancer. PLoS ONE 11: e0156546. PMI: 27244445

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Healey et al. 2016. Prevalent mutator genotype identified in fungal pathogen Candida glabrata promotes multi-drug resistance

The fungal pathogen Candida glabrata has emerged as a major health threat since it readily acquires resistance to multiple drug classes, including triazoles and/or echinocandins. Thus far, cellular mechanisms promoting the emergence of resistance to multiple drug classes have not been described in this organism. Here we demonstrate that a mutator phenotype caused by a mismatch repair defect is prevalent in C. glabrata clinical isolates. Strains carrying alterations in mismatch repair gene MSH2 exhibit a higher propensity to breakthrough antifungal treatment in vitro and in mouse models of colonization, and are recovered at a high rate (55% of all C. glabrata recovered) from patients. This genetic mechanism promotes the acquisition of resistance to multiple antifungals, at least partially explaining the elevated rates of triazole and multi-drug resistance associated with C. glabrata. We anticipate that identifying MSH2 defects in infecting strains may influence the management of patients on antifungal drug therapy.

Healey KR, Zhao Y, Perez WB, Lockhart SR, Sobel JD, Farmakiotis D, Kontoyiannis DP, Sanglard D, Taj-Aldeen SJ, Alexander BD, Jimenez-Ortigosa C, Shor E, Perlin DS (2016) Prevalent mutator genotype identified in fungal pathogen Candida glabrata promotes multi-drug resistance. Nat Commun 7: 11128. PMI: 27020939

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Vir et al. 2015. Single-cell cytokine gene expression in peripheral blood cells correlates with latent tuberculosis status

RNA flow cytometry (FISH-Flow) achieves high-throughput measurement of single-cell gene expression by combining in-situ nucleic acid hybridization with flow cytometry. In this work, Marila Gennaro’s group showed that the antigen-specific T-cell responses detected by FISH-Flow correlated with latent tuberculosis infection (LTBI). Induction of IFNG and IL2 genes, which was detected by FISH-Flow in a quarter the time required to see release of the corresponding proteins by ELISA, occurred primarily in activated CD4+ T cells via T-cell receptor engagement. NK cells also contributed to IFNG gene induction. These results show that antigen-driven induction of T-cell cytokine mRNA is a measurable single-cell parameter of the host responses associated with latent tuberculosis. FISH-Flow read-outs contribute a multi-scale dimension to the immunophenotyping afforded by antibody-based flow cytometry.

Figure: FISH-Flow detection of antigen-specific T cell responses associated with latent tuberculosis infection. PBMCs from 65 LTBI+ and LTBI- donors were cultured +/- PPD stimulation, stained with FITC-labeled alpha-CD3 antibody, hybridized with Cy5-labeled nucleic acid probes specific for IFNG, IL2, and no target control (GFP) and analyzed by flow cytometry. (a) Representative contour plots showing results with cells from an LTBI+ (top row) and an LTBI- (bottom row) donor, unstimulated and PPD-stimulated, as indicated. The rightmost panels in both rows show IFNG induction with PMA/Ionomycin stimulation as positive control. The upper right quadrant of each plot shows the frequencies of mRNA+CD3+ cells. (b) Frequencies of mRNA+CD3+ cells in the 65-donor population, by LTBI and stimulation status. The box plots show lower quartile, median, and upper quartile of the distribution. The lower whisker is the minimum, while the upper limit of the whisker represents the median + 1.5 x the interquartile range (values exceeding the upper limit of the whisker are shown as circles). Additionally, the mean is shown as (+) symbol.

Vir P, Arrigucci R, Lakehal K, Davidow AL, Pine R, Tyagi S, Bushkin Y, Lardizabal A, Gennaro ML (2015) Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status. PLoS One 10: e0144904. PMI: 26658491

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Pandey et al. 2015. MntR(Rv2788) a transcriptional regulator that controls manganese homeostasis in Mycobacterium tuberculosis

In this work, Dr. Rodriguez' group identified a transcriptional regulator essential to maintain manganese homeostasis in M. tuberculosis and discovered a new manganese transporter essential for replication of M. tuberculosis in macrophages. Their results suggest that Manganese is limiting in the host environment and that targetting Mn transport could be an effective way to attenuate M. tuberculosis.

Pandey R, Russo R, Ghanny S, Huang X, Helmann J, Rodriguez GM (2015) MntR(Rv2788): a transcriptional regulator that controls manganese homeostasis in Mycobacterium tuberculosis. Mol Microbiol. PMI: 26337157

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Prideaux et al. 2015. The association between sterilizing activity and drug distribution into tuberculosis lesions

Finding new treatment-shortening antibiotics to improve tuberculosis cure rates and curb the alarming emergence of drug resistance is the major objective of TB drug development. Using a MALDI mass spectrometry imaging suite in a biosafety containment facility, Veronique Dartois' group has shown that the key sterilizing drugs rifampicin and pyrazinamide efficiently penetrate the sites of TB infection in lung lesions. Rifampicin even accumulates in necrotic caseum, a critical lesion site where persisting tubercle bacilli reside. We also suggest that such differential spatial distribution and kinetics of accumulation in lesions may create temporal and spatial windows of monotherapy in specific niches, allowing the gradual development of multidrug resistant TB. We propose an alternative working model to prioritize new TB drug regimens based on quantitative and spatial distribution of TB drugs in the major lesion types found in human lungs. The finding that lesion penetration contributes to treatment outcome has wide implications not only for TB and infectious diseases, but for the discovery of new medicines against any disease with distinct pathology.

Figure Legend: Contrasting distribution of rifampicin and clofazimine in tuberculosis pulmonary lesions 24h after drug administration (at the end of the dosing interval). Two ion maps of rifampicin and clofazimine in thin sections of lung lesions are shown on the right; the signal intensity is fixed for each drug and indicated by the rainbow color scale bar to the right. Hematoxylin and Eosin (H E) staining of one adjacent section is shown on the left, with a zoomed-in inset containing two representative lesions. Black/white contour lines highlight the necrotic center of each lesion. Rifampicin accumulates in necrotic caseum, a critical lesion site where persisting tubercle bacilli reside.

Prideaux B, Via LE, Zimmerman MD, Eum S, Sarathy J, O'Brien P, Chen C, Kaya F, Weiner DM, Chen PY, Song T, Lee M, Shim TS, Cho JS, Kim W, Cho SN, Olivier KN, Barry CE, 3rd, Dartois V (2015) The association between sterilizing activity and drug distribution into tuberculosis lesions. Nature Medicine, in press PMI: 26343800

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Subbian et al. 2015. Lesion-Specific Immune Response in Granulomas of Patients with Pulmonary Tuberculosis: A Pilot Study

The formation and maintenance of granulomas is central to the host response to Mycobacterium tuberculosis (Mtb) infection. It is widely accepted that the lungs of patients with tuberculosis (TB) usually contain multiple infection foci, and that the granulomas evolve and differentiate independently, resulting in considerable heterogeneity. Although gene expression profiles of human blood cells have been proposed as biomarkers of Mtb infection and/or active disease, the immune profiles of discrete lesion types has not been studied extensively. Using histology, immunopathology and genome-wide transcriptome analysis, we explored the immunological profile of human lung TB granulomas. We show that although the different granulomas share core similarities in their immunological/inflammatory characteristics, they also exhibit significant divergence. Despite similar numbers of CD68+ macrophages in the different lesions, the extent of immune reactivity, as determined by the density of CD3+ T cells in the macrophage rich areas, and the extent of fibrosis, shows considerable variation. Both quantitative and qualitative differences among significantly differentially expressed genes (SDEG) were noted in each of the lesion types studied. Further, network/pathway analysis of SDEG revealed differential regulation of inflammatory response, immune cell trafficking, and cell mediated immune response in the different lesions. Our data highlight the formidable challenges facing ongoing efforts to identify peripheral blood biomarkers due to the diversity of lesion types and complexity of local immune responses in the lung.

Subbian S, Tsenova L, Kim MJ, Wainwright HC, Visser A, Bandyopadhyay N, Bader JS, Karakousis PC, Murrmann GB, Bekker LG, Russell DG, Kaplan G (2015) Lesion-Specific Immune Response in Granulomas of Patients with Pulmonary Tuberculosis: A Pilot Study. PLoS One 10: e0132249. PMI: 26133981

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Kurthkoti et al. 2015. The mycobacterial iron dependent regulator IdeR induces ferritin (bfrB) by alleviating Lsr2 repression

IdeR is an essential regulator in Mycobacterium tuberculosis. IdeR controls iron homeostasis by repressing iron uptake and inducing iron storage under conditions of iron sufficiency. IdeR-mediated induction of ferritin (BfrB) is necessary for adaptation to iron limitation, resistance to oxidative stress and tolerance to antibiotics. In this manuscript Dr. Rodriguez' group elucidate the molecular mechanism by which IdeR positively regulates bfrB. They found that IdeR induces bfrB expression by antagonizing the repressor function of the histone-like protein, Lsr2.

Kurthkoti K, Tare P, Paitchowdhury R, Gowthami VN, Garcia MJ, Colangeli R, Chatterji D, Nagaraja V, Rodriguez GM (2015) The mycobacterial iron-dependent regulator IdeR induces ferritin (bfrB) by alleviating Lsr2 repression. Mol Microbiol. PMI: 26268801

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Subbian et al. 2015. Vaccination with an Attenuated Ferritin Mutant Protects Mice against Virulent Mycobacterium tuberculosis

In this study, Dr. Marcela Rodriguez group, in collaboration with Dr. Selvakumar Subbian, examined whether immunization with a bfrB mutant could confer protection against subsequent infection with virulent M. tuberculosis in a mouse model. The results show that the protection elicited by immunization with the bfrB mutant is comparable to BCG vaccination with respect to reduction of bacterial burden. However, significant distinctions in the disease pathology and host genome-wide lung transcriptome suggest improved containment of Mtb infection in animals vaccinated with the bfrB mutant, compared to BCG.

Subbian S, Pandey R, Soteropoulos P, and Rodriguez M (2015) Vaccination with an Attenuated Ferritin Mutant Protects Mice against Virulent Mycobacterium tuberculosis. Journal of Immunology Research, in press.

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Datta et al. 2015. The Psp system of Mycobacterium tuberculosis integrates envelope stress sensing and envelope preserving functions


Interactions between envelope-stress signaling MprAB-sigmaE network and envelope-preserving ClgR-PspA-Rv2743c-Rv2742c system of Mycobacterium tuberculosis. Protein location relative to the line plot (proxy of ClgR activity) depicts the proposed course of events.

The bacterial envelope integrates essential stress-sensing and adaptive functions; thus, envelope-preserving functions are important for survival. In Gram-negative bacteria, envelope integrity during stress is maintained by the multi-gene Psp response. Mycobacterium tuberculosis was thought to lack the Psp system, since it encodes only pspA and no other psp ortholog. In M. tuberculosis, pspA maps downstream from the surface-stress-responsive transcription factor clgR. We discovered that a four-gene operon -- clgR, pspA (rv2744c), rv2743c, rv2742c – of M. tuberculosis that expresses Psp-like functions, based on regulatory and protein-protein interactions within the set and a requirement of the four genes for functions associated with envelope integrity and surface-stress tolerance. Among Actinobacteria, the four-gene module occurred only in tuberculous mycobacteria and was required for intra-macrophage growth, suggesting links between its function and mycobacterial virulence. Additionally, the four-gene module was required for full activity of stress-signaling MprAB-sigmaE network. The positive feedback between envelope-stress-sensing and envelope-preserving functions allows sustained responses to multiple, envelope-perturbing signals during chronic infection, making the system uniquely suited to tuberculosis pathogenesis.

Datta P, Ravi J, Guerrini V, Chauhan R, Neiditch MB, Shell SS, Fortune SM, Hancioglu B, Igoshin O, and Gennaro ML (2015) The Psp system of Mycobacterium tuberculosis integrates envelope stress sensing and envelope preserving functions.Mol Microbiol On-line April 21

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Bushkin et al. 2015. Profiling T cell activation using single-molecule fluorescence in situ hybridization and flow cytometry


Characterizing the “activated” status of stimulated T cells that fight infections, or provide a “memory” response to prior vaccinations and infections, typically relies on detection of induced proteins. Shifting the analysis to detection of induced mRNAs provides several significant advantages, which are illustrated by a new host immunity-based platform for detection of infections that is described in a recent publication by Bushkin et al. Production of intercellular messengers including canonical cytokines IL-2, IFNγ and TNFα is one of the major responses by stimulated T cells. The report shows that cytokine mRNAs synthesized in response to ex vivo stimulation with pathogen-specific antigens can be detected in T cells with single molecule-fluorescence in situ hybridization followed by flow cytometry (FISH-Flow). Background from pre-existing in vivo analytes is lower for mRNAs than for proteins, allowing greater sensitivity for detection of low frequency cells. Moreover, mRNA analysis reveals kinetic differences in cytokine expression (i.e., temporal expression profiles) that are not apparent at the protein level but provide novel insights into gene expression programs expected to define different T cell subsets. The utility of probing immunological memory of infections is demonstrated by detecting T cells that recognize mycobacterial and viral antigens in donors exposed to the respective pathogens. The FISH-Flow method provides a new tool that can be extended to detection of many different RNAs induced in various types of immune cells as a means to identify a response to vaccination, the stage of an infection, or the effectiveness of a therapy for a wide range of infectious diseases and pathological conditions.

Bushkin Y, Radford F, Pine R, Lardizabal A, Mangura BT, Gennaro ML, Tyagi S (2015) Profiling T cell activation using single-molecule fluorescence in situ hybridization and flow cytometry. J Immunol 194: 836-841. PMI: 25505292

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Mustaev et al., 2014. Fluoroquinolone-Gyrase-DNA Complexes: Two Modes of Drug Binding

Fluoroquinolones are widely used antibiotics that act by forming complexes with DNA gyrase and DNA such that the DNA is broken. These reversible complexes block DNA replication and bacterial growth. To understand details of fluoroquinolone action, the complexes have been crystallized and examined by X-ray crystallography. The drugs bind with one end, the C3/C4 end, near the GyrA subunit. The other end, called C7, locates in the GyrB region. PHRI investigators Arkady Mustaev and Karl Drlica led a team that investigated fluoroquinolone binding to gyrase using chemical crosslinking. As expected from X-ray crystallography, the C7 end of fluoroquinolone crosslinked with GyrB. Surprisingly, the same active drug also crosslinked its C7 end to GyrA. Since the GyrA and GyrB binding sites are far apart, fluoroquinolones must bind in two different ways to gyrase. The novel GyrA crosslink behaved as expected with cultured bacterial cells, showing exceptional activity with a resistant mutant and irreversible blockage of DNA replication. The existence of two drug-binding modes raises the possibility that multiple fluoroquinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.

Mustaev A, Malik M, Zhao X, Kurepina N, Luan G, Oppegard LM, Hiasa H, Marks KR, Kerns RJ, Berger JM, Drlica K (2014) Fluoroquinolone-gyrase-DNA complexes: two modes of drug binding. J Biol Chem 289: 12300-12312. PMI: 24497635

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Drlica 2014. Bypassing Fluoroquinolone Resistance with Quinazolinediones: Studies of Drug-Gyrase-DNA Complexes Having Implications for Drug Design

The fluoroquinolones are important antimicrobials whose activity is being severely eroded by bacterial resistance. Recent work revealed that fluoroquinolone activity depends largely on one end of the drug molecule forming a metal-water bridge with the target protein. Clinical resistance correlates with changes in the target protein eliminating the drug-metal-water-protein bridge. A new fluoroquinolone-like molecule called a dione bypasses existing fluoroquinolone resistance by working without forming a metal-water bridge. But the absence of the bridge makes diones weaker than fluoroquinolones. A new study by Karl Drlica and associates, soon to be published in the journal ACS Chemical Biology, shows how changes in the structure of diones can strengthen their activity while still bypassing existing resistance. The work also establishes the functional relevance of crystal structures of complexes formed by binding of fluoroquinolone to target protein and DNA, explains resistance due to another target change, and addresses crossover activity of diones with human topoisomerase II, an enzyme related to the bacterial targets of diones.

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Dartois 2014. The Path of Anti-Tuberculosis Drugs: From Blood to Lesions to Mycobacterial Cells

For the successful treatment of pulmonary tuberculosis (TB), drugs need to penetrate complex lung lesions and permeate the mycobacterial cell wall in order to reach their cellular targets. However, most currently used anti-tuberculosis drugs were introduced into clinical use without considering the pharmacokinetic and pharmacodynamic (PK/PD) properties that influence drug distribution, and this has contributed to the long duration and limited success of current therapies. Dr. Veronique Dartois, a PHRI faculty member, is an internationally recognized expert in studying the PK/PD of anti-tuberculosis drugs. Dr. Dartois has written a review on this important area of research, entitled “The path of anti-tuberculosis drugs: from blood to lesions to mycobacterial cells” that recently appeared in Nature Reviews Microbiology (volume 12 March 2014 pp. 159-167). In this article, Dr. Dartois describes new methods to quantify and image drug distribution in infected lung tissue and in mycobacterial cells, and she explores how this technology could be used to design optimized multidrug regimens. To download or request a PDF copy of the article, please visit the web site of Nature Nature Reviews Microbiology at www.nature.com

Dr. Dartois has demonstrated that the distribution of antibiotics in the lesions of TB infected individuals is not uniform. In another review, in press, entitled “Heterogeneity in tuberculosis pathology, microenvironments and therapeutic reponses”, she and her co-authors present evidence that the prevailing view which claims that all TB induced lesions in an individual react similarly to the systemic immune response and to antibiotic therapies is not valid. In fact, host pathogen interactions within lesions are a dynamic process and result in a spectrum of TB lesions in each infected individual. This new paradigm will be helpful to TB researchers who are studying the pathogenesis of tuberculosis and its treatment. To download a PDF copy of the article, please follow this link.

Dr. Véronique Dartois is Associate Professor at PHRI and the department of Medicine at the New Jersey Medical School, Rutgers, the State University of New Jersey

Dartois V (2014) The Path of Anti-Tuberculosis Drugs: From Blood to Lesions to Mycobacterial Cells. Nature Reviews Microbiology. 12: 159 - 167 PMI: 24487820

Salamon et al. 2014. Cutting Edge: Vitamin D Regulates Lipid Metabolism in Mycobacterium tuberculosis Infection

Vitamin D has long been linked to resistance to tuberculosis, an infectious respiratory disease that is increasingly hard to treat because of multidrug resistance. Previous work established that vitamin D induces macrophage antimicrobial functions against Mycobacterium tuberculosis. In this article, we report a novel, metabolic role for vitamin D in tuberculosis identified through integrated transcriptome and mechanistic studies. Transcriptome analysis revealed an association between vitamin D receptor (VDR) and lipid metabolism in human tuberculosis and infected macrophages. Vitamin D treatment of infected macrophages abrogated infection-induced accumulation of lipid droplets, which are required for intracellular M. tuberculosis growth. Additional transcriptomics results showed that vitamin D downregulates the proadipogenic peroxisome proliferator-activated receptor gamma (PPARgamma) in infected macrophages. PPARgamma agonists reversed the antiadipogenic and the antimicrobial effects of VDR, indicating a link between VDR and PPARgamma signaling in regulating both vitamin D functions. These findings suggest the potential for host-based, adjunct antituberculosis therapy targeting lipid metabolism.

Salamon H, Bruiners N, Lakehal K, Shi L, Ravi J, Yamaguchi KD, Pine R, Gennaro ML (2014) Cutting Edge: Vitamin D Regulates Lipid Metabolism in Mycobacterium tuberculosis Infection. Journal of immunology. PMI: 24899504

Prados-Rosales et al. 2014. Role for Mycobacterium tuberculosis Membrane vesicles in iron acquisition

Mycobacterium tuberculosis releases membrane vesicles packed with molecules that can modulate the immune response. Because environmental conditions often influence the production and content of bacterial vesicles, this study examined M. tuberculosis microvesicles released under iron limitation, a common condition faced by pathogens inside the host. The findings indicate that M. tuberculosis increases microvesicle production in response to iron restriction and these microvesicles contain mycobactin which can serve as an iron donor and supports replication of iron-starved mycobacteria. Consequently, the results revealed a role of microvesicles in iron acquisition in M. tuberculosis, which can be critical for survival in the host. The figure shows Mycobacterium tuberculosis producing microvesicles (yellow) that carry the siderophore mycobactin.

Prados-Rosales R, Weinrick BC, Pique DG, Jacobs WR, Jr., Casadevall A, Rodriguez GM (2014) Role for Mycobacterium tuberculosis membrane vesicles in iron acquisition. J Bacteriol 196: 1250-1256. PMI: 24415729

Pandey R and Rodriguez GM. Molecular Microbiology. 2014. IdeR is required for iron homeostasis and virulence in Mycobacterium tuberculosis

Iron is an essential but potentially harmful nutrient, poorly soluble in aerobic conditions, and not freely available in the human host. To acquire iron, bacteria have evolved high affinity iron acquisition systems that are expressed under iron limitation often in conjunction with virulence determinants. Because excess iron can be dangerous, intracellular iron must be tightly controlled. In mycobacteria, IdeR functions as a global iron dependent transcriptional regulator, but because inactivation of ideR is lethal for Mycobacterium tuberculosis, it has not been possible to use genetics to fully characterize this protein's function or examine the requirement of iron regulation during tuberculosis infection. In this work, a conditional M. tuberculosis ideR mutant was generated and used to study the basis of IdeR's essentiality. This investigation uncovered positive regulation of iron storage as a critical aspect of IdeR's function in regular culture and a prominent factor for survival under stresses associated with life in macrophages. Moreover, this study demonstrates that IdeR is indispensable in the mouse model of tuberculosis, thereby linking iron homeostasis to virulence in M.tuberculosis.

Pandey R and Rodriguez GM (2014) IdeR is required for iron homeostasis and virulence in Mycobacterium tuberculosis. Molecular Microbiology 91: 98-109.

Follow this link to access the journal's web site and to download a PDF copy of this article.

Shah K and Tyagi S. Molecular Systems Biology. 2013. Barriers to transmission of transcriptional noise in a c-fos c-jun pathway

In their paper “Barriers to Transmission of Transcriptional Noise in a c-fos c-jun Pathway,” Khyati Shah and Sanjay Tyagi show that in higher eukaryotes chromatin is simultaneously responsible for creating gene expression noise and for insulating genes from deleterious effects of noise form upstream sources.

Follow this link to access the journal's web site and to download a PDF copy of this article.

Subbian S. et al, Cell Communication and Signaling. 2013. Early innate immunity determines outcome of Mycobacterium tuberculosis pulmonary infection in rabbits

Pulmonary infection of humans by Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), results in active disease in 5-10% of individuals, while asymptomatic latent Mtb infection (LTBI) is established in the remainder. The host immune responses that determine this differential outcome following Mtb infection are not fully understood. Using a rabbit model of pulmonary TB, we have shown that infection with the Mtb clinical isolate HN878 (a hyper-virulent W-Beijing lineage strain) leads to progressive cavitary disease similar to what is seen in humans with active TB. In contrast, infection with Mtb CDC1551 (a hyper-immunogenic clinical isolate) is efficiently controlled in rabbit lungs, with establishment of LTBI, which can be reactivated upon treatment with immune-suppressive drugs. We hypothesize that the initial interaction of Mtb with the cells of the host response in the lungs determine later outcome of infection. To test this hypothesis, we used our rabbit model of pulmonary TB and infected the animals with Mtb HN878 or CDC1551. At 3 hours, with similar lung bacillary loads, HN878 infection caused greater accumulation of mononuclear and polymorphonuclear leukocytes (PMN) in the lungs, compared to animals infected with CDC1551. Using whole-genome microarray gene expression analysis, we delineated the early transcriptional changes in the lungs of HN878- or CDC1551-infected rabbits at this time and compared them to the differential response at 4 weeks of Mtb-infection. Our gene network and pathway analysis showed that the most significantly differentially expressed genes involved in the host response to HN878, compared to CDC1551, at 3 hours of infection, were components of the inflammatory response and STAT1 activation, recruitment and activation of macrophages, PMN, and fMLP (N-formyl-Methionyl-Leucyl-Phenylalanine)-stimulation. At 4 weeks, the CDC1551 bacillary load was significantly lower and the granulomatous response reduced compared to HN878 infection. Moreover, although inflammation was dampened in both Mtb infections at 4 weeks, the majority of the differentially expressed gene networks were similar to those seen at 3 hours. Based on the results, we propose that differential regulation of the inflammation-associated innate immune response and related gene expression changes seen at 3 hours determine the long term outcome of Mtb infection in rabbit lungs.

Follow this link to download a PDF copy of this article.

Manca C et al, PLoS One. 2013. Host Targeted Activity of Pyrazinamide in Mycobacterium tuberculosis Infection

Pyrazinamide (PZA) is one of the first line antibiotics used for the treatment of tuberculosis (TB). In the present study, we have used in vitro and in vivo systems to investigate whether PZA, in addition to its known anti-mycobacterial properties, modulate the host immune response during Mycobacterium tuberculosis (Mtb) infection. In vitro we have examined the effect of PZA on cytokine and chemokine release by Mtb-infected or Toll-like receptor (TLR) -stimulated primary human monocytes. In vivo, we have investigated at the transcriptional levels using genome-wide microarray gene expression analysis, whether PZA treatment of Mtb-infected mice alters the host immune response to Mtb infection in the lungs. Here, we report that PZA treatment of Mtb-infected human monocytes and mice significantly reduces the release of pro-inflammatory cytokines and chemokines, including IL-1Beta, IL-6, TNF-alpha and MCP-1 at the protein and at the gene transcription levels, respectively. Data from microarray analysis also reveal that PZA treatment of Mtb-infected mice significantly alters the expression level of genes involved in the regulation of the pro-inflammatory mediators, lung inflammatory response and TLR signaling networks. Specifically, genes coding for adenylate cyclase and Peroxisome-Proliferator Activated Receptor (PPAR), molecules known for their anti-inflammatory effect, were found to be up-regulated in the lungs of PZA-treated Mtb-infected mice. Based on the microarray findings, we propose that PZA treatment modulates the host immune response to Mtb infection by reducing pro-inflammatory cytokine production, probably through PPAR- and NF-kB- dependent pathways. In addition, our results suggest that inclusion or exclusion of PZA in the TB treatment regimen could potentially affect the biomarker signature detected in the circulation of TB patients.

Follow this link to download a PDF copy of this article.

Mavrianos J. et al, Eukaryotic Cell 2013. Mitochondrial Two-Component Signaling Systems in Candida albicans

Two-component signal transduction pathways are one of the primary means by which microorganisms respond to environmental signals. These signaling cascades originated in prokaryotes and were inherited by eukaryotes via endosymbiotic lateral gene transfer from ancestral cyanobacteria. In a recent issue of Eukaryotic Cell PHRI investigator Neeraj Chauhan and his collaborators report that the nuclear genome of pathogenic fungus Candida albicans contains elements of a two-component signaling pathway that seem to be targeted to the mitochondria. In this study they provide a link between regulation of mitochondrial function, such as apoptosis, and two-component signal transduction pathways in C. albicans.

Follow this link to download a PDF copy of this article.

Liu T. et al, PLOS Path 2013. Brain Inositol Is a Novel Stimulator for Promoting Cryptococcus Penetration of the Blood-Brain Barrier

Cryptococcus neoformans is the most common cause of fungal meningitis, with high mortality and morbidity. The reason for the frequent occurrence of Cryptococcus infection in the central nervous system (CNS) is poorly understood. The facts that human and animal brains contain abundant inositol and that Cryptococcus has a sophisticated system for the acquisition of inositol from the environment suggests that host inositol utilization may contribute to the development of cryptococcal meningitis. In this study, PHRI investigator Chaoyang Xue and his co-workers found that inositol plays an important role in Cryptococcus traversal across the blood-brain barrier (BBB) both in an in vitro human BBB model and in in vivo animal models. The capacity of inositol to stimulate BBB crossing was dependent upon fungal inositol transporters, indicated by a 70% reduction in transmigration efficiency in mutant strains lacking two major inositol transporters, Itr1a and Itr3c. Upregulation of genes involved in the inositol catabolic pathway was evident in a microarray analysis following inositol treatment. In addition, inositol increased the production of hyaluronic acid in Cryptococcus cells, which is a ligand known to binding host CD44 receptor for their invasion. These studies suggest an inositol-dependent Cryptococcus traversal of the BBB, and support our hypothesis that utilization of host-derived inositol by Cryptococcus contributes to CNS infection.

Follow this link to download a PDF copy of this article.

Salamon H. et al, J. Immunol 2013. Evidence for Postinitiation Regulation of mRNA Biogenesis in Tuberculosis

M. tuberculosis infection drives substantial changes in host gene expression, but the regulatory basis for those changes is poorly understood. To address this gap in knowledge, the regulation than controls expression of transcription factors STAT1 and IRF1 was studied, since they are essential for host defense against M. tuberculosis. We report discordance of changes in their nascent transcript and total nuclear RNA abundance, together with lack of effect on their RNA half-lives, in human THP-1 cells infected with M. tuberculosis and stimulated with IFN-gamma. The results indicate that negative post-initiation regulation of mRNA biogenesis limits the expression of these factors. Transcriptome analysis reveals down-regulation of post-initiation mRNA biogenesis processes and pathways by infection, with and without IFN-gamma stimulation. Clinical relevance for regulation of post-initiation mRNA biogenesis is demonstrated by analysis of transcriptome data from human samples showing that post-initiation mRNA biogenesis pathways are repressed in latent tuberculosis infection compared to cured disease and in active tuberculosis compared to ongoing treatment or to latent tuberculosis. Collectively, the molecular and bioinformatic analyses point toward down-regulation of post-initiation mRNA biogenesis pathways as a means by which M. tuberculosis infection limits expression of immunologically essential transcription factors. Thus, negative regulation of post-initiation mRNA biogenesis may constrain the macrophage response to infection and overall host defense against tuberculosis. This report has been highlighted in the "In This Issue" section of the March 15, 2013 issue of the Journal of Immunology.

This article (Salamon H. et al, 2013, J. Immunol) can be accessed at www.jimmunol.org.

Subbian S. et al, J Cell Commun Signal 2013. Molecular immunologic correlates of spontaneous latency in a rabbit model of pulmonary tuberculosis

Pulmonary infection by Mycobacterium tuberculosis (Mtb) results in latent tuberculosis infection (LTBI) in more than 90% of humans, with no symptoms of active disease. The World Health Organization estimates that 1.5 billion people have LTBI, which can reactivate in the setting of waning host immunity, posing a threat to global TB control. However, the host immune factors involved in establishing LTBI are poorly understood. Among various animal models used to understand the pathogenesis of TB, rabbits are the only animal model, besides nonhuman primates, that fully recapitulates the pathological features of human TB. We defined the molecular immunological correlates of LTBI establishment in a rabbit model of pulmonary infection with Mtb CDC1551. After aerosol infection, exponential bacterial growth was noted in the lungs for 4 weeks, followed by a significant decline by 12 weeks, resulting in the absence of cultivable bacilli by 24 weeks. We used microarrays to profile the Mtb-infected rabbit lung transcriptome at various stages of infection. At 2 weeks post-infection, the gene networks involved in natural killer (NK) and dendritic cell (DC) activation and macrophage antimicrobial activities were highly upregulated. This was followed by upregulation of gene networks involved in macrophage and T cell activation and autophagy, peaking at 4 to 8 weeks. Concomitantly, host Th1, but not Th2 or inflammatory, immune response genes were significantly upregulated. Thus, the expression kinetics of genes involved in cross-talk between innate and adaptive immunity over the first 8 weeks post-infection were consistent with early efficient control of infection in the lungs. Interestingly, expression of many genes of the host innate and adaptive immune response pathways was downregulated at 12 weeks, suggesting that immune activation did not persist once bacilli began to clear from the infected lungs. Our results suggest that an early activation of host innate immunity prior to efficient activation of T cell-mediated adaptive immunity is essential for establishment of LTBI in Mtb CDC1551-infected rabbits. We also show that T cell activation and the host adaptive immune response networks are dampened once bacterially load is cleared, resulting in spontaneous LTBI.

This article can be accessed at www.biosignaling.com.

Xue C., PLOS Pathogens 2012. Cryptococcus and beyond - Inositol utilization and Its implications for the emergence of fungal virulence

There are over one million fungal species in nature, however only a handful of them cause human diseases. One central question is what leads these fungi to become pathogenic. A variety of distinct factors have been identified to aid the virulence of fungi in their transition from environmental reservoirs to mammals. In a review article, recently published in PLoS Pathogens, Chaoyang Xue presented a new theory. Based on his group's research and work of others, Chaoyang argues that the ability to utilize the carbohydrate inositol by a yeast pathogen Cryptococcus neoformans, likely evolved by associating with its natural hosts, contributes to its virulence in mammals, especially during infections in brains.

This review (Xue, 2012, PLOS Pathogens, Volume 8, Issue 9, e1002869) can be accessed at www.plospathogens.org.

Batish M. et al, PNAS 2012. Neuronal mRNAs travel singly into dendrites

In a study, just published in the Early Edition of the Proceedings of the National Academy of Sciences of the USA, Sanjay Tyagi and colleagues show that neuronal mRNAs travel into dendrites as the solitary cargo of RNA transport granules. Their single-molecule imaging shows that two molecules of the same or different mRNA species do not assemble in common structures. This model of mRNA transport affords a finer control of mRNA content within a synapse for synaptic plasticity then previous models did.

The study (Batish et al 2012, PNAS, doi:10.1073 / pnas.1111226109) can be accessed at www.pnas.org.

Vargas D.Y. et al, Cell 2011. Single-molecule imaging of transcriptionally coupled and uncoupled splicing

Introns are usually removed from pre-mRNAs during transcription while the pre-mRNA is still tethered to the gene locus via RNA polymerase. In a recent issue of Cell, PHRI investigator Sanjay Tyagi and his group describes circumstances under which splicing is uncoupled from transcription. They show that proteins that ensure that only a particular splice variant is expressed during alternative splicing, function by slowing the splicing to such an extent, that it is concluded post-transcriptionally after the release of pre-mRNA from the gene locus. This insight was gleaned using a unique single-molecule imaging approach that the group has developed earlier.

Their work - Vargas DY, Shah K, Batish M, Levandoski M, Sinha S, Marras SA, Schedl P, Tyagi S (2011) Single-molecule imaging of transcriptionally coupled and uncoupled splicing. Cell 147: 1054-1065 - appeared as a featured article, which can be accessed at www.cell.com/

An interview conducted by Cell's editorial staff with Sanjay Tyagi about this work can be listened to at www.cell.com/../paperclip.mp3

Shi L. et al, 2010. Carbon flux rerouting during M. tuberculosis growth arrest

Mycobacterium tuberculosis is the causative agent of tuberculosis, an infectious disease that claims almost 2 million human lives every year. A hallmark of the life cycle of this pathogen is its ability to switch between replicative and non-replicative states in response to host immunity. A research team led by Lanbo Shi and Marila Gennaro at PHRI/UMDNJ reports in Molecular Microbiology the results of a multi-scale approach to the study of M. tuberculosis metabolism in vivo (mouse), in silico, and in vitro (broth culture). Transcription profiling of tubercle bacilli during infection of mouse lungs led the PHRI/UMDNJ investigators with their collaborators at the Veterans Affairs Medical Center (Long Beach, California), University of Surrey (Guildford, Surrey, United Kingdom) and the Trudeau Institute (Saranac Lake, New York) to a novel model of M. tuberculosis metabolic adaptation during infection. In this model, carbon flow redistributes at the phosphoenolpyruvate/pyruvate-oxaloacetate node as growth stops, switching from the generation of energy and biosynthetic precursors to the synthesis of storage compounds that include triacylglycerols and glutamate. The in vivo data provided conditions to perform in silico simulations of metabolic flux patterns associated with M. tuberculosis growth arrest using a genome-scale metabolic network of the tubercle bacillus. Metabolic fluxes predicted that utilization of different carbon sources would result in similar flux solutions. This prediction was tested in vitro using M. tuberculosis cultures subjected to bacteriostatic stress in media containing different carbon sources. It was found that transcriptional changes of key metabolic genes during M. tuberculosis growth arrest were independent of stress signal and carbon source. The work indicates that M. tuberculosis persistence shares critical metabolic features with bacterial and yeast sporulation, plant seed formation, and animal hibernation. Thus, mycobacterial dormancy can be viewed as a variation of the broader stress-response theme developed in nature. Moreover, the results lead to the novel view that the metabolic changes of M. tuberculosis are part of a developmental decision rather than a response to changed nutrient availability.

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Kunnath-Velayudhan et al, 2010. Dynamic antibody responses to the Mycobacterium tuberculosis proteome

Considerable effort has been directed toward controlling tuberculosis (TB), which kills almost two million people yearly. High on the research agenda is the discovery of biomarkers of active TB for diagnosis and for monitoring treatment outcome. Rational biomarker discovery requires understanding host-pathogen interactions leading to biomarker expression. An international, multi-center research team led by Marila Gennaro at PHRI/UMDNJ reports in the Proceedings of the National Academy of Sciences of the USA (Open Access on Publisher's web site) a systems immunology approach to TB biomarker discovery. The group integrated clinical data, bacterial metabolic and regulatory information with high-throughput detection in human serum of antibodies to the entire Mycobacterium tuberculosis proteome. Sera collected from more than 500 TB suspects world-wide (11 countries) recognized about 10% of the bacterial proteome. This result defines the M. tuberculosis immunoproteome, which is rich in membrane-associated and extracellular proteins. Additional analyses revealed that during active tuberculosis, (i) antibody responses focused on a about 0.5% of the proteome enriched for extracellular proteins, (ii) relative target preference varied among patients, and (iii) responses correlated with bacillary burden. These results indicate that the B-cell response tracks the evolution of infection and the pathogen burden and replicative state, and suggest functions associated with B-cell-rich foci seen in tuberculous lung granulomas. The integrated proteome-scale approach taken by the UMDNJ investigators, together with their partners at AbaSci, LLC (Albany, California), Istituto San Raffaele (Milan, Italy), Antigen Discovery Inc. (Irvine, California), and the Foundation for Innovative and New Diagnostics (Geneva, Switzerland), is applicable to other chronic infections characterized by diverse antibody target recognition.

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Hernandez et al., 2010. Immunogenicity and protection induced by a Mycobacterium tuberculosis sigE mutant in a BALB/c mouse model of progressive pulmonary tuberculosis

Tuberculosis is still one of the main challenges to human global health, leading to about two million deaths every year. One of the reasons for its success is the lack of efficacy of the widely used vaccine Mycobacterium bovis BCG. In this newly published paper, the authors analyzed the potential use of an attenuated mutant of Mycobacterium tuberculosis H37Rv lacking the sigma factor σE as a live vaccine. It was demonstrated that mice infected by this mutant strain showed significant higher survival and less tissue damage than animals infected with the parental or complemented mutant strains. Although animals infected with the sigE mutant had low bacillary loads, their lungs showed significantly higher production of the protective factors IFNγ, TNFα, iNOS and β-defensins than those of animals infected with the parental or complemented mutant strains. When the sigE mutant was used as a subcutaneous vaccine, it was able to induce a higher level of protection than did BCG with both H37Rv and the highly virulent Beijing strain of M. tuberculosis. Moreover, it was shown that the sigE mutant, when inoculated subcutaneously, was more attenuated than BCG in immunodeficient nude mice. Taken together, these findings suggest that the sigE mutant is a very promising candidate for the development of a novel, live attenuated vaccine strain against tuberculosis. This study was published in Infection and Immunity, July 2010 78(7):3168-76.

To download a copy of this publication, please follow this link

Xue et al., 2010. Role of an expanded inositol transporter repertoire in Cryptococcus neoformans sexual reproduction and virulence

The importance of fungal inositol acquisition system on the development and virulence of Cryptococcus neoformans, an AIDS-associated human fungal pathogen, has been discovered by a team of researchers from multiple institutions, lead by Chaoyang Xue, a principal investigator at PHRI. Their results are published in the inauguration issue of the new ASM press journal, mBio. C. neoformans is the leading cause of fungal meningitis in immunosuppressed patients that causes over 1 million cases of meningitis annually. How this fungus completes its sexual cycle and produce infectious spores in nature and why it frequently infects central nervous system to cause fatal meningitis are critical questions that remain to be understood. Dr. Xue’s previous study revealed that inositol is important for the sexual development of C. neoformans. The existence of abundant inositol in the human central nervous system promoted him to hypothesize that inositol acquisition could also be important for the development of cryptococcal meningitis. In this study, Dr. Xue and his collaborators demonstrate that inositol acquisition is important not only for fungal sexual reproduction, but also for fungal virulence. They identified an expanded inositol transporter gene family that contains over ten members in C. neoformans compared to only two in most fungi studied, and is important for both fungal sexual reproduction and virulence. This work contributes to our understanding on how fungi respond to the environmental inositol availability and its impact on sexual reproduction and virulence. These results will appear in mBio, May 2010, volume 1, issue 1.

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Several media groups have reported on this upcoming publication. Their editorial comments and reports can be found on the following sites: www.dukehealth.org, www.emaxhealth.com, and www.scientistlive.com.

Ibrahim et al, 2009. Structure of the C-terminal domain of transcription factor IIB from Trypanosoma brucei
Proc Natl Acad Sci USA 106: 13242 - 13247.

The first structure solved using data obtained from the PHRI X-ray Crystallography Core Facility was published in the Proceedings of the National Academy of Sciences. The laboratory of Dr. David Wah solved the structure of the C-terminal domain of Transcription Factor IIB (TFIIB) from the infectious parasite Trypanosoma brucei, the causative agent of African Sleeping sickness. T. brucei TFIIB is essential for the viability of this parasite. Strikingly, the putative DNA binding surface of T. brucei is different from the DNA binding surface of human TFIIB, suggesting that T. brucei TFIIB binds DNA in a different way from human TFIIB. These results give insights into transcriptional regulation in trypanosomes. A more thorough mechanistic understanding of the trypanosome transcriptional machinery will allow us to develop new therapeutic tools against these parasites. The results are published in Proc Natl Acad Sci USA 2009, Volume 106, Number 32, pages 13242-13247.

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Fontán and Smith, 2009. M. tuberculosis Sigma E protects against environmental stress, immune responses.
Microbe 4: 119 - 123.

The Mycobacterium tuberculosis transcription factor sigma E regulates expression of several bacterial components, including those that maintain the cell envelope and others that enable it to respond to environmental stresses. Sigma E is also essential for M. tuberculosis survival in macrophages and in the lungs of infected animals. Recent work, discussed by PHRI scientists Patricia Fontán and Issar Smith in the accompanying review, indicate that several bacterial genes are not expressed when the sigma E mutant strain infects macrophage infections and also that the products of these genes in the wild type bacterium suppress host innate immune responses. Further, these results suggest that the M. tuberculosis sigma E mutant, because it elicits elevated immune responses during infections, could serve as an effective live, attenuated vaccine strain. This review, "M. tuberculosis Sigma E protects against environmental stress, immune responses", was published in Microbe, Volume 4, Number 3, 2009, pages 119-123.

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Raj et al, 2008. Imaging individual mRNA molecules using multiple singly labeled probes.
Nature Methods 5: 877 - 879.

Sanjay Tyagi and Patrick van den Bogaard at the PHRI center in collaboration with MIT scientists Arjun Raj, Scott Rifkin and Alexander van Oudenaarden have developed a new method to image individual mRNA molecules in fixed cells. Their findings are published in the October issue of Nature Methods (Raj et al, Imaging individual mRNA molecules using multiple singly labeled probes, Nature Methods, 5, 877 - 879). In their study, they use multiple singly labeled probes to render each mRNA molecule visible as a computationally identifiable fluorescent spot in fluorescence microscopy. The number of mRNA molecules present in each cell can be explicitly counted. They demonstrate simultaneous detection of three mRNA species in cultured cells, yeasts, nematodes, fruit fly wing discs, mammalian cell lines and neurons.


                                                     Elt-2 mRNA molecules visible as magenta spots in
                                                     the gut region of an early stage nematode embryo.

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Fontan et al, 2008. Mycobacterium tuberculosis SigE regulon modulates the host inflammatory response.
Journal of Infectious Diseases 198: 877-885.

The first line of defense of the humans against Mycobacterium tuberculosis (Mtb) infection is the macrophage, a phagocytic cell that is part of the innate immune response and has mechanisms to control the growth of infection pathogens. However Mtb usually subverts these bactericidal mechanisms and can grow intracellularly in macrophages. An understanding of how Mtb adapts to the hostile environment of these cells is relevant for the development of new treatments for tuberculosis. Sigma factors are important bacterial transcription activators that confer transcriptional specificity, and these proteins play a major role in bacterial adaptation and in pathogenicity. Previous work from the Smith laboratory has shown that sigma factor E (SigE) is essential for the ability of Mtb to grow in macrophages and in animal models of infection. To find which genes controlled by SigE are essential for virulence, the Smith group, in collaboration with the laboratories of Drs. Richard Pine and Patricia Soteropoulos at PHRI and the facilities of the PHRI Center for Applied Genomics, has identified the SigE regulon during infection of macrophages and has analyzed the impact of this regulon on the transcriptional response of the infected macrophages. The newly published results of these experiments (P. Fontan et al “Mycobacterium tuberculosis SigE Regulon Modulates the Host Inflammatory Response” Journal of Infectious Diseases 198:877-885. 2008). indicate that SigE regulates the expression of genes involved in the maintenance of Mtb cell envelope integrity and function during macrophage infection. Analysis of the macrophage transcriptional response to Mtb infection indicates that the SigE regulon is involved in the down modulation of the early inflammatory response.

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