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Scientific Overview Research Interest Summary Principal Investigators    Yuri Bushkin, Ph.D.
   Theresa Chang, Ph.D.
   Neeraj Chauhan, Ph.D.
   Véronique Dartois, Ph.D.
   Thomas Dick, Ph.D.
   Karl Drlica, Ph.D.
   David Dubnau, Ph.D.
   Eliseo A. Eugenin, Ph.D.
   Marila Gennaro, M.D.
   Gilla Kaplan, Ph.D.
   Fred Kramer, Ph.D.
   Barry Kreiswirth, Ph.D.
   Min Lu, Ph.D.
   Leonard Mindich, Ph.D.
   Arkady Mustaev, Ph.D.
   Jyothi Nagajyothi, Ph.D.
   David Perlin, Ph.D.
   Richard Pine, Ph.D.
   Abraham Pinter, Ph.D.
   Marcela Rodriguez, Ph.D.
   Selvakumar Subbian, Ph.D.
   Sanjay Tyagi, Ph.D.
   Christopher Vinnard, M.D.
   Chaoyang Xue, Ph.D.
   Xilin Zhao, Ph.D.

   Research Faculty
   Liang Chen, Ph.D.
   Eugenie Dubnau, Ph.D.
   Jeanette Hahn, Ph.D.
   Salvatore Marras, Ph.D.
   Lanbo Shi, Ph.D.
   Yanan Zhao, Ph.D.

Emeritus Faculty Recent Publications
 
Marila Gennaro, M.D.

Research Summary  |  Selected Publications  |  C.V.
 

Professor of Medicine
Public Health Research Institute Center
New Jersey Medical School - Rutgers, The State University of New Jersey
225 Warren Street
Newark, New Jersey 07103

Phone: (973) 854-3210
e-mail: gennarma@njms.rutgers.edu


Research Summary

The Gennaro laboratory studies infection with Mycobacterium tuberculosis, which still causes almost 10 million new cases of tuberculosis (TB) and 2 million deaths per year worldwide (http://www.who.int/tb/). In the immunocompetent host, tubercle bacilli survive expression of adaptive immunity by entering a dormant state. The ensuing stage of infection is asymptomatic (latent infection). About one-third of the world population bears latent M. tuberculosis infection. Most infected persons remain symptom-free for their entire life because their immune system keeps the infection in check. When host immunity falters, however, tubercle bacilli can resume growth and cause disease. The diseased individual sheds tubercle bacilli by coughing or sneezing, thereby transmitting infection to new hosts. Thus, the ability of the microorganism to switch between growth states determines its persistence in the individual host and in the host population.

The switch between replicative and non-replicative states operated by the tubercle bacillus can be investigated by use of in vitro and in vivo models. One area of research in our laboratory is the characterization of the remodeling of the M. tuberculosis transcriptome during infection. We have shown that arrest of M. tuberculosis growth induced by expression of lung immunity in mice is characterized by transcriptional profiles indicative of upregulation of a “dormancy” regulon, changes in respiratory metabolism, rerouting of the carbon flow, and remodeling of the mycobacterial cell wall. Carbon flow rerouting was marked by a switch from metabolic pathways generating energy and biosynthetic precursors in growing bacilli to pathways for storage compound synthesis during growth arrest. We have also seen that growth and persistence of M. tuberculosis in an animal model are associated with different profiles of bacterial antigens, a finding that may have profound implications on new vaccine design. We are currently moving toward genome-scale explorations of the dormant tubercle bacillus to obtain an integrated knowledge of the transcriptional and metabolic changes associated with M. tuberculosis dormancy. Understanding the changing physiology of M. tuberculosis with growth state is key to the development of new drugs and new vaccines.

The outcome of infection with M. tuberculosis is the result of a complex, multicellular interplay that includes the pathogen and both innate and adaptive immune cells. While much is known about transcriptional programs and phenotypes of the individual cell types during infection, the dynamics of their interaction are still poorly understood. This poses a multi-scale problem that spans the cellular and molecular scales, with molecular changes mediating cellular interactions between individual cell types. Studying interactions between two different cell types should provide a relatively tractable system for multi-scale modeling of host-pathogen intercellular networks that departs from most current approaches focusing on a single cell type. A second area of research in our laboratory focuses on the critical interaction between tubercle bacilli and the macrophages that carry them. We hypothesize that any outcome of M. tuberculosis infection reflects reciprocal, likely iterative, interactions by which macrophage and tubercle bacillus change each other’s molecular landscapes and cellular phenotypes. These dynamics are governed by networks of mutual signaling that affect gene expression and downstream cellular activities of host cell and pathogen. The goal of our ongoing work is to uncover and mechanistically understand how these networks govern entry into and exit from latency by combining i) statistical pathway analyses and ii) bottom-up and top-down modeling strategies applied to ex vivo infection of human primary lung macrophages with M. tuberculosis. We expect to identify critical host-pathogen interactions leading to the development of novel therapeutic combination regimens targeting both host and pathogen functions, e.g., by disrupting both arms of an intercellular feedback loop. This program - which utilizes experimental methods, modeling approaches, and bioinformatics - establishes the Gennaro group as one of five NHLBI-funded TB systems biology centers in the United States.

Proper drug treatment not only cures the diseased persons but also drastically decreases the time they are contagious. Thus, early and accurate diagnosis of disease leading to timely treatment is a key element of tuberculosis control. A third area of research in the laboratory is identifying immune biomarkers of M. tuberculosis infection and disease, primarily for diagnostic purposes. Bacterial proteins characterized in our laboratory over the years have become part of multi-protein, diagnostic tests for latent infection and active disease that are commercially available or are being developed. To expand our current knowledge of antibody markers of disease, our laboratory has engaged in proteome-scale interrogation of the antibody response to M. tuberculosis infection and disease. We have adopted a systems immunology approach integrating clinical data and bacterial metabolic and regulatory information with high-throughput detection in human serum of antibodies to the entire M. tuberculosis proteome (~4,000 proteins). We found that sera from worldwide TB suspects recognized approximately 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 an approximately 0.5% of the proteome enriched for extracellular proteins, (ii) relative target preference varied among patients, and (iii) responses correlated with bacillary burden. On-going work focuses on evaluating the diagnostic potential of TB-associated proteins identified by the proteome screen and to investigate the cellular basis of the dynamics of the antibody response during the evolution of the disease.

The advent of the IFN-gamma release assays (IGRA) have tremendlously advanced diagnosis of latent M. tuberculosis infection (LTBI). IGRA however cannot distinguish between LTBI and active tuberculosis, and take at least one day from peripheral blood collection to final readout. In collaboration with Yuri Bushkin, Sanjay Tyagi, and Richard Pine, a novel platform for blood-based diagnostic assays has been developed in which peripheral blood T cells are stimulated ex vivo with M. tuberculosis-specific antigens, and induction of key Th1 cytokine genes is detected by fluorescence in situ hybridization (FISH) and flow cytometry. By combining nucleic acid detection and flow cytometry, we expect to be able to detect the response to ex vivo antigen stimulation rapidly and with high sensitivity. Moreover, multiparameter flow cytometry is uniquely suited to detect low frequency events. We are currently leading a multidisciplinary effort toward a rapid and accurate single-parameter assay for LTBI diagnosis, and we plan to move the FISH-Flow platform through preclinical product development in collaboration with an industrial partner. We are also currently exploring multiparameter readouts distinguishing LTBI from active TB, and detecting responses to TB vaccination.





Selected Publications

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

Radford F, Tyagi S, Gennaro ML, Pine R, Bushkin Y (2016) Flow cytometric characterization of antigen-specific T cells based on RNA and its advantages in detecting infections and immunological disorders. Critical Reviews in Immunology 5: 359-378. PMI:

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

Chauhan R, Ravi J, Datta P, Chen T, Schnappinger D, Bassler KE, Balazsi G, Gennaro ML (2016) Reconstruction and topological characterization of the sigma factor regulatory network of Mycobacterium tuberculosis. Nat Commun 7: 11062. PMI: 27029515

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

Shi L, Salamon H, Eugenin EA, Pine R, Cooper A, Gennaro ML (2015) Infection with Mycobacterium tuberculosis induces the Warburg effect in mouse lungs. Sci Rep 5: 18176. PMI: 26658723

Datta P, Ravi J, Guerrini V, Chauhan R, Neiditch MB, Shell SS, Fortune SM, Hancioglu B, Igoshin OA, Gennaro ML (2015) The Psp system of Mycobacterium tuberculosis integrates envelope stress-sensing and envelope-preserving functions. Mol Microbiol 97: 408-422. PMI: 25899163

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

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. J Immunol 193: 30-34. PMI: 24899504

Skerry C, Pinn ML, Bruiners N, Pine R, Gennaro ML, Karakousis PC (2014) Simvastatin increases the in vivo activity of the first-line tuberculosis regimen. J Antimicrob Chemother 69: 2453-2457. PMI: 24855121

Salamon H, Qiao Y, Cheng JC, Yamaguchi KD, Soteropoulos P, Weiden M, Gennaro ML, Pine R (2013) Evidence for Postinitiation Regulation of mRNA Biogenesis in Tuberculosis. J Immunol. PMI: 23378427

Pine R, Bushkin Y, and Gennaro ML (2013) Immunological Biomarkers for Tuberculosis: Potential for a Combinatorial Approach, in Systems Biology of Tuberculosis, JJ McFadden, D Beste and A Kierzek, eds., Springer, London, pp. 193-219.

Miotto P, Forti F, Ambrosi A, Pellin D, Veiga DF, Balazsi G, Gennaro ML, Di Serio C, Ghisotti D, Cirillo DM (2012) Genome-Wide Discovery of Small RNAs in Mycobacterium tuberculosis. PLoS One 7: e51950. PMI: 23284830

Kunnath-Velayudhan S, Davidow AL, Wang HY, Molina DM, Huynh VT, Salamon H, Pine R, Michel G, Perkins MD, Xiaowu L, Felgner PL, Flynn JL, Catanzaro A, Gennaro ML (2012) Proteome-Scale Antibody Responses and Outcome of Mycobacterium tuberculosis Infection in Nonhuman Primates and in Tuberculosis Patients. J Infect Dis 206: 697-705. PMI: 22732925

Balázsi G, Igoshin O and Gennaro ML (2012) The transcriptional regulatory network of Mycobacterium tuberculosis, in Bacterial Gene Regulation and Transcriptional Networks, M Madan Babu, ed., Horizon Scientific Press, pp. 181-193.

Kunnath-Velayudhan S, Gennaro ML (2011) Immunodiagnosis of tuberculosis: a dynamic view of biomarker discovery. Clin Microbiol Rev 24: 792-805. PMI: 21976609

Datta P, Shi L, Bibi N, Balazsi G, Gennaro ML (2011) Regulation of central metabolism genes of Mycobacterium tuberculosis by parallel feed-forward loops controlled by sigma factor E (sigma(E)). J Bacteriol 193: 1154-1160. PMI: 21193605

Tiwari A, Balazsi G, Gennaro ML, Igoshin OA (2010) The interplay of multiple feedback loops with post-translational kinetics results in bistability of mycobacterial stress response. Phys Biol 7: 036005. PMI: 20733247

Shi L, Sohaskey CD, Pfeiffer C, Datta P, Parks M, McFadden J, North RJ, Gennaro ML (2010) Carbon flux rerouting during Mycobacterium tuberculosis growth arrest. Mol Microbiol 78: 1199-1215. PMI: 21091505

Kunnath-Velayudhan S, Salamon H, Wang HY, Davidow AL, Molina DM, Huynh VT, Cirillo DM, Michel G, Talbot EA, Perkins MD, Felgner PL, Liang X, Gennaro ML (2010) Dynamic antibody responses to the Mycobacterium tuberculosis proteome. Proc Natl Acad Sci U S A 107: 14703-14708. PMI: 20668240

Hussain S, Malik M, Shi L, Gennaro ML, Drlica K (2009) In vitro model of mycobacterial growth arrest using nitric oxide with limited air. Antimicrob Agents Chemother 53: 157-161. PMI: 18955516

Shi L, Sohaskey CD, North RJ, Gennaro ML (2008) Transcriptional characterization of the antioxidant response of Mycobacterium tuberculosis in vivo and during adaptation to hypoxia in vitro. Tuberculosis (Edinb) 88: 1-6. PMI: 17928268

Khan IH, Ravindran R, Yee J, Ziman M, Lewinsohn DM, Gennaro ML, Flynn JL, Goulding CW, DeRiemer K, Lerche NW, Luciw PA (2008) Profiling antibodies to Mycobacterium tuberculosis by multiplex microbead suspension arrays for serodiagnosis of tuberculosis. Clin Vaccine Immunol 15: 433-438. PMI: 18077619

Bothamley G, Gennaro ML (2008) In Kaufmann SHE and Britton W (eds.), TB Handbook. Willey-VCH, pp. 227-244.

Balazsi G, Heath AP, Shi L, Gennaro ML (2008) The temporal response of the Mycobacterium tuberculosis gene regulatory network during growth arrest. Mol Syst Biol 4: 225. PMI: 18985025

Gennaro ML, Affouf M, Kanaujia GV, Brusasca PN, Mangura B, Reichman L (2007) Antibody markers of incident tuberculosis among HIV-infected adults in the USA: a historical prospective study. Int J Tuberc Lung Dis 11: 624-631. PMI: 17519093

Azzurri A, Kanaujia GV, Sow OY, Bah B, Diallo A, Del Prete G, Gennaro ML (2006) Serological markers of pulmonary tuberculosis and of response to anti-tuberculosis treatment in a patient population in Guinea. Int J Immunopathol Pharmacol 19: 199-208. PMI: 16569358

Shi L, Sohaskey CD, Kana BD, Dawes S, North RJ, Mizrahi V, Gennaro ML (2005) Changes in energy metabolism of Mycobacterium tuberculosis in mouse lung and under in vitro conditions affecting aerobic respiration. Proc Natl Acad Sci U S A 102: 15629-15634. PMI: 16227431

Kanaujia GV, Lam PK, Perry S, Brusasca PN, Catanzaro A, Gennaro ML (2005) Integration of microscopy and serodiagnostic tests to screen for active tuberculosis. Int J Tuberc Lung Dis 9: 1120-1126. PMI: 16229223

Davidow A, Kanaujia GV, Shi L, Kaviar J, Guo X, Sung N, Kaplan G, Menzies D, Gennaro ML (2005) Antibody profiles characteristic of Mycobacterium tuberculosis infection state. Infect Immun 73: 6846-6851. PMI: 16177363

Spencer JS, Kim HJ, Marques AM, Gonzalez-Juarerro M, Lima MC, Vissa VD, Truman RW, Gennaro ML, Cho SN, Cole ST, Brennan PJ (2004) Comparative analysis of B- and T-cell epitopes of Mycobacterium leprae and Mycobacterium tuberculosis culture filtrate protein 10. Infect Immun 72: 3161-3170. PMI: 15155617

Shi L, North R, Gennaro ML (2004) Effect of growth state on transcription levels of genes encoding major secreted antigens of Mycobacterium tuberculosis in the mouse lung. Infect Immun 72: 2420-2424. PMI: 15039373

Lewinsohn DA, Gennaro ML, Scholvinck L, Lewinsohn DM (2004) Tuberculosis immunology in children: diagnostic and therapeutic challenges and opportunities. Int J Tuberc Lung Dis 8: 658-674. PMI: 15137550

Kanaujia GV, Motzel S, Garcia MA, Andersen P, Gennaro ML (2004) Recognition of ESAT-6 sequences by antibodies in sera of tuberculous nonhuman primates. Clin Diagn Lab Immunol 11: 222-226. PMI: 14715573

Goulding CW, Apostol MI, Gleiter S, Parseghian A, Bardwell J, Gennaro M, Eisenberg D (2004) Gram-positive DsbE proteins function differently from Gram-negative DsbE homologs. A structure to function analysis of DsbE from Mycobacterium tuberculosis. J Biol Chem 279: 3516-3524. PMI: 14597624

Silva VM, Kanaujia G, Gennaro ML, Menzies D (2003) Factors associated with humoral response to ESAT-6, 38 kDa and 14 kDa in patients with a spectrum of tuberculosis. Int J Tuberc Lung Dis 7: 478-484. PMI: 12757050

Shi L, Jung YJ, Tyagi S, Gennaro ML, North RJ (2003) Expression of Th1-mediated immunity in mouse lungs induces a Mycobacterium tuberculosis transcription pattern characteristic of nonreplicating persistence. Proc Natl Acad Sci U S A 100: 241-246. PMI: 12506197

Rolinck-Werninghaus C, Magdorf K, Stark K, Lyashchenko K, Gennaro ML, Colangeli R, Doherty TM, Andersen P, Plum G, Herz U, Renz H, Wahn U (2003) The potential of recombinant antigens ESAT-6, MPT63 and mig for specific discrimination of Mycobacterium tuberculosis and M. avium infection. Eur J Pediatr 162: 534-536. PMI: 12715165

Kanaujia GV, Garcia MA, Bouley DM, Peters R, Gennaro ML (2003) Detection of early secretory antigenic target-6 antibody for diagnosis of tuberculosis in non-human primates. Comp Med 53: 602-606. PMI: 14727807

Brusasca PN, Peters RL, Motzel SL, Klein HJ, Gennaro ML (2003) Antigen recognition by serum antibodies in non-human primates experimentally infected with Mycobacterium tuberculosis. Comp Med 53: 165-172. PMI: 12784850

Brown RM, Cruz O, Brennan M, Gennaro ML, Schlesinger L, Skeiky YA, Hoft DF (2003) Lipoarabinomannan-reactive human secretory immunoglobulin A responses induced by mucosal bacille Calmette-Guerin vaccination. J Infect Dis 187: 513-517. PMI: 12552438

Goulding CW, Parseghian A, Sawaya MR, Cascio D, Apostol MI, Gennaro ML, Eisenberg D (2002) Crystal structure of a major secreted protein of Mycobacterium tuberculosis-MPT63 at 1.5-A resolution. Protein Sci 11: 2887-2893. PMI: 12441386

Chandramuki A, Lyashchenko K, Kumari HB, Khanna N, Brusasca P, Gourie-Devi M, Satishchandra P, Shankar SK, Ravi V, Alcabes P, Kanaujia GV, Gennaro ML (2002) Detection of antibody to Mycobacterium tuberculosis protein antigens in the cerebrospinal fluid of patients with tuberculous meningitis. J Infect Dis 186: 678-683. PMI: 12195355

Amadori M, Lyashchenko KP, Gennaro ML, Pollock JM, Zerbini I (2002) Use of recombinant proteins in antibody tests for bovine tuberculosis. Vet Microbiol 85: 379-389. PMI: 11856587

Landowski CP, Godfrey HP, Bentley-Hibbert SI, Liu X, Huang Z, Sepulveda R, Huygen K, Gennaro ML, Moy FH, Lesley SA, Haak-Frendscho M (2001) Combinatorial use of antibodies to secreted mycobacterial proteins in a host immune system-independent test for tuberculosis. J Clin Microbiol 39: 2418-2424. PMI: 11427548

Johnson S, Brusasca P, Lyashchenko K, Spencer JS, Wiker HG, Bifani P, Shashkina E, Kreiswirth B, Harboe M, Schluger N, Gomez M, Gennaro ML (2001) Characterization of the secreted MPT53 antigen of Mycobacterium tuberculosis. Infect Immun 69: 5936-5939. PMI: 11500477

Brusasca PN, Colangeli R, Lyashchenko KP, Zhao X, Vogelstein M, Spencer JS, McMurray DN, Gennaro ML (2001) Immunological characterization of antigens encoded by the RD1 region of the Mycobacterium tuberculosis genome. Scand J Immunol 54: 448-452. PMI: 11696195

Lyashchenko KP, Singh M, Colangeli R, Gennaro ML (2000) A multi-antigen print immunoassay for the development of serological diagnosis of infectious diseases. J Immunol Methods 242: 91-100. PMI: 10986392

Gomez M, Johnson S, Gennaro ML (2000) Identification of secreted proteins of Mycobacterium tuberculosis by a bioinformatic approach. Infect Immun 68: 2323-2327. PMI: 10722636

Colangeli R, Spencer JS, Bifani P, Williams A, Lyashchenko K, Keen MA, Hill PJ, Belisle J, Gennaro ML (2000) MTSA-10, the product of the Rv3874 gene of Mycobacterium tuberculosis, elicits tuberculosis-specific, delayed-type hypersensitivity in guinea pigs. Infect Immun 68: 990-993. PMI: 10639479





C.V.

M.D. University of Palermo, Italy, M.D., 1977; London School of Hygiene & Tropical Medicine, M.S., 1981; Istituto Superiore di Sanita, Rome, 1978-1986; PHRI, 1984-present.


 
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