Lab Page Link

Neal Silverman, Ph.D.

Academic Role: Associate Professor

Faculty Appointment(s) In:
   Infectious Diseases and Immunology
   Medicine

Other Affiliation(s):
   Center for AIDS Research
   Interdisciplinary Graduate Program
   Program in Immunology and Virology

Immune Signaling Pathways

The main goal of our lab is to decipher the molecular mechanisms responsible for transmitting a signal from the site of infection to the nucleus of an immune responsive cell. We are interested in how pathogens are distinguished, how related signaling pathways maintain specificity, and how various signals are integrated to produce the proper response. Research will focus on the immune response of the experimentally powerful fruit fly, Drosophila melanogaster. We are particularly interested in the mechanisms used in Drosophila that allow distinct pathogenic challenges to lead to specific immune responses by activating different signaling pathways and transcription factors. The immune signaling pathways in Drosophila have much in common with the pathways required for the activation of the mammalian innate immune response. In fact, the Toll-like receptor (TLR) family, which was discovered in Drosophila, plays a central role in pathogen recognition in both mammals and insects. A deeper understanding of these pathways in insects will undoubtedly lead to further advances in related mammalian fields.

The Drosophila antibacterial signaling pathway

In flies, infection causes the rapid production of a host of powerful antimicrobial peptides that are produced in the fat body (the insect liver) and ciruculate throughout the body. Pathogens are known to activate two separate and specific immune response signaling pathways, an antibacterial and an antifungal pathway, each of which culminates in the activation of different Drosophila NF-kB transcription factors. Fungal infection leads to the activation of Toll, which initiates a signal transduction cascade leading to the proteasome-mediated degradation of Cactus (the fly IkB), the nuclear translocation of two Drosophila NF-kB transcription factors, Dif and Dorsal, and the rapid expression of antifungal peptide genes. The Toll signaling pathway is also essential for the dorsoventral patterning of the developing embryo. The third Drosophila NF-kB protein, Relish, is required for antibacterial immunity. Relish is initially synthesized as a bipartite protein with an N-terminal NF-kB-like domain and C-terminal IkB-like domain, which inhibits its own nuclear translocation. Upon infection Relish is cleaved, freeing the NF-kB module to translocate to the nucleus where it activates antibacterial peptide gene expression.

Our lab is focused upon understanding the molecular mechanisms responsible for the activation of these two NF-kB pathways during the insect immune response. The antibacterial pathway, which is controlled by Relish, requires the Drosophila IkB kinase complex (IKK), a high molecular weight complex which contains a catalytic subunit, DmIKKb, and regulatory subunit, DmIKKg. Upon infection, the Drosophila IKK complex is activated and is required for the cleavage (and activation) of Relish. We are interested to know what lies upstream of the Drosophila IKK complex, what receptors are used to recognize pathogens and how does activation of these receptors, in turn, lead to the activation of the IKK complex. The mechanism of DmIKK-stimulated Relish cleavage is also a major focus in the laboratory.

The Drosophila Toll/antifungal signaling pathway

The antifungal pathway, which relies on the classic Toll signaling pathway, is also a focus of our research. Although we know a great deal about this signaling pathway, many important questions remain. In particular, we are interested in mechanisms of signal-induced Cactus degradation. Like mammalian IkBs, Cactus is phosphorylated upon signaling and this signaling leads to its proteasome mediated degradation. However, unlike IkB, Cactus degradation does not require the IKK complex, and the identity of the Cactus kinase is currently unknown.

Our goal is to uncover the molecular mechanism used by the innate immune system to recognize dangerous microbes and to rapidly mount a potent and specific response against them. Using Drosophila, with its powerful genetic, molecular, and biochemical tools, will enable us to gain a thorough understanding of the signal transduction pathways used by eukaryotes to fend-off their adversaries. This research has potential medical benefits beyond its primary goal of basic scientific understanding. A deeper understanding of the insect immune response will enable the design of new methods to combat the spread of infectious microbes by insects. Moreover, the similarities between the insect immune response and mammalian innate immunity will open-up new and exciting avenues of research into the mechanisms which control our more complicated immune response.

Representative Publications

Silverman, N. and Maniatis, T. (2001) NF-kB signaling pathways in mammalian and insect innate immunity. Genes & Dev. 15: 2321-2342.

Rutschmann, S., Jung, A.C., Zhou, R., Silverman, N., Hoffmann, J.A., Ferrandon, D. (2000) Involvement of the Drosophila IKKg/NEMO homologue in an innate immune response pathway independent of the Toll pathway. Nature Immunity 1: 342 - 347.

Silverman, N., Zhou, R., Stöven, S., Pandey, N., Hultmark, D., Maniatis, T. (2000) A Drosophila IkB kinase complex required for Relish cleavage and antibacterial immunity. Genes & Dev. 14: 2461-2471.

Rotation Projects

Intro:

Like us, insects recognize pathogenic microorganisms and respond with potent antimicrobial defenses.  Insects have only a primitive immune system which relies solely on germline encoded receptors to recognize various microbial derived substances.  Mammals have similar system, known as the innate immune response, that plays a critical role in recognizing dangerous pathogens and activating the more complicated adaptive immune response involving antigen presentations and T- and B-cell receptors.  Insect and mammalian innate immunity have much in common.  For example, they both use the NF-kB /Rel family of transcription factors to rapidly activate immune inducible gene expression.  Drosophila has three NF-kB homologs, all of which are involved in immunity.  One Drosophila NF-kB, known as Relish, is activated by proteolytic cleavage in response to infection with gram-negative bacteria.  The other two NF-kB  homologs, Dorsal and Dif, are activated during a fungal (or gram postive bacterial) infection when their inhibitor, known as Cactus (IkB) is degraded.  The focus of the lab is to understand the underlying molecular mechanisms responsible for activation of specific NF-kB transcription factors in response to different pathogens.

1. Goal:  To examine the role of all seven Drosophila caspase proteases in the activation of immunity.  Relish is activated by caspase-mediated proteolysis, but the protease that cleaves Relish is not yet identified. This experiment will use RNAi to inhibit caspase gene function in an immune responsive Drosophila cell line, followed by analysis of the immune-inducible gene expression.  This project is designed to identify which caspase proteases are required for Relish activation.  Further experiments will be designed to demonstrate the direct cleavage of Relish by caspase proteases.



2. Goal:  Genome wide analysis of the insect immune response.  In this project we will utilize Drosophila cDNA microarrays to study the changes in gene expression induced by immunostimulatory molecules.  In particular, we will analyze the activity of various microbial derived products to illicit specific immune responses. Also, we will examine if the role of different signaling pathways in the immune response, especially the JNK and NF-kB signaling pathways.



3. Goal:  To genetically characterize the function of the second Drosophila IKK homolog, IKKe.  The function of human and Drosophila IKKe remains controversial.  Drosophila IKKe mutants do not survive to adulthood.  This project will focus on studying the role of Drosophila IKKe in the developing embryo, by studying its expression pattern and by making germliine clones.  In the long term, we hope to determine the role, if any, of DmIKKe in the activation of NF-kB family transcription factors in Drosophila.



4. Goal:  To use Drosophila genetics to understand the pathogenesis of the plague.  In this project, we will establish transgenic flies that express  YopJ, a protein from the plague pathogen Yersinia pestis.  In mammals, YopJ blocks important signaling pathways, such as NF-kB, and thus prevents immune activation.  Although, YopJ has been proposed to be a ubiquitin-like protein protease (ULP), the molecular mechanisms by which YopJ functions are unclear.  We will demonstrate in flies that YopJ blocks immune activation of NF-kB.  Further genetic experiments will be designed to test the possibility that YopJ acts as a ULP.  Ultimately, forward genetic screens will be performed to identify genes required for YopJ function.

Laboratory Staff

Peihong Guan, Research Assistant
Deniz Hasdemir, graduate student
Takashi Koneko, postdoc
Nicholas Paquette, rotation student

Academic Background

Office: LRB 313
Phone: 508-856-5826
Fax: 508-856-5463
E-mail: Neal.Silverman@umassmed.edu
Keywords: Immunology, Genetics, Organisms - Drosophila, Infectious Disease, Gene Regulation

More on Neal Silverman's Research Research | Publications | Rotations | Personnel | Biography View All Sections on One Page