Research Projects

Innate immunity relies on the general recognition of non-self characteristics of pathogens and the induction of effector mechanisms that will dispose of the pathogens. The ability to recognize general features of foreign microbes distinguishes it from adaptive immunity, which selects for specific antibodies or receptors to mediate its response. The innate and adaptive immune systems converge in their use of common effector mechanisms, e.g., complement, phagocytosis, and cytolytic factors. The innate immune response may also be important to prime the adaptive immune response for an effective response.

Signaling pathways used in innate immunity are highly conserved through evolution.  We study the innate immune response in the model genetic organism Drosophila melanogaster.  The advantages of using Drosophila include (1) the ability to use genetics to identify and characterize new mutations, (2) the genomic resources which make it easier to clone the corresponding genes and manipulate them in vivo and (3) the fact that many biological processes are conserved between flies and humans.  The Drosophila immune response has both humoral and cellular components.  The humoral immune response results in the secretion of antimicrobial peptides and is important for the defense against bacteria and fungi.  In the cellular immune response, the hemocytes (blood cells) can phagocytose microbes and encapsulate parasites.  We are doing genetic and molecular screens to identify genes and characterize mutations important for the immune response to different types of pathogens.  We hope to better understand the molecular mechanisms important in the innate immune response.

Characterization of genes important for the antibacterial humoral response

The signaling pathways that regulate expression of the antimicrobial peptide (AMP) genes are highly conserved through evolution. The two main signaling pathways, Toll and IMD, use Toll receptors, Peptidoglycan Recognition Proteins (PGRPs), kinases and NF-kappaB/Rel transcription factors that have homologs in mammalian immune signaling pathways. Through our genetic and molecular screens, we have identified several genes that affect expression of the AMP genes. We are characterizing these mutants to understand the role these genes play in the fly immune response.

Using Genetics to Identify New Genes. Our first genetic screen focused on the IMD pathway. We identified mutants (immune response defective or ird) that were unable to induce an AMP (Diptericin)-lacZ reporter gene. From this screen, mutations for IKK-beta (ird5) and PGRP-LC (ird7) were isolated. We have identified a third gene, ird1, that is necessary for activating the IMD pathway. ird1 is a homolog of Vps15, a serine/threonine kinase important for phagosome maturation and starvation-induced autophagy in yeast and mammalian cells. ird1 animals are more susceptible to both E. coli and M. luteus bacterial infection suggesting impairment of both the IMD and Toll pathways.  To gain insight into ird1's function, we examined how amino acid starvation affects the immune signaling pathways in Drosophila.  Starvation, in the absence of infection, leads to expression of AMP genes and this response is dependent on ird1 and the IMD pathway.  Starvation, in addition to bacterial infection suppresses this AMP response in wildtype animals and reduces the ability to survive M.luteus infection.  Our results suggest that starvation can influence innate immune signaling. Our current genetic screens have focused on phagocytosis and responses against viruses (see below). We find that some of our new mutants from these screens also affect the Toll and/or IMD pathways.

Using Microarrays to Identify Potential Immune Response Genes. To take full advantage of Drosophila genomics, we have used Affymetrix and spotted cDNA microarrays to identify genes affected during an immune response to bacterial or fungal infection.  It is usually assumed, but not actually known, whether genes induced in an immune response play a role in that response. To extend our understanding beyond a descriptive list of genes, we examined 160 Drosophila lines containing P-element transposon insertions near upregulated genes, for defects in the activation of the Imd and Toll signaling pathways.  We find that a large number of lines are defective in the induction of the Toll pathway-regulated antimicrobial peptide gene, Defensin. Some of these genes are also affected in their response to bacteria (M. luteus), fungi (M. anisopliae), or viruses (DXV). These results suggest that genes affecting the Toll pathway may be specifically upregulated during infection.  We are characterizing these mutants to determine the roles these genes play in bacterial, fungal and/or viral infections. We have also used microarrays to explore how the three different Rel transcription factors are involved in regulation of these immune response genes. These experiments were done in collaboration with the CBR Microarray Core Facility.

Innate Immune Response against a Virus

Drosophila X Virus (DXV) is a double-stranded RNA virus in the Birnaviridae family that causes anoxia sensitivity and death in Drosophila.  One advantage of this system is that both pathogen and host are amenable to genetic manipulation. Less is known about the innate immune responses against viruses. It is also likely that the immune response against a virus, an intracellular pathogen, is distinct from the immune responses against extracellular bacteria and fungi. We have found that the Toll pathway and RNA interference are important for the host response to this virus. We are doing a forward genetic screen to identify novel genes important for the antiviral immune response. We have mutants that are more susceptible or more resistant to DXV infection. We find that some of our mutants show genetic interactions with RNAi genes. We hope to gain insight into the genes important for antiviral immune responses and viral pathogenesis. This project is an ongoing collaboration with Vik Vakharia (also of CBR, UMBI). 

Phagocytosis

Using an in vivo phagocytosis assay (developed by David Schneider's group), we have screened for mutations affecting phagocytosis of E. coli or S. aureus. We find that catalytic peptidoglycan recognition proteins are important for this response. picky (PGRP-SC1a) mutants are unable to activate the Toll pathway or phagocytose S. aureus particles. A catalytically dead PGRP-SC1a is able to rescue some of the picky phenotypes (Toll signaling) but is defective in its ability to clear S. aureus from the fly. This defect can be rescued with the addition of free peptidoglycan, suggesting that the fly cleaves peptidoglycan and that the byproduct is important for potentiating phagocytosis of the bacteria. We have identified other mutants that affect phagocytosis of E.coli and hope that these will give insight into how a fly recognizes Gram-negative bacteria.

Contact Us:

5115 Plant Sciences Bldg.
Center for Biosystems Research
University of Maryland Biotechnology Institute
College Park, MD 20742
Phone: 301-405-4949 Fax: 301-314-9075