Joseph T. Barbieri, PhD
Microbiology and Molecular Genetics
Medical College of Wisconsin
Director, Medical Scientist Training Program
Research Focus: Microbial Pathogenesis
PhD: University of Massachusetts, Amherst (1980) Microbiology
I am interested in understanding the molecular basis of microbial pathogenesis in an area of study termed Cellular Microbiology where components of both the pathogen and host are analyzed to understand the pathogenic process. My research involves the study of bacterial toxins. Currently two families of toxins are under investigation in my laboratory: the botulinum neurotoxins and ExoS, a type III cytotoxin of Pseudomonas aeruginosa. Studies on bacterial toxins have evolved from understanding their mechanism of action to develop novel strategies to produce vaccines and therapies against their action.
We are also characterizing the mechanism that tetanus toxin (TeNT) and botulinum toxin (BoNT) elicit unique pathology in humans. Analyzing the heavy chain receptor binding domains (HCR) of these neurotoxins, TeNT and BoNT/A were found to enter neurons by unique mechanisms. While synaptic vesicle cycling was required for HCR/A entry into neurons, HCR/T entered neurons by two pathways, one independent of stimulated synaptic vesicle cycling and one by synaptic vesicle cycling, but independent of SV2. These studies support a role for intracellular entry and trafficking in contributing to the unique pathology elicited by these neurotoxins. We utilize Total Internal Reflection Fluorescence (TIRF) microscopy and confocal microscopy to analyze toxin entry. TIRF microscopy provides an opportunity to visualize fluorescence within ~100 nm of the host cell plasma membrane, allowing the analysis of entry events that are difficult to resolve by standard fluorescence microscopy. TIRF is especially useful for quantitative measurement of co-localization between two fluorescent molecules. Figure 1 shows the colocalization of the receptors binding domains of BoNT/A (HCR/A) and TeNT (HCR/T) (bottom) where two populations of TeNT were resolved: one population co-localized with BoNT/A and another population localized independent of BoNT/A. Two standards were used that showed the co-localization of two fluorescent antibodies bound to a single protein, synaptophysin 1, (upper) and the segregation of synaptophysin 1, a marker protein in synaptic vesicles, and transferrin, a marker protein in endosomes, within a neuron (middle). Understanding the entry mechanisms of these neurotoxins may allow the development of targeted therapies against specific neurological diseases.
Figure 1. Entry of the receptor binding domains of BoNT/ A and TeNT in neurons of the central nervous system. Top panels show the co-localization of two antibodies that bind to synaptophysin. Middle panels show the segregation of synaptophysin 1 (red) and transferrin (green). Lower panels show the entry of HCR/A (red) and HCR/T (green) into primary cortical neurons. Data from TIRF images (three right most panels) were analyzed by ImageJ software (two left most panels).
ExoS is a bi-functional type III cytotoxin and includes a RhoGAP domain and an ADP-ribosyltransferase domain that inactive Rho GTPases and Ras GTPases, respectively. The outcome of ExoS action on mammalian cells is to inactivate the actin cytoskeleton to interfere with host cells phagocytosis of P. aeruginosa. Our current research involves the study of two aspects of ExoS. First, we are interested in understanding how ExoS traffics within the host cells. We have recently determined that ExoS possesses a localization domain that allows the toxin to usurp the host endocytic machinery to traffic to both Rho- and Ras- GTPases. Without this trafficking, ExoS can not locate these targets within the cell. A model for how ExoS traffics on vesicles in mammalian cells is shown in Figure 2. We are also interested in understanding how ExoS and a related toxin, ExoT, recognize their specific substrates. Although ExoS and ExoT share 76% primary amino acid homology, the two protein target completely different host proteins.
Figure 2. Trafficking of ExoS in mammalian cells (from Deng, Zhang, and Barbieri, Traffic, 2007).
The Clostridium neurotoxins (BoNTs) are the most potent protein toxins for humans and are Category A toxins. There are seven serotypes of BoNTs, termed A-G, with serotypes A, B, E, and F responsible for most natural human disease. BoNTs are single chain A-B toxins that are organized into three specific domains. The N-terminal zinc protease domain is link by a disulfide bond to the C-terminal domain heavy chain which comprises a translocation domain and a C-terminal receptor binding domain. BoNT toxicity for neurons is due to toxin's affinity for neuron specific receptors and for the substrates that the toxin cleaves either v- or t- SNAREs. Cleavage of v- or t- SNAREs yields flaccid paralysis that is observed in botulism. We are interested in understanding how BoNT interacts with neurons and how the BoNT recognize their specific substrates. These studies use biochemical and cell biological approaches. We are studying how BoNTs bind peripheral neurons and have observed that BoNTs bind to a neuronal synaptic vesicle protein complex, which included SV2, synaptotagmin, synaptophysin, VAMP, and a vacuolar proton pump. We are characterizing the interaction of the BoNTs with these neuronal proteins. We are also studying how BoNTs recognize and cleave their v- and t- SNARE substrates. We have identified a pocket model for the recognition of BoNT/A for SNAP25. A model for the recognition steps of BoNT/A for SNAP25 is shown in Figure 3. We are currently refining this model and addressing how other BoNT serotypes recognize their substrates. These studies will develop novel strategies for vaccine and therapies against botulism.
Figure 3. How BoNT/A recognizes SNAP25 (from Chen, Kim, and Barbieri, Journal of Biological Chemistry, 2006).