Characterization of the Agrobacterium tumefaciens VirB2 pilin of the VirB/D4 Type IV Secretion System

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) delivers oncogenic T-DNA and effector proteins to susceptible plant cells. This leads to the formation of tumors termed Crown Galls. The VirB/D4 T4SS is comprised of 12 subunits (VirB1 to VirB11 and VirD4), which assemble to form two structures, a secretion channel spanning the cell envelope and a T-pilus extending from the cell surface. In A. tumefaciens, the VirB2 pilin subunit is required for assembly of the secretion channel and is the main subunit of the T-pilus. The focus of this thesis is to define key reactions associated with the T4SS biogenesis pathway involving the VirB2 pilin. Topology studies demonstrated that VirB2 integrates into the inner membrane with two transmembrane regions, a small cytoplasmic loop, and a long periplasmic loop comprised of covalently linked N and C termini. VirB2 was shown by the substituted cysteine accessibility method (SCAM) to adopt distinct structural states when integrated into the inner membrane and when assembled as a component of the secretion channel and the T-pilus. The VirB4 and VirB11 ATPases were shown by SCAM to modulate the structural state of membrane-integrated VirB2 pilin, and evidence was also obtained that VirB4 mediates extraction of pilin from the membrane. A model that VirB4 functions as a pilin dislocase by an energy-dependent mechanism was further supported by coimmunoprecipitation and osmotic shock studies. Mutational studies identified two regions of VirB10, an N-terminal transmembrane domain and an outer membrane-associated domain termed the antennae projection, that contribute selectively to T-pilus biogenesis. Lastly, characterization of a VirB10 mutant that confers a ‘leaky’ channel phenotype further highlighted the role of VirB10 in gating substrate translocation across the outer membrane as well as T-pilus biogenesis. Results of my studies support a working model in which the VirB4 ATPase catalyzes dislocation of membrane-integrated pilin, and distinct domains of VirB10 coordinate pilin incorporation into the secretion channel and the extracellular T-pilus.

AN ASCENDING SEROTONERGIC PAIN MODULATION SYSTEM

This dissertation describes an ascending serotonergic pain modulation system projecting from the dorsal raphe (DR) nucleus of the midbrain to the parafascicularis (PF) nucleus of the thalamus. Previous studies by other investigators have led to the hypothesis that the DR would modulate responses to noxious stimuli in the PF by using 5HT. These other studies have shown that the DR contains serotonergic (5HT) cell bodies which project to many areas of the forebrain including the PF, that the PF is involved in pain perception, that electrical stimulation of the DR causes analgesia, and 5HT is necessary for this type of analgesia. One theory of the mechanisms of an endogenous pain modulation system is that brainstem nuclei have a decsending projection to the spinal cord to inhibit responses to noxious input at this level. The present study tests the hypothesis that there is also an ascending pain modulation pathway from the brainstem to the thalamus.^ To test this hypothesis, several types of experiments were performed on anesthetised rats. The major results of the experiments are as follows: (1) Three types of spontaneously active PF neurons were found: slow units firing at 1-10 spikes/sec, bursting units firing 2-5 times in 10-20 msec, pattern repeating every 1-2 sec, and fast units firing at 15-40 spikes/sec. The first two groups showed similar results to the treatments and were analysed together. The fast firing units did not respond to any of the treatments. (2) Noxious stimuli primarily increased neuronal firing rates in the PF, where as DR stimulation primarily decreased neuronal activity. DR stimulation applied simultaneously with noxious stimuli decreased the responses to the noxious stimuli as recorded in the PF units. (3) Microiontophoretically applied 5HT in the PF decreased spontaneous activity in the PF in a dose dependent manner and decreases responses to noxious stimuli in the PF. (4) Reduction of brain 5HT by 5,7 dihydroxytryptamine, a potent 5HT neurotoxin, caused PF units to be hypersensitive to both noxious and non noxious stimuli, reversed the effects of DR stimulation so that DR stimulation increased single units activity in the PF, and prolonged and intensified the depressant action of microiontophoretically applied 5HT. The results of this study are consistent with the hypothesis that the DR uses 5HT in a direct ascending pathway to the PF to modulate pain in the thalamus. ^