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Parvoviruses are among the simplest of viruses, with capsids composed of variants of a single structural protein. These capsids mediate many of the processes required for infection and are remarkably stable. However, antibody binding or mutations to the capsid can disrupt these processes and block infection. This dissertation discusses mutations to the canine parvovirus (CPV) capsid that result in loss of infection, the interaction between CPV and the transferrin receptor (TfR), and parvovirus endogenous viral elements (EVEs), which contain ancient parvovirus gene sequences. I found that point mutations to CPV VP2 residues 270, 272, 273, and 299/300 result in loss of viral infectivity. Mutation of residue 270 results in loss of a sub-molar proteolytic cleavage event in VP2 and increases capsid stability, residue 272 mutation causes loss of capsid assembly, and residue 273 mutation results in assembled capsids being trapped in the nucleus. Mutation of VP2 residues 299 and 300, which are associated with TfR binding, to lysine disrupts the interaction between CPV and the TfR, inhibiting infection but still allowing receptor binding and uptake into cells. I also found that CPV has different interactions with TfRs from different host species, binding strongly to some TfRs and very weakly to others, even though each TfR can mediate a successful infection. TfRs from different species also had varying levels of occupancy on CPV capsids, with up to 12 black-backed jackal TfRs, but only 1-2 feline TfRs binding to each capsid, and it is possible that the feline TfR induces a conformational change in CPV that inhibits binding of additional TfRs. Antibody binding could also disrupt the CPV/TfR binding interaction, suggesting a possible mechanism of virus neutralization. To examine the structure and function of ancient parvovirus capsids, I expressed the VP2 gene from three different parvovirus EVEs. VP2 expressed from an EVE in the M. spretus genome assembled into capsids, and I determined that these capsids were highly stable, could bind to N-Acetylneuraminic acid, and were endocytosed into murine cells. The results in this dissertation provide new information about parvovirus infection, receptor binding interactions, and evolution, and further the understanding of how infection occurs and may be disrupted.

Whooping cough, also known as pertussis, is caused by the bacterium Bordetella pertussis. Since widespread vaccination with heat-killed whole cell vaccines (wP) in the 1950s, the number of cases dropped dramatically. However, there has been a consistent resurgence in the past two decades, coinciding with the switch from wP vaccines to acellular vaccines (aP). US CDC estimates 16 million cases and 195,000 deaths worldwide per year. Accumulating evidence show that aP vaccines provide short protection against the symptoms but not against subclinical infection and transmission of the disease. Changing the adjuvant to induce more protective immunity or inclusion of additional protective antigens are some of the strategies to improve the efficacy of aP vaccines. Adenylate cyclase toxin (ACT) is a 177 kDa protein produced by B. pertussis and related species. ACT mainly targets leukocytes through [alpha] [subscript M] [beta] ₂ integrin and translocates its N-terminal cyclase domain into the cytosol, generating supraphysiological level of cAMP. Studies have shown that ACT-deficient stains are less pathogenic and passive immunization with polyclonal antibodies or active immunization with ACT protected mice against bacterial challenges. However, ACT is not included in any of the current aP vaccines, due to a lack of understanding of its protective epitopes and its poor solubility and stability. We aimed to identify potent neutralizing antibodies (nAbs) as therapeutic candidates, and map their epitopes to guide the design of vaccine antigen overcoming the solubility and stability issues. Two nAbs, M2B10 and M1H5, were discovered from antibody phage display libraries from ACT-immunized mice. They bind non-overlapping conformational epitopes in the C-terminal RTX domain of ACT. Our data suggest their mode of action is interrupting the interaction between the toxin and its cellular receptor. On the other hand, individual domains of ACT were expressed in E. coli and purified. The catalytic and RTX domains retained antigenicity but are biophysically superior to full-length ACT. We further showed that the RTX domain elicited similar level of neutralizing antibody response to ACT in mice. These antibodies, together with those neutralizing other major B. pertussis toxins, may constitute a therapy for severe pertussis infection, and the epitopes provide the basis for structure-based antigen design for superior stability and immunogenicity.

Bacteria engage in social behavior by communicating through a variety of mechanisms. One method of communication is contact-dependent growth inhibition (CDI), a phenomenon in which one bacterium binds and delivers a toxin to a closely related target cell, using the proteins CdiB and CdiA. This toxin blocks cell growth unless the target cell contains an immunity protein, CdiI. The CDI pathway is the process by which toxins are delivered and activated, using distinct target-cell proteins such as outer membrane receptors and inner membrane transporters. The first chapter of this thesis focuses on the CDI system from Burkholderia pseudomallei 1026b. We identify three genes whose products appear to be necessary for growth inhibition, and describe a potential CDI pathway for this system. The second chapter discusses the mechanism by which Escherichia coli EC93 binds target cells. Mutations in BamA, the CdiAEC93 receptor, are described. These mutations confer resistance to CDI and block cell-cell binding. Both chapters demonstrate the variety and species specificity of CDI growth inhibition pathways.

The aim of this dissertation was to provide some biochemical evidence relevant to previous functional studies by measuring muscarinic receptor binding and choline acetyltransferase (ChAT) activity following cochlear ablation and intense tone exposure. The ChAT activity in the rat cochlear nucleus (CN) had a characteristic distribution pattern. Significant gradients of ChAT activity were found in all dissected regions. These chemical gradients suggest that descending cholinergic input to the CN is widely distributed and tonotopically organized. After cochlear ablation, bilateral increases in ChAT were found in the anteroventral CN (AVCN) and dorsolateral granular region (G-AVCN) 1 mo after lesion. However, there was no change in the dorsal CN (DCN). In the lateral superior olive, there was a sustained decrease ipsilateral to the lesion after cochlear ablation, By contrast, ChAT activity in the ventral nucleus of the trapezoid body had a tendency to decrease bilaterally in 1 mo and 2 mo survival animals. Except for similar ChAT activities in granular regions, ChAT activity was 2-3-fold lower in the hamster CN than in the rat. Eight days after tone exposure, significant increases over control were found in the AVCN and fusiform soma layer of DCN on the exposed side. In addition, ChAT activities in the G-AVCN, in AVCN, and in deep layer of DCN (DCNd) ipsilateral to the exposure were higher than contralateral values. Two months after tone exposure, ChAT activity in the ipsilateral DCNd was still higher than the contralateral value. Changes of ChAT in the CN may underlie pathophysiological conditions. Analysis of 1-[N-methyl-3H]scopolamine binding in the ventral CN after cochlear ablation demonstrated significant asymmetry in muscarinic receptor binding in 2 mo survival rats, with higher density on the lesion side. The lesion side was 67% higher in the AVCN and 70% higher in the posteroventral CN. In the DCN, increased binding on the lesion side relative to contralateral was significant in DCNf and DCNd in the 1 mo and 2 mo survival animals. The asymmetry was more obvious in the DCNd. The [3H]NMS binding pattern in the DCNf changed from a patchy pattern in control rats to a more diffuse pattern on the lesion side.