The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. <i>Escherichia coli</i> which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49].<p> Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy.<p> To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyers patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation <i>in vitro</i>. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2.<p> To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.

Ribosome-inactivating proteins (RIPs) such as Shiga-like toxin 1 (SLT-1) halt protein synthesis in eukaryotic cells by depurinating a single adenine base in the sarcin-ricin loop of 28S rRNA. The molecular details involved in the ER lumenal escape and subsequent site-specific depurination are lacking, despite a general understanding of the biochemical basis of SLT-1 toxicity. Using a combination of yeast-2-hybrid and HeLa lysate pull-down followed by LC-MS/MS we have discovered yeast and human proteins that interact with the catalytic A1 chain of SLT-1. Yeast-2-hybrid library screens followed by the expression of full-length protein candidates and pull-down experiments yielded Cue2 as the only yeast cellular component that binds to the SLT-1 A1 chain. Further truncational analysis revealed that the known protein domains (two Cue domains and a Smr domain) within the primary sequence of Cue 2 were not essential for the interaction. Cue2 is a yeast monoubiquitin binding protein of no known function that is structurally homologous to the human ubiquitin-associated domain which has been implicated in intracellular routing and ER-associated degradation. Pull-down experiments indicated that the mechanism by which the catalytic domain of RIPs cleaves its substrate involves initial docking interactions with the ribosomal stalk by virtue of a conserved acidic C-terminal peptide domain common to all three stalk proteins P0, P1, and P2. The A1 chain of SLT-1 transiently binds to this peptide with a modest binding constant and rapid on and off rates. Mutagenesis of charged residues within the A1 chain identified a cationic surface that interacts with the peptide motif. In addition, phage-display was used to rapidly probe the importance of each residue within this C-terminal ribosomal peptide. The analysis revealed a complementary acidic surface and an additional hydrophobic motif involved in the interaction. Moreover, deletion mutagenesis performed on the ribosomal protein P0 revealed that the A1 chain binds to an alternate site on P0 in proximity to the contact sites for P1/P2 heterodimers. These results demonstrate that the catalytic chain of RIPs such as SLT-1 dock on ribosomes using two classes of binding sites located within the ribosomal stalk which may aid in orienting their catalytic domain in close proximity to the depurination site.

Control of mRNA stability is an important aspect in the regulation of gene expression. A well studied signal for rapid transcript decay in mammalian cells is the AU-rich element (ARE), which is found in the 3’ untranslated region (UTR) of many labile transcripts. These sequence elements confer destabilization of transcripts by binding to AU-binding proteins (AUBPs) that can recruit cellular decay enzymes. The stability of ARE-containing mRNAs can be regulated by extracellular stimuli, which allows for cells to adapt to the changing environment. AREs are found in many transcripts that encode for inflammatory genes, including TNF, GM-CSF, and IL-8. Pathogens evolve and devise mechanisms to subvert the immune response of the host to aid in its infection. Bacillus anthracis is one such infectious agent that can disable numerous arms of the host immune response. Its secreted toxin, anthrax lethal toxin (LeTx), causes the accelerated decay of the IL-8 mRNA. IL-8 is a dual function cytokine and chemokine that can recruit and activate neutrophils at the site of infection. Through the inactivation of MAPK pathways, LeTx activity causes the destabilization of IL-8 transcripts through its ARE. In this thesis, I show that an AUBP, TTP, is dephosphorylated by LeTx and MAPK inhibitors, and knock-down of its expression stabilized IL-8 transcripts. LeTx activity also increased the colocalization of TTP to P-bodies, cytoplasmic sites concentrated with RNA decay enzymes. This suggests that the post-translational modification of TTP induced by LeTx led to its enhanced destabilization function. Identified TTP-associated proteins, non-muscle myosin heavy chain 9 (myosin-9) and HSC-70, were examined for their role in IL-8 transcript decay. Knock-down of each protein led to a slower rate of IL-8 mRNA destabilization. However, treatment of LeTx continued to mediate accelerated destabilization of IL-8 in these siRNA-transfected cells. This suggests that LeTx, myosin-9, and HSC-70 modulate the destabilization function of TTP independently.

Ribosome-inactivating proteins (RIPs) such as Shiga-like toxin 1 (SLT-1) halt protein synthesis in eukaryotic cells by depurinating a single adenine base in the sarcin-ricin loop of 28S rRNA. The molecular details involved in the ER lumenal escape and subsequent site-specific depurination are lacking, despite a general understanding of the biochemical basis of SLT-1 toxicity. Using a combination of yeast-2-hybrid and HeLa lysate pull-down followed by LC-MS/MS we have discovered yeast and human proteins that interact with the catalytic A1 chain of SLT-1. Yeast-2-hybrid library screens followed by the expression of full-length protein candidates and pull-down experiments yielded Cue2 as the only yeast cellular component that binds to the SLT-1 A1 chain. Further truncational analysis revealed that the known protein domains (two Cue domains and a Smr domain) within the primary sequence of Cue 2 were not essential for the interaction. Cue2 is a yeast monoubiquitin binding protein of no known function that is structurally homologous to the human ubiquitin-associated domain which has been implicated in intracellular routing and ER-associated degradation. Pull-down experiments indicated that the mechanism by which the catalytic domain of RIPs cleaves its substrate involves initial docking interactions with the ribosomal stalk by virtue of a conserved acidic C-terminal peptide domain common to all three stalk proteins P0, P1, and P2. The A1 chain of SLT-1 transiently binds to this peptide with a modest binding constant and rapid on and off rates. Mutagenesis of charged residues within the A1 chain identified a cationic surface that interacts with the peptide motif. In addition, phage-display was used to rapidly probe the importance of each residue within this C-terminal ribosomal peptide. The analysis revealed a complementary acidic surface and an additional hydrophobic motif involved in the interaction. Moreover, deletion mutagenesis performed on the ribosomal protein P0 revealed that the A1 chain binds to an alternate site on P0 in proximity to the contact sites for P1/P2 heterodimers. These results demonstrate that the catalytic chain of RIPs such as SLT-1 dock on ribosomes using two classes of binding sites located within the ribosomal stalk which may aid in orienting their catalytic domain in close proximity to the depurination site.

Control of mRNA stability is an important aspect in the regulation of gene expression. A well studied signal for rapid transcript decay in mammalian cells is the AU-rich element (ARE), which is found in the 3’ untranslated region (UTR) of many labile transcripts. These sequence elements confer destabilization of transcripts by binding to AU-binding proteins (AUBPs) that can recruit cellular decay enzymes. The stability of ARE-containing mRNAs can be regulated by extracellular stimuli, which allows for cells to adapt to the changing environment. AREs are found in many transcripts that encode for inflammatory genes, including TNF, GM-CSF, and IL-8. Pathogens evolve and devise mechanisms to subvert the immune response of the host to aid in its infection. Bacillus anthracis is one such infectious agent that can disable numerous arms of the host immune response. Its secreted toxin, anthrax lethal toxin (LeTx), causes the accelerated decay of the IL-8 mRNA. IL-8 is a dual function cytokine and chemokine that can recruit and activate neutrophils at the site of infection. Through the inactivation of MAPK pathways, LeTx activity causes the destabilization of IL-8 transcripts through its ARE. In this thesis, I show that an AUBP, TTP, is dephosphorylated by LeTx and MAPK inhibitors, and knock-down of its expression stabilized IL-8 transcripts. LeTx activity also increased the colocalization of TTP to P-bodies, cytoplasmic sites concentrated with RNA decay enzymes. This suggests that the post-translational modification of TTP induced by LeTx led to its enhanced destabilization function. Identified TTP-associated proteins, non-muscle myosin heavy chain 9 (myosin-9) and HSC-70, were examined for their role in IL-8 transcript decay. Knock-down of each protein led to a slower rate of IL-8 mRNA destabilization. However, treatment of LeTx continued to mediate accelerated destabilization of IL-8 in these siRNA-transfected cells. This suggests that LeTx, myosin-9, and HSC-70 modulate the destabilization function of TTP independently.

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.

The discovery of the pore-forming toxins produced by Bacillus thuringiensis, which are toxic to insects but not to mammalians, has provided a new successful means to control harmful plant-feeding insects biologically. The toxins are also used on insects that don’t feed on plants, for example on Anopheles. The Bacillus thuringiensis toxins fall into two structural families, named cry and cyt. All of these toxins act by damaging the cell membranes in the mid gut of the insect. In this study, a reliable system for expression and purification of the recombinant Cyt2Aa1 toxin has been developed. The recombinant Cyt2Aa1 toxin has been produced, characterized, followed by the construction of the cysteine mutants V186C and L189C by site directed mutagenesis. The new expression system yields 0.4 g of protein per litre of culture. The activated Cyt2Aa1 toxin is active in the hemolysis assay. Of note, the hemolytic activity of the V186C mutant exceeds that of wild type Cyt2Aa1 toxin and of the L189C mutant. Calcein release assay experiments have been done to examine the activity of the toxin with different artificial liposomes. It was found that Cyt2Aa1 toxin is very active with DMPC, DMPC+DMPG unilamellar liposomes. Surprisingly, however, Cyt2Aa1 toxin showed no activity with liposomes containing cholesterol. With both erythrocytes and sensitive liposomes, the toxin shows a “pro-zone effect”, that is the activity decreases at very high concentrations. The findings are discussed in the context of the toxin’s putative mode of action.

The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. <i>Escherichia coli</i> which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49].<p> Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy.<p> To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyers patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation <i>in vitro</i>. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2.<p> To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.

The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. Escherichia coli which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49]. Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy. To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyer’s patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation in vitro. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2. To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.

Bacillus anthracis, the etiological agent of anthrax, is a spore- forming, toxin- producing bacterium. Currently, treatment of B. anthracis infections requires a 60- day antibiotic regimen. However, better therapeutics are required. Therefore, this study looked at the effect of levofloxacin and linezolid on B. anthracis cell viability, toxin production and spore formation using in vitro static models and a pharmacodynamic model. It was hypothesized that the combination would be the most effective at preventing toxin and spore production resulting in greater bacterial killing. However, these studies suggest otherwise. Nevertheless, clinically, the combination therapy may be more effective in rapid killing of vegetative B. anthracis and may be able to reduce the duration of therapy (by reducing the likelihood of spore survival). Therefore, the clinical benefit of combined therapy on long-term recurrence cannot be determined from these in vitro models. Further investigation with combination therapy is warranted. / October 2015