Cholera is a diarrhoeal disease caused by the infection of the intestine with bacterium vibrio cholera. The diarrhoeal disease is a recurrent feature of Zimbabwe's post 1990s history. From 1993 to 2018, the country has experienced several cholera outbreaks in both rural and urban areas. The country's worst cholera outbreak occurred in 2008/2009 and resulted in over 4000 deaths. The dissertation analyses three global health actors' responses to persistent cholera outbreaks in Harare, the capital city of Zimbabwe. Building on previous scholarship of water, cholera and politics, the dissertation compares global health actors' responses to the 2008/2009 and September to November 2018 cholera outbreaks. The dissertation used the qualitative research method including analysis of existing academic literature, Zimbabwean national legislation, non-governmental organisation (NGO) publications and conference reports, news articles and Zimbabwean government policy documents. In-depth interviews with personnel from the World Health Organisation, United Nations Children's Fund, Médecins Sans Frontierès (Doctors without Borders) and the Harare City Council Health Department were conducted in Harare, Zimbabwe from June to July 2019. The dissertation demonstrates that the scope and speed of global health actors' emergency cholera interventions in Harare are shaped by Zimbabwe's political climate and the state of Harare's health, water and sanitation infrastructure.

AB-type protein toxins such as cholera toxin (CT) consist of a catalytic A subunit and a cell-binding B subunit. CT proceeds through the secretory pathway in reverse, termed retrograde trafficking, and is delivered to the endoplasmic reticulum (ER). In order for the catalytic A1 subunit to become active it must separate from the rest of the holotoxin, and this dissociation event occurs in the ER lumen. CTA1 assumes an unfolded conformation upon dissociation from the holotoxin and is recognized by ERassociated degradation (ERAD), a quality control system that recognizes and exports misfolded proteins to the cytosol for degradation by the 26S proteasome. CTA1 is not degraded by the 26S proteasome because it has few sites for poly-ubitiquination, which is recognized by the cap of the 26S proteasome for degradation. Thus, CTA1 escapes the degradation of ERAD while at the same time using it as a transport mechanism into the cytosol. It was originally proposed that CTA1 is thermally stable and that ER chaperones actively unfolded CTA1 for translocation to the cytosol. In contrast, we hypothesized that the dissociated CTA1 subunit would unfold spontaneously at 37°C. This study focused on the three conditions linked to CTA1 instability and translocation: (i) CTA1 dissociation from the holotoxin, (ii) the translocation-competent conformation of CTA1, and the extraction of CTA1 from the ER into the cytosol. Disruption of any of these events will confer resistance to the toxin. The original model suggested that PDI actively unfolds CTA1 to allow for translocation. However, Fourier transform infrared iv spectroscopy (FTIR) and surface plasmon resonance (SPR) data we have gathered demonstrated that PDI dislodges CTA1 from the rest of the holotoxin without unfolding CTA1. Once released by the holotoxin, CTA1 spontaneously unfolds. PDI is thus required for the toxicity of CT, but not as an unfoldase as originally proposed. CTA1 must maintain an unfolded conformation to keep its translocation-competent state. Based on our model, if CTA1 is stabilized then it will not be able to activate the ERAD translocation system. Our SPR and toxicity results demonstrated that treatment with 4- phenylbutyrate (PBA), a chemical chaperone, stabilizes the structure of CTA1. This stabilization resulted in a decrease in translocation from the ER to the cytosol and a block of intoxication, which makes it a viable candidate for a therapeutic. Because CTA1 exits the ER in an unfolded state, there must be a driving force for this translocation. We hypothesized that Hsp90, a cytosolic chaperone, is responsible for the translocation of CTA1 across the membrane. Previous research had shown Hsp90 to be present on the cytosolic face of the ER and had also shown that Hsp90 will refold exogenously added proteins that enter the cytosol. Using drug treatments and RNAi, we found that Hsp90 is required for the translocation of CTA1 from the ER lumen to the cytosol, a brand new function for this chaperone. We have provided evidence to support a new, substantially different model of CTA1 translocation. CTA1 does not masquerade as a misfolded protein in order to utilize ERAD for entry into the cytosol; it actually becomes misfolded and is treated as any other ERAD substrate. The spontaneous unfolding of CTA1 is the key to its v recognition by ERAD and ultimately its translocation into the cytosol. Host factors play very important roles in intoxication by AB toxins and are targets for blocking intoxication.

Cholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much of the toxin is transported to the lysosomes for degradation, but a secondary pool of toxin is diverted to the Golgi apparatus and then to the ER. Here the A1 subunit detaches from the rest of the toxin and enters the cytosol. The disordered conformation of free CTA1 facilitates toxin export to the cytosol by activating a quality control mechanism known as ER-associated degradation. The return to a folded structure in the cytosol allows CTA1 to attain an active conformation for modification of its Gsα target through ADP-ribosylation. This modification locks the protein in an active state which stimulates adenylate cyclase and leads to elevated levels of cAMP. A chloride channel located in the apical enterocyte membrane opens in response to signaling events induced by these elevated cAMP levels. The osmotic movement of water into the intestinal lumen that results from the chloride efflux produces the characteristic profuse watery diarrhea that is seen in intoxicated individuals. The current model of intoxication proposes only one molecule of cytosolic toxin is required to affect host cells, making therapeutic treatment nearly impossible. However, based on emerging evidence, we hypothesize a threshold quantity of toxin must be present within the cytosol of the target cell in order to elicit a cytopathic effect. Using the method of surface plasmon resonance along with toxicity assays, I have, for the first time, directly measured the efficiency of toxin delivery to the cytosol and correlated the levels of cytosolic toxin to toxin iv activity. I have shown CTA1 delivery from the cell surface to the cytosol is an inefficient process with only 2.3 % of the surface bound CTA1 appearing in the cytosol after 2 hours of intoxication. I have also determined and a cytosolic quantity of more than approximately .05ng of cytosolic CTA1 must be reached in order to elicit a cytopathic effect. Furthermore, CTA1 must be continually delivered from the cell surface to the cytosol in order to overcome the constant proteasome-mediated clearance of cytosolic toxin. When toxin delivery to the cytosol was blocked, this allowed the host cell to de-activate Gs, lower cAMP levels, and recover from intoxication. Our work thus indicates it is possible to treat cholera even after the onset of disease. These findings challenge the idea of irreversible cellular toxicity and open the possibility of postintoxication treatment options.

The endoplasmic reticulum (ER) is the site of co- and post-translational modification for secretory proteins. In order to prevent vesicular transport and secretion of misfolded or misassembled proteins, a highly regulated mechanism called ER-associated degradation (ERAD) is employed. This pathway recognizes misfolded proteins in the ER lumen and targets them to the cytosol for ubiquitination and subsequent degradation via the 26S proteasome. Sec61 and Derlin-1 are ER pores through which export occurs. AB-type protein toxins such as cholera toxin (CT), Shiga toxin (ST), exotoxin A (ETA), and ricin have evolved means of exploiting the ERAD pathway in order to reach their cytosolic targets. AB-type protein toxins consist of a catalytic A-subunit and a cell-binding B-subunit. The B-subunit recognizes cell surface receptors for the toxin. This begins a series of vesicle trafficking events, collectively termed retrograde trafficking, that lead to the ER. Dissociation of the A and B subunits occurs in the ER, and only the A subunit enters the cytosol. The exact mechanism of A subunit translocation from the ER to the cytosol is unknown. Toxin translocation occurs through a pore in the ER membrane. Exit through the pore requires the toxin to be in an unfolded conformation. The current model for toxin translocation proposes that ER chaperones actively unfold the toxin A chain for translocation. After the translocation event, the toxin spontaneously refolds to an active conformation. Our model suggests that unfolding in the ER is spontaneous and refolding in the cytosol is dependent upon cytosolic chaperones. Based on our model, we hypothesize that blockage of the A subunit unfolding and/or the ERAD translocation step will confer a phenotype of non-harmful multi-toxin resistance to cells. In support of this model, we have shown that, at 37[degrees]C, the isolated catalytic subunit of cholera toxin (CTA1) is in an unfolded and protease sensitive confirmation that identifies the toxin as misfolded by the ERAD pathway. Stabilization of CTA1 via glycerol inhibits the loss of its tertiary structure. This stabilization results in decreased translocation from the ER to the cytosol and increased secretion of CTA1 to the extracellular medium. Treatment with glycerol also prevents CTA1 degradation by the 20S proteasome in vitro. These data indicate that the thermal stability of CTA1 plays an important role in intoxication. These data also suggest that stabilization of CTA1 tertiary structure is a potential target for therapeutic agents. Our model asserts that CTA1 behaves as a normal ERAD substrate upon dissociation from the holotoxin. In support of this model, we have shown that the ER luminal protein HEDJ, known to be involved in ERAD, interacts with CTA1. The interactions between HEDJ and CTA1 occur only at temperatures in which the toxin is in an unfolded conformation. We have also shown that HEDJ does not affect the thermally stability of CTA1 since there is no alteration in its pattern of temperature-dependent protease sensitivity. Alteration of the normal HEDJ-CTA1 interaction via a dominant-negative HEDJ construct resulted in decreased translocation from the ER to the cytosol and, as a result, decreased intoxication. Our work demonstrated toxin resistance can result through effects on toxin structure or ERAD chaperones. To identify other potential inhibitors, we developed a novel assay to detect the activity of other AB toxins and compared it with an established toxicity assay. We generated a Vero cell line that expressed a destabilized variant of enhanced green fluorescent protein (EGFP). These cells were used to monitor the Stx-induced inhibition of protein synthesis by monitoring the loss of EGFP fluorescence from cells. We screened a panel of 13 plant compounds, and indentified grape seed extract and grape pomace extract as inhibitors of Stx activity. Grape seed extract and grape pomace extract were also shown to block the toxic activities of ETA and ricin, providing the basis for a future high-throughput screen for multi-toxin inhibitors.

Pasteurella multocida is an important multi-host animal and zoonotic pathogen that is capable of causing respiratory and multi-systemic diseases, bacteremia, and infections resulting from bite wounds. The glycosaminoglycan capsule (CPS) of P. multocida is an essential virulence factor, protecting the bacterium from host defenses. However, chronic infections such as bovine respiratory disease (BRD) and avian cholera may be associated with biofilm formation. Biofilm formation was inversely related to capsule production (determined by uronic acid and N-acetylglucosamine assays), and was confirmed with capsule-deficient mutants of mucoid strains. Capsule-deficient mutants formed biofilms with a larger biomass that was much thicker and smoother than encapsulated strains. Gas chromatography-mass spectrometry, nuclear magnetic resonance, and enzymatic digestion demonstrated that the matrix material of the biofilm was composed predominately of a glycogen exopolysaccharide (EPS). Therefore, CPS may interfere with biofilm formation by blocking adherence to a surface or by preventing the EPS matrix to encase large numbers of bacterial cells. Chemical mutagenesis was performed on P. multocida strain P1059, resulting in isolation of an acapsular mutant designated as P1059-R8. A uridyltransferase encoded by gene P1059_01979 was mutated in such a way that a polar amino acid was changed to a non-polar amino acid near the active site. The protein product of P1059_01979 is important for the biosynthesis of the CPS subunit N-acetylglucosamine. CPS quantification revealed that the subunit glucuronic acid was produced in equal concentrations to the parent, but the CPS subunit N-acetylglucosamine was not detected in the chemical mutant. Biofilm formation in the chemical mutant was significantly higher than in WT P1059 and the capsule-deficient mutant. We hypothesize that P1059_01979 is essential for CPS production in P. multocida serogroup A. Histophilus somni and Pasteurella multocida cause bovine respiratory disease (BRD) and systemic infections in cattle. Following respiratory infection of calves with H. somni, P. multocida is also often isolated from the lower respiratory tract. Because H. somni normally forms a biofilm during BRD, we suspected that P. multocida may co-exist with H. somni in a polymicrobial biofilm. Interactions between the two species in the biofilm were characterized and quantified by fluorescence in situ hybridization (FISH), and the biofilm matrix of each species examined by fluorescently-tagged lectins (FTL), confocal scanning laser microscopy of in vitro biofilms and bovine pulmonary tissue following dual H. somni and P. multocida infection. FISH and FTL were used to show that P. multocida and H. somni were evenly distributed in the in vitro biofilm, and both species contributed to the polymicrobial biofilm matrix. COMSTAT z-stack image analysis revealed that the average biomass and biofilm thickness of the individual and polymicrobial biofilms were greatest when both species were present. Encapsulated P. multocida isolates not capable of forming a biofilm still formed a polymicrobial biofilm with H. somni, but only the EPS of H. somni could be detected by FTL staining of bovine tissues from which both species were isolated. Bacteria within a biofilm are more quiescent than during planktonic growth and induce less of an inflammatory response, indicating encapsulated P. multocida may take advantage of the H. somni biofilm to persist in the host during less severe, but more chronic, BRD. These results may have important implications for the management of BRD. Acute avian cholera is associated with encapsulated P. multocida, while chronic and asymptomatic cases of avian cholera are associated with acapsular P. multocida isolates. We hypothesize that biofilm formation is present and an important factor for chronic and asymptomatic avian cholera. Experimental infections of chickens with biofilm deficient P. multocida strain WT X73, proficient biofilm forming P. multocida strain X73ΔhyaD, and proficient biofilm forming clinical isolates 775 and 756 showed that virulence inversely correlated with biofilm formation. Histopathological analysis showed that biofilm forming isolates induced little inflammation in the lungs, heart, and liver, while biofilm deficient isolates induced greater inflammation. Biofilm material was located in pulmonary tissues of chickens diagnosed with chronic avian cholera using FTL staining.. Quantitative real-time PCR for expression of cytokine genes in the spleens of infected chickens indicated that P. multocida induced Th1 and Th17 immune responses during acute and chronic avian cholera. Chickens that succumbed to acute avian cholera after experimental challenge with WT X73 had high levels of INF-ƴ, IL-1β, IL-6, IL-12A, IL-22, IL-17A, and IL-17RA expression in the spleen compared to all other experimental groups. Antibody titers were low, indicating that antibodies may be less important in managing and clearing P. multocida infections. / Ph. D.

Thesis (MPH.) -- University of Limpopo, 2015 / Cholera is an acute enteric infection caused by the ingestion of bacterium Vibrio cholerae present in faecally contaminated water or food. Primarily linked to insufficient access to safe water and proper sanitation, its impact can be even more dramatic in areas where basic environmental infrastructures are disrupted or have been destroyed. The aim of the study was to investigate the factors contributing to the prevalence of cholera and the environmental risk factors associated with cholera in the Vhembe district of Limpopo province between 2008 and 2012. The objectives of the study were to identify environmental risk factors for cholera and to determine the number of cholera cases in the Vhembe district. The study used a quantitative, retrospective and cross-sectional research method. The records of 317 patients who met the study criteria were reviewed using an audit tool. The Statistical Package for Social Sciences (SPSS) version 22 was used to analyze the data. The results revealed that lack of adequate hygiene practices, limited access to safe drinking water, lack of safe food preparation and handling, and inadequate sanitation system are risk factors associated with cholera. The study recommends prevention, control of cholera outbreak and case management. Keywords: Cholera, outbreak, Vibrio cholerae 01 and 0139, Watery diarrhea (ricewater), Prevalence, Risk factors.

The primary objective of this thesis is to differentiate between the risk factors for pre-and post-monsoon cholera epidemics in rural Bangladesh by analyzing the complex interaction between select environmental, cultural/behavioral, and socioeconomic variables over space and time. In rural Bangladesh, cholera epidemics correspond with the annual monsoon: the first, and smallest, occurs between March and June, while the larger cholera peak occurs between September and December. The differences between the spatial and temporal patterns of seasonal cholera are analyzed, and the risk factors are calculated for pre-and post-monsoon cholera epidemics. The theoretical approach that underlies this medical geographical study is disease ecology, which espouses that risk of disease is caused by an interaction between people and their environment. This thesis is structured around a holistic understanding that human-environment interactions are inseparable. In Bangladesh, the monsoon season typically starts between May and June. The 1992 and 1993 cholera peaks occurred just before the monsoon in April and March respectively, while the 1994 cholera peak occurred between April and June. In 1992 and 1993 cholera incidence increased in the post-monsoon period, and peaked in October. The 1994 post-monsoon cholera peak occurred in November. There is a regular temporal pattern to cholera, as the peaks followed a seasonal pattern with the smaller epidemic occurring in the pre-monsoon period and the larger epidemic occurring in the post-monsoon period. This study shows that there are different risks associated with pre-monsoon cholera epidemics and post-monsoon cholera epidemics. The two main risk factors associated with cholera incidence pre-monsoon were bari population (i.e., crowding) and a house located within the flood controlled area. These two variables were even more strongly associated with post-monsoon cholera incidence to a greater degree, along with a number of other variables including water use, sanitation practices, and socioeconomic status.

The spread of cholera is always associated with contaminated food or water and this is the reason this disease has been endemic in developing countries for centuries due to their lack of proper sanitation facilities and poor or no infrastructure for sewage systems. Cholera can spread quickly and sporadically after any natural disaster that destroys the sewage system or safe drinking water supply of both developed and undeveloped countries. In Southeast Asia in December 2004 and in Pakistan and Haiti 2010, cholera outbreaks followed the natural disasters; with most of the cholera victims being children. Although it is known that the best way to prevent cholera outbreak is the development of the infrastructure, provision of a safe drinking water supply and proper sanitation, this is a very long-term process, and most of the developing countries cannot afford such improvements. These situations can be made worse by natural disasters. Therefore there is a pressing need for the development of a cholera vaccine and there have been numerous research projects working towards this end for several decades. A few of them have been successful to date but because of the severe side effects and narrow range of protection, more effective and wider range vaccine development is still ongoing. In this study, crystallographic and enzymatic studies have been carried out on several novel proteins involved in the control of the production of the factors required for quorum sensing. Quorum sensing is a process in which bacterial cells communicate among themselves by the synthesis, release and detection of small chemical compounds called autoinducers. In this work, structural analysis was carried out on proteins involved in the synthesis and detection of the major autoinducer of Vibrio cholerae, named CAI-1. The crystal structure of CqsA involved in CAI-1 synthesis has been successfully solved and its enzymatic properties have been characterized. The structure of one domain of the cytoplasmic region of the CAI-1 receptor CqsS was also elucidated, and other domains were expressed. The crystal structure of another enzyme (VCA0859, an aldo-keto reductase) thought to have been involved in the synthesis of CAI-1 was also determined. Another protein named VCA0939 was also studied, due to its importance in biofilm development, and its ability to control quorum-sensing in an alternative pathway in the mutated version of pathogenic strains of V. cholerae that were responsible for the seventh cholera pandemic. The aim of this project was to understand the three dimensional structure of some proteins that are involved in quorum sensing and control of the expression of virulence genes for the pathogenesis of V. cholerae. Understanding the three dimensional structure of the proteins and the mode of autoinducer binding to its specific receptor could be highly valuable in the development of a chemical compound that could lead to the discovery of a novel drug with the ability to target cross species specification.

Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (M.tb), remains a global health problem and although BCG offers some protection against childhood disseminated disease and other mycobacterial infections, its efficacy against pulmonary TB varies between 0 and 80%. Modified Vaccinia virus Ankara expressing antigen 85A (MVA85A) is a novel TB vaccine designed to boost mycobacterium-specific CD4+ T cell response primed by BCG. MVA85A induces strong interferon (IFN)-γ responses, a cytokine known to be essential for protection following M.tb infection. A strong IFN-γ response is not a correlate of protection and in terms of the adaptive response, interleukin (IL)-17 is emerging as an important cytokine following vaccination as it is thought to help boost IFN-γ production by CD4+ T cells. This thesis shows that MVA85A induces IL-17 in PBMC and whole blood of human BCG – MVA85A vaccinees. This is replicated in mice receiving BCG – MVA85A intranasally. The administration of cholera toxin (CT) with BCG enhances IL-17 and confers improved protection following M.tb challenge, which is partially dependent on IL-17 and on the mucosal route of administration. Since CT is not a suitable adjuvant in humans, an alternative IL-17-inducing pathway was investigated. In human BCG – MVA85A-vaccinated volunteers, blocking the hydrolysing ability of the CD39, an apyrase responsible for hydrolysing pro-inflammatory ATP, enhances IL-17 production. Challenge of BCG – MVA85A-vaccinated CD39-/- mice with M.tb slightly improved the protective capacity of the vaccine, suggesting that a pathway dependent on ATP-driven inflammation may be a target for improving the immunogenicity of a vaccine against M.tb disease. Overall, this thesis has confirmed the important role of IL-17 in vaccine-induced protection against TB disease and identifies a possible target pathway for improvement of a novel vaccine.

Doctor of Philosophy / Department of Anatomy and Physiology / Bruce D. Schultz / The first aim of this project is to define the cellular mechanisms that account for the low Na[superscript]+ concentration in human milk. MCF10A cells, which were derived from human mammary epithelium and grown on permeable supports, exhibit amiloride- and benzamil-sensitive short circuit current (I[subscript]sc), suggesting activity of the epithelial Na[superscript]+ channel, ENaC. When cultured in the presence of cholera toxin (Ctx), MCF10A cells exhibit greater amiloride sensitive I[subscript]sc at all time points tested, an effect that is not reduced with Ctx washout for 12 hours or by cytosolic pathways inhibitors. Ctx increases the abundance of both beta and gamma-ENaC in the apical membrane and increases its monoubiquitination but without changing total protein and mRNA levels. Additionally, Ctx increases the levels of both the phosphorylated and the nonphosphorylated forms of Nedd4-2, a ubiquitin-protein ligase that regulates ENaC degradation. The results reveal a novel mechanism in human mammary gland epithelia by which Ctx regulates ENaC-mediated Na[superscript]+ transport. The second project aim is to develop a protocol to isolate mammary gland epithelia for subsequent in vitro culture. Caprine (1[superscript]0CME) and bovine mammary epithelia (1[superscript]0BME) were isolated and cultured on permeable supports to study hormone- and neurotransmitter-sensitive ion transport. Both 1[superscript]0CME and 1[superscript]0BME cells were passed for multiple subcultures and all passages formed electrically tight barriers. 1[superscript]0CME were cultured in the presence of hydrocortisone and exhibited high electrical resistance and amiloride-sensitive I[subscript]sc, suggesting the presence of ENaC-mediated Na[superscript]+ transport. 1[superscript]0BME were grown in a complex media in the presence or absence of dexamethasone. In contrast to 1[superscript]0CME, 1[superscript]0BME exhibited no detectable amiloride-sensitive I[subscript]sc in either culture condition. However, 1[superscript]0BME monolayers responded to an adrenergic agonist, norepinephrine, and a cholinergic agonist, carbamylcholine, with rapid increases in I[subscript]sc. Thus, this protocol for isolation and primary cell culture can be used for future studies that focus on mammary epithelial cell regulation and functions. In conclusion, the results from these projects demonstrate that mammary epithelial cells form electrically tight monolayers and can exhibit neurotransmitter- and/or hormone-induced net ion transport. The mechanisms that regulate Na[superscript]+ transport across mammary gland may provide clues to prevent or treat mastitis.