Physiological adaptations contributing to stress survival in the foodborne pathogen Campylobacter jejuni
SUPERVISOR: Dr. Heidi Schraft (Biology)
ABSTRACT: In spite of being considered fragile and fastidious, the zoonotic pathogen Campylobacter jejuni remains the leading cause of foodborne bacterial gastroenteritis in the developed world. Lacking many of the stress responses common to other enteric pathogens, C. jejuni employs the survival strategies, biofilm formation and entry into the viable but non-culturable (VBNC) state, which have not been well characterized. Recent studies have indicated that these strategies are likely related at the molecular level. The purpose of this thesis was threefold: 1) to characterize entry into the VBNC state for planktonic and biofilm cells of C. jejuni with starvation at 4°C; 2) to evaluate a novel PMAqPCR method to quantify viable cells (both culturable and viable but non-culturable) in planktonic and biofilm cells of C. jejuni during starvation at 4°C; and 3) to investigate changes in gene expression of selected genes involved in biofilm formation and entry into the VBNC state. The three strains C. jejuni NCTC 11168 V1, C. jejuni NCTC V26 and C. jejuni 16-2R were included in all studies to compare variation based on strains. Cells were considered VBNC when there was no growth with enrichment, but cells scored as viable based on membrane integrity. Biofilm cells which became VBNC in some cases after 10 days of stress were found to enter the VBNC state earlier than planktonic cells by 10 to 50 days. Additionally, no significant reductions occurred in viable cell counts over the course of the experiments, confirming that the loss of culturability was not due to cell death (p<0.05). To date, no methods have been used to quantify viable but non-culturable biofilm cells of C. jejuni. The novel method PMAqPCR which has been successful for the enumeration of planktonic C. jejuni as well as for biofilm cells of other species was validated for quantifying C. jejuni biofilm cells in late log phase (20 h) and once cells had entered the VBNC state. The genes that affect both entry into a VBNC state and the ability to form biofilm in C. jejuni were upregulated during biofilm formation. Gene expression prior to stress treatment was 5 to 37 fold higher in biofilm cells than in their planktonic counterparts for all three strains IV (p<0.001). For the planktonic samples, only one of the 3 strains showed significant changes in gene expression during the transition to the VBNC state. In this case, all 4 target genes were significantly upregulated 4-6 fold just prior to cells becoming VBNC (p<0.05). At present food and drinking water safety in Canada continues to be assessed primarily using culture-based methodology. As validated in this thesis, the ability to quantify both culturable and viable but non-culturable C. jejuni cells in both planktonic and biofilm forms will allow for improved evaluation of quality control methods in both research and industries where these pathogens are a concern. Also, the understanding of the interaction between biofilm formation and entry into the VBNC state at the molecular level described herein provides information which can be used to develop appropriate interventions and reduce the incidence of campylobacteriosis.
Adventures in immunochemistry: Towards a fully synthetic vaccine
SUPERVISOR: Dr. Zi Hua Justin Jiang (Chemistry)
ABSTRACT: Lipid A, a unique disaccharide glycolipid, is the active principle of Gram-negative bacterial lipopolysaccharide in activating the innate immune response via Toll-like receptor 4 (TLR4). Given the important role that TLR4 plays in innate immunity, and ultimately, the development of an adaptive immune response, ligands that can modulate TLR4-mediated signalling have great therapeutic potential as both vaccine adjuvants, and anti-sepsis agents. In attempting to develop novel ligands which can successfully modulate TLR4-mediated signalling in a well defined fashion, simplified structures which aim to mimic the natural lipid A structure have shown great promise. The notion of cancer immunotherapy, in which the vast power of the immune system is tapped to prevent and/or eradicate the disease has begun to garner considerable attention. Tumour associated carbohydrate antigens, carbohydrate containing epitopes which are either unique of over-expressed by cancer cells, are viable targets of said immunotherapy. A major limitation, however, is the low antigenicity displayed by these carbohydrate epitopes. Studies have shown that the inclusion of adjuvant structures, especially when directly chemically conjugated to the antigen, improve the success of anti-cancer vaccination efforts. The primary goal of this study has been aimed at the development of novel vaccine adjuvants, specifically the design of novel molecular frameworks to mimic the structure of lipid A in the activation of TLR4. A secondary goal of this study has aimed at the application of successful novel lipid A mimics as the immunostimulatory component of self-adjuvanting carbohydrate antigens for use in therapeutic cancer vaccines. One novel molecular framework that has been designed and synthesized employs a flexible, acyclic diethanolamine-based scaffold to mimic one of the sugar moieties natural to the lipid A disaccharide. Several structural variations of this framework were generated for structure-activity relationship studies in an effort to maximize immunostimulatory potency. The mimics were evaluated in vitro for their ability to induce TLR4-mediated cytokines. All variations showed confirmed TLR4 stimulatory activity, the potency of which was dependent on the functionalization of the terminal ethanol moiety of the diethanolamine-based acyclic scaffold. In vivo studies evaluating the adjuvant potential of this novel family of lipid A mimics are currently underway. As part of an industrial partnership aimed at the development of novel vaccine adjuvants, a second lipid A mimic framework was designed and synthesized, in which an aromatic residue has been incorporated into the structural backbone. Two structural variations of the framework were generated which vary in the functionalization of the phenolic hydroxyl of the aromatic-based backbone. Several in vivo studies have shown that both mimics exhibit potent TLR4 immunostimulatory activity, and successful adjuvant properties. In an effort to construct a fully synthetic, self-adjuvanting tumour associated carbohydrate antigen for eventual use in therapeutic cancer vaccines, the immunostimulatory activity of the diethanolamine-based lipid A mimic framework designed herein, was tapped. As such, a conjugate structure in which the lipid A mimic framework and the Thomsen-Friedenreich carbohydrate antigen are directly linked via a flexible chemical linker was designed and synthesized. Future studies will determine the ability of the conjugate to induce an effective antibody response towards the carbohydrate epitope.
H2S as a novel biomarker and therapeutic target for Asthma
SUPERVISOR: Dr. Rui Wang (formerly with Chemistry)
ABSTRACT: Asthma is a chronic inflammatory disease with hyper-responsive bronchoconstriction and airway remodeling, leading to extensive airway narrowing. The pathogenic mechanisms for asthma remain unclear. Studies in the literature have shown that cystathionine gamma lysase (CSE)/ hydrogen sulfide (H2S) system participates in the regulation of airway contractility and immune response. In this PhD thesis study, we found that CSE was a major enzyme responsible for endogenous H2S synthesis in the lung and spleen as CSE gene knock out (CSE-KO) dramatically decreased H2S production rates in these two organs. In asthma model established via ovalbumin (OVA) sensitization and challenge, lung resistance of CSE-KO mice (12-16 weeks old), in response to aerosolized methacholine (MCh) at 12.5 mg/ml, was two times higher than that of wild type (WT) mice (12-16 weeks old). CSE-KO mice also developed more peri-bronchial inflammation and had higher levels of type 2 helper T cell (TH2) cytokines in bronchoalveolar lavage fluid (BALF). As allergic asthma is more prevalent among children than in adults, we next used young (3-4 weeks old) and old (7-8 months old) mice to observed if CSE/H2S were involved in the onset of childhood asthma. With the same intensity and duration of OVA treatments, WT young mice developed much more severe asthma with greater lung resistance, higher levels of eosinophils and TH2 cytokines in BALF, and more peri-bronchial inflammation than did WT old mice. This age-dependent propensity of immunoreaction and asthma development resulted from lower levels of CSE expression and H2S production in splenocytes from young mice, which was reversed by H2S donor treatment. Human umbilical cord blood mononuclear cells also had lower level of CSE proteins than that of peripheral blood mononuclear cell from adult people. CSE-KO mice had more severe asthma than WT mice, but without the age-dependent asthma propensity. Lower endogenous level of CSE/H2S in WT young mice and in CSE-KO mice at all ages than in old WT mice promoted the differentiation of splenocytes into type 2 helper T cytokines-generating cells, which was suppressed by H2S donor. CSE/H2S-induced inhibition of type 2 immunity is not mediated by STAT-6 activation. Instead, H2S caused S-sulfhydration of GATA3 in spleen cells and decreased GATA3 nuclear translocation, leading to the inhibition of type 2 immunity. We also found that CSE expression in the airway of WT mice was increased in an age-dependent manner. Lower abundance of CSE in young WT mice or absence of CSE in young/old CSE-KO mice aggravated airway responsiveness to MCh challenge, in the absence of allergen exposure, by more than two times compared to old WT mice. In conclusion, CSE/H2S in peripheral lymph tissues and the lung suppresses allergen-induced type 2 immunity, airway responsiveness and the consequential asthma. Lower activity of CSE/H2S pathway renders higher incidence of allergic asthma in childhood.
Dr. Young Jun Ju
H2S S-sulfhydration of pyruvate carboxylase in gluconeogenesis and its regulation by Trx1
SUPERVISOR: Dr. G. Yang (formerly in Chemistry)
ABSTRACT: Hydrogen sulfide (H2S), regarded as the third gasotransmitter, plays diverse physiological and pathological roles in the body along with another two gasotransmitters, including nitric oxide (NO) and carbon monoxide (CO). S-sulfhydration, a novel post-translational modification of proteins, is now considered as an important mechanism of H2S effects under various physiological and pathological conditions. In the liver, cystathionine gamma-lyase (CSE) is the main enzyme to generate H2S. The functions of H2S in the liver are largely unknown. Pyruvate carboxylase (PC), a mitochondrial protein, is an enzyme in the first step of gluconeogenesis in the liver. PC plays a critical role in tricarboxylic acid (TCA) cycle in mitochondria and in gluconeogenesis in the liver. PC is also involved in diverse metabolic pathways, such as lipogenesis and the biosynthesis of neurotransmitters. Here we found that H2S regulates gluconeogenesis through PC S-sulfhydration. First, PC activity and glucose production were decreased in liver of CSE-knockout (KO) mice comparing to the liver of wild type (WT) littermates. PC S-sulfhydration was lower in the liver tissue of CSE-KO mice than WT mice. NaHS treatment induces glucose production and PC activity in the primary liver cells. PC S-sulfhydration was increased by NaHS treatment in a time–dependent manner. Through mutation study, we further confirmed that the cysteine residue 265 is responsible for H2S S-sulfhdyration of PC. In cysteine 265 mutant-transfected cells, PC activity was significantly decreased and PC S-sulfhydration was disappeared. Thioredoxin 1 (Trx1), a well-known redox protein, is reported to be involved in transnitrosylation and/or denitrosylation. Here we first demonstrated that Trx1 acts as a S-desulfhydrase. Overexpressed Trx1 diminished H2S induced S-sulfhydration of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and PC proteins. The S-desulfhydration activity of Trx1 was inhibited by PX12, an inhibitor of Trx1, and DNCB, an iii inhibitor of thioredoxin reductase (TrxR). Furthermore, the cysteine 32 in Trx1 is required for the direct interaction of Trx1 with GAPDH or PC, and mutation of cysteine 32 abolished the S-desulfhydration activity of Trx1. Taken together, our studies demonstrated that H2S induces the gluconeogenesis through PC S-sulfhydration, and Trx1 acts as a S-desulfhydrase. Our findings will help to understand the mechanisms of H2S regulation of metabolic pathways via S-sulfhydration. These understandings will also extend the knowledge of physiological and pathological roles of H2S in both health and diseases.