The development and characterisation of a murine model of type 2 diabetes and burkholderia pseudomallei infection

Hodgson, Kelly Ann (2013) The development and characterisation of a murine model of type 2 diabetes and burkholderia pseudomallei infection. PhD thesis, James Cook University.

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Infections with Burkholderia pseudomallei, the causative agent of melioidosis, have been increasing steadily for the past two decades, earning its classification as an emerging tropical disease. Melioidosis is highly endemic in Northern Australia and Northeast Thailand, where annual incidence is approaching 50 cases per 100,000 individuals. In Northeast Thailand, melioidosis is the most common cause of death after infection with human immunodeficiency virus (HIV) and tuberculosis. Although rapid diagnosis, the availability of appropriate antibiotic therapy and intensive care facilities has recently improved outcomes from melioidosis in Australia, mortality rates remain high in many rural regions. Furthermore, therapeutic management of melioidosis involves prolonged antibiotic regimes, which do not always clear B. pseudomallei infection, frequently leading to relapse or reactivation of melioidosis following cessation of treatment. Therefore, the course of melioidosis is often protracted and can involve a range of clinical sequelae due to the protean manifestations of the infection.

A close association exists between melioidosis and type 2 diabetes (T2D). T2D has consistently been identified as the most significant risk factor predisposing to melioidosis. More than half of patients with melioidosis have pre-existing T2D and of those that do not, almost half have risk factors for pre-diabetes, including hypertension, dyslipidaemia and increased body mass index (BMI). Undoubtedly, the continued global prevalence of T2D will contribute to the increasing emergence of melioidosis as a tropical disease of significance. There is no vaccine available for melioidosis and no vaccine candidates have produced sterilising immunity, despite ongoing research. Furthermore, acquired antibiotic resistance by many bacteria, including B. pseudomallei, would have a significant impact on current therapeutic regimes, which rely on the availability of effective antibiotics and could subsequently result in increased mortality rates.

Future improvements in the therapeutic management of melioidosis will depend heavily on fundamental research to understand the host-pathogen interactions contributing to disease progression and how these are altered by T2D, leading to increased susceptibility. In vivo experiments are essential for this research, due to the complexity of interactions between the metabolic and immune systems. While in vitro experiments can be useful for understanding individual cell functions and interactions with bacteria, it is difficult to interpret the overall effect of these processes in the dynamic physiological environment of the host without comparative in vivo studies. Therefore, an animal model of T2D and comorbid B. pseudomallei infection is essential for the investigation of fundamental early immune responses leading to the divergence in disease progression between individuals with and without T2D. To this end, several murine models of T2D were investigated in this study to develop a suitable animal model that can be used to identify the mechanisms contributing to comorbid B. pseudomallei infection.

At the time of this study, the leptin signalling deficient Leptᵈᵇ monogenic model of T2D was widely used in research to understand disease pathogenesis of T2D. Leptᵈᵇ mice are available on either a C57BL/6 or BKS genetic background, believed to result in distinct metabolic phenotypes. Therefore, metabolic parameters including body mass, blood glucose, lipid profiles and glucose tolerance were compared. Although the Leptᵈᵇ mutation resulted in comparable body mass and glucose intolerance between background strains, BKS homozygous (db/db) mice developed severe hyperglycaemia, while C57BL/6 db/db mice tended to maintain moderate blood glucose levels. Both strains of db/db mice were more susceptible to subcutaneous infection with B. pseudomallei than heterozygous (db/+) mice and the increased severity of infection was associated with decreased blood glucose, a phenomenon that frequently occurs in sepsis. Importantly, the BKS Leptᵈᵇ monogenic model of T2D is widely available and requires less time to develop overt hyperglycaemia relative to other polygenic murine models of T2D, enhancing its utility for this research.

Growth of B. pseudomallei and mortality from the infection was greater in db/db (diabetic) compared to db/+ (non-diabetic) mice. However, these results need to be interpreted with caution due to the confounding complications of leptin signalling deficiency, which could be implicated in their increased susceptibility to infection. This is arguably the most significant limitation of this model. It is also a poor model in terms of the aetiology of T2D in humans, which is rarely caused by leptin deficiency and other single gene mutations. Therefore, we sought to develop and characterise a murine model more reflective of the natural aetiopathology of T2D and subsequent infection with B. pseudomallei. A polygenic, diet-induced model of T2D is more reflective of the aetiopathology of T2D in humans and would arguably provide a more clinically relevant and comprehensive understanding of the immunological basis of melioidosis and comorbid T2D. Developing a murine model of diet-induced T2D was complicated by the vast inconsistencies in the literature, possibly owing to the variability and incomplete description of diets, genetic backgrounds, genders, age and duration of feeding regimes.

In this study, male mice were found to be more susceptible to metabolic complications induced by consuming a high fat diet (HFD) in comparison to female mice. This is similar to the gender susceptibility that has been documented in clinical T2D, possibly related to the involvement of sex hormones in regulating insulin sensitivity, though the mechanisms are incompletely understood. Despite the increased glucose intolerance in male B6D2F1 mice, blood glucose levels were only moderately elevated after 20 weeks of consuming a HFD. Hyperglycaemia is the most important clinical criteria for diagnosis of T2D in humans. Therefore, metabolic parameters were compared to male C57BL/6 mice on an identical HFDfeeding regime. Whilst body weight gain in B6D2F1 mice exceeded that in C57BL/6 mice fed a HFD, glucose intolerance was exacerbated in C57BL/6 mice. The genetic predisposition towards development of T2D in C57BL/6 mice at a lower degree of adiposity is clinically significant since most melioidosis patients, particularly in Asia, have T2D without extreme obesity. Optimisation of the HFD-feeding regime led to further increases in blood glucose levels in C57BL/6 mice, which was associated with abnormal albumin/creatinine ratio and increased baseline levels of inflammation, consistent with clinical T2D.

In the current study, diet-induced T2D in C57BL/6 mice was found to be the most reflective model of clinical T2D, in terms of aetiology and disease pathogenesis, and as such was selected for subsequent experiments that investigated differences in cytokine responses and disease progression of B. pseudomallei infection. Diabetic mice were significantly more susceptible to B. pseudomallei infection compared to non-diabetic littermates. Increased mortality in diabetic mice was associated with greater dissemination and growth of B. pseudomallei in the spleen and liver within the first 24 hours post-infection and bacteraemia from day 3 post-infection, preceding death. Fatal outcome in diabetic mice was paralleled with delayed cytokine responses compared to non-diabetic littermates. At 12 hours post-infection, levels of TNF-α, IL-12 and IFN-γ were reduced in spleen of diabetic mice relative to non-diabetic mice. Impaired killing of B. pseudomallei by macrophages from diabetic mice was also observed following in vitro studies, compared to macrophages from non-diabetic mice. Given the essential role of macrophages in immune defence against B. pseudomallei infection, impaired macrophage function may contribute to increased B. pseudomallei growth and poor disease outcome observed in diabetic mice.

Macrophage function is influenced by the intracellular concentration of antioxidants such as glutathione (GSH), which regulates the cellular redox balance by cycling between reduced (GSH) and oxidised states (GSSG). In addition to the ability of GSH to regulate immune responses, there is also evidence that GSH has direct antimicrobial toxicity. The mechanisms for this and the sensitivity of B. pseudomallei to GSH are not known. Oxidative stress, defined by decreased GSH/GSSG levels, is central to the pathogenesis of T2D. However, the role of this in susceptibility to bacterial infections is unclear. Therefore, GSH concentrations were measured in blood and tissues of diabetic and non-diabetic mice prior to, and following, infection with B. pseudomallei. At baseline (uninfected), the GSH/GSSG ratio was significantly lower in diabetic mice compared to non-diabetic mice, primarily due to increased GSSG, indicative of oxidative stress.

The intracellular balance of GSH/GSSG levels in macrophages and other immune cells regulates many signalling pathways, including cytokine production. It was proposed that altered GSH/GSSG redox balance could be involved in the dysregulation of cytokine responses described in diabetic mice following infection with B. pseudomallei. GSH modulators were used to deplete or restore GSH in nondiabetic and diabetic mice, respectively, to determine the effect on disease progression of B. pseudomallei infection. The depletion of GSH in non-diabetic mice, prior to infection with B. pseudomallei and during the first 24 hours postinfection, did not increase susceptibility to infection. However, it is not known if repletion of GSH occurred following the first 24 hours post-infection, which may have been sufficient to control the infection. Further research should investigate whether increased duration of treatment or use of other GSH modulators to deplete GSH influences susceptibility to B. pseudomallei infection. Furthermore, diabetic mice treated with a cysteine derivative to restore GSH did not improve overall disease outcome from melioidosis, nor did this reduce B. pseudomallei growth. However, it is again recognised that treatment schedules may require further optimisation, particularly since a variety of synthetic pathways involved in GSH metabolism appear to be altered in T2D. Further research is warranted to investigate the potential for GSH derivatives as more suitable treatment options.

In summary, this thesis describes and characterises the first polygenic murine model of T2D and comorbid B. pseudomallei infection. This novel model will be useful for further fundamental and translational research exploring the pathogenic mechanisms that link these two significant diseases. Identifying the mechanisms responsible for the increased susceptibility of people with T2D to melioidosis will enable the development of improved therapeutic strategies to reduce morbidity and mortality in a highly susceptible population. In addition to the value of this murine model for understanding the pathogenesis of the most important comorbidity of melioidosis, it may also be invaluable for research focusing on other infectious comorbidities of T2D, such as tuberculosis.

Item ID: 40326
Item Type: Thesis (PhD)
Keywords: animal models; burkholderia; cellular immunology; cytokine responses; diabetes; Diabetes mellitus; diet-induced; hypoglycemia; in vivo; infectious diseases; medical bacteriology; melioidosis; mice; mouse models; murine models; non-insulin-dependent diabetes; pseudomallei; pseudomonas mallei; type 2 diabetes; type two diabetes
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For this thesis, Kelly Hodgson received the Dean's Award for Excellence 2015.

Publications arising from this thesis are available from the Related URLs field. The publications are:

Hodgson, Kelly A., Govan, Brenda L., Walduck, Anna K., Ketheesan, Natkunam, and Morris, Jodie L. (2013) Impaired early cytokine responses at the site of infection in a murine model of type 2 diabetes and melioidosis comorbidity. Infection and Immunity, 81 (2). pp. 470-477.

Hodgson, K., Govan, B., Ketheesan, N., and Morris, J. (2013) Dietary composition of carbohydrates contributes to the development of experimental type 2 diabetes. Endocrine, 43 (2). pp. 447-451.

Morris, Jodie L., Hodgson, Kelly A., and Ketheesan, Natkunam (2012) Development of protection. In: Ketheesan, Natkunam, (ed.) Melioidosis: a century of observation and research. Elsevier, The Netherlands, pp. 282-299.

Hodgson, Kelly A., Morris, Jodie L., Feterl, Marshall L., Govan, Brenda L., and Ketheesan, Natkunam (2011) Altered macrophage function is associated with severe Burkholderia pseudomallei infection in a murine model of type 2 diabetes. Microbes and Infection, 13 (14-15). pp. 1177-1184.

Hodgson, Kelly, Engler, Cathy, Govan, Brenda, Ketheesan, Natkunam, and Norton, Robert (2009) Comparison of routine bench and molecular diagnostic methods in identification of Burkholderia pseudomallei. Journal of Clinical Microbiology, 47 (5). pp. 1578-1580.

Date Deposited: 02 Sep 2015 04:44
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1107 Immunology > 110704 Cellular Immunology @ 50%
11 MEDICAL AND HEALTH SCIENCES > 1108 Medical Microbiology > 110801 Medical Bacteriology @ 50%
SEO Codes: 92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920109 Infectious Diseases @ 40%
92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920104 Diabetes @ 60%
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