Mucosal delivery of ESX-1-expressing BCG strains provides superior immunity against tuberculosis in murine type 2 diabetes.

Tuberculosis (TB) claims 1.5 million lives per year. This situation is largely due to the low efficacy of the only licensed TB vaccine, Bacillus Calmette-Guérin (BCG) against pulmonary TB. The metabolic disease type 2 diabetes (T2D) is a risk factor for TB and the mechanisms underlying increased TB susceptibility in T2D are not well understood. Furthermore, it is unknown if new TB vaccines will provide protection in the context of T2D. Here we used a diet-induced murine model of T2D to investigate the underlying mechanisms of TB/T2D comorbidity and to evaluate the protective capacity of two experimental TB vaccines in comparison to conventional BCG. Our data reveal a distinct immune dysfunction that is associated with diminished recognition of mycobacterial antigens in T2D. More importantly, we provide compelling evidence that mucosal delivery of recombinant BCG strains expressing the Mycobacterium tuberculosis (Mtb) ESX-1 secretion system (BCG::RD1 and BCG::RD1 ESAT-6 ∆92-95) are safe and confer superior immunity against aerosol Mtb infection in the context of T2D. Our findings suggest that the remarkable anti-TB immunity by these recombinant BCG strains is achieved via augmenting the numbers and functional capacity of antigen presenting cells in the lungs of diabetic mice.

Disparate Effects of Metformin on Mycobacterium tuberculosis Infection in Diabetic and Nondiabetic Mice.

Comorbid type 2 diabetes poses a great challenge to the global control of tuberculosis. Here, we assessed the efficacy of metformin (MET), an antidiabetic drug, in mice infected with a very low dose of Mycobacterium tuberculosis In contrast to diabetic mice, infected nondiabetic mice that received the same therapeutic concentration of MET presented with significantly higher disease burden. This warrants further studies to investigate the disparate efficacy of MET against tuberculosis in diabetic and nondiabetic individuals.

Group G Streptococcus Induces an Autoimmune Carditis Mediated by Interleukin 17A and Interferon γ in the Lewis Rat Model of Rheumatic Heart Disease.

Acute rheumatic fever and rheumatic heart disease (ARF/RHD) have long been described as autoimmune sequelae of Streptococcus pyogenes or group A streptococcal (GAS) infection. Both antibody and T-cell responses against immunodominant GAS virulence factors, including M protein, cross-react with host tissue proteins, triggering an inflammatory response leading to permanent heart damage. However, in some ARF/RHD-endemic regions, throat carriage of GAS is low. Because Streptococcus dysgalactiae subspecies equisimilis organisms, also known as ß-hemolytic group C streptococci and group G streptococci (GGS), also express M protein, we postulated that streptococci other than GAS may have the potential to initiate or exacerbate ARF/RHD. Using a model initially developed to investigate the uniquely human disease of ARF/RHD, we have discovered that GGS causes interleukin 17A/interferon γ-induced myocarditis and valvulitis, hallmarks of ARF/RHD. Remarkably the histological, immunological, and functional changes in the hearts of rats exposed to GGS are identical to those exposed to GAS. Furthermore, antibody cross-reactivity to cardiac myosin was comparable in both GGS- and GAS-exposed animals, providing additional evidence that GGS can induce and/or exacerbate ARF/RHD.

Increased Neurotropic Threat from Burkholderia pseudomallei Strains with a B. mallei-like Variation in the bimA Motility Gene, Australia.

Neurologic melioidosis is a serious, potentially fatal form of Burkholderia pseudomallei infection. Recently, we reported that a subset of clinical isolates of B. pseudomallei from Australia have heightened virulence and potential for dissemination to the central nervous system. In this study, we demonstrate that this subset has a B. mallei-like sequence variation of the actin-based motility gene, bimA. Compared with B. pseudomallei isolates having typical bimA alleles, isolates that contain the B. mallei-like variation demonstrate increased persistence in phagocytic cells and increased virulence with rapid systemic dissemination and replication within multiple tissues, including the brain and spinal cord, in an experimental model. These findings highlight the implications of bimA variation on disease progression of B. pseudomallei infection and have considerable clinical and public health implications with respect to the degree of neurotropic threat posed to human health.

Perturbation of the two-component signal transduction system, BprRS, results in attenuated virulence and motility defects in Burkholderia pseudomallei.

BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis, a severe invasive disease of humans and animals. Initial screening of a B. pseudomallei signature-tagged mutagenesis library identified an attenuated mutant with a transposon insertion in a gene encoding the sensor component of an uncharacterised two-component signal transduction system (TCSTS), which we designated BprRS. RESULTS: Single gene inactivation of either the response regulator gene (bprR) or the sensor histidine kinase gene (bprS) resulted in mutants with reduced swarming motility and reduced virulence in mice. However, a bprRS double mutant was not attenuated for virulence and displayed wild-type levels of motility. The transcriptomes of the bprS, bprR and bprRS mutants were compared with the transcriptome of the parent strain K96243. Inactivation of the entire BprRS TCSTS (bprRS double mutant) resulted in altered expression of only nine genes, including both bprR and bprS, five phage-related genes and bpss0686, encoding a putative 5, 10-methylene tetrahydromethanopterin reductase involved in one carbon metabolism. In contrast, the transcriptomes of each of the bprR and bprS single gene mutants revealed more than 70 differentially expressed genes common to both mutants, including regulatory genes and those required for flagella assembly and for the biosynthesis of the cytotoxic polyketide, malleilactone. CONCLUSIONS: Inactivation of the entire BprRS TCSTS did not alter virulence or motility and very few genes were differentially expressed indicating that the definitive BprRS regulon is relatively small. However, loss of a single component, either the sensor histidine kinase BprS or its cognate response regulator BprR, resulted in significant transcriptomic and phenotypic differences from the wild-type strain. We hypothesize that the dramatically altered phenotypes of these single mutants are the result of cross-regulation with one or more other TCSTSs and concomitant dysregulation of other key regulatory genes.

Immunological mechanisms contributing to the double burden of diabetes and intracellular bacterial infections.

Diabetes has been recognized as an important risk factor for a variety of intracellular bacterial infections, but research into the dysregulated immune mechanisms contributing to the impaired host-pathogen interactions is in its infancy. Diabetes is characterized by a chronic state of low-grade inflammation due to activation of pro-inflammatory mediators and increased formation of advanced glycation end products. Increased oxidative stress also exacerbates the chronic inflammatory processes observed in diabetes. The reduced phagocytic and antibacterial activity of neutrophils and macrophages provides an intracellular niche for the pathogen to replicate. Phagocytic and antibacterial dysfunction may be mediated directly through altered glucose metabolism and oxidative stress. Furthermore, impaired activation of natural killer cells contributes to decreased levels of interferon-γ, required for promoting macrophage antibacterial mechanisms. Together with impaired dendritic cell function, this impedes timely activation of adaptive immune responses. Increased intracellular oxidation of antigen-presenting cells in individuals with diabetes alters the cytokine profile generated and the subsequent balance of T-cell immunity. The establishment of acute intracellular bacterial infections in the diabetic host is associated with impaired T-cell-mediated immune responses. Concomitant to the greater intracellular bacterial burden and potential cumulative effect of chronic inflammatory processes, late hyper-inflammatory cytokine responses are often observed in individuals with diabetes, contributing to systemic pathology. The convergence of intracellular bacterial infections and diabetes poses new challenges for immunologists, providing the impetus for multidisciplinary research.

Neurotropic threat characterization of Burkholderia pseudomallei strains.

The death rate for neurologic melioidosis is high. Whether certain Burkholderia pseudomallei strains are more likely than other strains to cause central nervous system infection and whether route of infection influences the neurotropic threat remain unclear. Therefore, we compared the virulence and dissemination of Australian clinical isolates collected during October 1989-October 2012 from patients with neurologic and nonneurologic melioidosis after intranasal and subcutaneous infection of mice in an experimental model. We did not observe neurotropism as a unique characteristic of isolates from patients with neurologic melioidosis. Rather, a distinct subset of B. pseudomallei strains appear to have heightened pathogenic potential for rapid dissemination to multiple tissues, including the central nervous system, irrespective of the infection route. This finding has valuable public health ramifications for initiating appropriate and timely therapy after exposure to systemically invasive B. pseudomallei strains. Increasing understanding of B. pseudomallei pathology and its influencing factors will further reduce illness and death from this disease.

Impaired early cytokine responses at the site of infection in a murine model of type 2 diabetes and melioidosis comorbidity.

Bacterial infections are a common and serious complication of type 2 diabetes (T2D). The prevalence of melioidosis, an emerging tropical infection caused by the Gram-negative bacterium Burkholderia pseudomallei, is increased in people with T2D. This is the first study to compare murine models of T2D and melioidosis. Susceptibility and disease progression following infection with B. pseudomallei were compared in our diet-induced polygenic mouse model and a leptin receptor-deficient monogenic model of T2D. The metabolic profile of mice with diet-induced diabetes, including body weight, blood glucose, cholesterol, triglycerides, insulin resistance, and baseline levels of inflammation, closely resembled that of clinical T2D. Following subcutaneous infection with B. pseudomallei, bacterial loads at 24 and 72 h postinfection in the blood, spleen, liver, lungs, and subcutaneous adipose tissue (SAT) at the site of infection were compared in parallel with the expression of inflammatory cytokines and tissue histology. As early as 24 h postinfection, the expression of inflammatory (interleukin-1ß [IL-1ß], tumor necrosis factor alpha [TNF-α], and IL-6) and T(H)1 (IL-12 and gamma interferon [IFN-γ]) cytokines was impaired in diabetic mice compared to nondiabetic littermates. Early differences in cytokine expression were associated with excessive infiltration of polymorphonuclear neutrophils (PMN) in diabetic mice compared to nondiabetic littermates. This was accompanied by bacteremia, hematogenous dissemination of bacteria to the lungs, and uncontrolled bacterial growth in the spleens of diabetic mice by 72 h postinfection. The findings from our novel model of T2D and melioidosis comorbidity support the role of impaired early immune pathways in the increased susceptibility of individuals with T2D to bacterial infections.

Design and Development of an Internationally Applicable Educational Video to Increase Community Awareness in Regions with High Prevalence of Melioidosis and Diabetes.

Melioidosis is a neglected tropical disease that causes high morbidity and mortality. Public health awareness is essential for both prevention and early detection of the infection. This project aimed to develop an internationally applicable educational tool to increase community awareness in regions with high prevalence of diabetes and melioidosis. The animation was created with international collaboration. Sixty-four delegates from different cultural backgrounds participated in the survey to evaluate the animation. Feedback was positive, with 85% agreeing that they would use this video for public education and 82% agreeing that the video was culturally appropriate to them in the context of their region. The animation was refined after feedback. To supplement the 3-minute animation, a 13-minute film footage of interviews with clinicians, researchers and patients was also created. These materials have been made available online through the International Melioidosis Network and can be readily downloaded or subtitled in any language using publicly available software, demonstrating the utility of developing low-cost adaptable health education material targeted for widespread use internationally.

Q fever - immune responses and novel vaccine strategies.

Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii. It is an occupational risk for employees of animal industries and is associated with contact with wildlife and domestic animals. Although Q fever infection may be asymptomatic, chronic sequelae such as endocarditis occur in 5% of symptomatic individuals. Disease outcomes may be predicted through measurement of immune correlates. Vaccination is the most efficient method to prevent Q fever. Currently, Q-VAX is the only licenced human vaccine. Q-VAX is highly effective; however, individuals previously exposed to C. burnetii are at risk of adverse reactions. This review examines the immunological responses of acute and chronic Q fever and the efforts to provide a safer and cost-effective Q fever vaccine.

Q fever is a disease that is spread by some animals, such as sheep and cattle, to humans. Although most people will recover if they get Q fever, some become very ill. There is a vaccine for Q fever (Q-VAX), but it can cause a reaction when given to some people. Research is ongoing into how the human immune system reacts to the bacteria that causes Q fever. A small number of people who get Q fever will develop a prolonged disease that can be serious and affect the heart, which is why there is also research into developing new vaccines for this disease. This research will look at those parts of the germ that causes Q fever that can be used for a new vaccine.

Burkholderia pseudomallei triggers altered inflammatory profiles in a whole-blood model of type 2 diabetes-melioidosis comorbidity.

Melioidosis is a potentially fatal disease caused by the bacterium Burkholderia pseudomallei. Type 2 diabetes (T2D) is the most common comorbidity associated with melioidosis. B. pseudomallei isolates from melioidosis patients with T2D are less virulent in animal models than those from patients with melioidosis and no identifiable risk factors. We developed an ex vivo whole-blood assay as a tool for comparison of early inflammatory profiles generated by T2D and nondiabetic (ND) individuals in response to a B. pseudomallei strain of low virulence. Peripheral blood from individuals with T2D, with either poorly controlled glycemia (PC-T2D [n = 6]) or well-controlled glycemia (WC-T2D [n = 8]), and healthy ND (n = 13) individuals was stimulated with B. pseudomallei. Oxidative burst, myeloperoxidase (MPO) release, expression of pathogen recognition receptors (TLR2, TLR4, and CD14), and activation markers (CD11b and HLA-DR) were measured on polymorphonuclear (PMN) leukocytes and monocytes. Concentrations of plasma inflammatory cytokine (interleukin-6 [IL-6], IL-12p70, tumor necrosis factor alpha [TNF-α], monocyte chemoattractant protein 1 [MCP-1], IL-8, IL-1ß, and IL-10) were also determined. Following stimulation, oxidative burst and MPO levels were significantly elevated in blood from PC-T2D subjects compared to controls. Differences were also observed in expression of Toll-like receptor 2 (TLR2), CD14, and CD11b on phagocytes from T2D and ND individuals. Levels of IL-12p70, MCP-1, and IL-8 were significantly elevated in blood from PC-T2D subjects compared to ND individuals. Notably, differential inflammatory responses of PC-T2D, WC-T2D, and ND individuals to B. pseudomallei occur independently of bacterial load and confirm the efficacy of this model of T2D-melioidosis comorbidity as a tool for investigation of dysregulated PMN and monocyte responses to B. pseudomallei underlying susceptibility of T2D individuals to melioidosis.

Identification of defective early immune responses to Burkholderia pseudomallei infection in a diet-induced murine model of type 2 diabetes.

Co-occurrence of bacterial infections with type 2 diabetes (T2D) is a global problem. Melioidosis caused by Burkholderia pseudomallei is 10 times more likely to occur in patients with T2D, than in normoglycemic individuals. Using an experimental model of T2D, we observed that greater susceptibility in T2D was due to differences in proportions of infiltrating leucocytes and reduced levels of MCP-1, IFN-γ and IL-12 at sites of infection within 24 h post-infection. However, by 72 h the levels of inflammatory cytokines and bacteria were markedly higher in visceral tissue and blood in T2D mice. In T2D, dysregulated early immune responses are responsible for the greater predisposition to B. pseudomallei infection.

A murine model of tuberculosis/type 2 diabetes comorbidity for investigating the microbiome, metabolome and associated immune parameters.

Tuberculosis (TB) is one of the deadliest infectious diseases in the world. The metabolic disease type 2 diabetes (T2D) significantly increases the risk of developing active TB. Effective new TB vaccine candidates and novel therapeutic interventions are required to meet the challenges of global TB eradication. Recent evidence suggests that the microbiota plays a significant role in how the host responds to infection, injury and neoplastic changes. Animal models that closely reflect human physiology are crucial in assessing new treatments and to decipher the underlying immunological defects responsible for increased TB susceptibility in comorbid patients. In this study, using a diet-induced murine T2D model that reflects the etiopathogenesis of clinical T2D and increased TB susceptibility, we investigated how the intestinal microbiota may impact the development of T2D, and how the gut microbial composition changes following a very low-dose aerosol infection with Mycobacterium tuberculosis (Mtb). Our data revealed a substantial intestinal microbiota dysbiosis in T2D mice compared to non-diabetic animals. The observed differences were comparable to previous clinical reports in TB patients, in which it was shown that Mtb infection causes rapid loss of microbial diversity. Furthermore, diversity index and principle component analyses demonstrated distinct clustering of Mtb-infected non-diabetic mice vs. Mtb-infected T2D mice. Our findings support a broad applicability of T2D mice as a tractable small animal model for studying distinct immune parameters, microbiota and the immune-metabolome of TB/T2D comorbidity. This model may also enable answers to be found to critical outstanding questions about targeted interventions of the gut microbiota and the gut-lung axis.

Increased susceptibility to Mycobacterium tuberculosis infection in a diet-induced murine model of type 2 diabetes.

Tuberculosis (TB)-type 2 diabetes mellitus (T2D) comorbidity is re-emerging as a global public health problem. T2D is a major risk factor for increased susceptibility to TB infection and reactivation leading to higher morbidity and mortality. The pathophysiological mechanisms of T2D contributing to TB susceptibility are not fully understood, but likely involve dysregulated immune responses. In this study, a diet-induced murine model that reflects the cardinal features of human T2D was used to assess the immune responses following an intravenous Mycobacterium tuberculosis (Mtb) infection. In this study, T2D significantly increased mortality, organ bacillary burden and inflammatory lesions compared to non-diabetic controls. Organ-specific pro-inflammatory cytokine responses were dysregulated as early as one day post-infection in T2D mice. Macrophages derived from T2D mice showed reduced bacterial internalization and killing capacity. An early impairment of antimycobacterial functions of macrophages in diabetes is a key mechanism that leads to increased susceptibility of T2D.

Anti-streptococcal antibody and T-cell interactions with vascular endothelial cells initiate the development of rheumatic carditis.

The role of group A streptococcal and Streptococcus dysgalactiae subspecies equisimilis M-protein specific Abs and T-cells in endothelial cell activation was investigated using cultured rat aortic endothelial cells, and in a rat model of autoimmune valvulitis. Heat inactivated serum and mononuclear cells from streptococcal M-protein immunized rats independently induced upregulation of the endothelial cell adhesion molecules, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 in cultured cells. We also observed T-cell migration across endothelial cell monolayers incubated with serum from M-protein-immunized rats. Furthermore, we observed VCAM-1 and ICAM-1 expression in the myocardium of rats injected with M-protein compared to control animals. These observations support the contention that initial interactions between streptococcal M-protein specific Abs and/or T-cells with the heart endothelium lead to endothelial cell activation followed by transmigration of M-protein specific T-cells into heart tissue leading to an inflammatory process that leads to carditis in rheumatic fever and rheumatic heart disease.

B- and T-cell responses in group a streptococcus M-protein- or Peptide-induced experimental carditis.

The etiology of rheumatic fever and rheumatic heart disease (RF/RHD) is believed to be autoimmune, involving immune responses initiated between streptococcal and host tissue proteins through a molecular mimicry mechanism(s). We sought to investigate the humoral and cellular responses elicited in a Lewis rat model of group A streptococcus M-protein- or peptide-induced experimental valvulitis/carditis, a recently developed animal model which may, in part, represent human rheumatic carditis. Recombinant streptococcal M5 protein elicited opsonic antibodies in Lewis rats, and anti-M5 antisera recognized epitopes within the B- and C-repeat regions of M5. One peptide from the streptococcal M5 protein B-repeat region (M5-B.6, amino acids 161 to 180) induced lymphocytes that responded to both recombinant M5 and cardiac myosin. Rats immunized with streptococcal M5 protein developed valvular lesions, distinguished by infiltration of CD3(+), CD4(+), and CD68(+) cells into valve tissue, consistent with human studies that suggest that RF/RHD are mediated by inflammatory CD4(+) T cells and CD68(+) macrophages. The current study provides additional information that supports the use of the rat autoimmune valvulitis model for investigating RF/RHD.

Comparison of routine bench and molecular diagnostic methods in identification of Burkholderia pseudomallei.

This study compared the identification of Burkholderia pseudomallei with that of related organisms. Bench tests and latex agglutination were compared with molecular identification. Using bench tests and latex agglutination alone, 100% (30/30) of B. pseudomallei isolates were correctly identified. Amoxicillin-clavulanate susceptibility testing was also a good and simple discriminatory test.

Survey of local fauna from endemic areas of Northern Queensland, Australia for the presence of Mycobacterium ulcerans.

BACKGROUND: Buruli ulcer (BU), regionally known as the Daintree ulcer or Bairnsdale ulcer is caused by the environmental pathogen Mycobacterium ulcerans (MU). This disease is characterized by extensive and painless necrosis of skin and soft tissue with the formation of large ulcers and has been reported in >33 countries worldwide. This organism is geographically restricted and in Australia, the disease has been reported primarily in coastal Victoria and the Mossman-Daintree areas of northern Queensland. Australia is the only country where nonhuman cases of BU have been confirmed. The common ringtail possums and mountain brushtail possums have been suggested as potential animal reservoirs of MU in coastal Victoria, Australia. The exact mode of transmission of this disease remains unknown. METHODS: In this study, we surveyed local fauna from endemic areas of northern Queensland, Australia, for the presence of MU in scat samples. We collected 140 bandicoot, four white-tailed rats, and two possum scat samples from 56 overnight trapping sessions. Samples were examined for the presence of MU DNA by the polymerase chain reaction. RESULTS: Two out of five samples did not contain a sufficient amount of DNA to detect IS2606 and the ketoreductase B (KR) domain of the mycolactone polyketide synthase gene, which is represented by higher cycle threshold (Ct) values for IS2404 shown in table below. Despite of having desired Ct values for IS2404, one IS2404 positive sample possibly contained DNA of closely related M. ulcerans subspecies with lower copy number of IS2606 that do not commonly cause disease in human. All three targets: IS2404, IS2606 and KR were detected from the remaining two scat samples. CONCLUSION: We confirm the presence of M. ulcerans DNA in the scat samples collected from a Buruli ulcer endemic region of Northern Queensland, Australia.

A survey on Mycobacterium ulcerans in Mosquitoes and March flies captured from endemic areas of Northern Queensland, Australia.

Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU). This nontuberculous mycobacterial infection has been reported in 34 countries worldwide. In Australia, the majority of cases of BU have been recorded in coastal Victoria and the Mossman-Daintree areas of north Queensland. Mosquitoes have been postulated as a vector of M. ulcerans in Victoria, however the specific mode of transmission of this disease is still far from being well understood. In the current study, we trapped and analysed 16,900 (allocated to 845 pools) mosquitoes and 296 March flies from the endemic areas of north Queensland to examine for the presence of M. ulcerans DNA by polymerase chain reaction. Seven of 845 pools of mosquitoes were positive on screening using the IS2404 PCR target (maximum likelihood estimate 0.4/1,000). M. ulcerans DNA was detected from one pool of mosquitoes from which all three PCR targets: IS2404, IS2606 and the ketoreductase B domain of mycolactone polyketide synthase gene were detected. None of the March fly samples were positive for the presence of M. ulcerans DNA.