In this study, a Burkholderia mallei tonB mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection ...models of murine glanders and melioidosis.
Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 10(4) CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001.
Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis.
Prophylactic administration of CpG oligodeoxynucleotides (CpG ODNs) is known to confer protection against lethal sepsis caused by Burkholderia pseudomallei in the mouse model. The mechanisms whereby ...CpG regulates the innate immune response to provide protection against B. pseudomallei, however, are poorly characterized. In the present study, we demonstrate that intranasal treatment of mice with Class C CpG, results in recruitment of inflammatory monocytes and neutrophils to the lung at 48 h post-treatment. Mice infected with B. pseudomallei 48 h post-CpG treatment had reduced organ bacterial load and significantly altered cytokine and chemokine profiles concomitant with protection as compared to control animals. CpG administration reduced the robust production of chemokines and pro-inflammatory cytokines in blood, lung and spleen, observed following infection of non-treated animals. Death of control animals coincided with the time of peak cytokine production (day 1-3), while a moderate; sustained cytokine production in CpG-treated animals was associated with survival. In general, CpG treatment resulted in diminished expression of cytokines and chemokines post-infection, though IL-12p40 was released in larger quantities in CpG treated animals. In contrast to CpG-treated animals, the lungs of infected control animals were infiltrated with leukocytes, especially neutrophils, and large numbers of necrotic lesions were observed in lung sections. Therapeutic treatment of B. pseudomallei-infected animals with CpG at 24 h post-infection did not impact survival compared to control animals. In summary, protection of CpG-treated animals was associated with recruitment of inflammatory monocytes and neutrophils into the lungs prior to infection. These responses correspond with early control of bacterial growth, a dampened inflammatory cytokine/chemokine response, reduced lung pathology, and greatly increased survival. In contrast, a delay in recruitment of inflammatory cell populations, despite a robust production of pro-inflammatory cytokines, was associated with poorly controlled bacterial growth, severe lung pathology, and death of control animals.
, the etiologic agent of glanders, are Category B select agents with biothreat potential, and yet effective therapeutic treatments are lacking. In this study, we showed that CpG administration ...increased survival, demonstrating protection in the murine glanders model. Bacterial recovery from infected lungs, liver and spleen was significantly reduced in CpG-treated animals as compared with non-treated mice. Reciprocally, lungs of CpG-treated infected animals were infiltrated with higher levels of neutrophils and inflammatory monocytes, as compared to control animals. Employing the
bioluminescent strain CSM001 and the Neutrophil-Specific Fluorescent Imaging Agent, bacterial dissemination and neutrophil trafficking were monitored in real-time using multimodal
whole body imaging techniques. CpG-treatment increased recruitment of neutrophils to the lungs and reduced bioluminescent bacteria, correlating with decreased bacterial burden and increased protection against acute murine glanders. Our results indicate that protection of CpG-treated animals was associated with recruitment of neutrophils prior to infection and demonstrated, for the first time, simultaneous real time
imaging of neutrophils and bacteria. This study provides experimental evidence supporting the importance of incorporating optimized
imaging methods to monitor disease progression and to evaluate the efficacy of therapeutic treatment during bacterial infections.
Co-infection with HIV increases the morbidity and mortality associated with tuberculosis due to multiple factors including a poorly understood microbial synergy. We developed a novel small animal ...model of co-infection in the humanized mouse to investigate how HIV infection disrupts pulmonary containment of Mtb. Following dual infection, HIV-infected cells were localized to sites of Mtb-driven inflammation and mycobacterial replication in the lung. Consistent with disease in human subjects, we observed increased mycobacterial burden, loss of granuloma structure, and increased progression of TB disease, due to HIV co-infection. Importantly, we observed an HIV-dependent pro-inflammatory cytokine signature (IL-1β, IL-6, TNFα, and IL-8), neutrophil accumulation, and greater lung pathology in the Mtb-co-infected lung. These results suggest that in the early stages of acute co-infection in the humanized mouse, infection with HIV exacerbates the pro-inflammatory response to pulmonary Mtb, leading to poorly formed granulomas, more severe lung pathology, and increased mycobacterial burden and dissemination.
Abstract
In HIV-infected patients with latent or subclinical tuberculosis (TB), the development of immune reconstitution inflammatory syndrome (IRIS) following restoration of T cell populations by ...anti-retroviral therapy (ART) can produce a life threatening inflammatory reaction. The objective of this study was to use our humanized mouse model of Mycobacterium tuberculosis (Mtb) and HIV co-infection to develop a small animal model of IRIS. Humanized BLT mice (HuMice) were infected i.n. with Mtb H37Rv, and following development of TB disease were given TB chemotherapy (rifampicin and isoniazid) by oral gavage. Bacteria were not detected in the lung following TB chemotherapy, but low numbers of Mtb were detected two months after stopping drug treatment, which mimics subclinical TB. When Mtb-infected and TB drug-treated HuMice were infected i.v. with HIV-1 Ada, the animals developed HIV-1 co-infection as determined by the presence of the HIV p24 capsid protein in peripheral blood by ELISA. ART was performed to reduce viral load in blood plasma and restore T lymphocyte function in animals with subclinical TB. In ongoing studies, the development of inflammatory responses, Th1 cell activation and lung pathology are being determined to understand how immune recovery, due to ART, drives inflammation in the setting of subclinical TB. This model will facilitate investigations to understand the mechanistic basis for IRIS and identify targets for host-directed therapies.
Melioidosis is an endemic disease caused by the bacterium Burkholderia pseudomallei. Concerns exist regarding B. pseudomallei use as a potential bio-threat agent causing persistent infections and ...typically manifesting as severe pneumonia capable of causing fatal bacteremia. Development of suitable therapeutics against melioidosis is complicated due to high degree of genetic and phenotypic variability among B. pseudomallei isolates and lack of data establishing commonly accepted strains for comparative studies. Further, the impact of strain variation on virulence, disease presentation, and mortality is not well understood. Therefore, this study evaluate and compare the virulence and disease progression of B. pseudomallei strains K96243 and HBPUB10303a, following aerosol challenge in a standardized BALB/c mouse model of infection. The natural history analysis of disease progression monitored conditions such as weight, body temperature, appearance, activity, bacteremia, organ and tissue colonization (pathological and histological analysis) and immunological responses. This study provides a detailed, direct comparison of infection with different B. pseudomallei strains and set up the basis for a standardized model useful to test different medical countermeasures against Burkholderia species. Further, this protocol serves as a guideline to standardize other bacterial aerosol models of infection or to define biomarkers of infectious processes caused by other intracellular pathogens.
Background In this study, a Burkholderia mallei tonB mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational ...infection models of murine glanders and melioidosis. Methodology/Principal Findings Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 104 CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001. Conclusions/Significance Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis.
Abstract
Co-infection with human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (Mtb) promotes aggressive disease through mechanisms that are poorly understood. A recent study suggests ...an association between peripheral blood neutrophil count and Mtb load in the sputum of human subjects with HIV co-infection. We used our humanized BLT mouse (HuMouse) model of HIV/Mtb co-infection as an investigative tool to study the role of neutrophils in co-infection pathobiology. Humanized mice were infected intravenously with HIV-1 for 3 weeks and then co-infected intranasally with Mtb. Compared to HuMice with Mtb or HIV mono-infections, increased neutrophil numbers were observed in the lungs of co-infected animals, as determined by detection of myeloperoxidase using immunohistochemistry. Consistent with the role of neutrophils to promote immune-mediated pathology in TB disease, greater tissue necrosis and increased mycobacterial burden was observed in the lungs of co-infected animals. In support of these findings, an increased pulmonary production of cytokines and chemokines associated with neutrophil recruitment including IL-8, CXCL5, fractalkine, and IL-17 were also observed in co-infected animals. These studies identify mechanisms for immune-mediated pathology due to HIV/Mtb co-infection that can be targeted to restore immune balance in the lung and complement antimycobacterials.
Tuberculosis (TB) has become a global health threat in the wake of the Human Immunodeficiency Virus (HIV) pandemic and is the leading cause of death in people with HIV/AIDS. Treatment of patients ...with Mycobacterium tuberculosis (Mtb)/HIV co-infection is complicated by drug interactions and toxicity that present huge challenges for clinical intervention. Discovery efforts to identify novel compounds with increased effectiveness and decreased drug-drug interactions against Mtb, HIV-1, or both, would be greatly aided by the use of a co-infection model for screening drug libraries. Currently, inhibitors of Mtb are screened independently in mycobacterial cell cultures or target based biochemical screens and less often in macrophages or peripheral blood leukocytes. Similarly, HIV-1 drugs are screened in vitro independently from anti-mycobacterial compounds. Here, we describe an in vitro model where primary human peripheral blood mononuclear cells or monocyte-derived macrophages are infected with Mycobacterium bovis BCG and HIV-1, and used to evaluate drug toxicity and activity in a co-infection setting. Our results with standard compounds (e.g. Azidothymidine, Rifampicin) demonstrate the utility of this in vitro model to evaluate drug effectiveness relevant to cellular toxicity, HIV-1 replication, and intracellular mycobacterial growth, through the use of ELISA, bacterial enumeration, and multi-variate flow cytometry. This model and associated assays have great value in accelerating the discovery of compounds for use in Mtb /HIV-1 co-infected patients.
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