80 research outputs found
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Further Understanding Mycobacterial Biofilms and Their Interaction with the Host
Respiratory infections caused by nontuberculous mycobacteria (NTM), especially Mycobacterium avium, can lead to progressive, recurrent disease that is refractory to therapy. Bacterial biofilms are intrinsically resistant to a variety of stressors and pressures, including host killing mechanisms and antibiotic therapy. Though it is becoming increasingly evident that NTM biofilms are important for infection, it is not currently known how these biofilms evade the immune response in an immunocompetent individual. Furthermore, what distinguishes biofilm-associated bacteria from planktonic bacteria is also mostly unknown. The main goals of this dissertation were to investigate the interaction between the host and the NTM biofilm, as well as to further identify and characterize the biofilm itself.
In Chapter 2, I developed an in vitro model to investigate the interaction between an established MAH biofilm and surveilling macrophages, mimicking a scenario of how these cells would encounter NTM in the airway. Using this model, I was able to assess multiple aspects of this interaction including bacterial killing, activation of macrophages, and apoptosis. My results show the biofilm eliciting a unique hyper-stimulation from the macrophages that results in attenuation of bacterial killing and early, atypical TNFα-driven apoptosis. Interestingly, UV-killed biofilms elicited similar responses from macrophages, suggesting that acellular biofilm matrix components could be contributing to this unique response.
In Chapter 3, I discovered that M. avium and other NTM can contain substantial amounts of extracellular DNA (eDNA) in their biofilm matrix and supernatant. Utilizing scanning electron microscopy and immuno-gold labelling, I was able to visualize the eDNA in these biofilms, yielding insight into the structural scaffolding-like properties the eDNA possesses. I determined that the eDNA was genomic in origin, and did not contain a specific exported sequence. By using DNA targeted enzymatic digestion with DNase I, I showed that eDNA is integral for biofilm establishment, persistence, and tolerance to clinically used antibiotics.
The work in Chapter 3 led me to hypothesize that eDNA is potentially secreted by active mechanisms, which contrasts the widely accepted idea that eDNA in bacterial biofilms results from cell autolysis. In Chapter 4, I conducted an in-depth analysis into the regulation and production of eDNA. I designed a model for fluorescently quantifying eDNA export in real time in undisturbed biofilms, which allowed investigation into triggers responsible for eDNA. When these results were combined with biofilm surface proteomics and a comprehensive transposon library screen for eDNA deficient mutants, my data suggests bicarbonate as a novel trigger for eDNA and identified many genes involved with the mechanism of eDNA export. Interesting components include an undescribed FtsK/SpoIIIE-like DNA exporting pore, multiple bicarbonate-interacting carbonic anhydrases, and a unique genomic region in MAH and other eDNA-producing NTM that could be implicated in biofilm formation and eDNA production.
Cumulatively, the work presented in this dissertation significantly advances the information known about NTM biofilms, and how they interact with and persist in the host respiratory tract. Current therapies can be ineffective at treating NTM respiratory infections, even after very long, multidrug treatment regimens. Since there is evidence to be implicated between NTM biofilms and respiratory infection, the more we can learn about the physiology of these biofilms, the more targets we can have to direct new anti-virulence therapies toward. Both bicarbonate sensing and eDNA production could become valuable anti-virulence targets for NTM infection
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Mycobacterium avium Biofilm Attenuates Mononuclear Phagocyte Function by Triggering Hyperstimulation and Apoptosis during Early Infection
Mycobacterium avium subsp. hominissuis is an opportunistic human pathogen that has been shown to form biofilm in vitro and
in vivo. Biofilm formation in vivo appears to be associated with infections in the respiratory tract of the host. The reasoning behind
how M. avium subsp. hominissuis biofilm is allowed to establish and persist without being cleared by the innate immune
system is currently unknown. To identify the mechanism responsible for this, we developed an in vitro model using THP-1 human
mononuclear phagocytes cocultured with established M. avium subsp. hominissuis biofilm and surveyed various aspects of
the interaction, including phagocyte stimulation and response, bacterial killing, and apoptosis. M. avium subsp. hominissuis
biofilm triggered robust tumor necrosis factor alpha (TNF-α) release from THP-1 cells as well as superoxide and nitric oxide
production. Surprisingly, the hyperstimulated phagocytes did not effectively eliminate the cells of the biofilm, even when pre-stimulated
with gamma interferon (IFN-γ) or TNF-α or cocultured with natural killer cells (which have been shown to induce
anti-M. avium subsp. hominissuis activity when added to THP-1 cells infected with planktonic M. avium subsp. hominissuis).
Time-lapse microscopy and the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay
determined that contact with the M. avium subsp. hominissuis biofilm led to early, widespread onset of apoptosis, which is not
seen until much later in planktonic M. avium subsp. hominissuis infection. Blocking TNF-α or TNF-R1 during interaction with
the biofilm significantly reduced THP-1 apoptosis but did not lead to elimination of M. avium subsp. hominissuis. Our data collectively
indicate that M. avium subsp. hominissuis biofilm induces TNF-α-driven hyperstimulation and apoptosis of surveilling
phagocytes, which prevents clearance of the biofilm by cells of the innate immune system and allows the biofilm-associated infection
to persist
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Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics
Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA) has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH). In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain) and MAH 104 (reference strain) were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections
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Delivery of Aerosolized Liposomal Amikacin as a Novel Approach for the Treatment of Nontuberculous Mycobacteria in an Experimental Model of Pulmonary Infection
Pulmonary infections caused by nontuberculous mycobacteria (NTM) are an increasing problem in individuals with chronic lung conditions and current therapies are lacking. We investigated the activity of liposomal amikacin for inhalation (LAI) against NTM in vitro as well as in a murine model of respiratory infection. Macrophage monolayers were infected with three strains of Mycobacterium avium, two strains of Mycobacterium abscessus, and exposed to LAI or free amikacin for 4 days before enumerating bacterial survival. Respiratory infection was established in mice by intranasal inoculation with M. avium and allowing three weeks for the infection to progress. Three different regimens of inhaled LAI were compared to inhaled saline and parenterally administered free amikacin over a 28 day period. Bacteria recovered from the mice were analyzed for acquired resistance to amikacin. In vitro, liposomal amikacin for inhalation was more effective than free amikacin in eliminating both intracellular M. avium and M. abscessus. In vivo, inhaled LAI demonstrated similar effectiveness to a ∼25% higher total dose of parenterally administered amikacin at reducing M. avium in the lungs when compared to inhaled saline. Additionally, there was no acquired resistance to amikacin observed after the treatment regimen. The data suggest that LAI has the potential to be an effective therapy against NTM respiratory infections in humans
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The Environment of “Mycobacterium avium subsp. hominissuis” Microaggregates Induces Synthesis of Small Proteins Associated with Efficient Infection of Respiratory Epithelial Cells
“Mycobacterium avium subsp. hominissuis” is an opportunistic environmental pathogen that causes respiratory illness in immunocompromised patients, such as those with cystic fibrosis as well as other chronic respiratory diseases. Currently, there is no efficient approach to prevent or treat M. avium subsp. hominissuis infection in the lungs. During initial colonization of the airways, M. avium subsp. hominissuis forms microaggregates composed of 3 to 20 bacteria on human respiratory epithelial cells, which provides an environment for phenotypic changes leading to efficient mucosal invasion in vitro and in vivo. DNA microarray analysis was employed to identify genes associated with the microaggregate phenotype. The gene encoding microaggregate-binding protein 1 (MBP-1) (MAV_3013) is highly expressed during microaggregate formation. When expressed in noninvasive Mycobacterium smegmatis, MBP-1 increased the ability of the bacteria to bind to HEp-2 epithelial cells. Using anti-MBP-1 immune serum, microaggregate binding to HEp-2 cells was significantly reduced. By far-Western blotting, and verified by coimmunoprecipitation, we observed that MBP-1 interacts with the host cytoskeletal protein vimentin. As visualized by confocal microscopy, microaggregates, as well as MBP-1, induced vimentin polymerization at the site of bacterium-host cell contact. Binding of microaggregates to HEp-2 cells was inhibited by treatment with an antivimentin antibody, suggesting that MBP-1 expression is important for M. avium subsp. hominissuis adherence to the host cell. MBP-1 immune serum significantly inhibited M. avium subsp. hominissuis infection throughout the respiratory tracts of mice. This study characterizes a pathogenic mechanism utilized by M. avium subsp. hominissuis to bind and invade the host respiratory epithelium, suggesting new potential targets for the development of antivirulence therapy
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Mycobacterium tuberculosis Alters the Metalloprotease Activity of the COP9 Signalosome
Inhibition of apoptotic death of macrophages by Mycobacterium tuberculosis represents an important mechanism of virulence that results in pathogen survival both in vitro and in vivo. To identify M. tuberculosis virulence determinants involved in the modulation of apoptosis, we previously screened a transposon bank of mutants in human macrophages, and an M. tuberculosis clone with a nonfunctional Rv3354 gene was identified as incompetent to suppress apoptosis. Here, we show that the Rv3354 gene encodes a protein kinase that is secreted within mononuclear phagocytic cells and is required for M. tuberculosis virulence. The Rv3354 effector targets the metalloprotease (JAMM) domain within subunit 5 of the COP9 signalosome (CSN5), resulting in suppression of apoptosis and in the destabilization of CSN function and regulatory cullin-RING ubiquitin E3 enzymatic activity. Our observation suggests that alteration of the metalloprotease activity of CSN by Rv3354 possibly prevents the ubiquitin-dependent proteolysis of M. tuberculosis-secreted proteins.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the American Society for Microbiology. The published article can be found at: http://mbio.asm.org/
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Mycobacterium avium subsp hominissuis effector MAVA5_06970 promotes rapid apoptosis in secondary-infected macrophages during cell-to-cell spread
Mycobacterium avium subsp, hominissuis is an opportunistic intracellular pathogen associated with disease in patients either immunosuppression or chronic lung pathology. Once in the host, M. avium preferentially infects and replicates within the phagocytic cells. The host driven macrophage apoptosis appears to be an essential aspect of innate immunity during bacterial infection; however, the existing evidence suggests that M. avium has evolved adaptive approaches to trigger the phagocyte apoptosis, exit apoptotic cells or via ingestion of infected apoptotic bodies subsequently infect neighboring macrophages. By evaluating 4,000 transposon mutants of M. avium in THP-1 cells, we identified clones that can trigger a new form of early host cell apoptosis, which is only observed upon entry into the "secondary-infected" macrophages. Inactivation of MAVA5_06970 gene lead to significant attenuation in intracellular growth within macrophages and mice, and impaired M. avium to induce rapid apoptosis in the "secondary-infected" cells as measured by Annexin V-FITC detection assay. Complementation of MAVA5_06970 gene corrected the attenuation as well as apoptotic phenotypes. The MAVA5_06970 gene encodes for a secreted protein. Using the pull-down assay and then confirmed with the yeast two-hybrid screen, we found that MAVA5_06970 effector interacts with the Secreted Phosphoprotein 1, the cytokine also known as Osteopontin. This interaction enhances the THP-1 cell apoptosis and, consequently, restricts the production of interleukin-12 that likely may limit the activation of the type I immunity pathway in vivo. This work identified a key virulence effector of M. avium that contributes to the cell-to-cell spread of the pathogen
Environmental Effects of Stratospheric Ozone Depletion, UV Radiation, and interactions with Climate Change: 2022 Assessment Report
The Montreal Protocol on Substances that Deplete the Ozone Layer was established 35 years ago following the 1985 Vienna Convention for protection of the environment and human health against excessive amounts of harmful ultraviolet-B (UV-B, 280-315 nm) radiation reaching the Earth’s surface due to a reduced UV-B-absorbing ozone layer. The Montreal Protocol, ratified globally by all 198 Parties (countries), controls ca 100 ozone-depleting substances (ODS). These substances have been used in many applications, such as in refrigerants, air conditioners, aerosol propellants, fumigants against pests, fire extinguishers, and foam materials.
The Montreal Protocol has phased out nearly 99% of ODS, including ODS with high global warming potentials such as chlorofluorocarbons (CFC), thus serving a dual purpose. However, some of the replacements for ODS also have high global warming potentials, for example, the hydrofluorocarbons (HFCs). Several of these replacements have been added to the substances controlled by the Montreal Protocol. The HFCs are now being phased down under the Kigali Amendment. As of December 2022, 145 countries have signed the Kigali Amendment, exemplifying key additional outcomes of the Montreal Protocol, namely, that of also curbing climate warming and stimulating innovations to increase energy efficiency of cooling equipment used industrially as well as domestically.
As the concentrations of ODS decline in the upper atmosphere, the stratospheric ozone layer is projected to recover to pre-1980 levels by the middle of the 21st century, assuming full compliance with the control measures of the Montreal Protocol. However, in the coming decades, the ozone layer will be increasingly influenced by emissions of greenhouse gases and ensuing global warming. These trends are highly likely to modify the amount of UV radiation reaching the Earth\u27s surface with implications for the effects on ecosystems and human health.
Against this background, four Panels of experts were established in 1988 to support and advise the Parties to the Montreal Protocol with up-to-date information to facilitate decisions for protecting the stratospheric ozone layer. In 1990 the four Panels were consolidated into three, the Scientific Assessment Panel, the Environmental Effects Assessment Panel, and the Technology and Economic Assessment Panel.
Every four years, each of the Panels provides their Quadrennial Assessments as well as a Synthesis Report that summarises the key findings of all the Panels. In the in-between years leading up to the quadrennial, the Panels continue to inform the Parties to the Montreal Protocol of new scientific information
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Evaluation of eight live attenuated vaccine candidates for protection against challenge with virulent Mycobacterium avium subspecies paratuberculosis in mice
Johne’s disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which results in serious economic losses worldwide in farmed livestock such as cattle, sheep, and goats. To control this disease, an effective vaccine with minimal adverse effects is needed. In order to identify a live vaccine for Johne’s disease, we evaluated eight attenuated mutant strains of MAP using a C57BL/6 mouse model. The persistence of the vaccine candidates was measured at 6, 12, and 18 weeks post vaccination. Only strains 320, 321, and 329 colonized both the liver and spleens up until the 12-week time point. The remaining five mutants showed no survival in those tissues, indicating their complete attenuation in the mouse model. The candidate vaccine strains demonstrated different levels of protection based on colonization of the challenge strain in liver and spleen tissues at 12 and 18 weeks post vaccination. Based on total MAP burden in both tissues at both time points, strain 315 (MAP1566::Tn5370) was the most protective whereas strain 318 (intergenic Tn5367 insertion between MAP0282c andMAP0283c) had the most colonization. Mice vaccinated with an undiluted commercial vaccine preparation displayed the highest bacterial burden as well as enlarged spleens indicative of a strong infection. Selected vaccine strains that showed promise in the mouse model were moved forward into a goat challenge model. The results suggest that the mouse trial, as conducted, may have a relatively poor predictive value for protection in a ruminant host such as goats.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the Frontiers Research Foundation. The published article can be found at: http://www.frontiersin.org/Cellular_and_Infection_Microbiology.Keywords: Johne’s disease, Mouse model, Attenuated, Mycobacterium, Vaccines, Genomic
Ozone depletion, ultraviolet radiation, climate change and prospects for a sustainable future
Changes in stratospheric ozone and climate over the past 40-plus years have altered the solar ultraviolet (UV) radiation conditions at the Earth's surface. Ozone depletion has also contributed to climate change across the Southern Hemisphere. These changes are interacting in complex ways to affect human health, food and water security, and ecosystem services. Many adverse effects of high UV exposure have been avoided thanks to the Montreal Protocol with its Amendments and Adjustments, which have effectively controlled the production and use of ozone-depleting substances. This international treaty has also played an important role in mitigating climate change. Climate change is modifying UV exposure and affecting how people and ecosystems respond to UV; these effects will become more pronounced in the future. The interactions between stratospheric ozone, climate and UV radiation will therefore shift over time; however, the Montreal Protocol will continue to have far-reaching benefits for human well-being and environmental sustainability.Peer reviewe
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