Browsing by Subject "Mycobacteria"

Now showing 1 - 5 of 5
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    Isolation and characterization of novel mycobacteria species from Sarracenia purpurea
    (2015-03) Tran, Minh Phuong Thi
    Several fast to intermediate growing acid-fast, scrotochromogenic bacteria were isolated from Sarracenia purpurea pitcher waters in Minnesota sphagnum peat bogs. The isolates were identified by a polyphasic approach using morphological, biochemical, and bioinformatic techniques. Phylogenetic studies indicate the strains are located in the Mycobacterium genus with no obvious relation to any characterized type strains of mycobacteria. Two distinct clades emerged among the pitcher plant isolates, therefore two type strains (DL734 and DL739) were designated to represent the two clades. Phenotypic characterization revealed neither strain is similar to known genus type strains in collective properties of growth, pigmentation, or enzyme synthesis. Fatty acid methyl ester (FAME) profiles were unique for each type strain. The two isolates showed the unique ability to survive temperatures as low as 8°C. DL734 appeared to enter a viable but non-culturable state while DL739 appeared to be psychrotolerant. This could indicate that mycobacteria are more plastic in their ability to withstand different abiotic factors than previously recognized. The names Mycobacterium purpureae sp. nov. and Mycobacterium helvus sp. nov. are proposed for type strains DL734 (=JSM 30395 =NCCB 100519T) and DL739 (=JSM 30396=NCCB 100520T), respectively.
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    Strain dependent variations in iron metabolism of Mycobacterium avium subsp. paratuberculosis
    (2010-06) Janagama, Harish Kumar
    Johne’s disease is a major animal health problem of ruminant species worldwide and imposes significant economic losses to the industry. Our ability to culture the causative agent--Mycobacterium avium subsp. paratuberculosis (MAP)--and therefore its rapid diagnosis and our understanding of its virulence is limited. MAP is difficult to culture because of its unusually strict iron requirements. For optimal growth in laboratory media, MAP requires a siderophore (mycobactin) supplementation that makes MAP fastidious, often requiring eight to sixteen weeks to produce colonies in culture – a major hurdle in timely diagnosis and therefore implementation of optimal control measures. Understanding iron regulatory networks in the pathogen in vitro is therefore of great importance. Several microbiological and genotyping studies and clinical observations suggest that Johne’s in certain hosts such as sheep, goats, deer, and bison is caused by a distinct set of strains that show a relatively high degree of host preference. At least two microbiologically distinct types of MAP have been recognized. A less readily cultivable type is the common, but not invariable, cause of paratuberculosis in sheep (type I), while another readily cultivable type is the most common cause of the disease in cattle (type II). In addition, since the MAP genome sequence was published in 2005, very little research has focused on iron physiology and its contribution to metabolic networks of this fastidious organism. Based on these observations, I hypothesize that iron dependent gene regulation is different between type I and type II MAP strains. Iron dependent Regulator (IdeR), a transcription factor, is an essential gene in MAP and differentially controls the expression of genes involved in iron physiology in the two strain types of MAP. We identified polymorphisms in the IdeR open reading frame (ORF) and the promoters of putative IdeR regulated genes between the type I and type II strains of MAP. Structure-function association studies revealed repression of an iron storage gene, bfrA in the presence of iron by type I MAP strain alone. In contrast, bfrA was upregulated in the presence of iron in type II MAP strain. This leads us to propose that type I MAP strains may experience iron toxicity when excess iron is provided in the medium. The rationale is that excess free iron is detrimental to the cells and must be stored in bacterioferritins, a feature that type I strains lack. Transcriptional and proteomic profiling of these MAP strains under iron-replete or –deplete conditions revealed that iron-sparing response to iron limitation was unique to the type II strain as evidenced by repression of non-essential iron utilization enzymes (aconitase and succinate dehydrogenase) and upregulation of proteins of essential function (iron transport, [Fe-S] cluster biogenesis and cell division). Under iron-replete conditions, type II MAP alone increased expression of BfrA (bacterioferritin) and MhuD (mycobacterial heme utilization, degrader) protein, which is intricately involved in iron recycling. These findings further supported the contention that type I MAP strains are metabolically inept under iron-replete conditions. The intracellular lifestyle of MAP in the intestines and lymph nodes of natural infection revealed that MAP deployed genes involved in maintaining iron homeostasis under iron stress in the tissues of infected animals. There was a clear dichotomy in in vitro infected macrophages and natural infection in the expression profiles of both iron acquisition genes and other virulence factors involved in MAP survival inside the host. In summary, our studies revealed that IdeR of type II strain regulates mycobactin synthesis and iron storage genes, similar to the function of IdeR in M. tuberculosis (MTB), while the type I strain is deficient in iron storage function. Given our inability to delete ideR, it appears that this is an essential gene (as in MTB) for MAP survival. MAP IdeR regulon studies led us to define a novel operon carrying genes encoding a potential secretory apparatus (ESX-3/type VII secretory system). Functional analysis of the iron-induced proteome also identified novel ESAT-6 (early secreted antigenic target) family of proteins belonging to ESX-5, which have been identified as major virulence factors in MTB. We also established that, type I MAP strains are more sensitive to fluctuations of environmental iron due to defective regulation of bfrA and may grow better under lower iron levels in the culture media. Taken together, our studies suggest that MAP employs a sophisticated repertoire of proteins that are inter-connected and function in response to environmental stress.
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    Survival strategies of Mycobacterium avium subsp. paratuberculosis in a variety of microenvironments.
    (2012-06) Lamont, Elise A.
    Mycobacteria, specifically Mycobacterium avium subsp. paratuberculosis (MAP), are extreme strategists and as a rule live by deception. Mycobacteria represent a group of closely related acid-fast bacilli that encompass a wide-range of host tropisms and diseases. Mycobacteria can be divided into two complexes: the Mycobacterium tuberculosis complex and the Mycobacterium avium complex (MAC). The MAC is comprised of M. avium subsp. avium (M. avium), MAP, M. intracellulare and M. avium subsp. hominissuis (M. hominissuis), all of which share an over 90 percent nucleotide similarity. Despite its genetic similarity, MAC elicits different diseases in both animals and humans including infections of the lung, lymph nodes, bones, skin and gastrointestinal tract. MAP is a unique member of MAC as it infects and establishes itself within the intestine of ruminants and other wildlife. Furthermore, MAP lives in a quiescent state in soil and aquatic environments. Since MAP encounters numerous environments, including those with unfavorable conditions, it has developed several strategies to survive. However, the mechanisms by which MAP survival is achieved remains incompletely understood. The goal of these studies was to determine how MAP may survive and disseminate under unfavorable conditions, which included nutrient starvation and host pressures. We have identified the development of a new MAP morphotype under prolonged nutrient starved conditions. This novel MAP morphotype resembles a spore-like structure and contains dipicolinic acid, which is used to protect DNA located within the core. These novel structures are heat resistant at 70oC and can be enriched for in multiple MAP strains. Furthermore, we describe an unrecognized mechanism by which MAP takes advantage of host responses at the epithelium interface to recruit macrophages to the site of initial infection. MAP is able to safely enter into macrophages and consequently ensures its establishment, survival and dissemination throughout the host. Lastly, we demonstrate the importance of host physiological relevant temperature on successful disease progression. Infection utilizing the temperature of MAP’s natural host, the cow, enhances the speed of infection as well as host and pathogen transcriptomic profiles. Taken together, data generated from these studies will provide the basis for understanding MAP persistence and survival in diverse conditions. The mechanisms by which MAP establishes, disseminates and/or survives difficult conditions may impact new programs to control JD as well as rational vaccine/therapeutic design and the way in which we view other mycobacterioses.
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    Targeting Mycobacterium tuberculosis intrinsic resistance mechanisms to potentiate antitubercular drug action
    (2019-06) Thiede, Joshua
    The work presented herein has focused on understanding the mycobacterial metabolic responses that contribute to intrinsic resistance to the antitubercular drugs p-aminosalicylic acid and pyrazinamide. The interrogation of these responses has led to improved understanding of the underlying mechanisms conferring intrinsic resistance to each drug. Leveraging our observations, we were able to improve the antitubercular action of p-aminosalicylic acid by several orders of magnitude as well as circumvent a known mechanism of resistance. Further, we were able to decipher the long observed, but unknown mechanism responsible for conditional pyrazinamide susceptibility. Finally, we developed a method to drive pyrazinamide susceptibility in M. tuberculosis through co-treatment with peptidoglycan targeting drugs. These studies deepen our understanding of how M. tuberculosis withstands drug treatment and offers novel strategies to improve our ability to combat the disease.
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    Targeting Two Late-Stage Enzymes of the Mycobacterium tuberculosis Biotin Biosynthetic Pathway
    (2018-09) Bockman, Matthew
    Mycobacterium tuberculosis (Mtb), responsible for both latent and symptomatic tuberculosis (TB), remains the leading cause of mortality among infectious diseases worldwide. The rise and propagation of drug-resistant TB remains a global health crisis and has prompted researchers to investigate novel mechanisms of action for the development of antitubercular agents. Chapter 1 discusses the biotin biosynthetic pathway as a target for the development of antibiotics targeting Mtb, providing both chemical and genetic validation evidence of inhibiting this pathway in Mtb infections. This chapter thoroughly examines each enzyme in the biotin biosynthetic pathway by reviewing: the reaction it catalyzes, its mechanism of action, structural and sequence analysis, and catalogue of inhibitors known for each enzyme. The late-stage biotin synthase (BioB) and biotin protein ligase (BPL) proteins are elaborated on and will be the focus of this thesis. Mycobacterial biotin protein ligase (MtBPL) is an essential enzyme in Mtb and regulates lipid metabolism through the post-translational biotinylation of acyl coenzyme A carboxylases. Chapter 2 reports the synthesis and evaluation of a systematic series of potent nucleoside-based bisubstrate inhibitors of MtBPL that contain modifications to the ribofuranosyl ring of the nucleoside. All compounds were characterized by isothermal titration calorimetry (ITC) and shown to bind potently with KDs ≤ 2 nM. Additionally, this chapter discusses the structural interactions between the inhibitors and MtBPL using the highly-resolved x-ray co-crystal structures. Despite relatively uniform biochemical potency, the whole-cell Mtb activity varied greatly with minimum inhibitory concentrations (MICs) ranging from 0.78 to >100 uM. Cellular accumulation studies showed a nearly ten-fold enhancement in accumulation of a C-2′-a-fluoro analogue over the corresponding C-2′-b-fluoro analogue, consistent with their differential whole-cell activity. The parent compound, Bio-AMS, was also evaluated for its pharmacokinetic (PK) parameters, and although it shows stability toward plasma and liver microsomes, Bio-AMS is rapidly cleared form CD-1 mice. From chapter 2, the potent compound Bio-AMS was shown to possess selective activity against MtBPL. However, Mtb develops spontaneous resistance to Bio-AMS with a frequency of resistance (FOR) of at least 1 x 10-7 by overexpression of Rv3406, a type II sulfatase that enzymatically inactivates Bio-AMS. In an effort to circumvent this resistance mechanism, chapter 3 describes the strategic modification of the Bio-AMS at the 5’-position to prevent enzymatic inactivation. The new analogues retain subnanomolar potency to MtBPL, and the 5′R-C-methyl derivative exhibited identical antimycobacterial activity toward: Mtb H37Rv, MtBPL overexpression, and an isogenic Rv3406 overexpression strain (MIC = 1.56 uM). Moreover, this compound was not metabolized by recombinant Rv3406 and resistant mutants to this compound could not be isolated (FOR < 1.4 x 10-10) demonstrating it successfully overcame Rv3406-mediated resistance. The natural product acidomycin, discovered in 1952 and isolated from Streptomyces spp., was originally shown to have selective antibiotic activity against Mtb grown in the absence of biotin, implying it is an antimetabolite of the biotin biosynthetic pathway. Chapter 4 fully investigates the mechanism of action and selectivity of acidomycin. Acidomycin was evaluated against an array of drug susceptible and drug resistant Mtb strains, as well as a panel of gram-positive and gram-negative pathogens, and showed remarkable selectivity to Mtb with MICs ranging from 0.096 – 6.2 uM for the Mtb strains and >100 M for the other microorganisms. Acidomycin was also shown to be a reversible, competitive inhibitor of E. coli biotin synthase (EcBioB), with a Ki of 1.5 uM, and a homology model shows substantial sequence alignment in the mycobacterial enzyme (MtBioB). The selectivity of acidomycin against E. coli versus Mtb is due to differential levels of cellular accumulation, with a 30-fold increase in the amount of acidomycin accumulated in Mtb over E. coli. In vivo, acidomycin was shown to be rapidly eliminated from CD-1 mice, is a half-life of 9.6 min, but exhibited remarkable plasma and microsomal stability. A brief series of acidomycin analogues showed a very tight SAR window for modifications, with the primary amide analogue being the best analogue with an MIC less than two-fold of acidomycin.