The adaptive response of mycobacterium avium to anoxia and nitric oxide

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John Edward Pearl

THE ADAPTIVE RESPONSE OF MYCOBACTERIUM AVIUM TO

ANOXIA AND NITRIC OXIDE

Dissertação de candidatura ao grau de

Doutor em Ciências Biomédicas submetida

ao Instituto de Ciências Biomédicas de

Abel Salazar (ICBAS) da Universidade do

Porto

Orientador- Doutor Rui Appelberg

Categoria: Professor Catedrático

Afiliação: ICBAS

Co-orientador- Doutor Keith Derbyshire

Categoria: Professor

Afiliação: Department of Biomedical

Sciences, University at Albany, USA

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Abstract

Mycobacterium avium is a widespread environmental bacterium that is capable of causing

disease in humans and other vertebrate hosts. M. avium is related to M. tuberculosis, an obligate pathogen and the causative agent for tuberculosis disease in humans.

Experimental murine infection by M. avium exhibits several important similarities to human infection by M. tuberculosis, including the generation of a hypoxic pulmonary lesion and the development of a necrotic granuloma. It is already known that M. avium is resistant to one of the primary host-defensive anti-mycobacterial compounds, nitric oxide, which is produced in locally high concentrations by the infected host. It is known that in mice deficient in nitric oxide (nos2-/-_{), M. avium grows less well relative to control mice. We}

reasoned that due the wide ecological distribution of M. avium, it would have an enhanced capability to respond to oxygen restriction and nitrosative stress, both conditions likely encountered in the environment. In order to investigate the basis for these observations regarding the resistance of M. avium to nitric oxide and the reduced growth in the nos2

-/-mice, we examined the stress response of M. avium through inactivation of genes known to participate in the stress response to oxygen restriction (narG) as well as perform an unbiased gene expression screen using bacteria mRNA combined with a

mass-spectrometric identification of mycobacterial proteins. We made several novel findings, among which was the demonstration that M. avium was capable of logarithmic growth in the absence of oxygen in vitro and that long term anoxia required narG. In addition we defined two stress-specific operons, one induced by anoxia, the other induced by nitric oxide. We also examine the nature of the host response in the absence of nitric oxide (nos2-/-_{) and found that effector T cells in mycobacterial granulomata are not a uniform}

Th1 population but exist in distinct subsets with potentially different functions and differential susceptibilities to the effects of nitric oxide.

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Resumo

O Mycobacterium avium é uma bactéria encontrada no ambiente, capaz de causar doença em seres humanos e outros hospedeiros vertebrados. O M. avium é relacionado com o M. tuberculosis, um patogénio obrigatório e agente etiológico da tuberculose humana. A infecção experimental em murganho pelo M. avium tem numerosas

semelhanças com a infecção humana pelo M. tuberculosis, incluíndo a geração de lesões pulmonares hipóxicas e o desenvolvimento de granulomas necróticos. É já conhecida a resistência de M. avium a um dos mais importantes compostos anti-micobacterianos de defesa do hospedeiro, o óxido nítrico produzido localmente em concentrações elevadas no hospedeiro infectado. Sabe-se que o M. avium prolifera menos em animais deficientes em sintase do óxido nítrico (nos2-/-_{) que em animais controlo. Postulámos que, dada a}

vasta distribuição ecológica do M. avium, a bactéria deverá possuir uma capacidade aumentada de responder a uma limitação de oxigénio e ao stress nitrosativo, ambas condições prováveis no meio ambiente. De modo a estudar a base da maior resistência do M. avium ao óxido nítrico e ao crescimento reduzido em murganhos nos2-/_,

examinámos a resposta de stress do M. avium através da inactivação de genes que se sabem participar na resposta de stress à restrição de oxigénio (narG) bem como executámos um screen não viesado da expressão genética usando mRNA combinado com a identificação de proteínas micobacterianas por espectrometria de massa. Fizemos várias observações originais estando entre elas a demonstração que M. avium é capaz de crescimento logarítmico in vitro na ausência de oxigénio e que a anóxia prolongada necessita de narG. Adicionalmente, definimos dois operões específicos de stress, um induzido pela anóxia e o outro induzido pelo óxido nítrico. Também examinámos a natureza da resposta do hospedeiro na ausência de óxido nítrico (nos2-/-_{) e descobrimos} que as células T efectoras no granuloma micobacteriana não são uma população Th1 uniforme, mas existem em subconjuntos distintos com funções potencialmente diferentes e susceptibilidades diferenciais para o efeitos de óxido nítrico.

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Chapter 1. Mycobacterium avium

The genus Mycobacterium encompasses many species ranging from those that cause significant human disease to those that exist in the environment and have no capacity to colonize the vertebrate host. Among these species are important human pathogens;

Mycobacterium tuberculosis and M. bovis are obligate pathogens of human or mammalian

hosts, others such as M. avium are present in the environment and are capable of causing disease in humans. The environment encountered by infectious species of Mycobacterium within an immunocompetent host can be toxic; however, the toxicity generated by the host is necessarily constrained by the need to maintain host tissue function, such as gas exchange in the lung. In contrast, the harsh conditions encountered by environmental

Mycobacteria can be more diverse and are not constrained by the needs of a host. Since

infectious species of Mycobacterium are thought to have evolved from those living in the environment, it is possible that some of the adaptive mechanisms needed to counter environmental hazards have been retained by infectious species of Mycobacterium to counter the host-generated defenses. We reason that it would be informative to

investigate the adaptive responses of an environmental species of Mycobacterium, which is an effective murine pathogen, with the goal of identifying mechanisms that provide infectious species of Mycobacterium the ability to counter the defensive mechanisms of the human host. We feel that increased understanding of the relationship between pathogen and host will help identify novel drug and vaccine targets that can be used to combat those species of Mycobacterium that infect humans.

The pathogenic environmental Mycobacterium species chosen for these studies is M.

avium. This bacillus is an appropriate subject for this work because it is has a high

disease incidence compared to other infectious environmental Mycobacterial species. In addition, M. avium has been studied extensively and has similarities to the significant human Mycobacterial pathogen M. tuberculosis, which causes serious disease but which

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lacks the ability to persist in the environment without a vertebrate host. Importantly, substantial differences have been identified between M. avium and M. tuberculosis; among the most relevant for this work is the ability of M. avium to tolerate much higher levels of nitric oxide compared to M. tuberculosis. This reactive oxide of nitrogen is

normally produced at low levels by the vertebrate host and functions as a regulatory factor but is also generated at high concentrations in response to Mycobacterial infection by the macrophage, often in conjunction with the acidification of the Mycobacterium-containing phagosome. The production of nitrogen, oxygen and peroxynitrate radicals represents one of the primary anti-bacterial defense mechanisms of the host. Investigation of how M.

avium combats the damaging effects of nitric oxide will therefore provide insight into the

adaptive mechanisms of this pathogen and may provide new targets for intervention.

Mycobacterium avium

Environmental distribution and host diversity

M. avium is a widely dispersed environmental bacterium, existing as a free-living member

of the microbiotic community [1]. It has been identified in many diverse environments that serve as natural reservoirs from which humans and other hosts may become colonized.

The most common environment for M. avium is fresh surface water [2] which

encompasses rivers and streams [3] as well as lakes and ponds [4-6]. M. avium is more prevalent when conditions favor slowly flowing water that is acidic and brown with

generally warmer temperatures, lower pH, lower concentrations of dissolved oxygen, and elevated concentrations of soluble zinc, humic acid and fulvic acid [7]. These conditions are often found in flood plains, swamps, lowlands or wetlands and may be associated with an elevated incidence of non-tuberculosis Mycobacterial infection in the surrounding populations of people or animals [8]. M. avium has also been identified from brackish water, i.e. ~1% NaCl, and less frequently from estuary or oceanic water, i.e. >3% NaCl [9, 10]. Thus, M. avium is widely distributed throughout many geographic regions with a

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greater affinity for warmer, more humid climes. Importantly, the exposure of M. avium to a diverse range of minerals, salts and transition metals in these aqueous and slightly acidic environments has selected the genetic tools necessary for survival within these growth restrictive conditions. This may be especially relevant in regard to the response of M.

avium to nitrogen oxides, including nitrate, nitrite and nitric oxide, all of which are present

in the environment [11].

M. avium has also been found in drinking water [12] as well as its distribution systems [13]

and has been reported to colonize shower heads [14] [15], hot-tub baths [16] and other spa-type environments [17]. M. avium has been shown to persist as a biofilm within drinking water systems where pipe material, water temperature and available organic matter contribute to the number of effluvial bacilli [18]. It has been shown to be highly resistant to the chlorine, chloramine, chlorine dioxide, and ozone disinfection techniques used in municipal water purification systems [19]. M. avium shows a remarkable ability to grow within non-permissive environments.

In addition to aquatic environments, M. avium is also found in a wide variety of soils, both natural [20] as well as those commercially processed for gardening [21]. It is thought that environmental Mycobacteria serve as an ecological reservoir for the infection of livestock [22] and that once Mycobacterium is introduced into the soil, they move through the biome, often contaminating grass and the upper layers of soil [23].

As a result of M. avium in the local soil or water [24], the colonization of people, wildlife and livestock is more likely [25, 26] and illustrates how M. avium is capable of exploiting a highly diverse terrestrial ecology. In addition to the colonization of mammals, insects, phagocytic protozoa and amoeba have also been reported to harbor M. avium [27]. Examples include the syrphid flies potentially infecting pig [28] and cattle herds [29], insectivorous rodents [30, 31] as well as birds and their ticks [32]. In one final example, M.

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avium has been found residing in free living amoeba [33] within water distribution

systems.

Taken together, these examples highlight the ecological and host range of M. avium. It is clear that M. avium resides within the environment, inhabiting wet or humid locales with a preference for slightly acidic conditions in the presence of organic matter. Although only mildly infectious to individuals, it is broadly resistant to host-defensive measures

suggesting that adaptive responses to conditions encountered in its natural habitat have selected tools by which the bacteria can counter the immune response of the host. To summarize, M. avium has taxonomic and structural similarities to M. tuberculosis but is very different in that it colonizes not only humans but also other organisms and many environmental niches.

Infection, disease and pathogenesis

M. avium does not kill as many people as tuberculosis. It does, however, cause a diverse

range of diseases and death, including a progressive pulmonary infection in healthy adults [34], lymphadenitis in children [35, 36] and disseminated infection in immunocompromised individuals [37]. Less commonly, it is found to infect the skin and may cause soft tissue infections [38]. In immunocompromised people with a defective CD4 T cell compartment, especially those infected with HIV and suffering from AIDs, M. avium is an important opportunistic pathogen [37]. Much clinical research into the pathogenesis of M. avium infection has been focused on opportunistic infection of HIV/AIDs patients [39, 40].

Immunity to Mycobacterial infection is founded on the intersection between the bacteria-intrinsic capabilities of growth, adaptation and host resistance mediated by defined characteristics such as nitric oxide [41]. Bacterial features that influence the development of immunity include the route of inoculation, the infectious and antigenic dose received, and the specific strain(s) of Mycobacteria. The age of the host, their overall health status, and prior immunological experience with Mycobacteria are the host-intrinsic features that

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determine the quality, type and effectiveness of resulting immunity [42]. In addition, genetic determinants influence susceptibility to Mycobacterial disease, including the IL-12 and interferon- pathway resulting in macrophage activation [43].

In the case of M. avium, its widespread environmental distribution heavily influences the quality and type of immunity generated in the host [25]. As described earlier, M. avium has been found free-living in the environment, associated with protozoa and amoebae. It has been suggested that these non-mammalian host-pathogen interactions have played a role in the selection of pathogenic traits [44]. In humans and experimental systems using murine models of infection, immunity to Mycobacteria relies on both innate and acquired responses [45].

Innate immunity to Mycobacterial infection involves surveillance of various danger- or non-self-signals at the primary physical barriers of the host, especially at the pulmonary and intestinal mucosa as well as the skin [46]. Innate immunity links the response of these surveillance systems with inflammation to provide the appropriate environment to support the evolution of the acquired response. The basis of innate immunity is the interaction between the bacilli and the cells of first contact, thought to be phagocytes of myeloid origin [39]. These cells are stimulated through pattern recognition receptors, which detect conserved pathogen-associated molecular patterns [47]. These cells then phagocytose the bacilli and rapidly advertise their newly acquired intracellular guest by secretion of TNF-, lymphotoxin, interleukin (IL) IL-6, IL-1 and –1 [48, 49] as well as by the expression of Mycobacterial antigen presented within context of MHC [50]. These cytokines then trigger the expression of chemokines that subsequently recruit

inflammatory cells, including neutrophils, inflammatory macrophages and NK cells [51, 52]. This inflammatory focus serves as the nascent center for the formation of the

Mycobacterial lesion as T-cells migrate to the lesion to drive the macrophages toward

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interferon- (IFN-) and tumor necrosis factor (TNF) secreted from CD4+_{T cells that}

stimulate phagocytes to generate toxic reactive oxygen and nitrogen radicals including nitric oxide and superoxide [54, 55] which then restrict bacterial growth. Despite

sequestering the bacilli inside a phagosomal compartment and subjecting them to potent antimicrobial compounds, this intracellular compartment is not isolated. In fact, the

Mycobacteria-containing vacuole has been found to be fusogenic with the endosomal

pathways [56], a mechanism that may be responsible for bypassing the nutriprivitive response of the infected cell [57].

It is generally assumed that the initial stages of infection with M. avium progress similarly to M. tuberculosis [58]. Resident macrophages are thought to be the target of initial

infection and initial intracellular bacterial replication [48]. Dendritic cells are then thought to traffic bacteria and antigen to the draining lymph nodes where naive T cells are activated and clonally expand [54]. The interaction between M. avium and its host cell is mediated through a variety of molecules including the complement receptors 1, 3 and 4, mannose receptors, CD14, Fc receptors [59] and fibronectin or vitronectin receptors [39]. M. avium is capable of immunomodulating its host using cell wall glycopeptidolipids that act via TLR2 of the macrophage which trigger MAPK activation, leading to NF-κB nuclear translocation and cytokine secretion [60]. Thus, Mycobacteria are capable of modifying the host immune response.

The bacillus is often spread via the lymphatics to the hilar lymph nodes whereupon hematogenous seeding may occur [61]. Some organs and tissues appear more resistant to infection than others; the bone marrow, liver, and spleen are almost always infected, but most often control Mycobacterial growth, while the upper lobes of the lung, kidneys, bones, and brain fail to control Mycobacterial growth [62]. Within the granuloma, bacilli tend to be localized in or around the predominantly myeloid center of the granuloma, which is often necrotic and hypoxic [63-66]. Most people control Mycobacterial disease at this stage where bacterial growth slows as T-cell mediated immunity peaks [67].

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One key difference between M. avium and M. tuberculosis is the respective

susceptibilities of each to host-derived nitric oxide where M. avium is resistant [68] while

M. tuberculosis is not [69]. The effect of this difference has been observed in infection

models of mice lacking the gene for the inducible nitric oxide synthase (Nos2) gene, nos2, in which M. tuberculosis exhibits unrestrained bacterial growth which becomes quickly fatal [70]. In contrast, the growth of M. avium in the nos2 deficient mouse is either unaffected or impaired [71]. This dichotomy in response to Mycobacterial infection is the basis for this work. There are several possible explanations for the observed difference in susceptibility between M. avium and M. tuberculosis to the effects of nitric oxide. The first possibility is that due to the environmental origin of M. avium, this bacterium has a greater capacity to detoxify nitric oxide because it commonly encounters high concentrations of nitrates and nitrites, the chemical equilibrium partners of nitric oxide, within its natural milieu [72, 73]. The second possible explanation is that M. avium has the capacity to use nitric oxide or a redox product as a growth factor or metabolite when subjected to immune stress by the host. The third possible explanation is that the presence of nitric oxide within the host regulates the immune response to M. avium and that this lack of regulation results in decreased growth of the bacterium [68]. We will investigate the effect of nitrate and nitrite on M. avium (Chapter 2), the response of the bacterium to anoxic and nitric oxide stress (Chapter 3) and examine the impact of nitric oxide on the immune response to M. avium (Chapter 4).

Mycobacterium avium adaptation to the host response

Importantly, two clinically recognized features of human tuberculosis, which are absent in mouse models of tuberculosis [74], are observed in the mouse model of M. avium

disease. These two features of disease are the development of caseous necrosis within both the pulmonary and hepatic lesion [64, 75], as well as the development of regions of hypoxia within the granuloma [75]. Considering the latter, the murine model of M. avium disease offers a tool to examine the effect of hypoxia on Mycobacteria in the presence of

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a competent immune response. This aspect of human tuberculosis not well suited for study using M. tuberculosis in the mouse. More broadly, and within the context of the ability of M. avium to resist the toxic effects of nitric oxide, the murine model of M. avium disease provides insight into human tuberculosis because human tuberculosis evokes an altered nitric oxide response compared to that of tuberculosis in the mouse [76].

As described, besides the generation of nitric oxide, the murine Mycobacterial granuloma can become hypoxic and this can limit bacterial viability. In human M. tuberculosis, the granuloma has been found to have low oxygen availability [77] and M. tuberculosis

counters hypoxic stress with nitrate reductase as an alternative terminal electron acceptor [78]. This activity has been shown to depend on the nitrate reductase gene narG [79]. In the absence of narG, the bacteria are susceptible to increased damage by acids and nitrogen radicals under hypoxic conditions as well as to suffer reduced proton motive force [80]. It is unknown whether M. avium uses a similar strategy of nitrate reductase

respiration to counter oxygen restriction within the lesion. However, the observed ability of

M. avium to thrive within a hypoxic lesion in the presence of nitric oxide suggests that M. avium may have an enhanced capacity to counter this challenging environment [77],

perhaps due to its environmental origin and concomitant selection.

Taken together, the combination of oxygen restriction and exposure of nitric oxide considerably narrow the scope of physiological adaptive mechanisms available to M.

avium. In the hypothetical model presented hereafter, M. avium infects the vertebrate host

and initially encounters a permissive growth environment. The host then develops a complex immune response over time transforming normal tissue into an inflammatory lesion and eventually into a necrotic and hypoxic granuloma by infiltration of cells including macrophages, neutrophils and lymphocytes and by the deposition of

extracellular material including collagen and fibrin. The lesion also becomes toxic through the production of nitric oxide (NO) and other anti-mycobacterial compounds, including superoxide (Figure 1.1). As hypoxia is detected and antibacterial nitric oxide suffuses the

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environment, the bacteria respond to by shifting from away from its primarily oxygen-based respiration that uses NADH dehydrogenase encoded by the nuo genes (NDH-1), succinate dehydrogenase (Sdh), and the cytochrome bc1-aa3 supercomplex encoded by

the cta and qcr genes (Figure 1.1). Upon oxygen restriction and the presence of nitric oxide, the bacteria shift respiration to cytochrome bd oxidase encoded by the cyd genes. Ferredoxins are induced and oxygen loss drives induction of the nitrate transporter (NarK2) and the start of narG-dependent nitrate respiration. In M. tuberculosis, this

transition produces non-replicative persistent (NRP) bacteria (Figure 1.1). We hypothesize that M. avium may be able to adapt to severe oxygen and nitric oxide stress due to the selective pressures of their natural environment. In this thesis, the role of nitrate reductase in growth of M. avium under low oxygen conditions will be examined in Chapter 2. The ability of M. avium to grow under anoxia and nitric oxide stress will be addressed in Chapter 3. The ability of host-derived nitric oxide to regulate the immune response and environment of M. avium will be assessed in Chapter 4. Figure 1.1 illustrates these bacterial and host factors and some of the relevant physiological aspects of the bacillus necessary to counter the host immune response.

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environment. In addition, the presence of nitric oxide and especially its redox products have been shown to influence bacterial physiology.

Nitric oxide

Nitric oxide plays a pivotal role in eukaryotic physiology, immunology and cell signaling [81]. It is a freely diffusible gaseous radical possessing one free electron that readily forms covalent bonds with a variety of reactants. The 1-10 second biological half-life of nitric oxide reflects this reactivity [82]. While nitric oxide is the preferred IUPAC name for the molecule, other synonyms are used, including nitrogen monoxide, nitrogen oxide and nitrosyl radical.

Oxides of nitrogen and their reactive chemistry

In normal air, gaseous dioxygen reacts with nitric oxide to form gaseous nitrogen dioxide, which can then exist at equilibrium with dinitrogen tetroxide. At physiological

temperatures, this equilibrium strongly favors nitrogen dioxide. In water, nitric oxide reacts with oxygen to form nitrous acid, HNO2 (Eq. 1.) Nitrous acid spontaneously decomposes

into nitrogen dioxide, NO2, and nitric oxide (Eq. 2.) This nitrogen dioxide product then

disproportionates into nitric acid, HNO3, and nitrous acid, HNO2 (Eq. 3.) Thus at

physiological conditions, the net reduction of aqueous nitric oxide in the presence of dioxygen yields nitric and nitrous acid (Eq. 4.)

4 NO· + O2 + 2 H2O ⇀ 4 HNO2 (Eq. 1)

4 HNO2 ⇀ 2 NO2 + 2 NO· + 2 H2O (Eq. 2)

2 NO2 + H2O ⇀ HNO3 + HNO2 (Eq. 3)

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Stoichiometricly, the net reduction of nitric oxide consumes half of the available NO· while generating one molecule each of nitric and nitrous acid. This sequence of aqueous

reactions contribute to nitric oxide’s relatively long bioactive half-life within the context of other free radicals such as hydroxyl (HO·) which has a half-life of 10-9_{seconds or alkoxyl}

radical (RO·) with a half-life of 10-6 seconds [82]. Reduction of nitric oxide within biological systems is more complicated due to the formation of nitrosyl adducts and the buffering properties of biological fluids [83].

To complicate our understanding of the nature of the chemical species outlined above, many of these reaction products also auto-ionize under physiological conditions and therefore exist at equilibra with their non-ionized counterparts. Principally among these is nitric acid, HNO3, produced by the oxidation of either NO2 (Eq. 3) or HNO2 (Eq. 2) which

spontaneously ionizes into nitrate, NO−3_{, and hydronium, H}

3O+, under physiological

conditions.

HNO3 + H2O ⇌ H3O+ + NO−3 (Eq. 5)

In addition, nitrous acid can also ionize under physiological conditions.

HNO2 + H2O ⇌ H3O+ + NO−2 (Eq. 6)

Many of these equilibrium products can also undergo spontaneous auto-oxidation (Eq. 7) or protonation (Eq. 8-9.) The balance of each equilibra is governed by its particular acid dissociation, pKa, and thus affected by the local pH and the presence of electron donors and acceptors.

4 NO· + O2 + 2 H2O ⇌ 4 NO2- + 4 H+ (Eq. 7)

NO2- + H+ ⇌ HNO2 (Eq. 8)

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In summary, the biochemistry of nitric oxide is strongly influenced by local conditions, especially pH and available molecular oxygen. The net results of nitric oxide production within a biological system generate a range of nitrogen oxides, most prevalently nitrate and nitrate. Within the context of nitric oxide, nitrate and nitrite anions are particularly important in two respects. First, nitrate in the mammalian host is quickly reduced to nitrite by natural flora which then is rapidly absorbed [84] and used for the production of both inducible and constitutive nitric oxide [85]. Second, both nitrate and nitrite participate in the assimilatory nitrogen acquisition pathway via ammonia for the biosynthesis of

nitrogenous compounds for M. avium [86, 87]. In addition to an assimilatory function, the presence of nitrate reductase in Mycobacteria has also been implicated in respiratory function under hypoxic conditions, as described above and portrayed in Figure 1.1.

Nitric oxide in the oxygen-restricted environment

The reactivity of nitric oxide in the absence of oxygen has been examined in detail from the context of anaerobic nitrification [88] and anaerobic ammonium oxidation [89] in soils and water, the same environmental niche as M. avium is known to inhabit. Both of these biological processes generate nitric oxide, to which M. avium may be exposed [90]. The best studied example of the generation of nitric oxide under conditions of little or no oxygen involves nitrite decomposition, denitrification, which produces nitric oxide [91] among other nitrogen species, most often in anaerobic soil. Such soils include wetlands, which are generally anoxic and thus support the production of trace gases, including nitric oxide by anaerobic denitrifies [92]. Thus it appears environmental Mycobacteria are likely exposed to nitric oxide within their biome.

Host-derived nitric oxide and Mycobacterium avium

As discussed, nitric oxide exists in a pH-dependent equilibrium with nitrite, (NO2) and

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forms highly reactive peroxynitrate, ONOO-_{, which has been shown to interact with}

tyrosine residues involved in regulatory protein phosphorylation [93]. In addition to these regulatory activities, nitric oxide can also function as a terminal electron acceptor for bacterial anaerobic respiration [94]. The ability to metabolize nitric oxide is not limited to obligate anaerobes, as an environmental Pseudomonas species has been identified that can oxidize nitric oxide to nitrite under a range of oxygen concentrations [95].

Importantly, nitric oxide has been shown to play a key role in the killing of M. tuberculosis resident within the activated macrophage [69]. The evolution of this host-derived nitric oxide is illustrated in Figure 1.1. As described, some strains of M. avium are highly resistant to the effect of bactericidal effects of nitric oxide [71]. One possible explanation for this resistance may be the presence of a predicted gene encoding nitric oxide

reductase (mav_4011) in the genome of M. avium, although its function has not yet been ascertained.

In summary, nitric oxide is produced by a variety of host cells and is generated

enzymatically by nitric oxide synthase acting on the substrate arginine [96]. The ability to use nitric oxide under conditions lacking dioxygen might be advantageous to

Mycobacteria. Indeed, a possible respiratory function for nitric oxide under anaerobic

conditions may explain why some strains of M. avium do less well in the nitric oxide-deficient murine host. Thus, determining whether nitric oxide is utilized by M. avium under anaerobic conditions as a growth factor is one goal of our current work.

Mycobacterium avium strain 25291

We have chosen to use M. avium strain 25291 in this project because our previous work demonstrated that: (i) it is not compromised by the presence of host-derived nitric oxide; (ii) that it is virulent in vivo; and (iii) that in the absence of host-derived nitric oxide the number of bacteria is less when compared to its nitric-oxide competent control [66, 68, 97]. These characteristics, along with the fact that nitric oxide modulates the immune

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response to this pathogen and that this pathogen can induce necrotic and hypoxic lesions in the mouse model [66, 77, 98] make this strain of M. avium an excellent target for our investigation into the unique stress response of M. avium. In order to investigate the ability of M. avium to combat various stress conditions in an unbiased manner it is essential that we have access to modern genomic tools. The development of these tools for M. avium has lagged behind that for other Mycobacterial species, so a secondary goal for this work is develop the necessary molecular biology tools needed to perform genetic manipulation of the strain.

The genome of Mycobacterium avium

Since the main subject of this work, M. avium sub-species avium 25291, does not yet have a fully annotated genomic sequence completed, but instead has only a whole shotgun sequenced draft genomic sequence, much of the following examination of the genomic features will focus on its nearest annotated taxonomic neighbor, M. avium 104.

Genome features and genome equivalency

The size of M. avium 25291’s genome is 4,857,995, which is somewhat less than that of its nearest taxonomic neighbor, M. avium 104, at 5,475,491 bases [99]. This difference in size is likely an artifact of the draft status versus the fully annotated reference sequence of

M. avium 104.

M. avium has more protein encoding genes, more genes involved in lipid metabolism, and

more regulatory genes then other members of Mycobacteriaceae. It has more unique genes than other members of Mycobacteriaceae. The extent of the genome may be a reflection of the wide environmental conditions that M. avium is able to exploit. The large genome also makes this species an excellent tool with which to investigate the stress response of the genus Mycobacterium as it is likely to have the greatest variety of

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responses available to it. We will investigate the breadth of the transcriptomic response of

M. avium to oxygen and nitric oxide stress in Chapter 3.

This work makes extensive use of genetic and protein homology. Genes are described in the genome records maintained by NCBI for M avium subspecies avium strain 104 as a fully sequenced and annotated genome and at EMBL for M avium subspecies avium strain 25291 as a draft whole shotgun. Since the experimental subject of this work, M.

avium subspecies avium strain 25291 does not yet have an annotated genome, all

description of genes and their function must be based on the closest taxonomic neighbor,

M. avium subspecies avium 104. This substitution also extends to proteins; this work will

discuss the translation products of the experimental strain 25291 bacillus in terms of the proteins originating from strain 104. While not ideal, this substitution is the only way to address bacterial gene function and metabolism in strain 25291.

Metabolism and respiration

As discussed above M. avium inhabits a range of diverse environments and requires metabolic adaptation in order to survive. This is especially true during infection of the vertebrate host, specifically the mouse.

Bacterial respiration is a modular solution to one of the fundamental requirements of life, that is, the extraction of metabolic energy from the environment and its conversion into potential energy used to drive various thermodynamically unfavorable reactions. M. avium is classified as a chemoheterotroph in that it uses organic molecules for both the

derivation of energy and as a source of carbon, which is wholly consistent with its

environmental distribution. For M. avium, respiration requires an external terminal electron acceptor that is derived from an inorganic substrate. When the terminal electron acceptor is oxygen, this process is aerobic. Anaerobic respiration uses compounds other than oxygen, including compounds such as nitrate or sulfate, as a terminal acceptor.

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not considered as anaerobes. Many Mycobacteria, however, have evolved a capacity to respire under microaerobic conditions [100] which necessitates the use of an alternate terminal electron acceptor used in combination with an oxygen scavenging system [101]. The alternate electron acceptor is understood to be nitrate [102]. These respiratory configurations are shown schematically in Figure 1.1.

One might suggest that the basis for the wide environmental distribution of M. avium is its respiratory capacity, which functions under a variety of challenging conditions. These various habitats contain a multitude of interacting organisms engaged in the competitive acquisition of resources, energy and reproductive space. Simultaneously, M. avium is engaged in the competitive exclusion of abiotic factors, including toxic metals and halides as well as toxic secondary metabolites used by other organisms to aid in their acquisition of resources, energy and space. Thus, the local environment experienced by M. avium is rich in both organic compounds and inorganic molecules and we reason that M. avium will have been evolutionarily selected to exploit these resources.

Nitrogen metabolism

The nitrogen metabolism of M. avium allows the bacteria to utilize inorganic sources of nitrogen, principally nitrate and nitrite that are converted into ammonia, which is the nitrogen source of for biosynthesis of more complex nitrogen-containing molecules

including amino acids and nucleotides. In this regard, two species of Mycobacterium have been shown to have a functional nitrogen assimilatory pathway that reduces nitrate to nitrite [87, 103]. These observations have been generalized through genome sequence analysis of the other Mycobacterium species, suggesting that nitrogen metabolism uses many of the same genetic components within Mycobacteriaceae [86]. Many of the observed differences in nitrogen utilization between the species, for example, the

difference between M. tuberculosis and M. bovis in their ability to reduce nitrate to nitrite, arise from differential regulation of homologous genes [104]. Mycobacteria possess genes

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encoding nitrate and nitrite reductases, various anionic nitrate/nitrite transporters and the necessary regulatory components for the maintenance of an intracellular ammonia pool supporting downstream production of glutamate and glutamine as the main nitrogen assimilatory path [105, 106].

In addition to supporting the assimilatory demands of the bacilli, several components of the nitrogen metabolic system are also used for energy production under low-oxygen conditions. The use of an inorganic terminal electron acceptor under conditions of oxygen restriction is widespread and relatively common and has been described in Wolinella

succinogenes and both pathogenic Campylobacter and Helicobacter species [107]. The

nature of this metabolic response to oxygen limitation has been well characterized [108-110] and is triggered by stress conditions comparable to those observed within the mycobacterial lesion [111].

In Mycobacteria the reduction of nitrate to nitrite by nitrate reductase has been shown to accompany hypoxic stress [79]. It has been suggested that nitrate reductase can function as the terminal electron acceptor during periods of dormancy in M. tuberculosis infections [112]. In support of this, it has been found that nitrate reductase is required for in vitro growth under hypoxia for M. bovis BCG [113] and M. tuberculosis [77] but not for in vivo growth [77]. Although not essential for in vivo growth, possibly due to the inability of the mouse to generate hypoxic lesions when infected with M. tuberculosis, expression of nitrate reductase and the nitrate transporter NarK2 are upregulated during the chronic infection by M. tuberculosis in the mouse [78]. Generalizing these observations, as a terminal electron acceptor, nitrate reductase would receive electrons from a charge carrier. Figure 1.1 illustrates an example of this metabolic configuration.

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Electron transport

The electron transport chain transfers electrochemical charges between electron donors, including NADH and succinate, with electron acceptors, including the terminal acceptor oxygen or an intermediate acceptor menaquinone. This transfer is manifest through the movement of protons across a membrane, which produces an electrochemical gradient. This gradient is then used to generate chemical energy through the regeneration of adenosine triphosphate. Electron transport chains are the cellular mechanisms used for extracting energy from redox reactions.

Bacterial electron transport in is considered to be branched, inducible and modular [114]. The hallmark of bacterial electron transport is the simultaneous use of multiple electron donors and acceptors; this branched architecture allows maximum energetic efficiency under non-permissive conditions. Mycobacterial electron transport uses a variety of electron donors and acceptors. The electron transport chain harvests electrons using nicotinamide adenine dinucleotide (NADH) dehydrogenase and other dehydrogenases, oxidoreductases, hydrogenases and reductases [115]. M. avium has genes encoding some or all of the proteins comprising these enzymes, although their functionality is in some cases unknown as are the conditions that may trigger induction. Figure 1.1 shows a generalized schematic of Mycobacterial electron transport operating initially under aerobic conditions, then under oxygen-restricted conditions.

NADH and succinate dehydrogenase

NADH and succinate represent two charge carriers capable of donating an electron within the electron transport chain. NADH dehydrogenase, the enzyme responsible for this charge transference, is present in two distinct forms in M. avium and other Mycobacteria. It directs the transfer of electrons from NADH to the downstream components of the

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respiratory chain. In M. tuberculosis, it has been shown that the expression of the NAD dehydrogenases is affected by the availability of oxygen [78].

Type I NAD dehydrogenase (NDH-1), encoded by the nuo operon, is comprised of fourteen genes. The product of this operon is a membrane-bound enzyme complex that oxidizes both NADH to NAD+ and FADH to FAD+ while expelling protons into the

extracellular space. NDH-1 is non-essential in vitro, but necessary for the virulence of M.

tuberculosis in the mouse [116]. Genes encoding NDH-1 are present in M. avium.

The second form of NADH dehydrogenase is the type 2 non-proton-pumping (NDH-2), which is encoded by ndh genes. In M. avium, four putative ndh genes have been identified by homology (mav_1130, mav_2867, mav_4103 and mav_4772.) NDH-2 is essential for

M. tuberculosis survival and is thought to provide the majority of NADH oxidation in Mycobacteria during aerobic growth [117].

Succinate dehydrogenase is a membrane-bound enzyme complex that also participates in energy metabolism. Its function is to provide an electron carrier in the form of fumarate, which is generated by oxidation from succinate. In M. avium, this enzyme is composed of

sdhABCD genes and one accessory gene, an iron-sulfur subunit, mav_4910. The roles of

both succinate dehydrogenase and NADH dehydrogenase are illustrated in Figure 1.1.

Menaquinone

Electrons generated by NADH and succinate dehydrogenase are transferred the menaquinon-menaquinol pool. Menaquinol, also known as vitamin K2, is essential to

bacterial electron transport [118]. It has been proposed that menaquinone links the NDH-1 and succinate dehydrogenase with the cytochrome bc1-aa3 supercomplex in order to

efficiently reduce oxygen to water under conditions of normal oxygen [115] as illustrated in Figure 1.1.

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Cytochrome bc1-aa3 supercomplex

Menaquinol transfers reducing equivalents to the cytochrome bc1-aa3 supercomplex,

[119]. Structurally, cytochrome bc1 is a multi-subunit complex encoded by qcrCAB that

includes a Rieske iron-sulfur protein. In M. avium, the cytochrome bc1 complex is encoded

by qcrA (mav_2297), petB (mav_2296) and cyt1 (mav_2298.) The second component of the supercomplex is cytochrome aa3 oxidase is encoded by cyoE (mav_3326), ctaD

(mav_3908), coxB (mav_2291), coxC (mav_2299) and ctaA (mav_3324). This cytochrome bc1-aa3 supercomplex is the primary electron path used during aerobiosis in vitro to which

the terminal electron acceptor is molecular oxygen [115]. The presence of nitric oxide has been shown to inhibit the oxidoreductase activity this cytochrome supercomplex [78] as depicted in Figure 1.1.

Cytochrome bd oxidase

In contrast to cytochrome bc1-aa3 supercomplex, cytochrome bd oxidase is the preferred

terminal oxidase under conditions of reduced oxygen availability [78]. It has a relatively high affinity for dioxygen as has been shown to associate with acts as a terminal oxidase in both M. tuberculosis and M smegmatis [120]. It has been suggested that this

supercomplex may participate in oxygen-restricted bacterial dormancy [101]. The genes for this oxidase are encoded by cydAB and are present in M. avium, at loci mav_3164 and mav_3165 as illustrated in Figure 1.1.

Hydrogenases

Hydrogenases are a relatively new class of hypoxia-induced redox enzymes observed in

Mycobacteria whose function appears to be the reversible oxidation and reduction of

hydrogen [115]. Such functionality under oxygen-restriction would allow the use of alternative charge carriers such as ferredoxins to be used as electron transporters [121].

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The advantage of such an alternative electron transfer path would allow for a more flexible and inducible pathway for electrons to their terminal acceptor.

Ferredoxin

The possibility of an alternate charge carrier, such as ferredoxin, operating under conditions of oxygen restriction has been proposed [115]. Ferredoxins are electron carriers that contain iron and sulfur, are water-soluble and typically function in two distinct contexts. The first context is similar to that of the menaquinone pool, in which these mobile charge carriers transit between the less mobile complexes embedded in the plasma membrane [122]. The second context is that in which these molecules participate in electron transport from within the macromolecular complexes of NADH dehydrogenase and the oxidoreductases [123]. Induction of ferredoxins and the components necessary to regenerate the redox state are found in Mycobacteria undergoing hypoxic stress [115]. Figure 1.1 illustrates how ferredoxins might provide an alternate electron transport system in under hypoxic conditions.

Conclusion

M. avium is a widely distributed environmental bacterium capable of causing human

disease. Within its ecological niche it is exposed to a variety of environmental challenges including restricted oxygen, exposure to acidic conditions and naturally produced nitrogen oxides, including nitric oxide. When the infected mammalian host evolves a T-cell

mediated granulomatous immune response, aspects of the Mycobacterial lesion resemble its native environment, particularly oxygen limitation and the presence of nitric oxide. M.

tuberculosis, an obligate pathogen, can adapt to hypoxia using the alternate terminal

electron acceptor, nitrate, but the role of this activity in vivo has not been clarified. M.

avium with its environmentally selected survival traits, has the genetic capacity to utilize

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in vivo and in vitro [71]. As such, M. avium offers a unique tool with which to probe the

interface between the host immune response and the adaptation of the pathogen in a manner that could provide insight into human tuberculosis. Our focus encompasses the effect of nitrate and nitrites on M. avium (Chapter 2), the response of the bacterium to anoxic and nitric oxide stress (Chapter 3) and have defined the impact of nitric oxide on the immune response to M. avium (Chapter 4).

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References

1 Falkinham, J. O., 3rd, Surrounded by mycobacteria: nontuberculous mycobacteria in the human environment. J Appl Microbiol 2009. 107: 356‐367. 2 Lee, E. S., Lee, M. Y., Han, S. H. and Ka, J. O., Occurrence and molecular differentiation of environmental mycobacteria in surface waters. J Microbiol Biotechnol 2008. 18: 1207‐ 1215. 3 Bland, C. S., Ireland, J. M., Lozano, E., Alvarez, M. E. and Primm, T. P., Mycobacterial ecology of the Rio Grande. Appl Environ Microbiol 2005. 71: 5719‐5727. 4 Pickup, R. W., Rhodes, G., Arnott, S., Sidi‐Boumedine, K., Bull, T. J., Weightman, A., Hurley, M. and Hermon‐Taylor, J., Mycobacterium avium subsp. paratuberculosis in the catchment area and water of the River Taff in South Wales, United Kingdom, and its potential relationship to clustering of Crohn's disease cases in the city of Cardiff. Appl Environ Microbiol 2005. 71: 2130‐2139. 5 Pickup, R. W., Rhodes, G., Bull, T. J., Arnott, S., Sidi‐Boumedine, K., Hurley, M. and Hermon‐Taylor, J., Mycobacterium avium subsp. paratuberculosis in lake catchments, in river water abstracted for domestic use, and in effluent from domestic sewage treatment works: diverse opportunities for environmental cycling and human exposure. Appl Environ Microbiol 2006. 72: 4067‐4077. 6 Whittington, R. J., Marsh, I. B. and Reddacliff, L. A., Survival of Mycobacterium avium subsp. paratuberculosis in dam water and sediment. Appl Environ Microbiol 2005. 71: 5304‐5308. 7 Kirschner, R. A., Jr., Parker, B. C. and Falkinham, J. O., 3rd, Epidemiology of infection by nontuberculous mycobacteria. Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in acid, brown‐water swamps of the southeastern United States and their association with environmental variables. Am Rev Respir Dis 1992. 145: 271‐275. 8 Brooks, R. W., George, K. L., Parker, B. C., Falkinham, J. O., 3rd and Gruff, H., Recovery and survival of nontuberculous mycobacteria under various growth and decontamination conditions. Can J Microbiol 1984. 30: 1112‐1117.

(34)

9 George, K. L., Parker, B. C., Gruft, H. and Falkinham, J. O., 3rd, Epidemiology of infection by nontuberculous mycobacteria. II. Growth and survival in natural waters. Am Rev Respir Dis 1980. 122: 89‐94. 10 Gruft, H., Loder, A., Osterhout, M., Parker, B. D. and Falkinham, J. O., 3rd, Postulated sources of Mycobacterium intracellulare and Mycobacterium scrofulaceum infection: isolation of mycobacteria from estuaries and ocean waters. Am Rev Respir Dis 1979. 120: 1385‐1388. 11 Holloway, J. M. and Dahlgren, R. A., Nitrogen in rock: Occurrences and biogeochemical implications. Global Biogeochem. Cycles 2002. 16: 1118. 12 Vaerewijck, M. J., Huys, G., Palomino, J. C., Swings, J. and Portaels, F., Mycobacteria in drinking water distribution systems: ecology and significance for human health. FEMS Microbiol Rev 2005. 29: 911‐934. 13 Falkinham, J. O., 3rd, Norton, C. D. and LeChevallier, M. W., Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other Mycobacteria in drinking water distribution systems. Appl Environ Microbiol 2001. 67: 1225‐1231. 14 Feazel, L. M., Baumgartner, L. K., Peterson, K. L., Frank, D. N., Harris, J. K. and Pace, N. R., Opportunistic pathogens enriched in showerhead biofilms. Proc Natl Acad Sci U S A 2009. 106: 16393‐16399. 15 Falkinham, J. O., 3rd, Iseman, M. D., de Haas, P. and van Soolingen, D., Mycobacterium avium in a shower linked to pulmonary disease. J Water Health 2008. 6: 209‐213. 16 Aksamit, T. R., Hot tub lung: infection, inflammation, or both? Semin Respir Infect 2003. 18: 33‐39. 17 Lumb, R., Stapledon, R., Scroop, A., Bond, P., Cunliffe, D., Goodwin, A., Doyle, R. and Bastian, I., Investigation of spa pools associated with lung disorders caused by Mycobacterium avium complex in immunocompetent adults. Appl Environ Microbiol 2004. 70: 4906‐4910. 18 Norton, C. D., LeChevallier, M. W. and Falkinham, J. O., 3rd, Survival of Mycobacterium avium in a model distribution system. Water Res 2004. 38: 1457‐1466.

(35)

19 Taylor, R. H., Falkinham, J. O., 3rd, Norton, C. D. and LeChevallier, M. W., Chlorine, chloramine, chlorine dioxide, and ozone susceptibility of Mycobacterium avium. Appl Environ Microbiol 2000. 66: 1702‐1705. 20 Maekawa, K., Ito, Y., Hirai, T., Kubo, T., Imai, S., Tatsumi, S., Fujita, K., Takakura, S., Niimi, A., Iinuma, Y., Ichiyama, S., Togashi, K. and Mishima, M., Environmental risk factors for pulmonary Mycobacterium avium‐intracellulare complex disease. Chest 2011. 21 De Groote, M. A. and Huitt, G., Infections due to rapidly growing mycobacteria. Clin Infect Dis 2006. 42: 1756‐1763. 22 Norby, B., Fosgate, G. T., Manning, E. J., Collins, M. T. and Roussel, A. J., Environmental mycobacteria in soil and water on beef ranches: association between presence of cultivable mycobacteria and soil and water physicochemical characteristics. Vet Microbiol 2007. 124: 153‐159. 23 Salgado, M., Collins, M. T., Salazar, F., Kruze, J., Bolske, G., Soderlund, R., Juste, R., Sevilla, I. A., Biet, F., Troncoso, F. and Alfaro, M., Fate of Mycobacterium avium subsp. paratuberculosis after application of contaminated dairy cattle manure to agricultural soils. Appl Environ Microbiol 2011. 77: 2122‐2129. 24 Biet, F., Boschiroli, M. L., Thorel, M. F. and Guilloteau, L. A., Zoonotic aspects of Mycobacterium bovis and Mycobacterium avium‐intracellulare complex (MAC). Vet Res 2005. 36: 411‐436. 25 Primm, T. P., Lucero, C. A. and Falkinham, J. O., 3rd, Health impacts of environmental mycobacteria. Clin Microbiol Rev 2004. 17: 98‐106. 26 Reed, C., von Reyn, C. F., Chamblee, S., Ellerbrock, T. V., Johnson, J. W., Marsh, B. J., Johnson, L. S., Trenschel, R. J. and Horsburgh, C. R., Jr., Environmental risk factors for infection with Mycobacterium avium complex. Am J Epidemiol 2006. 164: 32‐40. 27 Rowe, M. T. and Grant, I. R., Mycobacterium avium ssp. paratuberculosis and its potential survival tactics. Lett Appl Microbiol 2006. 42: 305‐311. 28 Fischer, O. A., Matlova, L., Dvorska, L., Svastova, P., Bartos, M., Weston, R. T. and Pavlik, I., Various stages in the life cycle of syrphid flies (Eristalis tenax; Diptera: Syrphidae) as

(36)

potential mechanical vectors of pathogens causing mycobacterial infections in pig herds. Folia Microbiol (Praha) 2006. 51: 147‐153. 29 Fischer, O. A., Matlova, L., Dvorska, L., Svastova, P., Bartos, M., Weston, R. T., Kopecna, M., Trcka, I. and Pavlik, I., Potential risk of Mycobacterium avium subspecies paratuberculosis spread by syrphid flies in infected cattle farms. Med Vet Entomol 2005. 19: 360‐366. 30 Fischer, O., Matlova, L., Bartl, J., Dvorska, L., Melicharek, I. and Pavlik, I., Findings of mycobacteria in insectivores and small rodents. Folia Microbiol (Praha) 2000. 45: 147‐152. 31 Durnez, L., Eddyani, M., Mgode, G. F., Katakweba, A., Katholi, C. R., Machang'u, R. R., Kazwala, R. R., Portaels, F. and Leirs, H., First detection of mycobacteria in African rodents and insectivores, using stratified pool screening. Appl Environ Microbiol 2008. 74: 768‐773. 32 Kovalev, G. K., The role of wild birds and their ectoparasites (ticks) in the circulation and distribution of M. avium and possible formation of natural foci of avian tuberculosis. J Hyg Epidemiol Microbiol Immunol 1983. 27: 281‐288. 33 Ben Salah, I. and Drancourt, M., Surviving within the amoebal exocyst: the Mycobacterium avium complex paradigm. BMC Microbiol 2010. 10: 99. 34 Chitty, S. A. and Ali, J., Mycobacterium avium complex pulmonary disease in immunocompetent patients. South Med J 2005. 98: 646‐652. 35 Esther, C. R., Jr., Henry, M. M., Molina, P. L. and Leigh, M. W., Nontuberculous mycobacterial infection in young children with cystic fibrosis. Pediatr Pulmonol 2005. 40: 39‐44. 36 Pierre‐Audigier, C., Ferroni, A., Sermet‐Gaudelus, I., Le Bourgeois, M., Offredo, C., Vu‐ Thien, H., Fauroux, B., Mariani, P., Munck, A., Bingen, E., Guillemot, D., Quesne, G., Vincent, V., Berche, P. and Gaillard, J. L., Age‐related prevalence and distribution of nontuberculous mycobacterial species among patients with cystic fibrosis. J Clin Microbiol 2005. 43: 3467‐3470.

(37)

37 Corti, M. and Palmero, D., Mycobacterium avium complex infection in HIV/AIDS patients. Expert Rev Anti Infect Ther 2008. 6: 351‐363. 38 Wagner, D. and Young, L. S., Nontuberculous mycobacterial infections: a clinical review. Infection 2004. 32: 257‐270. 39 Bermudez, L. E., Immunobiology of Mycobacterium avium infection. Eur J Clin Microbiol Infect Dis 1994. 13: 1000‐1006. 40 Mycobacterioses and the acquired immunodeficiency syndrome. Joint Position Paper of the American Thoracic Society and the Centers for Disease Control. Am Rev Respir Dis 1987. 136: 492‐496. 41 Turenne, C. Y., Wallace, R., Jr. and Behr, M. A., Mycobacterium avium in the postgenomic era. Clin Microbiol Rev 2007. 20: 205‐229. 42 Cooper, A. M., Host versus pathogen: two sides of the same challenge in the TB world. Eur J Immunol 2009. 39: 632‐633. 43 Filipe‐Santos, O., Bustamante, J., Chapgier, A., Vogt, G., de Beaucoudrey, L., Feinberg, J., Jouanguy, E., Boisson‐Dupuis, S., Fieschi, C., Picard, C. and Casanova, J. L., Inborn errors of IL‐12/23‐ and IFN‐gamma‐mediated immunity: molecular, cellular, and clinical features. Semin Immunol 2006. 18: 347‐361. 44 Segal, G. and Shuman, H. A., Legionella pneumophila utilizes the same genes to multiply within Acanthamoeba castellanii and human macrophages. Infect Immun 1999. 67: 2117‐ 2124. 45 Pelletier, M., Forget, A., Bourassa, D., Gros, P. and Skamene, E., Immunopathology of BCG infection in genetically resistant and susceptible mouse strains. J Immunol 1982. 129: 2179‐2185. 46 Demangel, C. and Britton, W. J., Interaction of dendritic cells with mycobacteria: where the action starts. Immunol Cell Biol 2000. 78: 318‐324. 47 Noss, E. H., Pai, R. K., Sellati, T. J., Radolf, J. D., Belisle, J., Golenbock, D. T., Boom, W. H. and Harding, C. V., Toll‐like receptor 2‐dependent inhibition of macrophage class II MHC

(38)

expression and antigen processing by 19‐kDa lipoprotein of Mycobacterium tuberculosis. J Immunol 2001. 167: 910‐918. 48 Cooper, A. M., Appelberg, R. and Orme, I. M., Immunopathogenesis of Mycobacterium avium infection. Front Biosci 1998. 3: e141‐148. 49 Kaufmann, S. H. and Flesch, I. E., The role of T cell‐‐macrophage interactions in tuberculosis. Springer Semin Immunopathol 1988. 10: 337‐358. 50 Ramachandra, L., Chu, R. S., Askew, D., Noss, E. H., Canaday, D. H., Potter, N. S., Johnsen, A., Krieg, A. M., Nedrud, J. G., Boom, W. H. and Harding, C. V., Phagocytic antigen processing and effects of microbial products on antigen processing and T‐cell responses. Immunol Rev 1999. 168: 217‐239. 51 Korbel, D. S., Schneider, B. E. and Schaible, U. E., Innate immunity in tuberculosis: myths and truth. Microbes Infect 2008. 10: 995‐1004. 52 Holland, S. M., Host defense against nontuberculous mycobacterial infections. Semin Respir Infect 1996. 11: 217‐230. 53 Schreiber, H. A. and Sandor, M., The role of dendritic cells in mycobacterium‐induced granulomas. Immunol Lett 2010. 130: 26‐31. 54 Cooper, A. M., T cells in mycobacterial infection and disease. Curr Opin Immunol 2009. 21: 378‐384. 55 Appelberg, R., Pathogenesis of Mycobacterium avium infection: typical responses to an atypical mycobacterium? Immunol Res 2006. 35: 179‐190. 56 Russell, D. G., Dant, J. and Sturgill‐Koszycki, S., Mycobacterium avium‐ and Mycobacterium tuberculosis‐containing vacuoles are dynamic, fusion‐competent vesicles that are accessible to glycosphingolipids from the host cell plasmalemma. J Immunol 1996. 156: 4764‐4773. 57 Appelberg, R., Macrophage nutriprive antimicrobial mechanisms. J Leukoc Biol 2006. 79: 1117‐1128.

(39)

58 Dunlap, N. E., John Bass, J., Fujiwara, P., Hopewell, P., Horsburgh, C. R., Jr, Salfinger, M. and Simone, P. M., Diagnostic Standards and Classification of Tuberculosis in Adults and Children. This official statement of the American Thoracic Society and the Centers for Disease Control and Prevention was adopted by the ATS Board of Directors, July 1999. This statement was endorsed by the Council of the Infectious Disease Society of America, September 1999. Am J Respir Crit Care Med 2000. 161: 1376‐1395. 59 Irani, V. R. and Maslow, J. N., Induction of murine macrophage TNF‐alpha synthesis by Mycobacterium avium is modulated through complement‐dependent interaction via complement receptors 3 and 4 in relation to M. avium glycopeptidolipid. FEMS Microbiol Lett 2005. 246: 221‐228. 60 Gomes, M. S., Sousa Fernandes, S., Cordeiro, J. V., Silva Gomes, S., Vieira, A. and Appelberg, R., Engagement of Toll‐like receptor 2 in mouse macrophages infected with Mycobacterium avium induces non‐oxidative and TNF‐independent anti‐mycobacterial activity. Eur J Immunol 2008. 38: 2180‐2189. 61 Freeman, A. F., Olivier, K. N., Rubio, T. T., Bartlett, G., Ochi, J. W., Claypool, R. J., Ding, L., Kuhns, D. B. and Holland, S. M., Intrathoracic nontuberculous mycobacterial infections in otherwise healthy children. Pediatr Pulmonol 2009. 44: 1051‐1056. 62 Nobrega, C., Cardona, P. J., Roque, S., Pinto do, O. P., Appelberg, R. and Correia‐Neves, M., The thymus as a target for mycobacterial infections. Microbes Infect 2007. 9: 1521‐ 1529. 63 Aly, S., Laskay, T., Mages, J., Malzan, A., Lang, R. and Ehlers, S., Interferon‐gamma‐ dependent mechanisms of mycobacteria‐induced pulmonary immunopathology: the role of angiostasis and CXCR3‐targeted chemokines for granuloma necrosis. J Pathol 2007. 212: 295‐305. 64 Florido, M. and Appelberg, R., Genetic control of immune‐mediated necrosis of Mycobacterium avium granulomas. Immunology 2006. 118: 122‐130. 65 Florido, M. and Appelberg, R., Granuloma necrosis during Mycobacterium avium infection does not require tumor necrosis factor. Infect Immun 2004. 72: 6139‐6141.