Luận án tiến sĩ: Modulation of macrophage biology and host immune response by mycobacterial lipids

MODULATION OF MACROPHAGE BIOLOGY AND HOST IMMUNE RESPONSE BY MYCOBACTERIAL LIPIDS A Dissertation Submitted to the Graduate School of the University of Notre Dame in Partial Fulfillment o

INTRODUCTION

Background

Mycobacteria are intracellular pathogens that can survive within a host cell and thereby evade many of the host immune response A number of mycobacterial species are important human pathoges and are aetological agents of leprosy and tuberculosis In recent years, mycobacteria have emerged in the United Sates as major opportunistic pathogens in immuno-suppressed patients, particularly people with late-stage human immunodeficiency virus, chronic lung diseases or terminal renal disease and kidney transplantation Many of these patients develop disseminated mycobacterial infections, and a surprisingly high proportion of these isolates have been shown to be M avium or

Classification of mycobacteria

Mycobacteria have been conventionally classified into four or five groups based on the following general criteria: pathogenicity for humans and animals, rate of growth at optimum temperatures, and effect of visible light on pigment production Mycobacteria are slim rod shaped, non-motile and non-spore forming bacteria that are 1-10àm long Based on the classification given by Runyon and others in early 1950’s mycobacteria can be sub-categorized in two groups: atypical and typical mycobacteria The typical mycobacterial group has two prominent members: Mycobacterium tuberculosis and

Mycobacterium leprae The Mycobacterium avium Complex (MAC) is the member of the second group also termed as “mycobacteria other then tuberculosis” (MOTT) or “non- tuberculous mycobacteria” (NTM) MAC is further classified as acid-fast slow growing bacilli that may produce a yellow pigment in the absence of light and the fast growing bacilli that may or may not produce pigment Figure 1.1 summarizes some of the disease causing species of mycobacteria according to their groups

The M tb complex consists of M tuberculosis, M africanum, M canettii, M bovis, and M microti, sharing > 99% identity at the nucleotide level at some loci

However, these related members of typical group differ greatly in morphology, biochemistry, host range and disease patterns in experimental animals Currently, tuberculosis is the leading health problem, with two million deaths and nine million new cases emerging annually (Amin 2006) Since early 90s, the number of cases and deaths due to tuberculosis are decreasing in the developed countries; however TB remains a major problem in the developing countries Ninety percent of the estimated nine million new cases of TB each year occur in developing countries, which comprise 85% of the world’s population (Hopewell and Pai 2005)

TB is typically a disease of the lung and spreads through inhalation of cough droplets containing minute numbers of mycobacteria These inhaled mycobacteria are engulfed by alveolar macrophages and eventually transported to the draining lymph nodes However, only one in ten people infected by the bacilli actually develop disease

Figure 1.1 Mycobacterial species known to cause human disease The mycobacteria are listed according to their groups

Skin Pulmonary Dissemination Bone and joint

Dissemination Bone and joint Catheter-related

Catheter-related over a life time, as a healthy immune system keeps the infection controlled However, latent TB can reactivate years or decades later, usually due to weakened immune system

In recent years, more dramatic figures for TB patients have emerged due to the dangerous liaison between tuberculosis and acquired immunodeficiency syndrome (AIDS) Half a million people die every year because of the co-infection with mycobacteria and HIV (Harries, Chimzizi et al 2006) This problem is further complicated by increasing numbers of multi-drug resistant (MDR) strains These strains are posing huge problem and a serious health threat, particularly in Eastern Europe and Central Asia

1.2.2 Mycobacterium marinum and Mycobacterium ulcerans

M marinum causes a systemic tuberculosis-like granulamatous infection and disease in fish and frogs M marinum is one of the closest relatives of the M tuberculosis-complex organisms and is emerging as an interesting model for M tuberculosis pathogenesis

M ulcerans is the causative agent of Buruli ulcers, which are on the rise in certain areas of the tropics such as West Africa The bacterium secretes a cytotoxic polyketide toxin that produces extensive, painless, necrotic, and non-inflammatory ulcers where the bacteria grow extra-cellularly

This species is the causative agent of leprosy, a highly variable disease with a spectrum of clinical manifestations Leprosy infection ranges from the mildest intermediate form to the most severe lepromatous form of leprosy The intermediate leprosy is the earliest form of the disease with very few cutaneous lesions The tuberculoid form of leprosy usually presents with large lesions and infected nerve often thickens and results in loss of function Progression can lead to borderline type leprosy in which patients develop numerous cutaneous lesions Lepromatous leprosy develops in patient with no or very little immune resistance to M leprae, mainly lacking the cell mediated immunity The early lesions of lepromatous leprosy are multiple, symmetrically distributed, erythromatous ill-defined macules and papules Nerve damage associated with leprosy is a consequence of immune mediated killing of Schwann cells and subsequent loss of the myelin sheath, responsible for protecting nerve axons

The M avium complex (MAC) is a group of related environmental mycobacteria, including M avium subspecies avium, paratuberculosis, and silvaticum, and M intracellulare The M avium complex consists of at least 31 different serotypes that can be readily distinguished from one another both on the basis of their animal virulence patterns and their serology (Chatterjee and Khoo 2001) M avium was first isolated from birds but has long been recognized as virulent in both humans and animals Members of the MAC complex in pre-AIDS era caused only pulmonary infections in the elderly people or localized lymphadenitis in young children but with the advent of HIV, the MAC emerged as the major opportunistic infection in the immuno-comprised individuals

Up to 50% of the AIDS patients have been reported to have systemic MAC infections at later stages of disease, mostly involving serotypes 1, 4 and 8 (Kiehn, Edwards et al 1985) The most prominent risk factor for the development of the disseminated MAC infection are a CD4 + -cell count of less than 100/àl and previous colonization of mucosal surfaces with MAC (Mapother and Songer 1984; Klatt, Jensen et al 1987; Horsburgh 1991) M avium paratuberculosis is a significant pathogen of livestock causing Johne’s disease and has been implicated in the Crohn’s disease, a debilitating inflammation of the intestines in humans

Another interesting feature of the MAC is the occurrence of the colony type variations M avium may appear in three different colony variants: (i) smooth, opaque, and domed; (ii) smooth, transparent, and flat; and (iii) rough Most often, clinical isolates of MAC usually appear as smooth transparent and flat suggesting a higher pathogenicity for this morphotype Isogenic colony variants of MAC differ in their virulence, susceptibility to antibiotics, stimulation of oxygen radicals and cytokines The virulent smooth transparent colony variants are more frequently isolated from the AIDS patients, more efficient in mucosal colonization, and adhere more efficiently to epithelial cells as compared to the less virulent smooth opaque variants (Reddy, Luna-Herrera et al 1996) Despite the apparent relationship between colony morphotype and antimicrobial resistance as well as pathogenicity, very little is known about the genetics and the regulation of colony type variation.

Mycobacterial cell wall

One of the best-studied aspects of mycobacteria is the structure and function of the mycobacterial cell wall which confers upon these unusual gram positive bacteria their resistance to most common antibiotics and chemotherapeutic reagents The chemical model was proposed by Minnikin (Minnikin 1991) and further elaborated by studies of Brennan and Nikaido (Brennan and Nikaido 1995) The architecture of mycobacterial cell wall is based on electron microscopy studies and is composed of a variety of soluble proteins, carbohydrates, and lipids and basically three unique insoluble macromolecular components: arabinogalactan, peptidoglycan and mycolic acid Essentially, four major layers can be envisioned for the cell envelope of mycobacteria, as shown in figure 1.2 The first layer, plasma membrane, is similar to those found in other bacteria, consisting of a permeable lipid bilayer The second layer is the peptidoglycan/ arabinogalactan rich layer that makes up the basic structural component of the cell wall Adjacent to this is an electron transparent layer rich in mycolic acids followed by the outermost layer rich in many complex and strain specific lipids including lipoarabinomannan (LAM), phosphoinositol-mannoside (PIM), glycopeptidolipids (GPL), and 19kDa lipoprotein Some of these complex and unique lipids are summarized in figure 1.3 along with their biological roles

Mycolic acids are high-molecular-weight α-alkyl, β-hydroxy fatty acids which are present mostly as bound esters of arabinogalactan However, the mycobacterial mycolic acids are distinguishable from those of the other genera such as Corynebacterium,

Nocardia and Rhodococcus because of the increased length and complexity

Another ubiquitous lipid constituent of the mycobacterial cell wall is the lipoarabinomannan (LAM) LAMs and its related precursors, lipomannan (LM) and phosphatidyl-myo-inositol mannosides (PIMs) are major lipoglycans that are non- covalently attached to the plasma membrane through their phosphatidyl-myo-inositol (PI) anchor and extend to the exterior of the cell wall (Besra, Morehouse et al 1997) In

Figure 1.2 A chemical model of the mycobacterial cell wall Adapted from

Mycobacterial lipoarabinomannan: an extraordinary lipoheteroglycan with profound physiological effects, Delphi Chatterjee and Kay-Hooi Khoo

COMMON MYCOBACTERIAL LIPIDS AND THEIR FUNCTIONS

Figure 1.3 List of some common mycobacterial lipids present on mycobacterial cell wall

• Stimulates fusion between phagosome and early endosomes (Vergne et al., 2004)

• Contribute to the avoidance of phagosomal acidification (Vergne et al., 2004)

• Inhibits phagosome maturation (Fratti et al., 2003)

• Inhibits recruitment of EEA1 (Fratti et al., 2001)

• Inhibits Ca 2+ rise (Vergne et al., 2003)

• Enhance resistance to degradation (Hines et al., 1995)

• Induce TNF-α, IL-6 and IL-1β (Barrow et al.,)

• GPL coated staphylococcus cells showed enhanced phagocytosis (Takegaki et al., 2000)

• Promote entry of M leprae into Schwann cells (Ng et al., 2000)

• Suppress superoxide production (Vachula et al., 1989)

Mycolic acids • Induce granuloma formation

(Hines et al., 1995) addition to the PI anchor, LAM possesses a mannan core with a branched arabinan polymer and in some cases, this structure is further decorated by cap motifs that includes either mannosyl or phosphoinositide residues Based on the capping motif, LAM can be classified in three different groups as follows: “ManLAM” when Mannosylated or

“PILAM” when containing phosphoinositide caps or “AraLAM” when uncapped Generally, most pathogenic, slow-growing species of mycobacteria contain ManLAM, whereas the fast-growing, non-pathogenic species M smegmatis, M fortuitum contain

PILAM and AraLAM has recently been identified in M chelonae (Guerardel, Maes et al 2002) Considerable studies have been done in identifying the role of LAM in the pathogenesis of mycobacteria and its affect on the host immune response LAM has been shown to inhibit the IFN-γ signaling (Knutson, Hmama et al 1998), M tuberculosis infection-induced apoptosis of macrophages (Rojas, Barrera et al 1997) and the secretion of IL-12 induced by LPS in DCs (Nigou, Gilleron et al 2002) LAM also induced abrogation of T cell activation (Kaplan, Gandhi et al 1987), scavenging of potentially cytotoxic oxygen free radicals (Chan, Fan et al 1991) and induced large array of cytokines associated with macrophages like TNF-α, IL-12, Il-1 and IL-6 (Roach, Barton et al 1993); (Barnes, Chatterjee et al 1992) Moreover, antibodies against LAM have been detected in M tuberculosis infected individuals (Julian, Matas et al 1997), suggesting that in vivo, LAM may be important in disease progression

Another distinguishing lipid present in the mycobacterial cell wall is the phenolic glycolipids (PGL) Phenolic glycolipids are found in several obligate and opportunistic mycobacterial pathogens; these include members of the M tuberculosis complex, M leprae, M kansasii, M marinum, M haemophilum, and M ulcerans The lipid core of

PGL is composed of phenol-phthiocerol and relatives (long chain C33-C41 beta-diols) which are esterified by two multi-methyl-branched C27-C34 fatty acids (mycocerosic or phthioceranic acids) The sugar moiety of PGL is species specific and consists of one to four O-methylated sugars (principally deoxysugars) These glycolipids were first isolated from Mycobacterium leprae and shown to be involved in the immnunogenicity and the pathogenicity of the mycobacteria (Hunter and Brennan 1981) Mycobacterial PGLs have been shown to suppress cytokine release from human monocytes (Silva, Faccioli et al 1993) and lymphoproliferative response (Brett, Lowe et al 1984) and interfere with the antigen presentation function (Hashimoto, Maeda et al 2002) Recently, PGLs from M tb (W-Beijing family) have been shown to inhibit the innate immune response (Reed, Domenech et al 2004)

Another class of surface mycobacterial glycolipids is called glycopeptidolipids (GPLs) also referred as C-mycosides or Shaefer antigens GPLs are the major cell surface antigens of the M avium, M intracellulare, M scrofulaceum group and are used to subcategorize these mycobacterial species into 31 distinct serotypes based on the serospecific GPLs Most GPLs are the alkali-stable C-type GPLs; however variant forms of GPL have been reported like alkali-labile serine containing GPLs of the M xenopi (Riviere, Auge et al 1993) As shown in figure 1.2, the GPLs consist of a tripeptide amino alcohol (D-Phe-D-allo Thr-D-Ala-l-alaninol) core modified with an amide-linked 3-hydroxy and 3-methoxy C26-34 fatty acid, a methylated rhamnose (Rhap) and a 6- deoxytalose GPLs vary in the extent of glycosylation of the allo Thr and/or alaninol

Figure 1.4 Structure of glycopeptidolipid Adapted from Mycobacterial lipids: A Historical Perspective; Jean Asselineau and Gilbert Lanéelle

GPLs are divided into two different groups: (i) polar GPLs and (ii) apolar GPLs Apolar GPLs are non-serovar specific GPLs (nsGPLs), which are singly glycosylated The apolar GPLs contain a 3-O-methoxy-6-deoxy-Tal attached to the allo- Thr and either a 3-O- methoxy-Rha or a 3, 4-di-O-Me-Rha attached to the alaninol Addition of different oligosaccharides to nsGPLs, leads to synthesis of serovar specific GPLs (ssGPLs) In the ssGPLs, oligosaccharides were found to be attached to the allo Thr Each serovar of the MAC is distinguished by the oligosaccharide linked to the D-allo Thr of its ssGPL The figure 1.3 summarizes different serovars showing oligosaccharides attached to the core structure of M avium GPLs

Experiment with human peripheral blood monocytes (PBMCs) have established that GPLs from M avium can induce TNF-α, IL-6 and IL-1β (Barrow, de Sousa et al 1993) Studies by Vergne et al., demonstrated the ability of mycobacterial GPLs to become inserted into phospholipids monolayer suggesting that GPLs can alter host responsiveness by interaction with cell membranes (Vergne, Prats et al 1995) Certain serovars are known to be associated more with active disease such as serovars 1, 4 and 8 which have been isolated frequently in humans infected with HIV (Horgen, Barrow et al 2000) Also, serovar 4 GPL coated staphylococcal cells showed enhanced phagocytosis and marked inhibition of phagosome-lysosome fusion (Takegaki 2000) However, most studies published prior to our work have used purified mycobacterial GPLs for illustrating the affect of GPL on host immune response This introduces complexity in interpreting the data regarding the GPL-receptor interaction at the cell surface and uptake of GPL We have addressed this problem by defining the role of GPL in the context of whole mycobacteria in this dissertation We utilized mycobacteria modified to lack GPL

TYPES OF OLIGOSACCHARIDE CHAIN ATTACHED TO ns-GPL

Figure 1.5 Different serovars showing oligosaccharides attached to the core structure of GPLs

Serovar Oligosaccharide structure linked to D - allo -threonine

2 - L -6-dtal-(2 1)- - L -Rha-(3 1)-2,3-di-O-Me- - L -Fuc

3 - L -6-dtal-(2 1)- - L -Rha-(3 1)-2,3-di-O-Me- - L -Fuc-(4 1)

9 - L -6-dtal-(2 1)- - L -Rha-(3 1)-2,3-di-O-Me- - L -Fuc-(4 1)-

- D -GlcA-(4 1)-4-O-Ac-2,3-di-O-Me- - L -Fuc

14 4-formamido-4,6-dideoxy-2-O-Me-3-C-Me-a-L-Manp-(1-3)-2-O-

19 3,4-di-O-Me-β-D-GlcpA-(1-3)-3-C-Me-2,4-di-O-Me-a-L-Rhap-

25 2-O-Me-a-D-FucpNAc-(1-4)-β-D-GlcpA-(1-4)-2-O-Me-a-L-Fucp-

26 2,4-di-O-Me-a-L-Fucp-(1-4)-β-D-GlcpA-(1-4)-2-O-Me-a-L-

Fucp-(1-3) expression at the cell surface either naturally or by genetic manipulation to establish the role of GPL in mycobacteria pathogenesis.

Biology of macrophages

Intracellular pathogens reside within a niche of the host where they can multiply and avoid the host cellular and humoral defense mechanisms The host cell of choice for many pathogens including mycobacteria is the macrophage Macrophages can ingest invading microorganisms and other foreign materials by phagocytosis Phagocytosis is the uptake by the cell of relatively large particles (>0.5àm) into vacuoles and is a central mechanism in the tissue remodeling, inflammation, and defense against infectious agent Unfortunately, phagocytosis is also a mechanism by which pathogens including mycobacteria invade host cells The phagocytic process can be dissected into the following steps: 1) binding of the particle to the phagocyte cell surface; 2) invagination of the plasma membrane to accommodate the incoming particle; 3) closure of the nascent phagosome and detachment from the plasma membrane; 4) maturation of the phagosome The phagosome maturation involves a series of fusion/ fission processes with vesicles in the cell to eventually form phagolysosome The phagolysosome is an acidic compartment enriched in hydrogen peroxide, oxygen free radicals, peroxidase, lysozymes and hydrolytic enzymes that creates hostile environment for the microbe enclosed in the phagolysome.

Mycobacterial entry in macrophage

Binding and phagocytosis of mycobacteria can be mediated by different kinds of receptors on the host cell including complement receptors (CR), mannose receptors, surfactant receptors, scavenger receptors and GPI-anchored receptors such as CD14

Among the phagocyte receptors that recognize mycobacteria, the complement receptor 3 (CR3), also named integrin αMβ2, or CD11b/CD18, plays an important role in the phagocytosis of the bacterium CR3 is a hetero-dimeric surface receptor that is expressed on neutrophils, monocytes, macrophages, and natural killer cells (Dana, Fathallah et al 1991) Some of the ligands interacting with CR3 include complement fragment iC3b, intracellular adhesion molecule-1 (ICAM-1), and bacterial products (Bilsland, Diamond et al 1994) Complement-opsonized phagocytosis of mycobacteria plays an important role in the pathogenesis of mycobacteria because it affects the phagosome-lysosome fusion and permits the survival of the bacterium Uptake by complement receptor is not accompanied by inflammatory responses (Aderem and Underhill 1999) Mycobacteria can be opsonized by C3b and iC3b after activation of the complement pathway and interact with complement receptors CR1, CR3 and CR4 (Schlesinger, Bellinger-Kawahara et al 1990) However, CR3 mediates approximately 80% of complement-opsonized M tuberculosis phagocytosis (Schlesinger, Bellinger-Kawahara et al 1990) Owing to multiple recognition sites, CR3 can also mediate non- opsonized phagocytosis (Le Cabec, Carreno et al 2002)

1.5.2 Mannose receptor mediated uptake of mycobacteria

The mannose receptor on macrophage recognizes mannose and fucose on the surfaces of pathogens and mediates phagocytosis of the organisms The MR is a single chain receptor with a short cytoplasmic tail and an extracellular domain including 8 lectin-like carbohydrate-binding domains The cytoplasmic tail is crucial to both the endocytic and phagocytic functions of the receptor There is evidence showing a role for the MR in mediating the uptake of both pathogenic and non-pathogenic mycobacteria (Astarie-Dequeker, N'Diaye et al 1999) Further, entry through MR constitutes a safe portal as MR-mediated phagocytosis is not coupled to NADPH oxidation or phagosome fusion with lysosome (Astarie-Dequeker, N'Diaye et al 1999)

Scavenger receptors (SRs) are a family of structurally diverse receptors having broad ligand specificity that includes LDL, phosphatidylserine and polyanionic compounds Different types of SRs have been identified, but two members of the family play a prominent role in internalizing microbes Scavenger receptor A (SR-A) is a transmembrane homo-trimer with an extended extracellular domain composed of a collagenous triple-helix It is expressed on most macrophages and bind whole mycobacteria as well as microbial cell wall components

MARCO (macrophage receptor with collagenous structure), another member of the scavenger family receptor, also participates in the phagocytosis of microbes MARCO is expressed constitutively on macrophage and binds a variety of particles including gram-positive and gram-negative bacteria Recently, macrophage infection by BCG showed up-regulation of MARCOS on macrophages (van der Laan, Dopp et al 1999)

Membrane CD14 is expressed in mature myeloid cells, and to varying extents in most human tissue macrophages and in gingival fibroblasts Since, CD14 lacks transmembrane and intracellular domains, it has been suggested that CD14 functions in concert with some other receptors CD14 has been reported to be a membrane receptor for various mycobacterial components such as peptidoglycan and lipoarabinomannan; although exact signaling pathways are yet to be elucidated (Dziarski, Ulmer et al 2000).

Mycobacterial invasion of macrophage

Acute mycobacterial infections are controlled by the healthy immune system through secretion of various immune mediators like TNF-α, IFN-γ, IL-12, IL-23, lymphotoxins, CD40 and nitric oxide On the other hand mycobacteria have also devised mechanisms to evade these potent immune mediators Consequently, one mandatory requirement is to maintain a constant immunological pressure by activation of phagocytes, primarily through T cells This continuous activation can also occur through the interaction of different mycobacterial components released by the sequestered bacilli with pattern recognition receptors (PRRs) The macrophage has a number of receptors which include complement receptors, the immunoglobulin receptors (FcR) and the pattern-recognition receptor (PRR) such as the mannose receptor (MR), CD14 and the Toll-like receptors (TLR) Figure 1.6 summarizes different types of PRRs and the ligands which interact with them

Mammalian TLRs represent a structurally conserved family of membrane receptors, which have homology to the Drosophila Toll system TLRs are type I membrane proteins containing an extra-cellular domain with 19-25 tandem copies of

• TLR2 Bacterial lipoproteins, lipomannan, arabinosylated lipoarabinomannan (AraLAM), phosphoinosylated LAM, phosphoinositolmannoside

Peptidoglycans, muramyl dipeptides, diamino pimelated containing N-acetyl glucosamine-N-acetyl muramic acid Galectin-3 L major-specific polygalactose epitopes Scavenger Receptor LPS, teichoic acid, peptidoglycan

Mannose Receptor Terminal mannose and fucose residues on microbial glycoproteins, lysosomal proteases, glycosidases and peroxidases

DC-SIGN HIV-1 envelope glycoprotein gp120,

CD44 Receptor for binding of M tb on macrophage and for phagocytosis Complement Receptor Zymosan, bacterial lectins

Figure 1.6 List of pattern recognition receptors and some of their respective pathogen associated molecular patterns (PAMPS) leucine-rich repeats (LRR) and a cytoplasmic Toll/IL-1 receptor (TIR) domain similar to that of the interleukin 1 (IL-1) receptor family (Lasker and Nair 2006) TLR signal transduction, initiated by the ligands-receptor interaction, is mediated by the binding of the adaptor protein MyD88 to the TIR domain of the receptors This is followed by the recruitment of IL-1 receptor associated kinases (IRAK), TNF receptor associated factor (TRAF 6), TGFβ-activated protein kinase 1 (TAK1), mitogen-activated protein kinases (MAPK) and NF-κB activation (West, Koblansky et al 2006) Recently, MyD88 independent toll-like receptor activation have been reported occurring through either Mal/TIRAP or TIR-containing adaptor molecule-1 (TICAM-1) (Kawai, Takeuchi et al 2001)

Several receptors other than TLRs have been shown to recognize ligands on the mycobacterial surface Nucleotide-binding oligomerisation domain (NOD) proteins belong to a TLR-related protein family with LRR domains NOD proteins recognize ligands such as peptidoglycans, muramyl dipeptides and diaminopimelate-containing N- acetylglucosamine-N-acetyl-muramic acid tripeptide Other group of receptors unrelated to TLR include CD14, scavenger and complement receptor, pulmonary surfactant protein

A, dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN), CD40 and CD44 The cross-talk between different PRRs and other non-PRRs receptors can further boost the immune response and therefore accentuate the protection against the mycobacteria.

Mycobacterial lifestyle inside macrophages

Normally, macrophage activation results in a series of events designed to induce killing of engulfed microorganisms These include: (i) a gradual acidification of the phagosome due to the activity of a protein-ATPase pump located in the phagosomal membrane, (ii) phagosome-lysosome fusion, which loads the resulting phagolysosome with proteolytic enzymes, (iii) induction of reactive oxygen and nitrogen intermediates and (iv) antigen processing Once mycobacterium enters the host, it undergoes a rapid phagocytosis, mediated by different receptors For the survival of many intracellular pathogens, the ability to avoid or delay the acidification of the phagosome is essential for their successful survival inside the cell Similarly, persistence of the mycobacterium in the host cell and the subsequent infectious potential depends on its ability to inhibit phagosome maturation It was D’Arcy Hart’s work which showed for the first time that in a cell infected with the M tb, the fusion between the phagosome and the lysosome is suppressed (Hart, Young et al 1987) However, this arrest of phagosome maturation is still not well understood but recent work has provided some insight into this process The mycobacteria containing phagosome has been shown to fuse with the surrounding early endosomes and thus acquires endosome associated markers such as Rab5 and the transferrin receptors while remaining negative for lysosomal markers such as lysosome associated membrane protein 1 (LAMP1) and proton ATPases (Sturgill-Koszycki, Schlesinger et al 1994; Sturgill-Koszycki, Schaible et al 1996; Sturgill-Koszycki, Haddix et al 1997) Without lysosomal interaction, the phagosome does not acidify and the bacillus remains intact and metabolically active within the early endocytic environment (Deretic, Via et al 1997; Fratti, Vergne et al 2000; Kelley and Schorey 2003; Vergne, Chua et al 2003; Kelley and Schorey 2004) Thus, mycobacteria containing phagosomes do not participate in the normal series of fusion and fission events which result in the acquisition and elimination of markers associated with the maturation of a phagosome into a phagolysosome.

Immunity to mycobacteria

As critical as it is for mycobacteria to reside inside the macrophage to perpetuate infection, it is more important from the host perspective to eliminate the pathogen via the activation of microbicidal mechanisms Further, only one in ten people exposed to M tb, actually develops the disease, again strengthening the fact that a healthy immune system is able to contain the mycobacterial infection According to the most recent research in basic immunology and microbiology, immunity to mycobacteria is based on a complex interaction between macrophage and several T cell subsets This concerted action is mediated and directed by cytokines and chemokines

Phagocytosis and processing of mycobacterial antigens by macrophages trigger a specific cellular immune response, including the activation of T helper cells, macrophages, T-cytotoxic cells and natural killer (NK) cells Following mycobacterial infection, processed mycobacterial antigens are presented on cell surface, in association with MHC molecules T cell subsets including CD4 + and CD8 + T cells have been proposed to play a critical role in the immune response against mycobacteria The main function of CD4 + T cells is thought to induce IFN- γ production, which is essential for macrophage activation CD8 + T cells can also produce cytokines and promote cytolytic functions directed against mycobacteria infected macrophages Finally, NK cells are believed to be involved as a first line of defense and exhibit non-MHC restricted cytotoxic activity (Scharton and Scott 1993)

Beside cellular immunity, another mode of controlling the mycobacterial infection involves the secretion of immune mediators This includes the release of numerous cytokines and chemokines by activated immune cells Two of the most important cytokines in macrophage activation are interferon γ (IFN- γ) and TNF-α generated by TH-

1 cells TH-2 cells on the other hand are a source of IL-4 and IL-10 The counter regulatory activity of TH-2 cells is mediated by IL-4 resulting in a down regulation of TH-

1 responses (Mosmann and Coffman 1989) Another cytokine playing an important role in the immunity against mycobacteria is IL-12 which is also essential for a TH-1 response Following IL-12 secretion, TH-1 cells are recruited to the site of infection and are stimulated to produce IFN-γ (Flynn, Chan et al 1993) Large amounts of chemokine like MCP-1, RANTES, and MIP are also secreted to increase the influx of immune cells at the site of infection

The activation of the macrophage greatly enhances its bactericidal and bacteriostatic capabilities with the production of reactive oxygen intermediates, such as hydrogen peroxide and nitric oxide Hydrogen peroxide, one of the ROI generated by the macrophages via the oxidation burst, was the first identified effector molecule that mediated mycobacterial effects of mononuclear phagocytes (Walker and Lowrie 1981) Phagocytes upon activation by IFN-γ and TNF-α generate nitric oxide and related RNI via inducible nitric oxide synthase (iNOS) High level expression of NOS2 has been detected in macrophages of patients with active pulmonary TB (Nicholson, Bonecini-Almeida Mda et al 1996).

Trafficking of Mycobacterial lipids

Besides providing a habitable environment, the mycobacteria containing phagosome can also act as a platform for the accumulation and sorting of mycobacterial components to different compartments in the infected cell The trafficking of mycobacterial lipids is of particular interest as mycobacteria produce copious amounts of complex lipids, constituting almost 60% of the cell wall (Lee, Brennan et al 1996; Daffe and Etienne 1999) The transport of the lipids out of the phagosome will be dependent on their release from the mycobacteria as well as the membrane kinetics between the phagosome and the vesicular compartments Studies by David Russell demonstrated the trafficking of hydrazide-labeled mycobacterial lipids in the mycobacteria infected cells indicating their release from the mycobacterial cell wall (Beatty, Rhoades et al 2000) There is evidence showing LAM integration into the plasma membrane of murine and lymphomonocytic cells (Ilangumaran, Arni et al 1995) As a result of this insertion, the membrane domains or signaling complexes could be disrupted, affecting vesicle trafficking and cellular signaling Indeed, Man-LAM has been implicated in disrupting phagosome-lysosome fusion (Hmama, Sendide et al 2004) as well as promoting SHP-1 phosphatase activity (Knutson, Hmama et al 1998) Studies by Beatty et al showed some lipids like Man-LAM distributed throughout the cells, whereas lipids like PIM showed a more selective localization in the cells (Beatty, Rhoades et al 2000) Therefore, the ability of mycobacterial lipids to leave the mycobacterial phagosome and traffic to other compartments within the infected cell and their potential to traffic outside the infected cells may play a noteworthy role in mycobacterial pathogenesis and host response

Glycopeptidolipids have been shown to play an important role in the pathogenesis of M avium (Krzywinska, Bhatnagar et al 2005; Bhatnagar and Schorey 2006) We explored the hypothesis that GPL can be released from the M avium surface and therefore accumulate inside the mycobacteria containing phagosome Based on the ability of mycobacterial lipids to insert in the membranes and also the continuous membrane movement in the host endocytic pathway, we investigated the trafficking of GPL in the

Macrophage endocytic pathway

Eukaryotic cells secrete proteins from the biosynthetic pathway by constitutive exocytosis of secretory vesicles or by regulated release of secretory/storage granules upon appropriate stimulation However, recently the endocytic pathway has also been demonstrated to be an alternative secretory pathway (van Niel, Porto-Carreiro et al 2006) Electron microscopy showed endocytic compartments with numerous intra luminal membrane bound vesicles (ILV), collectively called the multivesicular bodies (MVB) MVBs are part of the pleiomorphic endosomal system, which consists of primary endocytic vesicles, early endosomes (EEs), late endosomes (LEs) and lysosomes Early endosomes are the entry site for any endocytosed material whereas LEs are the site for accumulating newly synthesized lysosomal hydrolase directly from trans-golgi network Lysosomes are the final destination in the endocytic pathway and the site for ultimate degradation of protein and lipid The MVB is an intermediate compartment that is formed from endosomes by invagination of the limiting endosomal membrane MVBs were first described by Pan and Johnstone in the erythrocytes (Pan and Johnstone 1983), but only recently the presence of MVBs have been established in different cell types; including B lymphocytes (Raposo, Nijman et al 1996), dendritic cells (Zitvogel, Regnault et al 1998), platelets (Heijnen, Schiel et al 1999), epithelial cells (van Niel, Raposo et al

2001), reticulocytes (Johnstone, Adam et al 1987) and neurons (Marzesco, Janich et al 2005)

Although the formation of MVB was proposed almost forty years ago, the underlying mechanism for the biogenesis of MVB is not yet well defined So far, two mechanisms are described to play an important role in the biogenesis of MVBs in mammalian cells (i) annexin II is part of the core machinery that regulates the MVB biogenesis and associates with the endosomes forming cholesterol-rich platforms This organizes the membrane of early endosomes and initiates the internal budding process leading to MVB formation (Harder, Kellner et al 1997; Oliferenko, Paiha et al 1999) (ii) an endosomal coatomer protein (COP) complex and ARF1 (ADP-ribosylation factor-1) are proposed to have a role in the MVB biogenesis from the late endosomes (Aniento and Gruenberg 1995; Whitney, Gomez et al 1995; Gu, Aniento et al 1997) Figure 1.7 shows a schematic of formation of MVB and its subsequent fate in the cell

Unlike the complexity in defining the formation and origin of MVBs, the function of the MVBs has received considerable attention over the years because of their central role in the cellular endocytic trafficking In most cell types, the MVB are incorporated into the lysosomal compartment and thus shuttling the MVB cargoes for degradation But in recent years several other functions have been attributed to MVBs, including the down regulation of signal transduction by plasma membrane receptors (Kramer 2002) MVB are reported to be a sorting compartment for lipids such as lysobisphaospatidic acid (LBPA) (Kobayashi, Startchev et al 2001) LBPA rich membranes are suggested to regulate intracellular cholesterol transport (Laulagnier, Motta et al 2004) MVB

Figure 1.7 Schematic of formation of MVB and its subsequent fate in the cell comprise distinct organelle in certain cell types including Weibel-Palade bodies (secretory organelle) in human endothelial cells (Romani de Wit, Rondaij et al 2003), platelet dense granule in platelets (Heijnen, Debili et al 1998) and melanosomes in melanocytes (Huizing, Anikster et al 2001) Interestingly, MVBs also serve as a storage site for major histocompatibility complex II (MHCII) and are therefore a potential site for antigen presentation (Kleijmeer, Escola et al 2001) MVB have been shown to fuse with the plasma membrane and release the ILVs as “exosomes” (Denzer, Kleijmeer et al 2000) The exosomes thus released from a cell can act as a messenger from one cell to the other neighboring cell and mediate an important function in intercellular communication

Exosomes are a homogenous population of membrane vesicles with a diameter of 50-100 nm that are secreted by many cell types into the extracellular milieu Exosomes were also defined as micro vesicles containing 5’-nucleotidase activity that were released from the neoplastic cell lines (Trams, Lauter et al 1981), but they are distinct from the more recently described ribonuclease complex that is also called exosome (Mitchell, Petfalski et al 1997) Electron-microscopic studies have shown the release of ILVs by the fusion of limiting membrane of MVB with the plasma membrane in different cell types of hematopoietic origin, such as cytotoxic T lymphocytes (CTLs) (Kast, Offringa et al 1989), Epstein-Barr virus (EBV) transformed B cells (Raposo, Nijman et al 1996), mastocytes (Raposo, Tenza et al 1997), dendritic cells (DCs) (Zitvogel, Regnault et al 1998; Thery, Regnault et al 1999), platelets (Heijnen, Schiel et al 1999), macrophages (Savina, Fader et al 2005) and non-hematopoietic origin like neurons (Marzesco, Janich et al 2005)

The protein composition of exosomes has been very well characterized The presence of various proteins on exosomes has been analyzed by western blotting and fluorescence-activated cell-sorting (FACS) Although the protein and lipid profile on the exosome varies between cells types, a general picture of different cargoes carried on the exosomes have emerged by proteomics analysis The cytosolic proteins present on exosomes include tubulins, actin and actin binding proteins, annexins and Rab proteins helping in the exosome biogenesis, docking and the membrane fusion events (Harder, Kellner et al 1997) A series of molecules involved in the signal transduction like protein kinases, 14-3-3 and heterotrimeric G proteins as well as the metabolic enzymes including peroxidases, pyruvate and lipid kinases, and enolase-1 are also reported to be present on the exosomes (Hegmans, Bard et al 2004) Exosomes also contain heat shock protein HSP70 and HSP90 facilitating the loading of peptides on MHCI and MHCII complexes (Gastpar, Gehrmann et al 2005) The characteristic feature of exosomes is the transmembrane tetraspannins which include CD9, CD63, CD81 and CD82 Exosomes also carry some cell specific proteins like MHCII molecules on exosomes from antigen presenting cells (APCs) and CD86 (Segura, Amigorena et al 2005) and milk-fat-globule EGF-factor VIII (MFGE8)/lactadherin on DCs exosomes (Veron, Segura et al 2005) Lipid components like LBPA, phosphatidylserine (PS) and cholesterol rich domains are also present on exosomes (de Gassart, Geminard et al 2003)

Although different proteins have been recognized on the exosome, there is still a significant gap in our understanding of the signals required to target these proteins to exosomes for elimination However, two theories have become popular over these years (i) since many proteins are common to most cell types, it has been suggested that proteins which are not attached to the cytoskeleton are targeted for externalization via exosomes

(de Gassart, Geminard et al 2004) (ii) a second hypothesis suggests that protein targeted for exosomes require mono-ubiquitination (Stoorvogel, Kleijmeer et al 2002; Buschow, Liefhebber et al 2005)

The biological function of exosomes is dictated by the molecules exposed on the exosomes and the cell type of the origin Earlier the major function defined for exosomes was the elimination of the waste –an economical alternative to lysosomal degradation But since exosomes can bear spectrum of surface molecules, they can provide a mechanism for engaging different cell receptors simultaneously and exchange material between cells without the cells coming in contact Therefore, it is tempting to hypothesize that exosomes can function to facilitate communication between cells In the pioneering studies by Raposo et al., she showed that exosomes secreted by EBV- transformed B cells can stimulate human CD4+ T-cell clones in an antigen specific manner (Raposo, Nijman et al 1996) Exosomes produced by mouse DCs pulsed with tumor peptides induced the rejection of established tumors in a T cell dependent fashion (Zitvogel, Regnault et al 1998) Exosomes can also transfer antigens from the tumor cell to DCs (Wolfers, Lozier et al 2001) There is evidence suggesting that exosomes function in cross-presentation of antigens Like DC- derived exosomes, exosomes from tumor cells carry MHC molecules along with tumor antigens like melan-A/MART1 (melanoma tumor), which are recognized by T cells in the presence of DCs Exosomes from bone-marrow derived DC can prolong rat heart allograft survival (Peche, Heslan et al 2003) Further, an in vitro test showed significant decrease of CD4+ T cells in the exosome treated recipient, suggesting immuno-tolerance effects (Peche, Heslan et al 2003) Other than the antigen presentation, exosomes have been shown to be involved in some other immune regulation Mast-cell derived exosomes induce blast formation, cell proliferation and the production of IL-2 and IFN-γ by spleen cells (Skokos, Le Panse et al 2001)

Exosomes are not only beneficial for the host cells but pathogens have also devised mechanisms to utilize exosomes for enhancing their survival Recent studies with retroviruses have revealed the ability of viruses to hijack the intracellular machinery of MVB for their budding at the cell surface (Booth, Fang et al 2006) HIV utilizes MVBs as the major site for budding and accumulation in human macrophages (Pelchen- Matthews, Raposo et al 2004) Once inside the MVBs, viruses bud off as exosomes and thus escape the host surveillance, hence favoring the spread of infection However, one of the caveats in summarizing the physiological exosome function is that the studies published so far are done in vitro and the significance of exosomes is yet to be established in vivo We explore in this dissertation, the hypothesis that mycobacterial components that are trafficked to MVBs in the infected macrophage are released via exosomes These exosomes carrying mycobacterial lipids and proteins can activate the immune surveillance system of the host

I Investigate the role of glycopeptidolipids as virulence factors by studying the modulation of macrophage response to mycobacteria expressing GPL in comparison to the morphotypes lacking or expressing modified GPL The response to the modified mycobacteria is evaluated in context of:

1 Signaling cascades activated in bone-marrow derived macrophages

2 Virulence studies using mouse infection model

3 The mechanism of attenuation of virulence e.g., cytokines or chemokines

Previous to this work, most studies done to address the role of GPL in virulence were done with purified GPLs Our studies will define the role of GPL as virulence factor in the context of mycobacteria and thus the results will mimic more closely the physiological picture

II GPL transport/trafficking in the macrophage infected by GPL sufficient mycobacteria

1 To understand the trafficking of GPLs in macrophages infected with M avium

2 Characterize the cellular compartment to which GPLs are localizing

3 Understanding the mechanism of cell to cell transfer of glycopeptidolipids

The mycobacteria cell wall is composed of almost 60% lipids and these lipids can be shed and released in the mycobacteria infected cells We wish to address the release and trafficking of GPLs in the M avium infected macrophages These studies will help us to understand the role of GPL in modulating the host membrane properties and its affect on the immune response

III Defining whether exosomes which carry mycobacterial components function in macrophage activation

There is significant evidence indicating the transport of mycobacterial components into MVBs, with subsequent release of these components into the extracellular environment as part of the exosomes Our studies will help define the role of these exosomes, which carry mycobacterial lipids and proteins, in modulating the response of the neighboring cells to the mycobacterial infection We hypothesize that the exposure of the bystander cells to these modified exosomes will act as a warning signal and thus prepare the surrounding cells to control the mycobacterial infection.

Mycobacterium avium 104 deleted of the methyltransferase D gene by

Introduction

Mycobacteria have a long history as infectious organisms and are the aetiologic agents of numerous human diseases One such disease is caused by the Mycobacterium avium complex (MAC), which consists of two mycobacterial species: M avium and M intracellulare MAC is one of the most common opportunistic pathogens found in AIDS patients particularly in individuals with low CD4 + cell counts (~50–100 cells mm-3) and is associated with increased morbidity and mortality in these patients (Horsburgh 1999;Abrams 2000) Although both species of mycobacteria classified under MAC have been isolated from AIDS patients, M avium appears to account for the majority of infections, up to 90% in one study (Yajko, Chin et al 1995) This may reflect the ubiquitous nature of M avium in the environment where it can be found in the water, soil and house dust (Yakrus and Good 1990; von Reyn, Maslow et al 1994) Introduction of highly active antiretroviral therapy (HAART) has resulted in a decreased incidence of MAC infection in HIV patients from ~ 10 individuals per 100 person-years in 1992 to ~ 2 in 1998 (Kaplan, Hanson et al 2000) However, a new disease designated MAC lymphadenitis has been found in HAART patients (Race, Adelson-Mitty et al 1998) Furthermore, pulmonary MAC infection in the United States appears to have increased over the past half century in non-AIDS patients (Rusin, Rose et al 1997) At present it is unclear if this reflects an increased incidence of infection or increased ability to recognize MAC-infected individuals

The study of M avium pathogenicity is complicated by the variability among different strains in both their genetic composition and morphology M avium can be isolated as three major morphotypes: smooth-opaque (SmO), smooth-transparent (SmT) and rough (Rg) (Belisle and Brennan 1994) Although for many M avium isolates the genetic and biochemical basis for the different morphotypes is unclear, one characterized distinction is the lack of glycopeptidolipids (GPLs) in some Rg M avium variants (Barrow and Brennan 1982)

The GPLs are major surface components of M avium, M intracellulare, M scrofulaceum, M smegmatis, M chelonae and M fortuitum The GPLs consist of a tripeptide amino alcohol core modified with an amide-linked fatty acid, a methylated rhamnose (Rhap) and a 6-deoxytalose The MAC GPLs can be further modified in length and composition of sugars attached to the 6-deoxytalose (6-dTal) residue This variation has been used to classify MAC isolates into 31 serotypes using antibodies specific for the different GPLs (Chatterjee and Khoo 2001) The genetic locus responsible for glycosylation and methylation of the GPL lipopeptide core has been mapped to the M avium ser gene cluster (Belisle, Klaczkiewicz et al 1993; Eckstein, Inamine et al 2000)

What is less clear about GPLs is their importance in M avium pathogenesis Some serotypes have been isolated with high frequency from AIDS patients (i.e serotypes 1, 4 and 8) suggesting an increased exposure to or increased virulence of these serotypes (Chatterjee and Khoo 2001) Although many of the Rg morphotypes lack GPLs or serotype-specific GPLs (ssGPLs), most Rg strains have not been characterized for virulence (Torrelles, Ellis et al 2002) Further, many of these Rg morphotypes lack genes not involved in GPL biosynthesis again making experimental interpretation difficult Therefore defining what role individual genes play in the formation of ssGPLs required targeted gene deletions within the ser gene cluster This study describes the inactivation of the M avium 104 methyltransferase D (mtfD) from the ser gene cluster by homologous recombination For a number of years homologous recombination has been possible for other pathogenic mycobacteria including M tuberculosis (Balasubramanian, Pavelka et al 1996) but only recently has targeted allelic replacement been achieved in M avium ((Maslow, Irani et al 2003; Otero, Jacobs et al 2003; Irani, Lee et al 2004) Our results suggest that the mtfD functions in M avium 104 (Serotype 1) as a 3-O-methyltransferase of the 3,4-di-O-methyl Rhap Interestingly, the disruption of mtfD in M avium 104 resulted in bacilli that induced an elevated pro-inflammatory response by macrophages in vitro and whose virulence was attenuated in mice These results strongly support a role for GPLs in M avium pathogenesis.

Materials and Methods

2.2.1 Bacterial strains and growth conditions

Mycobacterium avium strain 104 (generously provided by Dr Eric Brown,

University of California, San Francisco, CA) was grown on liquid Middlebrook 7H9 broth (Difco, Detroit, MI) supplemented with glucose, oleic acid, albumin, Tween 20 and NaCl, or on solid supplemented Middlebrook 7H10 medium (Difco) Escherichia coli DH10B, used as a host strain for plasmids throughout the study, was grown on liquid or solid Luria–Bertani medium When required, antibiotics were included at the following concentration: kanamycin (20 mg ml -1 ), gentamycin (5 mg ml -1 ) and hygromycin (50 mg ml -1 ) Ten per cent sucrose was added to supplement Middlebrook medium for selection of allelic exchanged mutants To generate M avium strain stocks for animal experiments, bacteria were passaged through a mouse to ensure virulence, and a single colony was used to inoculate supplemented Middlebrook 7H9 broth Bacteria were grown for 1 week at 37 o C with vigorous shaking and re-suspended in supplemented Middlebrook media containing 15% glycerol, and then the preparations were divided into aliquots and stored at -70 o C Frozen stocks were quantitated by serial dilution on supplemented Middlebrook agar

2.2.2 Gene exchange by homologous recombination

This work was done by Dr Elzbieta Krzywinska using the replicative pPR27 plasmid containing a temperature-sensitive mycobacterial origin of replication as described previously (Pelicic et al., 1997) In brief, M avium transformed with pPR27: mtfD::Kan was grown in Middlebrook broth with kanamycin at 32 o C with slow rotation for 6 days Cultures were pelleted, re-suspended in Middlebrook medium containing 15% glycerol, aliquot and stored at -80 o C Frozen stocks were quantitated by serial dilution on Middlebrook agar plates Approximately 6*10 6 -*10 7 cfu were plated on counter-selective Middlebrook plates containing sucrose and kanamycin and incubated at 39 o C for 10 days

Gene replacement was verified by PCR and sequencing across the mtfD gene using mtfD and Kanamycin specific primers

Bone marrow-derived macrophages (BMMΦs) were isolated from 6- to 8-week- old 129 S6/SvEv mice as previously described (Roach and Schorey 2002) The isolated primary macrophages were cultured on 100 mm non-tissue culture plates in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY) supplemented with 20 mM HEPES (Mediatech Cellgro, Herndon, VA), 10% fetal bovine serum (FBS) (Life Technologies), 100 U ml -1 penicillin and 100 mg ml -1 streptomycin (BioWhittaker, Walkersville, MD), 1X L-glutamine (Mediatech cellgro), and 20% L-cell supernatant as a source of macrophage colony-stimulating factor (M-CSF medium) The macrophages were used 7–14 days after isolation or frozen after 7 days of culture in freezing media [50% Dulbecco’s modified Eagle’s medium, 40% FBS and 10% endotoxin-tested dimethyl sulfoxide (DMSO; SIGMA, St Louis, MO)] Thawed or fresh macrophages were cultured on non-tissue culture plates for 3–7 days and then re-plated at 3*10 5 cells per 35 mm tissue culture plate The cells were allowed to adhere for 24 h prior to mycobacterial infection The BMMΦs in culture media minus antibiotics were infected for 4 h, washed to remove unattached mycobacteria and incubated for an additional 5 or

20 h with fresh media All tissue culture reagents were found to be negative for the endotoxin contamination by the E-Toxate assay (SIGMA)

Appropriate concentration of mycobacteria were suspended in macrophage culture media containing 10% normal horse serum as a source of complement components and incubated for 2 h at 37 o C (Roach and Schorey, 2002) Infection ratios of 80:1, 80:1 and 60:1 (mycobacteria to BMMΦ) were used for WT, mtfD mutant and complemented M avium 104 strains to obtain approximately 80% of the macrophages infected with two to three mycobacteria per cell

2.2.5 Chemokine/cytokine profile of macrophages infected with M avium 104

Semi-quantitative levels of various inflammatory mediators secreted by macrophages following infection with the mtfD mutant and WT M avium 104 isogenic strains were obtained using mouse cytokine array membranes (RayBio®, RayBiotech Norcross, GA, USA) according to the manufacturer’s protocol Briefly, the antibody coated membranes were incubated with the culture media from macrophages infected for

24 h Culture media from uninfected cells was used as a control in these experiments The membranes were treated with the primary and secondary antibodies provided by the manufacturer The bound antibodies were detected using Super Signal West Femto enhanced chemiluminescence’s reagents (Pierce)

The infection studies were performed with 6- to 8-week-old BALB/c and 129S6/SvEvTac (129/Sv) mice (Harlam, Indianapolis, IN, USA) The infection experiments complied with the Institutional Animal Care and Use Committee guidelines Groups of mice were infected retro-orbitally with 106 cfu of M avium strains and the infections were allowed to continue for 1 day or for 1, 5 or 9 weeks At this time the mice were killed by cervical dislocation and their spleens and portions of their liver and lung were aseptically removed, homogenized in 5 ml of 1% NP40 solution in PBS, and serial dilutions of the homogenates were plated onto Middlebrook plates

Liver and lung sections from infected mice were fixed in 10% buffered formalin for 4–6 h and transferred to 70% ethanol Samples were embedded in paraffin and stained with haematoxylin and eosin and with Ziehl-Neelsen by the Holburn Biomedical Corporation (Bowmanville, Ontario, Canada)

The levels of TNF-α (BD Pharmingen, San Diego, CA), IFN-γ (eBioscience, San Diego, CA), RANTES and IL-12p40 (Biosource, Camarillo, CA) secreted into the culture medium or present in the spleen, lung or liver homogenates were measured by ELISA according to the manufacturer’s protocol One milliliter of spleen, lung and liver homogenate from each mouse was centrifuged at 10,000 r.p.m for 15 min to remove tissue debris TNF-α activity was determined in 1:2 diluted lysates while IFN-γ activity was determined in undiluted and 1:2 diluted lysates Organ lysates were analyzed for cytokines according to the manufacturer’s protocol, and the cytokines concentrations were determined against RANTES, IL-12p40, IFN-γ and TNF-α standard curves

Data obtained from independent experiments were analyzed by a one-tailed Student t-test Differences were considered significant for P < 0.05.

Results

2.3.1 Disruption of the M avium 104 mtfD gene by homologous recombination

The mtfD gene in M avium 104 genome was disrupted by inserting a kanamycin (Kan) resistance cassette using a replicative plasmid pPR27 (Pelicic, Jackson et al 1997) by Elzbieta Krzywinska This plasmid has two counter-selection markers: a sacB that when expressed by mycobacteria confer susceptibility to sucrose and a temperature- sensitive mycobacterial origin of replication that is non-functional at the non-permissive temperature of 39 o C The pPR27: mtfD::Kan was electroporated into M avium 104 and the transformants were selected for Kan resistance The transformed M avium was grown for 6 days at 32 o C in the presence of Kan to allow for homologous recombination to occur Approximately 10 7 cfu of the transformed M avium were grown at 39 o C on Middlebrook plates containing Kan and 10% sucrose and colonies that grew on the selective media were screened by PCR using the mtfD specific primers (Fig 2.1) Disruption of the mtfD by homologous recombination was confirmed by PCR analysis using primers with target sites in the Kan gene and in the mtfC and dhgA genes, located upstream and downstream of the mtfD respectively (Fig 2.1 and data not shown)

Figure 2.1 Schematic organization of the mtfD disruption construct (A) Black and hatched regions represent mtfD and Kan resistance cassette, respectively Primer pairs (depicted by the black arrows) mtfDF/mtfDR, mtfCH/Kan1, and Kan2/dhgA were used for PCR amplification and sequencing of the genomic region subjected to homologous recombination

BamHI mtfCH mtfDF Kan1 dhgA Kan2 mtfDR mtfC mtfD::Km dhgA BamHI

2.3.2 In vitro macrophage infections with WT, mtfD mutant and complemented M avium 104 isolates

Murine bone marrow-derived macrophages (BMMΦs) isolated from the 129/Sv mouse strain were infected with the WT, mtfD mutant or complemented M avium 104 isogenic strain as described in the materials and methods We confirmed by fluorescent microscopy that equal infection levels were obtained with the different complement opsonized strains We isolated culture supernatants from the infected BMMΦs at 8 and

24 h post infection and assayed for tumor necrosis factor-a (TNF-α ) and IL-12p40 by ELISA As shown in Fig 2.2A, TNF-α level were significantly higher in BMMΦs infected with the mtfD mutant relative to cells infected with WT or complemented M avium 104 No difference in IL-12p40 was detected (data not shown) Mouse studies have shown TNF-α to be required to control an M avium infection in vivo (Appelberg and Orme 1993).To obtain a more global picture of the macrophage activation state following the infections with the different M avium strains, we used a mouse cytokine array to obtain semi-quantitative levels of 32 different growth factors, cytokines and chemokines (data not shown) Although, the levels for most factors appeared similar between the different infections, the data did indicate differences in TNF-α (confirmed by ELISA) and RANTES, an important chemokine in facilitating T cell migration (Luther and Cyster, 2001) The increased production of RANTES by mtfD-infected macrophages was validated by ELISA (Fig 2.2B)

Figure 2.2 BMMΦs were infected for 8 or 24 h with M avium 104 WT, mtfD mutant or mtfD mutant complemented with intact mtfD Culture supernatants from infected

BMMΦs were assayed for TNF-a (A) or for RANTES after 24 h (B) by ELISA Shown are the means plus standard deviation for triplicate wells and are representative of at least three experiments RANTES levels were below detection in non-infected cells; therefore, no bar is shown *Cytokine concentrations were significantly higher (P < 0.05) compared to mice infected with wild-type M avium 104

2.3.3 Mouse infections with the WT, mtfD mutant and complemented M avium 104 isolates

BALB/c and 129/Sv mice, which are relatively susceptible and resistant to M avium infections, respectively, were used in these studies 129/Sv mice were selected for the experiments as their increased resistance to M avium infection could amplify the attenuation of the mtfD mutant Mice were infected retro-orbitally with WT, mtfD mutant and mtfD complemented mutant To confirm that we infected with similar bacterial numbers and to observe the distribution of the mycobacteria during the initial stages of infection, we harvested the liver, spleen and lung 1 day post infection Similar colony forming units (cfu) were obtained for all three isogenic strains in both the liver and spleen (Fig 2.3A)

The infection level in the lung was below detection for all strains at this time Bacterial loads were also determined in spleens and livers after a 1- and 5-week infection and lungs after a 1-, 5- and 9-week infection After 5 weeks, 5- and 10-fold fewer cfu were isolated, respectively, from the livers and lungs of 129/Sv mice following an infection with the mtfD deficient mutant compared to WT 104 (Fig 2.3A) By 9 weeks the lungs from mice infected with the mtfD mutant had a 20-fold lower bacterial load compared to WT Importantly, the mtfD mutant complemented with an intact mtfD exhibited an infection level similar to WT M avium 104 We did observe a small but significant increase in bacterial numbers isolated from the spleen in mice infected with the complemented strain relative to WT This ~2.5-fold difference may be attributed to a slight change in GPL expression between WT and complemented mtfD mutant No difference was observed for the three isogenic strains after a 1-week infection Similar growth characteristics in broth culture between the three M avium 104 strains indicate

Figure 2.3 Bacterial loads in the livers (per gram) or spleens (A) or lungs (per gram) (B) following an infection of 129/Sv mice with M avium 104 WT, mtfD deficient mutant or mtfD complemented mutant Mice infected retro-orbitally for 1 day, 1 week and 5 weeks (A) or 1, 5 and 9 weeks (B) Shown are the means plus standard deviation and are representative of three to four experiments with four to five mice per group *Colony counts were significantly different (P < 0.05) compared to mice infected with wild-type

M avium 104 that the in vivo results are likely not attributed to differences in growth rates (data not shown) A decrease in cfu obtained from livers and spleens was also observed in BALB/c mice following a 5-week infection with the mtfD mutant compared to WT; however, the difference was only twofold (data not shown) ELISAs on the liver and spleen homogenates were performed to determine if mice infected with the mtfD deficient and

WT M avium 104 strains differed in the production of TNF-α and IFN-γ; two cytokines known to be essential for controlling an M avium infection (Appelberg, Sarmento et al 1995; Ehlers, Benini et al 1999) Differences in their production could account, at least in part, for the cfu data We observed a significant increase in IFN-γ levels in the 1-week liver homogenate from mtfD-infected compared to WT- or complemented-infected mice (Fig 2.4A) Although the total levels were lower and the differences not statisticallysignificant, the same trend was observed for TNF-α (Fig 2.4B) Of note is the observed difference in liver IFN-γ levels occurs prior to any difference in bacterial load, indicating that changes in IFN-γ predate changes in the mycobacterial infection levels

No differences in TNF-α or IFN-γ were observed in the lungs of infected mice at any time point tested (i.e 1, 5 or 9 weeks) (data not shown) As the colonization of the lung occurred with delayed kinetics relative to liver (see Fig 2.3), it is possible that the lung immune response including cytokine production was also delayed No difference in cytokine levels was observed in the spleen after the 1- or 5-week infections (data not shown) Granuloma formation is a hallmark of mycobacterial infections and plays an essential role in controlling an infection Therefore, a histological analysis was performed on the liver and lung sections isolated from the infected mice Haematoxylin and eosin and acid fast staining of tissue samples did not indicate any significant differences in

Figure 2.4 Liver homogenates from mice infected for 1 week with M avium 104 WT, mtfD mutant or mtfD complemented mutant were assayed by ELISA for IFN-γ (A) or

TNF-α (B) Shown are the means plus standard deviation from a minimum of three infected mice and are representative of two experiments *IFN-γ concentration was significantly different (P < 0.05) compared to mice infected with wild-type M avium

104 granuloma number, size or general appearance between mice infected with mtfD mutant and WT 104 (data not shown).

Discussion

The understanding of M avium pathogenesis has been hampered by the lack of genetic tools Most notable is the difficulties with creating gene knockouts in M avium by homologous recombination In a recent report, Maslow and colleagues (Maslow, Irani et al 2003;Irani, Lee et al 2004) were able to generate homologous recombinants in M avium 724 The mutation in the mtfD gene, generated by Elzbieta Krzywinska, also demonstrates the feasibility of producing homologous recombinants in M avium GPLs in M avium can be modified into serotype-specific oligosaccharides that contribute towards the immnunogenicity of these glycolipids (Chatterjee and Khoo 2001).Interestingly, the deletion of mtfD results in a M avium 104 that expresses only a precursor of the nsGPL as the mutant lacks methylation of the Rhap linked to the alaninol

The function that GPLs play in M avium pathogenesis is currently unknown These glycolipids are present in copious amounts on the cell surface and it has been speculated that the abundance of these molecules make cryptic many of the other cell wall lipoglycans (e.g lipoarabinomannan, lipomannan and phosphatidylinositol mannosides) Ambiguous results have been obtained using rough morphotypes of M avium Pedrosa and colleagues (Pedrosa, Florido et al 1994) determined that some Rg variants of M avium were avirulent compared to their isogenic SmT isolate while others were of similar virulence The differences in GPL composition between these Rg and SmT morphotypes were not defined The M avium 2151 Rg variants have been studied extensively for genomic deletions resulting in either a complete loss of GPLs or an absence of glycosylation but retention of the lipopeptide core (Belisle, Pascopella et al 1991; Belisle, McNeil et al 1993) However, the virulence of these different 2151 GPL mutants has not been published Our in vitro BMMΦ experiments and the mouse infection studies using the mtfD deficient M avium 104 and complemented strain imply a role for GPLs in M avium pathogenesis The glycolipid analysis indicates only alterations in the GPL methylation pattern and the absence of Rhap attached to the 6- dTal This is particularly important as large changes in GPL or loss of GPLs may have other effects on the mycobacteria cell wall and thus complicate experimental interpretation This is suggested by the increased permeability of GPL deficient M smegmatis to chenodeoxycholate, a negatively charged hydrophobic molecule often used to study mycobacteria cell wall fluidity (Etienne, Villeneuve et al 2002)

There is an interesting difference between the liver, lung and spleen in their ability to control mutant and WT M avium 104 infections This disparity was apparent at

5 and 9 weeks but not 1 week A number of possibilities could account for our observation including differences in: (i) T cell infiltration/activation, (ii) granuloma formation, (iii) macrophage numbers and their activation state and (iv) intrinsic differences in the ability of resident macrophages within the liver, lung and spleen to control an infection The in vitro infection studies indicate that BMMΦs respond with a greater pro-inflammatory response to infection with the mtfD mutant compared to WT Previous studies have shown that GPLs can modulate macrophage-signaling responses but this reaction may vary depending on the GPL serotype For example, GPLs from serotype 8 induce high levels of PGE2 in treated human peripheral blood mononuclear cells while GPLs from serotype 4 and 20 failed to induce PGE2 production (Barrow,

Davis et al 1995) PGE2 is a potent negative regulator of many signaling pathways (Takayama, Garcia-Cardena et al 2002) Together, these data suggest that an initial macrophage response in vivo to M avium strains with different GPL structure may also vary Indeed, we observed increased IFN-γ and a suggestive increase in TNF-α from liver, but not spleen, homogenates following an infection with the M avium mtfD mutant

In contrast, we did not observe a difference in these cytokines in the lung homogenates after the different M avium infections despite the differences in cfu observed 5 and 9 weeks post infection However, as the colonization of the lung occurred with delayed kinetics compared to liver and spleen (see Fig 2.3A), the 1-week time point where we observed differences in liver cytokine levels may not hold for lung Additional infection time points between 1 and 5 weeks may address this possibility

It is also interesting that different strains of mice show varied ability to control an infection with mtfD deficient compared to WT M avium 104 The increased resistance of 129/Sv mice to M avium infections compared to BALB/c mice may in part be attributed to a functional Nramp1 in the 129/Sv mouse strain Nramp1 has been associated with a plethora of functions linked to controlling mycobacterial infections (Govoni and Gros 1998) Whether there is a link between GPLs and Nramp1 expression awaits investigations using Nramp1 knockout mice However, it must be cautioned that there are numerous differences between 129/Sv and BALB/c mice, which might also account for our observation

In summary these studies have shown that gene knockouts created by homologous recombination can be used to define M avium virulence factors In these experiments, allelic replacement was used to disrupt the mtfD gene in M avium 104 and suggest that mtfD encodes for a 3-Omethyltransferase responsible for methylating the Rhap

Moreover, these are the first studies to indicate that maintaining an intact GPL profile is important for M avium pathogenicity.

Elevated MAP kinase signaling and increased macrophage activation in

Introduction

Mycobacteria are the etiological agents of numerous human diseases The

Mycobacterium avium complex (MAC), which consist of two species: M avium and M intracellulare, is one of the most common opportunistic pathogen s in AIDS patients and is associated with increased morbidity and mortality in these individuals (Abrams, 2000; Horsburgh, 1999) Although both MAC species have been isolated from AIDS patients,

M avium appears to account for the majority of infections (Yajko et al., 1995) This may reflect the ubiquitous nature of M avium in the environment where it can be found in the water, soil and house dust (von Reyn et al., 1994; Yakrus and Good, 1990) Moreover, an increased number of pulmonary MAC infections has been reported over the past half century among non-AIDS patients in the United States (Rusin et al., 1997) However, it is unclear if this reflects an increased incidence of infection or an increased ability to recognize MAC infected individuals

The study of M avium pathogenicity is complicated by the variability among strains in both their genetic composition and morphology M avium can be isolated as three major morphotypes; smooth-opaque (SmO), smooth-transparent (SmT), and rough (Rg) (Belisle and Brennan, 1994) Although in many cases the genetic and biochemical basis for the different morphotypes is unclear, one characterized distinction is the lack of glycopeptidolipids (GPLs) in some rough M avium variants (Barrow and Brennan, 1982) The GPLs are major surface components of M avium and M intracellulare and consist of a tripeptide amino alcohol core modified with an amide-linked fatty acid, a methylated rhamnose (Rha) and a 6-deoxytalose (6dTal) Together, this constitutes the apolar or non-serotype specific GPLs The MAC GPLs can be further modified in the length and composition of sugars attached to the 6dTal residue This variation has been used to classify MAC isolates into 28 serotypes using antibodies specific for the different GPLs (reviewed in (Chatterjee and Khoo, 2001)

What is less clear about GPLs is their importance in M avium pathogenesis These glycolipids are present in copious amounts on the cell surface and it has been speculated that the abundance of these molecules make cryptic many of the other cell wall lipoglycans (lipoarabinomannan, lipomannan, and phosphatidylinositol mannosides) Studies have shown some serotypes of GPLs to be isolated with higher frequency from AIDS patients than others (i.e serotypes 1, 4 and 8) suggesting an increased exposure or increased virulence of these serotypes (Chatterjee and Khoo, 2001) GPLs have also been associated with drug resistance (Khoo et al., 1999) In addition there is evidence that purified GPLs can modulate macrophage signaling pathways GPLs from serotype 8 induce high levels of PGE2 in treated human peripheral blood mononuclear cells while GPLs from serotype 4 and 20 failed to induce PGE2 production (Barrow et al., 1995) However, in this study the receptors engaged by the GPLs and the macrophage signaling pathways initiated were not elucidated GPLs can insert into phospholipid membranes but vary in this ability depending on the number of carbohydrate residues (Vergne et al., 1995) Therefore, GPLs could potentially modulate macrophage signaling by insertion into the phagosomal or plasma membrane

To address the effect of GPLs on macrophage activation and signaling responses, we infected murine bone marrow-derived macrophages with the Rg and SmT isolates of

M avium 2151 Studies by John Belisle and colleagues have shown this Rg mutant to lack both serotype specific and apolar GPLs (Belisle et al., 1993a) In our studies we found that macrophages produce a markedly different signaling response upon infection with the M avium 2151 Rg morphotype compared to SmT Our data also indicates that macrophages were more activated, as assessed by increased production of various inflammatory mediators, after being infected with the GPL deficient compared to the GPL sufficient M avium 2151 Our results suggest that surface exposed GPLs may function to limit the macrophage’s ability to illicit a strong initial response to a M avium infection.

Materials and Methods

Bone marrow derived macrophages (BMMΦs), were isolated from 6- to 8-week- old BALB/c mice as previously described (Roach and Schorey, 2002) The isolated primary macrophages were cultured on 100-mm petri dishes in Dulbecco’s modified

Eagle’s medium (Life Technologies, Grand Island, N.Y.) supplemented with 20 mM HEPES (Mediatech Cellgro,Herndon,VA), 10% fetal bovine serum (FBS) (Life Technologies), 100-U/ml penicillin and 100-àg/ml streptomycin (BioWhittaker, Walkersville, MD), 1X L-glutamine (Mediatech cellgro), and 20% L-cell supernatant as a source of macrophage colony-stimulating factor (BMMΦ medium) The macrophages were used 7 to 14 days after isolation or frozen after 7 days of culture in freezing media (50% Dulbecco’s modified Eagle’s medium, 40% FBS, and 10% endotoxin-tested dimethyl sulfoxide (DMSO) (SIGMA, St Louis, MO) Thawed or fresh macrophages were cultured on petri dishes for 3 to 7 days and then re-plated at 3x10 5 cells per 35-mm tissue culture plate The cells were allowed to adhere for 24 hrs prior to mycobacteria infection or inhibitor treatment The plates were then incubated at 37 o C in 5% CO2 for times indicated For time points which extended beyond 4 hours, BMMΦs were washed to remove non-ingested mycobacteria and fresh BMMΦ media without L-cell supernatant was added All tissue culture reagents were found to be negative for the endotoxin contamination by the E-Toxate assay (SIGMA)

The M avium 2151 isogenic stocks (Rg, SmO and SmT) used in this study were previously described (Tse et al., 2002; Belisle et al., 1993a) and generously provided by Julia Inamine (Colorado State University, Fort Collins, CO) Middlebrook 7H11 plates containing the different morphotypes were sent to us and the mycobacteria were resuspended directly from the plates into freezing media (Middlebrooks 7H9, 10% glycerol and OADC) and stored at -80 o C until needed All the stocks were quantitated by the serial dilution (Bohlson et al., 2001) BMMΦs were infected with different concentrations of complement opsonized M avium 2151 as described (Roach and Schorey, 2002) to determine the infection ratio required to obtain approximately 80% of macrophages infected For complement opsonization, appropriate concentrations of mycobacteria were suspended in BMMΦ media without L-cell supernatant but containing 10% normal human serum (NHS) as a source of complement components and incubated for 2 hrs at 37 o C (Bohlson et al., 2001) The NHS came from the same donor for all the experiments For some experiments, the NHS was heat-inactivated at 56 o C for 30 minutes prior to its incubation with the mycobacteria

3.2.3 Preparation of surface-exposed material from M avium

M avium surface lipids were removed as previously described (Etienne et al.,

2002) In brief, SmT, SmO and Rg M avium 2151 (1 gm of wet weight) removed from Middlebrook plates and resuspended in distilled water (50ml/flask) were incubated with

5 gm of glass beads (4-mm diameter) for 1 min with constant shaking Bacilli were removed by filtration through a 0.22àm-pore-size sterile filter (Corning, NY 14831) The crude filtrate, which contained material removed by the glass bead extraction, was then concentrated using Millipore Series 8000 Stirred Cells with ultra-filtration membranes (NMWL 1000, Millipore, MA)

3.2.4 Extraction and purification of GPLs

Total lipids, from 1 gram of wet weight of mycobacteria or the concentrated samples containing surface exposed material, were extracted with CHCl3:CH3OH (2:1 v/v) at 37 o C overnight and insoluble material removed by centrifugation The GPLs were partially purified as described (Torrelles et al., 2002) In brief, the lipid extracts were dried and subjected to alkaline methanolysis with 0.2M NaOH in CH3OH at 37 o C for 40 minutes, neutralized and subjected to a biphasic extraction The alkali-stable lipids were subjected to Folch wash The organic layers were separated and dried, re-suspended in equal amounts of CHCl3:CH3OH (2:1) Alkali stable lipids which primarily consist of GPLs were separated by thin layer chromatography (TLC) using silica gel 60 plates (EMD Chemicals Inc., NJ) with chloroform/ methanol/water (30:8:1 v/v/v) as the developing solvent The GPLs were visualized using α-napthol/sulfuric acid as the spray reagent

The different inhibitors used in this study were purchased from Calbiochem, La Jolla, CA and reconstituted with sterile endotoxin tested DMSO Macrophages were treated with: the p38 MAPK specific inhibitor SB203580 (10àM) 30 min prior infection, the MEK1- specific inhibitor PD98059 (20àM) 1 hr before infection, the CaMK inhibitor KN62 (10àM) 30 min prior to infection or the NFκB inhibitor, CAPE (14.8àg/ml) 1 hr before infection DMSO was used as a vehicle control in all studies The inhibitors did not affect BMMΦ viability or their ability to phagocytose M avium 2151 (Fig 3.4B and (Yadav et al., 2004) The concentrations chosen were based on our previous dose- response experiments (Yadav et al., 2004; Roach and Schorey, 2002) and on prior studies (Fitzpatrick et al., 2001)

At the times indicated, the infected BMMΦs were placed on ice, culture media was collected and the cells were washed thrice with ice-cold PBS containing 1mM pervandate The BMMΦs were lysed by treating the cells for 10-15 min with ice-cold lysis buffer (150mM NaCl, 1mM PMSF, 1àg/ml aprotinin, 1àg/ml leupeptin, 1 1àg/ml pepstatin, 1mM pervandate, 1mM EDTA, 1% IGEPAL, 0.25% deoxycholic acid, 1mM NaF, and 50mM Tris-HCL (pH 7.4) The cell lysates were stored at -20 o C For Western blots, equal concentration of protein lysates, as quantitated by the Micro BCA Protein Assay (Pierce, Rockford, IL), were loaded on 10% SDS-PAGE gels, electrophoresed, and transferred onto polyvinylidene difluoride membrane (Milipore, Bedford, MA) The membranes were probed for phospho-p38, phospho-ERK1/2, or total ERK1/2 as described (Roach and Schorey, 2002)

The levels of TNF-α, IL10, RANTES, IL-6, and IL-12p40 secreted by macrophages into the culture medium were measured by ELISA according to manufacturer’s protocol (BD Pharmingen, San Diego, CA, eBioscience and Endogen, Woburn, MA)

3.2.8 Chemokine/Cytokine profile of macrophages infected with M avium 2151

Semi-quantitative levels of various inflammatory mediators secreted by macrophages following infection with the M avium 2151 isogenic strains were obtained using mouse cytokine array membranes (RayBio ® , RayBiotech, Inc.) according to the manufacturer’s protocol Briefly, the antibody coated membranes were incubated with the culture media from macrophages infected for 24 or 48 hours Culture media from uninfected cells was used as a control in these experiments The membranes were treated with the primary and secondary antibodies provided by the manufacturer The bound antibodies were detected using Super Signal West Femto enhanced chemiluminescence’s reagents (Pierce) Densitometry was performed on the same blots using the Quantity One, Version 4.4.1 with GS-800 software (BioRad Laboratories, Hercules, CA)

Bone marrow derived macrophages from Balb/c mice were plated at 3x10 5 cells per 35-mm tissue culture plate and infected with M avium Rg and SmT 2151 at the MOI of 10:1 Cells supernatant were collected after 4 hours, 2, 4, 6 and 8 days post infection and cells were lysed with 1 % IGEPAL in PBS for 20 min The mycobacterial load was quantitated by serial dilutions of lysates and supernatant after each time point Three separate infections were done for each time point and mean colony counts were calculated

Statistical significance was determined with the paired two-tailed Student t test at p