participation of AMs in host defense, inflammatory processes and immune mechanisms has been amply documented (Schlesinger, 1997). In general, their primary function is the intracellular breakdown and disposal of particulate elements. In this regard, they contain a wide variety of hydrolases such as abundant lipase, acid phosphatase, cathepsin, lysozyme, esterase, acid ribonuclease, and -glucuronidase activities [on a specific activity basis] (Cohn & Wiener, 1963;Sorber *et al.*, 1973). Interestingly, dead BCG-stimulated AMs exhibited up to a 4-fold increase in the activities for lipases, acid phosphatases and lysozyme compared to control (Cohn & Wiener, 1963) indicating an up-regulation of these hydrolases within the AMs in the presence of antigen. This observation was corroborated in AMs obtained from live BCG-vaccinated rabbits, where acid phosphatase, lipase and lysozyme activities increased up to 40-fold when compared to AMs from control rabbits (Sorber *et al.*, 1973). How these hydrolases are regulated during *M.tb* infection and their role in redecorating the

Mononuclear phagocytes enter the lung both constitutively to maintain AM and dendritic cell populations, and during lung inflammation (Srivastava *et al.*, 2005). The role of monocyte accumulation in the lung in acute and chronic pulmonary inflammation is largely unknown, although these cells are accessible by bronchoaleolar lavage (Maus *et al.*, 2001). In the mouse, alveolar deposition of a stimulus provoked a significant influx of monocytes into the interstitium of the alveolar compartment along with a characteristic recruitment of neutrophils (Gunn *et al.*, 1997;Li *et al.*, 1998;Ulich *et al.*, 1991). This was confirmed by studies showing that circulating leukocytes could be recruited across the endothelial and epithelial barriers into the alveolar space under both non-inflammatory and highly inflammatory conditions (Li *et al.*, 1998;Maus *et al.*, 2001). Given the capacity of monocytes to produce hydrolases, reactive oxygen species, or inflammatory cytokines (Van Furth, 1988), their accumulation has been implicated in several inflammatory diseases in the pulmonary system (Antoniades *et al.*, 1992). As is the case for AMs, how monocytes and their secreted

products affect the cell wall of *M.tb* within the alveolar space remains unknown.

hydrolases affect the integrity of the cell wall of *M.tb* during infection.

**4.1.3 Alveolar epithelial cells**

Neutrophils may play an important role in controlling *M.tb* infection (Pedrosa *et al.*, 2000;Seiler *et al.*, 2003). In the bronchoalveolar lavage fluid, they normally represent less than 2% of all cells, however, during inflammation a massive influx of neutrophils occurs (Mizgerd, 2002;P. Zhang *et al.*, 2000). Neutrophils eliminate microbes by a number of oxidative and nonoxidative mechanisms (P. Zhang *et al.*, 2000) including secreted hydrolases such as *N*-acetyl- glucosaminidase, -glucuronidase, -mannosidases, and lysozyme. Neutrophils can kill *M.tb* through both oxidative and non-oxidative processes (Brown *et al.*, 1987;Jones *et al.*, 1990). Although neutrophils may interact with *M.tb* cell wall components during alveolar deposition [such as the 19-KDa lipoglycoprotein, SL-1 and PGLs (Faldt *et al.*, 1999;Neufert *et al.*, 2001;L. Zhang *et al.*, 1991)], there is no information regarding how alveolar neutrophil-derived

Alveolar epithelium lines the alveoli air sacs of the lung and is comprised predominantly of two specialized cell types (type I and type II). Alveolar type I cells function in gas exchange.

deposited in the terminal bronchioles and alveoli, as well as, following release from lysed macrophages and in cavities in reactivated TB, *M.tb* bacilli are in intimate contact with lung surfactant hydrolases. We recently demonstrated that hydrolases present in the human lung surfactant (Mason, 2006;Williams, 2003), at their relevant concentrations *in vivo*, dramatically alter the cell wall of *M.tb* during infection (Arcos *et al.*, 2011). As a result of these cell wall modifications, a significant decrease in association of *M.tb* with human macrophages was observed followed by an increase in phagosome-lysosome fusion (35%), which translated to a significant decrease in *M.tb* intracellular survival within these cells and an increase in inflammatory cytokine production leading to better control of infection (Arcos *et al.*, 2011). Importantly, we demonstrated that minimal contact time (15 min) with human lung surfactant hydrolases significantly reduced the cell surface exposure of two major *M.tb*  virulence factors, ManLAM and TDM (Arcos *et al.*, 2011). As mentioned above, both, ManLAM and TDM have been shown to play important roles in the intracellular survival of *M.tb* in the host by blocking the phagosome maturation process (Axelrod *et al.*, 2008;Kang *et al.*, 2005). Thus, below we will address which are the sources of these hydrolases in the alveolar space.

#### **4.1 The** *M. tuberculosis* **infection pathway and the alveolar environment: The potential role of human lung surfactant hydrolases**

The first interaction between *M.tb* and the human host takes place in the lung. The respiratory epithelium is actively involved in inflammation and host defense in multiple ways: providing a physical barrier, constituting the structural basis of mucociliary clearance aimed at the physical removal of inhaled bacteria; recognizing PAMPs/MAMPs by PPRs expressed on epithelial and myeloid cells, and secreting a variety of pro- and antiinflammatory mediators, including a large variety of hydrolases (Nicod, 2005). When *M.tb* bacilli reach the alveolar space, AMs, monocytes, and neutrophils represent the array of innate immune myeloid cells that will participate in host defense.

#### **4.1.1 The alveolar macrophage**

The AM is the first professional phagocyte to encounter inhaled *M.tb* bacilli. AMs are placed in a unique location within the alveolar surfactant film, the latter of which is produced by type II alveolar epithelial cells and is composed of phospholipids and proteins (Jonsson *et al.*, 1986). AMs are at the interface between air and lung tissue, and represent the first line of defense against inhaled *M.tb* found in the air (Lohmann-Matthes *et al.*, 1994). AMs possess a high phagocytic and clearance potential. In a normal healthy individual, they represent more than 90% of the cells in bronchoalveolar lavage fluid (Reynolds, 1987).

Many studies have demonstrated that resident AMs can phagocytose large numbers of microbes through both opsonic and non-opsonic receptors (Fels & Cohn, 1986;Lohmann-Matthes *et al.*, 1994;Palecanda & Kobzik, 2001;Serrano-Gomez *et al.*, 2004;Stephenson and Shepherd, 1987;Tailleux *et al.*, 2005;Taylor *et al.*, 2002;F.X. Zhang *et al.*, 1999). Though AMs have high phagocytic activity, their microbicidal capacity is less well-defined. Efficient microbial phagocytosis followed by slow intracellular killing may be sufficient to control infection with many routinely encountered extracellular pathogens. Intracellular pathogens like *M.tb*, however, may take advantage of the reduced microbial activity of the AM by residing and multiplying within these cells (Ferguson & Schlesinger, 2000). The participation of AMs in host defense, inflammatory processes and immune mechanisms has been amply documented (Schlesinger, 1997). In general, their primary function is the intracellular breakdown and disposal of particulate elements. In this regard, they contain a wide variety of hydrolases such as abundant lipase, acid phosphatase, cathepsin, lysozyme, esterase, acid ribonuclease, and -glucuronidase activities [on a specific activity basis] (Cohn & Wiener, 1963;Sorber *et al.*, 1973). Interestingly, dead BCG-stimulated AMs exhibited up to a 4-fold increase in the activities for lipases, acid phosphatases and lysozyme compared to control (Cohn & Wiener, 1963) indicating an up-regulation of these hydrolases within the AMs in the presence of antigen. This observation was corroborated in AMs obtained from live BCG-vaccinated rabbits, where acid phosphatase, lipase and lysozyme activities increased up to 40-fold when compared to AMs from control rabbits (Sorber *et al.*, 1973). How these hydrolases are regulated during *M.tb* infection and their role in redecorating the cell envelope of *M.tb* are currently unknown.

#### **4.1.2 Monocytes and neutrophils in the alveolar space**

96 Understanding Tuberculosis – Analyzing the Origin of Mycobacterium Tuberculosis Pathogenicity

deposited in the terminal bronchioles and alveoli, as well as, following release from lysed macrophages and in cavities in reactivated TB, *M.tb* bacilli are in intimate contact with lung surfactant hydrolases. We recently demonstrated that hydrolases present in the human lung surfactant (Mason, 2006;Williams, 2003), at their relevant concentrations *in vivo*, dramatically alter the cell wall of *M.tb* during infection (Arcos *et al.*, 2011). As a result of these cell wall modifications, a significant decrease in association of *M.tb* with human macrophages was observed followed by an increase in phagosome-lysosome fusion (35%), which translated to a significant decrease in *M.tb* intracellular survival within these cells and an increase in inflammatory cytokine production leading to better control of infection (Arcos *et al.*, 2011). Importantly, we demonstrated that minimal contact time (15 min) with human lung surfactant hydrolases significantly reduced the cell surface exposure of two major *M.tb*  virulence factors, ManLAM and TDM (Arcos *et al.*, 2011). As mentioned above, both, ManLAM and TDM have been shown to play important roles in the intracellular survival of *M.tb* in the host by blocking the phagosome maturation process (Axelrod *et al.*, 2008;Kang *et al.*, 2005). Thus, below we will address which are the sources of these hydrolases in the

**4.1 The** *M. tuberculosis* **infection pathway and the alveolar environment: The potential**

The first interaction between *M.tb* and the human host takes place in the lung. The respiratory epithelium is actively involved in inflammation and host defense in multiple ways: providing a physical barrier, constituting the structural basis of mucociliary clearance aimed at the physical removal of inhaled bacteria; recognizing PAMPs/MAMPs by PPRs expressed on epithelial and myeloid cells, and secreting a variety of pro- and antiinflammatory mediators, including a large variety of hydrolases (Nicod, 2005). When *M.tb* bacilli reach the alveolar space, AMs, monocytes, and neutrophils represent the array of

The AM is the first professional phagocyte to encounter inhaled *M.tb* bacilli. AMs are placed in a unique location within the alveolar surfactant film, the latter of which is produced by type II alveolar epithelial cells and is composed of phospholipids and proteins (Jonsson *et al.*, 1986). AMs are at the interface between air and lung tissue, and represent the first line of defense against inhaled *M.tb* found in the air (Lohmann-Matthes *et al.*, 1994). AMs possess a high phagocytic and clearance potential. In a normal healthy individual, they represent

Many studies have demonstrated that resident AMs can phagocytose large numbers of microbes through both opsonic and non-opsonic receptors (Fels & Cohn, 1986;Lohmann-Matthes *et al.*, 1994;Palecanda & Kobzik, 2001;Serrano-Gomez *et al.*, 2004;Stephenson and Shepherd, 1987;Tailleux *et al.*, 2005;Taylor *et al.*, 2002;F.X. Zhang *et al.*, 1999). Though AMs have high phagocytic activity, their microbicidal capacity is less well-defined. Efficient microbial phagocytosis followed by slow intracellular killing may be sufficient to control infection with many routinely encountered extracellular pathogens. Intracellular pathogens like *M.tb*, however, may take advantage of the reduced microbial activity of the AM by residing and multiplying within these cells (Ferguson & Schlesinger, 2000). The

alveolar space.

**role of human lung surfactant hydrolases**

**4.1.1 The alveolar macrophage**

innate immune myeloid cells that will participate in host defense.

more than 90% of the cells in bronchoalveolar lavage fluid (Reynolds, 1987).

Mononuclear phagocytes enter the lung both constitutively to maintain AM and dendritic cell populations, and during lung inflammation (Srivastava *et al.*, 2005). The role of monocyte accumulation in the lung in acute and chronic pulmonary inflammation is largely unknown, although these cells are accessible by bronchoaleolar lavage (Maus *et al.*, 2001). In the mouse, alveolar deposition of a stimulus provoked a significant influx of monocytes into the interstitium of the alveolar compartment along with a characteristic recruitment of neutrophils (Gunn *et al.*, 1997;Li *et al.*, 1998;Ulich *et al.*, 1991). This was confirmed by studies showing that circulating leukocytes could be recruited across the endothelial and epithelial barriers into the alveolar space under both non-inflammatory and highly inflammatory conditions (Li *et al.*, 1998;Maus *et al.*, 2001). Given the capacity of monocytes to produce hydrolases, reactive oxygen species, or inflammatory cytokines (Van Furth, 1988), their accumulation has been implicated in several inflammatory diseases in the pulmonary system (Antoniades *et al.*, 1992). As is the case for AMs, how monocytes and their secreted products affect the cell wall of *M.tb* within the alveolar space remains unknown.

Neutrophils may play an important role in controlling *M.tb* infection (Pedrosa *et al.*, 2000;Seiler *et al.*, 2003). In the bronchoalveolar lavage fluid, they normally represent less than 2% of all cells, however, during inflammation a massive influx of neutrophils occurs (Mizgerd, 2002;P. Zhang *et al.*, 2000). Neutrophils eliminate microbes by a number of oxidative and nonoxidative mechanisms (P. Zhang *et al.*, 2000) including secreted hydrolases such as *N*-acetyl- glucosaminidase, -glucuronidase, -mannosidases, and lysozyme. Neutrophils can kill *M.tb* through both oxidative and non-oxidative processes (Brown *et al.*, 1987;Jones *et al.*, 1990). Although neutrophils may interact with *M.tb* cell wall components during alveolar deposition [such as the 19-KDa lipoglycoprotein, SL-1 and PGLs (Faldt *et al.*, 1999;Neufert *et al.*, 2001;L. Zhang *et al.*, 1991)], there is no information regarding how alveolar neutrophil-derived hydrolases affect the integrity of the cell wall of *M.tb* during infection.

#### **4.1.3 Alveolar epithelial cells**

Alveolar epithelium lines the alveoli air sacs of the lung and is comprised predominantly of two specialized cell types (type I and type II). Alveolar type I cells function in gas exchange.

Broadening Our View About the Role of

**4.1.4 Pulmonary surfactant**

**5. Conclusions**

in the alveoli, altering its cell envelope and metabolism.

hydrolases alter the *M.tb* cell wall (Arcos *et al.*, 2011).

*Mycobacterium tuberculosis* Cell Envelope Components During Infection: A Battle for Survival 99

This, in turn, increases the probability that secreted hydrolases will impact the *M.tb* bacillus

Pulmonary surfactant prevents alveoli from collapsing at low lung volumes by reducing the surface tension in the alveolar space. Dipalmitoylphosphatidylcholine comprises almost 50% of total surfactant and is its major surface-active component (van Golde, 1985). The exact function of the remaining lipid components, such as unsaturated phosphatidylcholines, phosphatidylglycerols, phosphatidylethanolamines, phosphatidyl-inositols, and cholesterol are still uncertain (King, 1982;van Golde, 1985). The surfactant protein fraction comprises a highly variable amount of serum proteins, a wide variety of specific hydrolases (Griese, 1999) and four apoproteins (the surfactant proteins termed SP-A, -B, -C, and –D) that contribute to its specific function (Weaver & Whitsett, 1991). As mentioned above, surfactant contains several products secreted by alveolar myeloid and epithelial cells, some of them already defined in host defense, such as the bacteriolytic lysozyme (Haller *et al.*, 1992;Singh *et al.*, 1988). The wide variety of hydrolases secreted into surfactant resemble but are distinct from lysosomal enzymes (Hook and Gilmore, 1982). The surfactant resident

There remain many fundamental questions about how the alveolar environment influences *M.tb* pathogenesis. We recently showed that secreted hydrolases involved in surfactant homeostasis affect the *M.tb* cell wall (Arcos *et al.*, 2011) and/or trigger *M.tb* endogenous hydrolases to modify its own cell wall prior to contacting its natural niche, the AM, or after its lysis from the cell. Exposure of '*de novo*' motifs on the surface of *M.tb* after alveolar enzymatic processing will provide insight into the real nature of the *M.tb* cell envelope during infection. Since components of the *M.tb* cell envelope dictate the innate immune response against the bacillus via their interaction with surface receptors on myeloid cells, the identification of hydrolases that shape the surface of the *M.tb* cell envelope will enable more predictive *in vitro* models to be developed and novel drug targets to be identified.

The interface between *M.tb* and the host depends of many factors. *M.tb* strains differ in their cell wall components exposed on their surface. Even within the same strain, it is likely that some bacilli differ, and thus bacilli may interact different with the host. Thus, at a given infection we may find a mixture of events. On one hand, and depending on the cell wall components exposed on their surface, we may find bacilli that interact with a receptor inducing pro-inflammation and promoting *M.tb* killing, and on the other hand, we may find bacilli interacting with another receptor inducing anti-inflammation and *M.tb* intracellular survival. From the host perspective, genetic predisposition and living conditions dictate the predilection for *M.tb* infection. Even in the context of the same infected person and within the same host cell population, differences amongst cells in the expression of cell surface receptors, signaling, trafficking and innate and adaptive function exist. These differences in the host get enhanced even more when we compare host cells from different model systems. Our current studies on the impact of the alveolar space in the *M.tb* infection outcome also

These cells have an extremely thin cytoplasm extending away from the nuclear body and contain a large number of plasmalemmal invaginations termed caveolae (Gil *et al.*, 1981; Williams, 2003). Caveolae regulate removal of endogenous and exogenous particulates from the alveolar space by regulating activities of receptors, hydrolase secretion to the alveolar lumen and signaling molecules (Gumbleton, 2001;Marx, 2001;Razani & Lisanti, 2001). Importantly, these caveolae contain lipid phosphate phosphohydrolase, a critical enzyme in hydrolyzing a variety of phospholipids to produce diacylglycerol (Nanjundan & Possmayer, 2003). Type I cells (as well as AMs) also produce carboxypeptidase which increases during bacterial deposition in the alveolar space and functions in the processing of many peptides (Skidgel & Erdos, 1998). Other epithelial cell enzymes are related to the regulation of ion transport to the alveolar space (Johnson *et al.*, 2002).

Alveolar type II cells are considerably smaller than the type I cells and are richly endowed with organelles and microvilli on their apical membrane. These cells are located in the corners of the alveolus where their physiological functions include surfactant production, secretion and recycling (Fehrenbach, 2001). Surfactant is released to the alveolar space by exocytosis from intracellular storage organelles termed lamellar bodies which contain the majority of the components of the surfactant. During active secretion of contents from the lamellar bodies to the alveoli and during the surfactant recycling process, a variety of hydrolases have been related to these organelles (de Vries *et al.*, 1985; DiAugustine, 1974; Edelson *et al.*, 1988; Gilder *et al.*, 1981; Hook & Gilmore, 1982; S.L. Young *et al.*, 1993), many of which have lysosomal-type degradative functions. The presence of hydrolases within the lamellar bodies implies that if the contents of these structures are secreted, then hydrolases should also be secreted with surfactant. It has been shown that surfactant contains substantial quantities of hydrolases (Hook, 1978), where some hydrolases are highly active (*i.e.* -mannosidase and -*N*-acetylglucosaminidase) while others much less so (*i.e.* glucuronidase and arylsulfatase). Thus, the lamellar bodies provide a vehicle for the release of hydrolases into the alveoli, and their influence on host defense is being to be elucidated by our laboratory. In general, studies on epithelial cells have focused on phospholipases as second messengers in signaling, however, these same phospholipases are also degradative hydrolases with great potential to redecorate any microbial cell wall. For example, phospholipase A2 can release the fatty acids from phospholipids (Dennis, 2000). The action of phophatidylinositol phospholipase C provides both diacylglycerol and inositol trisphosphate. Other hydrolases include secreted and extracellular membrane-associated phosphatidic acid phosphatases which act on 1,2-diacyl,*sn*-glycerol phosphate to produce diacylglycerol and inorganic phosphate (Brindley & Waggoner, 1998), however, the membrane-associated hydrolases also act on a variety of phospholipids to generate anionic and/or neutral lipids (Nanjundan & Possmayer, 2003). These hydrolases may also be active against the rich phospholipid content of the cell wall of *M.tb.* (*i.e.* PIMs, cardiolipid, phosphatidylethanolamine, etc.) (Arcos *et al.*, 2011). To what extent *M.tb* bacilli directly interact with epithelial cells and their secreted products remains unknown. However, the location of the alveolar epithelium, as well as, the relatively large alveolar epithelial surface area estimated at 100 to 140 m2, makes it likely that *M.tb* will interact with components of the alveolar space prior to and following its residence in the AM. The low alveolar fluid volume relative to the alveolar epithelial surface area (7- 20 ml per 100 m2) likely increases the local concentration of released hydrolases when compared to other tissue compartments. This, in turn, increases the probability that secreted hydrolases will impact the *M.tb* bacillus in the alveoli, altering its cell envelope and metabolism.

#### **4.1.4 Pulmonary surfactant**

98 Understanding Tuberculosis – Analyzing the Origin of Mycobacterium Tuberculosis Pathogenicity

These cells have an extremely thin cytoplasm extending away from the nuclear body and contain a large number of plasmalemmal invaginations termed caveolae (Gil *et al.*, 1981; Williams, 2003). Caveolae regulate removal of endogenous and exogenous particulates from the alveolar space by regulating activities of receptors, hydrolase secretion to the alveolar lumen and signaling molecules (Gumbleton, 2001;Marx, 2001;Razani & Lisanti, 2001). Importantly, these caveolae contain lipid phosphate phosphohydrolase, a critical enzyme in hydrolyzing a variety of phospholipids to produce diacylglycerol (Nanjundan & Possmayer, 2003). Type I cells (as well as AMs) also produce carboxypeptidase which increases during bacterial deposition in the alveolar space and functions in the processing of many peptides (Skidgel & Erdos, 1998). Other epithelial cell enzymes are related to the regulation of ion

Alveolar type II cells are considerably smaller than the type I cells and are richly endowed with organelles and microvilli on their apical membrane. These cells are located in the corners of the alveolus where their physiological functions include surfactant production, secretion and recycling (Fehrenbach, 2001). Surfactant is released to the alveolar space by exocytosis from intracellular storage organelles termed lamellar bodies which contain the majority of the components of the surfactant. During active secretion of contents from the lamellar bodies to the alveoli and during the surfactant recycling process, a variety of hydrolases have been related to these organelles (de Vries *et al.*, 1985; DiAugustine, 1974; Edelson *et al.*, 1988; Gilder *et al.*, 1981; Hook & Gilmore, 1982; S.L. Young *et al.*, 1993), many of which have lysosomal-type degradative functions. The presence of hydrolases within the lamellar bodies implies that if the contents of these structures are secreted, then hydrolases should also be secreted with surfactant. It has been shown that surfactant contains substantial quantities of hydrolases (Hook, 1978), where some hydrolases are highly active (*i.e.* -mannosidase and -*N*-acetylglucosaminidase) while others much less so (*i.e.* glucuronidase and arylsulfatase). Thus, the lamellar bodies provide a vehicle for the release of hydrolases into the alveoli, and their influence on host defense is being to be elucidated by our laboratory. In general, studies on epithelial cells have focused on phospholipases as second messengers in signaling, however, these same phospholipases are also degradative hydrolases with great potential to redecorate any microbial cell wall. For example, phospholipase A2 can release the fatty acids from phospholipids (Dennis, 2000). The action of phophatidylinositol phospholipase C provides both diacylglycerol and inositol trisphosphate. Other hydrolases include secreted and extracellular membrane-associated phosphatidic acid phosphatases which act on 1,2-diacyl,*sn*-glycerol phosphate to produce diacylglycerol and inorganic phosphate (Brindley & Waggoner, 1998), however, the membrane-associated hydrolases also act on a variety of phospholipids to generate anionic and/or neutral lipids (Nanjundan & Possmayer, 2003). These hydrolases may also be active against the rich phospholipid content of the cell wall of *M.tb.* (*i.e.* PIMs, cardiolipid, phosphatidylethanolamine, etc.) (Arcos *et al.*, 2011). To what extent *M.tb* bacilli directly interact with epithelial cells and their secreted products remains unknown. However, the location of the alveolar epithelium, as well as, the relatively large alveolar epithelial surface area estimated at 100 to 140 m2, makes it likely that *M.tb* will interact with components of the alveolar space prior to and following its residence in the AM. The low alveolar fluid volume relative to the alveolar epithelial surface area (7- 20 ml per 100 m2) likely increases the local concentration of released hydrolases when compared to other tissue compartments.

transport to the alveolar space (Johnson *et al.*, 2002).

Pulmonary surfactant prevents alveoli from collapsing at low lung volumes by reducing the surface tension in the alveolar space. Dipalmitoylphosphatidylcholine comprises almost 50% of total surfactant and is its major surface-active component (van Golde, 1985). The exact function of the remaining lipid components, such as unsaturated phosphatidylcholines, phosphatidylglycerols, phosphatidylethanolamines, phosphatidyl-inositols, and cholesterol are still uncertain (King, 1982;van Golde, 1985). The surfactant protein fraction comprises a highly variable amount of serum proteins, a wide variety of specific hydrolases (Griese, 1999) and four apoproteins (the surfactant proteins termed SP-A, -B, -C, and –D) that contribute to its specific function (Weaver & Whitsett, 1991). As mentioned above, surfactant contains several products secreted by alveolar myeloid and epithelial cells, some of them already defined in host defense, such as the bacteriolytic lysozyme (Haller *et al.*, 1992;Singh *et al.*, 1988). The wide variety of hydrolases secreted into surfactant resemble but are distinct from lysosomal enzymes (Hook and Gilmore, 1982). The surfactant resident hydrolases alter the *M.tb* cell wall (Arcos *et al.*, 2011).

There remain many fundamental questions about how the alveolar environment influences *M.tb* pathogenesis. We recently showed that secreted hydrolases involved in surfactant homeostasis affect the *M.tb* cell wall (Arcos *et al.*, 2011) and/or trigger *M.tb* endogenous hydrolases to modify its own cell wall prior to contacting its natural niche, the AM, or after its lysis from the cell. Exposure of '*de novo*' motifs on the surface of *M.tb* after alveolar enzymatic processing will provide insight into the real nature of the *M.tb* cell envelope during infection. Since components of the *M.tb* cell envelope dictate the innate immune response against the bacillus via their interaction with surface receptors on myeloid cells, the identification of hydrolases that shape the surface of the *M.tb* cell envelope will enable more predictive *in vitro* models to be developed and novel drug targets to be identified.

#### **5. Conclusions**

The interface between *M.tb* and the host depends of many factors. *M.tb* strains differ in their cell wall components exposed on their surface. Even within the same strain, it is likely that some bacilli differ, and thus bacilli may interact different with the host. Thus, at a given infection we may find a mixture of events. On one hand, and depending on the cell wall components exposed on their surface, we may find bacilli that interact with a receptor inducing pro-inflammation and promoting *M.tb* killing, and on the other hand, we may find bacilli interacting with another receptor inducing anti-inflammation and *M.tb* intracellular survival. From the host perspective, genetic predisposition and living conditions dictate the predilection for *M.tb* infection. Even in the context of the same infected person and within the same host cell population, differences amongst cells in the expression of cell surface receptors, signaling, trafficking and innate and adaptive function exist. These differences in the host get enhanced even more when we compare host cells from different model systems. Our current studies on the impact of the alveolar space in the *M.tb* infection outcome also

Broadening Our View About the Role of

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indicate that environmental host factors, such as alveolar hydrolases, play important roles in the establishment of the infection. With this in mind, what are the necessary elements that result in a succesful *M.tb* infection? Is *M.tb* infection just chance or a perfect combination of bacterial and host elements? Upon the successful establishment of *M.tb* infection, the ultimate goal of the host is to reduce inflammation and tissue destruction and in this scenario *M.tb* has learned to evolve, adapt, and survive.

What do we know about the *M.tb* cell wall adaptation to the host? The most revealing attribute of the *M.tb* cell wall is its complexity. Studies have been focused on depicting the composition, structure, biosynthesis, and the spatial conformation of the *M.tb* cell wall and its components for decades. Currently, researchers are focusing to reveal how the cell wall of *M.tb* is during infection (*in vivo* and *in vitro*). The development of new technologies and/or the use of known technologies already successfully applied in other fields (such as cancer research) are moving fast into the field of TB. Thus, studies using scanning electron and atomic force microscopy revealed that the *M.tb* cell wall from MDRand XDR-strains differs from susceptible strains (Velayati *et al.*, 2009a, 2009b, 2010). Experiments analyzing infected granulomas by using high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS-NMR) showed that we are capable of analyzing the cell wall of *M.tb* when inside of the granuloma without further manipulation (Somashekar *et al.*, 2011). The introduction of novel reporters that can be used for selective labeling of the cell wall of *M.tb* during infection *in vitro* and *in vivo* is already allowing us to see how the cell wall of *M.tb* is remodeling during infection (Backus *et al.*, 2011). Efforts in improving purification techniques are also allowing us to be able to purify *M.tb* directly from infected tissues. The use of novel *state-of-the art* mass spectrometry techniques such as LC/MS/MS (Sartain *et al.*, 2006, 2011), ESI/MS (Barry *et al.*, 2011) and MALDI-Tissue Imaging (Prideaux *et al.*, 2011) in drug discovery will allow us to obtain new information about the cell wall composition from a single *M.tb* bacillus isolated from tissue and also to see how *M.tb* cell wall components, and other biomarkers and drugs are distributed within the infected tissue. These are few of many other novel biotechniques that are starting to be applied in the field of TB. But this is only the beginning, more efforts improving the protocols and the development of new technology will allow us to move quickly to solve the "mystery" involving how *M.tb* adapted to the host and became such a successful infection currently affecting one third of the world population and taking away ~2 million lives every year.

#### **6. Acknowledgements**

This work was partially supported by NIH/NIAID AI-073856 and the Parker B. Francis Fellowship Program.

#### **7. References**

Andersen, A. B. & Brennan P.J. (1994) Proteins and antigens of *M. tuberculosis*. In *Tuberculosis: Pathogenesis, Protection, and Control*. Bloom BR (ed.), pp. 307-332, American Society for Microbiology, ISBN<PHONE_NUMBER>, Washington, DC.

indicate that environmental host factors, such as alveolar hydrolases, play important roles in the establishment of the infection. With this in mind, what are the necessary elements that result in a succesful *M.tb* infection? Is *M.tb* infection just chance or a perfect combination of bacterial and host elements? Upon the successful establishment of *M.tb* infection, the ultimate goal of the host is to reduce inflammation and tissue destruction and in this

What do we know about the *M.tb* cell wall adaptation to the host? The most revealing attribute of the *M.tb* cell wall is its complexity. Studies have been focused on depicting the composition, structure, biosynthesis, and the spatial conformation of the *M.tb* cell wall and its components for decades. Currently, researchers are focusing to reveal how the cell wall of *M.tb* is during infection (*in vivo* and *in vitro*). The development of new technologies and/or the use of known technologies already successfully applied in other fields (such as cancer research) are moving fast into the field of TB. Thus, studies using scanning electron and atomic force microscopy revealed that the *M.tb* cell wall from MDRand XDR-strains differs from susceptible strains (Velayati *et al.*, 2009a, 2009b, 2010). Experiments analyzing infected granulomas by using high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS-NMR) showed that we are capable of analyzing the cell wall of *M.tb* when inside of the granuloma without further manipulation (Somashekar *et al.*, 2011). The introduction of novel reporters that can be used for selective labeling of the cell wall of *M.tb* during infection *in vitro* and *in vivo* is already allowing us to see how the cell wall of *M.tb* is remodeling during infection (Backus *et al.*, 2011). Efforts in improving purification techniques are also allowing us to be able to purify *M.tb* directly from infected tissues. The use of novel *state-of-the art* mass spectrometry techniques such as LC/MS/MS (Sartain *et al.*, 2006, 2011), ESI/MS (Barry *et al.*, 2011) and MALDI-Tissue Imaging (Prideaux *et al.*, 2011) in drug discovery will allow us to obtain new information about the cell wall composition from a single *M.tb* bacillus isolated from tissue and also to see how *M.tb* cell wall components, and other biomarkers and drugs are distributed within the infected tissue. These are few of many other novel biotechniques that are starting to be applied in the field of TB. But this is only the beginning, more efforts improving the protocols and the development of new technology will allow us to move quickly to solve the "mystery" involving how *M.tb* adapted to the host and became such a successful infection currently affecting one third of the world

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**5**

Elsa Anes

*Portugal*

**For Host Factors Weddings and a Koch's**

**Maturation and Inflammasome Activation**

*Universidade de Lisboa /Faculdade de Farmácia/ Centro de Patogénese*

*Molecular-URIA-Instituto de Medicina Molecular*

**Bacillus Funeral: Actin, Lipids, Phagosome**

When a particle or microorganism enters the host, is readily phagocytosed by the local macrophages and dendritic cells. If the microorganism is a non-pathogenic bacteria such as *Mycobacterium smegmatis*, the bacteria containing phagosomes will fully mature. Maturation refers to a multi-dimensional and complex process that integrates biochemical alterations on the phagosomal membranes, resulting as a direct response from the external stimuli and indirectly from the immediate response at the gene expression level in the form of transcriptional programs, as well as from its regulation. This, in turn, leads to modifications at the intracellular architecture, with the reorganization of the organelle dynamics, dependent on the assembly of actin, and resulting ultimately in the fusion of phagosomes with lysosomes. Not only several biochemical pathways have been implicated, but also physical and mechanical events will determine the faith of the bacteria containing phagosome. At the biochemical level, acidic hydrolases and the proton ATPase will reach the phagosomes while maturing and promote the lowering of the intraphagosomal pH, thereafter contributing to the killing of the bacteria. The production of nitric oxide (NO) by the inducible nitric oxide synthase (iNOS) and the reactive oxygen species (ROS) by phagosome oxidases (phox) is also known to be important for the bacterial killing. NO and cytokines are an important part of the inflammatory response which is downstream of

When the pathogenic *Mycobacterium tuberculosis* enters the macrophage phagosome, the maturation process is blocked. We have shown that at least three distinct processes are targeted: actin assembly, fusion with lysosomes and acidification (Anes et al., 2003, Anes et al., 2006, Castandet et al., 2005). It has been suggested that specific *M. tuberculosis* lipids such as the sulfolipids (Goren et al., 1976), and phenolic glicolipids (Rhoades et al., 2003), and secreted peptides (Walburger et al., 2004) have been implicated as effectors involved in blocking the phagosome maturation to phagolysosome. These factors, which have been reported to be transferred to different host membranes in infected cells, are likely to be the cause of the well-described inhibition of pro-inflammatory responses, including the

transcription factors such as nuclear factor-B (NF-B).

inhibition of TNF- secretion, and the restriction of phagosome motility.

**1. Introduction**

### **For Host Factors Weddings and a Koch's Bacillus Funeral: Actin, Lipids, Phagosome Maturation and Inflammasome Activation**

Elsa Anes

*Universidade de Lisboa /Faculdade de Farmácia/ Centro de Patogénese Molecular-URIA-Instituto de Medicina Molecular Portugal*

#### **1. Introduction**

122 Understanding Tuberculosis – Analyzing the Origin of Mycobacterium Tuberculosis Pathogenicity

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macrophages binding and signal transduction in the absence of serum. *Infect*

T., Yang, R. D., Chu, T. S., Zhang, C., Zhang, L., Han, J. W., Yu, G. Q., Quan, C., Yu, Y. X., Zhang, Z., Shi, B., Zhang, L. H., Cheng, H., Wang, C. Y., Lin, Y., Zheng, H. F., Fu, X. A., Zuo, X., Wang, Q., Long, H., Sun, Y. P., Cheng, Y. L., Tian, H. Q., Zhou, F., Liu, H. X., Lu, W. S., He, S. M., Du, W. L., Shen, M., Jin, Q. Y., Wang, Y., Low, H., Erwin, T., Yang, N., Li, J. Y., Zhao, X., Jiao, Y., Mao, L., Yin, G., Jiang, Z., Wang, X., Yu, J., Hu, Z., Gong, C., Liu, Y., Liu, R., Wang, D., Wei, D., Liu, J., Cao, W., Cao, H., Li, Y., Yan, W., Wei, S., Wang, K., Hibberd, M., Yang, S., Zhang, X., & Liu, J. J. (2009) Genomewide association study of leprosy. *N Engl J Med,* Vol. 361, No. 27,

> When a particle or microorganism enters the host, is readily phagocytosed by the local macrophages and dendritic cells. If the microorganism is a non-pathogenic bacteria such as *Mycobacterium smegmatis*, the bacteria containing phagosomes will fully mature. Maturation refers to a multi-dimensional and complex process that integrates biochemical alterations on the phagosomal membranes, resulting as a direct response from the external stimuli and indirectly from the immediate response at the gene expression level in the form of transcriptional programs, as well as from its regulation. This, in turn, leads to modifications at the intracellular architecture, with the reorganization of the organelle dynamics, dependent on the assembly of actin, and resulting ultimately in the fusion of phagosomes with lysosomes. Not only several biochemical pathways have been implicated, but also physical and mechanical events will determine the faith of the bacteria containing phagosome. At the biochemical level, acidic hydrolases and the proton ATPase will reach the phagosomes while maturing and promote the lowering of the intraphagosomal pH, thereafter contributing to the killing of the bacteria. The production of nitric oxide (NO) by the inducible nitric oxide synthase (iNOS) and the reactive oxygen species (ROS) by phagosome oxidases (phox) is also known to be important for the bacterial killing. NO and cytokines are an important part of the inflammatory response which is downstream of transcription factors such as nuclear factor-B (NF-B).

> When the pathogenic *Mycobacterium tuberculosis* enters the macrophage phagosome, the maturation process is blocked. We have shown that at least three distinct processes are targeted: actin assembly, fusion with lysosomes and acidification (Anes et al., 2003, Anes et al., 2006, Castandet et al., 2005). It has been suggested that specific *M. tuberculosis* lipids such as the sulfolipids (Goren et al., 1976), and phenolic glicolipids (Rhoades et al., 2003), and secreted peptides (Walburger et al., 2004) have been implicated as effectors involved in blocking the phagosome maturation to phagolysosome. These factors, which have been reported to be transferred to different host membranes in infected cells, are likely to be the cause of the well-described inhibition of pro-inflammatory responses, including the inhibition of TNF- secretion, and the restriction of phagosome motility.

For Host Factors Weddings and a Koch's Bacillus Funeral:

that may resemble a simple cold.

lifetime (Kaufmann & Parida, 2007).

(Russell, 2007).

Actin, Lipids, Phagosome Maturation and Inflammasome Activation 125

replication. The first contact with these phagocytic cells will display a series of innate immune mechanisms that may lead to the complete clearance of the bacilli. These include the release of NO, ROS, the fusion of the phagosome with the lysosome with the simultaneously bacteria digestion by acidic hydrolases and antimicrobial peptides (Cooper et al., 2000, Liu et al., 2007, MacMicking et al., 1997). Ultimately the programmed cell death

Furthermore the initially infected cells release pro-inflammatory cytokines which leads to recruitment of more DC, monocytes and neutrophils from the blood stream and the infected DC become mature and migrate to the local lymph node where they activate specific T cells. The cytokines IL-12 and IL-18 from the infected cells induce natural killer cell (NK) activity, and the NK cells in turn produce IFN-γ, which activates the macrophages to produce TNF-α and more microbicidal effectors (Korbel et al., 2008). In fact resting phagocytic cells are designed to uptake and clear bacteria by phagolysosome fusion with full bacteria digestion, while activated macrophages are reprogrammed to produce more free radicals, and decrease the extend of bacteria digestion in order to produce antigens for immune cross presentation. Through cytokine and chemokine signaling, other immune cells are recruited and the pathological hallmark of TB, the granuloma, is formed. In the granuloma, macrophages differentiate further into epitheloid cells or foamy macrophages, or fuse to form giant cells, and become surrounded by lymphocytes and an outer cuff of fibroblasts and extracellular matrix proteins. At this stage infected people may complaint for symptoms

Thereby, the bacilli are contained until the granuloma fails due to immunosuppression (Russell, 2007). For a long time, the granuloma was viewed as beneficial only for the host – it coincided with the onset of adaptive immunity and reduction of bacterial growth in the lung – but recent studies in zebrafish embryos infected with a close relative of *M. tuberculosis*, *Mycobacterium marinum*, have indicated that mycobacteria also use the granuloma for their benefit upon initial infection, recruiting new macrophages to allow spread between host cells (Davis & Ramakrishnan, 2009). This stage were the centre of the granuloma is formed by infected macrophages usually is referred as a latent infection without disease symptoms and according to the World Health Organization (WHO reports 2008) corresponds to one third of the world infected people. From these only 5-10% will develop disease during their

When the granuloma centre becomes caseous containing necrotic macrophages following necrotic programme cell death, latent infection becomes in active disease, which in advanced TB form cavities in the lung. Historical texts identify the disease as "consumption," "wasting away," "king's evil," "lupus vulgaris," "the white plague" or "phthisis" based on its clinical manifestations (Donoghue, 2009). Although mycobacteria are able to survive and proliferate within phagocytes, it should be noted that during active pulmonary tuberculosis they are preferentially found in the extracellular space. The necrotic centers of infection sites during active disease contain liquefied, so-called caseating, debris. These lesions are rich in cellular detritus, membrane phospholipids and cholesterol, which serve as an important carbon source for mycobacteria. Spillage of infectious bacilli into the airways occurs when the structure ruptures, and this allows spread to new individuals

of the infected host cell will kill the reminiscent infecting bacteria (Park et al., 2006).

These blocking mechanisms, which are still yet poorly understood, can somehow, be reversed. Our published data show that phagosome maturation is directly linked to actin assembly on membranes (Defacque et al., 2000). This will consequently lead the organelles to aggregate before they fuse with lysosomes (Jahraus et al., 2001). We also described the addition of several pro-inflammatory lipids to *M. tuberculosis* infected cells with surprisingly strong killing effects (Anes et al., 2003, Gutierrez et al., 2009, Jordao et al., 2008). These selected lipids can manipulate the phagolysosome maturation by interfering with the actin machinery. In addition to these signaling lipids, ATP, the P2X7 receptor and cAMP were shown to be involved in actin assembly and the killing/survival of pathogenic mycobacteria (Kalamidas et al., 2006, Kuehnel et al., 2009, Kuehnel et al., 2009b). Furthermore, some lipid effectors for actin assembly also control NF-kB (Gutierrez et al., 2009). Using microarray analysis, we linked NF-kB in the regulation of many lysosomal enzymes and membranetrafficking regulators, including cathepsins, LAMP-2 and Rab34, during infection (Gutierrez et al., 2008).

Important classes of lipids that are effectors for actin assembly and for NF-kB control include eicosanoids. Eicosanoides produced from arachidonic acid such lipoxin X4 (LX4) as well ATP, and the P2X7 R are all additionally involved in a necrotic-programmed cell death (Chen et al., 2008). Distinct molecules were found to play opposite roles either inducing apoptosis or necrosis during infection. In fact while virulent *M. tuberculosis* promotes necrotic cell death and inhibits apoptosis, the non-virulent strain H37Ra induces apoptosis that results in lower bacterial viability. Pyroptosis in the context of tuberculosis is controversial: from one side it leads to potent pro-inflammation that drives tuberculosis; from other side it allows the release of intracellular bacteria and their escape to hydrolytic digestion. This type of programmed cell death depends on the assembly of a specific inflammasome leading to caspase 1 activation and IL-1 and IL-18 secretion. Some groups claimed that *M. tuberculosis* might block the inflammasome activation (Master et al., 2008). We have shown that *M. tuberculosis* in human macrophages, not only activates caspase 1 and IL1- secretion but also that different inflammasomes are assembled during infection (Mishra et al., 2010).

So we propose in this chapter to present an overview of host factors that may be manipulated in order to reverse phagosome maturation arrest, NF-kB translocation, inflammasome activation and therefore boosting the macrophage killing abilities to Koch's bacilli.

#### **2. The disease**

Tuberculosis is usually a lung infection caused by inhalation of Koch's bacilli within microdroplets, but it can affect any organ. In the lung, bacilli are envisaged to be engulfed by cell patrolling the alveolar surface such as alveolar macrophages and tissue dendritic cells (DC). These cells transport the pathogens to the lung interstitium and draining lymph nodes. At both sites, primary lesions develop that rarely cause disease. These professional phagocytes that are devoted to maintain the lungs cleaned from particles, produce a series of bacterial insults designed to kill pathogens. However *M. tuberculosis* developed capacities to subvert the killing mechanisms of phagocytes to allow their intracellular survival and/or

These blocking mechanisms, which are still yet poorly understood, can somehow, be reversed. Our published data show that phagosome maturation is directly linked to actin assembly on membranes (Defacque et al., 2000). This will consequently lead the organelles to aggregate before they fuse with lysosomes (Jahraus et al., 2001). We also described the addition of several pro-inflammatory lipids to *M. tuberculosis* infected cells with surprisingly strong killing effects (Anes et al., 2003, Gutierrez et al., 2009, Jordao et al., 2008). These selected lipids can manipulate the phagolysosome maturation by interfering with the actin machinery. In addition to these signaling lipids, ATP, the P2X7 receptor and cAMP were shown to be involved in actin assembly and the killing/survival of pathogenic mycobacteria (Kalamidas et al., 2006, Kuehnel et al., 2009, Kuehnel et al., 2009b). Furthermore, some lipid effectors for actin assembly also control NF-kB (Gutierrez et al., 2009). Using microarray analysis, we linked NF-kB in the regulation of many lysosomal enzymes and membranetrafficking regulators, including cathepsins, LAMP-2 and Rab34, during infection (Gutierrez

Important classes of lipids that are effectors for actin assembly and for NF-kB control include eicosanoids. Eicosanoides produced from arachidonic acid such lipoxin X4 (LX4) as well ATP, and the P2X7 R are all additionally involved in a necrotic-programmed cell death (Chen et al., 2008). Distinct molecules were found to play opposite roles either inducing apoptosis or necrosis during infection. In fact while virulent *M. tuberculosis* promotes necrotic cell death and inhibits apoptosis, the non-virulent strain H37Ra induces apoptosis that results in lower bacterial viability. Pyroptosis in the context of tuberculosis is controversial: from one side it leads to potent pro-inflammation that drives tuberculosis; from other side it allows the release of intracellular bacteria and their escape to hydrolytic digestion. This type of programmed cell death depends on the assembly of a specific inflammasome leading to caspase 1 activation and IL-1 and IL-18 secretion. Some groups claimed that *M. tuberculosis* might block the inflammasome activation (Master et al., 2008). We have shown that *M. tuberculosis* in human macrophages, not only activates caspase 1 and IL1- secretion but also that different inflammasomes are assembled during infection

So we propose in this chapter to present an overview of host factors that may be manipulated in order to reverse phagosome maturation arrest, NF-kB translocation, inflammasome activation and therefore boosting the macrophage killing abilities to Koch's

Tuberculosis is usually a lung infection caused by inhalation of Koch's bacilli within microdroplets, but it can affect any organ. In the lung, bacilli are envisaged to be engulfed by cell patrolling the alveolar surface such as alveolar macrophages and tissue dendritic cells (DC). These cells transport the pathogens to the lung interstitium and draining lymph nodes. At both sites, primary lesions develop that rarely cause disease. These professional phagocytes that are devoted to maintain the lungs cleaned from particles, produce a series of bacterial insults designed to kill pathogens. However *M. tuberculosis* developed capacities to subvert the killing mechanisms of phagocytes to allow their intracellular survival and/or

et al., 2008).

(Mishra et al., 2010).

**2. The disease**

bacilli.

replication. The first contact with these phagocytic cells will display a series of innate immune mechanisms that may lead to the complete clearance of the bacilli. These include the release of NO, ROS, the fusion of the phagosome with the lysosome with the simultaneously bacteria digestion by acidic hydrolases and antimicrobial peptides (Cooper et al., 2000, Liu et al., 2007, MacMicking et al., 1997).