Multiple Intracellular Microbial Strategies

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When macrophages ingest particulate material, such as a microorganism, an intracellular vacuole, called a phagosome, is constituted, that undergoes a step-wise maturation. This process consists of a progressive acidification and several fusion events, finally resulting in fusion with a lysosome to form a phagolyso-some. This organelle contains enzymes and the acidic environment necessary to degrade the phagocytosed material. This pathway is an important component of the macrophage's defence against invading pathogens. For an obligate or facultative intracellular pathogen to be successful, i.e. to survive and to multiply within the macrophage, it has to evolve one or more strategies to escape or to combat this phagosomal maturation process (Garcia-del-Portillo and Finlay, 1995). Schematically (and in a simplified manner), these strategies are: (i) to escape from the phagosome into the cytoplasm and spread directly from cell to cell; (ii) to inhibit phagosomal acidification and phagosome-lysosome fusion. Other evasion strategies that help microorganisms to survive within macrophages, and that will not be further discussed here, are: (iii) adaptation to the hostile phagolysosome conditions; (iv) modulation of macrophage apoptosis (e.g. several viral genomes encode for proteins that exert an antiapoptotic effect, which may ensure a sustained infection); (v) modulation of macrophage cytokine production, i.e. suppression or lack of induction of activating cytokines, or induction of deactivating cytokines; (vi) inhibition of antigen presentation and T-cell stimulation. These multiple and complex microbial strategies to counteract the cellular defences have been categorized in to three (anthropomorphic) attitudes: stealth, sabotage and exploitation (Brodsky, 1999). Detailed discussion of these microbial strategies is beyond the scope of this chapter. However, it is worth concentrating on some of the evasion manoeuvres that impact directly upon microbial iron availability. When the first strategy, that of escape to the cytosol, is operative, the microorganism probably finds its iron source within the so-called 'labile iron pool' (LIP), where the metal is thought to be in its reduced Fe(II) form and bound to low-molecular-weight molecules (see Chapter 7). It is possible that Listeria monocytogenes, which escapes from the phagosome to the cytoplasm, uses its reductase and Fe(II) uptake system to capture iron from the low-molecular-weight iron ligands of the LIP. In the case of the second strategy, where the microbe blocks the maturation of the phagosome, we could well ask at which stage in phagosome maturation it would be most effective for the block to occur. As we saw in Chapter 5, the uptake of transferrin-bound iron occurs in the early endosome, and so it might be useful for such microorganisms to arrest phagosome maturation precisely at this stage, before fusion with lysosomes, which should facilitate access to the metal. This may be the case for Mycobacterium tuberculosis, whose dual 'mycobactin T' siderophore system (De Voss et al., 1999) is well equipped to acquire iron from transferrin. Mycobacterium tuberculosis arrests the maturation of its phagosome at a stage at which the phagosome interacts with early and late endosomes, but not yet with lysosomes (Clemens and Horwitz, 1996). It is therefore possible that 'freezing' of the M. tuberculosis-containing vacuole at the early endosome stage evolved in part to facilitate iron acquisition. How this 'freezing' occurs is not fully established, although the persistence of the Rab5 protein on the M. tuberculosis-containing phagosome may help in maintaining its early endosomal properties (Clemens et al., 2000).

During the invasion of macrophages by a microorganism, both the host and the pathogen may elicit mechanisms that alter iron metabolism within the macrophage. The interested reader is referred to a recent elegant review on this topic by Eugene Weinberg, who for decades has pioneered research on iron and infection (Weinberg, 2000). One of the major weapons utilized by the macrophage against intracellular parasitism is associated with its stimulation by g-interferon. This T-cell derived cytokine promotes several activities that are relevant to iron metabolism. First, it induces a decreased expression of the number of transferrin receptors at the surface of the macrophage, resulting in a limited cellular iron uptake and impaired iron acquisition by microorganisms such as Legionella pneumophila that escapes into the cytosol and are likely to be dependent on the LIP for growth (Byrd and Horwitz, 1989). The same authors recently reported an 'experiment of nature', namely a patient, whose non-activated monocytes were uniquely non-permissive for L. pneumophila growth. This was associated with abnormally low levels of transferrin receptor expression, possibly as a result of systemic inflammation due to chronic periodontal disease, as the abnormality resolved after definitive cure of the gingivitis (Byrd and Horwitz, 2000). As this transferrin pathway of iron acquisition may be essential to certain pathogens, they-not astonishingly - have evolved opposing strategies, consisting in upregulating host cell expression of the transferrin receptor mRNA, resulting in a selective accumulation of diferric-transferrin. This is reported for Coxiella burnettii (Howe and Mallavia, 1999) and Ehrlichia chaffeensis. In the latter case, this upregulation of TfR mRNA may be secondary to a direct activation of IRP-1 (Barnewall et al, 1999).

A second way by which the T-helper 1 lymphocyte-derived g-interferon can alter iron metabolism in the macrophage is by inducing synthesis of NO by the cytokine-inducible isoform of nitric oxide synthase (iNOS, NOS2). There is now ample evidence that iNOS is expressed in human and not just in rodent mononuclear macrophages (Weinberg, 1999). The interactions of NO and iron metabolism are discussed in more detail in Chapters 7 and 10. Of importance for the present topic are: (i) NO and its complexes with other cellular compounds ('reactive nitrogen intermediates' such as peroxynitrite) exert a potent antimicrobial effect on an impressive number of pathogens by virtue of their reaction with non-haem iron prosthetic groups of enzymes involved in DNA synthesis and electron transport, eventually resulting in damage to or even death of the pathogens; (ii) NO production results in efflux of iron from the cell, while iron loading of the cell results in a suppression of nitric oxide production (Weiss etal., 1994). In view of the importance of NO as key effector molecule, it could be anticipated that some pathogens might try to subvert this machinery. We give two examples. The first concerns Leishmania major, an obligate intracellular parasite of macrophages in mammalian hosts that has proved so useful for studying the immune components of the parasite/host interactions. Lipophosphoglycan, a surface molecule of Leishmania promastigotes, was shown to reduce the expression of nitric oxide synthase mRNA in murine macrophages, at least when present prior to activation of the host cell by g-interferon (Proudfoot et al., 1996). The second concerns the ability of Cryptococcus neoformans to produce an unknown compound that can capture NO, allowing the fungus to evade the host defence mechanism (Trajkovic et al, 2000).

It is likely that g-interferon also uses other pathways to modulate iron metabolism during microbial cell invasion. This cytokine is reported to enhance the synthesis by the host macrophage of a protein (Nramp1) to be discussed later in this chapter, that may act by modulating iron transport at the phagosomal membrane. Finally, g-interferon was found to inhibit Toxoplasma gondii replication in a non-macrophage type of cell (the primary rat enterocyte) by limiting the availability of intracellular iron by an unidentified mechanism, unrelated to those just discussed, namely suppression of host-cell transferrin receptors or enhanced NO formation (Dimier and Bout, 1998). This is of interest, as enterocytes may be viewed as one of the first lines of defence against pathogen invasion.

Recently, another interaction was reported between an intracellular pathogen and the key regulator system of intracellular iron concentration, i.e. the degree of binding of the IRPs to IRE (see Chapter 7). In extracts from Leishmania tarentolae, a protein has been detected that binds specifically to the mammalian IRE. However, the exact nature and function of this protein is unclear up to now. In contrast to mammalian IRPs, the L. tarentolae IRE-binding activity was not induced by growth in iron-depleted medium (Meehan et al., 2000).

The Impact of Chronic Inflammation/Infection on Iron Metabolism 11.2.6 Comment

The reader should realize that the schematic subdivisions of methods of iron acquisition by microorganisms, even if useful for didactic reasons, are oversimplifications. For instance, many microbes utilize - in an apparently redundant manner - several of the outlined strategies according to their site of growth within or outside of the body. To give just two examples of very common pathogens: Candida albicans can produce a hydroxamate siderophore, can lyse erythrocytes and bind haemoglobin, and can produce a ferric reductase; Staphylococcus aureus can bind transferrin, can produce siderophores staphyloferrin A and B and can digest haemoglobin and haem.

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