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Life and Death in a Macrophage: Role of Glyoxylate Cycle in Virulence

Life and Death in a Macrophage: Role of Glyoxylate Cycle in Virulence


Michael C. Lorenz and Gerald R. Fink


"Phagocytic cells of the innate immune system, such as macrophages and neutrophils, are a primary line of defense against microbial infections. Patients with defects in innate immunity, such as those with chronic granulomatous disease or neutropenia, are extremely sensitive to a variety of infections. When a phagocyte recognizes the presence of an invading cell, it engulfs the microbe with its membrane to form the phagosome, an intracellular compartment containing the microbe. This compartment matures by fusion with lysosomes to create the phagolysosome, an organelle replete with antimicrobial compounds and an acidic pH. Internalization creates a hostile environment for the microorganism, which, of course, is the intent. The phagolysosome is a precarious neighborhood even before the onslaught of antimicrobial compounds. Engulfment by the macrophage thrusts the microorganism into an alien milieu, one devoid of key nutrients necessary for metabolism and division. Surviving the antimicrobial assault in the phagolysosome depends on the microbe's ability to synthesize the proteins and other cellular components necessary to counteract these stresses. Thus, a pathogen must find the requisite nutrients to provide the building blocks for these complex macromolecules and the energy with which to synthesize them.

In this article we consider the initial responses of several microbes to nutrient deprivation inside the macrophage. The first of these, Mycobacterium tuberculosis, the bacterium that causes tuberculosis, resides for prolonged periods within the macrophage, in which it can proliferate and subsequently spread throughout the body. The second, the yeast Saccharomyces cerevisiae, is killed efficiently by the macrophage. The third, the opportunistic fungal pathogen Candida albicans, survives ingestion by changing rapidly from a yeast to a filamentous morphology, lysing the macrophage from the inside out. Once free, C. albicans cells are able to disseminate through the body. The interaction of C. albicans with the macrophage is transient, as opposed to the long-term persistence of M. tuberculosis. Although the outcomes of this macrophage capture are quite different among the three microbes, the initial responses of all three to the internal environment are remarkably similar: induction of the glyoxylate cycle, a pathway that permits the utilization of compounds with two carbons (C2 compounds), such as acetate, to satisfy cellular carbon requirements.

Systemic fungal infections have increased dramatically in prevalence and severity over the last few decades, in concert with the number of patients living for extended periods with significant immune dysfunction. AIDS, cancer chemotherapy, and organ transplantation have all contributed to this rise, as has the widespread use of antibiotics. The most common systemic fungal infection is candidiasis, which accounts for well over half of these invasive mycoses (3). A single species, C. albicans, causes the majority of these infections. C. albicans, which also causes oropharyngeal thrush and vaginitis, is normally a commensal of the mammalian gastrointestinal tract, in which it lives without adverse effects on the host. Both C. albicans and S. cerevisiae are readily phagocytosed by cultured macrophages in the presence of serum. While the macrophages efficiently kill S. cerevisiae, engulfment induces C. albicans cells to grow in a filamentous morphology. These hyphal filaments can penetrate through the membrane of the phagocytic cell, releasing the fungal cell back into the extracellular medium while killing the macrophage in the process. The different outcomes are not surprising; C. albicans is a common pathogen while S. cerevisiae is rarely found in human hosts.

The primary function of the glyoxylate cycle is to permit growth when C2 compounds, such as ethanol and acetate, are the only sources of carbon. The glyoxylate pathway (also dubbed the glyoxylate shunt, for clear reasons) bypasses these decarboxylations, allowing C2 compounds to serve as carbon sources in gluconeogenesis and to be incorporated into glucose and, from there, into amino acids, DNA, and RNA. Glucose, as the preferred carbon source in most organisms, can be both converted into five-carbon sugars (such as ribose and deoxyribose) via the pentose phosphate pathway and catabolized to acetyl-CoA via glycolysis. In microorganisms, however, glucose is frequently not available, and simple carbon compounds provide the only accessible carbon.

With the population of immunocompromised people on the rise, the frequency of invasive fungal infections continues to increase, making the need for effective treatments more imperative."


C. albicans is a yeast related to the brewer's yeast S. cerevisiae. Brewer's yeast is not normally pathogenic, though it can be isolated from patients on rare occasions. Because of the plethora of molecular genetic tools available for this organism, it is frequently used to model various aspects of the behavior of C. albicans.

Both C. albicans and S. cerevisiae are readily phagocytosed by cultured macrophages in the presence of serum. While the macrophages efficiently kill S. cerevisiae, engulfment induces C. albicans cells to grow in a filamentous morphology. These hyphal filaments can penetrate through the membrane of the phagocytic cell, releasing the fungal cell back into the extracellular medium while killing the macrophage in the process. The different outcomes are not surprising; C. albicans is a common pathogen while S. cerevisiae is rarely found in human hosts.

We utilized a genomic approach to understand the fungal response to phagocytosis. To do this, we used S. cerevisiae as a model, since genomic microarrays have been available for S. cerevisiae for several years, whereas the genome sequence of C. albicans has only recently been completed (24). Using a coculture system, we isolated S. cerevisiae cells from the macrophage phagolysosome after 3 h of contact, a time at which their C. albicans counterparts, phagocytosed but not killed by the macrophage, were beginning to escape from the macrophage. RNA from this population was analyzed with genomic microarrays and compared to RNA from cells grown in medium alone (11).

The primary response to phagocytosis in S. cerevisiae was the induction of gene products related to the glyoxylate cycle. Both isocitrate lyase (the ICL1 product) and malate synthase (the MLS1 product), the key enzymes of the glyoxylate cycle, were highly induced (both were induced 22-fold above the levels in the control population), as were malate dehydrogenase and citrate synthase. Further, the products of genes with functions associated with the glyoxylate cycle were also induced, such as acetyltransferases and carrier proteins which transport metabolites between organelles, acetyl coenzyme A (acetyl-CoA) synthase, and the gluconeogenic enzyme fructose-1,6-bisphosphatase. In total, 11 of the 15 genes whose products were most highly induced in macrophages encode proteins whose function is related to the glyoxylate cycle. Other transcripts, most notably those of the tricarboxylic acid (TCA) cycle, were not induced under these conditions.

Based on these observations for Saccharomyces, we analyzed the glyoxylate pathway in C. albicans when this organism is inside the macrophage. The C. albicans homologs of isocitrate lyase (products of the CaICL1MLS1 genes) are also induced upon phagocytosis, as determined by Northern blotting. The C. albicans {Delta}icl1/{Delta}icl1 mutant strain has no in vitro phenotypes other than the expected inability to utilize C2 is markedly less virulent in a mouse model of systemic candidiasis (11). Further, the mutant strain is less persistent in internal organs such as the kidney and liver than is the wild-type strain (our unpublished observations). Thus, in these fungi and in M. tuberculosis, the glyoxylate cycle is both induced during phagocytosis by the macrophage and required for full virulence in C. albicans. and Ca carbon sources, but it

S. cerevisiae is not a pathogen in immunocompetent humans or in the mouse model described earlier. However, Goldstein and McCusker have developed an animal model that allows them to assess the ability of different Saccharomyces strains to survive in vivo (4). This assay uses immunodeficient mice lacking part of the complement cascade and is done as a competition, with two Saccharomyces strains being injected into the same mouse. The relative levels of abundance of the two strains in the tissues of the animal (they focused on the brain) at time points several weeks postinjection are a measure of the in vivo fitness of the two strains. Using this system, they determined that an {Delta}icl1 mutant strain persists nearly as well in the brain as the wild-type strain; hence, the glyoxylate cycle does not significantly affect the ability of S. cerevisiae to survive in vivo (4).

The role of the glyoxylate cycle in one other fungal pathogen, the yeast C. neoformans, has been studied. This yeast, taxonomically quite distant from C. albicans and S. cerevisiae, can cause a fungal pneumonia, but its most serious manifestation is a central nervous system (CNS) meningitis. This infection is one of the leading causes of death in AIDS patients (8, 20). J. Perfect and colleagues found that isocitrate lyase (the product of ICL1) is upregulated in C. neoformans cells at the site of infection (the CNS), as determined by differential display technology. Unlike the earlier examples, this population was from an in vivo infection and therefore was not necessarily exposed to phagocytic cells. Mutations in ICL1, however, do not affect the virulence of this fungus in an animal model of cryptococcal meningitis (18a). As for S. cerevisiae, this finding raises the question of why the glyoxylate cycle is induced if it does not affect the organism's fitness in vivo.


Full Article:

http://ec.asm.org/cgi/content/full/1/5/657







Keywords: Life Death Macrophage Glyoxylate Cycle Virulence Phagocytic neutrophils drjefftop advanced

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