Nutritional Supply and Pathogenesis The Glyoxylate Cycle Case

Genes encoding enzymes of the glyoxylate cycle have been implicated in both fungal and bacterial pathogenesis (Munoz-Elias & McKinney, 2005; McKinney et al., 2000; Lorenz & Fink, 2001). The glyoxylate cycle involves two critical steps catalysed by the enzymes isocitrate lyase and malate synthase, which bypass the two decarboxylation steps of the TCA cycle. Therefore, the glyoxylate cycle is required for growth on gluconeogenic carbon sources, such as acetate and fatty acids, and it is activated under conditions of nutrient (glucose) deprivation. Such conditions are believed to occur inside macrophages and pose particular challenges to intracellular pathogens (Lorenz & Fink, 2002). In C. albicans, the isocitrate lyase gene icl1 is upregulated during growth inside macrophages. Strains deficient for icl1 are less virulent than wild-type strains in a mouse pathogenicity model (Lorenz & Fink, 2001). Therefore the ability to utilize fatty acids as carbon sources appears to be important for pathogenesis (Lorenz et al., 2004; Piekarska et al., 2006; Ramirez & Lorenz, 2006). Plant pathogens have also been shown to require the glyoxylate cycle for pathogenicity (Idnurm & Howlett, 2002; Solomon et al., 2004; Wang et al., 2003). However, this is not universal amongst all pathogens as it has been shown that loss of isocitrate lyase in C. neoformans, for example, has no effect in on pathogenicity (Rude et al., 2002). It is likely that host niche in which pathogens operates will dictate the requirement for the various nutrient assimilation pathways.

Despite the importance of the glyoxylate cycle in virulence, the molecular mechanisms which regulate the isocitrate lyase encoding genes in pathogens are poorly understood. In the non-pathogenic fungus A. nidulans, expression of the isocitrate lyase encoding acuD gene is regulated by a group of Zn(II)2Cys6 DNA-binding proteins: FacB, in response to acetate (Todd et al., 1997); FarA, in response to long-chain fatty acids; and ScfA and FarB, in response to short-chain fatty acids (Hynes et al., 2006). In S. cerevisiae the glyoxylate cycle genes are also activated by the Zn(II)2Cys6 DNA-binding transcriptional activators Cat8 and Sip4 in response to glucose limitation (Schuller, 2003). In addition to the acetate and fatty acid induction of the glyoxylate cycle genes, their expression is also subject to glucose-mediated repression by CreA in A. nidulans (Bowyer et al., 1994;

De Lucas et al., 1994) or Migl in S. cerevisiae (Schuller, 2003). In P. marneffei, acuD is independently regulated by acetate and temperature and a combination of both inducing conditions result in the highest level of expression, suggesting additive relationship (Figure 9.5) (Canovas & Andrianopoulos, 2006). acuD is induced at 37°C even in the presence of a repressing carbon source such as glucose. Regulation of acuD expression by temperature in P. marneffei is dependent on specific cis elements and trans-acting factors, as determined by reciprocal promoter exchange and heterologous expression studies using P. marneffei and A. nid-ulans, showing a unique evolutionary path for acetate and fatty acid regulation in this dimorphic pathogen. The Zn(II)2Cys6 DNA-binding motif transcriptional activator FacB is responsible for carbon source-dependent, but not temperature-dependent induction of acuD in P. marneffei (Canovas & Andrianopoulos, 2006). acuD is also independently regulated by the dimorphic switching developmental programme and part of this control is through the developmental transcriptional activator AbaA. However, deletion of abaA does not completely eliminate temperature-dependent induction, suggesting that acuD and the glyoxylate cycle are regulated by a complex network of factors in P. marneffei which may contribute to its pathogenicity (Canovas & Andrianopoulos, 2006). The P. braziliensis acuD orthologue was also shown to be differentially expressed during the dimorphic transition (Goldman et al., 2003) and is highly expressed in the pathogenic yeast growth form at 37°C (Felipe et al., 2005).

Recently it has been reported that the glyoxylate cycle genes in C. albicans are repressed by the physiological concentrations of glucose found in the bloodstream (Barelle et al., 2006). However, these genes are induced upon phagocytosis by macrophages or neutrophils, emphasizing the importance of carbon metabolism during pathogenesis. In P. marneffei, high concentrations of glucose are not enough

Figure 9.5 Transcriptional regulation of the glyoxylate bypass gene acuD encoding isocitrate lyase. The acuD promoter is shown (bottom) with the predicted DNA-binding sites depicted for the morphogenetic regulator AbaA (arrowhead), acetate regulator FacB (rectangle), and fatty acid regulators FarA/FarB (oval). Above the promoter are depictions of the AbaA and FacB regulators (centre) showing the environmental cues (left) which trigger their activity on the acuD promoter and the relative strength of these signals on transcriptional activation (right)

Figure 9.5 Transcriptional regulation of the glyoxylate bypass gene acuD encoding isocitrate lyase. The acuD promoter is shown (bottom) with the predicted DNA-binding sites depicted for the morphogenetic regulator AbaA (arrowhead), acetate regulator FacB (rectangle), and fatty acid regulators FarA/FarB (oval). Above the promoter are depictions of the AbaA and FacB regulators (centre) showing the environmental cues (left) which trigger their activity on the acuD promoter and the relative strength of these signals on transcriptional activation (right)

to repress the expression of acuD and temperature induction overrides glucose repression. After passing the primary line of defence (the macrophages), P. marneffei spreads throughout the body and yeast cells have been found in blood and fluids (Vanittanakom et al., 2006). Therefore, it is important to test the role of P. marneffei acuD during the early stages of infection and through progression to systemic infection in an appropriate pathogenic model.

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