The flexible loop of Staphylococcus aureus IsdG is required for its degradation in the absence of heme

The ability of bacterial pathogens to sense and respond to changing environmental conditions is imperative for their survival. Transcriptional and translational mechanisms of regulation have been studied in great detail; however these pathways require synthesis of new proteins, which is a time consuming process. The most rapid strategy to alter cellular composition in order to deal with changing stressors is through protein degradation. Recent studies have suggested that regulated protein degradation is as important to cellular homeostasis as classical transcriptional and translational regulation (27). In order to avoid uncontrolled protein degradation, protease substrate specificity is critical. This fact is highlighted by the recent identification of acyldepsipeptides, a novel class of antibiotics that exerts toxicity by rendering an intracellular protease constitutively active (3). However, the mechanisms by which bacteria recognize specific proteins and target them for proteolytic degradation remain ill-defined. Bacterial pathogens such as Staphylococcus aureus must adapt to a variety of host environments in order to cause infection. For instance, upon host entry bacteria encounter an environment that is virtually devoid of available iron. In the vertebrate host 80% of iron is found as a component of heme, the majority of which is bound to hemoglobin (5). In order for S. aureus to acquire iron for use as a nutrient source, proteins of the iron-regulated surface determinant (Isd) system bind host hemoglobin, remove the heme cofactor, and transport heme into the cytoplasm (25). The cytoplasmic components of this system, IsdG and IsdI, are paralogous heme oxygenases that degrade heme to release nutrient iron (24). IsdG and IsdI are each required for growth on heme as a sole iron source, an environment which is predicted to mimic conditions experienced by the bacterium within the host. Accordingly, IsdG and IsdI are required for staphylococcal pathogenesis in a murine model of systemic infection (21). IsdG and IsdI are 64% identical at the amino acid level and their three-dimensional structures can be superimposed with a root mean square deviation of less than 2 A (14, 24). Moreover, IsdG and IsdI both degrade heme to the chromophore staphylobilin (22). Although their structures and catabolic mechanisms are nearly identical, IsdG and IsdI are not functionally redundant enzymes. In addition, IsdG and IsdI are differentially regulated by iron and heme availability (21). Under low iron conditions Fur-mediated transcriptional repression is released and isdG and isdI are transcribed. IsdG is also regulated at the post-transcriptional level such that IsdG is stabilized in the presence of heme. Therefore, IsdI is most abundant in low iron conditions, while IsdG levels are maximal in iron-deplete environments containing heme (21). This differential regulation likely allows the bacteria to fine-tune the expression of heme oxygenase activity in order to adapt to specific environments. However, the mechanism by which IsdG is specifically stabilized by heme has not been elucidated. We sought to utilize the differential heme-dependent regulation of IsdG and IsdI in order to investigate the mechanism by which IsdG is specifically targeted for degradation. Here we show that IsdG catalytic activity is not required for heme-dependent IsdG stability. Furthermore, IsdG degradation is ATP-dependent, suggesting that an as-yet unidentified ATP-dependent protease may be responsible for IsdG degradation in the absence of heme. We also show that IsdG is targeted for degradation by an amino acid motif located internal in the primary sequence. This is a unique in vivo demonstration of a bacterial protein that is specifically targeted for degradation by a sequence not located within the N- or C-terminus. Finally, we identify the flexible loop of IsdG as a critical determinant of IsdG degradation. Combined, these results begin to elucidate the mechanism of heme-dependent IsdG stability. Moreover, these studies will lead to an increased understanding of targeted protein degradation in general, as the IsdG recognition sequence may be widely utilized in protein turnover in Gram positive pathogens.