The impact of metal sequestration on Staphylococcus aureus metabolism

Summary of recent advances

The Gram-positive pathogen Staphylococcus aureus poses a serious risk to public health due to its prevalence as a commensal organism, its ability to cause a multitude of diseases, and the increasing incidence of antibiotic resistant strains. S. aureus infects diverse niches within vertebrates despite being challenged by a robust immune response. The host-pathogen confrontation occurs in an environment nearly devoid of metals that are essential for bacterial proliferation. S. aureus is able to flourish in these conditions and often causes significant morbidity and mortality. This review highlights current themes pertaining to the process of host-mediated metal sequestration known as “nutritional immunity”, S. aureus metal acquisition strategies, and how proliferating within a metal restricted environment impacts bacterial physiology.

Introduction

Staphylococcus aureus is a leading cause of global morbidity and mortality [1-3]. This extracellular pathogen innocuously colonizes the anterior nares of one-third of the world’s population and is commonly associated with commensal colonization of the skin [4,5]. Vertebrates decrease the infectious capacity of invading microbes by limiting the availability of essential nutrients in a process known as nutritional immunity [6]. Once S. aureus breaches the epithelium, it survives within the bloodstream and disseminates to the peripheral organs despite limited access to nutrient metals [7,8]. A pathological hallmark of S. aureus infection is the formation of tissue lesions called abscesses (Fig. 1). Abscesses represent the extensive accumulation of neutrophils that surround invading staphylococci in an attempt to confine bacterial proliferation. In a mouse model of systemic infection, S. aureus colonizes peripheral tissues 1 to 3 hours post infection but abscess formation in the kidneys, heart and liver is not observed until 4 days post infection. This mimics the pathology of human disease and highlights the ability of S. aureus to colonize diverse niches within the host [7].

Figure 1. Staphylococcus aureus proliferates within the metal limited environment of tissues abscesses to cause disease.

The host response to S. aureus involves the extensive recruitment of neutrophils to the site of infection, leading to the formation of a tissue abscess. Lactoferrin (Lf) and Calprotectin (CP) are produced by neutrophils and severely limit the availability of iron and manganese to staphylococci. In addition, serum transferrin (Tf) further reduces the amount of iron within the host environment. Nonetheless, S. aureus has evolved metal acquisition systems that promote pathogenesis and disease.

Neutrophils restrict the growth of S. aureus by creating an environment devoid of available iron, manganese, and zinc. These metals are required cofactors for metabolic processes that facilitate staphylococcal growth [9]. Despite restricted access to these metals, S. aureus can cause life threatening disease. Understanding the mechanisms by which S. aureus survives within the metal-depleted environment of the host will provide an enhanced understanding of metabolic processes utilized by this pathogen during infection and will lead to new avenues for combating staphylococcal disease. Guided by this perspective, this review will (i) define the mechanisms by which vertebrates sequester iron, manganese, and zinc, (ii) describe the strategies S. aureus utilizes to overcome metal sequestration, and (iii) present ideas on how nutritional immunity may impact S. aureus physiology during infection.

Vertebrates restrict access to essential metals

Iron, manganese, and zinc are transition metals that act as cofactors for enzymes involved in many cellular processes including DNA synthesis, respiration, and defense against reactive oxygen and nitrogen species [10,11]. The absence of iron, manganese, or zinc severely impedes staphylococcal growth in vivo and in vitro [9,12]. Greater than 90% of iron in vertebrates is found within host cells, rendering it unavailable to S. aureus [13]. The intracellular localization of iron represents the first barrier of nutritional immunity that must be circumvented by extracellular pathogens. Additionally, host proteins that sequester iron, manganese, and zinc provide another barrier to S. aureus proliferation.

The host restricts the amount of available iron in the serum through the iron-binding activity of the glycoproteins lactoferrin and transferrin (Fig. 2A). However, the majority of iron (75 to 80%) is complexed to the tetrapyrrole ring of heme [13]. Most heme-iron is bound by hemoglobin that functions to transport oxygen to peripheral tissues within circulating erythrocytes (Fig. 2A). Hemoglobin is the most abundant heme-binding protein in vertebrates, making it an attractive iron source for pathogens that are able to liberate it from erythrocytes [14]. In the event of erythrocyte lysis, the host proteins hemopexin and haptoglobin sequester the liberated heme and hemoglobin, respectively, providing another barrier to heme acquisition [15,16]. S. aureus utilizes a multifaceted approach that targets transferrin and hemoglobin in an effort to satisfy its requirement for nutrient iron (Fig. 2B).

Figure 2. The struggle for metals during staphylococcal infection.

(A) The host proteins that sequester metals and participate in nutritional immunity. Hemoglobin is the major reservoir of heme-iron in vertebrates and is targeted by staphylococci in order to fulfill its requirement for iron. Haptoglobin and hemopexin bind hemoglobin and heme, respectively, and provide an additional barrier to staphylococcal heme-iron acquisition. The iron-binding glycoproteins transferrin (Tf) and lactoferrin (Lf) provide another obstacle to S. aureus iron (Fe⁺²) acquisition. The host protein calprotectin (CP) also sequesters Mn⁺² (manganese) and Zn⁺² (zinc) in an effort to restrict staphylococcal growth. (B) The metal acquisition systems of S. aureus. S. aureus circumvents nutritional immunity by two primary mechanisms. One mechanism involves heme-iron acquisition through the Isd system. Alternatively, staphyloferrin A (Sa) and staphyloferrin B (Sb) scavenge iron from host iron-containing proteins such as Tf and Lf. The MntABC system competes with CP for manganese in an effort to support the proliferation of S. aureus. (C) Metals are essential cofactors in many bacterial metabolic processes. Nutritional immunity limits bacterial growth by disrupting metabolic pathways necessary for bacterial proliferation. The functions of flavohemoglobin (Fh), superoxide dismutases and catalase contribute to the ability of S. aureus to withstand host-generated nitric oxide or superoxide. These proteins require metal cofactors and will have reduced function when S. aureus is unable to procure iron or manganese. The ability to generate ATP via respiration (PMF) will also be disrupted by a lack of metals.

In addition to restricting iron availability, the host also limits the availability of manganese and zinc (Fig. 2A). The host protein calprotectin inhibits the growth of S. aureus by sequestering manganese and zinc [9]. Calprotectin is a major protein constituent of the neutrophil cytoplasm and is found within abscesses at concentrations in excess of 1 mg/ml [17,18]. The observation that a ring of necrotic neutrophils surround the staphylococci within the abscess suggests that calprotectin is released from the neutrophils as a result of cell lysis [7]. Following intravenous challenge with S. aureus, calprotectin-deficient mice exhibit increased bacterial loads in the liver underscoring the importance of this host protein to protection against infection [9]. Although manganese levels are increased within abscesses of calprotectin-deficient animals compared to their wildtype counterparts, zinc levels are not changed suggesting a calprotectin-independent mechanism for zinc sequestration within abscesses. S. aureus is able to persist within tissue abscesses despite host-mediated metal chelation [7]. Given the importance of manganese and zinc to staphylococcal metabolism, it is likely that S. aureus is able to outcompete the host for these metals; however, the mechanisms by which manganese and zinc are acquired during infection are only beginning to emerge [19].

S. aureus metal acquisition strategies

The iron-limited environment of the host triggers the expression of S. aureus iron acquisition systems through alleviation of Fur-mediated transcriptional repression [20,21]. Fur represses the transcription of genes involved in iron acquisition when S. aureus is grown in iron-replete conditions. Transcription is activated when S. aureus encounters an iron-deplete environment resulting in the expression of the staphylococcal iron acquisition systems. These systems target the most abundant sources of iron, the iron-containing host proteins hemoglobin and transferrin. S. aureus obtains iron by importing host heme directly or by stealing iron from transferrin through the activity of iron-chelating siderophores.

The first step in staphylococcal heme-iron acquisition is the release of hemoglobin from erythrocytes through hemolysin-mediated erythrocyte lysis [22]. Upon release, hemoglobin is captured by the S. aureus iron-regulated surface determinant system (isd). The Isd system catalyzes the extraction of heme from hemoglobin and the passage of heme through the cell wall. The transfer of heme from the S. aureus hemoglobin receptor through the bacterial cell wall is mediated by proteins containing NEAT domains [23]. An isd-encoded ABC transporter facilitates the import of heme into the cytoplasm (Fig. 2B). Once in the cytoplasm, heme is degraded by isd-encoded heme oxygenases, facilitating the release of iron [24,25]. Heme is the preferred iron-source of S. aureus [26] and the importance of the isd system to pathogenesis is best exemplified by studies revealing that the staphylococcal hemoglobin receptor IsdB is required for full virulence [27]. IsdB is also required for renal abscess formation five days post infection [7]. These facts underscore the significance of isd-mediated heme-iron acquisition to S. aureus pathogenesis. Notably, the essentiality of iron has ensured that S. aureus has evolved additional mechanisms for acquiring this nutrient during infection.

Staphyloferrin A and staphyloferrin B are siderophores produced by S. aureus that function to scavenge iron in serum (Fig. 2B). The cognate lipoprotein receptors and associated import systems for staphyloferrin A and B are well characterized and have been reviewed elsewhere [20,28]. S. aureus strains unable to produce or utilize either staphyloferrin A or staphyloferrin B are attenuated in their ability to cause disease [29,30]. These siderophores provide an alternative source of iron for S. aureus if the bacteria are unable to acquire heme during infection. This approach to iron acquisition affords S. aureus the flexibility to meet the challenges of the iron-limiting environment of the host.

The transcriptional repressors MntR and Zur allow S. aureus to respond to manganese and zinc deplete environments, respectively, and regulate the expression of high affinity metal transporters [19,31]. In the absence of manganese and zinc, MntR- and Zur-mediated transcriptional repression is alleviated and the manganese and zinc transport systems are activated. The importance of the manganese transporter MntABC is evident when S. aureus is grown in the presence of calprotectin (Fig. 2B). Strains inactivated for mntA and mntB and hence are unable to transport manganese, are more sensitive to the effects of calprotectin than wildtype S. aureus [9]. The contribution of the Mnt system to staphylococcal pathogenesis is beginning to be resolved [9,19], but given the significance of manganese and zinc to S. aureus growth it is likely that the full arsenal of manganese and zinc acquisition pathways have yet to be uncovered.

The impact of nutritional immunity on S. aureus physiology

Bacterial metal acquisition has been referred to as the critical determinant for the outcome of infection [6]. However, little is known about the role of acquired metals in staphylococcal physiology during infection. Elucidating the protein recipients of host-derived iron, manganese, and zinc will uncover the metabolic pathways that are active during S. aureus pathogenesis. A clue to the identity of these proteins comes from a unique feature of the metals: distinctive redox potentials which make them ideal cofactors for enzymes involved in transferring electrons between molecules. The transfer of electrons is critical to S. aureus physiology during infection for at least two reasons: (i) the enzymatic reduction of reactive oxygen and nitrogen species detoxifies these poisonous molecules and (ii) the transfer of electrons is essential for the generation of a proton motive force (PMF) that increases energy production through respiration (Fig. 2C). As many a 30% of bacterial proteins require a metal cofactor, so it is likely that other metabolic processes will be affected by host-mediated metal sequestration [10,32,33].

The host immune response to bacterial infection includes the generation of the effector molecules superoxide and nitric oxide. Bacterial pathogens detoxify these molecules through the action of superoxide dismutase, catalase, and flavohemoglobin [19,34,35]. The genome of S. aureus encodes homologues of these metal-dependent enzymes and the importance of superoxide dismutase and flavohemoglobin to S. aureus pathogenicity has been established experimentally [34,36,37]. The sequestration of manganese by calprotectin increases the sensitivity of S. aureus to superoxide stress during infection. This is due to the reduced activity of the staphylococcal manganese-dependent superoxide dismutases, SodA and SodM [37]. Inactivation of sodA and sodM leads to reduced bacterial loads in the livers of systemically infected mice and in the abscesses of subcutaneously-infected mice, highlighting the importance of these enzymes to bacterial survival within the host [36,37].

Compared to other bacteria, S. aureus exhibits increased resistance to the effects of nitric oxide in part due to the expression of flavohemoglobin, which is encoded by the hmp gene (Fig. 2C). Flavohemoglobin mutants are less virulent in the systemic mouse model of infection [34]. Interestingly, flavohemoglobins typically require heme as a cofactor [38]. The heme requirement of S. aureus flavohemoglobin remains to be experimentally verified, but the fact that S. aureus can synthesize heme de novo is consistent with the possibility that Hmp is a hemoprotein. This de novo synthesis of heme requires iron and the gene products from the hemAXCDBL, hemEHY, hemN, and hemZ loci [39, for review see 40]. In addition to being used as a potential cofactor for flavohemoglobin, heme is an essential cofactor for S. aureus respiratory metabolism. The finding that inactivation of the heme-dependent cytochrome subunit qoxB leads to a significant reduction of bacterial loads in the liver emphasizes the importance of respiratory metabolism during staphylococcal infection [41]. The ability to both synthesize and acquire heme likely affords S. aureus and many other pathogenic bacteria the metabolic flexibility to overcome the challenges of colonizing diverse niches within the vertebrate host.

It is unknown how isd-mediated heme acquisition coordinates with heme synthesis pathways to maximize survival of S. aureus during infection. The iron status of the bacterium likely determines how exogenous heme is utilized. For example, when S. aureus encounters an environment where heme is the sole iron source, it is degraded to release iron. Alternatively, at sites of colonization where iron is available due to siderophore-mediated acquisition, exogenous heme might be more valuable as a cofactor. This contention is supported by in vitro experiments demonstrating that heme is segregated intact to the membrane when S. aureus is grown in the presence of alternative iron sources [26]. The significance of this finding is underscored by the fact that membrane-localized, heme-dependent cytochromes and reductases facilitate respiratory metabolism. These results suggest that S. aureus has the ability to alter exogenous heme trafficking in order to maximize respiration during infection.

Staphylococci that cannot respire are forced to produce energy through fermentation, which reduces both growth and hemolysin production [42,43]. However, fermentative enzymes such as alcohol dehydrogenases also require metal cofactors [42], supporting the possibility that the fermentative pathways of S. aureus are affected by the availability of metals during infection. Recent analysis of protein databases estimates that one third of all proteins require a metal cofactor [44-46]. Clearly, there is much to be learned regarding how alterations in metal availability impact S. aureus physiology during infection.

Concluding remarks

The transition metals iron, manganese, and zinc are vital for the proliferation of bacterial pathogens such as S. aureus. Vertebrates exploit this requirement through metal sequestration as a means to control bacterial growth. S. aureus has evolved strategies to overcome this nutritional immunity and is capable of causing significant morbidity and mortality. Given the importance of metals to S. aureus proliferation during infection, the pathways dedicated to metal acquisition and the protein recipients of these metals are appealing targets for therapeutic intervention. Inhibiting these pathways has the potential to restrict the capacity of S. aureus to generate energy and defend itself against reactive oxygen and nitrogen species. This possibility is particular attractive considering S. aureus has the capability to resist all currently available antimicrobials.

Highlights.

> The vertebrate host sequesters metals in an effort to reduce bacterial growth. > Metal depletion reduces bacterial growth. > S. aureus utilizes a multifaceted approach to overcome this metal sequestration. > The staphylococcal pathways that receive host-metals are unknown. > S. aureus utilizes host metals to maximize survival during pathogenesis.