MEDICINE

Combating Impervious Bugs

Richard P. Novick^*

Science 15 February 2008:

Staphylococcus aureus has always been a serious human pathogen, and during recent decades it has become more serious owing to its acquisition of antibiotic resistance. In the past few years, a new strain of methicillin-resistant S. aureus (MRSA), known as USA300, and its close relatives, have emerged that are not only resistant to antibiotics but are more virulent and highly contagious. MRSA is presently spreading throughout the world, in hospitals and also in community settings where people are in close contact (1-3). Indeed, in the United Sates, MRSA infections now account for more deaths each year than AIDS (4). But two reports, by Corbin et al. on page 962 in this issue (5), and by Liu et al. in Science Express (6), are cause for some cautious optimism about new therapeutic approaches to treat such infections.

Both studies describe possible strategies for interfering with the ability of S. aureus to thwart attacks that are mounted by the immune system during infection. Bacteria defend against lethal reactive oxygen species (ROS) produced by neutrophils, immune cells that are mobilized to sites of infection. Corbin et al. show that calprotectin, a wellknown mammalian calcium-binding protein, chelates manganese (Mn²⁺), which the bacterium requires for growth and for detoxifying ROS. Liu et al. report that certain cholesterol- lowering drugs have an entirely unexpected activity against S. aureus--blocking synthesis of staphyloxanthin, the pigment that imparts the organism's characteristic color (aureus means "golden" in Latin) and also chemically detoxifies ROS.

Staphylococcal infection is an especially serious health threat in individuals with weakened immune systems, impaired circulation (as with diabetics), and surgical wounds. In deep-tissue sites, staphylococci can be lifethreatening, even in otherwise healthy individuals. Staphylococcal abscesses form when the host immune system recognizes certain bacterial products, including cell wall components and chemotactic (neutrophil-attracting) enzymes (7). The host mounts an aggressive inflammatory response, walling off the infected area by forming a meshwork of fibrin protein packed with neutrophils and other immune cells. Neutrophils take up (phagocytose) bacteria and kill them with ROS and other lethal agents. In defense, staphylococci deploy cytotoxins and enzymes to degrade host tissue and kill neutrophils and other cells. Neutrophils, however, continue their antibacterial activities posthumously (see the figure). Upon lysis, they release bactericidal ROS as well as DNA that forms a network to entrap bacteria (8). Bacteria respond by secreting potent nucleases to degrade the DNA. Staphylococci also produce substances that detoxify ROS, including staphyloxanthin and superoxide dismutase. Although the role of staphyloxanthin seems clear (9), that of calprotectin seems complex.

Raging battle. S. aureus and immune cells (neutrophils) use an arsenal of agents and enzymes to mount and combat infection. Nonantibiotic-based therapeutics may focus on the functions of calprotectin and staphyloxanthin. CREDIT: ADAPTED BY P. HUEY/SCIENCE

Calprotectin is a calciumbinding heterodimer that constitutes about 40% of the soluble cytoplasmic protein of a neutrophil. Its widespread biological roles include signaling to other immune cells after tissue damage and/or inflammation (10) and, as Corbin et al. show, defending against bacterial infection by chelating Mn²⁺. Because this cation is a required trace element, its depletion could simply inhibit bacterial growth, as the authors observed in vitro. Moreover, mice with calprotectin deficiency had abscesses with higher bacterial loads and about three times the amount of Mn²⁺ as did those in wild-type mice. But bacteria can grow in tissues and form abscesses in the presence of low natural concentrations of Mn²⁺, so it seems more likely that abscess attenuation is due to the effects of Mn²⁺ depletion on bacterial defenses against ROS. Mn²⁺ is also an essential component of bacterial superoxide dismutase A, which inactivates ROS enzymatically (11).

However, the situation may not be quite so simple, because staphylococci express catalase and other antioxidant enzymes that are repressed by Mn²⁺ through a regulatory protein, PerR (12). Thus, calprotectin could enhance the expression of these enzymes, thus enabling bacteria to counter the effects of ROS, even when the Mn²⁺ concentration is low. The effects of iron (Fe²⁺) in bacteria complicate matters further. Fe²⁺ drives the Fenton reaction, which converts hydrogen peroxide (H₂O₂) to the highly bactericidal hydroxyl radical (13); but Fe²⁺ also relieves PerR repression of the catalase gene, katA. This induces the synthesis of catalase, which destroys H₂O₂ and blocks the Fenton reaction. Therefore, katA and the other genes under the control of PerR may help counter ROS even if Mn²⁺ concentration is high (14). Because the relative effects of these various reactions in an abscess are unknown, and the prediction that adding excess Mn²⁺ would negate the calprotectin-dependent antibacterial effects of neutrophils was not examined, more work is needed to reveal how chelation of Mn²⁺ by calprotectin attenuates a bacterial infection.

As for detoxification of ROS by the pigment staphyloxanthin, nonpigmented staphylococci are rapidly killed by neutrophil-generated ROS and consequently are deficient in skin abscess formation in mice (9). Liu et al. show that similarities exist in the biosynthetic pathways of staphyloxanthin and host cholesterol. Remarkably, presqualene diphosphate is an early intermediate in both pathways. Liu et al. determined that the structure of CrtM, the staphylococcal enzyme that catalyzes presqualene diphosphate synthesis, resembles that of mammalian squalene synthetase. Moreover, the staphylococcal enzyme is inhibited in vitro by cholesterollowering drugs that block squalene synthetase. The authors further show that these cholesterol- lowering compounds attenuated a systemic murine infection by a pigmented strain of S. aureus infection (though a nonpigmented strain was not examined).

Blocking ROS detoxification by inhibiting staphyloxanthin synthesis and chelating Mn²⁺ may be attractive, new antistaphylococcal treatments that do not involve conventional antibiotics. However, some unanswered questions give pause. The strategy of removing an essential nutrient to combat infection is one previously proposed by Mahan et al. (15), who found that bacteria unable to synthesize purines were avirulent because the in vivo supplies of purines were insufficient to support bacterial growth. Whether one could reduce an essential trace metal to a concentration low enough to block bacterial growth without also compromising the functions of host cells remains to be seen. It is also a matter of concern that nonpigmented S. aureus are often isolated from abscesses and other infections. For translation to the clinic, both strategies warrant further examination.

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