Bacillus anthracis -derived nitric oxide is essential for pathogen virulence and survival in macrophages

Abstract

Phagocytes generate nitric oxide (NO) and other reactive oxygen and nitrogen species in large quantities to combat infecting bacteria. Here, we report the surprising observation that in vivo survival of a notorious pathogen-Bacillus anthracis-critically depends on its own NO-synthase (bNOS) activity. Anthrax spores (Sterne strain) deficient in bNOS lose their virulence in an A/J mouse model of systemic infection and exhibit severely compromised survival when germinating within macrophages. The mechanism underlying bNOS-dependent resistance to macrophage killing relies on NO-mediated activation of bacterial catalase and suppression of the damaging Fenton reaction. Our results demonstrate that pathogenic bacteria use their own NO as a key defense against the immune oxidative burst, thereby establishing bNOS as an essential virulence factor. Thus, bNOS represents an attractive antimicrobial target for treatment of anthrax and other infectious diseases.anthrax ͉ bacterial NO-synthase ͉ oxidative stress T he spore-producing Gram-positive soil organism, Bacillus anthracis, is the causative agent of anthrax, an acute lifethreatening infection in humans and domestic animals. Inhalation of B. anthracis spores results in a high rate of mortality, because effective treatment must be provided within a very short time after exposure (1). Deliberate dispersal of anthrax spores through the United States Postal Service in 2001 emphasized the importance of developing effective treatments to combat this potential biological scourge.Although the innate immune response is the first line of defense against B. anthracis, its spores survive, germinate, and proliferate in macrophages, eventually bursting them to produce a lethal titer of infectious particles. The mechanism by which B. anthracis evades immune attack is not fully understood. Most studies have been focused on major virulence factors found on two plasmids (pXO1 and pXO2) that are responsible for exotoxins, capsule formation, and spore germination (2). These plasmid-borne virulence factors have been the prime candidates for anti-anthrax drug design. However, the ability of pathogens such as B. anthracis to survive in phagocytes also depends critically on the state of their oxidative stress defense system. Reactive oxygen species (ROS) play essential roles in innate immunity against many types of microorganisms (3-5). The antibacterial effects of ROS have been largely attributed to DNA and protein damage mediated by the Fenton reaction (6). This process generates hydroxyl radicals that react with DNA bases, sugar moieties, and amino acid side chains, causing various types of lesions (7). We showed that nonpathogenic B. subtilis utilizes its own nitric oxide (NO) to gain rapid protection against sudden oxidative damage (8). The mechanism of protection does not rely on transcriptional gene induction but rather on rapid suppression of DNA damage by preventing the Fenton reaction and direct activation of catalase (8). Here, we describe the key role of NO-synthase (bNOS)-derived ...

Endogenous nitric oxide regulates the recovery of the radiation-resistant bacterium Deinococcus radiodurans from exposure to UV light

“…In addition, NO activates a major B. subtilis catalase which further protects the cell against oxidative stress (18). Furthermore, NOS-derived NO was shown to protect the human pathogen B. anthracis from oxidative damage induced by macrophages (37). Given these observations, it was unexpected that Dr ⌬nos was not more susceptible to H 2 O 2 than wt and that peroxide treatment did not induce nos gene expression (Fig.…”

“…Both Streptomyces NOS (17) and Bacillus anthracis NOS (37) have been shown to produce NO in vivo. However, unlike bacilli, Dr does not appear to contain the biosynthetic enzymes necessary to produce the mammalian NOS cofactor H 4 B (10, 38).…”

NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum

“…More recently, bacterial NOSs have been reported (20)(21)(22)(23)(24)(25). Unlike eukaryotic NOSs, prokaryotic NOSs do not have a reductase domain as a contiguous polypeptide (20,21) and consequently NO detection has been mostly limited to single turnover experiments (22).…”

“…In the case of Bacillus subtilis, exogenous reductases (a combination of flavodoxin and flavodoxin reductase) were added to NOS, leading to NO formation (determined by the formation of an NOS porphyrinFe(III)NO complex and indirectly by nitrite formation) (23). Furthermore, studies with B. anthracis and B. subtilis which have a gene for a stand-alone oxygenase domain of NOS, indicate that oxygenase domains of bacterial NOSs generate NO by recruitment of intracellular reductases (24,25).…”

The Proximal Hydrogen Bond Network Modulates Bacillus subtilis Nitric-oxide Synthase Electronic and Structural Properties

“…This metabolic role seemed to extend to the Deinococcus radiodurans NOS, which seemed able to associate with the tryptophanyl-tRNA synthetase and to nitrate Trp amino acids (20,21). Following different hypotheses, Nudler and colleagues (22)(23)(24) proposed that bacNOSs could intervene in a series of functions related to host-pathogen interaction such as protection against oxidative stress (22), pathogen survival and virulence (23), or defense against antibiotics (24). Recently, Crane suggested that D. radiodurans NOS could intervene in the recovery processes of bacteria subject to a UV stress (25).…”