The innate immune system in humans consists of both cellular and humoral components that collaborate to eradicate invading bacteria from the body. Here, we discover that the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, does not grow in human serum. Fractionation of serum by gel filtration chromatography led to the identification of human transferrin as the inhibiting factor. Purified transferrin blocks growth of both the fully virulent encapsulated B. anthracis Ames and the non-encapsulated Sterne strain. Growth inhibition was also observed in serum of wild-type mice but not of hypotransferrinemic mice that only have ϳ1% circulating transferrin levels. We were able to definitely assign the bacteriostatic activity of transferrin to its iron-binding function: neither iron-saturated transferrin nor a recombinant transferrin mutant unable to bind iron could inhibit growth of B. anthracis. Additional iron could restore bacterial growth in human serum. The observation that other important Gram-positive pathogens are not inhibited by transferrin suggests they have evolved effective mechanisms to circumvent serum iron deprivation. These findings provide a better understanding of human host defense mechanisms against anthrax and provide a mechanistic basis for the antimicrobial activity of human transferrin.The Gram-positive bacterium Bacillus anthracis can infect humans and is notorious for its potential as a biological weapon (1). Depending on the route of infection, B. anthracis causes respiratory, cutaneous, or gastrointestinal anthrax (2). The most lethal form, respiratory anthrax, is caused by inhalation of dormant bacterial spores, which are taken up by alveolar macrophages and dendritic cells. These spores can germinate within macrophages and hijack them for transport of vegetative bacilli to the lymph nodes. Subsequent spread into the bloodstream often results in sepsis and death (1). The ability to divide within macrophages without inducing an inflammatory reaction depends on the plasmid-encoded tripartite anthrax toxin (pXO1) that blocks intracellular signaling in immune cells (3-5). Intradermal inoculation with B. anthracis spores results in cutaneous anthrax, a milder infection that normally remains localized and resolves spontaneously (6).Little is known about human innate immune defenses against B. anthracis (7). In cutaneous anthrax, neutrophils are essential for maintaining a localized infection (6). Recent studies have demonstrated that cathelicidin antimicrobial peptides, expressed by epithelial cells and phagocytes, are also an important component the innate immune response that targets B. anthracis (8 -9). Serum proteins provide additional defense functions by activation of the complement system. Complement-depleted or C5-deficient mice are much more susceptible to infection with B. anthracis (10). However, the poly-␥-D glutamic acid capsule (pXO2) makes B. anthracis highly resistant to complement-dependent clearance (11). Here we discover that serum also provides resis...

In the treatment of inhalational anthrax, the prolonged course of antibiotics required to achieve prophylaxis may not be necessary to prevent anthrax that results from the germination of retained spores after the discontinuation of antibiotics.

A transposon site hybridization (TraSH) assay was developed for functional analysis of the Bacillus anthracis genome using a mini-Tn10 transposon which permitted analysis of 82% of this pathogen's genes. The system, used to identify genes required for generation of infectious anthrax spores, spore germination, and optimal growth on rich medium, was predictive of the contributions of two conserved hypothetical genes for the phenotypes examined.The goal of obtaining a detailed understanding of Bacillus anthracis physiology has not been realized due to limited information concerning factors required for growth, sporulation, and virulence. For example, few factors or processes required for generation of the anthrax spore, which develops to enable long-term survival outside the host, have been described (28). Such information would be very valuable as the spore is the infectious form of the pathogen and has been the focus of several virulence and vaccine studies (1,5,15,16). Recently, a functional genomics assay termed the transposon site hybridization (TraSH) assay was described, and this assay has the potential to significantly increase our understanding of microbial physiology by directly identifying genes required for growth and survival in specific environments (18). The TraSH assay uses microarray technology to globally determine the locations of transposon insertions in a population of mutants and to compare the effects of each insertion on the representation of clones before and after exposure to an environmental stress ( Fig. 1) (19,20). Comprehensive TraSH screens depend on thorough distribution of transposon insertion mutations in the genome. Several investigators have noted that transposons commonly used in gram-positive bacteria (Tn916 and Tn917) appear to target preferentially one or both of the B. anthracis virulence plasmids and that few insertions are observed in the chromosome (9,11,24,26). Recently, a mariner-based transposon mutagenesis system which shows promise was described for B. anthracis; however, the utility of this system in global mutagenesis has not been demonstrated (23). Thus, such mutagenesis systems may not permit global genome-wide analysis of B. anthracis physiological systems in strains harboring one or both of the virulence plasmids. This presents a significant hurdle since strains that lack both virulence plasmids are completely attenuated and of limited use in genetic screens for virulence traits in animal models of disease (26). In this study we sought to adapt TraSH to functional genomic analyses of the B. anthracis genome using a mini-Tn10 transposon and whole-genome B. anthracis microarrays.Mini-Tn10 permits global mutagenesis of the B. anthracis genome. Plasmid pIC333, a mini-Tn10 vector encoding spectinomycin resistance and containing a tnpA allele with relaxed target specificity to increase the randomness of transposition (21), was used to mutagenize two B. anthracis strains, B. anthracis ⌬Ames harboring the capsule plasmid (pXO1 Ϫ pXO2 ϩ ) and a derivative of this strai...