Differential Proteomic Analysis of the Bacillus anthracis Secretome: Distinct Plasmid and Chromosome CO2-Dependent Cross Talk Mechanisms Modulate Extracellular Proteolytic Activities†

Bacillus anthracis is a gram-positive spore-forming bacterium that is the etiological agent of anthrax, a lethal disease sporadically affecting humans and animals, in particular herbivores. In its most severe manifestation, B. anthracis infection is initiated by inhalation of spores, which are taken up by alveolar macrophages and germinate into fast-dividing vegetative cells which secrete toxins and virulence factors during growth (81, 99). If untreated by prompt antibiotic administration, the bacteria invade the bloodstream, resulting in massive bacteremia and consequently generalized systemic failure and death. B. anthracis is considered to represent a potential biothreat agent, owing to the severity of the anthrax disease, the ease of respiratory contamination, and the perpetual environmental stability of the infective spores. The recent deliberate dissemination of B. anthracis (15) accelerated the efforts to identify new B. anthracis virulence-related determinants for the design of novel diagnostic, preventive, and/or therapeutic strategies. Fully virulent B. anthracis strains harbor two native plasmids, pXO1 and pXO2, which encode critical pathogenicity factors. The absence of either one of the two plasmids results in a pronounced attenuation of B. anthracis virulence. The pXO2 plasmid encodes proteins involved in the biosynthesis of the poly-d-glutamic acid capsule, which may inhibit phagocytosis of bacteria during infection; pXO1 encodes the three toxin components protective antigen (PA), lethal factor (LF) (a zinc-dependent metalloprotease which proteolytically inactivates protein kinase kinases 1 and 2), and edema factor (EF) (a calmodulin-dependent adenylate cyclase), which form two binary toxins, lethal toxin and edema toxin. PA, the common component of both toxins, is not toxic by itself, yet it plays the central role of binding a specific receptor on the host cells and translocating LF and EF into the cytosol of infected cells, where they exert their detrimental activities. Anthrax is acknowledged as a toxinogenic disease, owing to the lethality of pure toxin preparations (77); on the other hand, additional B. anthracis secreted proteins are most probably involved in the onset and course of the disease and in survival of the bacteria in the host. The regulatory circuits governing the virulence of B. anthracis are still to be fully deciphered, yet certain observations suggest that the virulence of the bacteria entails cross talk mechanisms which link expression of plasmid-encoded and chromosomally encoded genes. The regulatory AtxA protein, encoded by pXO1, is essential for expression of the toxin and capsule synthesis genes in vivo (a situation which can be mimicked by growing the bacteria in minimal medium under high bicarbonate-CO2 conditions) (75). Two additional regulatory proteins, AcpA and AcpB, encoded by pXO2, were suggested to act downstream of AtxA and to affect capsule synthesis (35, 36). AtxA was found also to influence expression of chromosomal genes, either directly or via AcpA and AcpB. In addition, the protein AbrB, which is a chromosomally encoded transition state regulator, was suggested to negatively control the activity of the toxin gene promoters (123, 131) via AtxA. Secreted proteins include factors involved in pathogenicity, in particular in gram-positive bacteria (79). Such proteins may serve as possible targets for diagnostic purposes and/or therapeutic intervention. Bacteria of the Bacillus cereus phylogenetic group (B. cereus and Bacillus thuringiensis), to which B. anthracis belongs, secrete a diversity of factors that are essential for virulence, including toxins, hemolysins, proteases, and lecithinases. Notably, in these bacteria the secretion of certain virulence factors is regulated by a pleiotropic regulator, PlcR (2, 82, 87, 110), which is inactive in B. anthracis (2). It has been suggested that the evolutionary inactivation of the PlcR regulon in B. anthracis was due to incompatibility with the AtxA-controlled regulon and reflects the fact that the PlcR target genes are not essential for anthrax pathogenicity (96). Several studies have postulated that secreted proteases, other than those belonging to the silenced PlcR regulon, are responsible for some clinical manifestations of anthrax (1, 9, 117, 140). Such proteases could damage host tissues, interfere with immune effectors of the host, and/or provide nutrients for bacterial survival (103). Some chromosomally encoded B. anthracis extracellular proteases were suggested to be controlled by Cot43, a novel regulatory gene encoded by pXO1 (9). The availability of the B. anthracis genomic DNA sequence (109, 121) paved the way for high-throughput genomic, transcriptomic, and proteomic analyses of B. anthracis (6, 7, 8, 17, 25, 41, 50, 64, 78, 85, 121, 144) in an effort to elucidate pathogenicity mechanisms by identification of novel virulence factors or in search for specific therapeutic and/or diagnostic targets. Indeed, bioinformatic surveys of the B. anthracis genome (7, 8, 121) suggested that proteins other than PA, LF, and EF, may participate in anthrax pathogenesis. Furthermore, many B. anthracis open reading frames (ORFs) encode potentially secreted or membrane-bound proteins exhibiting homology to known virulence factors from other bacteria (7, 8). A preliminary proteomic study carried out in our laboratory examined membrane proteins prepared from a nonvirulent B. anthracis strain and led to the recognition of a number of immunodominant exposed proteins (8, 25). Here, we document an extended proteomic study, focusing on B. anthracis secreted proteins, which expands the data set of expressed B. anthracis proteins from both virulent and nonvirulent strains (6, 25, 41, 50, 64, 78, 85, 144). Based on identification of more than 400 two-dimensional electrophoresis (2-DE)-separated protein spots, we report the expression of 64 proteins which represent the most abundant B. anthracis secreted proteins, many of which resemble factors involved in the virulence of other pathogens. Comparison of the relative abundances of proteins in pXO1- and pXO2-containing and plasmid-cured strains reveals about 30 ORFs which are either preferentially expressed or repressed in the virulent B. anthracis strain under conditions which are considered to simulate those encountered within the mammalian host. The pattern of expression of these specific proteins demonstrates that B. anthracis possesses distinct regulatory pathways which involve plasmid- or chromosome-encoded CO2-inducible responsive factors.