Bacillus anthracis is a spore-forming, nonmotile Gram-positive bacterium that is the causative agent of the zoonotic infectious disease, anthrax (Beyer and Turnbull 2009). It is an effective pathogen because the infectious agent of anthrax is the metabolically dormant and highly resistant spore. Upon ingestion by a professional phagocytic cell (e.g., activated macrophage), the spore undergoes germination and outgrowth to generate a vegetative cell that is capable of reproduction within the infected host, as it produces plasmid-encoded toxins and protective capsule material for evading immune capture and destruction (Fouet et al. 1999; Koehler 2009; Moayeri and Leppla 2009; Tournier et al. 2009). Germination and outgrowth in the macrophage takes place within a hostile environment, made so by the phagocyte's oxidative burst, which generates a toxic combination of reactive oxygen species (ROS), nitric oxide (NO), and hypochlorous acid (HOCl), as well as phospholipase, and antimicrobial peptides (Piris-Gimenez et al. 2005; Passalacqua and Bergman 2006; Passalacqua et al. 2006; Dawson and Liu 2008; Welkos et al. 2011). Successful establishment of infection involves mechanisms of oxidant resistance (Shatalin et al. 2008; Welkos et al. 2011). Such systems in bacteria are activated by encounters with a variety of toxic agents, not only components of the oxidative burst, but antibiotics and other chemical and physical insults (Gusarov et al. 2009; Mols and Abee 2011).
Much of what is known about the oxidative stress response in Bacilli has come from studies of Bacillus subtilis, a nonpathogen, which is a model genetic system used in studying Gram-positive physiology and the bacterial response to harsh conditions. Recent studies of oxidant sensitivity indicated that B. subtilis is more sensitive to the lethal effects of peroxide and superoxide-generating agents than is B. anthracis (Pohl et al. 2011). The findings suggest that more robust processes of oxidant detoxification and tolerance have evolved in the pathogen, which is in keeping with its developmental cycle involving reproduction within phagocytic hosts. Several regulatory proteins govern the oxidative stress response in B. subtilis, including the peroxide sensor PerR (Lee and Helmann 2006), organic hydroperoxide-sensing MarR family protein, OhrR (Fuangthong et al. 2001), HypR, which senses thiol-reactive HOCl stress (Palm et al. 2012), and SpxA (Zuber 2004). All of these proteins have orthologs in B. anthracis (PerR [BA0537], HypR [BA3379], OhrR [BA4699]). Both species possess the general stress response sigma subunit, σB, which controls a large regulon that becomes activated by starvation and reduced energy-generating capability, as well as by stress brought about through encounters with toxic chemical and physical agents (Hecker et al. 2007; van Schaik et al. 2007). Peroxide induces the σB regulon in B. subtilis, but σB is poorly activated by peroxide stress in B. anthracis, which is likely due to the different regulatory architectures that operate in the two organisms (Pohl et al. 2011; Tu et al. 2012b). The response of B. anthracis to superoxide stress, which is likely encountered within the infected macrophage, is the elevated expression of genes within the Fur (Ferric uptake regulator) regulon specifying iron uptake mechanisms, which is also observed in B. subtilis (Mostertz et al. 2004; Passalacqua et al. 2007; Tu et al. 2012a). The response seems maladaptive as it would expose macromolecules to potentially damaging, hydroxyl radicals generated by the Fenton reaction (Liochev and Fridovich 1999; Imlay 2008). Superoxide is known to cause decomposition of enzyme iron centers, which could trigger an iron starvation response through inactivation of the Fur transcriptional regulator and stimulation of the Fur regulon (Mostertz et al. 2004; Passalacqua et al. 2007). However, it has been proposed that superoxide is a germination signal for B. anthracis spores (Fisher and Hanna 2005), and accelerated iron uptake during subsequent outgrowth may assist in coping with the iron-poor environment that characterizes the infected host. In contrast to B. subtilis, the response of B. anthracis to superoxide is limited involving around 40 genes, which might reflect the signaling role of superoxide in B. anthracis infection rather than an agent of general stress generation (Tu et al. 2012b).
Peroxide stress induces over 200 genes in B. anthracis that specify a variety of activities related to detoxification, macromolecular damage repair, and disposal of damaged protein (Pohl et al. 2011). Thus, genes encoding DNA repair enzymes, and other members of the LexA regulon, are induced. Genes functioning in redox homeostasis, including those encoding components of the bacillithiol biosynthesis pathway, are also activated. A significant change in the B. anthracis transcriptome is evident from the observation that several genes encoding regulatory proteins are activated after peroxide treatment. These include genes specifying the PerR and SpxA transcriptional regulators (Bergman et al. 2007; Passalacqua et al. 2007; Pohl et al. 2011).
SpxA is a global regulator of the stress response that is activated upon thiol stress. Over 250 genes, or 144 operons, are controlled by SpxA in B. subtilis (Nakano et al. 2003; Rochat et al. 2012). The protein is a direct transcriptional activator through an interaction with the RNA polymerase alpha subunit in B. subtilis (Nakano et al. 2005; Newberry et al. 2005). Among the genes activated by SpxA are those required to establish the reduced state of thiols in the cytoplasm. Such genes include those that encode thioredoxin (trxA), thioredoxin reductase (trxB), methionine sulfoxide reductase (You et al. 2008), enzymes required for synthesis of the low-molecular-weight thiol bacillithiol (Gaballa et al. 2010; Chi et al. 2011; Gaballa and Helmann 2011), and genes that function in cysteine biosynthesis (Nakano et al. 2003; Choi et al. 2006; Zuber et al. 2011; Rochat et al. 2012). A null mutation in spx causes sensitivity to thiol reactive compounds, partial cysteine auxotrophy, and causes disruption of iron uptake control. The spx gene is under complex transcriptional control that is responsive to stress caused by variety of physical and chemical agents (Helmann et al. 2001; Petersohn et al. 2001; Jervis et al. 2007; Leelakriangsak et al. 2007; Eiamphungporn and Helmann 2008). In B. subtilis and in other Gram-positive species, spxA is transcriptionally induced by mechanisms responsive to cell envelope stress. SpxA can undergo stress-induced thiol oxidation of a CxxC disulfide center, which is necessary for its productive interaction with RNA polymerase (Nakano et al. 2005). The SpxA protein is under proteolytic control that requires the ATP-dependent protease, ClpXP, and a substrate recognition factor, YjbH (Larsson et al. 2007; Garg et al. 2009). SpxA, as might be expected given its role in regulating the oxidative stress response, has been found to be required for virulence in Streptococci and Enterococcus (Kajfasz et al. 2010, 2012; Chen et al. 2012).
Several of the low %GC Gram-positive bacteria possess multiple paralogs of Spx (Veiga et al. 2007; Turlan et al. 2009). The genome of B. anthracis bears two spxA genes, spxA1 and spxA2 (Fig. 1A). The spxA1-linked genes show syntenic similarity to those in B. subtilis, but B. anthracis also contains an additional paralogous spx gene in a part of the genome that shows no synteny with B. subtilis (Fig. 1B). Previous transcriptomic studies have shown that spxA1 is expressed in early log phase of a B. anthracis culture, while spxA2 transcript is detected during stationary phase (Bergman et al. 2006). The spxA2 gene is one of the most highly induced genes in B. anthracis cells following germination in the host macrophage (Bergman et al. 2007). The differences in expression patterns exhibited by the two paralogous genes suggest differences in their roles within the stress response network of B. anthracis.
To uncover the roles of the two paralogous genes of spxA in B. anthracis, a study was conducted to examine the phenotype conferred by null mutations in the genes encoding the two paralogs and to identify the genes that are regulated by SpxA1 and SpxA2. The work reported herein shows that the two paralogous SpxA proteins oversee two large overlapping regulons. While both function in the oxidative stress response, SpxA1 plays an essential role in the bacterium's resistance to peroxide and disulfide stress.