Influence of the phosphoenolpyruvate:carbohydrate phosphotransferase system on toxin gene expression and virulence in Bacillus anthracis

Abstract

AtxA, the master virulence gene regulator of Bacillus anthracis, is a PRD-Containing Virulence Regulator(PCVR) as indicated by the crystal structure, posttranslational modifications and activity of the protein. PCVRs are transcriptional regulators, named for PTS Regulatory Domains (PRDs) subject to phosphorylation by the phosphoenolpyruvate phosphotransferase system (PEP-PTS) and for their impact on virulence gene expression. Here we present data from experiments employing physiological, genetic and biochemical approaches that support a model in which the PTS proteins HPr and Enzyme I (EI) are required for transcription of the atxA gene, rather than phosphorylation of AtxA. We show that atxA transcription is reduced 2.5-fold in a mutant lacking HPr and EI, and that this change is sufficient to affect anthrax toxin production. Mutants harboring HPr proteins altered for phosphotransfer activity were unable to restore atxA transcription to parent levels, suggesting that phosphotransfer activity of HPr and EI is important for regulation of atxA. In a mouse model for anthrax, a HPr -EImutant was attenuated for virulence. Virulence was restored by expressing atxA from an alternative, PTS-independent, promoter. Our data support a model in which HPr transfers a phosphate to an unidentified downstream transcriptional regulator to influence atxA gene transcription.

Carbohydrate Metabolism Affects Macrophage-Mediated Killing of Enterococcus faecalis

“…Bacteria depend on the phosphotransferase system (PTS) to sense and transport extracellular carbohydrates. The PTS has been also reported to affect virulence gene expression and infection in Streptococcus sp., Bacillus anthracis , and Vibrio cholerae ( 32 – 34 ), and mannose metabolites can also act as signaling molecules to coordinate virulence gene expression and influence infection ( 25 ). Surface components, including teichoic acids, a capsule, and enterococcal polysaccharide antigen, play key roles for Enterococcus sp.…”

PRD-Containing Virulence Regulators (PCVRs) in Pathogenic Bacteria

“…In B. anthracis, addition of glucose increased the expression of pagA in an AtxA-mediated manner and it was noted that this also required a functional CcpA (Chiang et al, 2011). Conversely, Bier and colleagues reported no such alterations in atxA expression in the presence of glucose in the media, nor were they able to detect a difference in atxA expression between a WT and ccpA deletion strain (Bier et al, 2020). Thus, changes in the availability of glucose could alter both AtxA and Mga expression through CCR rather than through direct interaction with EI/HPr/EIIB proteins.…”

“…Taken together, these reports suggest that AtxA activity may be directly linked to the PTS and phosphorylation. However, a recent report by Bier and colleagues has challenged the hypothesis that AtxA is directly phosphorylated by PTS proteins ( Bier et al., 2020 ). They found that EI and HPr did not directly phosphorylate AtxA in vitro , but instead, an intermediate regulatory protein is likely phosphorylated by the PTS which in turn modulates the expression of atxA .…”

“…Although there is no evidence of direct PTS phosphorylation of Mga in vivo , Valdes and colleagues demonstrated that Mga does undergo variable phosphorylation in vivo ( Valdes et al., 2018 ). Unlike AtxA, Hondorp et al provided clear evidence that Mga from serotype M4 ( mga-2 ) and M1 ( mga-1 ) could be phosphorylated by the PTS in vitro using purified PEP, EI, and HPr ( Hondorp et al., 2013 ; Bier et al., 2020 ). Combinations of phospho-mimetic and -ablative mutations targeting the histidine residue codons in mga yielded a collection of mutants with differential Mga activity ( Figure 3 ).…”

BrnQ-type Branched-chain Amino Acid Transporters Influence Bacillus anthracis Growth and Virulence

“…Bacillus anthracis, the bacterium that causes anthrax, is well known for its robust proliferation in diverse niches of mammalian hosts. Infection can result in up to 10 8 colony-forming units (CFU) per gram of tissue, including various organs, blood, and cerebral spinal fluid, at the time of host death (1)(2)(3)(4)(5). While the roles of several secreted virulence factors, including the anthrax toxin proteins, poly-D-glutamic acid capsule, siderophores, and proteases, have been discerned for anthrax (2,(6)(7)(8)(9), the metabolic attributes of B. anthracis that permit rapid proliferation to high numbers in multiple mammalian tissues have not been established.…”