Loss of Dihydroxyacid Dehydratase Induces Auxotrophy in Bacillus anthracis

Abstract

Anthrax disease is caused by infection with the bacteria Bacillus anthracis which, if left untreated, can result in fatal bacteremia and toxemia. Current treatment for infection requires prolonged administration of antibiotics. Despite this, inhalational and gastrointestinal anthrax still result in lethal disease. By identifying key metabolic steps that B. anthracis uses to grow in host-like environments, new targets for antibacterial strategies can be identified. Here, we report that the ilvD gene, which encodes dihydroxyacid dehydratase in the putative pathway for synthesizing branched chain amino acids, is necessary for B. anthracis to synthesize isoleucine de novo in an otherwise limiting microenvironment. We observed that Δ ilvD B. anthracis cannot grow in media lacking isoleucine, but growth is restored when exogenous isoleucine is added. In addition, ΔilvD bacilli are unable to utilize human hemoglobin or serum albumin to overcome isoleucine auxotrophy, but can when provided with the murine forms. This species-specific effect is due to the lack of isoleucine in human hemoglobin. Furthermore, even when supplemented with physiological levels of human serum albumin, apotransferrin, fibrinogen, and IgG, the ilvD knockout strain grew poorly relative to non-supplemented wild-type. In addition, comparisons upon infecting humanized mice suggest that murine hemoglobin is a key source of isoleucine for both WT and Δ ilvD bacilli. Further growth comparisons in murine and human blood show that the auxotrophy is detrimental for growth in human blood, not murine. This report identifies ilvD as necessary for isoleucine production in B. anthracis , and that it plays a key role in allowing the bacilli to effectively grow in isoleucine poor hosts. Importance Anthrax disease, caused by B. anthracis , can cause lethal bacteremia and toxemia, even following treatment with antibiotics. This report identifies the ilvD gene, which encodes a dihydroxyacid dehydratase, as necessary for B. anthracis to synthesize the amino acid isoleucine in a nutrient-limiting environment, such as its mammalian host. The use of this strain further demonstrated a unique species-dependent utilization of hemoglobin as an exogenous source of extracellular isoleucine. By identifying mechanisms that B. anthracis uses to grow in host-like environments, new targets for therapeutic intervention are revealed.

BrnQ-Type Branched-Chain Amino Acid Transporters Influence Bacillus anthracis Growth and Virulence

“…The deletion of BCAA biosynthesis genes in other bacteria harboring apparent BCAA transporter genes, including L. monocytogenes and S. pneumoniae , also results in reduced virulence ( 20 , 63 ). We note that while the manuscript was in review, Jelinski et al reported work indicating that an ilvD -null mutant of B. anthracis strain 34F2 was not attenuated in a murine model using intranasal infection with spores ( 64 ). The bacterial strain and infection models differed from those employed in our study, and the basis for this conflicting result will be examined in future studies.…”

Handling of Outbreaks of Anthrax: Future Perspectives and Challenges

“…The spores are resilient to certain chemicals and elements, and they can survive in the soil for almost 50 years. The bacteria return to the disease-causing vegetative stage when the spores gain entry in an animal, generally by feeding infected grass or by respiration (Jelinski et al 2021).…”