Petrobactin Protects against Oxidative Stress and Enhances Sporulation Efficiency in Bacillus anthracis Sterne.

Ada K. Hagan,Zachary I. Mendel,Stephen R. Cendrowski,Ashootosh Tripathi,Yael M. Plotnick,Ryan E. Dingle,David H. Sherman,Philip C. Hanna,Howard A. Shuman

doi:10.1128/mbio.02079-18

Abstract

Bacillus anthracis is a Gram-positive bacillus that under conditions of environmental stress, such as low nutrients, can convert from a vegetative bacillus to a highly durable spore that enables long-term survival. The sporulation process is regulated by a sequential cascade of dedicated transcription factors but requires key nutrients to complete, one of which is iron. Iron acquisition by the iron-scavenging siderophore petrobactin is required for vegetative growth of B. anthracis under iron-depleted conditions and in the host. However, the extent to which petrobactin is involved in spore formation is unknown. This work shows that efficient in vitro sporulation of B. anthracis requires petrobactin, that the petrobactin biosynthesis operon (asbA to -F) is induced prior to sporulation, and that the siderophore itself associates with spores. Petrobactin is also required for oxidative stress protection during late-stage growth and for wild-type levels of sporulation in sporulation medium. Sporulation in bovine blood was found to be petrobactin dependent. Collectively, the in vitro contributions of petrobactin to sporulation as well as growth imply that petrobactin may be required for B. anthracis transmission via the spore during natural infections, in addition to its key known functions during active anthrax infections.IMPORTANCEBacillus anthracis causes the disease anthrax, which is transmitted via its dormant, spore phase. However, conversion from bacillus to spore is a complex, energetically costly process that requires many nutrients, including iron. B. anthracis requires the siderophore petrobactin to scavenge iron from host environments. We show that, in the Sterne strain, petrobactin is required for efficient sporulation, even when ample iron is available. The petrobactin biosynthesis operon is expressed during sporulation, and petrobactin is biosynthesized during growth in high-iron sporulation medium, but instead of being exported, the petrobactin remains intracellular to protect against oxidative stress and improve sporulation. It is also required for full growth and sporulation in blood (bovine), an essential step for anthrax transmission between mammalian hosts.

Highlights

Bacillus anthracis is a gram-positive, spore-forming bacillus, which causes the disease anthrax
Anthracis Sterne, an asb mutant strain, and a dhb mutant strain were incubated in iron-depleted medium supplemented with 1mM inosine (IDM+I) for one hour
To further explore our hypothesis that petrobactin plays a role in spore biology, we tested the ability of an asb mutant strain and a dhb mutant strain to sporulate relative to wild-type

Summary

Introduction

Bacillus anthracis is a gram-positive, spore-forming bacillus, which causes the disease anthrax. Anthrax can manifest in four ways depending on the route of exposure to B. anthracis spores: cutaneous, inhalational, gastrointestinal, or injectional [1, 2]. The spores, a metabolically dormant form of B. anthracis, are taken up by antigen presenting cells (APCs) such as macrophages and dendritic cells [3, 4]. APCs, a set of small molecules from the host initiate germination of spores into vegetative bacilli [3]. The bacilli rapidly initiate cellular functions and within 30 minutes begin transcription and translation of required proteins, including the toxins that both enable escape from the APC and cause anthrax pathologies [5]. If the APC is in transit to proximal lymph nodes when escape occurs, the bacilli are released directly into the blood or lymph to replicate, quickly reaching titers greater than 108 CFU/mL [6, 7]

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