Colonic Immune Suppression, Barrier Dysfunction, and Dysbiosis by Gastrointestinal Bacillus anthracis Infection

and Dysbiosis by Gastrointestinal Bacillus anthracis Infection. PLoS ONE 9(6): e100532. doi:10.1371/journal.pone.0100532 Colonic Immune Suppression, Barrier Dysfunction, and Dysbiosis by Gastrointestinal Bacillus anthracis Infection Yama L. Lightfoot 0 Tao Yang 0 Bikash Sahay 0 Mojgan Zadeh 0 Sam X. Cheng 0 Gary P. Wang 0 Jennifer L. Owen 0 Mansour Mohamadzadeh 0 Nupur Gangopadhyay, University of Pittsburgh, United States of America 0 1 Department of Infectious Diseases and Pathology, University of Florida, Gainesville, Florida, United States of America, 2 Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Florida, Gainesville, Florida, United States of America, 3 Division of Gastroenterology, Department of Pediatrics, University of Florida, Gainesville, Florida, United States of America, 4 Division of Infectious Diseases and Global Medicine, Department of Medicine, University of Florida, Gainesville, Florida, United States of America, 5 Department of Physiological Sciences, College of Veterinary Medicine, University of Florida , Gainesville, Florida , United States of America Gastrointestinal (GI) anthrax results from the ingestion of Bacillus anthracis. Herein, we investigated the pathogenesis of GI anthrax in animals orally infected with toxigenic non-encapsulated B. anthracis Sterne strain (pXO1+ pXO22) spores that resulted in rapid animal death. B. anthracis Sterne induced significant breakdown of intestinal barrier function and led to gut dysbiosis, resulting in systemic dissemination of not only B. anthracis, but also of commensals. Disease progression significantly correlated with the deterioration of innate and T cell functions. Our studies provide critical immunologic and physiologic insights into the pathogenesis of GI anthrax infection, whereupon cleavage of mitogen-activated protein kinases (MAPKs) in immune cells may play a central role in promoting dysfunctional immune responses against this deadly pathogen. - Funding: This work was supported by the National Institute of Allergy and Infectious Diseases RO1 AI093370 to MM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Gastrointestinal (GI) anthrax, named for its primary route of infection, is an acute infectious disease resulting from the ingestion of the spore-forming, Gram-positive bacterium, Bacillus anthracis [1]. Anthrax can also be contracted via inhalation or cutaneous exposure, with inhalation anthrax having the highest mortality rate of the three clinical subtypes [2]. Disease-causing B. anthracis spores primarily infect grazing animals, but humans may be exposed to anthrax through the handling of infected animals and animal products, the consumption of tainted meat, or through intentional exposure [1]. Independent of the route of entry, unchecked infection rapidly becomes systemic and death occurs due to septicemia and/or toxemia [3]. Within fully virulent B. anthracis strains, two large plasmids, pXO1 and pXO2, are composed of the genes needed for toxin production and capsule formation, respectively, and both plasmids are necessary for complete virulence [4,5]. The pXO1 encodes protective antigen (PA), lethal factor (LF), and edema factor (EF); lethal toxin (LT) comprises PA+LF, while edema toxin (ET) comprises PA+EF. Via these two toxins, B. anthracis evades and inhibits critical signals of the innate and adaptive immune systems [6]. The poorly immunogenic anthrax capsule is encoded on pXO2 and consists of poly-c-D-glutamic acid, which protects B. anthracis from phagocytosis and complement binding [7,8]. Several therapeutic strategies have targeted specific B. anthracis virulence factors [9,10]; however, development of next generation vaccines and therapeutics against B. anthracis requires a better understanding of disease pathogenesis in humans. In particular, insufficient data exist regarding the pathogenesis of GI anthrax [1113]. GI B. anthracis infection is not only a persistent and major problem in developing countries, but also poses a threat in biological warfare, whereby intentional contamination of food sources may occur [1]. Here, we report that GI B. anthracis spore infection results in swift morbidity and mortality and is associated with pathogen dissemination throughout visceral organs by induction of leakage in the intestinal barrier and significant changes in the guts microbial composition, all of which may orchestrate dysfunctional immune responses. A greater understanding of the pathogenesis of GI anthrax and molecular studies of the microorganismmammalian immune defense interface [14] is imperative and may result in improvement of a protective vaccine in man. Materials and Methods Mice and Ethics Statement A/J mice were purchased from the Jackson Laboratory and bred in-house in the animal facility at the College of Veterinary Medicine, University of Florida. For microbiota composition experiments, mice were tested after a minimum of two generations of in-house breeding. Mice were used at 68 weeks of age in accordance with the Animal Welfare Act and the Public Health Policy on Humane Care. All procedures were approved by the Institutional Animal Case and Use Committee (IACUC) at the University of Florida under protocol number 201107129, and all efforts were made to minimize animal suffering. Infected mice were monitored every 24 hours and were humanely euthanized when signs of advanced infection (e.g., difficulty breathing) were noted; in some cases, mice died as a direct result of the infection before euthanasia could take place. Euthanasia was performed by prolonged inhalation of isoflurane and confirmed by cervical dislocation. B. anthracis Spore Preparation and Mouse Infections Spores were prepared with a toxigenic non-encapsulated strain of B. anthracis (Sterne), as described previously [15] with the approval of the Institutional Biosafety Committee (IBC) at the University of Florida. To calculate final concentrations, serial dilutions were grown in triplicate on lysogeny broth agar plates and colonies counted. For survival studies, mice were orally infected with 105 spores (n = 10), 107 spores (n = 10), or 109 spores (n = 20) in a final volume of 100 mL with a reusable, 30 mm, 20 gauge, barrel-tipped feeding needle after fasting for 4 hours; infected mice were monitored, and deaths recorded. For immunologic and microbiota composition studies, A/J mice (n = 10/group) were orally infected with Sterne spores (109 spores/100 mL PBS/mouse) for the specified time points. Groups of A/J mice (n = 10/group) were also either orally gavaged or injected intraperitoneally (i.p.) with 125 mg LT (PA+LF) and monitored for morbidity and death. Histopathology Sterne-infected A/J mice were sacrificed at various days postinf (...truncated)