Anthrax Lethal Toxin Disrupts Intestinal Barrier Function and Causes Systemic Infections with Enteric Bacteria

A variety of intestinal pathogens have virulence factors that target mitogen activated protein kinase (MAPK) signaling pathways, including Bacillus anthracis. Anthrax lethal toxin (LT) has specific proteolytic activity against the upstream regulators of MAPKs, the MAPK kinases (MKKs). Using a murine model of intoxication, we show that LT causes the dose-dependent disruption of intestinal epithelial integrity, characterized by mucosal erosion, ulceration, and bleeding. This pathology correlates with an LT-dependent blockade of intestinal crypt cell proliferation, accompanied by marked apoptosis in the villus tips. C57BL/6J mice treated with intravenous LT nearly uniformly develop systemic infections with commensal enteric organisms within 72 hours of administration. LT-dependent intestinal pathology depends upon its proteolytic activity and is partially attenuated by co-administration of broad spectrum antibiotics, indicating that it is both a cause and an effect of infection. These findings indicate that targeting of MAPK signaling pathways by anthrax LT compromises the structural integrity of the mucosal layer, serving to undermine the effectiveness of the intestinal barrier. Combined with the well-described immunosuppressive effects of LT, this disruption of the intestinal barrier provides a potential mechanism for host invasion via the enteric route, a common portal of entry during the natural infection cycle of Bacillus anthracis

The Effects of Anthrax Lethal Toxin on Host Barrier Function

The pathological actions of anthrax toxin require the activities of its edema factor (EF) and lethal factor (LF) enzyme components, which gain intracellular access via its receptor-binding component, protective antigen (PA). LF is a metalloproteinase with specificity for selected mitogen-activated protein kinase kinases (MKKs), but its activity is not directly lethal to many types of primary and transformed cells in vitro. Nevertheless, in vivo treatment of several animal species with the combination of LF and PA (termed lethal toxin or LT) leads to morbidity and mortality, suggesting that LT-dependent toxicity is mediated by cellular interactions between host cells. Decades of research have revealed that a central hallmark of this toxicity is the disruption of key cellular barriers required to maintain homeostasis. This review will focus on the current understanding of the effects of LT on barrier function, highlighting recent progress in establishing the molecular mechanisms underlying these effects

A New Murine Model for Gastrointestinal Anthrax Infection.

The scientific community has been restricted by the lack of a practical and informative animal model of gastrointestinal infection with vegetative Bacillus anthracis. We herein report the development of a murine model of gastrointestinal anthrax infection by gavage of vegetative Sterne strain of Bacillus anthracis into the complement-deficient A/J mouse strain. Mice infected in this manner developed lethal infections in a dose-dependent manner and died 30 h-5 d following gavage. Histological findings were consistent with penetration and growth of the bacilli within the intestinal villi, with subsequent dissemination into major organs including the spleen, liver, kidney and lung. Blood cultures confirmed anthrax bacteremia in all moribund animals, with approximately 1/3 showing co-infection with commensal enteric organisms. However, no evidence of immune activation was observed during infection. Time-course experiments revealed early compromise of the intestinal epithelium, characterized by villus blunting and ulceration in the ileum and jejunum. A decrease in body temperature was most predictive of near-term lethality. Antibiotic treatment of infected animals 24 h following high-dose bacterial gavage protected all animals, demonstrating the utility of this animal model in evaluating potential therapeutics

Delayed antibiotic treatment prevents lethality in murine gastrointestinal anthrax.

<p>A/J mice were infected via gavage with 1.2×10⁹ CFU of vegetative BaS. Twenty four hours later, the infected mice were treated with amoxicillin and gentamicin or PBS alone for 3 consecutive days. Mice were followed for 10 days post infection. **P<0.01. The <i>p</i> value represents the significance of the difference in survival between the antibiotic treatment and PBS control groups, calculated using the log-rank (Mantel-Cox) Test.</p

Gastrointestinal anthrax has time-dependent effects on various clinical parameters.

<p>Mice were fasted for 16 h and subsequently treated with ∼10⁹ intragastric BaS or PBS control. Body weight (BW), body temperature (BT), and appearance/activity were assessed prior to the fasting period and were subsequently monitored every 12 h until the animals became moribund, died, or reached the termination of the experiment (120 h). BW values for each animal were normalized to their baseline weights, which were assigned the value of 100% (<b>A</b>). BTs for each animal were assessed via a digital rectal thermometer (<b>B</b>). Appearance/activity for each animal was scored as follows: no change: (0 points); decreased locomotion, ruffled fur (2 points); and isolation, minimal spontaneous locomotion (4 points) (<b>C</b>). Combined clinical scores for each animal are shown in <b>D</b>. These scores were calculated from the 3 parameters described above. BW scores were assigned as follows: <5% change (0 points), ≥5%, but <10% (2 points), and ≥10% (4 points). BT scores were scored as follows: rectal temperature ≥35°C (0 points), body temperature <35°C, but ≥30°C (2 points), and body temperature <30°C (4 points). These scores were added to the appearance/activity score to provide the combined clinical score, with 12 representing the maximal possible clinical score.</p

Murine gastrointestinal anthrax infection causes intestinal villus blunting and ulceration.

<p>Intestinal sections were obtained from moribund mice that had been infected with BaS or the vehicle control by gavage. Representative images were obtained at 4×, 20× or 40× magnifications as indicated (<b>A–F</b>). Representative H&E-stained sections from a control animal (<b>A</b>) or moribund BaS-treated animals (<b>B–F</b>) are shown. Black arrows indicate regions of epithelial destruction and bleeding (<b>B</b>). The block arrow points to a region of ulceration (<b>C</b>). Triangles identify sloughed epithelial cells (<b>D</b>) and/or inflammatory cell infiltration (<b>E</b>) found in the intestinal lumen. Image <b>F</b> shows a region within image <b>E</b> that was acquired at a higher magnification, revealing the presence of polymorphonuclear leukocytes. (<b>G–I</b>) Brown and Brenn-stained sections were assessed for evidence of anthrax infection. The section shown in <b>G</b> was obtained from a control non-infected mouse, whereas sections shown in <b>H</b> and <b>I</b> were from moribund mice. Gram-positive bacteria stain blue. Arrowheads identify clusters of Gram⁺ rods within the intestinal villi.</p

Summary of pathological findings^*.

*<p>The pathological scores of the intestinal specimens were classified into four grades as follows: no lesions seen (−); mild villous blunting and/or isolated hemorrhages (+); multifocal areas of moderate mucosal ulceration, villus blunting, and/or hemorrhage (++); severe mucosal ulceration characterized by severe structural destruction and abundant bacterial colonies in contact or within submucosal tissues and/or severe hemorrhage (+++).</p

Intragastric challenge of <i>B anthracis</i> Sterne strain causes lethal anthrax infection.

<p>(<b>A</b>) Cohorts of ten week old female A/J mice were gavaged with varying doses of vegetative bacteria or with the PBS vehicle alone as indicated. The percentage of surviving/non-moribund mice was assessed at 6 h intervals for a total of 10 d as shown [p values were calculated using the log-rank (Mantel-Cox) test, * P<0.05, **P<0.01]. (<b>B</b>) A photographic image was taken of a BHI agar culture of blood from a representative animal that developed a co-infection following BaS gavage. The arrowhead indicates a bacterial colony of BaS, whereas the arrow points colonies of <i>Enterococcus faecalis</i>. (<b>C</b>) This table summarizes blood culture and bacterial identification results from 19 moribund animals.</p

Gastrointestinal anthrax infection in mice is characterized by gross hemorrhage.

<p>Mice that were treated with intragastric BaS were euthanized when they became moribund, simultaneously with control mice administered the buffer control. Shown are representative gross autopsy findings noted upon exposure of the abdominal cavity (<b>A</b>) or following dissection of the gastrointestinal tract (<b>B</b>). Control animals are shown in the left panels, and BaS infected animals are shown in the right panels. Arrows indicate regions with blood evident within the intestinal lumen.</p

Murine gastrointestinal anthrax progresses to multi-organ infection.

<p>Tissue sections from multiple organs were collected from control or moribund GI-infected mice, and stained with H&E (<b>A</b>). The left columns show representative sections from untreated control mice, whereas columns on the right show representative sections from moribund infected mice. The star highlights proteinaceous material in the alveolar spaces, accompanied by alveolar destruction (lung section); white arrows indicate areas of hemorrhage and/or destruction of the normal tissue architecture (liver, spleen and kidney sections). Shown in <b>B</b> are sections stained with Brown and Brenn. Arrowheads indicate Gram+ rods, which were present throughout the interstitial areas of the tissues (lung, liver, spleen, and kidney) of infected animals (right column), but not in uninfected control animals (left column).</p