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Anthrax lethal toxin disrupts intestinal barrier function and causes systemic infections with enteric bacteria.

Sun C, Fang H, Xie T, Auth RD, Patel N, Murray PR, Snoy PJ, Frucht DM - PLoS ONE (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, United States Food and Drug Administration, Bethesda, Maryland, United States of America.

ABSTRACT
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.

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Dose-dependent effects of anthrax LT on the intestinal barrier.(A) C57BL/6J mice were injected intravenously with PBS (control, n = 5), 100 µg PA/40 µg LF (n = 10), or 200 µg PA/80 µg LF (n = 10). LT-treated animals were euthanized when they became moribund, each with a simultaneously euthanized PBS-treated control. Samples from the small intestines of these animals were analyzed by H&E staining. Representative sections from a control animal and animals from each of the two LT-dose cohorts are shown; all animals in each cohort showed similar histological findings. Aperio ScanScope-acquired images are shown at 5×. Arrows indicate mucosal ulcerations and asterisks identify the area of villous blunting. (B) C57BL/6J mice were injected intravenously with 200 µg PA/80 µg LF (n = 8) or 100 µg PA/40 µg LF (n = 8) as shown. Cardiac blood samples were collected when the mice became moribund. Bacterial count data are shown (mean ± SD; * p<0.001, Student's t-test).
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pone-0033583-g003: Dose-dependent effects of anthrax LT on the intestinal barrier.(A) C57BL/6J mice were injected intravenously with PBS (control, n = 5), 100 µg PA/40 µg LF (n = 10), or 200 µg PA/80 µg LF (n = 10). LT-treated animals were euthanized when they became moribund, each with a simultaneously euthanized PBS-treated control. Samples from the small intestines of these animals were analyzed by H&E staining. Representative sections from a control animal and animals from each of the two LT-dose cohorts are shown; all animals in each cohort showed similar histological findings. Aperio ScanScope-acquired images are shown at 5×. Arrows indicate mucosal ulcerations and asterisks identify the area of villous blunting. (B) C57BL/6J mice were injected intravenously with 200 µg PA/80 µg LF (n = 8) or 100 µg PA/40 µg LF (n = 8) as shown. Cardiac blood samples were collected when the mice became moribund. Bacterial count data are shown (mean ± SD; * p<0.001, Student's t-test).

Mentions: To resolve potential inconsistencies in the literature, we explored the contribution of toxin dose to the development of intestinal pathology. We routinely used a dose of 200 µg PA and 80 µg LF for experiments with C57BL/6J mice, representing a 2.7 molar ratio of PA/LF that slightly exceeds the 7∶3 molar ratio of the fully occupied toxin complex. Our dosing regimen differed with that used by the other group that reported minimal LT-induced pathology in the intestines of wild-type or heterozygous MyD88 +/− mice (100 µg PA and 100 µg LF) [10], [11]. We hypothesized that our regimen could represent a higher effective dose than the 1∶1 weight-based ratio used by the other group. Therefore, we investigated a lower dose to determine whether we could explain the findings of these other reports. These experiments revealed that mice that received a reduced dose of LT (100 µg PA and 40 µg LF) still developed lethal intoxication, but deaths in these animals were delayed relative to mice receiving the higher dose (>1 wk vs. ≤4 d post-treatment, data not shown). In addition, there was little evidence of intestinal pathology in mice receiving the reduced LT dose (Figure 3A). Moreover, most mice in the lower dose group were blood culture negative, whereas all of the animals receiving the full dose developed systemic infections with relatively high levels of circulating bacteria (Figure 3B and Table S3). Thus, LT-induced pathology and the development of systemic bacterial infections is dose-dependent, an observation that could explain apparent discrepancies in the literature.


Anthrax lethal toxin disrupts intestinal barrier function and causes systemic infections with enteric bacteria.

Sun C, Fang H, Xie T, Auth RD, Patel N, Murray PR, Snoy PJ, Frucht DM - PLoS ONE (2012)

Dose-dependent effects of anthrax LT on the intestinal barrier.(A) C57BL/6J mice were injected intravenously with PBS (control, n = 5), 100 µg PA/40 µg LF (n = 10), or 200 µg PA/80 µg LF (n = 10). LT-treated animals were euthanized when they became moribund, each with a simultaneously euthanized PBS-treated control. Samples from the small intestines of these animals were analyzed by H&E staining. Representative sections from a control animal and animals from each of the two LT-dose cohorts are shown; all animals in each cohort showed similar histological findings. Aperio ScanScope-acquired images are shown at 5×. Arrows indicate mucosal ulcerations and asterisks identify the area of villous blunting. (B) C57BL/6J mice were injected intravenously with 200 µg PA/80 µg LF (n = 8) or 100 µg PA/40 µg LF (n = 8) as shown. Cardiac blood samples were collected when the mice became moribund. Bacterial count data are shown (mean ± SD; * p<0.001, Student's t-test).
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Related In: Results  -  Collection

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pone-0033583-g003: Dose-dependent effects of anthrax LT on the intestinal barrier.(A) C57BL/6J mice were injected intravenously with PBS (control, n = 5), 100 µg PA/40 µg LF (n = 10), or 200 µg PA/80 µg LF (n = 10). LT-treated animals were euthanized when they became moribund, each with a simultaneously euthanized PBS-treated control. Samples from the small intestines of these animals were analyzed by H&E staining. Representative sections from a control animal and animals from each of the two LT-dose cohorts are shown; all animals in each cohort showed similar histological findings. Aperio ScanScope-acquired images are shown at 5×. Arrows indicate mucosal ulcerations and asterisks identify the area of villous blunting. (B) C57BL/6J mice were injected intravenously with 200 µg PA/80 µg LF (n = 8) or 100 µg PA/40 µg LF (n = 8) as shown. Cardiac blood samples were collected when the mice became moribund. Bacterial count data are shown (mean ± SD; * p<0.001, Student's t-test).
Mentions: To resolve potential inconsistencies in the literature, we explored the contribution of toxin dose to the development of intestinal pathology. We routinely used a dose of 200 µg PA and 80 µg LF for experiments with C57BL/6J mice, representing a 2.7 molar ratio of PA/LF that slightly exceeds the 7∶3 molar ratio of the fully occupied toxin complex. Our dosing regimen differed with that used by the other group that reported minimal LT-induced pathology in the intestines of wild-type or heterozygous MyD88 +/− mice (100 µg PA and 100 µg LF) [10], [11]. We hypothesized that our regimen could represent a higher effective dose than the 1∶1 weight-based ratio used by the other group. Therefore, we investigated a lower dose to determine whether we could explain the findings of these other reports. These experiments revealed that mice that received a reduced dose of LT (100 µg PA and 40 µg LF) still developed lethal intoxication, but deaths in these animals were delayed relative to mice receiving the higher dose (>1 wk vs. ≤4 d post-treatment, data not shown). In addition, there was little evidence of intestinal pathology in mice receiving the reduced LT dose (Figure 3A). Moreover, most mice in the lower dose group were blood culture negative, whereas all of the animals receiving the full dose developed systemic infections with relatively high levels of circulating bacteria (Figure 3B and Table S3). Thus, LT-induced pathology and the development of systemic bacterial infections is dose-dependent, an observation that could explain apparent discrepancies in the literature.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, United States Food and Drug Administration, Bethesda, Maryland, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus