Colonic Immune Suppression, Barrier Dysfunction, and Dysbiosis by Gastrointestinal <i>Bacillus anthracis</i> Infection

<div><p>Gastrointestinal (GI) anthrax results from the ingestion of <i>Bacillus anthracis</i>. Herein, we investigated the pathogenesis of GI anthrax in animals orally infected with toxigenic non-encapsulated <i>B. anthracis</i> Sterne strain (pXO1⁺ pXO2⁻) spores that resulted in rapid animal death. <i>B. anthracis</i> Sterne induced significant breakdown of intestinal barrier function and led to gut dysbiosis, resulting in systemic dissemination of not only <i>B. anthracis</i>, 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.</p></div

T Cell Responses in Sterne-Infected A/J Mice.

<p>A/J mice were orally gavaged with 10⁹ spores of the Sterne strain of <i>B. anthracis</i> and adaptive immune responses in the colon analyzed at various time points by flow cytometry. <b>A</b>. Gating strategy for the analysis of colonic T cells. <b>B</b>. Th1, Th17, and regulatory T cells responses were tested by flow cytometry. Representative plots indicate cytokine production of uninfected and day 5-infected mice. <b>C</b>. Surface expression of PD1 in colonic T cells. <b>D</b>. Gene expression profile of the distal colon of Sterne-infected A/J mice. Data represent observations from four independent experiments and are shown as mean +/− SEM. *P<0.05, **P<0.01, ***P<0.001 compared with PBS.</p

Dynamics of major NS3 variants in liver transplant recipients.

<p>Forward pyrosequence reads (∼337 nt, corresponding to coordinates 3342 to 3674 on the H77 genome, accession NC_004102) were clustered into operational taxonomic units (OTUs) at 97% sequence identity. The relative abundance of each major variant is shown by the black shading, where the extent of the black region from left to right within the gray bar indicates the proportion in the total viral population. The black double-headed arrow denotes the time of liver transplantation, and the number of days before and after liver transplantation are indicated at the bottom. Trees were generated using UPGMA. Shannon diversity values based on NS3 pyrosequence reads represent changes in NS3 diversity over time (Top panels). ALT: alanine aminotransferase. (A) The major variant that established re-infection post-LT was identical or closely related to the dominant variant pre-LT (B) Two minor variants pre-LT became dominant quasispecies post-LT.</p

Illumina paired-end sequencing identifies low frequency T54A+R155K double mutant in mock RNA populations.

<p>RNA transcripts were synthesized from WT, T54A, or T54A+R155K plasmid <i>in vitro</i>. Each mock population of RNA transcripts was constructed according to the proportions indicated in the “Expected” column. The proportion of paired-end reads that harbored T54A single mutant, T54A/R155K double mutant, or WT are shown in the “observed” column (%). While the double mutant at a level below 0.1% was detected (Population 3), it was below our experimentally determined threshold for background error rate of ∼0.2% (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069698#pone-0069698-t002" target="_blank">Table 2</a>).</p

Cleavage of MAPKs in DCs of Sterne-Infected A/J Mice.

<p>A/J mice were orally gavaged with 10⁹ spores of the Sterne strain of <i>B. anthracis</i> and DCs analyzed at various time points. <b>A</b>. Gating strategy for the analysis of CD45⁺MHCII^hiCD11c⁺F4/80⁻CD11b⁺ colonic DCs. <b>B</b>. Cell surface expression of CD86, CD80, and B7-H1 by colonic DCs was analyzed by flow cytometry. Gray tinted line = isotype control; black line = PBS group; green line = day 1 Sterne-infected A/J mice; magenta line = day 3 Sterne-infected A/J mice; blue line = day 5 Sterne-infected mice. <b>C</b>. Activity of MAPKs in colonic DCs of infected versus uninfected mice was analyzed by flow cytometry. Gray tinted line = isotype control; black line = PBS group; blue line = day 5 Sterne-infected mice. <b>D</b>. Colonic LP cells were isolated from uninfected A/J mice and incubated with 1 MOI of <i>B. anthracis</i> spores for 1, 3, or 6 hours. Activity of p38 and Erk1/2 was subsequently analyzed in colonic DCs. Gray tinted line = isotype control; black line = PBS group; green line = 1 hour treatment; magenta line = 3 hour treatment; blue line = 6 hour treatment. Data represent observations from three independent experiments and are shown as mean +/− SEM. *P<0.05, **P<0.01, ***P<0.001 compared with PBS.</p

Mutation linkage analyzed by Illumina paired-end sequencing.

<p>Each row corresponds to an individual sample (control transcripts or clinical samples).</p><p>Each column corresponds to single or double mutant variants associated with PI resistance (WT codons are listed), and their mutation frequency (defined as non-WT) is shown (%). Technical error rate was determined as 0.2% based on the WT control data.</p

Frequency of NS3 resistance-associated variants (RAVs) in (A) chronic HCV and (B) longitudinal liver transplant recipients as determined by Roche/454 pyrosequencing.

<p>The proportion of pyrosequencing reads that harbored authentic RAVs is indicated by the intensity of magenta shown in the heatmap (p≤0.05, chi-square test). RAVs detected in pyrosequence reads that were not significantly enriched compared to background technical error rates are shown in grey (p>0.05, chi-square test). The background error rate was determined using <i>in vitro</i> control transcripts of known sequence that were amplified and sequenced in parallel with the RNA extracted from HCV subjects. Each column is a different sample, and each row represents a different RAV detected by pyrosequencing. Asterisks denote RAVs detected by direct Sanger sequencing. In (A), each column is a different subject (14 genotype 1a and 6 genotype 1b), and in (B), longitudinal samples within each subject (as indicated by the subject code at the bottom) are ordered from left to right, and phylogenetic analysis demonstrates that temporally associated samples are more closely related within subjects than samples between subjects.</p

Deep sequencing strategy to determine abundance and mutation linkage of NS3 RAVs.

<p>(A) Roche/454 pyrosequencing. Primer binding sites for 454/Roche pyrosequencing primers are shown in yellow. The pyrosequencing primers are composites containing the required sequences for the Roche/454 titanium chemistry procedure at 5′ end (blue and green), a unique 8-base DNA barcode that indexes each sample (red), and HCV-specific primer sequences at the 3′ end (yellow). (B) Illumina paired-end sequencing. Partial NS3 gene segments were first amplified using gene-specific primers that contain HCV-specific sequences (yellow), a barcode sequence unique to each sample (red) and partial adapter sequences (green and blue). Amplified fragments were tailed with flow cell adaptors (green and blue arrows), then subjected to paired-end sequencing using the standard Illumina paired-end protocol. This protocol will generate non-overlapping forward (100 nt) and the reverse (100 nt) reads from each cluster, providing precise long-range positional and sequence information. After trimming and quality control, the paired-end reads allowed linkage analysis between V36-V55 and R155-I170. Sequences of all primers used in (A) and (B) are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069698#pone.0069698.s001" target="_blank">Supporting Information S1</a>.</p

Lethality and Systemic <i>B. anthracis</i> Sterne Dissemination in A/J Mice.

<p>A. A/J mice were orally gavaged with 10⁵, 10⁷, or 10⁹ spores of the Sterne strain of <i>B. anthracis</i>. Lethal infection was established within 3 days in A/J mice receiving 10⁹ spores; n = 10 mice/group (10⁵ and 10⁷), n = 20 mice/group (10⁹). Experiments were performed a minimum of three times. Statistical significance was calculated using the log-rank test. After 3 days of infection, A/J mice were sacrificed; both spores and vegetative bacilli (marked with *) were observed in the colon (<b>B</b>), MLNs (<b>C</b>), spleen (<b>D</b>, <b>E</b>), liver (<b>F</b>), kidneys (<b>G</b>), lungs (<b>H</b>), and in bronchoalveolar lavage (BAL) fluid (<b>I</b>). Bar = .</p

GI Epithelial Barrier Dysfunction Induced by Sterne Infection.

<p>A/J mice were orally gavaged with 10⁹ spores of the Sterne strain of <i>B. anthracis</i> and intestinal barrier integrity was analyzed <i>ex vivo</i> three days post-infection; n = 5 mice/group. TEER (<b>A</b>), trans-epithelial conductance (<b>B</b>), and short-circuit current (<b>C</b>) of Sterne-infected versus uninfected A/J mice. <b>D and E</b>. Delta current (Δ<i>I_SC</i>) before and after cholinergic (<b>D</b>) and histamine (<b>E</b>) challenges. <b>F</b>. Post-challenge secretory current of Sterne-infected versus uninfected A/J mice. Data are shown as mean +/− SEM. *P<0.05, **P<0.01, ***P<0.001 compared with PBS. <b>G</b>. Colonoscopies were performed in groups of uninfected and 10⁹ Sterne spores-infected A/J mice three days post infection with a Multi-Purpose Rigid Telescope attached to a TELE PACK X. <b>H</b>. Gross hemorrhage in the small intestines<b>. </b><b>I and J.</b> Hemorrhagic lesions in the colon (<b>I</b>) and small intestine (<b>J</b>) of Sterne-infected A/J mice. Bar = 200 µm.</p