The <i>Vibrio cholerae</i> Extracellular Chitinase ChiA2 Is Important for Survival and Pathogenesis in the Host Intestine

<div><p>In aquatic environments, <i>Vibrio cholerae</i> colonizes mainly on the chitinous surface of copepods and utilizes chitin as the sole carbon and nitrogen source. Of the two extracellular chitinases essential for chitin utilization, the expression of <i>chiA2</i> is maximally up-regulated in host intestine. Recent studies indicate that several bacterial chitinases may be involved in host pathogenesis. However, the role of <i>V. cholerae</i> chitinases in host infection is not yet known. In this study, we provide evidence to show that ChiA2 is important for <i>V. cholerae</i> survival in intestine as well as in pathogenesis. We demonstrate that ChiA2 de-glycosylates mucin and releases reducing sugars like GlcNAc and its oligomers. Deglycosylation of mucin corroborated with reduced uptake of alcian blue stain by ChiA2 treated mucin. Next, we show that <i>V. cholerae</i> could utilize mucin as a nutrient source. In comparison to the wild type strain, Δ<i>chiA2</i> mutant was 60-fold less efficient in growth in mucin supplemented minimal media and was also ∼6-fold less competent to survive when grown in the presence of mucin-secreting human intestinal HT29 epithelial cells. Similar results were also obtained when the strains were infected in mice intestine. Infection with the Δ<i>chiA2</i> mutant caused ∼50-fold less fluid accumulation in infant mice as well as in rabbit ileal loop compared to the wild type strain. To see if the difference in survival of the Δ<i>chiA2</i> mutant and wild type <i>V. cholerae</i> was due to reduced adhesion of the mutant, we monitored binding of the strains on HT29 cells. The initial binding of the wild type and mutant strain was similar. Collectively these data suggest that ChiA2 secreted by <i>V. cholerae</i> in the intestine hydrolyzed intestinal mucin to release GlcNAc, and the released sugar is successfully utilized by <i>V. cholerae</i> for growth and survival in the host intestine.</p></div

List of Primers used in construction of <i>chiA2</i> deleted <i>V. cholerae</i> strain and cloning <i>chiA2</i> in pBAD-TOPO TA expression vector.

<p>List of Primers used in construction of <i>chiA2</i> deleted <i>V. cholerae</i> strain and cloning <i>chiA2</i> in pBAD-TOPO TA expression vector.</p

Histological study of rabbit ileal tissues.

<p>Panels show photomicrographs of histology of rabbit ileal loop tissue after infection with <b>A</b>. wild type <i>V. cholerae</i> showing hemorrhagic fluid accumulation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103119#pone-0103119-g005" target="_blank">Fig. 5B</a>). Extensive damage of mucosa with damage to the villus structure and gross dilatation of villi with hemorrhage and necrotic changes in villus, lamina, propria, mucosa and sub-mucosal area was observed. <b>B</b>. Almost normal villous and mucosal structure was observed in ileal tissues infected with Δ<i>chiA2</i> mutant though some congestion and scattered hemorrhagic change was noticed in villus, lamina, propria and rest of the mucosa. <b>C</b>. complemented strain showing hemorrhagic fluid accumulation. Extensive damage of mucosa with damage to the villus structure and gross dilatation of villi with hemorrhage and necrotic changes in villus, lamina, propria, mucosa and sub-mucosal area was observed. <b>D</b>. PBS treated ileal tissue showed normal gut mucosa.</p

Role of ChiA2 in <i>V. cholerae in vitro</i> and <i>in vivo</i> proliferation.

<p><b>A</b>. Comparative study of the survival of wild type and Δ<i>chiA2</i> mutant in human intestinal HT29 cell line. The graph represents a dose dependent survival of wild type <i>V. cholerae</i> and Δ<i>chiA2</i> mutant and also a complemented strain. <b>B</b>. Comparative study of dose dependent mice intestinal survival of wild type <i>V. cholerae</i>, Δ<i>chiA2</i> mutantand the complemented strain. For the experiment, 5 days old suckling mice were fed with different dilutions of the mentioned strains and sacrificed after 16 hours. The viability of the strains was measured by cell count method. Bars: (□) PBS; (▪) wild type <i>V. cholerae</i>; ( ) Δ<i>chiA2</i> mutant () complemented strain. Each of the experiment was repeated thrice (n = 3) and the data expressed as means ± SEM.</p

Fluid accumulation studies.

<p><b>A</b>. This is the graphical representation of fluid accumulation ratio in infant mice model. For fluid accumulation studies 5 days old infant mice were infected with 100 µl of 1×10⁶ CFU/ml of wild type <i>V. cholerae</i>, Δ<i>chiA2</i> mutant, and the complemented strain. Mice were sacrificed after 3, 6, 12 and 18 hours. The fluid accumulation calculated and presented graphically. In all the above three experiments PBS was used as negative control and the data expressed as means ± SEM of three independent experiments (P<0.01). <b>B</b>. Rabbit ileal loop response of wild type <i>V. cholerae</i> cells (1×10⁹ CFU/ml) showing hemorrhagic fluid accumulation whereas, infection with Δ<i>chiA2</i> mutant (1×10⁹ CFU/ml) shows significant decrease in fluid accumulation. Infection with the complemented strain showed similar hemorrhagic fluid accumulation like the wild type strain. PBS was used as negative control. The experiment was repeated thrice (n = 3). <b>C</b>. This is the graphical representation of fluid accumulation ratio in rabbit ileal loop. The fluid accumulation ratio for the Δ<i>chiA2</i> mutant (FA/cm ratio: 0.04±0.01) was 40-fold less compared to the wild type <i>V. cholerae</i> (FA/cm ratio: 1.6±0.2). The fluid accumulation ratio for the complemented strain was similar with the wild type. PBS was used as negative control (FA/cm ratio: 0.02±0.008). The experiment was repeated thrice (n = 3) and the data expressed as means ± SEM.</p

Mucin deglycosylase activity of ChiA2.

<p><b>A</b>. This shows the graphical representation of % area density of the alcian blue stained spots (the above picture) of control, ChiA2-untreated and treated rabbit mucin respectively (from left). A 50-fold decrease in the % area density of the spot was observed for ChiA2-treated rabbit mucin compared to the untreated one. The mucin treatment was done as mentioned in materials and methods section. The % area density for each spot was calculated using Lab works software after alcian blue staining. <b>B</b>. This is the graphical representation of the amount of GlcNAc released by ChiA2 from in-vivo rabbit intestinal mucin (•) with respect to the control (Δ) with increasing incubation time. The graph shows a significant increase in reducing sugar concentration in the reaction loop containing active ChiA2 compared to the control loop containing the heat deactivated enzyme in phosphate buffer. The assay was done as mentioned in materials and methods. Each of the experiment was repeated thrice (n = 3) and the data expressed as means ± SEM.</p

HPLC analysis of end products of ChiA2-treated mucin.

<p>The mucin-ChiA2 Reactions were carried as mentioned in the materials and methods section. The end products were analyzed using a 4.6 nm×250, 5 µm Zorbax carbohydrate analysis column (Agilent Technologies, Waldbrown, Germany) connected to a Shimadzu Prominence 20A; HPLC system. <b>A</b>. This figure shows the HPLC chromatogram of the end products of ChiA2 treated mucin. The chromatogram shows four distinct peaks and an unresolved region. Peak 1 with retention time 5.4 minutes indicates presence of GlcNAc. Peak 2 indicates presence of (GlcNAc)2 which is divided into two separate peaks: peak 2A and peak 2B. Peak 2A with retention time 6.0 minutes probably is for the β enantiomer and peak 2B with retention time 6.57 minutes is for α enantiomer of (GlcNAc)₂. Peak 3 with retention time 7.6 minutes indicates the presence of (GlcNAc)₃. <b>B</b>. This is the HPLC chromatogram of standards of GlcNAc, (GlcNAc)₂, (GlcNAc)₃ and (GlcNAc)₆. The chromatogram shows 4 distinct peaks. Peak 1 with retention time 5.5 minutes is for GlcNAc. Peak 2 with retention time 6.6 minutes is for α-(GlcNAc)₂. Peak 3 with retention time 7.5 minutes is for (GlcNAc)₃ and peak 4 with retention time 11.1 minutes is for (GlcNAc)₆.</p

Utilization of mucin as nutrient for growth by <i>V. cholerae</i>.

<p><i>V. cholerae</i> wild type, Δ<i>chiA2</i> mutant and the complemented strain were separately grown in LB upto log phase. The log phase cultures of the wild type <i>V. cholerae</i>, Δ<i>chiA2</i> mutant and the complemented strain were centrifuged and washed in PBS. The minimal media supplemented with 2% (w/v) porcine mucin and fresh LB broth were inoculated separately with equal number of washed <i>V. cholerae</i> wild type and Δ<i>chiA2</i> mutant and complemented strain. The number of bacteria survived with increasing incubation time was detected by plate count method for all of the strains. The viable counts of bacteria were graphically represented. <b>A</b>. Viable count when mucin used as nutrient. <b>B</b>. Viable count when grown in LB broth. Each of the experiment was repeated thrice (n = 3) and the data expressed as means ± SEM.</p

HPLC analysis of the end products of ChiA2 treated mucin.

<p>Reactions were carried out in 20 mM phosphate buffer pH 7.4 with 1 mg/ml rabbit mucin as substrate and incubated at 37°C with 50 µg/ml of pure ChiA2 for 3 hours. The end products were analyzed using a 4.6 nm×250, 5 µm Zorbax carbohydrate analysis column (Agilent Technologies, Waldbrown, Germany) connected to a Shimadzu Prominence 20A; HPLC system. The reaction was stopped by 10% TCA loaded into the HPLC column and eluted using a gradient of 60–80% acetonitrile in water as mobile phase. Each experiment was conducted maintaining same conditions and repeated 3 times.</p><p>*Not Observed.</p><p>HPLC analysis of the end products of ChiA2 treated mucin.</p

ChiA2 can use mucin as a substrate.

<p><b>A</b>. This diagram shows the specific activity of ChiA2 when mucin was used as substrate. In the control experiment heat inactivated ChiA2 was used instead of active ChiA2. The assay was done as described in materials and methods. The experiment was repeated thrice (n = 3) and the data expressed as means ± SEM. <b>B</b>. This figure shows the Lineweaver Burk plot of ChiA2-mucin reaction. The plot was generated by plotting inverse of different substrate (mucin) concentrations (0.5 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml and 3 mg/ml) in the X-axis and inverse of each reaction velocity in Y axis.</p