The structure and biofilm-stabilizing activity of Enterococcal surface protein’s N-terminal region

Enterococcal surface protein (Esp) is a cell wall-attached virulence factor of the leading nosocomial pathogens Enterococcus faecalis and E. faecium and is found predominantly in disease-causing strains. The presence of esp is associated with increased bacterial burden in rodent models of enterococcal endocarditis and urinary tract infection. The mechanism by which Esp enhances colonization of tissues is unknown. The presence of Esp has also been found to enhance biofilms, as assayed by crystal violet staining, in some but not all strains of Enterococcus. The circumstances under and extent to which Esp enhances biofilms are not well understood, and conflicting data exist, leaving its role in biofilms undefined. To address the functional role of Esp, we sought to use X-ray crystallography to determine the structure of the unique N-terminal region of Esp, as structure is often indicative of function. We successfully determined the structure of the first 405 amino acids of Esp (Esp452), which revealed that this region is composed of two Ig-type domains typical of bacterial adhesins. While we did not detect binding to host factors, we did find that Esp452 strengthens enterococcal biofilms against mechanical and enzymatic challenge. Further examination revealed that this activity is correlated with the formation of amyloid-like fibrils in a pH-dependent manner. Interestingly, a larger fragment consisting of the entire N-terminal region of Esp, Esp743, did not demonstrate this activity, despite containing Esp452. We used a structural approach to explore the possibility of auto-inhibition, and found by small-angle X-ray scattering that the additional 291 amino acids contained in Esp743 appear to have close contacts with Esp452, supporting a role in structural stabilization of the protein and prevention of the formation of amyloid fibrils. Finally, we explored mechanisms of activation of the N-terminal region. We found that at pH 4.0, Esp743 was capable of strengthening biofilms, suggesting that extreme pH may be sufficient to generate activity of cell wall-attached protein. Alternatively, we found that a shorter truncation fragment of the N-terminal region, consistent with cleavage by a human protease, was also capable of strengthening enterococcal biofilms

Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins.

Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways

CdtC Mediates Cholesterol Dependency of Ec-CDT.

<p>CHO-A745 cells were seeded at 8 x 10³ cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with or without 5 mM MβCD and/or 12.5 μM EGA for 1 h then challenged with 1 μM Ec-CDT or Ec-CdtAB for 16 h. Intoxication was assessed by measuring pH₂AX by laser scanning cytometry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143977#pone.0143977.g002" target="_blank">Fig 2B</a>. Data were normalized against pH₂AX signal induced by Ec-CDT holotoxin (maximum signal) in each experiment. Graphs represent average values and SEM from three independent experiments, each performed in triplicate. All statistical analyses are from the pairwise post-test (Tukey’s) derived from one-way ANOVA. (Prism 5, GraphPad). Symbols above each column reflect comparison to Ec-CDT holotoxin (ns = not significant; * p < 0.001). Additional pairwise comparisons are indicated by brackets.</p

Ec-CdtC Dictates Resistance to EGA and Alters Intracellular Trafficking of Ec-CdtB.

<p>(A) CHO-A745 cells were intoxicated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143977#pone.0143977.g001" target="_blank">Fig 1</a> except that all wells were additionally treated with 12.5 μM EGA. (B) CHO-A745 cells were seeded at 8 x 10³ cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with 1μM Ec-CDT holotoxin or 1 μM Ec-CdtAB for 4 or 16 h. Phosphorylated H₂AX (anti-pH₂AX) was measured by laser scanning cytometry as described in Methods. Signal intensity for pH₂AX induced by Ec-CDT holotoxin was set at 100% and used to normalize signal from CdtAB for each time point. Graphs represent average values from three independent experiments, each performed at least 3 times. *p value = 0.0121 calculated by unpaired two-tailed t test (Prism 5, GraphPad). (C, D) CHO-A745 cells were seeded at 2 x 10⁴ cells/well on 8-well chambered slides and allowed to adhere overnight. The next day, cells were incubated on ice with 100 μM Ec-CDT holotoxin, Ec-CdtAB or Ec-CdtBC for 30 min, washed and incubated at 37°C for 60 minutes. Cells were then fixed, stained, and imaged as described in Methods [anti-Ec-CdtB (green) and EEA1 or Rab9 antibody (red)]. White scale bars at the left panel of each treatment indicate 10 μm and the right insert panel indicate 2 μm. Quantification of microscopy results was performed using Pearson's coefficient values indicating colocalization of the Ec-CdtB signal with the EEA1 or Rab9 enriched vesicles. Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during three independent experiments and error bars represent standard deviations.</p

Intoxication Mediated by CdtA and CdtC Subunits.

<p>Jurkat, HeLa, or CHO-A745 cells were seeded in clear-bottom 384-well plates, incubated overnight, then challenged with the indicated toxin concentrations. Holotoxin, black circles; CdtAB, red squares; CdtBC, blue triangles. Intoxication was allowed to proceed for 48 h (Jurkat) or 72 h (HeLa and CHO-A745). Cell viability was measured by ATPlite reagent (Perkin Elmer), and normalized to ATPlite signal from unintoxicated controls. Data represent average values from three independent experiments, each performed in triplicate, +/- standard deviation. Lines represent nonlinear curve fit calculated using Prism 5 (GraphPad).</p

Holotoxin Assembly Method Affects Sensitivity to EGA.

<p>CHO-A745 cells were intoxicated as above in the presence or absence of 12.5 μM EGA. Additionally, cells were challenged with a combination of purified CdtA, CdtB, and CdtC subunits that were combined at the time of intoxication without further purification of assembled holotoxin (Ec-ABC). Cell viability was measured by ATPlite and normalized as above. Data represent average values from three independent experiments, each performed in triplicate.</p

Tissue Culture LD₅₀ Values for Ec-Cdt Dimers and Trimers.

<p>Average values and standard deviation (+/-) were determined from at least three biological replicates, each performed in triplicate. NT, not tested; ND, value not determined due to lack of cytotoxicity.</p><p>Tissue Culture LD₅₀ Values for Ec-Cdt Dimers and Trimers.</p

Tissue Culture LD₅₀ Values for Hd-Cdt Dimers and Trimers.

<p>Average values and standard deviation (+/-) were determined from at least three biological replicates, each performed in triplicate. NT, not tested; ND, value not determined due to lack of cytotoxicity.</p><p>Tissue Culture LD₅₀ Values for Hd-Cdt Dimers and Trimers.</p