Structural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae

EndA is a sequence non-specific endonuclease that serves as a virulence factor during Streptococcus pneumoniae infection. Expression of EndA provides a strategy for evasion of the host's neutrophil extracellular traps, digesting the DNA scaffold structure and allowing further invasion by S. pneumoniae. To define mechanisms of catalysis and substrate binding, we solved the structure of EndA at 1.75 Å resolution. The EndA structure reveals a DRGH (Asp-Arg-Gly-His) motif-containing ββα-metal finger catalytic core augmented by an interesting ‘finger-loop’ interruption of the active site α-helix. Subsequently, we delineated DNA binding versus catalytic functionality using structure-based alanine substitution mutagenesis. Three mutants, H154A, Q186A and Q192A, exhibited decreased nuclease activity that appears to be independent of substrate binding. Glu205 was found to be crucial for catalysis, while residues Arg127/Lys128 and Arg209/Lys210 contribute to substrate binding. The results presented here provide the molecular foundation for development of specific antibiotic inhibitors for EndA

Keratoconus: A One-Sided Debate

Background. A 30-year old man diagnosed with refractive amblyopia OD, was referred for a contact lens fitting. After additional testing was completed the diagnosis was changed to unilateral keratoconus and the patient was successfully fit to a specialty gas permeable lens design OD. Case Report. Presenting symptoms included blurry and double vision with haloes around lights and intermittent tearing, OD. Unaided distance visual acuity (DVA) was 20/100 OD and 20/15 OS. Manifest refraction included a significant degree of myopic astigmatism OD and only a minimal amount OS. While performing biomicroscopy keratoconic signs of prominent corneal nerves and Vogt’s Striae were observed OD only. Keratometric mires were distorted OD and clear OS. Corneal mapping with the Humphrey Atlas Topographer revealed a moderately steep apex OD only with abnormal corneal irregularity (CIM) and shape factor (SF) measurements elicited.OD. On pachymetry the central corneal thickness was measured as 466μm OD and 523μm OS. A light feather touch fluorescein pattern with average edge clearance was seen with the prescribed contact lens OD. After adaptation, the DVA was 20/20- OD with the contact lens compared to 20/15 OS without correction, and a resolution of the patient’s presenting symptoms was reported. Conclusion. Keratoconus is sited as a clinically non-inflammatory corneal ectasia that is typically bilateral and asymmetric. The question of whether monocular keratoconus truly exists is controversial. This case reflects a rare circumstance where only the monitoring of progression will determine whether the initial diagnosis of unilateral keratoconus stands the test of time

Adhesion of Transferrin and Albumin to FDA Group II Omafilcon Contact Lenses

Objective:Tear protein deposits on contact lenses can cause irritation of the conjunctiva and are associated with bacterial infection. The adhesion of transferrin and albumin to Omafilcon contact lenses was investigated. The charges on protein molecules interact with the contact lens hydrogel polymer causing adhesion of the proteins to the lenses. Methods: Lenses were incubated for 5 days in a solution (2 mg/mL) of each protein. Bicinchoninic acid colorimetry was performed to determine protein concentration in the vials and protein adhesion to lenses. Results: The albumin concentration in the incubation vials remained fairly constant while the concentration of transferrin in the vials fluctuated. Albumin initially adhered to the lenses, declined slightly on subsequent days, and increased again on day five. Transferrin adhered to the lenses consistently, reaching peak deposition after four days of incubation, before declining on day five. The adhesion of transferrin to the Omafilcon lenses was similar to the adhesion of transferrin to Hilafilcon lenses (both FDA Group II: nonionic high water). Albumin adhesion to Omafilcon contact lenses resembled the adhesion of lysozyme to the same lenses. Conclusion: Human serum albumin has three domains that allows albumin to bind and release hydrophobic molecules while transferrin has numerous positive charges. Thus, the negatively charged hydrophilic polymer of the Omafilcon lenses facilitated transferrin adhesion but limited albumin adhesion. Supported by a NSU President’s Fa

Adhesion of Transferrin to Tisifilcon A Contact Lenses

Tear proteins accumulate on contact lenses causing lens deterioration and conjunctival irritation. We examined the adhesion of transferrin to contact lenses made of tisifilcon A, a rigid gas-permeable silicone hydrogel (FDA Group III). Lenses were incubated 2.0 mg/ml solutions of human holo-transferrin for 1, 2, 3, and 4 days, and protein adhesion was determined by bicinchoninic acid assay. Transferrin adhesion increased from day 0 to day 3 and then dropped after days 4 and 5. This pattern resembled transferrin adhesion to lenses made of polymacon (FDA Group I), alphafilcon (FDA Group II), omafilcon (FDA Group II) and balafilcon (FDA Group III) materials, but differed from the pattern of transferrin adhesion to etafilcon (FDA Group IV) material. Transferrin adhesion to tisifilcon A was greater than transferrin adhesion to all other materials except balafilcon, which had equivalent transferrin adhesion. These differences may be related to the positive charges on transferrin originating from arginine residues and the N-terminus. These cause transferrin to adhere better to the low water ionic Group III materials than to the non-ionic materials (FDA Groups I and II) and the high water ionic material (FDA Group IV)

Adhesion of Lysozyme and Transferrin to Omafilcon A Contact Lenses

Human tears contain ~60 different proteins that accumulate on contact lenses. We examine the adhesion of human transferrin to FDA Group II Omafilcon A contact lenses, fabricated from a biomimetic material whose interaction with tear proteins is not well characterized and for which the consequences of protein accumulation are unclear. Omafilcon A lenses were incubated in human transferrin for five days, and protein adhesion was determined by bicinchoninic acid colorimetry on a daily basis. Transferrin adhered to the Omafilcon A lenses to a lesser extent (~10%) than it did to Alphafilcon A lenses (also FDA Group II). Our previous data indicate the same is true for lysozyme adhesion, with Omafilcon A lenses adsorbing less lysozyme (~25%) than Alphafilcon A lenses. Levels of transferrin and lysozyme adhesion to Omafilcon A lenses are lower than their levels of adhesion to any other type of contact lenses in all four FDA groups, suggesting that Omafilcon A lenses are better able to resist protein adhesion than contact lenses fabricated from other materials

Adhesion of Albumin to FDA Group I Contact Lenses

Tear protein adhesion can contaminate contact lenses and reduce their effectiveness for the treatment of vision abnormalities.. Protein adhesion depends upon the type of contact lens material, and fluctuates with tear secretion rate and pathology. We examined the adhesion of albumin, a major tear protein, to FDA Group I contact lenses over a four day period. Never worn Optima FW contact lenses (Bausch and Lomb, Inc., Rochester, NY) were incubated in albumin dissolved in optical saline (0.2 mg/ml) contained in borosilicate glass vials. The concentration of albumin in the vials, and adhering to the lenses, was monitored with bicinchoninic acid (BCA). Albumin concentration in the vials decreased on day three, and then regained initial levels. After one day of incubation, albumin adhesion to lenses reached a plateau that was stable through the remainder of the incubation period. This pattern is in contrast to that of lysozyme, which adheres to lenses in an up-down-up-down pattern regardless of lens material. Both albumin and lysozyme adhered to a lesser degree to these lenses than to FDA group IV lenses. These results clearly indicate the need for further studies of this complex interaction

Adhesion of Albumin to FDA Group IV Contact Lenses

Contact lenses are essential for the treatment of vision abnormalities. Tear protein adhesion can contaminate lenses and reduce their effectiveness. This adhesion depends upon the type of contact lens material, and fluctuates with tear secretion rate and pathology. We examined the adhesion of albumin, a major tear protein, to FDA Group IV contact lenses over a four day period. Never worn AcuVue contact lenses (Bausch and Lomb, Inc., Rochester, NY) were incubated in albumin dissolved in optical saline (0.2 mg/ml) contained in borosilicate glass vials. The concentration of albumin in the vials, and adhering to the lenses, was monitored with bicinchoninic acid (BCA). Albumin concentration in the vials decreased after one day of incubation, and then regained initial levels. Albumin adhesion to lenses increased to a plateau during the first two days of incubation. In contrast, lysozyme adhesion follows a consistent up-down- up-down pattern over four days of incubation, regardless of the type of contact lens. Both albumin and lysozyme adhered to a greater degree to these lenses than to FDA group I lenses. Much about this dynamic interaction remains a mystery, indicating the need for further study

Adhesion of Lysozyme, Albumin and Transferrin to Two Types of FDA Group II Contact Lenses

Tears contain ~60 different proteins that adhere to contact lenses, causing lens deterioration and ocular pathology. We examined the adhesion of three tear proteins to two different types of FDA Group II contact lenses (hilafilcon and omafilcon). Lenses were incubated in 2.0 mg/ml solutions of human lysozyme, albumin and transferrin for 1–4 days. Protein adhesion was determined by bicinchoninic acid assay. Lysozyme adhered to hilafilcon lenses in an up-down pattern, with a maximum on day 3. Lysozyme adhesion to omafilcon lenses was high after 1 day and remained high on day 4. Albumin adesion to both types of lenses was high after 1 day, declined, and increased on day 4. Transferrin adhesion to both lenses was initally low, increasing to a maximum on day 3 and declining on day 4. These results are due to differences in lens material and tear protein structure. Hilafilcon, more negatively charged than Omafilcon, absorbed more lysozyme (+ charged at physiological pH). Omafilcon lenses are coated with phosphorylcholine, reducing adhesion by the hydrophobic domains of albumin. Human apo-transferrin has few positive charges and thus bound more reluctantly to both materials