Enhanced Antimicrobial Activity and Structural Transitions of a Nanofibrillated Cellulose-Nisin Biocomposite Suspension.

Resistance to antibiotics has posed a high demand for novel strategies to fight bacterial infections. Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. However, their poor solubility in water and sensitivity to degradation has limited their application. Here, we report the design of a smart, pH-responsive antimicrobial nanobiocomposite material based on the AMP nisin and 2,2,6,6-tetramethyl-1-piperidinyloxyl-oxidized nanofibrillated cellulose (TONFC). Morphological transformations of the nanoscale structure of nisin functionalized-TONFC fibrils were discovered at pH values between 5.8 and 8.0 using small-angle X-ray scattering. Complementary ζ potential measurements indicate that electrostatic attractions between the negatively charged TONFC surface and the positively charged nisin molecules are responsible for the integration of nisin. Modification of the pH level or increasing the ionic strength reduces the nisin binding capacity of TONFC. Biological evaluation studies using a bioluminescence-based reporter strain of Bacillus subtilis and a clinically relevant strain of Staphylococcus aureus indicated a significantly higher antimicrobial activity of the TONFC-nisin biocomposite compared to the pure nisin against both strains under physiological pH and ionic strength conditions. The in-depth characterization of this new class of antimicrobial biocomposite material based on nanocellulose and nisin may guide the rational design of sustainable antimicrobial materials.

Beneficial Oral Biofilms as Smart Bioactive Interfaces.

Periodontitis is a very common health problem caused by formation of pathogenic bacterial biofilm that triggers inflammation resulting in either reversible gingivitis or irreversible periodontal hard and soft tissue damages, leading to loss of teeth when left untreated. Commensal bacteria play an important role in oral health in many aspects. Mainly by colonizing oral tissues, they (i) contribute to maturation of immune response, and (ii) foreclose attachment of pathobiont and, therefore, prevent from infection. The main goal of the study was to investigate if blocking of receptors on a commensal biofilm can prevent or reduce the attachment of pathogenic strains. To do so, biofilm produced by commensal Streptococcus sanguinis was treated with whole cell lysate of pathobionts Fusobacterium nucleatum or Porphyromonas gingivalis, followed by incubation with respective strain(s). The study revealed significant reduction in pathobiont adhesion to lysate-treated commensal biofilm. Therefore, adhesion of pathobionts onto the lysate-blocked biofilm was hindered; however, not completely eliminated supporting the idea that such approach in the oral cavity would benefit the production of a well-balanced and healthy bioactive interface.

Core-Shell Silver Nanoparticles in Endodontic Disinfection Solutions Enable Long-Term Antimicrobial Effect on Oral Biofilms.

To achieve effective long-term disinfection of the root canals, we synthesized core-shell silver nanoparticles (AgNPs@SiO2) and used them to develop two irrigation solutions containing sodium phytate (SP) and ethylene glycol-bis(ß-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), respectively. Ex vivo studies with instrumented root canals revealed that the developed irrigation solutions can effectively remove the smear layer from the dentinal surfaces. Further in vitro experiments with single- and multispecies biofilms demonstrated for the first time that AgNPs@SiO2-based irrigation solutions possess excellent antimicrobial activities for at least 7 days, whereas the bare AgNPs lose the activity almost immediately and do not show any antibacterial activity after 2 days. The long-term antimicrobial activity exhibited by AgNPs@SiO2 solutions can be attributed to the sustainable availability of soluble silver, even after 7 days. Both solutions showed lower cytotoxicity toward human gingival fibroblasts compared to the conventionally used solution (3% NaOCl and 17% EDTA). Irrigation solutions containing AgNP@SiO2 may therefore be highly promising for applications needing a long-term antimicrobial effect.

Six hydrophobins are involved in hydrophobin rodlet formation in Aspergillus nidulans and contribute to hydrophobicity of the spore surface.

Hydrophobins are amphiphilic proteins able to self-assemble at water-air interphases and are only found in filamentous fungi. In Aspergillus nidulans two hydrophobins, RodA and DewA, have been characterized, which both localize on the conidiospore surface and contribute to its hydrophobicity. RodA is the constituent protein of very regularly arranged rodlets, 10 nm in diameter. Here we analyzed four more hydrophobins, DewB-E, in A. nidulans and found that all six hydrophobins contribute to the hydrophobic surface of the conidiospores but only deletion of rodA caused loss of the rodlet structure. Analysis of the rodlets in the dewB-E deletion strains with atomic force microscopy revealed that the rodlets appeared less robust. Expression of DewA and DewB driven from the rodA promoter and secreted with the RodA secretion signal in a strain lacking RodA, restored partly the hydrophobicity. DewA and B were able to form rodlets to some extent but never reached the rodlet structure of RodA. The rodlet-lacking rodA-deletion strain opens the possibility to systematically study rodlet formation of other natural or synthetic hydrophobins.

Engineering hydrophobin DewA to generate surfaces that enhance adhesion of human but not bacterial cells.

Hydrophobins are fungal proteins with the ability to form immunologically inert membranes of high stability, properties that makes them attractive candidates for orthopaedic implant coatings. Cell adhesion on the surface of such implants is necessary for better integration with the neighbouring tissue; however, hydrophobin surfaces do not mediate cell adhesion. The aim of this project was therefore to investigate whether the class I hydrophobin DewA from Aspergillus nidulans can be functionalized for use on orthopaedic implant surfaces. DewA variants bearing either one RGD sequence or the laminin globular domain LG3 binding motif were engineered. The surfaces of both variants showed significantly increased adhesion of mesenchymal stem cells (MSCs), osteoblasts, fibroblasts and chondrocytes; in contrast, the insertion of binding motifs RGD and LG3 in DewA did not increase Staphylococcus aureus adhesion to the hydrophobin surfaces. Proliferation of MSCs and their osteogenic, chondrogenic and adipogenic differentiation potential were not affected on these surfaces. The engineered surfaces therefore enhanced MSC adhesion without interfering with their functionality or leading to increased risk of bacterial infection.

The pneumococcal cell envelope stress-sensing system LiaFSR is activated by murein hydrolases and lipid II-interacting antibiotics.

In the Firmicutes, two-component regulatory systems of the LiaSR type sense and orchestrate the response to various agents that perturb cell envelope functions, in particular lipid II cycle inhibitors. In the current study, we found that the corresponding system in Streptococcus pneumoniae displays similar properties but, in addition, responds to cell envelope stress elicited by murein hydrolases. During competence for genetic transformation, pneumococci attack and lyse noncompetent siblings present in the same environment. This phenomenon, termed fratricide, increases the efficiency of horizontal gene transfer in vitro and is believed to stimulate gene exchange also under natural conditions. Lysis of noncompetent target cells is mediated by the putative murein hydrolase CbpD, the key effector of the fratricide mechanism, and the autolysins LytA and LytC. To avoid succumbing to their own lysins, competent attacker cells must possess a protective mechanism rendering them immune. The most important component of this mechanism is ComM, an integral membrane protein of unknown function that is expressed only in competent cells. Here, we show that a second layer of self-protection is provided by the pneumococcal LiaFSR system, which senses the damage inflicted to the cell wall by CbpD, LytA, and LytC. Two members of the LiaFSR regulon, spr0810 and PcpC (spr0351), were shown to contribute to the LiaFSR-coordinated protection against fratricide-induced self-lysis.