Team:WPI-Worcester/Background
Background
Biofilms
Biofilms are a way in which single-celled organisms can act in a multicellular manner through cooperation. They are strongly resistant to antimicrobials (Donlan and Costerton 2002), which means that combating them is an important step in the fight against healthcare-associated infections, which have many times been shown to be associated with medical devices or surgical sites that are colonized by biofilms (Abdallah et al. 2014), which are thought to be causative (Akers et al. 2015). Biofilms are also the primary source—and a persistent one—of food product contamination (Abdallah et al. 2014). Biofilms are largely made up of an extracellular matrix that is produced by the organisms. The matrix composes 90% of the biomass in a given biofilm, and is made up of extracellular polymeric substances, carbohydrate-binding proteins, pili, flagella, adhesive fibers, and extracellular DNA (Kostakioti et al. 2013).
Biofilm formation occurs in steps. The first step is introduction of bacteria to a surface, which is followed by the second step, adhesion. Motile bacteria have an advantage in this step; flagella allow bacteria to overcome forces that might otherwise prevent attachment (Kostakioti et al. 2013). Other extracellular appendages, and adhesins secreted by the bacteria, affect adhesion as well. However, initial adhesion is reversible based on repulsive or hydrodynamic forces, or the availability of nutrients. If the bacteria are capable of sticking despite those forces, they will become irreversibly attached. Irreversible attachment is facilitated by a variety of factors, including type 1 and type IV pili, curli fibers, and Antigen 43 (Kostakioti et al. 2013). Surface contact results in the up-regulation of genes that will make the bacteria become sessile—that is, anchored.
Following attachment, dispersal can occur. Dispersal can be passive (as a result of shear forces) or active, as in a bacterial response to detected environmental factors, such as oxygen levels or nutrient availability. Occasionally dispersal can occur in other ways; for example, in B. subtilis, fluctuations in certain amino acid levels over the course of the cell cycle can result in dispersal, and it’s been suggested that similar mechanisms could exist in other bacteria (Kostakioti et al. 2013).
In 2014, Heisig et al. found that an antifreeze glycoprotein from ticks, IAFGP, and a peptide derived from that protein, could inhibit the formation of S. aureus biofilms in a variety of in vitro and in vivo scenarios. Figure 1C from the study, below, shows the results of an in vitro biofilm inhibition assay, performed by staining with Safranin.
The current research suggests that the anti-virulent properties of IAFGP are based on structural elements of the protein that allow it to bind to microbes and disrupt biofilm formation (Heisig et al. 2014), however, very little is known about this, and only one study has been completed. More research is needed to find out whether other antifreeze proteins exhibit the same effect.
Antifreeze Proteins
Water crystallizes as it freezes, forming sharp structures capable of lysing cells. Organisms which regularly encounter subzero conditions have evolved a number of methods to prevent cell lysis from ice crystallization, including the production of antifreeze proteins. Antifreeze proteins, or AFPs, are a class of polypeptides with the unique property of binding and shaping ice crystals, preventing their growth into relatively large cell-lysing structures and allowing organism survival in colder environments (Davies and Sykes, 1997).
Over the past decade, scientists have discovered and isolated a variety of AFPs from different organisms. Five different classifications of antifreeze proteins have been discovered in fish: Type I, Type II, Type III, Type IV, and antifreeze glycoproteins, or AFGPs. Type I AFPs are long, single alpha-helices that exist as 3-5 kDa monomers. Within Type I, there are two subgroups that bind to different planes of ice; the repetitive Type I AFPs have clearly defined repeats of alanine and aspartate or threonine while the nonrepetitive Type I AFPs are amphipathic with lysine and arginine side chains (Davies and Sykes, 1997). Type II AFPs are 12-24 kDa homologs that form globular, calcium-dependent lectins and carbohydrate-recognition domains. Two different subgroups of Type II AFPs, calcium-dependent and calcium-independent, have been discovered and differ in regards to whether calcium is necessary for ice binding. Type III AFPs are 7 kDa globular proteins that have unique 3D folds. Type IV AFPs are 12.3 kDa proteins composed of mostly alpha helices (Davies and Sykes, 1997). AFGPs are 3.3 kDa disaccharides consisting of repeating glycopeptide sequences (Ala-Ala-Thr-galactosyl-N-acetyl galactosamine).
The discovery of antifreeze proteins in plants and insects created more classifications because of the differences in amino acid residues and thermal hysteresis activity. Plant AFPs exhibit lower thermal hysteresis than fish AFPs and function to recrystallize ice instead of preventing its initial formation (Griffith and Yaish, 2004). In insects, AFPs are classified into two families: Tenebrio and Dendroides. Tenebrio, also known as Type V AFPs, are found in beetles; Dendroides, also known as Choristoneura fumiferana AFPs, are found in butterflies and moths. Both AFPs have higher thermal hysteresis values than fish AFPs and contain cysteine residues within their protein structures (Graham et al, 1997).
While the specific mechanisms by which AFPs function are unclear, scientists have proposed a variety of different mechanisms based on structural features. They are thought to work by binding water molecules across their relatively flat ice-binding surfaces, maintaining those bonds primarily through van der Waals interactions, with some debate over whether hydrogen bonding or “entropic and enthalipc contributions from hydrophobic residues” keep the structure stability (Davies and Sykes, 1997). To inhibit ice crystal growth, AFPs are believed to adsorb to ice crystals. Normally when ice crystals form in an aqueous solution, the water forms an ice lattice while pushing solutes away. Adsorption restricts the lattice growth of ice crystals to areas between the proteins. This causes the crystals to grow in a way that is thermodynamically unfavorable for water molecules to continue adding to the ice lattice (Raymond and DeVries, 1977). Because of this, AFPs exhibit thermal hysteresis, a property that creates change in the freezing point of water without affecting the melting point. This allows organisms that produce AFPs to survive colder conditions by reducing the temperature at which dangerous ice crystals form (Jorov et al, 2004).
References
Abdallah, M., Djamel Benoliel C Fau - Drider, Pascal Drider D Fau - Dhulster, Nour-Eddine Dhulster P Fau - Chihib, and N. E. Chihib. 2014. Biofilm formation and persistence on abiotic surfaces in the context of food and medical environments. (1432-072X (Electronic)).
Akers, K. S., Joseph C. Cardile Ap Fau - Wenke, Clinton K. Wenke Jc Fau - Murray, and C. K. Murray. 2015. Biofilm formation by clinical isolates and its relevance to clinical infections. (0065-2598 (Print)).
Davies, P. L., and B. D. Sykes. 1997. Antifreeze proteins. (0959-440X (Print)).
Donlan, Rodney M., and J. William Costerton. 2002. Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms. Clinical Microbiology Reviews 15 (2):167-193.
Graham, Laurie A., Yih-Cherng Liou, Virginia K. Walker, and Peter L. Davies. 1997. Hyperactive antifreeze protein from beetles. Nature 388 (6644):727-728.
Griffith, M., D. S. Ala P Fau - Yang, W. C. Yang Ds Fau - Hon, B. A. Hon Wc Fau - Moffatt, and B. A. Moffatt. 1992. Antifreeze protein produced endogenously in winter rye leaves. (0032-0889 (Print)).
Heisig, M., N. M. Abraham, L. Liu, G. Neelakanta, S. Mattessich, H. Sultana, Z. Shang, J. M. Ansari, C. Killiam, W. Walker, L. Cooley, R. A. Flavell, H. Agaisse, and E. Fikrig. 2014. Antivirulence properties of an antifreeze protein. (2211-1247 (Electronic)).
Jorov, A., Daniel S. C. Zhorov Bs Fau - Yang, and D. S. Yang. Theoretical study of interaction of winter flounder antifreeze protein with ice. (0961-8368 (Print)).
Kostakioti, M., Scott J. Hadjifrangiskou M Fau - Hultgren, and S. J. Hultgren. 2013. Bacterial biofilms: development, dispersal, and therapeutic strategies in the dawn of the postantibiotic era. (2157-1422 (Electronic)).
Raymond, J. A., and A. L. DeVries. 1977. Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Proceedings of the National Academy of Sciences of the United States of America 74 (6):2589-2593.