Ɛ34 Phage Tailspike Protein is Resistant to Trypsin and Inhibits and Salmonella Biofilm Formation

Abstract

Salmonella can cause acute and chronic infections in humans. Salmonella species are known to cause food poisoning and other diseases in developing countries. Their role in the pathogenesis of these diseases has received increased international attention. Despite numerous advances in sanitation, they still can infect humans and cause outbreaks in developed countries. For example, Salmonella causes about 1.2 million illnesses in the US each year with over 450 deaths. Additionally, Salmonella outbreaks cause significant losses to chicken producers globally. The Salmonella species is also prone to acquiring resistance to various classes of antibiotics. Hence, the need for a paradigm shift from antibiotics to bacteriophages to manage, control and treat bacterial infections. The ɛ34 phage belongs to Podoviruses and categorized into the P22-like phages. The P22-like phages include ɛ34, ES18, P22, ST104, and ST64T. In this work, we investigated the antibacterial property of ɛ34 phage tailspike protein against Salmonella newington (S. newington). We demonstrate here that, the phage’s tailspike protein enzymatic property as a LPS hydrolase synergizes with Vero Cell culture supernatant in killing S. newington. Using decellularized cartilage scaffold as an ex vivo tissue model, the ɛ34 TSP protected the scaffold from S. newington biofilm formation. Computational analysis of the ɛ34 TSP interaction with membrane proteins of S. newington demonstrated a higher probability (0.7318) of binding to ompA of S. newington, and when docked to ompA extracellular component, it produced a high free energy of -11.3kcal/mol. We also demonstrate the resistance/sensitivity of the tailspike to the digestive enzyme trypsin. The data obtained in this work indicates that the trypsin resistant tailspike protein of Ɛ34 phage can be formulated as a novel antibacterial agent against S. newington.

Proteolytic Analysis of Epsilon 34 Phage Tailspike protein indicates Partial Sensitivity to Proteinase K

“…The predicted structure of Epsilon 34 TSP consists of a globular head binding domain, a solenoid-shape parallel beta-helix domain that is used in the LPS binding process as also found in the P22 phage TSP [9,14,15]. Phages or phage derived components can complement host cell defense against bacteria [3,16]. Purified bacteriophage Ɛ34 TSP can bind to S. newington via the binding site of the protein, which is the O antigens of the LPS of the bacterium [9].…”

“…Salmonella has been demonstrated to cause several acute and chronic infections in humans [1] . These bacteria is known to be responsible for over 1.2 million illnesses in the US each year [2, 3, 4, 5] . Salmonella species are prone to acquiring resistance to various classes of antibiotics this sparks the need to look for methods of treating salmonella related infections other than the use of antibiotics, thus calling for the application of bacteriophages to manage, control and treat bacterial infections [6, 7] .…”

“…The predicted structure of Epsilon 34 TSP consists of a globular head binding domain, a solenoid-shape parallel beta-helix domain that is used in the LPS binding process as also found in the P22 phage TSP [9, 14, 15] . Phages or phage derived components can complement host cell defense against bacteria [3, 16] .…”

“…There exist extensive research on the folding behavior of β-helix proteins, which also provides insight on how amyloid fibrils are generated since these proteins consist of parallel β-helix motifs [19, 20, 21, 22] . One of the most significantly studied system for investigating protein folding is the from the Salmonella bacteriophage P22 [3, 9, 23] . The major component of this protein is a right-handed parallel β-helix with 13 rungs [24] .…”

“…Our previous studies indicated that despite the similarities of the two proteins, P22 TSP shows higher resistance to proteases (e.g. trypsin) and heat compared to ε34 TSP [3] . Several common proteases that have been used in most published works include chymotrypsin, trypsin, enterokinase, proteinase K, factor Xa, Staphylococcus aureus protease, papain etc., in proteolytic analysis [25, 26, 27, 28, 29], and their data are demonstrated to be consistent with expected fragments in denatured substrates.…”