Thermoresponsive C22 phage stiffness modulates the phage infectivity

www.nature.com/scientificreports OPEN Thermoresponsive C22 phage stiffness modulates the phage infectivity Udom Sae‑Ueng1*, Anjana Bhunchoth1, Namthip Phironrit1, Alongkot Treetong2, Chaweewan Sapcharoenkun2, Orawan Chatchawankanphanich1, Ubolsree Leartsakulpanich1 & Penchit Chitnumsub1 Bacteriophages offer a sustainable alternative for controlling crop disease. However, the lack of knowledge on phage infection mechanisms makes phage-based biological control varying and ineffective. In this work, we interrogated the temperature dependence of the infection and thermoresponsive behavior of the C22 phage. This soilborne podovirus is capable of lysing Ralstonia solanacearum, causing bacterial wilt disease. We revealed that the C22 phage could better infect the pathogenic host cell when incubated at low temperatures (25, 30 °C) than at high temperatures (35, 40 °C). Measurement of the C22 phage stiffness revealed that the phage stiffness at low temperatures was 2–3 times larger than at high temperatures. In addition, the imaging results showed that more C22 phage particles were attached to the cell surface at low temperatures than at high temperatures, associating the phage stiffness and the phage attachment. The result suggests that the structure and stiffness modulation in response to temperature change improve infection, providing mechanistic insight into the C22 phage lytic cycle. Our study signifies the need to understand phage responses to the fluctuating environment for effective phage-based biocontrol implementation. Bacterial wilt disease caused by Ralstonia solanacearum has destroyed economic crops including potato, tomato, tobacco, and chili, costing about 1 billion US dollars per y ear1. Conventional practices such as soil disinfection, soil amendment, and crop rotation are labor-intensive and ineffective due to pathogenic bacteria’s ability to survive in the soil for a long time and propagate to nearby regions via water c hannels2,3. Using chemicals and pesticides to eliminate the bacteria inflicts harmful residues on consumable products, human health, and the environment. Therefore, biological control of Ralstonia solanacearum causing wilt disease using antagonistic agents has gained interest as a safe a lternative4,5. Among such agents, lytic bacteriophages or phages demonstrate promising results in controlling such damaging b acteria6–9. Phages target specific host bacterial cells. Once all host cells are eradicated, phages will no longer be replicated. Phages are generally recognized as safe (GRAS) and non-toxic to eukaryotes, rendering them an attractive choice for preventing and treating crop diseases, including bacterial wilt d isease10,11. Despite the phage advantages, phage-mediated biocontrol is underutilized due to its variable efficacy12–15. The success of phage biocontrol depends on the infection of the bacterial host cell by phage, which is governed by several surrounding factors such as pH, ions, and osmotic pressure16–18. An influential factor in all microenvironments is temperature. Phages used in biocontrol constantly experience fluctuating temperatures due to daily climate and seasonal variations in the agricultural fields. The temperature dependence of phages has been previously investigated. However, the exact role of the temperature on phage infection remains unclear. Some phages can infect better at their specific permissive t emperatures19–22. Lambda phage infecting Escherichia coli and the phages infecting Burkholderia pseudomallei infected their host cells more effectively and produced more progeny phages at about 35–40 °C than at about 20–35 °C20,22. The cell lysis study of Pseudomonas fluorescens by ɸS1 phage showed a higher infection rate at 26 °C than 37 °C23. On the other hand, some phages are insensitive to temperature. The T4 myovirus phage effectively lysed E. coli BL21 within a temperature range of 15 to 41 °C24. The P100 phage infecting Listeria monocytogenes remained infectious at 4–60 °C25,26. The MR5 phage infecting Pseudomonas syringae, causing bacterial canker, showed similar infectivity at 20, 27, and 37 °C27. Understanding how the temperature affects the phage infection can provide a guideline on phage biocontrol use when coupled 1 National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand. 2National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand. *email: Scientific Reports | (2022) 12:13001 | https://doi.org/10.1038/s41598-022-16795-y 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1.  Relative titers of C22 phage at 25, 30, 35, and 40 °C. The titer measurement was conducted in triplicate, and the error bar represented standard deviations (s.d.). with temperature forecast data and infection6,28. Therefore, we attempt to decipher the role of the temperature for phages in biocontrol use. The influence of temperature on phage infection is regulated by how structural components of phages molecularly respond to temperature changes. Infecting Listeria monocytogenes, the A511 phage was more stable at high temperatures (60–80 °C) than the P100 phage due to the slightly higher melting point of the A511phage’s tailcapsid connector p rotein25. A study on the T7 phage showed that high temperatures reduced the phage infection of the host tail since the high temperature caused the breaking of the phage tail from the phage capsid29. Some studies suggested that temperature increase improved infection since it provided more thermal energy to propel the genome into the host c ells30–32. Understanding the molecular interactions within phage proteins and structures in response to thermal change reveals the underlying mechanism of phage infection and survival. Therefore, insight at the molecular scale will be required to understand the effect of the temperature, which will offer tailor-made guidelines for using phages as a biocontrol agent. In this work, we revealed the role of temperature on a lytic C22 phage and its infection. The C22 podovirus isolated from a soil sample in Thailand can lyse pathogenic bacteria Ralstonia solanacearum (Rs), causing bacterial wilt disease in tomato and p epper33. The disease also destroys other Solanaceous crops such as potato, tobacco, and eggplant, leading to a global loss of one billion US dollars per year34. The C22 phage, therefore, presents a promising and urgently needed antibacterial agent for the bacterial wilt disease. We studied the temperature effect on the C22 phage using a plaque-based infectivity assay. We found that the C22 phage heated at 25–30 °C can infect the host cell better than the C22 phage incubated at 35–40 °C by about 44%. We investigated the direct effect of the temperature by examining the C22 phage particles incubated at different temperatures using atomic force microscopy (AF (...truncated)