C22 podovirus infectivity is associated with intermediate stiffness

www.nature.com/scientificreports OPEN C22 podovirus infectivity is associated with intermediate stiffness Udom Sae‑Ueng 1*, Anjana Bhunchoth1, Namthip Phironrit1, Alongkot Treetong2, Chaweewan Sapcharoenkun 2, Orawan Chatchawankanphanich1, Ubolsree Leartsakulpanich1 & Penchit Chitnumsub1 Bacteriophages have potential for use as biological control agents (biocontrols) of pathogenic bacteria, but their low stability is limiting for their utilization as biocontrols. Understanding of the conditions conducive to storage of phages in which infectivity is maintained over long periods will be useful for their application as biocontrols. We employed a nanomechanical approach to study how external environmental factors affect surface properties and infectivity of the podovirus C22 phage, a candidate for biocontrol of Ralstonia solanacearum, the agent of bacterial wilt in crops. We performed atomic force microscopy (AFM)-based nano-indentation on the C22 phage in buffers with varying pH and ionic strength. The infectivity data from plaque assay in the same conditions revealed that an intermediate range of stiffness was associated with phage titer that remained consistently high, even after prolonged storage up to 182 days. The data are consistent with the model that C22 phage must adopt a metastable state for maximal infectivity, and external factors that alter the stiffness of the phage capsid lead to perturbation of this infective state. Ralstonia solanacearum, the agent of bacterial wilt disease in crops such as potato, tomato, tobacco and chili, is ranked as the second most important bacterial pathogen worldwide, responsible for an estimated annual loss of crop value exceeding 950 million US dollars1,2. Chemical methods for controlling R. solanacearum are ineffective and environmentally damaging. Natural biological control agents, also known as biocontrols, represent an alternative pest control method that is sustainable, more effective and less detrimental to the environment. Bacteriophage viruses (phages) that naturally infect bacterial pathogens in soil are attractive biocontrol candidates. Phage biocontrol is sustainable because it does not introduce foreign materials into the e nvironment3. Secondly, phages can only replicate in specific host bacteria, meaning that once all host cells are lysed, phage replication will terminate4,5. Thirdly, phages are non-toxic to humans, rendering them appropriate for consumable p roducts6. Despite the advantages of phages for biocontrol, phages must remain viable and infectious for long periods, including storage and field a pplication7–9. The stability of phage is therefore a critical issue that compromises its practical use in agriculture. The optimal conditions to maintain phage infectivity are thus important for efficient phage usage in biocontrol. Phage survival and infectivity are dependent on the nanomechanical property of the phage genomic material encapsulated by the capsid protein s hell10,11. It has been shown that DNA p ackaging12, DNA ejection from the capsid13,14, and DNA transition inside the c apsid15 are correlated with phage nanomechanical property. We recently isolated a novel phage named C22 in the family of Podoviridae (podovirus) that can lyse R. solanacearum. The high lytic activity of this phage makes it an attractive candidate for biological control of R. solanacearum16. In this study, we characterized the nanomechanical property of C22 phage by measuring stiffness using atomic force microscopy (AFM). Employing a nanometer-size tip, AFM can acquire stiffness data and nanometer-resolution images of phage in a liquid m ilieu17–20. Therefore, the data reflect the phage’s properties in its natural infectious state. We found that C22 phage exhibited variable stiffness according to the conditions of buffer pH and ionic 1 National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand. 2National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), 111 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand. *email: Scientific Reports | (2020) 10:12604 | https://doi.org/10.1038/s41598-020-69409-w 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1.  Experimental schemes of AFM-based nano-indentation and plaque assay. (a) C22 virus particles (blue) were bound to a chemically treated mica surface (dark grey). An AFM cantilever (light grey) approaches the particle by moving downward and indents into the particle. The indentation on the particle (blue) and on a mica surface (red) yields force-distance curves for the stiffness calculation of the C22 phage. The separation between the two force-distance curves determines the indenting depth into the phage particle. (b) Plaque assay performed by the double agar layer technique. 1.5% agar CPG was overlaid with a top layer of 0.45% agar CPG containing Ralstonia solanacearum (Rs) and C22 phage. After overnight incubation, clear zones (plaques) from the bacterial lysis were observed and enumerated (black arrows). Figure 2.  Microscopic images of the C22 phage. AFM images are shown in parts (a–c). (a) Analysis of the cross section indicates the C22 phage diameter to be 40 nm. (b) and (c) Images showing the threefold and fivefold symmetry faces, respectively consistent with icosahedral geometry of the phage capsid (schematic drawing top right). The color scale bar underneath the images is the scale in the vertical (Z) axis for the AFM images. (d) TEM image of a C22 phage particle; scale bar = 50 nm. concentration, and that phage stiffness was associated with phage infectivity. The data suggest that intermediate stiffness, possibly representing a metastable state, is key for phage stability. Results Nanomechanical property of C22 phage. The nanomechanical property of phage can be measured by AFM-based nano-indentation, in which the phage properties are probed via the direct interaction between the AFM tip and the surface of the phage structure as shown in Fig. 1a. C22 phage images were captured using AFM Scientific Reports | Vol:.(1234567890) (2020) 10:12604 | https://doi.org/10.1038/s41598-020-69409-w 2 www.nature.com/scientificreports/ in the non-contact imaging mode (Fig. 2a–c). C22 particles exhibited icosahedral geometry with a diameter of 40 nm (Fig. 2a inset) consistent with the transmission electron microscope (TEM) image (Fig. 2d). The threefold and fivefold symmetry faces observed in AFM images (Fig. 2b,c) were consistent with the characteristic features of viral icosahedral g eometry21,22. The AFM images of C22 particles (Fig. 2a–c) appeared to map incompletely to the icosahedral outline (insets), which is likely due to occasional aberrant tip-sample interactions of soft biological samples in (...truncated)