Structural basis for Salmonella infection by two Microviridae phages

Abstract

The global resurgence of multidrug-resistant Salmonella species, responsible for millions of annual infections, underscores the urgent need for alternative antimicrobial strategies, such as phage therapy. Microviridae phages offer a promising model for studying phage-host interactions with their unique structural and infection mechanisms. Here, we identify two Microviridae phages, PJNS001 and PJNS002, with different host receptor dependencies, and determine their cryo-EM structures at 2.68 Å and 2.59 Å resolution, respectively. These icosahedral capsids with T = 1 symmetry exhibit a unique vertex reinforcement mechanism, stabilizing the viral assembly. The specific pentameric adaptations, coupled with DNA binding protein engagements and thermodynamic constraints, collectively preclude the formation of hybrid virions. Structural analysis and in situ visualization reveal spike protein features and host-attachment intermediates, informing host specificity. Together, these findings advance our understanding of Microviridae infection mechanisms and provide a structural framework for rational phage design against antibiotic-resistant pathogens.

Fig. 1. Phage origin, receptor specificity, and phylogenetic tree of PJNS001 and PJNS002.

a Schematic representation of LPS structural mutations recognized by PJNS001 and PJNS002. Sugar residues are labeled using standard abbreviations: Man, mannose; Rha, rhamnose; Gal, galactose; Glc, glucose; GlcNAc, N-acetylglucosamine; Hep, L-glycero-D-manno-heptose; Kdo, 2-keto-3-deoxyoctonate; EtNP, ethanolamine phosphate; P, phosphate. Dashed arrows indicate the specific sugar positions added by enzymes encoded by the rfaL, rfaJ, rfaI, and rfaG genes. Hollow arrows indicate the LPS truncation points associated with susceptibility to PJNS001 (ΔrfaL) and PJNS002 (ΔrfaJ), showing that each phage targets distinct regions of the LPS core. b Phage sensitivity assay for PJNS001 and PJNS002 with different mutations. Infection specificity of phages PJNS001 and PJNS002 toward distinct receptor-deficient strains was determined via spotting assays. c Phylogenetic tree of PJNS001 and PJNS002 (red star). Phylogenetic analysis was performed using VipTree software. A total of 155 phages representing distinct viral families were analyzed, including Inoviridae, Microviridae, Plectroviridae, Pleolipoviridae, and Paulinoviridae.

Fig. 2. Cryo-EM structures of PJNS001 and PJNS002.

a Cryo-EM electron density map of PJNS001 at a contour level of 1.0. Pentagons, triangles, and diamonds mark fivefold, threefold, and twofold symmetry axes, respectively. Capsid protein F, spike protein G, and DNA-binding protein J are colored in cornflower blue, light sea green, and gold, respectively. Due to the internal location of protein J, its density is not visible in the overall map. To visualize interactions with one protein G copy, proteins surrounding the fivefold axis are differentially colored (details in Fig. 3a). Insets show representative interacting residues between capsid subunits near each axis. b Cartoon representation of the ASU within the dashed triangle indicated in panel a, colored as cornflower blue (F), light sea green (G), and gold (J) to indicate the generality. c Cryo-EM electron density map of PJNS002 at a contour level of 0.65. Protein F, G, and J are shown in deep sky blue, turquoise, and lime, respectively, with subunits near the fivefold axis colored as Fig. 3b. d Cartoon representation of the ASU in panel a.

Fig. 3. Inter-subunit interactions surrounding the fivefold axis of PJNS001 and PJNS002.

a Fivefold symmetric assembly of PJNS001, shown with 40% surface transparency. Subunits are colored consistently with Fig. 2a: protein G2, G3, F2, F3, F4, and F5 are shown in tomato, cyan, teal, olive, magenta, and pale green, respectively. Other subunits are shown in light sea green (G), cornflower blue (F), and gold (J). Dashed black lines indicate hydrogen bonds. Three regions are boxed: Inset 1 highlights the interaction interface between G1 and G2; Inset 2 shows the interactions surrounding the N-terminus of G1; Inset 3 displays a cross-sectional electrostatic surface of the G pentamer. b Fivefold symmetric assembly of PJNS002, with 40% transparency. Subunits are colored as in Fig. 2c: protein G2, F2, F4, and F5 are colored chocolate, dark khaki, hot pink, and aquamarine, respectively. Other subunits follow the scheme of turquoise (G), deep sky blue (F), and lime (J).

Fig. 4. Electrostatic potential and interaction details of protein J in PJNS001 and PJNS002.

a Electrostatic potential surface of the PJNS001 fivefold assembly, viewed from the interior. The close-up is colored according to the scheme in Fig. 3a. b Electrostatic potential surface of PJNS002 fivefold assembly, shown from the interior. Single-stranded DNA (ssDNA) is colored as crimson in the close-up. c Interaction analysis between protein J and F in PJNS001. The density of protein J is displayed at 0.8 threshold with 40% transparency. Three boxed regions are magnified to show representative interacting residues, with hydrogen bonds indicated by dashed lines. d Interaction analysis between protein J and both protein F and ssDNA in PJNS002.