Receptor binding protein of prophage reversibly recognizes the low-molecular weight subunit of the surface-layer protein SlpA in Clostridioides difficile

Abstract

Receptor-binding proteins (RBPs) are located at the viral tail and mediate the initial recognition of phage to a specific bacterial host. Phage RBPs have co-evolved with numerous types of host receptors resulting in the formation of a diverse assortment of cognate pairs of RBP-receptors that function during the phage attachment step. Although several Clostridioides difficile bacteriophages have been discovered, their RBPs are poorly described. Using homology analysis, putative prophage-tail structure (pts) genes were identified from the prophage genome of the C. difficile HN10 strain. Competition and enzyme-linked immunosorbent assays, using recombinant PtsHN10M, demonstrated the interaction of this Pts to C. difficile cells, suggesting a role as a phage RBP. Gel filtration and cross-linking assay revealed the native form of this protein as a homotrimer. Moreover, truncated variants indicated that the C-terminal domain of PtsHN10M was important for binding to C. difficile cells. Interaction of PtsHN10M was also observed to the low-molecular weight subunit of surface-layer protein A (SlpA), located at the outermost surface of C. difficile cells. Altogether, our study highlights the function of PtsHN10M as an RBP and potentially paves the way toward phage engineering and phage therapy against C. difficile infection.

Clostridioides difficile S-Layer Protein A (SlpA) Serves as a General Phage Receptor

“…Recent studies pointed toward a role of the C. difficile surface layer protein SlpA in phage infection ( 35 , 37 , 38 , 40 ). However, in some cases, only indirect evidence based on engineered Avidocin-CD ( 35 ) or adsorption assays alone ( 37 , 38 ) are currently available.…”

“…More recent reports brought additional evidence that the S-layer acts as the receptor for two C. difficile myophages (ϕCD1801 and ϕHN10) ( 37 , 38 ) and a siphophage (CDHS-1) ( 39 ). One study provided direct evidence that SlpA, and more specifically the LMW fragment, is the receptor used by ϕHN10 ( 40 ). The report by Whittle et al involved adsorption assays with the myophage ϕCD1801 and C. difficile strain 630 coexpressing one of three different SLCTs in addition to its native S-layer ( 38 ).…”

A tale of two phage tails: Engineering the host range of bacteriophages infectingClostridioides difficile

“…Tail fibre genes are typically located between the genes encoding the tail tape measure protein and the endolysin 18,30,31 and fall within the structural tail gene module 32 .A BLAST query of genes located within the tail module revealed the presence of “tail collar fiber protein” (QVW56628.1, tcf ) in ΦCD1801 and “tail protein” (QVW56667.1, hereafter referred to as tail fibre protein – tfp ) in ΦCD2301; both ∼800bp in size. These were predicted to be the RBP for each phage through homology and location.…”

“…To address the issue of specificity, the underlying mechanisms dictating phage binding range require investigation. Phage receptor binding protein (RBP) and host range determining factors are poorly characterised in phages infecting C. difficile, although it has been established that the counterpart host receptor is encoded by the slpA gene [14][15][16][17][18] . Part of the phage host range is its binding range, dictated by the tail components, mainly the RBP [19][20][21][22] , which is recognised as the tail fibre (or spike in other species) 23 .…”

Engineering bacteriophages for enhanced host range and efficacy: insights from bacteriophage-bacteria interactions

“…Bioinformatics methods use this homology to help predict the RBPs of phages. In our study of the phage genome, we can select genomic annotation of TFs, TSPs or related genes that are vaguely annotated as “tail proteins” or “substrate proteins” for subsequent research ( Boeckaerts et al, 2021 ; Phetruen et al, 2022 ). Furthermore, homology modeling of genes with high homology can be performed, selecting models with high coverage and confidence in the target sequence to analyze possible docking structural domains and associated sites ( Yehl et al, 2019 ).…”