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

Therapeutic bacteriophages (phages) are being considered as alternatives in the fight against <i>Clostridioides difficile</i> infections. To be efficient, phages should have a wide host range, buthe lack of knowledge about the cell receptor used by <i>C. difficile</i> phages hampers the rational design of phage cocktails. Recent reports suggested that the <i>C. difficile</i> surface layer protein A (SlpA) is an important phage receptor, but available data are still limited. Here, using the epidemic R20291 strain and its FM2.5 mutant derivative lacking a functional S-layer, we show that the absence of SlpA renders cells completely resistant to infection by ϕCD38-2, ϕCD111, and ϕCD146, which normally infect the parental strain. Complementation with 12 different S-layer cassette types (SLCTs) expressed from a plasmid revealed that SLCT-6 also allowed infection by ϕCD111 and SLCT-11 enabled infection by ϕCD38-2 and ϕCD146. Of note, the expression of SLCT-1, -6, -8, -9, -10, or -12 conferred susceptibility to infection by 5 myophages that normally do not infect the R20291 strain. Also, deletion of the D2 domain within the low-molecular-weight fragment of SlpA was found to abolish infection by ϕCD38-2 and ϕCD146 but not ϕCD111. Altogether, our data suggest that many phages use SlpA as their receptor and, most importantly, that both siphophages and myophages target SlpA despite major differences in their tail structures. Our study therefore represents an important step in understanding the interactions between <i>C. difficile</i> and its phages. <b>IMPORTANCE</b> Phage therapy represents an interesting alternative to treat <i>Clostridioides difficile</i> infections because, contrary to antibiotics, most phages are highly species specific, thereby sparing the beneficial gut microbes that protect from infection. However, currently available phages against <i>C. difficile</i> have a narrow host range and target members from only one or a few PCR ribotypes. Without a clear comprehension of the factors that define host specificity, and in particular the host receptor recognized by phages, it is hard to develop therapeutic cocktails in a rational manner. In our study, we provide clear and unambiguous experimental evidence that SlpA is a common receptor used by many siphophages and myophages. Although work is still needed to define how a particular phage receptor-binding protein binds to a specific SLCT, the identification of SlpA as a common receptor is a major keystone that will facilitate the rational design of therapeutic phage cocktails against clinically important strains.