AUTHOR=Forrest Samantha , Ton Sarah , Sholes Samantha L. , Harrison Sarah , Plaut Roger D. , Verratti Kathleen , Wittekind Michael , Ettehadieh Elham , Necciai Bryan , Sozhamannan Shanmuga , Grady Sarah L. 

TITLE=Genetic evidence for the interaction between Bacillus anthracis-encoded phage receptors and their cognate phage-encoded receptor binding proteins

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1278791

DOI=10.3389/fmicb.2023.1278791

ISSN=1664-302X

ABSTRACT=<p>Bacteriophages such as γ and AP50c have been shown to infect strains of <italic>Bacillus anthracis</italic> with high specificity, and this feature has been exploited in the development of bacterial detection assays. To better understand the emergence of phage resistance, and thus the potential failure of such assays, it is important to identify the host and phage receptors necessary for attachment and entry. Using genetic approaches, the bacterial receptors of AP50c and γ have been identified as <italic>sap</italic> and GamR, respectively. A second AP50c-like phage, Wip1, also appears to use <italic>sap</italic> as a receptor. In parallel with this work, the cognate phage-encoded receptor binding proteins (RBPs) have also been identified (Gp14 for γ, P28 for AP50c, and P23 for Wip1); however, the strength of evidence supporting these protein–protein interactions varies, necessitating additional investigation. Here, we present genetic evidence further supporting the interaction between <italic>sap</italic> and the RBPs of AP50c and Wip1 using fluorescently tagged proteins and a panel of B. anthracis mutants. These results showed that the deletion of the <italic>sap</italic> gene, as well as the deletion of <italic>csaB</italic>, whose encoded protein anchors <italic>sap</italic> to the bacterial S-layer, resulted in the loss of RBP binding. Binding could then be rescued by expressing these genes in <italic>trans</italic>. We also found that the RBP of the γ-like prophage λBa03 relied on csaB activity for binding, possibly by a different mechanism. RBP<sub>λBa03</sub> binding to <italic>B. anthracis</italic> cells was also unique in that it was not ablated by heat inactivation of vegetative cells, suggesting that its receptor is still functional following incubation at 98°C. These results extend our understanding of the diverse attachment and entry strategies used by <italic>B. anthracis</italic> phages, enabling future assay development.</p>