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

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

doi:10.3389/fmicb.2023.1278791

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

Front Microbiol. 2023 Oct 31:14:1278791. doi: 10.3389/fmicb.2023.1278791. eCollection 2023.

Authors

Affiliations

¹ Johns Hopkins University Applied Physics Laboratory, Laurel, MD, United States.
² Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States.
³ Olympic Protein Technologies, Seattle, WA, United States.
⁴ Joint Program Executive Office for Chemical, Biological, Radiological and Nuclear Defense (JPEO-CBRND), Joint Project Lead for CBRND Enabling Biotechnologies, Frederick, MD, United States.
⁵ Joint Research and Development, Inc., Stafford, VA, United States.

Abstract

Bacteriophages such as γ and AP50c have been shown to infect strains of Bacillus anthracis 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 sap and GamR, respectively. A second AP50c-like phage, Wip1, also appears to use sap 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 sap 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 sap gene, as well as the deletion of csaB, whose encoded protein anchors sap to the bacterial S-layer, resulted in the loss of RBP binding. Binding could then be rescued by expressing these genes in trans. We also found that the RBP of the γ-like prophage λBa03 relied on csaB activity for binding, possibly by a different mechanism. RBP_λBa03 binding to B. anthracis 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 B. anthracis phages, enabling future assay development.

Keywords: Bacillus anthracis; S-layer; bacterial receptors; fluorescence detection; phage resistance; phages; receptor binding proteins.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. Funding for this work was provided by the Department of Defense (DoD) Joint Program Executive Office for Chemical, Biological, Radiological and Nuclear Defense (JPEO-CBRND) under NAVSEA contract number N00024-13-D-6400. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the JPEO-CBRND, the Departments of the Army, Navy, or Defense, nor the U.S. Government. References to non-federal entities do not constitute or imply Department of Defense or Army endorsement of any company or organization.