A Novel Staphylococcus Podophage Encodes a Unique Lysin with Unusual Modular Design Katie Cater, a Vidya Sree Dandu, a S. M. Nayeemul Bari, a Kim Lackey, a Gabriel F. K. Everett, b * Asma Hatoum-Aslan a Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama, USA a ; Center for Phage Technology, Texas A&M University and Texas A&M AgriLife, College Station, Texas, USA b ABSTRACT Drug-resistant staphylococci, particularly Staphylococcus aureus and Staphy- lococcus epidermidis, are leading causes of hospital-acquired infections. Bacterio- phages and their peptidoglycan hydrolytic enzymes (lysins) are currently being explored as alternatives to conventional antibiotics; however, only a limited di- versity of staphylococcal phages and their lysins has yet been characterized. Here, we describe a novel staphylococcal phage and its lysins. Bacteriophage Andhra is the first reported S. epidermidis phage belonging to the family Podo- viridae. Andhra possesses an 18,546-nucleotide genome with 20 open reading frames. BLASTp searches revealed that gene product 10 (gp10) and gp14 harbor putative catalytic domains with predicted peptidase and amidase activities, char- acteristic functions of phage lysins. We purified these proteins and show that both Andhra_gp10 and Andhra_gp14 inhibit growth and degrade cell walls of diverse staphylococci, with Andhra_gp10 exhibiting more robust activity against the panel of cell wall substrates tested. Site-directed mutagenesis of its pre- dicted catalytic residues abrogated the activity of Andhra_gp10, consistent with the presence of a catalytic CHAP domain on its C terminus. The active site loca- tion combined with the absence of an SH3b cell wall binding domain distin- guishes Andhra_gp10 from the majority of staphylococcal lysins characterized to date. Importantly, close homologs of Andhra_gp10 are present in related staphy- lococcal podophages, and we propose that these constitute a new class of phage-encoded lysins. Altogether, our results reveal insights into the biology of a rare family of staphylococcal phages while adding to the arsenal of antimicro- bials with potential for therapeutic use. IMPORTANCE The spread of antibiotic resistance among bacterial pathogens is in- citing a global public health crisis. Drug-resistant Staphylococcus species, especially S. aureus and S. epidermidis, have emerged in both hospital and community settings, underscoring the urgent need for new strategies to combat staphylococcal infec- tions. Bacterial viruses (phages) and the enzymes that they use to degrade bacterial cell walls (lysins) show promise as alternative antimicrobials; however, only a limited variety of staphylococcal phages and their lysins have yet been identified. Here, we report the discovery and characterization of a novel staphylococcal phage, Andhra. We show that Andhra encodes two lysins (Andhra_gp10 and Andhra_gp14) that in- hibit growth and degrade the cell walls of diverse staphylococci, including S. aureus and S. epidermidis strains. Andhra and its unique lysins add to the arsenal of antimi- crobials with potential for therapeutic use. KEYWORDS Staphylococcus, antimicrobial agents, bacteriophage lysis, bacteriophage therapy, bacteriophages Received 23 January 2017 Accepted 3 March 2017 Published 22 March 2017 Citation Cater K, Dandu VS, Bari SMN, Lackey K, Everett GFK, Hatoum-Aslan A. 2017. A novel Staphylococcus podophage encodes a unique lysin with unusual modular design. mSphere 2:e00040-17. https://doi.org/10.1128/ mSphere.00040-17. Editor Paul D. Fey, University of Nebraska Medical Center Copyright © 2017 Cater et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Asma Hatoum- Aslan, ahatoum@ua.edu. * Present address: Gabriel F. K. Everett, NCH Corporation, Irving, Texas, USA. K.C. and V.S.D. contributed equally to this work. A tiny virus adds to the arsenal of anti- staphylococcal agents. OBSERVATION Therapeutics and Prevention crossm March/April 2017 Volume 2 Issue 2 e00040-17 msphere.asm.org 1 on June 29, 2017 by guest http://msphere.asm.org/ Downloaded from