title: Genomic Analysis of Paenibacillus Larvae Bacteriophages
Creators: name: Stamereilers, Casey
Publisher: name: University of Nevada, Las Vegas
Year: 2021

description: American Foulbrood is the most destructive bacterial infection of the honeybee (Apis mellifera) and is caused by the Gram-positive, spore forming bacterium Paenibacillus larvae. Current treatment methods rely on antibiotics, but antibiotics treatments are experiencing a reduction in efficacy due to the recent rise in antibiotic resistant strains of P. larvae. This has been a major catalyst for exploration of alternative treatment methods. Phage therapy is an alternative treatment method that uses viruses that exclusively infect bacteria, known as bacteriophages (phages), to combat bacterial infections. Several experimental studies have shown that phages P. larvae phages are effective at lysing P. larvae and to thus serve as treatment agents. In addition to experimental studies, it is important to characterize the genomes of P. larvae phages to gain insight into their biology so as to guide future therapy. The first P. larvae phage genome was sequenced in 2013 and the number of sequenced P. larvae phage genomes stands at 49 as of 2021; 13 of these were isolated at UNLV in 2013-2014. P. larvae phages have been isolated in Portugal, Germany, Spain, and ten states in the United States, from sources such as honeybee hive interiors, soil samples underneath healthy honeybee hives, P. larvae lysogens, and commercial beeswax products. We classified sequenced P. larvae phage genomes into four clusters and two singletons based on average nucleotide identity. There exists a large disparity in the size of these clusters with one cluster having 30 members, while the other three clusters have eight, seven, and two members. Genome size correlates with DNA-packaging strategy; the 41 phages using the cohesive ends (cos) packaging strategy have genomes in the 35-46 kbp range and the eight direct terminal repeat phages (DTR) have genomes in the 50-56 kbp range. One cluster is comprised of all DTR phages, while the cos phages are split among the remaining three clusters and two singletons. The structural and assembly proteins located at the front of the genome tend to be conserved among clusters, but regulatory and replication proteins located in the middle and rear of the genome are not conserved even within the same cluster. Identification of an integrase, excisionase, or Cro/CI in all sequenced P. larvae phage genomes indicates that all sequenced P. larvae phages are temperate. All phages lyse P. larvae through cleavage of the peptidoglycan cell wall by means of a conserved N-acetylmuramoyl-L-alanine amidase. The P. larvae phage amidases were classified into two clusters based on amino acid sequence identity, which correlates with DNA-packaging strategy. A first of its kind investigation into the presence of CRISPR spacer sequences in sequenced P. larvae and P. larvae phage genomes revealed 384 unique spacers in P. larvae strains. The distribution of CRISPR spacer sequences is uneven in the P. larvae strains, with one strain having over 150 spacers and three strains having fewer than 20. Of the 384 unique spacers, 18 are found as protospacers in the genomes of 49 currently sequenced P. larvae phages. One P. larvae strain does not have any protospacers found in phages, while another has eight. Protospacer distribution in the phages is uneven, with two phages having up to four protospacers, while a third of phages have none. The differential lysing ability of P. larvae phages is likely a confluence of amino acid substitutions within the N-acetylmuramoyl-L-alanine amidase, differences in non-conserved regions of the genome, and the presence of CRISPR spacer sequences in P. larvae genomes. Understanding the genomic landscape of P. larvae phages will offer insights for future phage therapy studies by elucidating the mechanisms responsible for the differential lysing ability of P. larvae phages.