Proteomic Characterization of Bacteriophage Peptides from the Mastitis Producer Staphylococcus aureus by LC-ESI-MS/MS and the Bacteriophage Phylogenomic Analysis

Abstract

The present work describes LC-ESI-MS/MS MS (liquid chromatography-electrospray ionization-tandem mass spectrometry) analyses of tryptic digestion peptides from phages that infect mastitis-causing Staphylococcus aureus isolated from dairy products. A total of 1933 nonredundant peptides belonging to 1282 proteins were identified and analyzed. Among them, 79 staphylococcal peptides from phages were confirmed. These peptides belong to proteins such as phage repressors, structural phage proteins, uncharacterized phage proteins and complement inhibitors. Moreover, eighteen of the phage origin peptides found were specific to S. aureus strains. These diagnostic peptides could be useful for the identification and characterization of S. aureus strains that cause mastitis. Furthermore, a study of bacteriophage phylogeny and the relationship among the identified phage peptides and the bacteria they infect was also performed. The results show the specific peptides that are present in closely related phages and the existing links between bacteriophage phylogeny and the respective Staphylococcus spp. infected.

Keywords: LC-ESI-MS/MS; mass spectrometry; pathogen detection; phage peptide biomarker; proteomics.

Figure 1

Comparative representation of different types of phage proteins identified in this study for the different strains (represented by different colors). The number of each type of protein is shown in parentheses.

Figure 2

Phylogenomic tree generated by the Virus Classification and Tree Building Online Resource (VICTOR) using the complete genomic sequences of the determined Staphylococcus spp. phages. The access numbers of the determined phage genomes are shown in Table S2 in Supplemental Data 2. Genomes of the lambda (NC_001416.1), T4 (NC_000866.4) and T7 (NC_001604.1) phages were added for comparison purposes. The VICTOR phylogenetic tree construction was based on an intergenic distance analysis with the GBDP tool (Genome BLAST Distance Phylogeny). The significance of each branch is indicated by a pseudo-bootstrap value calculated as a percentage for 1000 subsets. Bar, 20 nt (nucleotides) substitutions per 100 nt. Clusters are represented by different colors: light blue, cluster A, red, cluster A.1, purple, cluster A.2, light green, cluster B, yellow, cluster B.1, pink, cluster B.2, black, cluster C and orange, cluster D. Specific cluster peptides are represented by different color forms: , yellow-filled diamond IQQLADYFNVPK (cluster A-specific), , brown-filled diamond HAGYVRC*KLF (cluster A-specific), , black-outlined diamond IYDRNSDTLDGLPVVNLK (cluster A.1-specific), , red=outlined diamond AVAELLKEINR (cluster A.2-specific), , pink-filled diamond KSNVEAFSNAVK (cluster A.1), , gray-filled diamond QKNVLNYANEQLDEQNKV (cluster A.1), , brown-outlined diamond MPVYKDGNTGKWYFSI (cluster A-specific), , dark gray-filled diamond KLYIIEEYVKQGM (cluster A.1-specific), , purple-outlined diamond EVPNEPDYIVIDVC*EDYSASK (cluster A.1-specific), , orange-filled diamond AYINITGLGFAK (cluster B.1-specific), , yellow-outlined diamond TSIELITGFTK (cluster B.2-specific), , red-filled diamond VSYTLDDDDFITDVETAK (cluster D-specific), , green-filled diamond LLHALPTGNDSGGDKLLPK (cluster D-specific), , black-filled diamond RVSYTLDDDDFITDVETAKELKL (cluster D-specific), , purple-filled diamond LYVGVFNPEATK (cluster D-specific, , blue-filled diamond ELAEAIGVSQPTVSNWIQQTK (cluster D-specific); , light green-filled diamond VLEMIFLGEDPK (cluster D-specific), , orange-outlined diamond KAMIKASPK (cluster D-specific) and , gray-outlined diamond GMPTGTNVYAVKGGIADK (cluster D-specific).