|Fagerquist, Clifton - Keith|
Submitted to: Meeting Abstract
Publication Type: Proceedings
Publication Acceptance Date: 6/2/2015
Publication Date: 8/1/2015
Citation: Fagerquist, C.K., Zaragoza, W.J. 2015. Enhanced detection and identification of Shiga toxin 1 and 2 from pathogenic bacteria by MALDI-TOF-TOF-MS/MS-PSD and top-down proteomic analysis. ASMS201525491.3685VER.1.pdf.
Technical Abstract: Shiga toxin producing Escherichia coli (STEC) represent a continuing threat to the Nation’s food supply and public health. Shiga toxin genes (stx) are encoded in lambda-like bacteriophages whose genome is inserted into the bacterial DNA. Environmental stress can trigger bacteriophage replication and Stx production. For example, DNA damaging antibiotics can trigger the bacterial SOS response leading to expression of bacteriophage-encoded proteins including Stx. Release of bacteriophage and toxin from the bacterial cell is caused by expression of bacteriophage-encoded lytic proteins. Previous sample preparation for MALDI-TOF-TOF analysis in our lab utilized mechanical cell lysis by bead-beating to release Stx. However, there might be an analytical advantage to relying exclusively on antibiotic-induced lysis (AIL) for Stx release, detection and identification. We observed a distinct enhancement of Stx1 and Stx2 signal in certain STEC strains when using only antibiotic-induced cell lysis (AIL). For example, E. coli O157:H7 strain EDL933 is an important clinical STEC isolate that has stx1a and stx2a genes. The stx1a gene is located in an incomplete prophage (cryptic phage) genome whereas the stx2a gene is located in a complete bacteriophage genome. Our previous experiments on this strain utilized mechanical lysis in which we detected and identified the highly abundant B-subunit of Stx2a but did not detect the much less abundant B-subunit of Stx1a. However, using AIL, we detected the B-subunits of both Stx1a and Stx2a at m/z 7688 and 7817, respectively. Stx1a is ~100-fold less abundant than Stx2a, however, the Stx1a ion signal was sufficient for MS/MS-PSD and top-down proteomic identification. We concluded that AIL enhances Stx (and other bacteriophage-encoded proteins) by reducing the number of bacterial proteins from non-induced bacterial cells. Another important clinical STEC is E. coli O91:H21 strain B2F1 that has two copies of a stx2d gene: one in a complete bacteriophage genome and the other in a cryptic phage genome. Previous experiments utilizing mechanical lysis resulted in detection of the B-subunit of Stx2d, but the weakness of the precursor ion signal did not allow for MS/MS-PSD. However, AIL of this strain allowed both MS detection of the B-subunit and MS/MS-PSD top-down identification. E. coli O26:H11 strain ECRC # 05.2217 is a clinical STEC isolate that has a single stx1a gene. AIL of this strain allowed detection of the B-subunit of Stx1a and its top-down proteomic identification by MS/MS-PSD. We observed that the advantages of AIL were strain dependent, and enhanced detection of Stx was not observed for every STEC strain. However, in some strains, AIL showed significant advantages over mechanical lysis.