Botulinum neurotoxins (BoNTs) are very potent toxins and category A biological threat agents. BoNT serotypes /C1 and /D affect birds and mammals and can be potentially lethal to humans. We have previously described the usefulness of the Endopep-MS method to detect the activity of BoNT A through G. This report was followed by the application of the method to clinical samples. The activity of the BoNT serotypes associated with human disease (/A, /B, /E, and /F) was successfully detected. However, BoNT/C and /D require different conditions for fast substrate cleavage, and a comprehensive description of a method to study BoNT/C and /D has not yet been reported. This work describes a new, optimized version of the Endopep-MS method to detect BoNTs /C1 and /DC either spiked directly in 20 mL of reaction buffer or spiked in a larger volume of buffer and further extracted using antibody-coated magnetic beads. It was found that the incubation temperature at 42 1C was more effective for both toxin serotypes, but each toxin serotype has an optimum cleavage pH. Additionally, we describe for the first time a proteomics study using a fast trypsin digestion method and label-free quantification of these toxin serotypes.

Matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometry (MS) is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI) tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA) which combines with lethal factor (LF) and edema factor (EF), forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS/MS. Pre-concentration/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase.

Bacillus cereus isolates have been described harboring Bacillus anthracis toxin genes, most notably B. cereus G9241, and capable of causing severe and fatal pneumonias. This report describes the characterization of a B. cereus isolate, BcFL2013, associated with a naturally occurring cutaneous lesion resembling an anthrax eschar. Similar to G9241, BcFL2013 is positive for the B. anthracis pXO1 toxin genes, has a multi-locus sequence type of 78, and a pagA sequence type of 9. Whole genome sequencing confirms the similarity to G9241. In addition to the chromosome having an average nucleotide identity of 99.98% when compared to G9241, BcFL2013 harbors three plasmids with varying homology to the G9241 plasmids (pBCXO1, pBC210 and pBFH_1). This is also the first report to include serologic testing of patient specimens associated with this type of B. cereus infection which resulted in the detection of anthrax lethal factor toxemia, a quantifiable serum antibody response to protective antigen (PA), and lethal toxin neutralization activity.