Advanced Proteomics as a Powerful Tool for Studying Toxins of Human Bacterial Pathogens

Abstract

Exotoxins contribute to the infectious processes of many bacterial pathogens, mainly by causing host tissue damages. The production of exotoxins varies according to the bacterial species. Recent advances in proteomics revealed that pathogenic bacteria are capable of simultaneously producing more than a dozen exotoxins. Interestingly, these toxins may be subject to post-transcriptional modifications in response to environmental conditions. In this review, we give an outline of different bacterial exotoxins and their mechanism of action. We also report how proteomics contributed to immense progress in the study of toxinogenic potential of pathogenic bacteria over the last two decades.

Keywords: B. cereus; bacterial toxins; human pathogens; proteomics.

Figure 1

Genetic organization of hbl and nhe operons in B. cereus and schematic representation of the formation of Hbl and Nhe PFTs. Hbl operon comprises three ORFs, hblCDA that encode Hbl-L2, L1, and B proteins, respectively. Nhe operon comprises three ORFs, nheABC, that encode NheA, NheB, and NheC proteins, respectively. All the Hbl and Nhe components are synthesized as precursors with an N-terminal Sec pathway signal peptide (red circle). Transport through the bacterial membrane is accompanied by the release of both peptide signal and mature polypeptide in the extracellular media. The three mature components of both Hbl and Nhe bind sequentially to form PFTs in the plasma membrane of target eukaryotic cells. The HblB locus that encodes Hbl B’ is also shown. Its role in Hbl-PFT formation is currently unknown.

Figure 2

Examples of proteomics-based strategies to decipher the toxigenic profile of a bacterial pathogen. Pathogens are grown in regulated batch cultures that mimic the conditions encountered in the host. Exoproteins are collected by centrifugation of the culture medium and then filtration (0.22 µm) of the resulting supernatant. Exoproteins are precipitated using trichloroacetic acid (TCA) or collected by other methods. In two-dimensional (2D) gel–based proteomics, exoproteins are resolved by 2D gel electrophoresis. Exoproteins of interest are excised from the gels and digested using trypsin. Tryptic peptides are analyzed on a Matrix Assisted Laser Desorption Ionisation-Time of Flight (MALDI-TOF) mass spectrometer. Protein identification relies on the comparison of the measured mass of the tryptic peptides with the predicted masses of tryptic peptides from database protein sequences. This approach is quite time consuming as mass spectrometry measurement should be done on each protein spot. In shotgun proteomics, the whole proteome is collected as a single band from a one-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE) gel, which is treated and in-gel proteolyzed with trypsin. Alternatively, the proteins may be in-solution proteolyzed in a gel-free approach. The resulting peptide mixture is injected into a reverse phase chromatography column coupled to a high-resolution mass spectrometer. The recorded tandem mass (MS/MS) spectra are processed against a protein sequence database using a search engine such as Mascot Daemon algorithm (Matrix Science). Exotoxin semi-quantification is simply but reliably evaluated by MS/MS spectral counts.

Figure 3

Relative abundances of B. cereus ATCC 14579 proteins in culture supernatant. The relative abundances were determined by averaging the Normalized Spectral Abundance Factor (NSAF) values of three biological samples harvested at the early exponential growth phase [65]. The diagrams represent the average abundance of (A) each functional protein group and (B) each protein belonging to the toxin group. CytK (BC1110): Cytotoxin K; NheA (BC1809), Non-hemolytic enterotoxin, lytic component A; NheB (BC1810): Non-hemolytic enterotoxin, lytic component B; NheC (BC1811): non-hemolytic enterotoxin, component C; HblB’ (BC3101): Hemolysin BL, putative binding component B′; HblB (BC3102): Hemolysin BL, binding component B; HblL1 (BC3103): Hemolysin BL, lytic component L1; HblL2 (BC3104): Hemolysin BL, lytic component L2; HlyII (BC3523): Hemolysin II; HlyI (BC5101): Cereolysin; EntA (BC5239): enterotoxin-like; EntB (BC2952): enterotoxin-like; EntC (BC0813): enterotoxin-like; EntD (BC3716): enterotoxin-like.

Figure 4

Strategy for phospho- and acetyl-proteomic analysis. The strategy for Post-Translational Modification (PTM) proteomics of bacterial proteins involves protein extraction, trypsin digestion of proteins, enrichment of PTM peptides using an appropriate method (here four methods are indicated), nano LC MS/MS analysis of the enriched PTM peptides, peptide identification, mapping PTM sites, and quantification. The most common methods for phosphopeptide enrichment and acetylpeptide enrichment are TiO2 chromatography and immunoenrichment, respectively. High-resolution tandem mass spectrometry is the most appropriate detection method as the site of modification can be delineated with precision.