Proteomic analysis of four Clostridium botulinum strains identifies proteins that link biological responses to proteomic signatures

Abstract

Microorganisms alter gene and protein expression in response to environmental conditions to adapt and survive. Whereas the genetic composition of a microbe represents an organism’s biological potential, the proteins expressed provide a functional readout of the organism’s response to the environment. Understanding protein expression patterns in response to specific environmental conditions furthers fundamental knowledge about a microbe, which can be especially useful for understudied organisms such as Clostridium botulinum examined herein. In addition, protein expression patterns that reproducibly occur in certain growth conditions hold potential in fields such as microbial forensics, in which determination of conditions in which an unknown possible biothreat sample had been grown may be important. To investigate the identity and reproducibility of protein profile patterns for varied strains, we defined the proteomic profiles of four Group I strains of Clostridium botulinum, a Category A biothreat agent and the organism responsible for the production of the botulinum neurotoxin (BoNT), in two different culture media grown for five days. The four C. botulinum strains produced one of three neurotoxins (BoNT/A, /B, or /F), and their protein profiles were compared to that of a fifth non-toxigenic strain of C. sporogenes. These strains each had DNA sequences available to assist in accurate protein identification. Differing culture growth phase, bacterial strain, and growth medium resulted in reproducible protein profiles, which were used to calculate relative protein abundance ratios as an internally normalized metric of microbial growth in varying conditions. The resulting protein profiles provide functional information about how four Group I C. botulinum strains and a C. sporogenes strain respond to the culture environment during growth and explores the feasibility of using these proteins to characterize unknown samples.

Proteomic Signatures of Antimicrobial Resistance in Yersinia pestis and Francisella tularensis

“…Proteomic results demonstrated that the effect of growth phase was significant on protein expression in all three Y. pestis and F. tularensis strain pairs examined, a phenomenon that also has been observed in other species ( 38 ). Figure 1 shows Principal Component Analyses (PCA) of all proteomics data from Y. pestis strain pair Yp4181 (AMS) and Yp4003 (AMR, +/– antibiotics; Figures 1A,D ), F. tularensis strain pair FSC013 (AMS) and FSC016 (AMR, +/– antibiotics; Figures 1B,E ), and F. tularensis strain pair FSC201 (AMS) and FSC232 (AMR, +/– antibiotics; Figures 1C,F ).…”

Methods used in Microbial Forensics and Epidemiological Investigations for Stronger Health Systems

“…For example, Deatherage Kaiser et al. [ 41 ] studied the reproducibility of bacterial protein profile patterns using Clostridium botulinum as a study system. The effect of bacterial strain, culture medium, and the growth phase on protein expression profiles was determined using LC and TMS.…”

Immunoproteomic analysis of Clostridium botulinum type B secretome for identification of immunogenic proteins against botulism

“…Culture filtrates were concentrated using 10 kDa cutoff membranes (Millipore, USA) and precipitated using 10% Ice-cold trichloroacetic acid (Sigma USA) after that incubated on ice for 3 h. Precipitate were centrifuged at 8000× g for 30 min at 4 °C and washed the pellet three times with cold acetone and left the room for air dried (Hirose et al 2000 ). Further, the pellets were resuspended in protein solubilization buffer (8 M urea, 2% CHAPS, and 2% carrier ampholytes pH 4–7) (Deatherage Kaiser et al 2018 ). The resultant proteins were cleaned using protein clean up kit (Bio-Rad, USA).…”