Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500

Ashish K Sharma,Stephen H Leppla,Andrei P Pomerantsev,Joseph Shiloach

doi:10.1038/s41598-018-34196-y

Abstract

Protective antigen (PA) of Bacillus anthracis is being considered as a vaccine candidate against anthrax and its production has been explored in several heterologous host systems. Since the systems tested introduced adverse issues such as inclusion body formation and endotoxin contamination, the production from B. anthracis is considered as a preferred method. The present study examines the effect of PA expression on the metabolism of B. anthracis producing strain, BH500, by comparing it with a control strain carrying an empty plasmid. The strains were grown in a bioreactor and RNA-seq analysis of the producing and non-producing strain was conducted. Among the observed differences, the strain expressing rPA had increased transcription of sigL, the gene encoding RNA polymerase σ54, sigB, the general stress transcription factor gene and its regulators rsbW and rsbV, as well as the global regulatory repressor ctsR. There were also decreased expression of intracellular heat stress related genes such as groL, groES, hslO, dnaJ, and dnaK and increased expression of extracellular chaperons csaA and prsA2. Also, major central metabolism genes belonging to TCA, glycolysis, PPP, and amino acids biosynthesis were up-regulated in the PA-producing strain during the lag phase and down-regulated in the log and late-log phases, which was associated with decreased specific growth rates. The information obtained from this study may guide genetic modification of B. anthracis to improve PA production.

Highlights

Protective antigen (PA) is 83-kDa protein, a component of anthrax exotoxin, which in addition to PA contains the lethal factor (LF) and edema factor (EF) proteins
In a study of production of an insulin-like growth factor I fusion protein (IGF-If) in E. coli, Choi et al found down regulation of prsA, a gene required for E. coli nucleotide and amino acid biosynthesis
Profiling showed that bottlenecks can develop in different pathways in E. coli depending on the type and behavior of the recombinant protein, e.g. interferon-β, xylanase, and GFP16

Summary

Introduction

Protective antigen (PA) is 83-kDa protein, a component of anthrax exotoxin, which in addition to PA contains the lethal factor (LF) and edema factor (EF) proteins. Protein production from these host systems can be associated with low productivity, inclusion body formation, contamination from lipopolysaccharide, etc.[4,5,6,7,8,9,10] Expressing this protein in a modified, nonpathogenic, B. anthracis host may offer an attractive strategy. The present study seeks to identify plausible bottlenecks restricting overexpression of PA protein by analyzing the whole genome transcriptional changes in producing and non-producing (control) recombinant BH500 strains grown in a bioreactor. The differences seen in essential pathways required for protein expression including: central carbon metabolism, amino acid biosynthesis, transcription, translation, folding and secretion, were evaluated to identify plausible bottlenecks. The genes identified provide targets for genetic engineering to increase the effectiveness of B. anthracis strains as production hosts[11]

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Conclusion