Laboratory strains of Bacillus anthracis exhibit pervasive alteration in expression of proteins related to sporulation under laboratory conditions relative to genetically related wild strains
December
Laboratory strains of Bacillus anthracis exhibit pervasive alteration in expression of proteins related to sporulation under laboratory conditions relative to genetically related wild strains
Owen P. LeiserID 0 1
Jason K. Blackburn 1
Ted L. Hadfield 1
Helen W. Kreuzer 0 1
David S. Wunschel 0 1
Cindy J. Bruckner-Lea 0 1
0 Chemical and Biological Signature Science, Pacific Northwest National Laboratory, Richland, Washington, United States of America, 2 Emerging Pathogens Institute, University of Florida, Gainesville, Florida, United States of America, 3 Spatial Epidemiology & Ecology Research Laboratory, Department of Geography, University of Florida , Gainesville, Florida , United States of America
1 Editor: Adam Driks, Loyola University Chicago , UNITED STATES
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Funding: This work was supported by internal
laboratory directed research and development
funding through PNNL to OPL, HWK, CJBL, and
DSW in collaboration with JKB and TLH. The
funder had no role in study design, data collection
The spore forming pathogen Bacillus anthracis is the etiologic agent of anthrax in humans
and animals. It cycles through infected hosts as vegetative cells and is eventually introduced
into the environment where it generates an endospore resistant to many harsh conditions.
The endospores are subsequently taken up by another host to begin the next cycle.
Outbreaks of anthrax occur regularly worldwide in wildlife and livestock, and the potential for
human infection exists whenever humans encounter infected animals. It is also possible to
encounter intentional releases of anthrax spores, as was the case in October 2001.
Consequently, it is important to be able to rapidly establish the provenance of infectious strains of
B. anthracis. Here, we compare protein expression in seven low-passage wild isolates and
four laboratory strains of B. anthracis grown under identical conditions using LC-MS/MS
proteomic analysis. Of the 1,023 total identified proteins, 96 had significant abundance
differences between wild and laboratory strains. Of those, 28 proteins directly related to
sporulation were upregulated in wild isolates, with expression driven by Spo0A, CodY, and AbrB/
ScoC. In addition, we observed evidence of changes in cell division and fatty acid
biosynthesis between the two classes of strains, despite being grown under identical experimental
conditions. These results suggest wild B. anthracis cells are more highly tuned to sporulate
than their laboratory cousins, and this difference should be exploited as a method to
differentiate between laboratory and low passage wild strains isolated during an anthrax
outbreak. This knowledge should distinguish between intentional releases and exposure to
strains in nature, providing a basis for the type of response by public health officials and
investigators.
and analysis, decision to publish, or preparation of
the manuscript.
Introduction
Bacteria growing in the laboratory experience dramatically different selective pressures than
those found in the environment. Bacillus anthracis cells respond to conditions outside of
mammalian hosts by forming a metabolically dormant endospore, capable of surviving
extended periods of harsh conditions [
1
]. Cells must overcome interspecies competition and
nutrient-limiting conditions to infect new hosts. In contrast to growth in the environment,
growth conditions in the laboratory are often stable, with abundant nutrients?conditions
tailored for optimum growth.
Intuitively, adaptation to different selective pressures between laboratory and
environmental conditions will result in measurable genotypic or phenotypic changes. Indeed, long-term
evolution has been studied extensively in an ongoing experiment in Escherichia coli [
2?5
], in
which cultures have been maintained for over 60,000 generations with pervasive genomic and
phenotypic changes observed. Additionally, Mikkola and Kurland [6], Eydallin et al. [
7
] and
Saxer et al. [
8
] examined genomic signatures of adaptation of wild E. coli to laboratory
conditions. However, far less is known about the mechanisms of wild pathogen adaptation to
laboratory conditions: Sjo?din et al. [
9
] investigated naturally occurring and laboratory strains of
Francisella tularensis using whole-genome sequencing, and Leiser et al. [
10
] investigated the
proteomic and genomic indicators of wild Y. pestis adaptation to laboratory conditions. Sjo?din
et al. examined very closely related strains of F. tularensis [
9
], and the laboratory-adapted
strains of Y. pestis examined by Leiser et al. [
10
] were direct descendants of the respective
starting wild strains. Systemic differences in gene/protein expression between wild and
laboratoryadapted strains can be elucidated using genetically similar (same clade) but distinct (wild type
or laboratory adapted) strains.
Previous work in our laboratory demonstrated the utility of proteomics to study
mechanisms of Y. pestis adaptation to laborato (...truncated)