How anthrax spores grow in cultured human tissues
Making conditions real
Most of what researchers know about anthrax comes from studying cancerous lung cells of both humans and rabbits because they are easy to grow in a lab. But cancer cells are very different from normal cells, which are referred to as primary cells.
For this study, PNNL researchers wanted to see if normal cells reacted differently. So, they carefully cultured primary rabbit lung cells on special inserts in petri dishes, coaxing them to form small pieces of 3-D lung tissue about the size of a quarter.
“The cells are fed with nutrients from below and we trick the top layer of cells into thinking they are at the air/liquid interface as they would be in a living lung,” said Josh Powell, a microbiologist at PNNL.
Researchers observed the top layer of cells producing sticky mucus, which traps the anthrax spores. This did not occur with cells completely submerged in the growth medium where the spores just float on top. This suggests that this mucus facilitates germination of the spores into bacteria.
“Byproducts secreted in the mucus by lung cells, in reaction to the anthrax, cause the spore to proliferate very quickly,” said Powell. “We don’t know what those byproducts are yet, but this is the first time it’s been shown that growth rate is impacted by these byproducts secreted by the lungs.”
Additional biochemical tests revealed that nutrients in the standard culture media provide an extra, unnatural fuel that makes spores germinate faster than would likely happen in the natural lung.
“These finding have implications for how we study pathogens within in vitro cell systems,” said Powell. “Understanding the impacts of the methodology ensures we get the best data we can from both species on specific rates of spore intake or dose, clearance, germination and proliferation in a lab setting.”
Researchers hope to reproduce this study using the more virulent strain at DHS’s National Biodefense Analysis and Countermeasures Center in Frederick, Maryland, rather than the similar but milder Sterne strain used in this study, which is virtually unable to cause illness in people or animals.
Predicting to protect
The release notes that in the next phase of the project, researchers will put this experimental data into a computational model to more accurately predict outcomes of anthrax exposure. For instance, a model based on primary cell data may calculate how much time doctors have to initiate treatment, how many spores are likely needed to cause disease or mortality in humans, or be able to determine if there is a “safe” level for exposure or a required level of cleanup of a contaminated area.
Once the models are refined with data from the latest experiments, those numbers will be checked against animal data to see if they are indeed predicting outcomes accurately. The models could also potentially speed future drug design.
Researchers hope these fundamental findings and models can be applied to other diseases related to inhaled pathogens, such as the flu or SARS coronavirus. “This is an investment that may eventually help officials triage, treat and influence drug discovery for these lung illnesses,” said Powell.
The Department of Homeland Security Science and Technology Directorate funded this research. All images were acquired with a specialized confocal microscope at EMSL, the Environmental Molecular Sciences Laboratory, a DOE Office of Science user facility at PNNL.
— Read more in Joshua D. Powell et al., “Bacillus anthracis spores germinate extracellularly at air-liquid-interface in an in vitro lung model under serum-free conditions,” Journal of Applied Microbiology (accepted manuscript, 15 June 2015)
