Within a population of bacteria, the individual bacterial cells can perform different tasks. Despite being genetically identical, certain genes are more active in some subsets of the population than others. When analyzing gene activity at the individual cell level, researchers have now discovered something astounding: bacteria of the species Clostridium perfringens produce their pathogenic toxin primarily when there is a lack of food. However, if they have enough food available, genes that enable the breakdown of food are activated instead of the poison genes. The knowledge could lead to new strategies to prevent bacterial infections.
Clostridium perfringens is one of the most common causes of foodborne illness. If we eat food in which the bacteria have multiplied, we have to reckon with severe gastrointestinal problems after a few hours. Because Clostridium perfringens produces toxins that perforate the cells in our intestines and lead to severe diarrhea. The bacterium is also feared in industrial poultry farming. It causes $6 billion in damage annually and makes it difficult to go without antibiotics on poultry farms. However, not all C. perfringens bacteria produce toxins.
Genetically identical and yet different
Researchers led by Ryan McNulty from the IFF Health and Biosciences in Wilmington in the USA have now developed a method with which they can analyze at the level of individual bacterial cells which genes are active in the respective cell and shape its function. “Bacteria behave very differently than we previously thought,” says McNulty’s colleague Adam Rosenthal. “Even if we study a community of bacteria that are all genetically identical, they don’t all behave in the same way. We wanted to find out why.”
When a gene is read, the cell first creates a kind of blueprint called messenger RNA (mRNA). This contains the blueprint for the gene product to be produced. In order to find out which genes are active, it is useful to analyze a cell’s transcriptome, i.e. the RNA molecules produced in the cell. There are already well-established methods for large amounts of cells at the same time. However, until now it was hardly possible to determine the individual mRNA profile for thousands of cells. The new method called ProBac-seq, which McNulty and his team have developed, now makes it possible to separate the bacterial cells into tiny droplets, mark each one individually and then automatically analyze the transcriptome. In this way, it can be determined whether different genes are active in certain subgroups of a bacterial population than in others.
Transcriptome of thousands of single cells analyzed
“We sequenced the transcriptome of thousands of individual bacterial cells per experiment and detected several hundred transcripts per cell on average,” reports the research team. To validate their method, McNulty and his colleagues first used the well-studied bacterial species Bacillus subtilis and Escherichia coli as model organisms. “Using ProBac-seq, we were able to identify known cellular states and also discovered several previously unknown transcriptional states in which genes relevant to specific metabolic pathways and physiological states of the bacterium are expressed,” the authors report.
“Next, we wanted to find out whether we could identify different subpopulations in a real pathogen,” explains the team. To do this, they chose Clostridium perfringens, whose toxins are responsible, among other things, for severe intestinal diseases in humans and animals. The analysis with ProBac-seq showed that although all bacteria produce toxins to a certain extent, only 43 percent of the cells produced particularly large amounts of them – precisely those that were supplied with little nutrients. “This led us to speculate whether toxin production and the proportion of toxin-producing cells could be regulated by adding nutrients such as acetate,” the researchers said.
More nutrients – less poison
So they added sodium acetate to the bacterial culture and then re-examined the transcriptome. And indeed: “The addition of acetate significantly reduced the proportion of cells in the primary toxin-producing state from 43 percent to 30 percent,” reports the team. And the remaining 30 percent also produced less venom than without acetate. Overall, this significantly reduced the toxin content in the bacterial culture. “Our results with C. perfringens show that the disease-causing toxin is differentially expressed by a specialized subpopulation of cells and that providing growth conditions that favor alternative cell states can reduce the proportion of virulent cells in a genetically identical bacterial population,” the scientists summarize together.
Future studies should clarify whether there are similar mechanisms in other pathogenic bacteria, such as salmonella and Staphylococcus aureus. This could help to develop strategies against bacterial infections and circumvent antibiotic tolerance. “We anticipate that our method will be used extensively to understand how the external environment modulates the virulence of pathogens at the single-cell level,” the researchers said.
Source: Ryan McNulty (IFF Health and Biosciences, Wilmington, Delaware, USA) et al., Nature Microbiology, doi: 10.1038/s41564-023-01348-4