Prokaryotic toxin-antitoxin (TA) systems (also known as addiction modules), are ubiquitous genetic modules first discovered due to their role in stabilising vertical transmission of plasmids. Generally they are two-gene systems encoding a stable toxin (Tx) and an unstable antitoxin (ATx). Loss of the TA module leads to rapid ATx degradation and depletion, leaving the Tx free to interact with cellular targets and inhibit growth. For plasmid encoded TA systems, this leads to the death of plasmid free daughter cells, ensuring plasmid maintenance in a population, and gives rise to the term "addiction module". More recently, the expansion in microbial genome data has highlighted the prevalence and diversity of TA systems, and demonstrated that they are common features of many bacterial chromosomes. In addition, metagenomic surveys have pointed to the enrichment of some TA families in particular microbial ecosystems; a prime example from surveys of the human gut microbiome and RelBE TA family. Collectively, these observations indicate a wider role for TA modules in bacterial function, with numerous roles for TA systems now hypothesised. These include: i) Stabilisation of TA associated chromosomal DNA during vertical transmission; ii) Formation of "persister" cells resistant to environmental stresses, and; iii) Population level resistance to bacteriophage attack. Additionally, some Tx components have shown activity in eukaryotic cells, raising the potential for a role in prokaryote-eukaryote interaction. Here we undertook a systematic study of Type II TA systems, to provide a comprehensive assessment of their distribution and relative abundance, to confirm activity of prevalent TA systems, and to understand putative roles these may play in gut associated bacteria and the gut microbiome.
A comparative genomic and metagenomic analysis of 3919 bacterial chromosomes, 4580 plasmids, 711 bacteriophage genomes, and 781 metagenomes encompassing 16 distinct habitats was conducted using all known Type II TA systems present in the Toxin Antitoxin Database (~10,100 TA genes ~1:1 Tx:ATx). Of the 817 Type II TA system homologues found in human gut datasets, 686 were observed to have significantly higher relative abundance in the human gut microbiome over other microbial ecosystems. In parallel to these in silico findings, PCR and qPCR surveys of microbiomes from 65 stool samples obtained from healthy volunteers, as well as those with polyps or colorectal cancer, were undertaken. This demonstrated a higher ATx presence than Tx or complete module, however no differences in Tx copy number between health groups was seen.
To confirm the activity of the most abundant TA system homologues identified in sequence surveys, ORFs were amplified from gut metagenomic DNA, and individual Tx or ATx cloned under the control of inducible promoters. Induction of Tx expression under normal growth conditions resulted in bacterial growth inhibition, while live dead staining showed entry into a viable but non-cultivatable state, commensurate with TA function. Experiments simulating environmental stresses encountered during colonisation of the GI tract (starvation, low pH, bile), indicated that expression of these TA systems could increase cell survival when carbon or nitrogen availability was limited (starvation). Since antibiotics are also commonly encountered by gut associated-bacteria (both as residents of the GI tract and during colonisation of other body sties) a role for gut associated TA systems in facilitating survival during antibiotic exposure was also explored. This revealed an increased number of cells surviving two hours post-treatment with β-lactams when Tx genes were expressed, and in keeping with an impact on cell growth.
To test the hypothesis that TA systems may stabilize associated regions of DNA, the composition of gene neighbourhoods surrounding TA systems were also explored. ORFs surrounding TA system homologues identified in metagenomic and genomic datasets were identified using the Metagene annotator, and ORF functions predicted based on searches of the Clusters of Orthlogous Groups (COG) database. This revealed significant increases in ORFs with functions related to replication/recombination/repair and those with unknown functions. It also identified a decrease in the proportion of ORFs encoding functions such as carbohydrate and lipid transport and metabolism in regions surrounding TA systems, suggesting involvement with stabilization of mobile elements.
Finally, we explored the potential for gut associated TA systems to modulate phage-microbe, and host-microbe interactions. In the case of phage-host interactions, TA systems have previously been shown to function as mediators of phage resistance at the population level, by directing cells towards a dormant state which prevents phage replication, and permits a sub-set of cells to survive phage attack. Our findings indicated the potential for gut associated TA systems to provide some degree of protection during particular host-phage interactions, but specific modules did not provide universal protection against phage. In the case of host-microbe interaction, some Type II TA system Tx components have been shown to be functional in cultured eukaryotic cells, promoting apoptosis when introduced and expressed in these cell types. However, no studies to date have examined the potential for bacterially expressed TA systems to influence eukaryotic cell health in co-culture models. To investigate this, we assessed the impact of bacterial TA system expression on the health of the intestinal epithelial cell line Caco-2 in co-culture systems specifically focusing on cell apoptosis and necrosis whilst in the presence of Escherichia coli expressing p22-RelBE.
|Date of Award||2016|