Humans are constantly exposed to different bacterial species that cause disease. Ideally, agents such as antibiotics would substantially protect against these infections. But what happens when these bacteria not only develop resistance against our beloved antibiotics but also establish the ability to create specialised resistant communities? Within these communities, bacteria are protected from chemical detergents, immunological responses, and antimicrobial drugs. These protective hubs have been deemed aggregates and biofilms, and they contribute to as much as 80% of bacterial persistent infections in humans. Hence, the mechanisms behind the formation of these threatening communities deserve special attention.

As scary as having community-forming pathogenic bacteria may sound, not much is known regarding the molecular mechanisms involved. One thing we do know however, is that aggregation and biofilm formation can be promoted by proteins located on the surface of bacteria, which belong to the autotransporter (AT) family. ATs are the largest group of secreted and outer-membrane proteins in Gram-negative bacteria. Within this family, a highly abundant member is Ag43, found in pathogenic strains such as enterohemorrhagic E. coli (EHEC) and uropathogenic E. coli (UPEC), which cause severe diarrhoeal diseases and UTIs, respectively.

Our research focussed on analysing Ag43 variants from three strains representative of EHEC and UPEC (Ag43^EDL933, Ag43^UTI89 and Ag43b). Our results have helped us understand how these proteins behave in different micro-environments, influencing bacterial ability to form the dreaded aggregates and biofilms and ultimately establish an infection.

In our quest to showcase the function of these three Ag43 variants at atomic detail, we required the use of various techniques. Our golden tool was X-ray crystallography, which provided information of the precise architecture of Ag43 homologues under study. To help us navigate the waters of this research, we used the previously characterised Ag43a homologue as reference. As expected, due to the high sequence identities amongst the Ag43 homologues, their structures showed strikingly similar L-shaped 3-stranded ß-helices. 

Combining the structural information with mutagenesis, as well as biophysical and cell-based assays we also revealed that Ag43 molecules self-associate via the long arm of the L-structure, while the short arm remains attached to the bacterial cell-surface. These “hand-shake”-like interactions between Ag43 molecules in neighbouring cells, bring bacteria together to promote aggregation and biofilm formation.

Although the overall Ag43 structures and self–association mechanisms all resembled each other, the regions involved in self-association display low sequence identities. These differing interfaces directly influence the number of interactions stabilising the Ag43-Ag43 associations (ie. different Ag43 homologues had different “hand-shake” strengths). Indeed, Ag43^EDL933 and the reference Ag43a have interfaces with the highest number of interactions, followed by Ag43b and Ag43^UTI89. This information is highly relevant, as Ag43 homologues showing high number of bonds in the self-adhesive interfaces also displayed more rapid formation of aggregates and biofilms.

So, what does all this tell us? For starters, what our work shows is that the specific amino acid sequences of the aggregation factors expressed by bacteria, directly influence the strength of the aggregates and biofilms formed. Bacteria are faced with a trade-off, whereby as aggregation is increased, more protection is afforded within the biofilm, but bacterial dissemination is reduced. By producing “weaker or stronger” aggregating factors, bacteria may optimise colonisation and persistence in different micro-environments.

Our research has established the molecular basis of how bacteria develop aggregates and biofilms. These results open new avenues that can be used in the search for novel ways to tackle these resilient bacterial communities. You can read all about our works with Ag43 and see more nice pictures of structures in our paper here.