Interplay between bacterial adhesion and antibiotic tolerance

Aggregation has been shown to play a key role when bacteria protect themselves from external stresses. Neisseria gonorrhoeae, a biofilm-forming pathogen, which is responsible for the second-most common sexually transmitted disease, gonorrhea, poses a critical public health concern due to its multidrug resistance. Tolerance, by means of prolonged survival times under high dose antibiotic treatment, promotes the emergence of resistance. However, the underlying mechanisms of tolerance remain unknown. Gonococci form aggregates by attractive interactions of single cells mediated by type IV pili (T4P). It is unclear how T4P control the physical properties of bacterial colonies and how these properties are linked to antibiotic tolerance. In this thesis, we investigated the effect of antibiotics on T4P dynamics and cell-to-cell interactions. To this end, we developed a fluorescence-based method for direct visualisation of T4P, as well as a laser tweezers-based approach to characterise T4P-mediated interaction dynamics under external stress conditions. Using a self-developed semi-automated image analysis pipeline, we found that, without stress, pili are produced at an impressively high rate of approximately 200 pili per minute. The application of external stress, including antibiotic treatment, reduced both the production rate and the dynamics of T4P. Optical tweezers experiments further revealed that attractive interactions between cells are modulated by antibiotic treatment. Specifically, an increase in binding and retraction probabilities leads to reduced colony fluidity, whereas a decrease in these parameters results in increased fluidity. We conclude that T4P dynamics and production rates are strongly affected by external stress and influence cell-to-cell interactions. The T4P filament is composed of subunits, the major pilin being PilE. The surface of the T4P is constantly varied as PilE undergoes antigenic variation, a mechanism also known to facilitate immune evasion. We investigated how antigenic variation of PilE affects bacterial lifestyle and antibiotic susceptibility. Image analysis of surface motility and transmission electron microscopy confirmed that all pilin variants support twitching motility and exhibit comparable levels of piliation. From structural predictions using AlphaFold, we predicted that different pilin variants have different stereochemistry, which affects cell-cell attraction. Indeed, double laser trap experiments confirmed that cell-to-cell interactions were altered. At the multicellular level, weakly interacting variants adopted a planktonic lifestyle, while strongly interacting variants formed aggregates. By analysing the survival fraction of bacteria when treated with antibiotics, we found that antibiotic tolerance to ceftriaxone and ciprofloxacin is significantly reduced for planktonic bacteria and that aggregating variants are protected from antibiotic treatment. In conclusion, the stereochemical properties of PilE modulate cell-cell interactions, thereby determining bacterial lifestyle and influencing antibiotic tolerance. We aimed to identify the molecular mechanisms that contribute to aggregation-mediated tolerance and hypothesised that genes upregulated by aggregation would protect bacteria from antibiotic treatment. Transcriptome analysis revealed differential gene expression of pilin variants exhibiting either a planktonic or an aggregating lifestyle. We identified a prominent upregulation of pro-phage genes and genes associated with a shift towards anaerobic respiration in aggregating strains, consistent with the reduced growth rate they exhibit compared to planktonic variants. To assess functional relevance in antibiotic tolerance, deletion of the most up-regulated genes was tested in relation to bacterial survival under antibiotic treatment with ceftriaxone and ciprofloxacin. By measuring the killing kinetics of the deletion strains in either a planktonic or an aggregating pilin variant background, five genes involved in antibiotic tolerance were identified. In summary, gonococcal aggregation leads to differential expression of genes involved in antibiotic tolerance. Finally, we investigated the transition from microcolonies to mature biofilms of N. gonorrhoeae. Flow chamber experiments revealed that late-stage colonies undergo eversion, characterized by a directed outward flow of cells from the centre to the periphery, transporting live and dead cells to the surface. Based on the hypothesis that oxygen depletion at the colony centre reduces T4P density and thus cohesion, we propose that this local weakening of cohesion is the trigger for colony eversion. To test this, we analysed the spatio-temporal dynamics of single cells during colony maturation and found that eversion is initiated by the emergence of an expanding shell of hyper-motile cells originating from the centre. We conclude that eversion is triggered by reduced cohesion in the colony core as a result of oxygen depletion towards the centre of the colony. To summarise, this study elucidates the interplay between T4P dynamics, antigenic variation and antibiotic treatment in shaping bacterial lifestyles and mechanical properties of N. gonorrhoeae colonies. In particular, it demonstrates that aggregation is a key determinant of antibiotic tolerance.