Berenike Maier | Paul Higgins - B 06

According to WHO, gonorrhea is currently the second most common STI worldwide. Vaccination is not available and the probability of failure of treatment with antibiotics is rising rapidly. Some aspects of (multi-) drug resistance are understood at the genetic level, but the effect of biofilm formation and its structure on efficiency of treatment remains poorly understood. This project aims at elucidating potential mechanisms of antibiotic tolerance in gonococcal biofilms.

Communities of bacterial cells can live together as a biofilm. This allows the bacteria to hide from external stresses including antibiotic treatment. The causative agent of gonorrhea, Neisseria gonorrhoeae, forms biofilms, but whether and how biofilm formation favors antibiotic tolerance remains largely unknown. We hypothesize that heterogeneous environments within biofilms induce different physiological states that cause antibiotic tolerance. In this project, we aim at deciphering molecular mechanisms causing tolerance by correlating structure of early biofilms with putative factors enabling tolerance. 1) We will develop time-resolved single cell methods for measuring growth and death rates and for reporting cellular state. Combining confocal microscopy with advanced image analysis, we aim at establishing lineage tracking, and characterization of cellular physiology. 2) We aim at defining factors that confer antibiotic tolerance. Antibiotic tolerance is usually associated with changes in cellular physiology. We will correlate death dynamics during drug treatment with these factors. 3) We will link cellular state and antibiotic tolerance to biofilm structure. We will build on previous biophysical projects and on a collection of clinical isolates with different biofilm structures to address the correlation between biofilm structure and antibiotic tolerance.

The major goal of the project will be to find out whether and how antibiotic tolerance develops in gonococcal biofilms. Specifically, we will;

1. develop methods for lineage tracking and reporting cellular state at the single cell level with temporal resolution
2. correlate death dynamics during antibiotic treatment with cellular physiology
3. characterize variability of biofilm structure across isolates and its relation to antibiotic tolerance

Bacterial type 4 pili govern colony structure and dynamics

The bacterial type 4 pilus performs so many different functions that it has been termed the bacterial "Swiss army knife". It is an extracellular polymer involved in adhesion to host cells and other surfaces, motility and force generation, colony formation, transformation and electron transport [1, 2]. We showed that the pilus may be used as a tool for controlling structure, dynamics, and materials properties of gonococcal biofilms.

We developed tools that allow linking molecular interaction kinetics of pili with structure and motility of gonococcal colonies [3](Fig. 1). The pilus polymer elongates and retracts continuously. When the pilus of one cell binds to a pilus of another cell and retracts, it generates a mechanical force. As a consequence, one bacterium attracts another bacterium until eventually the pilus-pilus bond ruptures. The attachment-detachment dynamics enable a tug-of-war within bacterial colonies generating high mobility of bacteria [3, 4]. These dynamics strongly depend on the interaction forces between the pili. We developed a method based on laser tweezers that enables us to measure the pilus-mediated interaction force between pairs of bacteria. We showed that pilus-mediated interactions between gonococci can be tuned by pilus density [5], pilin post-translational modification [6], and pilus retraction activity [3]. 

We link pilus-mediated interactions to colony architecture, dynamics, and bacterial fitness. Specifically, we found that different pilin post-translational modifications have severe effects on the viscosity of microcolonies [5, 6]. When different gonococcal strains are mixed, they sort themselves with respect to differential interaction forces. Remarkably, the colony morphologies can be predicted by the differential strength of adhesion hypothesis initially formulated to understand cell sorting during early embryonic development, suggesting similar physical principles underlying cell sorting. Cell sorting has important consequences for bacterial fitness. Cells residing close to the colony surface have best access to nutrients and experience no steric hindrance during cell division [6]. On the other hand, bacteria centrally located within the biofilm are likely to be protected from external stress including antibiotics. Pilin phase and antigenic variation generate a standing variation of pilin sequences with different interaction strengths that cause cell sorting. 

We propose that altering the local physical interactions within biofilms through phase and antigenic variation is an important and possibly common contributor to the dynamics of biofilm growth, dispersion and adaptation. This hypothesis will be addressed using a collection of clinical isolates. The isolates were collected from patients with symptoms of urethritis between November 2015 and May 2019 (Fig. 2). All isolates have had their susceptibility to penicillin, ciprofloxacin, tetracycline, azithromycin and ceftriaxone determined at the Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne. All isolates have had their genomes sequenced, resistome identified, and been genotyped to determine their molecular epidemiology.

1.  Craig, L., K.T. Forest, and B. Maier, Type IV pili: dynamics, biophysics and functional consequences. Nat Rev Microbiol, 2019.
2. Maier, B. and G.C.L. Wong, How Bacteria Use Type IV Pili Machinery on Surfaces. Trends Microbiol, 2015. 23(12): p. 775-788.
3. Welker, A., et al., Molecular Motors Govern Liquidlike Ordering and Fusion Dynamics of Bacterial Colonies. Phys Rev Lett, 2018. 121(11): p. 118102.
4. Marathe, R., et al., Bacterial twitching motility is coordinated by a two-dimensional tug-of-war with directional memory.Nat Commun, 2014. 5: p. 3759.
5. Oldewurtel, E.R., et al., Differential interaction forces govern bacterial sorting in early biofilms. Elife, 2015. 4.
6. Zollner, R., et al., Phase and antigenic variation govern competition dynamics through positioning in bacterial colonies. Sci Rep, 2017. 7(1): p. 12151.