Characterization of Pseudomonas aeruginosa mechanosensing through label-free imaging of type IV pili

Bacteria are ubiquitously found in all sorts of environment. They're found in the ocean, soil, or even in our guts or on our skin. Independently of their niche, they can transition from a planktonic state were they freely swim in an aqueous environment to a surface-associated state where they form multicellular communities known as biofilms. It is known that bacteria change their physiology when associated with surfaces. This is many times accompanied with a different transcriptional state compared to planktonic populations. Pseudomonas aeruginosa is an opportunistic pathogen often involved in nosocomial infections that can lead to chronic infection in immunocompromised patients. P. aeruginosa infections are often associated with a surface-associated biofilm lifestyle. In addition, it uses a host contact-dependent toxin secretion system during pathogenicity. It also possesses a surface-specific motility system known as twitching. These three examples are all physiological adaptation to surface contact. All these are a result of a change in gene expression upon surface contact.To power twitching motility, P. aeruginosa uses long and thin extracellular filaments called type IV pili (TFP). P. aeruginosa extends and retracts its TFP to pull itself forward on surfaces. Beyond their function in motility, TFP extension, attachment on a solid substrate and retraction are required for a surface-specific transcriptional response in a process called mechanosensing. A chemotaxis-like two component system called the Chp system also mediates this surface-dependent response. The activated Chp system upregulates transcription of virulence related genes including genes involved in TFP biogenesis. However, little is known about how the Chp system is activated by TFP, and how TFP respond to mechanosensing. The aim of this thesis is to characterize the mechanisms of mechanosensation in P. aeruginosa. In order to achieve this, one must first better characterize the mechanical input signal generated by TFP attachment events. This is an experimental challenge as TFP fibers cannot be imaged with conventional microscopes. In this thesis I present an approach to the investigation of P. aeruginosa mechanosensation by leveraging interferometric scattering microscopy (iSCAT) that I optimized for label-free detection of TFP during live cell imaging. I also show the power of iSCAT microscopy to detect and analyze TFP and their dynamic behavior on surfaces.By combining iSCAT to fluorescence microscopy, I could then track intracellular regulatory proteins and simultaneous TFP activity. Combining these measurements with single cell motility assays and protein localization, I was able to highlight signaling feedback loops between TFP and the Chp chemotaxis system that are critical to motility and mechanosensing. This feedback system allows for cell-polarization that directs twitching motility, which we term mechanotaxis.Finally, based on new biophysical and structural evidence, I propose a model for the mechanisms of mechanosensing by TFP and the Chp system. Here, my preliminary data show that the PilA monomers that are disassembled from TFP during retraction interact with the Chp chemosensor PilJ. Due to TFP attachment, the flux of PilA towards PilJ is different in liquid vs surfaces. I propose a mechanism wherein PilJ senses this flux imbalance.

Natural competence of the pathogen Acinetobacter baumannii: an elusive phenomenon

Nina Vesel

Natural competence for transformation is an important driver of horizontal DNA exchange between different organisms. This can result in accumulation of dangerous genetic features, such as antibiotic resistance genes, in a single organism. One example of an organism that frequently acquires antibiotic resistance genes is Acinetobacter baumannii, a Gram-negative opportunistic pathogen that has recently become problematic in hospital settings. Even though natural competence for transformation was demonstrated for some A. baumannii isolates, its competence regulon, the exact mechanism of DNA uptake, and the contribution of transformation to the acquisition of antibiotic resistance genes were largely unexplored at the beginning of this thesis. The aim of this thesis was therefore to better characterize optimal conditions of competence, the underlying competence regulon, and the necessary set of machinery proteins. Additionally, we aimed to identify the limiting factors that oppose transformation-mediated exchange of DNA in a small selection of strains. Here, we first addressed the general aspects of natural transformation, such as the competence window and the DNA uptake machinery. Our results showed that transformation is growth phase-dependent in A. baumannii and correlated with type IV pili (T4P) production. We demonstrated that T4P are essential for transformation and surface-associated motility but are only produced in a subfraction of the bacterial population. Furthermore, T4P production and assembly were under control of the PilSR two component system and the chemotaxis-like Pil-Chp system, respectively. These two regulatory systems were essential for competence development in A. baumannii, which is in contrast to what was observed for its close relative A. baylyi. We also investigated the reasons for non-transformability of certain A. baumannii isolates. We showed that comM interruption as well as the diversity of PilA protein sequences did not explain the strains' non-transformability. Instead, decreased expression of certain pilus genes was associated to the absence of transformability, which was also reflected in the protein level of the major component of T4P - PilA. Since the previously identified regulators could not explain the reduced transcript levels of the respective pilus genes, a transposon sequencing screen was performed to identify novel transformation-relevant genes. By comparison of transcriptional profiles of such genes between the transformable and non-transformable strains, we identified possible candidates that might explain non-transformability. Lastly, we explored whether the epigenome can influence A. baumannii's transformability. Our results showed that the source of transforming DNA has a significant effect on transformation of certain, yet not all tested strains. Specifically, for strain A118 the transformation levels were significantly decreased when non-self DNA was used as the donor DNA. Consequently, we identified a A118-specific restriction modification (RM) system that methylated specific DNA sequences in this strain and fostered the discrimination of self versus foreign DNA. Collectively, the findings of this thesis deciphered several important aspects of natural transformation in A. baumannii, which might ultimately help to better understand the spread of antibiotic resistance genes in this organism.

EPFL2022

Rhythmic and clock-dependent chromatin loops regulate circadian gene expression

Jérôme Mermet

In mammals the circadian clock drives daily behavioural and physiological changes that resonate with environmental cues, which can be observed, for example, in the intricate timing of rest during the night and activity during the day in humans. The circadian clock consists of a network of hierarchical clocks in which the central pacemaker is located in the suprachiasmatic nucleus, which is sensitive to external light and propagates time to the peripheral organ-based clocks. Within organs, clocks tick in each individual cell and are molecularly encoded in the genome as a network of dynamic feedback loops. By mediating the expression of the appropriate gene products at the correct moment of the day, the molecular clocks op-timize physiological functions of virtually every cell in our body, allowing us to synchronize with daily fluc-tuations in the environment. In mammalian cells the chromatin organization in the nucleus consists of a topological network, in which chromatin interactions between distal regulatory elements such as enhancers and target genes are essen-tial to regulate gene expression. Important questions remain: is the complex chromatin folding dynamic and if so on which genomic and temporal scales? In fact, dynamic rearrangements of the chromatin have been reported on multiple genomic and time scales, from the entire genome to enhancer-promoter con-tacts, across developmental transitions, cellular differentiation, and transcription cycles. With this per-spective in mind, the circadian oscillator provides a unique model to study the dynamics of genomic inter-actions in the context of fluctuating transcription. Here, we explored chromatin contacts of core-clock and clock-controlled gene promoters in the mouse. We aimed at evaluating the putative dynamics of chromatin contacts, at estimating their conservation across tissues, and at exploring the contribution of the circadian clock to this interaction network. To achieve this goal, we performed 4C-sequencing assays at two circadian time points in the liver and kidney of WT and clock-deficient animals. Overall, we observe that chromatin contacts are largely conserved across circadian time points and genetic backgrounds, and are tissue-specific. Our experiments reveal that the promoters of core-clock and clock-controlled genes contact distal genomic regions containing en-hancer chromatin signatures, suggesting a functional role of these distal sites in target gene regulation. Our data also suggest a clock-driven module of rhythmic genes that are colocalized in the nucleus. However, we also observe dynamic contacts between oscillating gene promoter and enhancer-like ge-nomic regions. In this thesis, we discuss in detail two examples of dynamic chromatin looping that are ob-served at the Cry1 and Gys2 loci. Our data reveal not only a dynamic coupling between the chromatin loop and the transcriptional state but also that the dynamic of chromatin looping depends on a functional clock. Furthermore, genome editing experiments using CRISPR-Cas9 tools reveal that the Cry1 distal site is essential for the transcriptional regulation of Cry1 and contributes to the circadian clock robustness both in-vivo and in cultured cells. These results demonstrate how local chromatin rearrangements take place in a 24-hour time-scale and set chromatin looping between gene promoters and distal regions as a new reg-ulatory layer for circadian gene expression.

EPFL2017

Natural competence of the pathogen Acinetobacter baumannii: an elusive phenomenon

Nina Vesel

EPFL2022

Rhythmic and clock-dependent chromatin loops regulate circadian gene expression

Jérôme Mermet

EPFL2017