How do pathogenic Gram-negative bacteria successfully adhere to host surfaces under hydrodynamic flow, and which key targets in the adhesion process should new drugs aim for?
Final Report Abstract
Multidrug-resistant bacterial infections are increasing at a high rate in both developing and developed countries. To tackle the problem of multidrug-resistant bacterial pathogens, we need to develop new effective methods, substances, and materials that can disarm pathogenic bacteria and prevent them from causing infections. However, to accomplish this goal we first need to find new targets in bacteria to approach and new strategies to embrace. Such discovery and development require a strengthening of our knowledge of bacterial infection processes from a variety of viewpoints. The aim of my proposed project was to focus on elucidating the bacterial adhesion mechanism, since adhesion of bacterial pathogens to host tissue is a prerequisite for infection. To infect a host, bacteria have developed smart and unique ways to adapt to their environment. For example, enterotoxigenic Escherichia coli (ETEC) which is an infectious agent that causes severe diarrhea, affecting 200 million humans per year. In their natural environment, the intestine, adhered ETECs are in general exposed to strong shear forces imposed by luminal fluid flows generated by peristaltic motion of the intestines. These fluidic flows cause drag forces and fluid velocities, sometimes of considerable magnitude, by which bacteria must sustain in order not to be flushed away. To survive in this tough environment, ETEC express 1-2 µm long filaments on their surface so called pili or fimbriae that establish the first contact to the host and act as active dampers against shear forces. The aim of my project was to investigate how bacterial infections can be disarmed if the adhesive abilities of pathogenic bacteria are attacked or blocked, e.g. by changing the biomechanical properties of the pili. To realize this aim using microfluidic and optical tweezers assisted dynamic force spectroscopy measurements, my coworkers in Umeå and I first developed a set of reliable and robust tracking algorithm to detect bacteria in microfluidic environments. These methods are based on digital in-line holographic microscopy (DIHM). DIHM is a noninvasive, quantitative phase contrast imaging technique for high resolution live bacteria observation and analysis. By acquiring holograms (in this case 2D images) of bacteria, their xyz-positions can be determined with high accuracy even if the bacteria are not in the focal plane of the microscope. To create a hologram, non-scattered light from the source interferes with scattered light from the object, requiring a monochromatic coherent light source. Usually, lasers are a good choice for such applications, but for illumination purposes their long coherence length is of disadvantage since they create speckle noise. To overcome this issue, we proposed a simple guide to build a cheap and robust speckle-free laser illumination setup. The centerpiece of the setup is a rotating ground glass diffuser mounted on a stepper motor and one objective to collect scattered light behind the diffuser. Using this setup, the normalized speckle contrast is still below 1 % even at frame rates up to 15 000 Hz, providing specklefree images with excellent signal-to-noise ratio. To investigate surface-attached bacteria by mimicking their natural environment, we designed biocompatible micro-fluidic flow chambers with predictable shape by embedding a 3D printed water-soluble channel scaffold in polydimethylsiloxane (PDMS) using a regular fused deposition modeling (FDM) 3D printer. Our proposed protocols facilitate an easy, fast and adaptable production of micro-fluidic channel designs that are cost-effective, biocompatible and air-permeable, do not require specialized training and can be used for a variety of cell and bacterial assays. To perform force measurements on pili, we developed a compact, stable and easy to align optical tweezers setup by using a simple 3-lens Cooke-Triplet design. After developing all required experimental techniques, I investigated the biochemical and adhesive properties of pili using optical tweezers experiments. For that purpose, I measured the biomechanical response of the F-pilus and YEH fimbriae. It turned out, that the required force to unfold the shaft subunits depend on the function of the respective pilus. For a pilus used to establish the initial contact to the host cell under strong microfluidic flows (e.g. YEH fimbriae), forces to unfold the shaft are significantly higher compared to a pilus that is only used to transfer genes from one individuum to another (e.g. F-pilus). In summary, the developed methods and algorithm in combination my results on biomechanical properties of pili will provide important insights into the binding mechanism of pathogenic bacteria to host cells. This specific knowledge might in turn be used to develop new or further develop existing anti-bacterial drugs.
Publications
- “Step-by-step guide to reduce spatial coherence of laser light using a rotating ground glass diffuser”, Applied Optics, Optical Society of America 2017, Vol. 56, (19): 5427-5435
Stangner, T.; Hanqing, Z.; Dahlberg, T.; Wiklund, K. and Andersson, M.
(See online at https://doi.org/10.1364/AO.56.005427) - “UmUTracker: a versatile MATLAB program for automated particle tracking of 2D light microscopy or 3D digital holography data”, Computer Physics Communications, Elsevier 2017, Vol. 219 : 390-399
Zhang, H.; Stangner, T.; Wiklund, K.Rodrigues, A. and Andersson, M.
(See online at https://doi.org/10.1016/j.cpc.2017.05.029) - “3D printed water-soluble scaffolds for rapid production of PDMS micro-fluidic flow chambers”, Scientific Reports, 2018, Vol. 8, (1)
Dahlberg, T.; Stangner, T.; Hanqing, Z.; Wiklund, K.; Lundberg, P.; Edman, L. and Andersson, M.
(See online at https://doi.org/10.1038/s41598-018-21638-w) - “Cooke-Triplet-Tweezers: More compact, robust and efficient optical tweezers”, Optics Letters, Vol. 43, (9), 2018: 1990-1993
Stangner, T.; Dahlberg, T.; Svenmarker, P.; Zakrisson, J.; Wiklund, K.; Oddershede, L. B. and Andersson, M.
(See online at https://doi.org/10.1364/OL.43.001990)
