Endospore pili: Flexible, stiff, and sticky nanofibers.

Species belonging to the Bacillus cereus group form endospores (spores) whose surface is decorated with micrometers-long and nanometers-wide endospore appendages (Enas). The Enas have recently been shown to represent a completely novel class of Gram-positive pili. They exhibit remarkable structural properties making them extremely resilient to proteolytic digestion and solubilization. However, little is known about their functional and biophysical properties. In this work, we apply optical tweezers to manipulate and assess how wild-type and Ena-depleted mutant spores immobilize on a glass surface. Furthermore, we utilize optical tweezers to extend S-Ena fibers to measure their flexibility and tensile stiffness. Finally, by oscillating single spores, we examine how the exosporium and Enas affect spores' hydrodynamic properties. Our results show that S-Enas (µm-long pili) are not as effective as L-Enas in immobilizing spores to glass surfaces but are involved in forming spore-to-spore connections, holding the spores together in a gel-like state. The measurements also show that S-Enas are flexible but tensile stiff fibers, which support structural data suggesting that the quaternary structure is composed of subunits arranged in a complex to produce a bendable fiber (helical turns can tilt against each other) with limited axial fiber extensibility. Finally, the results show that the hydrodynamic drag is 1.5 times higher for wild-type spores expressing S- and L-Enas compared with mutant spores expressing only L-Enas or "bald spores" lacking Ena, and 2 times higher compared with spores of the exosporium-deficient strain. This study unveils novel findings on the biophysics of S- and L-Enas, their role in spore aggregation, binding of spores to glass, and their mechanical behavior upon exposure to drag forces.

Monitoring bacterial spore metabolic activity using heavy water-induced Raman peak evolution.

Endospore-forming bacteria are associated with food spoilage, food poisoning, and infection in hospitals. Therefore, methods to monitor spore metabolic activity and verify sterilization are of great interest. However, current methods for tracking metabolic activity are time-consuming and resource intensive. This work investigates isotope labeling and Raman microscopy as a low-cost rapid alternative. Specifically, we monitor the Raman spectrum of enterotoxic B. cereus spores undergoing germination and cell division in D2O-infused broth. During germination and cell division, water is metabolized and deuterium from the broth is incorporated into proteins and lipids, resulting in the appearance of a Raman peak related to C-D bonds at 2190 cm-1. We find that a significant C-D peak appears after 2 h of incubation at 37 °C. Further, we found that the peak appearance coincides with the observed first cell division indicating little metabolic activity during germination. Lastly, the germination and cell growth rate of spores were not affected by adding 30% heavy water to the broth. This shows the potential for real-time monitoring of metabolic activity from a bacterial spore to a dividing cell. In conclusion, our work proposes tracking the evolution of the C-D Raman peak in spores incubated with D2O-infused broth as an effective and time-, and cost-efficient method to monitor the outgrowth of a spore population, simultaneously allowing us to track for how long the bacteria have grown and divided.

The F-pilus biomechanical adaptability accelerates conjugative dissemination of antimicrobial resistance and biofilm formation.

Conjugation is used by bacteria to propagate antimicrobial resistance (AMR) in the environment. Central to this process are widespread conjugative F-pili that establish the connection between donor and recipient cells, thereby facilitating the spread of IncF plasmids among enteropathogenic bacteria. Here, we show that the F-pilus is highly flexible but robust at the same time, properties that increase its resistance to thermochemical and mechanical stresses. By a combination of biophysical and molecular dynamics methods, we establish that the presence of phosphatidylglycerol molecules in the F-pilus contributes to the structural stability of the polymer. Moreover, this structural stability is important for successful delivery of DNA during conjugation and facilitates rapid formation of biofilms in harsh environmental conditions. Thus, our work highlights the importance of F-pilus structural adaptations for the efficient spread of AMR genes in a bacterial population and for the formation of biofilms that protect against the action of antibiotics.

Three structural solutions for bacterial adhesion pilus stability and superelasticity.

Bacterial adhesion pili are key virulence factors that mediate host-pathogen interactions in diverse epithelial environments. Deploying a multimodal approach, we probed the structural basis underpinning the biophysical properties of pili originating from enterotoxigenic (ETEC) and uropathogenic bacteria. Using cryo-electron microscopy we solved the structures of three vaccine target pili from ETEC bacteria, CFA/I, CS17, and CS20. Pairing these and previous pilus structures with force spectroscopy and steered molecular dynamics simulations, we find a strong correlation between subunit-subunit interaction energies and the force required for pilus unwinding, irrespective of genetic similarity. Pili integrate three structural solutions for stabilizing their assemblies: layer-to-layer interactions, N-terminal interactions to distant subunits, and extended loop interactions from adjacent subunits. Tuning of these structural solutions alters the biophysical properties of pili and promotes the superelastic behavior that is essential for sustained bacterial attachment.

Archaic chaperone-usher pili self-secrete into superelastic zigzag springs.

Adhesive pili assembled through the chaperone-usher pathway are hair-like appendages that mediate host tissue colonization and biofilm formation of Gram-negative bacteria1-3. Archaic chaperone-usher pathway pili, the most diverse and widespread chaperone-usher pathway adhesins, are promising vaccine and drug targets owing to their prevalence in the most troublesome multidrug-resistant pathogens1,4,5. However, their architecture and assembly-secretion process remain unknown. Here, we present the cryo-electron microscopy structure of the prototypical archaic Csu pilus that mediates biofilm formation of Acinetobacter baumannii-a notorious multidrug-resistant nosocomial pathogen. In contrast to the thick helical tubes of the classical type 1 and P pili, archaic pili assemble into an ultrathin zigzag architecture secured by an elegant clinch mechanism. The molecular clinch provides the pilus with high mechanical stability as well as superelasticity, a property observed for the first time, to our knowledge, in biomolecules, while enabling a more economical and faster pilus production. Furthermore, we demonstrate that clinch formation at the cell surface drives pilus secretion through the outer membrane. These findings suggest that clinch-formation inhibitors might represent a new strategy to fight multidrug-resistant bacterial infections.

Patient-specific brain arteries molded as a flexible phantom model using 3D printed water-soluble resin.

Visualizing medical images from patients as physical 3D models (phantom models) have many roles in the medical field, from education to preclinical preparation and clinical research. However, current phantom models are generally generic, expensive, and time-consuming to fabricate. Thus, there is a need for a cost- and time-efficient pipeline from medical imaging to patient-specific phantom models. In this work, we present a method for creating complex 3D sacrificial molds using an off-the-shelf water-soluble resin and a low-cost desktop 3D printer. This enables us to recreate parts of the cerebral arterial tree as a full-scale phantom model ([Formula: see text] cm) in transparent silicone rubber (polydimethylsiloxane, PDMS) from computed tomography angiography images (CTA). We analyzed the model with magnetic resonance imaging (MRI) and compared it with the patient data. The results show good agreement and smooth surfaces for the arteries. We also evaluate our method by looking at its capability to reproduce 1 mm channels and sharp corners. We found that round shapes are well reproduced, whereas sharp features show some divergence. Our method can fabricate a patient-specific phantom model with less than 2 h of total labor time and at a low fabrication cost.

Reactive oxygen species generated by infrared laser light in optical tweezers inhibits the germination of bacterial spores.

Bacterial spores are highly resistant to heat, radiation and various disinfection chemicals. The impact of these on the biophysical and physicochemical properties of spores can be studied on the single-cell level using optical tweezers. However, the effect of the trapping laser on spores' germination rate is not fully understood. In this work, we assess the impact of 1064 nm laser light on the germination of Bacillus thuringiensis spores. The results show that the germination rate of spores after laser exposure follows a sigmoid dose-response relationship, with only 15% of spores germinating after 20 J of laser light. Under anaerobic growth conditions, the percentage of germinating spores at 20 J increased to 65%. The results thereby indicate that molecular oxygen is a major contributor to the germination-inhibiting effect observed. Thus, our study highlights the risk for optical trapping of spores and ways to mitigate it.

Unveiling molecular interactions that stabilize bacterial adhesion pili.

Adhesion pili assembled by the chaperone-usher pathway are superelastic helical filaments on the surface of bacteria, optimized for attachment to target cells. Here, we investigate the biophysical function and structural interactions that stabilize P pili from uropathogenic bacteria. Using optical tweezers, we measure P pilus subunit-subunit interaction dynamics and show that pilus compliance is contour-length dependent. Atomic details of subunit-subunit interactions of pili under tension are shown using steered molecular dynamics (sMD) simulations. sMD results also indicate that the N-terminal "staple" region of P pili, which provides interactions with pilins that are four and five subunits away, significantly stabilizes the helical filament structure. These data are consistent with previous structural data, and suggest that more layer-to-layer interactions could compensate for the lack of a staple in type 1 pili. This study informs our understanding of essential structural and dynamic features of adhesion pili, supporting the hypothesis that the function of pili is critically dependent on their structure and biophysical properties.

pH-induced changes in Raman, UV-vis absorbance, and fluorescence spectra of dipicolinic acid (DPA).

Dipicolinic acid (DPA) is an essential component for the protection of DNA in bacterial endospores and is often used as a biomarker for spore detection. Depending upon the pH of the solution, DPA exists in different ionic forms. Therefore, it is important to understand how these ionic forms influence spectroscopic response. In this work, we characterize Raman and absorption spectra of DPA in a pH range of 2.0-10.5. We show that the ring breathing mode Raman peak of DPA shifts from 1003 cm-1 to 1017 cm-1 and then to 1000 cm-1 as pH increases from 2 to 5. The relative peak intensities related to the different ionic forms of DPA are used to experimentally derive the pKa values (2.3 and 4.8). We observe using UV-vis spectroscopy that the changes in the absorption spectrum of DPA as a function of pH correlate with the changes observed in Raman spectroscopy, and the same pKa values are verified. Lastly, using fluorescence spectroscopy and exciting a DPA solution at between 210-330 nm, we observe a shift in fluorescence emission from 375 nm to 425 nm between pH 2 and pH 6 when exciting at 320 nm. Our work shows that the different spectral responses from the three ionic forms of DPA may have to be taken into account in, e.g., spectral analysis and for detection applications.

Laser induced degradation of bacterial spores during micro-Raman spectroscopy.

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was ∼60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results.

Synthetic NAC 71-82 Peptides Designed to Produce Fibrils with Different Protofilament Interface Contacts.

Alpha-synucleinopathies are featured by fibrillar inclusions in brain cells. Although α-synuclein fibrils display structural diversity, the origin of this diversity is not fully understood. We used molecular dynamics simulations to design synthetic peptides, based on the NAC 71-82 amino acid fragment of α-synuclein, that govern protofilament contacts and generation of twisted fibrillar polymorphs. Four peptides with structures based on either single or double fragments and capped or non-capped ends were selected for further analysis. We determined the fibrillar yield and the structures from these peptides found in the solution after fibrillisation using protein concentration determination assay and circular dichroism spectroscopy. In addition, we characterised secondary structures formed by individual fibrillar complexes using laser-tweezers Raman spectroscopy. Results suggest less mature fibrils, based on the lower relative ß-sheet content for double- than single-fragment peptide fibrils. We confirmed this structural difference by TEM analysis which revealed, in addition to short protofibrils, more elongated, twisted and rod-like fibril structures in non-capped and capped double-fragment peptide systems, respectively. Finally, time-correlated single-photon counting demonstrated a difference in the Thioflavin T fluorescence lifetime profiles upon fibril binding. It could be proposed that this difference originated from morphological differences in the fibril samples. Altogether, these results highlight the potential of using peptide models for the generation of fibrils that share morphological features relevant for disease, e.g., twisted and rod-like polymorphs.

Optical design for laser tweezers Raman spectroscopy setups for increased sensitivity and flexible spatial detection.

We demonstrate a method to double the collection efficiency in laser tweezers Raman spectroscopy (LTRS) by collecting both the forward-scattered and backscattered light in a single-shot multitrack measurement. Our method can collect signals at different sample volumes, granting both the pinpoint spatial selectivity of confocal Raman spectroscopy and the bulk sensitivity of non-confocal Raman spectroscopy simultaneously. Further, we display that our approach allows for reduced detector integration time and laser power. To show this, we measure the Raman spectra of both polystyrene beads and bacterial spores. For spores, we can trap them at 2.5 mW laser power and acquire a high signal-to-noise ratio power spectrum of the calcium-dipicolinic acid peaks using an integration time of ${2} \times {30}\;{\rm s}$. Thus, our method will enable the monitoring of biological samples sensitive to high intensities for longer times. Additionally, we demonstrate that by a simple modification, we can add polarization sensitivity and retrieve extra biochemical information.

Step-by-step guide to 3D print motorized rotation mounts for optical applications.

Motorized rotation mounts and stages are versatile instruments that introduce computer control to optical systems, enabling automation and scanning actions. They can be used for intensity control, position adjustments, etc. However, these rotation mounts come with a hefty price tag, and this limits their use. This work shows how to build two different types of motorized rotation mounts for $1^{\prime \prime}$ optics, using a 3D printer and off-the-shelf components. The first is intended for reflective elements, such as mirrors and gratings, and the second for transmissive elements, such as polarizers and retarders. We evaluate and compare their performance to commercial systems based on velocity, resolution, precision, backlash, and axis wobble. Also, we investigate the angular stability using Allan variance analysis. The results show that our mounts perform similarly to systems costing as much as $\$ 2500\,\rm USD $, while also being quick to build and costing less than $\$ 220\,\rm USD$. As a proof of concept, we show how to control lasers used in an optical tweezers and Raman spectroscopy setup. When used for this, the 3D printed motorized rotational mounts provide intensity control with a resolution of 0.03 percentage points or better.

Impact of an alpha helix and a cysteine-cysteine disulfide bond on the resistance of bacterial adhesion pili to stress.

Escherichia coli express adhesion pili that mediate attachment to host cell surfaces and are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow forces, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that class 1a pili of uropathogenic E. coli (UPEC), as well as class 1b of enterotoxigenic E. coli (ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC class 5 pili. Examining structural and steered molecular dynamics simulation data, we find that this difference in class 1 pili subunit behavior originates from an α-helical motif that can unfold when exposed to force. A disulfide bond cross-linking ß-strands in class 1 pili stabilizes subunits, allowing them to tolerate higher forces than class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche, since resident bacteria are exposed to stronger, more transient drag forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of class 5 ETEC pili.

Mode of Action of Disinfection Chemicals on the Bacterial Spore Structure and Their Raman Spectra.

Contamination of toxic spore-forming bacteria is problematic since spores can survive a plethora of disinfection chemicals and it is hard to rapidly detect if the disinfection chemical has inactivated the spores. Thus, robust decontamination strategies and reliable detection methods to identify dead from viable spores are critical. In this work, we investigate the chemical changes of Bacillus thuringiensis spores treated with sporicidal agents such as chlorine dioxide, peracetic acid, and sodium hypochlorite using laser tweezers Raman spectroscopy. We also image treated spores using SEM and TEM to verify if we can correlate structural changes in the spores with changes to their Raman spectra. We found that over 30 min, chlorine dioxide did not change the Raman spectrum or the spore structure, peracetic acid showed a time-dependent decrease in the characteristic DNA/DPA peaks and ∼20% of the spores were degraded and collapsed, and spores treated with sodium hypochlorite showed an abrupt drop in DNA and DPA peaks within 20 min and some structural damage to the exosporium. Structural changes appeared in spores after 10 min, compared to the inactivation time of the spores, which is less than a minute. We conclude that vibrational spectroscopy provides powerful means to detect changes in spores but it might be problematic to identify if spores are live or dead after a decontamination procedure.

Cooke-Triplet tweezers: more compact, robust, and efficient optical tweezers.

We present a versatile three-lens optical design to improve the overall compactness, efficiency, and robustness for optical tweezers based applications. The design, inspired by the Cooke-Triplet configuration, allows for continuous beam magnifications of 2-10×, and axial as well as lateral focal shifts can be realized without switching lenses or introducing optical aberrations. We quantify the beam quality and trapping stiffness and compare the Cooke-Triplet design with the commonly used double Kepler design through simulations and direct experiments. Optical trapping of 1 and 2 µm beads shows that the Cooke-Triplet possesses an equally strong optical trap stiffness compared to the double Kepler lens design but reduces its lens system length by a factor of 2.6. Finally, we demonstrate how a Twyman-Green interferometer integrated in the Cooke-Triplet optical tweezers setup provides a fast and simple method to characterize the wavefront aberrations in the lens system and how it can help in aligning the optical components perfectly.

3D printed water-soluble scaffolds for rapid production of PDMS micro-fluidic flow chambers.

We report a novel method for fabrication of three-dimensional (3D) biocompatible micro-fluidic flow chambers in polydimethylsiloxane (PDMS) by 3D-printing water-soluble polyvinyl alcohol (PVA) filaments as master scaffolds. The scaffolds are first embedded in the PDMS and later residue-free dissolved in water leaving an inscription of the scaffolds in the hardened PDMS. We demonstrate the strength of our method using a regular, cheap 3D printer, and evaluate the inscription process and the channels micro-fluidic properties using image analysis and digital holographic microscopy. Furthermore, we provide a protocol that allows for direct printing on coverslips and we show that flow chambers with a channel cross section down to 40 µm × 300 µm can be realized within 60 min. These flow channels are perfectly transparent, biocompatible and can be used for microscopic applications without further treatment. Our proposed protocols facilitate an easy, fast and adaptable production of micro-fluidic channel designs that are cost-effective, do not require specialized training and can be used for a variety of cell and bacterial assays. To help readers reproduce our micro-fluidic devices, we provide: full preparation protocols, 3D-printing CAD files for channel scaffolds and our custom-made molding device, 3D printer build-plate leveling instructions, and G-code.

Step-by-step guide to reduce spatial coherence of laser light using a rotating ground glass diffuser.

Wide field-of-view imaging of fast processes in a microscope requires high light intensities motivating the use of lasers as light sources. However, due to their long spatial coherence length, lasers are inappropriate for such applications, as they produce coherent noise and parasitic reflections, such as speckle, degrading image quality. Therefore, we provide a step-by-step guide for constructing a speckle-free and high-contrast laser illumination setup using a rotating ground glass diffuser driven by a stepper motor. The setup is easy to build, cheap, and allows a significant light throughput of 48%, which is 40% higher in comparison to a single lens collector commonly used in reported setups. This is achieved by using only one objective to collect the scattered light from the ground glass diffuser. We validate our setup in terms of image quality, speckle contrast, motor-induced vibrations, and light throughput. To highlight the latter, we record Brownian motion of micro-particles using a 100× oil immersion objective and a high-speed camera operating at 2000 Hz with a laser output power of only 22 mW. Moreover, by reducing the objective magnification to 50×, sampling rates up to 10,000 Hz are realized. To help readers with basic or advanced optics knowledge realize this setup, we provide a full component list, 3D-printing CAD files, setup protocol, and the code for running the stepper motor.