Biology subjects

Depelteau, J. S.

Publications and source records attributed to Depelteau, J. S..

6 recordsLinked to original sources

bioRxiv2026

Identification of a C3b-specific nanobody that does not bind C3 and blocks alternative pathway convertases

The human complement system is a protein network in blood and other body fluids that fights invading pathogens and is involved in maintaining homeostasis. A central step in the complement cascade is the conversion of complement protein C3 to C3b by convertase enzymes. Upon cleavage of C3, the nascent C3b molecule undergoes a large conformational change which exposes new epitopes. Molecules that discriminate between C3b and C3 could provide a powerful tool to selectively bind surface-bound complement activation products while leaving the circulating precursors untouched. In this study, we developed C3b-specific nanobodies by generating phage libraries from llamas immunized with purified and surface-bound C3b. We describe UNbC3b-1 as a high-affinity binder that recognizes soluble and surface-bound C3b molecules but does not bind to C3 in its native state. A 4.2 [A] structure of the UNbC3b-1:C3b complex generated by cryogenic electron microscopy shows that UNbC3b-1 binds C3b at the interface of MG3, MG4, and MG6, overlapping with the complement receptor immunoglobulin (CRIg) binding site. Functionally, we show that UNbC3b-1 inhibits complement activity in the alternative pathway (AP), likely by preventing association between substrate C3 and the AP C3 convertase (C3bBb). Altogether, nanobody UNbC3b-1 is a valuable tool to specifically bind C3b molecules and to selectively inhibit the AP convertases of the complement system.

immunology↗

bioRxiv2025

ICP1 bacteriophage treatment antagonizes colonization of the zebrafish larval intestine by Vibrio cholerae

Outbreaks of cholera pose a major threat to human health. Currently, antibiotics are the most effective treatment against the causative agent, the bacterium Vibrio cholerae. However, the use of antibiotics eventually leads to the emergence of resistant strains, which necessitates the need for alternative approaches. The use of bacteriophages to target the infection by antibiotic resistant bacteria is one promising alternative. While clearance of Vibrio cholerae with the use of phages has been performed on several animal models, none of these models are natural hosts of V. cholerae. Therefore, we set out to investigate the interaction between V. cholerae and bacteriophage ICP1 both in vitro and in vivo in a natural host, the zebrafish model, Danio rerio. To study the interplay between host, bacteria and phages we used a combination of light and ultrastructural imaging techniques, including confocal fluorescence microscopy, serial block face scanning electron microscopy (EM) imaging and cryogenic EM, which allowed us to investigate both the colonization process by V. cholerae and clearance by the ICP1 bacteriophage. In addition, we determined the effects of the microbiome on this treatment by using germ-free, conventionalized and monoassociated zebrafish larvae as a host. Independent of the presence and composition of microbiomes used here, V. cholerae efficiently colonized the larval intestine. Finally, we demonstrate significant in vivo clearance of V. cholerae N16961-dsRED by ICP1, underscoring the role of phage-bacteria dynamics in shaping pathogen colonization within the zebrafish larval host. SIGNIFICANCECholera remains a life-threatening disease that causes recurring outbreaks and significant mortality, particularly in developing and conflict-affected regions. As antimicrobial resistance continues to rise, there is an urgent need to better understand the ecological and microbial dynamics that govern Vibrio cholerae colonization and persistence. This research investigates how V. cholerae interacts with bacteriophages, the host environment, and the resident microbiota within a natural vertebrate host, offering new insights into the factors that influence pathogen clearance and shaping of the gut ecosystem during infection. The powerful combination of serial block-face scanning and cryogenic electron microscopy, fluorescence microscopy, and traditional colony/plaque counting methods revealed previously unobserved aspects of the interplay between host, pathogen, phages, and the microbiome, highlighting phage-driven clearance of V. cholerae during colonization.

microbiology↗

bioRxiv2021

Transition of Vibrio cholerae through a natural host induces resistance to environmental changes

The pandemic-related strains of Vibrio cholerae are known to cause diarrheal disease in animal hosts. These bacteria must overcome rapid changes in their environment, such as the transition from fresh water to the gastrointestinal system of their host. To study the morphological adjustments during environmental transitions, we used zebrafish as a natural host. Using a combination of fluorescent light microscopy, cryogenic electron tomography and serial block face scanning electron microscopy, we studied the structural changes that occur during the infection cycle. We show that the transition from an artificial nutrient-rich environment to a nutrient-poor environment has a dramatic impact on the cell shape, most notably membrane dehiscence. In contrast, excreted bacteria from the host retain a uniform distance between the membranes as well as their vibrioid shape. Inside the intestine, V. cholerae cells predominantly colonized the anterior to mid-gut, forming micro-colonies associated with the microvilli as well as within the lumen. The cells retained their vibrioid shape but changed their cell-length depending on their localization. Our results demonstrate dynamic changes in morphological characteristics of V. cholerae during the transition between the different environments, and we propose that these structural changes are critical for the pathogens ability to colonize host tissues.

microbiology↗

bioRxiv2021

The VarA-CsrA regulatory pathway influences cell shape in Vibrio cholerae

Despite extensive studies on the curve-shaped bacterium Vibrio cholerae, the causative agent of the diarrheal disease cholera, its virulence-associated regulatory two-component signal transduction system VarS/VarA is not well understood. This pathway, which mainly signals through the downstream protein CsrA, is highly conserved among gamma-proteobacteria, indicating there is likely a broader function of this system beyond virulence regulation. In this study, we investigated the VarA-CsrA signaling pathway and discovered a previously unrecognized link to the shape of the bacterium. We observed that varA-deficient V. cholerae cells showed an abnormal spherical morphology during late-stage growth. Through peptidoglycan (PG) composition analyses, we discovered that these mutant bacteria contained an increased content of disaccharide dipeptides and reduced peptide crosslinks, consistent with the atypical cellular shape. The spherical shape correlated with the CsrA-dependent overproduction of aspartate ammonia lyase (AspA) in varA mutant cells, which likely depleted the cellular aspartate pool; therefore, the synthesis of the PG precursor amino acid meso-diaminopimelic acid was impaired. Importantly, this phenotype, and the overall cell rounding, could be prevented by means of cell wall recycling. Collectively, our data provide new insights into how V. cholerae use the VarA-CsrA signaling system to adjust its morphology upon unidentified external cues in its environment. Significance StatementResponsible for the diarrheal disease cholera, the bacterium Vibrio cholerae tightly regulates its virulence program according to external stimuli. Here, we discovered that a sensing-response mechanism involved in the regulation of virulence also controls bacterial shape. We show that V. cholerae lacking this system lose their normal comma shape and become spherical due to an abnormal cell wall composition caused by metabolic changes that reduce available cell wall building blocks. Our study therefore sheds new light on how V. cholerae modulates its morphology based on environmental changes.

microbiology↗

bioRxiv2021

UVC inactivation of pathogenic samples suitable for cryoEM analysis

Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples, from individual proteins to whole cells. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of human pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the delicate ultrastructure of the sample. Alternatively, researchers use in vitro or ex vivo models, which are non-pathogenic but lack the complexity of the pathogen of interest. Here we show that ultraviolet-C (UVC) radiation at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of two model organisms, a pathogenic bacterium (Vibrio cholerae) and a virus-like particle (the ICP1 bacteriophage). We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography (cryo-ET) followed by sub-tomogram averaging (STA), and single particle analysis (SPA). Additionally, we applied the UVC irradiation to the protein apoferritin (ApoF), which is a widely used test sample for high resolution SPA studies. The UVC-treated ApoF sample resulted in a 2.1 [A] structure that did not reveal any discernable structural damage. Together, these results show that the UVC irradiation dose that effectively inactivates cryo-EM samples does not negatively impact their structure. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.

molecular biology↗

bioRxiv2017

An open-source cryo-storage solution

The field of cryo-electron microscopy is a rapidly growing method in structural biology. With this development, access to cryo-EM facilities becomes a bottleneck that results in long wait times between sample preparation and data acquisition. To improve sample storage, we developed a cryo-storage system with a more efficient and larger storage capacity that enables cryo-sample storage in a highly organized manner. This system is simple to use, cost-effective and easily adaptable for any type of grid box and storage dewar and any size cryo-EM laboratory.

molecular biology↗