Jamie S. Depelteau

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 Å 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.

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 naturalistic 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 naturalistic 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. IMPORTANCE: Cholera 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 selected microsymbionts, highlighting phage-driven clearance of V. cholerae during colonization.

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. SIGNIFICANCE: Cholera 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.

Staphylococcus aureus is the major cause of healthcare-associated infections, including life-threatening conditions as bacteremia, endocarditis, and implant-associated infections. Despite adequate antibiotic treatment, the mortality of S. aureus bacteremia remains high. This calls for different strategies to treat this infection. In past years, sequencing of Ab repertoires from individuals previously exposed to a pathogen emerged as a successful method to discover novel therapeutic monoclonal Abs and understand circulating B cell diversity during infection. In this paper, we collected peripheral blood from 17 S. aureus bacteremia patients to study circulating plasmablast responses. Using single-cell transcriptome gene expression combined with sequencing of variable heavy and light Ig genes, we retrieved sequences from >400 plasmablasts revealing a high diversity with >300 unique variable heavy and light sequences. More than 200 variable sequences were synthesized to produce recombinant IgGs that were analyzed for binding to S. aureus whole bacterial cells. This revealed four novel monoclonal Abs that could specifically bind to the surface of S. aureus in the absence of Ig-binding surface SpA. Interestingly, three of four mAbs showed cross-reactivity with Staphylococcus epidermidis. Target identification revealed that the S. aureus–specific mAb BC153 targets wall teichoic acid, whereas cross-reactive mAbs BC019, BC020, and BC021 target lipoteichoic acid. All mAbs could induce Fc-dependent phagocytosis of staphylococci by human neutrophils. Altogether, we characterize the active B cell responses to S. aureus in infected patients and identify four functional mAbs against the S. aureus surface, of which three cross-react with S. epidermidis.

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.

Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples. 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 pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the 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 applied at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of Vibrio cholerae and the bacterial virus, the ICP1 bacteriophage. We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography followed by sub-tomogram averaging, 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 Å structure indistinguishable from an untreated published map. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.

Summary: 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 pathogen’s ability to colonize host tissues.

Visualizing biological structures and cellular processes in their native state is a major goal of many scientific laboratories. In the past 20 years, the technique of preserving samples by vitrification has greatly expanded, specifically for use in cryogenic electron microscopy (cryo-EM). Here, we report on improvements in the design and use of a portable manual cryogenic plunge freezer that is intended for use in laboratories that are not equipped for the cryopreservation of samples. The construction of the instrument is economical, can be produced by a local machine shop without specialized equipment, and lowers the entry barriers for newcomers with a reliable alternative to costly commercial equipment. The improved design allows for successful freezing of isolated proteins for single particle analysis as well as bacterial cells for cryo-electron tomography. With this instrument, groups will be able to prepare vitreous samples whenever and wherever necessary, which can then be imaged at local or national cryo-EM facilities.

Cryo-electron microscopy (cryo-EM) enables the study of biological structures in situ in great detail and to solve protein structures at Ångstrom level resolution. Due to recent advances in instrumentation and data processing, the field of cryo-EM is a rapidly growing. Access to facilities and national centers that house the state-of-the-art microscopes is limited due to the ever-rising demand, resulting in long wait times between sample preparation and data acquisition. To improve sample storage, we have developed a cryo-storage system with an efficient, high storage capacity that enables sample storage in a highly organized manner. This system is simple to use, cost-effective and easily adaptable for any type of grid storage box and dewar and any size cryo-EM laboratory.