Lentz, A. | Search bioRxiv

bioRxiv2024

Discovery of three novel neutralizing antibody epitopes on the human astrovirus capsid spike and mechanistic insights into virus neutralization

Human astroviruses (HAstVs) are a leading cause of viral childhood diarrhea that infect nearly every individual during their lifetime. Although human astroviruses are highly prevalent, no approved vaccine currently exists. Antibody responses appear to play an important role in protection from HAstV infection, however knowledge about the neutralizing epitope landscape is lacking, as only 3 neutralizing antibody epitopes have previously been determined. Here, we structurally define the epitopes of 3 uncharacterized HAstV-neutralizing monoclonal antibodies: antibody 4B6 with X-ray crystallography to 2.67 [A], and antibodies 3H4 and 3B4 simultaneously with single-particle cryogenic-electron microscopy to 3.33 [A]. We assess the epitope locations relative to conserved regions on the capsid spike and find that while antibodies 4B6 and 3B4 target the upper variable loop regions of the HAstV spike protein, antibody 3H4 targets a novel region near the base of the spike that is more conserved. Additionally, we found that all 3 antibodies bind with high affinity, and they compete with receptor FcRn binding to the capsid spike. These studies inform which regions of the HAstV capsid can be targeted by monoclonal antibody therapies and could aid in rational vaccine design. ImportanceHuman astroviruses infect nearly every child in the world, causing diarrhea, vomiting, and fever. Despite the prevalence of human astroviruses, little is known about how antibodies block virus infection. Here, we determined high-resolution structures of the astrovirus capsid protein in complex with three virus-neutralizing antibodies. The antibodies bind distinct sites on the capsid spike domain. We find that the antibodies block virus attachment to human cells and prevent capsid spike interaction with the human neonatal Fc receptor. These findings support the use of the human astrovirus capsid spike as an antigen in a vaccine to prevent astrovirus disease.

microbiology↗

bioRxiv2020

Developing Cyclic Peptomers as Broad-Spectrum Gram negative Bacterial Type III Secretion System Inhibitors

Antibiotic resistant bacteria are an emerging global health threat. New antimicrobials are urgently needed. The injectisome type III secretion system (T3SS), required by dozens of Gram-negative bacteria for virulence but largely absent from non-pathogenic bacteria, is an attractive antimicrobial target. We previously identified synthetic cyclic peptomers, inspired by the natural product phepropeptin D, that inhibit protein secretion through the Yersinia Ysc and Pseudomonas aeruginosa Psc T3SSs, but do not inhibit bacterial growth. Here we describe identification of an isomer, 4EpDN, that is two-fold more potent (IC50 4 M) than its parental compound. Furthermore, 4EpDN inhibited the Yersinia Ysa and the Salmonella SPI-1 T3SSs, suggesting that this cyclic peptomer has broad efficacy against evolutionarily distant injectisome T3SSs. Indeed, 4EpDN strongly inhibited intracellular growth of Chlamydia trachomatis in HeLa cells, which requires the T3SS. 4EpDN did not inhibit the unrelated Twin arginine translocation (Tat) system, nor did it impact T3SS gene transcription. Moreover, although the injectisome and flagellar T3SSs are evolutionarily and structurally related, the 4EpDN cyclic peptomer did not inhibit secretion of substrates through the Salmonella flagellar T3SS, indicating that cyclic peptomers broadly but specifically target the injestisome T3SS. 4EpDN reduced the number of T3SS basal bodies detected on the surface of Y. enterocolitica, as visualized using a fluorescent derivative of YscD, an inner membrane ring with low homology to flagellar protein FliG. Collectively, these data suggest that cyclic peptomers specifically inhibit the injectisome T3SS from a variety of Gram-negative bacteria, possibly by preventing complete T3SS assembly. IMPORTANCETraditional antibiotics target both pathogenic and commensal bacteria, resulting in a disruption of the microbiota, which in turn is tied to a number of acute and chronic diseases. The bacterial type III secretion system (T3SS) is an appendage used by many bacterial pathogens to establish infection, but is largely absent from commensal members of the microbiota. In this study, we identify a new derivative of the cyclic peptomer class of T3SS inhibitors. These compounds inhibit the T3SS of the nosocomial ESKAPE pathogen Pseudomonas aeruginosa and enteropathogenic Yersinia and Salmonella. The impact of cyclic peptomers is specific to the T3SS, as other bacterial secretory systems are unaffected. Importantly, cyclic peptomers completely block replication of Chlamydia trachomatis, the causative agent of genital, eye, and lung infections, in human cells, a process that requires the T3SS. Therefore, cyclic peptomers represent promising virulence blockers that can specifically disarm a broad spectrum of Gram-negative pathogens.

microbiology↗