Biology subjects

Willsey, G. G.

Publications and source records attributed to Willsey, G. G..

6 recordsLinked to original sources

bioRxiv2025

A Broadly Conserved Protective Epitope on the Lyme Disease Vaccine Antigen, OspA

Lyme disease, caused by the spirochete, Borreliella burgdorferi sensu latu (Bbsl), is a debilitating tickborne infection of increasing incidence in North America, Europe and Asia. While vaccines based on Outer surface protein A (OspA) have proven highly efficacious at blocking Bbsl tick-to-human transmission, the high degree of antigenic variability among the major OspA serotypes (ST) has made the development of a broadly cross protective vaccine difficult. Recent profiling of protective human monoclonal antibodies (mAbs) has suggested the existence of conserved epitopes situated within OspAs central {beta}-sheet (CBS), although direct comparisons of cross-serotype functionality has been hindered by biological differences among the major Bbsl genospecies. To address these issues, we developed a panel of isogenic B. burgdorferi viability reporter strains expressing the seven major OspA serotypes (ST1-7) and probed them with CBS-targeting mAbs to evaluate their complement-dependent borreliacidal activity. The mAbs segregated into three distinct classes with varying degrees of borreliacidal activity: class 1 mAbs exhibited potent killing against all seven OspA serotypes, while classes 2 and 3 had restricted or no activity against two of the seven serotypes. Structural analysis of Fabs derived from each class of mAbs in complex with OspA ST1 showed that they target overlapping epitopes spanning {beta}-strands 6-10 and involve contact with largely invariant residues. Further analysis of B. burgdorferi reporter strains expressing OspA variants from 17 additional Bbsl genospecies identified Lys-107 as a determinant of susceptibility for nearly all CBS mAbs. Taken together, these findings raise the prospect of structure-based design of a broadly protective monovalent Lyme disease vaccine. AUTHOR SUMMARYLyme disease, caused by the spirochete, Borreliella burgdorferi sensu latu (Bbsl), is a potentially debilitating tickborne infection of increasing incidence in North America, Europe and Asia. While vaccines based on Outer surface protein A (OspA) have proven highly efficacious at blocking Bbsl tick-to-human transmission, the genetic and serological heterogeneity of OspA across Borrelia genospecies has complicated matters. In this report, we use a collection of transmission-blocking human monoclonal antibodies to delineate a protective region (epitope) within the central core of OspA that is conserved across all major OspA serotypes. These results have important implications for engineering a broadly reactive Lyme disease vaccine.

immunology↗

bioRxiv2025

An Fc-silent OspA monoclonal antibody passively protects mice from tick and intradermal Borrelia burgdorferi challenge

The monoclonal antibody, LA-2, has played a pivotal role in the development of Outer surface protein A (OspA)-based vaccines for Lyme disease, a multisystem illness caused by the tick-borne spirochete, Borrelia burgdorferi sensu lato. Of particular significance was the demonstration more than three decades ago that LA-2 equivalent antibody titers, defined by a competitive-inhibition ELISA, serve as a reliable correlate of vaccine-induced protection across different species, including humans. In vitro characterization of LA-2 has identified both complement-dependent and -independent activities, although which of these attributes contribute to protection against B. burgdorferi remains unresolved. To address this issue, we generated and characterized an "Fc-silent" version of LA-2 IgG1 carrying so-called LALAPG substitutions (L234A, L235A, P329G). We demonstrate that LA-2 LALAPG retained OspA binding activity but was severely attenuated in in vitro complement deposition and complement-dependent borreliacidal assays. Nonetheless, LA-2 LALAPG was as effective as LA-2 at passively protecting C3H mice against nymphal tick-mediated B. burgdorferi challenge. LA-2 LALAPG was also equivalent to LA-2 in passively protecting BALB/c mice against intradermal B. burgdorferi challenge. In the intradermal challenge model, viable spirochetes were not recoverable 24 h after injection from skin biopsies of mice treated with LA-2 or LA-2 LALAPG, and an influx of pro-inflammatory cytokines and chemokines to the injection site was abrogated. Collectively, these results suggest that LA-2s primary mode of action involves direct physical interactions with the spirochete rather than complement-dependent killing. Elucidating these mechanisms may have implications for understanding the mechanistic correlates of OspA-based vaccine-induced immunity in humans.

immunology↗

bioRxiv2024

A Salmonella enterica serovar Typhimurium Genome-wide CRISPRi Screen Reveals a Role for Type 1 Fimbriae in Evasion of Antibody-Mediated Agglutination

The O5-specific monoclonal IgA antibody, Sal4, mediates the conversion of Salmonella enterica serovar Typhimurium (STm) from virulent, free-swimming cells to non-motile, multicellular aggregates in solution ("flocs") as well as biofilm-like associations at the air-liquid interface (ALI). We hypothesize that the rapid transition from an invasive to a non-invasive state reflects an adaptation of STm to Sal4 IgA exposure. In this report, we performed a genome-wide CRISPRi screen to identify STm genes that influence multicellular aggregate formation in response to Sal4 IgA treatment. From a customized library of >36,000 spacers, [~]1% (373) were enriched at ALI after two consecutive rounds of Sal4 IgA treatment. The enriched spacers mapped to a diversity of targets, including genes involved in O-antigen modification, cyclic-di-GMP metabolism, outer membrane biosynthesis/signaling, and invasion/virulence, with the most frequently targeted gene being fimW, which encodes a negative regulator of Type 1 Fimbriae (T1F) expression. Generation of a STm {Delta}fimW strain confirmed that the loss of FimW results in a hyperfimbriated phenotype and evasion of Sal4 IgA-mediated agglutination in solution. Closer examination of the fimW mutant revealed its propensity to form biofilms at the ALI specifically in response to Sal4 exposure, suggesting that T1F "primes" STm to transition from a planktonic to a sessile state possibly by facilitating bacterial attachment to abiotic surfaces. These findings shed light on the mechanism by which protective secretory IgA antibodies influence STm virulence in the intestinal environment.

microbiology↗

bioRxiv2024

Structure of a human monoclonal antibody in complex with Outer surface protein C (OspC) of the Lyme disease spirochete, Borreliella burgdorferi

Lyme disease is a tick-borne, multisystem infection caused by the spirochete, Borreliella burgdorferi. Although antibodies have been implicated in the resolution of Lyme disease, the specific B cell epitopes targeted during human infections remain largely unknown. In this study, we characterized and defined the structural epitope of a patient-derived bactericidal monoclonal IgG ("B11") against Outer surface protein C (OspC), a homodimeric lipoprotein necessary for B. burgdorferi tick-mediated transmission and early-stage colonization of vertebrate hosts. High-resolution epitope mapping was accomplished through hydrogen deuterium exchange-mass spectrometry (HDX-MS) and X-ray crystallography. Structural analysis of B11 Fab-OspCA complexes revealed the B11 Fabs associated in a 1:1 stoichiometry with the lateral faces of OspCA homodimers such that the antibodies are essentially positioned perpendicular to the spirochetes outer surface. B11s primary contacts reside within the membrane proximal regions of -helices 1 and 6 and adjacent loops 5 and 6 in one OspCA monomer. In addition, B11 spans the OspCA dimer interface, engaging opposing -helix 1, -helix 2, and loop 2-3 in the second OspCA monomer. The B11-OspCA structure is reminiscent of the recently solved mouse transmission blocking monoclonal IgG B5 in complex with OspCA, indicating a mode of engagement with OspC that is conserved across species. In conclusion, we provide the first detailed insight into the interaction between a functional human antibody and an immunodominant Lyme disease antigen long considered an important vaccine target.

immunology↗

bioRxiv2021

Creatine utilization as a sole nitrogen source in Pseudomonas putida KT2440 is transcriptionally regulated by CahR

Glutamine amidotransferase-1 domain-containing AraC-family transcriptional regulators (GATRs) are present in the genomes of many bacteria, including all Pseudomonas species. The involvement of several characterized GATRs in amine-containing compound metabolism has been determined, but the full scope of GATR ligands and regulatory networks are still unknown. Here, we characterize Pseudomonas putidas detection of the animal-derived amine compound, creatine, a compound particularly enriched in muscle and ciliated cells by a creatine-specific GATR, PP_3665, here named CahR (Creatine amidohydrolase Regulator). cahR is necessary for transcription of the gene encoding creatinase (PP_3667/creA) in the presence of creatine and is critical for P. putidas ability to utilize creatine as a sole source of nitrogen. The CahR/creatine regulon is small and electrophoretic mobility shift demonstrates strong and specific CahR binding only at the creA promoter, supporting the conclusion that much of the regulon is dependent on downstream metabolites. Phylogenetic analysis of creA orthologs associated with cahR orthologs highlights a strain distribution and organization supporting likely horizontal gene transfer, particularly evident within the genus Acinetobacter. This study identifies and characterizes the GATR that transcriptionally controls P. putida metabolism of creatine, broadening the scope of known GATR ligands and suggesting GATR diversification during evolution of metabolism for aliphatic nitrogen compounds.

microbiology↗

bioRxiv2019

Functional characterization of Ralstonia insidiosa, a bona fide resident at the maternal-fetal interface

Controversy about whether there are microbes in the placenta and if they have any functional importance during pregnancy and for neonatal health is ongoing. Previous work has demonstrated that the basal plate (BP), comprising maternal and fetal derived cells harbors intracellular bacteria. 16S sequencing and bacterial species-specific analysis of term placentas revealed that the gram-negative bacillus Ralstonia insidiosa, native to aqueous environments and an effective biofilm promoter, comprises the most abundant species in the BP. Here, we demonstrate whether R. insidiosa cells home to a particular niche in the BP, how they may arrive there, and whether they are associated with adverse outcomes. We developed methods to detect and study cell-specific localization of R. insidiosa using ex vivo and in vitro models. Additionally, we studied potential routes of R. insidiosa entry into the placenta. We show that R. insidiosa is a bona fide resident in human placental BP. It can access trophoblast cells in culture and within basal plate tissues where it localizes to intracellular single-membrane vacuoles and can replicate. However, the presence of R. insidiosa does not cause cell death and does not induce a pro-inflammatory immune response suggesting that it is not harmful in and of itself. Finally, we show that in a pregnant mouse model, R. insidiosa traffics to the placenta via the intrauterine route but does not induce preterm labor or preterm birth. Together, our findings provide a foundation for understanding non-pathogenic placental cell-microbe interactions and the functional importance of R. insidiosa in placental health and physiology.

microbiology↗