Biology subjects

Picking, W. L.

Publications and source records attributed to Picking, W. L..

5 recordsLinked to original sources

bioRxiv2026

Eco-tank Housing Maintains Wild-Type Microbiota and Rewilds the Laboratory Mouse Gut Microbiome to Restore Natural Immune Tone

Laboratory mice housed under individually ventilated cage (IVC) conditions harbor simplified gut microbiota and immune phenotypes that diverge substantially from those shaped by environmental exposure, limiting translational relevance. To reintroduce controlled ecological complexity while maintaining biosafety and reproducibility, we developed the Eco-tank, a pathogen-monitored semi-natural housing system incorporating environmental substrates and dietary diversity. Longitudinal 16S rRNA sequencing revealed that even wild-caught Mus musculus rapidly lose microbial richness and predicted metabolic breadth under IVC housing. Eco-tank conditions stabilized diversity and preserved elements of wild-associated community structure during extended captivity. In parallel, standardized C57BL/6 mice housed in Eco-tanks underwent rewilding-like restructuring, with increased richness and community shifts toward a wild-associated configuration. Functional inference analyses indicated expansion of predicted pathways linked to short-chain fatty acid production, amino acid metabolism, and environmental substrate utilization. Eco-tank housing enhanced baseline resistance to pulmonary Pseudomonas aeruginosa (Pa) infection without compromising vaccine-induced protection, indicating that restoration of environmental microbial signals does not impair adaptive immunity. Together, these findings identify housing ecology as a dominant determinant of microbiome structure and functional potential. The Eco-tank provides a scalable and tractable framework for integrating environmental microbial complexity into laboratory models to better align preclinical immunology with ecologically conditioned immune systems. ImportanceLaboratory mice are foundational models for immunology, yet their specific pathogen-free rearing and housing environments impose ecological constraints that reshape the gut microbiome and immune tone. This study introduces a scalable, pathogen-monitored Eco-tank system that restores environmental microbial complexity while preserving experimental control. By demonstrating that housing ecology reshapes microbiome functional potential and modulates baseline immune resistance without compromising vaccine responsiveness, this work highlights environmental context as a critical experimental variable in preclinical immunology and offers a tractable framework for improving translational relevance.

microbiology↗

bioRxiv2026

The pod components of the Shigella T3SS sorting platform accommodate multiple copies of Spa33 (SctQ)

The bacterial type III secretion system (T3SS) uses a membrane-embedded injectisome assembly to export effector proteins into host cells. While atomic-level structural details have been revealed for much of the T3SS apparatus, the model of the cytoplasmic sorting platform remains largely low-resolution. A central structural element of the sorting platform is the so-called "pod" protein, SctQ, which anchors the sorting platform to the inner membrane via interaction with the adaptor protein SctK, and connects to the central ATPase via the spoke protein SctL. SctQ proteins also interact with alternatively translated homodimers of their C-terminal SPOA2 domains. Low resolution electron density maps have provided an outline of the sorting platform architecture, and fluorescence microscopy studies have suggested a 1:4:2 SctK:SctQ:SctL stoichiometry. While there are experimental and AlphaFold structures of the individual components or complexes of sorting platform pod, there is currently no model for the pod structure that adequately fits the electron density or accounts for the proposed stoichiometry. Here we use AlphaFold to generate a model of the Shigella pod complex in which two copies of the SctQ protein, Spa33, bind the adaptor protein MxiK, with each copy of Spa33 bound to an alternately translated SPOA2-SPOA2 domain. We show through mutation of energetically critical interface residues, predicted by computational mutant scanning, that both Spa33 binding sites on MxiK are required for T3SS activity in Shigella flexneri, as well as binding of Spa33 to the SPOA2-SPOA2 homodimer. We find that this model fits well to the upper two-thirds of the pod electron density, albeit in a manner that places the protein components slightly closer to the inner membrane than traditionally presented. Further, cryogenic electron tomography of mutant injectisomes reveals the lack of a complete sorting platform and may suggest an alternative model featuring four copies of Spa33 that still fits the upper pod electron density but better explains unfilled density in the lower third of the pod.

biochemistry↗

bioRxiv2025

A High-throughput Multi-Species Platform Using Biolayer Interferometry Immunosorbent Assay (BLI-ISA) as an Alternative to Indirect ELISA for Vaccine Development

In vaccine development, the ELISA (Enzyme-Linked Immunosorbent Assay) is commonly used to compare the antibody titers of samples from several treatment groups. This often requires extensive sample preparation, manual labor, and long incubation and processing times. Biolayer Interferometry (BLI) has emerged as an alternative to the ELISA for the detection and quantification of antigen-specific antibodies in biological samples. However, the implementation of BLI as a replacement for the ELISA in vaccine development requires that experimental parameters are established for accurate and reproducible results. Here we give a general protocol for a biolayer interferometry immunosorbent assay (BLI-ISA) for the comparison of antigen-specific antibody levels in treatment group sera that uses secondary antibody binding responses as replacement for ELISA endpoint titers. We also validate that this BLI-ISA yields the same results as the ELISA endpoint titer while requiring far less time and effort.

immunology↗

bioRxiv2021

Composition and Biophysical Properties of the Sorting Platform Pods in the Shigella Type III Secretion System

Shigella flexneri, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisomes cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. The SP consists of oligomeric Spa33 "pods" that associate with the basal body via MxiK and connect to the Spa47 ATPase via MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33C). The structure of Spa33C is known, but the precise makeup and structure of the pods in situ remains elusive. We show here that recombinant wild-type Spa33 can be prepared as a heterotrimer that forms distinct stable complexes with MxiK and MxiN. In two-hybrid analyses, association of the Spa33 complex with these proteins occurs via the full-length Spa33 component. Furthermore, these complexes each have distinct biophysical properties. Based on these properties, new high-resolution cryo-electron tomography data and architectural similarities between the Spa33 and flagellar FliM-FliN complexes, we provide a preliminary model of the Spa33 heterotrimers within the SP pods. From these findings and evolving models of SP interfaces and dynamics in the Yersinia and Salmonella T3SS, we suggest a model for SP function in which two distinct complexes come together within the context of the SP to contribute to form the complete pod structures during the recruitment of T3SS secretion substrates.

microbiology↗

bioRxiv2020

The Structures of SctK and SctD from Pseudomonas aeruginosa Reveal the Interface of the Type III Secretion System Basal Body and Sorting Platform

Many Gram-negative bacterial pathogens use type III secretion systems (T3SS) to inject effector proteins into eukaryotic cells to subvert normal cellular functions. The T3SS apparatus (injectisome) shares a common overall architecture in all systems studied thus far, comprising three major components - the cytoplasmic sorting platform, the envelope-spanning basal body and the external needle with controlling tip complex. The sorting platform consists of an ATPase (SctN) connected to "pods" (SctQ) having six-fold symmetry via radial spokes (SctL). These pods interface with the 24-fold symmetric SctD inner membrane ring (IR) via an adaptor protein (SctK). Here we report the first high-resolution structure of a SctK protein family member, PscK from Pseudomonas aeruginosa, as well as the structure of its interacting partner, the cytoplasmic domain of PscD (SctD). The cytoplasmic domain of PscD forms a forkhead-associated (FHA) fold, like that of its homologues from other T3SS. PscK, on the other hand, forms a helix-rich structure that does not resemble any known protein fold. Based on these structural findings, we present the first model for an interaction between proteins from the sorting platform and the IR. We also test the importance of the PscD residues predicted to mediate this electrostatic interaction using a two-hybrid analysis. The functional need for Arg96 in vivo was then confirmed by monitoring secretion of the effector ExoU. These structures will contribute to the development of atomic-resolution models of the entire sorting platform and to our understanding of the mechanistic interface between the sorting platform and the basal body of the injectisome. HighlightsO_LIThe structures of Pseudomonas aeruginosa PscD (SctD) and PscK (SctK) were solved C_LIO_LIThe interface between the T3SS basal body and sorting platform was modeled C_LIO_LIThe crystal structure of PscK is the first for any SctK family member C_LIO_LIPscK represents a novel protein fold C_LIO_LISite-directed mutagenesis supports a computational model of the PscD-PscK interface C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/308627v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1efcf14org.highwire.dtl.DTLVardef@1043d81org.highwire.dtl.DTLVardef@161f21corg.highwire.dtl.DTLVardef@15e4357_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. The first reported structure for a T3SS SctK protein family member was solved for PscK from Pseudomonas aeruginosa and this allowed for modeling of the interface between this sorting platform protein and the cytoplasmic domain of PscD (a SctD protein family member). This allowed for identification of amino acid residues that may play a role in the interaction between these proteins. The interface appears to be dominated by electrostatic interactions and mutagenesis confirmed the importance of key residues in driving their interaction based on two-hybrid analysis. C_FIG

biophysics↗