Biology subjects

Clubb, R. T.

Publications and source records attributed to Clubb, R. T..

8 recordsLinked to original sources

bioRxiv2026

Cryo-EM provides insight into how the Staphylococcus aureus IsdH receptor removes hemin from the hemoglobin:haptoglobin complex

Staphylococcus aureus extracts hemin from human hemoglobin (Hb) to overcome host-imposed iron limitation. How it recovers Hb-bound hemin from the hemoglobin:haptoglobin (Hb:Hp) complex, the major circulating form of Hb outside red blood cells, remains unclear. Here we use cryo-electron microscopy, biophysical measurements, and solution kinetics to define how the S. aureus IsdH surface receptor extracts hemin from Hb:Hp. A 3.1 [A] cryo-EM structure of Hb:Hp bound by full-length IsdH reveals that its N-terminal NEAT domain (N1) anchors it to Hb, whereas its downstream N2N3 extraction unit engages {beta}Hb to remove its hemin. The receptor engages Hb:Hp differently than isolated Hb, because N-linked glycans on haptoglobin bias the extraction unit toward {beta}Hb, sterically occluding its access to Hb while still permitting engagement by N1. Kinetic assays show that IsdH actively accelerates hemin release from Hb:Hp. Three-dimensional variability analysis indicates that this likely occurs via a dynamic interface in which receptor motions reposition the extraction unit relative to {beta}Hb, collectively supporting a model in which IsdH transiently perturbs the F-helix to promote hemin extraction. Alignment of that model with a previously determined CD163:Hb:Hp structure shows how IsdH may disrupt Hb:Hp recognition by macrophage and monocyte CD163 receptors, helping to explain how it may hinder clearance of Hb:Hp from circulation. In aggregate, these results help define the structural basis for hemin extraction from Hb:Hp and how IsdH may subvert receptor-mediated clearance of the Hb:Hp complex. SignificanceStaphylococcus aureus scavenges hemin from host hemoglobin to proliferate, yet most extracellular hemoglobin is sequestered in hemoglobin:haptoglobin (Hb:Hp) complexes, which are rapidly cleared from circulation. To clarify how bacteria access hemin in this context, we now show that the IsdH surface receptor is structurally adapted to extract hemin from Hb:Hp. Our results indicate that IsdH uses distinct NEAT domains to anchor to Hb and selectively extract hemin from {beta}Hb. This {beta}Hb selectivity is shaped by haptoglobin N-glycans and enhances microbial access to iron. These findings provide a mechanistic framework for targeting heme acquisition as an anti-virulence strategy.

microbiology↗

bioRxiv2026

AlphaFold-Driven Structural Proteomics Reveals Extensive Cellulosome Machinery in Human Ruminococcal Symbionts

Cellulosomes are large, surface-displayed enzyme complexes that enable anaerobic bacteria to degrade recalcitrant plant polysaccharides, yet cellulosome-expressing bacteria are thought to be rare in the human gut. Here we show that extensive sequence divergence obscures the detection of many ruminococcal cellulosomes by conventional sequence homology-based methods. Using proteome-scale AlphaFold2 structural predictions, we uncovered a substantially expanded set of cellulosome-producing Ruminococcus species, including six previously unrecognized human symbionts. Structure-based clustering identifies several novel cohesin families that retain conserved folds despite extreme sequence divergence and define distinct, phylogenetically conserved cellulosome architectures. The analysis reveals R. callidus and related human symbionts encode elaborate cellulosomes that are invisible to sequence-based annotation. Similarly, R. difficilis, a human gut symbiont, is found to produce an atypical cohesin-based assembly enriched in amylases and related starch-binding proteins that may enable this microbe to degrade resistant starches that evade digestion in the upper gastrointestinal tract. Together, these findings reveal that ruminococcal cellulosomes are far more prevalent and diverse than previously appreciated and demonstrate the power of structural proteomics to uncover deeply divergent functional systems in the gut microbiome. Significance StatementPlant cell wall polysaccharides are a major dietary carbon source, yet their degradation relies on rare, highly specialized microbial enzyme assemblies known as cellulosomes, which have long appeared uncommon in the human gut. Using proteome-scale structure prediction combined with experimental validation, we show that cellulosomes are far more widespread and structurally diverse in human-associated Ruminococcus species than previously appreciated. We identify multiple new cohesin families and reveal distinct cellulosome architectures likely adapted to degrade different dietary substrates. Together, these findings redefine the distribution and evolution of cellulosomes in gut microbes and demonstrate the power of structural proteomics to uncover deeply diverged biological systems.

microbiology↗

bioRxiv2026

Native Mass Spectrometry-Based Proteomics Reveals the Mechanism of Hemophore Release by Pathogenic Corynebacterium diphtheriae

Iron is an essential micronutrient for nearly all forms of life, including pathogenic microbes that must acquire it from their host during infection. At the host-pathogen interface, humans restrict microbial access to iron through nutritional immunity, which many pathogens overcome by secreting hemophores that scavenge extracellular heme (iron protoporphyrin IX). However, identifying hemophores and other ligand-binding proteins in complex proteomes remains challenging using conventional peptide-based bottom-up mass spectrometry (MS). Here, we introduce ProteoMIX (Proteome Analysis by Mixing), a function-based native top-down proteomics workflow that combines slow-mixing mode native MS with charge reduction to identify ligand-binding proteins directly from complex mixtures. Applying ProteoMIX to the Corynebacterium diphtheriae exoproteome identified ChtA30-314, an abundant soluble hemophore generated by proteolytic processing of the surface-exposed ChtA heme receptor. Using native top-down MS sequencing, cell fractionation, and gene deletion, we show that the protease DIP2069 releases ChtA30-314 by removing ChtAs transmembrane helix, producing a soluble proteoform that delivers heme to support microbial growth. In contrast, the related paralog ChtC is not processed, enabling C. diphtheriae to generate localization-specific heme-binding proteoforms from related gene products. Extending this approach to Staphylococcus aureus, ProteoMIX also revealed soluble IsdA hemophores that coexist with surface-anchored variants, demonstrating the generality of the method and suggesting that protease-mediated hemophore release may operate across gram-positive pathogens.

microbiology↗

bioRxiv2024

The exoproteome and surfaceome of toxigenic Corynebacterium diphtheriae 1737 and its response to iron-restriction and growth on human hemoglobin

Toxin-producing Corynebacterium diphtheriae strains are the etiological agent of the severe upper respiratory disease, diphtheria. A global phylogenetic analysis revealed that biotype gravis is particularly lethal as it produces diphtheria toxin and a range of other virulence factors, particularly when it encounters low levels of iron at sites of infection. To gain insight into how it colonizes its host we have identified iron-dependent changes in the exoproteome and surfaceome of C. diphtheriae strain 1737 using a combination of whole-cell fractionation, intact cell surface proteolysis, and quantitative proteomics. In total, we identified 1,425 of the predicted 2,265 (63%) proteins encoded by its reference genome. For each protein we quantified its degree of secretion and surface-exposure, revealing that exoproteases and hydrolases predominate in the exoproteome, while the surfaceome is enriched with adhesins, particularly DIP2093. Our analysis provides insight into how components in the heme-acquisition system are positioned, showing pronounced surface-exposure of the strain-specific ChtA/ChtC paralogues and high secretion of the species-conserved heme-binding HtaA protein suggesting it functions as a hemophore. Profiling the response of the exoproteome and surfaceome after microbial exposure to human hemoglobin and iron limitation reveals potential virulence factors that may be expressed at sites of infection. Data are available via ProteomeXchange with identifier PXD051674.

biochemistry↗

bioRxiv2023

The Basal and Major Pilins in the Corynebacterium diphtheriae SpaA Pilus Adopt Similar Structures that Competitively React with the Pilin Polymerase

Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the CdSrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that CdSrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (NSpaA) that is also crosslinked by CdSrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed "latch" mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting NSpaA for access to a shared thioester enzyme-substrate reaction intermediate.

biochemistry↗

bioRxiv2021

Directed inter-domain motions enable the IsdH Staphylococcus aureus receptor to rapidly extract heme from human hemoglobin

Pathogenic Staphylococcus aureus actively acquires iron from human hemoglobin (Hb) using the IsdH surface receptor. Heme extraction is mediated by a tridomain unit within the receptor that contains its second (N2) and third (N3) NEAT domains joined by a helical linker domain. Extraction occurs within a dynamic complex, in which receptors engage each globin chain; the N2 domain tightly binds to Hb, while substantial inter-domain motions within the receptor enable its N3 domain to transiently distort the globins heme pocket. Using molecular simulations, Markov modeling, and quantitative measurements of heme transfer kinetics, we show that directed inter-domain motions within the receptor play a critical role in the extraction process. The directionality of N3 domain motion and the rate of heme extraction is controlled by amino acids within a short, flexible inter-domain tether that connects the N2 and linker domains. In the wild-type receptor directed motions originating from the tether enable the N3 domain to populate configurations capable of distorting Hbs pocket, whereas mutant receptors containing altered tethers are less able to adopt these conformers and capture heme slowly via indirect processes in which Hb first releases heme into the solvent. Thus, our results show inter-domain motions within the IsdH receptor play a critical role in its ability to extract heme from Hb and highlight the importance of directed motions by the short, unstructured, amino acid sequence connecting the domains in controlling the directionality and magnitude of these functionally important motions.

biophysics↗

bioRxiv2020

Secreted and cell surface proteome analysis of pathogenic Corynebacterium diphtheriae reveals proteins relevant to virulence

Corynebacterium diphtheriae is responsible for the severe upper respiratory tract disease, diphtheria, which can be fatal in healthy individuals without proper treatment. Its interaction with the infected host is crucial to its virulence, secreting diphtheria toxin and a variety of machinery to acquire nutrients for further import into the cell. Additionally, it shares a conserved iron import mechanism with related pathogenic actinobacteria such as Mycobacterium tuberculosis, and contains sortase-mediated anchored cell wall proteins similar to other gram-positive bacteria. Information obtained from secreted and cell-surface proteomes are relevant for the study of diphtheria and related bacterial infections. Mass spectrometry-based proteomics measurements on samples of pathogenic Corynebacterium diphtheriae culture supernatant and cell-surface digested proteins identified greater than 3 times more proteins than a previous similar study. The diphtheria toxin was identified, as well as pathologically relevant proteins involved in iron-uptake and cell adhesion. For instance, 17 proteins predicted to be on the iron-regulated DtxR promoter were identified in cells grown under different iron concentrations, opening the door to performing comparative quantitative proteomics studies on samples where iron sources are modulated. Results of this study serve as a repository for future studies on pathogenic C. diphtheriae uptake.

microbiology↗

bioRxiv2018

Structure and mechanism of TagA, a novel membrane-associated glycosyltransferase that produces wall teichoic acids in pathogenic bacteria

Staphylococcus aureus and other bacterial pathogens affix wall teichoic acids (WTAs) to their surface. These highly abundant anionic glycopolymers have critical functions in bacterial physiology and their susceptibility to {beta}-lactam antibiotics. The membrane-associated TagA glycosyltranserase (GT) catalyzes the first-committed step in WTA biosynthesis and is a founding member of the WecB/TagA/CpsF GT family, more than 6,000 enzymes that synthesize a range of extracellular polysaccharides through a poorly understood mechanism. Crystal structures of TagA from T. italicus in its apo- and UDP-bound states reveal a novel GT fold, and coupled with biochemical and cellular data define the mechanism of catalysis. We propose that enzyme activity is regulated by interactions with the bilayer, which trigger a structural change that facilitates proper active site formation and recognition of the enzymes lipid-linked substrate. These findings inform upon the molecular basis of WecB/TagA/CpsF activity and could guide the development of new anti-microbial drugs.\n\nAUTHOR SUMMARYGram-positive bacteria cause thousands of deaths in the United States each year and are a growing health concern because many bacterial strains have become resistant to commonly used antibiotics. One of the most abundant polymers displayed on the surface of Gram-positive bacteria is wall teichoic acid (WTA), a negatively charged carbohydrate polymer that has critical functions in cell division, morphology, adhesion and pathogenesis. The WTA biosynthetic pathway has drawn significant interest as a drug target because clinically important methicillin-resistant S. aureus (MRSA) strains that lack WTA are defective in host colonization and re-sensitized to {beta}-lactam antibiotics. To understand how bacteria produce WTA, we determined the structure and deduced the enzymatic mechanism of TagA, an important enzyme that is required for WTA synthesis. This research reveals a new method for enzyme regulation, whereby peripheral membrane association enables TagA to adopt its active form as a monomer. As TagA enzymes are highly conserved in bacteria, they can be expected to operate through a similar mechanism. The results of this work provide insight into WTA biosynthesis and could lead to innovative approaches to treat infections caused by pathogenic bacteria.

biochemistry↗