Biology subjects

Kiessling, L. L.

Publications and source records attributed to Kiessling, L. L..

16 recordsLinked to original sources

bioRxiv2026

A TmaT-AftD interaction is required for the CmpL4 mycomembrane biogenesis pathway in Corynebacterium glutamicum.

Bacteria in the Mycobacteriales order like mycobacteria and corynebacteria surround themselves with a multilayered cell envelope. Their cytoplasmic membrane is fortified by a peptidoglycan cell wall that is decorated with branched arabinogalactan (AG) polymers. The AG glycans are further modified with mycolic acids to form an outer membrane. Biogenesis of this mycomembrane requires the transport of mycolates from their site of synthesis in the cytoplasmic membrane to the cell surface. How mycolate transport is controlled and coordinated with AG synthesis has remained unclear. Mycolate transport is mediated by the essential RND-family transporter MmpL3 in mycobacteria and a pair of related, partially redundant transporters called CmpL1 and CmpL4 in corynebacteria. The acetyltransferase TmaT has also been implicated in mycolate transport in both types of bacteria. In corynebacteria, it is required for the production of acetylated mycolates, and this modification has been proposed to promote mycolate transport via both CmpL transporters in the model organism Corynebacterium glutamicum (Cglu). Here, we reinvestigated the function of TmaT in Cglu and found that it and several factors encoded in the tmaT locus are specifically required for mycolate transport via the CmpL4 transporter pathway. Notably, one of these additional genes encodes the arabinosyltransferase AftD involved in AG biogenesis. TmaT and AftD were found to interact, and our results indicate that this interaction is required for acetylated mycolate production, mycolate transport via the CmpL4 pathway, and normal arabinan synthesis. Thus, the TmaT-AftD interaction may serve as a regulatory link connecting mycolate transport with AG biogenesis. SIGNIFICANCEMycobacteriales bacteria, including pathogens like Mycobacterium tuberculosis (Mtb), have a complex cell surface comprising an inner membrane, a cell wall modified with arabinogalactan (AG), and an outer mycomembrane made of mycolic acids linked to AG polymers. Because these surface biogenesis pathways are targeted by frontline anti-Mtb drugs, there is great interest in elucidating their underlying mechanisms. Here, we identify an interaction between factors involved in mycomembrane (TmaT) and AG biosynthesis (AftD) in the model organism Corynebacterium glutamicum. We show that this interaction is important for proper surface biogenesis and may therefore function to coordinate mycomembrane assembly with AG synthesis. This and other potential regulatory connections controlling envelope biogenesis represent attractive targets for future antibiotic development.

microbiology↗

bioRxiv2026

Astrocyte-driven small vessel disease is an early, amyloid-independent feature of PSEN1 E280A familial Alzheimer's disease

Cerebral Small vessel disease (cSVD) is a prevalent feature of Alzheimers disease (AD) pathology. Whether this pathology is a late consequence of amyloid and tau accumulation or an early, direct effect of PSEN1 dysfunction has remained unresolved. We found that it is more severe in familial AD (FAD) caused by E280A mutation in presenilin 1 (PSEN1). These cases present with a distinctive proteomic signature, associated with pathological features, more dysregulated in the occipital cortex (OC) compared to the frontal cortex (FC), and characterized by multiple dysregulated proteins involved in extracellular matrix (ECM) and RNA-associated processes. This proteomic fingerprint was associated with abnormal collagen build up, ECM disorganization, and signatures of aberrant angiogenesis. Six months old transgenic knock-in mice homozygous for Psen1 E280A mutation (PSEN1Ki) also showed a similar phenotype with microvascular tortuosity and proteomic changes. Critically, these mice develop neither A{beta} plaques nor tau tangles, indicating that the shared microvascular and RNA-associated changes are direct consequences of PSEN1 dysfunction rather than downstream effects of amyloid pathology. Remarkably, dysregulated RNA-associated protein networks overlapped between FAD and PSEN1Ki mice. Cerebral microvessels microstructure in PSEN1Ki mice at two months and six months showed abnormal astrocytic end-feet with lamellar deposits implicating blood-brain barrier damage. Finally, single nuclei transcriptomic analysis of AD patients and controls showed similar abnormal astrocytes in both sporadic and familial variants, but FAD astrocytes expressed dysregulated genes identified in the proteomic analyses, such as GLUL, APOE, and CLU. Our findings suggest that cSVD is an early pathological event in PSEN1 FAD and that is driven by abnormal RNA-associated processes and astrocytic dysfunction.

neuroscience↗

bioRxiv2026

Biological recognition of mirror-image glycans

Recent synthesis of essential enzymes, such as DNA and RNA polymerases with opposite chirality, has boosted the feasibility of creating mirror-image life. Such life, if ever produced, will undoubtedly be coated by a dense display of glycans (glycoproteins, glycolipids, polysaccharides) built from enantiomers of common monosaccharides. Recognition of mirror-image glycans by extant glycan-binding proteins (GBPs) may be critical for colonization by or immune response to mirror life organisms. We evaluated recognition of enantiomers of common glycans by a diverse set of purified GBPs (plant and human derived), antibodies (including IgM from human plasma), mammalian cells (including immune cells), and organs in live animals. We found that GBP binding to enantiomers of naturally prevalent glycans is widespread. Notably, L-glucose and L-galactose interact with fucose-binding lectins, including DC-SIGN, a C-type lectin expressed on immune cells. These interactions can be inhibited by soluble "natural" glycan ligands and enantiomeric ones confirming specificity. Binding of L-glycans to diverse immune cell repertoires revealed preferences for specific glycan enantiomers. IgM antibodies from human serum showed donor-specific recognition of L-glycans. We propose that the recognition of L-glycans by extant GBPs arises from their co-evolution over millennia with the L-glycans that are present in the glycocalyx of many microorganisms.

biochemistry↗

bioRxiv2026

Principles of in situ protein sequencing: expansion microscopy-adapted Edman degradation and amino acid recognition

The ability to map protein identity, with resolution sufficient to infer interactions, would support analysis of how proteins work together, or malfunction, in biological processes and diseases. Although several emerging technologies aim towards single-molecule protein sequencing, they require proteins to be removed from the nanoscale spatial context of cells and tissues. Expansion microscopy (ExM) has facilitated a diversity of chemical analyses by isotropically separating molecules throughout a specimen after permeation via a charged hydrogel, followed by gel swelling. Here, we adapt key protein sequencing steps - Edman degradation and amino acid recognition - to the ExM gel context. Using testbed peptides in ExM gels, we show that N-terminal amino acids can be recognized over multiple cycles of in-gel Edman degradation. This principle-oriented study demonstrates sequencing chemistry on defined synthetic constructs, rather than endogenous proteins in biological samples. These results establish principles of in situ protein sequencing and provide a framework for future in situ protein sequencing developments, including the development of higher specificity and affinity amino acid binders.

biochemistry↗

bioRxiv2025

The phenotypic landscape of the mycobacterial cell

The Mycobacteriales are an order of diverse bacteria that thrive in many environmental and host-associated niches. Because the most notorious member of this clade, Mycobacterium tuberculosis, is a major human pathogen, research on Mycobacteriales has focused on pathogenesis, and, as a consequence, many fundamental aspects of Mycobacterial biology remain understudied. Here, we address this gap by performing a genome-wide CRISPRi chemical genomics screen using a diverse set of >35 antibiotics, detergents, and other anti-microbials predominantly targeting the cell envelope of Mycobacterium smegmatis, a saprophytic model Mycobacterium. We highlight new information derived from this screen, including the identification of novel functions for previously uncharacterized conserved and essential genes (in mycolic acid and arabinogalactan synthesis), the discovery of a new drug scaffold/protein target pair, and insights into the mechanism of action of two commonly used antibiotics. These data are also a valuable resource for the mycobacterial research community, as they provide thousands of novel phenotypes for uncharacterized genes and meaningful phenotypic correlations between annotated and uncharacterized genes.

microbiology↗

bioRxiv2025

Intelectin-2 is a broad-spectrum antimicrobial lectin

Mammals regulate the localization, composition, and activity of their native microbiota at colonization sites. Lectins residing at these sites influence microbial populations, but their individual functions are often unclear. Intelectins are found in chordates at mucosal barriers, but their functions are not well characterized. We found that mouse intelectin-2 (mItln2) and human intelectin-2 (hItln2) engage and crosslink mucins via carbohydrate recognition. Moreover, both lectins recognize microbes within native microbial communities, including gram-positive and gram-negative isolates from the respiratory and gastrointestinal tracts. This ability to engage mammalian and microbial glycans arises from calcium-coordinated binding of carbohydrate residues within mucus and microbial surfaces. Microbes, but not human cells, bound by mItln2 or hItln2, suffer a loss of viability. These findings underscore the crucial antimicrobial role of mammalian intelectin-2 in mucosal defense, where it plays offensive (microbial killing) and defensive (mucus crosslinking) roles in regulating microbial colonization.

microbiology↗

bioRxiv2025

Synthetic Mucins as Glycan-Defined Prebiotics

The human microbiome contains at least as many bacterial cells as human cells. The mucosal layer that lines all epithelial cells organizes, cultivates, and regulates these bacterial inhabitants. Some commensal bacteria offer benefits, like improving gut barrier function, suppressing pathobiont growth, and modulating host immunity. These health benefits have fueled the popularity of probiotics, but their retention is often hindered by their low colonization efficiency and mucosal adhesion. Mucins, the primary structural components of the mucosal layer, are essential for the organization and regulation of microbial populations, promoting growth and offering sites for adhesion through their multivalent presentation of O-glycosylation. The molecular mechanisms of mucin- probiotic interactions remain understudied due, in part, to the inability to incisively manipulate native mucin sequences or the glycans they bear. In this investigation, we developed synthetic mucins with defined glycan presentations to interrogate glycan-dependent interactions between mucus and probiotic Lactobacillus species. Though synthetic mucins can dampen the effects of pathogens, toxins, and viruses, their impact on probiotic bacteria as prebiotics or binding sites is unclear. We synthesized mucin surrogates that bind to three investigated Lactobacillus species. The nutrient conditions under which bacteria were cultured influenced glycan binding preferences, suggesting mucin-probiotic interactions change with nutrient availability. The addition of synthetic mucins to native mucin increased Lactobacillus fermentum adherence. Additionally, an increase in Lactobacillus glycosidase activity indicated that native and synthetic mucins both function as prebiotics, as probiotic bacteria can cleave the displayed O-glycans. Thus, synthetic mucins can cultivate target probiotic bacteria and increase adhesion as binding sites, highlighting their value as tools for elucidating native mucin functions and as promising agents for promoting human health.

biochemistry↗

bioRxiv2024

Glycan analysis probes inspired by human lectins for investigating host-microbe crosstalk

Human lectins are critical carbohydrate-binding proteins that recognize diverse glycoconjugates from microorganisms and can play a key role in host-microbe interactions. Despite their importance in immune recognition and pathogen binding, the specific glycan ligands and functions of many human lectins remain poorly understood. Using previous proof-of-concept studies on selected lectins as the foundation for this work, we present ten additional glycan analysis probes (GAPs) from a diverse set of human soluble lectins, offering robust tools to investigate glycan-mediated interactions. We describe a protein engineering platform that enables scalable production of GAPs that maintain native-like conformations and oligomerization states, equipped with functional reporter tags for targeted glycan profiling. We demonstrate that the soluble GAP reagents can be used in various applications, including glycan array analysis, mucin- binding assays, tissue staining, and microbe binding in complex populations. These capabilities make GAPs valuable for dissecting interactions relevant to understanding host responses to microbes. The tools can be used to distinguish microbial from mammalian glycans, which is crucial for understanding the cross-target interactions of lectins in a physiological environment where both glycan types exist. GAPs have potential as diagnostic and prognostic tools for detecting glycan alterations in chronic diseases, microbial dysbiosis, and immune-related conditions.

biochemistry↗

bioRxiv2024

Massively parallel combination screen reveals small molecule sensitization of antibiotic-resistant Gram-negative ESKAPE pathogens

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces. Here, we deployed a high-throughput combinatorial screening platform, DropArray, to evaluate the interactions of over 30,000 compounds with up to 22 antibiotics and 6 strains of Gram-negative ESKAPE pathogens, totaling to over 1.3 million unique strain-antibiotic-compound combinations. In this dataset, compounds more frequently exhibited synergy with known antibiotics than single-agent activity. We identified a compound, P2-56, and developed a more potent analog, P2-56-3, which potentiated rifampin (RIF) activity against Acinetobacter baumannii and Klebsiella pneumoniae. Using phenotypic assays, we showed P2-56-3 disrupts the outer membrane of A. baumannii. To identify pathways involved in the mechanism of synergy between P2-56-3 and RIF, we performed genetic screens in A. baumannii. CRISPRi-induced partial depletion of lipooligosaccharide transport genes (lptA-D, lptFG) resulted in hypersensitivity to P2-56-3/RIF treatment, demonstrating the genetic dependency of P2-56-3 activity and RIF sensitization on lpt genes in A. baumannii. Consistent with outer membrane homeostasis being an important determinant of P2-56-3/RIF tolerance, knockout of maintenance of lipid asymmetry complex genes and overexpression of certain resistance-nodulation-division efflux pumps - a phenotype associated with multidrug-resistance - resulted in hypersensitivity to P2-56-3. These findings demonstrate the immense scale of phenotypic antibiotic combination screens using DropArray and the potential for such approaches to discover new small molecule synergies against multidrug-resistant ESKAPE strains. Significance StatementThere is an unmet need for new antibiotic therapies effective against the multidrug-resistant, Gram-negative ESKAPE pathogens. Combination therapies have the potential to overcome resistance and broaden the spectrum of existing antibiotics. In this study, we use DropArray, a massively parallel combinatorial screening tool, to assay more than 1.3 million combinations of small molecules against the Gram-negative ESKAPE pathogens, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. We discovered a synthetic small molecule potentiator, P2-56, of the antibiotic rifampin effective in A. baumannii and K. pneumoniae. We generated P2-56-3, a more potent derivative of P2-56, and found that it likely potentiates rifampin by compromising the outer membrane integrity. Our study demonstrates a high-throughput strategy for identifying antibiotic potentiators against multidrug-resistant bacteria.

microbiology↗

bioRxiv2024

Glycan-costumed virus-like particles promote type 1 anti-tumor immunity

Cancer vaccine development is inhibited by a lack of strategies for directing dendritic cell (DC) induction of effective tumor-specific cellular immunity. Pathogen engagement of DC lectins and toll-like receptors (TLRs) shapes immunity by directing T cell function. Strategies to activate specific DC signaling pathways via targeted receptor engagement are crucial to unlocking type 1 cellular immunity. Here, we engineered a glycan-costumed virus-like particle (VLP) vaccine that delivers programmable peptide antigens to induce tumor-specific cellular immunity in vivo. VLPs encapsulating TLR7 agonists and decorated with a selective mannose-derived ligand for the lectin DC-SIGN induced robust DC activation and type 1 cellular immunity, whereas VLPs lacking this key DC-SIGN ligand failed to promote DC-mediated immunity. Vaccination with glycan-costumed VLPs generated tumor antigen-specific Th1 CD4+ and CD8+ T cells that infiltrated solid tumors, inhibiting tumor growth in a murine melanoma model. Thus, VLPs employing lectin-driven immune reprogramming provide a framework for advancing cancer immunotherapies.

biochemistry↗

bioRxiv2022

Lectin-Seq: a method to profile lectin-microbe interactions in native communities

Soluble human lectins are critical components of innate immunity. Genetic models suggest lectins influence host-resident microbiota, but their specificity for commensal and mutualist species is understudied. Elucidating lectins roles in regulating microbiota requires understanding which microbial species they bind within native communities. To profile human lectin recognition, we developed Lectin-Seq. We apply Lectin-Seq to human fecal microbiota using mannose-binding lectin (MBL) and intelectin-1 (hItln1). The microbial interactomes of MBL and hItln1 differ in composition and diversity. MBL binding is highly selective for a small subset of species commonly associated with humans. In contrast, hItln1s interaction profile encompasses a broad range of lower-abundance species. Thus, human lectins have evolved to recognize distinct species of commensals, suggesting they directly influence microbiome composition. Lectin-Seq offers a new means of annotating microbial communities. One-Sentence SummarySoluble human lectins bind distinct bacterial species in fecal microbiota.

biochemistry↗

bioRxiv2022

Rapid Single-Shot Synthesis of the 217 Amino Acid-Long N-Terminal Domain of Pyocin S2

The impermeable outer membrane of Pseudomonas aeruginosa is bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials against Pseudomonas infections. Their production and modification, however, remains challenging. To address this limitation, we employed automated fast-flow peptide synthesis (AFPS) for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2NTD) was synthesized in under 10 hours and purified to yield milligrams quantities of the desired product. To our knowledge, the 217 amino acid sequence of PyS2NTD is among the longest peptides produced from a "single-shot" synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2NTD with circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2NTD binds to P. aeruginosa expressing the cognate ferripyoverdine receptor (FpvA) and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching (FRAP). These modified, synthetic S-type pyocins domains can be used to probe import mechanisms of P. aeruginosa and leveraged to develop selective antimicrobial agents that bypass the outer membrane.

synthetic biology↗

bioRxiv2022

Antibiotic action revealed by real-time imaging of the mycobacterial membrane

The current understanding of mycobacterial cell envelope remodeling in response to antibiotics is limited. Chemical tools that report on phenotypic changes with minimal cell wall perturbation are critical to understanding such time-dependent processes. We employed a fluorogenic chemical probe to image how antibiotics perturb mycobacterial cell envelope assembly in real-time. Time-lapse microscopy revealed that differential antibiotic treatment elicited unique cellular phenotypes, providing a platform for simultaneously monitoring cell envelope construction and remodeling responses. Our data show that rifampicin, which does not directly inhibit cell wall biosynthesis, affords a readily detected mycomembrane phenotype. The fluorogenic probe revealed the production of extracellular vesicles in response to antibiotics, and analyses of these vesicles indicate that antibiotic treatment elicits the release of agents that attenuate macrophage activation.

microbiology↗

bioRxiv2021

Bifunctional Peptide that Anneals to Damaged Collagen and Clusters TGF-β Receptors Enhances Wound Healing

Transforming growth factor-{beta} (TGF-{beta}) plays important roles in wound healing. The activity of TGF-{beta} is initiated upon binding of the growth factor to extracellular domains of its receptors. We sought to facilitate activation by clustering these extracellular domains. To do so, we used a known peptide that binds to TGF-{beta} receptors without diminishing their affinity for TGF-{beta}. We conjugated this peptide to a collagen-mimetic peptide that can anneal to damaged collagen in a wound bed. We find that the conjugate enhances collagen deposition and wound closure in mice in a manner consistent with the clustering of TGF-{beta} receptors. This strategy provides a means to upregulate the TGF-{beta} signaling pathway without adding exogenous TGF-{beta} and could inspire means to treat severe wounds. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/461420v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@a0c248org.highwire.dtl.DTLVardef@332b3forg.highwire.dtl.DTLVardef@15c3533org.highwire.dtl.DTLVardef@a8b75a_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Discovery of small molecule pathway regulators by image profile matching

Identifying chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual profile-based screening approach instead identifies compounds that match to pathways based on phenotypic information in public cell image data, created using the Cell Painting assay. Our straightforward correlation-based computational strategy retrospectively uncovered the expected, known small molecule regulators for 32% of positive-control gene queries. In prospective, discovery mode, we efficiently identified new compounds related to three query genes, and validated them in subsequent gene-relevant assays, including compounds that phenocopy or pheno-oppose YAP1 overexpression and kill a Yap1-dependent sarcoma cell line. This image profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds. One sentence summaryIf a genetic perturbation impacts cell morphology, a computational query can reveal compounds whose morphology "matches".

bioinformatics↗

bioRxiv2021

Biosynthetic Glycan Labeling

Glycans are ubiquitous and play important biological roles, yet chemical methods for probing their structure and function within cells remain limited. Strategies for studying other biomacromolecules, such as proteins, often exploit chemoselective reactions for covalent modification, capture, or imaging. Unlike amino acids that constitute proteins, glycan building blocks lack distinguishing reactivity because they are composed primarily of polyol isomers. Moreover, encoding glycan variants through genetic manipulation is complex. Therefore, we formulated a new, generalizable strategy for chemoselective glycan modification that directly takes advantage of cellular glycosyltransferases. Many of these enzymes are selective for the products they generate yet promiscuous in their donor preferences. Thus, we designed reagents with bioorthogonal handles that function as glycosyltransferase substrate surrogates. We validated the feasibility of this approach by synthesizing and testing probes of D-arabinofuranose (D-Araf), a monosaccharide found in bacteria and an essential component of the cell wall that protects mycobacteria, including Mycobacterium tuberculosis. The result is the first probe capable of selectively labeling arabinofuranose-containing glycans. Our studies serve as a platform for developing new chemoselective labeling agents for other privileged monosaccharides. This probe revealed an asymmetric distribution of D-Araf residues during mycobacterial cell growth and could be used to detect mycobacteria in THP1-derived macrophages.

biochemistry↗