Biology subjects

Garcia, B. L.

Publications and source records attributed to Garcia, B. L..

10 recordsLinked to original sources

bioRxiv2026

Developmental dysregulation of chandelier cell excitability in a mouse model of Dravet Syndrome

Chandelier cells (ChCs) are a rare and highly specialized sub-type of inhibitory neuron in the cerebral cortex that specifically and exclusively form synapses onto the axon initial segment of excitatory neurons and thereby exert powerful control of excitability. We applied a newly developed, cell type-specific enhancer element to label ChCs and assess their electrophysiological and morphological properties across development in Dravet Syndrome (Scn1a+/- mice), a prominent neurodevelopmental disorder defined by epilepsy and features of autism spectrum disorder. Dravet Syndrome is known to predominantly impact GABAergic interneurons. We found that ChCs from juvenile Scn1a+/- mice (postnatal day, P18-21) exhibit impaired excitability, and that deficits largely persist in young adulthood (P35-56). These findings are distinct from prior observations in the more common subtype of parvalbumin-positive interneurons, basket cells. We found no differences in the axonal or dendritic morphology of ChCs at either time point. Our results suggest a role for ChCs in the pathogenesis of Dravet Syndrome and constitute the first targeted study of ChC function in a specific genetically-defined neurodevelopmental disorder.

neuroscience↗

bioRxiv2025

Discrete interneuron subsets participate in GluN1/GluN3A excitatory glycine receptor (eGlyR)-mediated regulation of hippocampal network activity throughout development and evolution.

Decades of studies implicating GluN3A N-methyl-D-aspartate receptor (NMDAR) subunits in physiological and pathological function have largely been interpreted through direct regulation of conventional glutamatergic NMDARs. However, emerging evidence indicates that GluN3A frequently assembles with GluN1 forming unconventional glutamate-insensitive NMDARs that operate as native excitatory glycine receptors (eGlyRs). Here we demonstrate that hippocampal somatostatin and neurogliaform interneurons (Sst-INs and NGFCs) express functional eGlyRs from early postnatal through adult ages. In the developing hippocampus eGlyR-mediated excitation of NGFCs with ambient glycine dramatically increases GABAergic tone, with consequences for the generation of giant depolarizing potentials (GDPs). In the mature hippocampus, eGlyR- mediated excitation of Sst-INs regulates sharp wave ripples (SWRs). Finally, we reveal evolutionary conservation of hippocampal Sst-IN eGlyRs and eGlyR- mediated SWR regulation in non-human primates confirming functional eGlyR availability for therapeutic potential in higher species. Our findings underscore that eGlyR mediated regulation of cell and circuit excitability through both cell autonomous and cell non-autonomous mechanisms must be considered to understand GluN3A roles in brain development, plasticity, and disease.

neuroscience↗

bioRxiv2025

A novel, evolutionarily conserved inhibitory circuit selectively regulates dentate gyrus mossy cell function.

The mammalian dentate gyrus contributes to mnemonic function by parsing similar events and places. The disparate activity patterns of mossy cells and granule cells is believed to enable this function yet the mechanisms that drive this circuit dynamic remain elusive. We identified a novel inhibitory interneuron subtype, characterized by VGluT3 expression, with overwhelming target selectivity for mossy cells while also revealing that CCK, PV, SOM and VIP interneurons preferentially innervate granule cells. Leveraging pharmacology and novel enhancer viruses, we find that this target-specific inhibitory innervation pattern is evolutionarily conserved in non-human primates and humans. In addition, in vivo chemogenetic manipulation of VGluT3+ interneurons selectively alters the activity and functional properties of mossy cells. These findings establish that mossy cells and granule cells have unique, evolutionarily conserved inhibitory innervation patterns and suggest selective inhibitory circuits may be necessary to maintain DG circuit dynamics and enable pattern separation across species.

neuroscience↗

bioRxiv2024

Heterologous Surface Display Reveals Conserved Complement Inhibition and Functional Diversification of Borrelia burgdorferi Elp Proteins

Lyme disease is a tick-borne spirochetosis with diverse clinical manifestations. Phenotypic variation among Borrelia burgdorferi strains correlates with variable manifestations of Lyme disease in humans; this diversity is attributed in part to variation in surface-exposed lipoproteins, which are targets of the human antibody response and contribute to tissue adhesion, immune evasion, and other host interactions. Many B. burgdorferi lipoproteins are encoded as multi-copy gene families including the OspE/F- like leader peptide (Elp) protein family. To characterize Elp allelic variants, we adapted the Pseudomonas syringae ice nucleation protein (INP) system to present B. burgdorferi lipoproteins on the surface of Escherichia coli. We identified interactions with classical complement proteins and mapped binding regions in the E. coli system. We validated interactions using recombinant proteins and B. burgdorferi surface display. By assessing potential interactions with extracellular matrix components, we identified a novel interaction between Elp proteins and perlecan, a component of mammalian basement membranes, and revealed the bifunctional nature of Elps. Our findings reveal that Elps have undergone functional diversification while maintaining classical complement inhibition mediated by potent and conserved C1s binding and demonstrate that E. coli surface display offers an efficient, cost-effective, and relatively high throughput approach to characterize B. burgdorferi lipoproteins.

microbiology↗

bioRxiv2024

The dual activity of BBK32: Implications for simultaneous inhibition of borrelial-specific antibody-dependent complement activation and fibronectin binding

Complement inhibition is exploited by extracellular pathogens to combat clearance. Borreliella burgdorferi, the causative agent of Lyme disease, harnesses complement evasion techniques to establish and maintain infection in mammalian hosts. B. burgdorferi encodes bbk32, a surface lipoprotein that binds extracellular matrix (ECM) components, specifically glycosoaminoglycans (GAGs) and fibronectin (Fn) within its amino terminus. In addition to its ECM-binding functions, the carboxy terminal half of BBK32 binds to the C1r protease and prevents complement activation. This classical complement inhibitory activity protects B. burgdorferi from complement-mediated killing following the addition of normal human serum. Herein we demonstrate that full length BBK32 binds both Fn and C1 concurrently, indicating that binding of these macromolecules do not sterically hinder their simultaneous interaction. Given the link of antibody dependence to the classical pathway, we tested how the presence of BBK32 would protect B. burgdorferi from antibody mediated, complement dependent killing. The presence of BBK32 provided protection against borrelial-specific antibody binding and concomitant complement activation in vitro. We also demonstrated, using both fluorescence microscopy and flow cytometry, that the presence of BBK32 was required for reduced C4 deposition on the surface of borrelial cells. This work demonstrates the potential for BBK32 to simultaneously bind to both C1r and Fn and contributes to the broader understanding of the ability of B. burgdorferi to evade antibody-dependent complement-mediated killing. We contend that these observations ostensibly provide B. burgdorferi with coincident dissemination and immune evasion activities needed for optimal survival during infection. AUTHOR SUMMARYLyme disease, caused by Borreliella burgdorferi and other related species, is the most common arthropod-borne infection in the United States. As an extracellular pathogen, B. burgdorferi is exposed to the complement system--a soluble proteolytic cascade that clears invaders. Complement is defined by three pathways known as the alternative, lectin, and classical. The classical complement cascade is activated by the binding of antibodies to a foreign or damaged cell. For B. burgdorferi, the BBK32 surface protein is known to mute the classical pathway by binding and inhibiting the initiating protease C1r. However, no studies have addressed how BBK32 protects infectious B. burgdorferi from borrelial-specific antibody binding and clearance. Here we show that native BBK32 protects infectious B. burgdorferi from antibody-dependent, complement mediated killing. Given BBK32s other activity--namely its known adherence to fibronectin--we were interested to test if BBK32 could bind the C1 complex, which contains C1r, together with fibronectin. Our results suggest that these complex macromolecules can bind BBK32 simultaneously. These observations suggest that the dual activity of BBK32, namely fibronectin binding and C1r inhibition, are not mutually exclusive and contribute to B. burgdorferis ability to establish infection and evade antibody-based host clearance, respectively.

microbiology↗

bioRxiv2024

Novel roles for pirin proteins and a 2-ketoglutarate:ferredoxin oxidoreductase ortholog in Bacteroides fragilis central metabolism and comparison of metabolic mutants susceptibility to antimicrobials metronidazole and amixicile.

The regulation of the central metabolism and fermentation pathways and its effect on antimicrobial susceptibility in the anaerobic pathogen Bacteroides fragilis is not completely understood. In this study we show that B. fragilis encodes for two iron-dependent redox-sensitive regulatory pirin protein genes, pir1 and pir2, whose mRNA expression are upregulated following oxygen exposure and growth in iron-limiting conditions.{Delta} pir1 and{Delta} pir2 mutants showed altered short-chain fatty acids production compared to the parent strain. Overexpression of Pir1 and Pir2 increased susceptibility to metronidazole (MTZ), and to amixicile (AMIX), a novel inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobes. We hypothesized these observations were a result of a modulatory effect of Pir1 and Pir2 proteins. Consistent with this, we showed that Pir1 forms direct protein-protein interactions with PFOR. In addition, AlphaFold2-based structural analysis predicts that Pir1 and Pir2 form stable interactions with several enzymes of the central metabolism including the 2-ketoglutate:ferredoxin oxidoreductases (KFOR), Kor1AB and Kor2CDAEBG. A series of metabolic mutants and electron transport chain inhibitors were used to show a wide-ranging effect of bacterial metabolism on MTZ and AMIX susceptibility. There was no cross-resistance between MTZ resistant strains with AMIX susceptibility. Furthermore, we show that AMIX is an effective antimicrobial against B. fragilis in an experimental model of intra-abdominal infection. This investigation led to the discovery that the kor2AEBG genes are essential for growth, and we present evidence that kor2AEBG genes have dual-functions including the reductive synthesis of 2-ketoglutarate via reverse TCA cycle. Support for a novel Kor2AEBG activity comes from the findings showing that addition of compounds containing oleic acid stimulated growth of the{Delta} kor2AEBG mutant. However, the metabolic activity that bypasses KorAEBG function remains to be defined. Collectively our investigation reveals new information on B. fragilis central metabolism and its modulatory control by pirin proteins. These data provide new genetic and metabolic knowledge which may be leveraged for the future development of new narrow-spectrum antimicrobials.

microbiology↗

bioRxiv2023

Conformational dynamics of C1r inhibitor proteins from Lyme disease and relapsing fever spirochetes

Borrelial pathogens are vector-borne etiological agents of Lyme disease, relapsing fever, and Borrelia miyamotoi disease. These spirochetes each encode several surface-localized lipoproteins that bind to components of the human complement system. BBK32 is an example of a borrelial lipoprotein that protects the Lyme disease spirochete from complement-mediated attack. The complement inhibitory activity of BBK32 arises from an alpha helical C-terminal domain that interacts directly with the initiating protease of the classical pathway, C1r. Borrelia miyamotoi spirochetes encode BBK32 orthologs termed FbpA and FbpB, and these proteins also inhibit C1r, albeit via distinct recognition mechanisms. The C1r-inhibitory activities of a third ortholog termed FbpC, which is found exclusively in relapsing fever spirochetes, remains unknown. Here we report the crystal structure of the C-terminal domain of B. hermsii FbpC to a limiting resolution of 1.5 [A]. Surface plasmon resonance studies and assays of complement function demonstrate that FbpC retains potent BBK32-like anti-complement activities. Based on the structure of FbpC, we hypothesized that conformational dynamics of the complement inhibitory domains of borrelial C1r inhibitors may differ. To test this, we utilized the crystal structures of the C-terminal domains of BBK32, FbpA, FbpB, and FbpC to carry out 1 {micro}s molecular dynamics simulations, which revealed borrelial C1r inhibitors adopt energetically favored open and closed states defined by two functionally critical regions. This study advances our understanding of how protein dynamics contribute to the function of bacterial immune evasion proteins and reveals a surprising plasticity in the structures of borrelial C1r inhibitors.

microbiology↗

bioRxiv2022

Outer surface lipoproteins from the Lyme disease spirochete exploit the molecular switch mechanism of the complement protease C1s

Proteolytic cascades comprise several important physiological systems, including a primary arm of innate immunity called the complement cascade. To safeguard against complement-mediated attack, the etiologic agent of Lyme disease, Borreliella burgdorferi, produces numerous outer surface-localized lipoproteins that contribute to successful complement evasion. Recently, we discovered a pair of B. burgdorferi surface lipoproteins of the OspEF-related protein family - termed ElpB and ElpQ - that inhibit antibody-mediated complement activation. In this study, we investigate the molecular mechanism of ElpB and ElpQ complement inhibition using an array of biochemical and biophysical approaches. In vitro assays of complement activation show that an independently folded homologous C-terminal domain of each Elp protein maintains full complement inhibitory activity and selectively inhibits the classical pathway. Using surface plasmon resonance, Alpha bead-based technology, and C1s enzyme assays, we show that binding of Elp proteins to activated C1s blocks C4 cleavage by competing with C1s/C4 binding without occluding the active site. C1s-mediated C4 cleavage is dependent on activation-induced binding sites, termed exosites. To test whether these exosites are involved in Elp/C1s binding, we performed site-directed mutagenesis which showed that ElpB- and ElpQ-binding require C1s residues in the anion-binding exosite located on the serine protease domain of C1s. Based on these results, we propose a model whereby ElpB and ElpQ exploit activation-induced conformational changes that are normally important for C1s-mediated C4 cleavage. Our study expands the known complement evasion mechanisms of microbial pathogens and reveals a novel molecular mechanism for selective C1s inhibition by Lyme disease spirochetes.

biochemistry↗

bioRxiv2021

Lipoproteome screening of the Lyme disease agent identifies novel inhibitors of antibody-mediated complement killing

Spirochetal pathogens such as the causative agent of Lyme disease, Borrelia burgdorferi sensu lato, encode an abundance of lipoproteins; however, due in part to their evolutionary distance from more well-studied bacteria such as Proteobacteria and Firmicutes, very few spirochetal lipoproteins have assigned functions. Indeed, B. burgdorferi devotes almost 8% of its genome to lipoprotein genes and interacts with its environment primarily through the production of at least eighty surface-exposed lipoproteins throughout its tick vector-vertebrate host lifecycle (57). Several B. burgdorferi lipoproteins have been shown to serve diverse roles, such as cellular adherence or immune evasion, but the functions for most B. burgdorferi surface lipoproteins remain unknown. In this study, we developed a B. burgdorferi lipoproteome screening platform utilizing intact spirochetes that enables the identification of previously unrecognized host interactions. As spirochetal survival in the bloodstream is essential for dissemination, we targeted our screen to C1, the first component of the classical (antibody-mediated) complement pathway. We identified two high-affinity C1 interactions by the paralogous lipoproteins, ErpB and ErpQ. Using biochemical, microbiological, and biophysical approaches, we demonstrated that ErpB and ErpQ inhibit the activated forms of the C1 proteases, C1r and C1s, and represent a new mechanistic class of C1 inhibitors that protect the spirochete from antibody-mediated complement killing by allosteric regulation. In addition to identifying a novel mode of complement inhibition, our study establishes a lipoproteome screening methodology as a discovery platform for identifying direct host-pathogen interactions that are central to the pathogenesis of spirochetes, such as the Lyme disease agent. Significance StatementSpirochetal pathogens encode an abundance of lipoproteins that can provide a critical interface with the host environment. For example, Borrelia burgdorferi, the model species for spirochetal biology, must survive an enzootic life cycle defined by fluctuations between vector (tick) and vertebrate host. While B. burgdorferi expresses over eighty surface lipoproteins-- many of which likely contribute to host survival--the B. burgdorferi lipoproteome is poorly characterized. Here, we generated a platform to rapidly identify targets of B. burgdorferi surface lipoproteins and identified two orthologs that allosterically inhibit complement C1 subcomponents, conferring resistance to classical complement killing. This work expands our understanding of complement evasion mechanisms and points towards a discovery approach for identifying host-pathogen interactions that are central to spirochete pathogenesis.

microbiology↗

bioRxiv2021

A structural basis for inhibition of the complement initiator protease C1r by Lyme disease spirochetes

Complement evasion is a hallmark of extracellular microbial pathogens such as Borreliella burgdorferi, the causative agent of Lyme disease. Lyme disease spirochetes express nearly a dozen outer surface lipoproteins that bind complement components and interfere with their native activities. Among these, BBK32 is unique in its selective inhibition of the classical pathway. BBK32 blocks activation of this pathway by selectively binding and inhibiting the C1r serine protease of first component of complement, C1. To understand the structural basis for BBK32-mediated C1r inhibition, we performed co-crystallography and size exclusion chromatography-coupled small angle x-ray scattering experiments, which revealed a molecular model of BBK32-C in complex with activated human C1r. Structure-guided site-directed mutagenesis was combined with surface plasmon resonance binding experiments and assays of complement function to validate the predicted molecular interface. The studies reported here, for the first time, provide a structural basis for classical pathway-specific inhibition by a human pathogen.

microbiology↗