Biology subjects

Bosch, A.

Publications and source records attributed to Bosch, A..

13 recordsLinked to original sources

bioRxiv2026

A cold-adapted Cas9 nuclease from the deep ocean

The abundance of CRISPR systems in bacteria and archaea fuels the search for Cas nucleases that perform where established enzymes fail. Deep Ocean 1 Cas9 (DO1Cas9) is to our knowledge the first Cas9 nuclease characterized from the deep ocean, recovered from a metagenomic sample at 1,500 m depth in the Atlantic Ocean. DO1Cas9 shows cold-adapted structural flexibility, thermal susceptibility, salt-dependent stability, higher specificity than SpCas9 and cleaves double-stranded DNA efficiently under cold conditions where mesophilic SpCas9 retains only residual activity. DO1Cas9 edits genomes in diverse organisms such as Caenorhabditis elegans, zebrafish, the marine tunicate Oikopleura dioica, and the malaria parasite Plasmodium falciparum. In the marine bacterium Marinomonas mediterranea, DO1Cas9 programmed against an invading phage reduced plaquing efficiency about 10-fold more than SpCas9. These results expand the CRISPR-Cas9 toolbox with a nuclease for cold-chain editing and marine biotechnology.

genetics↗

bioRxiv2026

Noncanonical CD11b on CD8+ T cells promotes cytotoxic effector function

Cancer immunosurveillance largely relies on effector CD8+ T cells, yet surface determinants of cytotoxic competence within tumor-reactive populations remain incompletely defined. Using single-cell surface proteomics in a mouse breast cancer model, we identified a tumor-enriched subset of antigen-experienced CD8+ T cells expressing CD11b, a prototypic myeloid integrin. CD11b CD8 T cells were detected across mouse tumor models and human solid tumors and, within tumor-reactive populations, CD11b marked cells with greater cytotoxic potential. Following activation, naive CD8+ T cells induced ITGAM transcription and CD18-dependent CD11b surface expression. Mechanistically, CD11b localized to the peripheral adhesive ring of the immune synapse and promoted lytic-granule secretion. Accordingly, CD11b+ T cells exhibited enhanced TCR- and CAR-mediated tumor cell killing, whereas CD11b deletion impaired T cell antitumor activity. Together, these findings identify CD11b as an inducible, noncanonical component of the T cell immune synapse that marks cytotoxic competence and promotes antitumor T cell function.

immunology↗

bioRxiv2026

pH-Dependent Evolution of Delafloxacin and Ciprofloxacin Resistance in Pseudomonas aeruginosa from cystic fibrosis (CF) and non-CF-patients

Delafloxacin (DLX) is a novel fluoroquinolone with enhanced antibacterial activity in acidic environments, a property that may be advantageous for treating Pseudomonas aeruginosa infections in cystic fibrosis (CF), where airway surface liquid pH is typically reduced (pH 5.5-6.7). However, the propensity for resistance development and the underlying mechanisms in P. aeruginosa remain incompletely defined. We conducted serial passage experiments on six clinical P. aeruginosa isolates (three CF-derived and three non-CF-derived) exposed to sub-inhibitory concentrations of DLX or ciprofloxacin (CIP) at pH 6.0 and 7.3 over nine days. Susceptibility was assessed by broth microdilution (BMD), and resistance mechanisms were characterized by whole-genome sequencing (WGS), efflux pump expression analysis (qRT-PCR), and functional validation using CRISPR/Cas9-mediated genome editing and complementation assays. DLX minimal inhibitory concentrations (MICs) rose only 10.1- to 28.5-fold over 9 days, compared with 77.6- to 97.8-fold for CIP, indicating a substantially higher genetic barrier to resistance. This barrier was most pronounced under acidic conditions: only 38.9% of DLX-passaged samples crossed the resistance breakpoint, compared with 94.4% at neutral pH, whereas CIP resistance reached 100% regardless of pH. Cross-resistance was asymmetric: exposure to DLX consistently selected for CIP cross-resistance (97.2% of samples), whereas exposure to CIP induced DLX cross-resistance efficiently at neutral pH but only partially under acidic conditions. A previously undescribed gyrA mutation (p.Ala51Val) conferred a 4-fold increase in DLX MIC when introduced by CRISPR/Cas9, and upregulation of the MexEF-OprN efflux pump, reversible by mexS complementation, emerged as a prominent resistance mechanism. Overall, DLX exhibited a markedly higher genetic barrier to resistance than CIP in P. aeruginosa, particularly under the acidic conditions characteristic of the CF airway. However, its use may co-select for CIP cross-resistance through efflux upregulation, underscoring the need for careful stewardship in CF.

microbiology↗

bioRxiv2025

Glycans modulate the adsorption of RBD Glycoproteins on polarizable surfaces

The complex interplay between glycans and protein conformational dynamics during adsorption onto polarizable surfaces opens several routes to exploring the glycans potential as molecular interactions modulators. Molecular simulations are able to dissect the interactions of Receptor Binding Domain (RBD) glycoproteins for different SARS-CoV-2 variants of concern (VoC), in both open and closed conformations, with polarizable planar interfaces. Advanced analysis projected on 2D revealed distinct adsorption mechanisms depending on the initial loci of the glycan within the protein wall. Hydrophobic surfaces facilitated stable adsorption for both RBD conformations. Conversely, hydrophilic surfaces exhibited reduced adsorption, particularly for the closed-RBD, where glycans predominantly formed hydrogen bonds. Glycans significantly modulated closed-RBD adsorption, either enhancing it by permanent tethering or impeding it depending on the two initial conformations and protein mutations (omicron). Results for the individual RBDs are shown to be consistent with simulations for the complete S1 spike glycoprotein. Our findings unveil novel glycan-mediated adsorption phenomena and provide fundamental insights into glycoprotein-surface interactions, paving the way for understanding glycan roles in protein aggregation and recognition at polarizable biological interfaces.

biophysics↗

bioRxiv2025

The unique molecular recognition features of Siglec-10: structural insights into sialoglycan and antibody interactions

Siglec-10 is a sialic acid-binding immunoglobulin-like lectin implicated in immune regulation, yet the molecular basis for ligand recognition and how this is functionally linked to immune modulation remains poorly defined. Herein, we present a multidisciplinary study encompassing structural, biochemical, and cellular approaches to elucidate Siglec-10-carbohydrate interactions and their functional consequences. The crystal structure of the extracellular domain of Siglec-10 in complex with 2-6 sialyllactose revealed the presence of two key arginine residues within the Siglec-10 binding site that interact with the carboxyl group of sialic acid, the canonical R119 and R127, suggesting potential dual contributions to ligand engagement. Saturation Transfer Difference (STD)-Nuclear Magnetic Resonance (NMR) confirmed that R119 is essential for sialoglycan binding in solution, whereas R127 appears dispensable for interactions with glycans under these conditions. In contrast, cell-based binding assays using primary human T cells and engineered monocytic lines demonstrated that both arginine residues (R119 and R127) are critical for cellular recognition, revealing a context-dependent interaction. By obtaining direct images at a molecular resolution of 6-7 nm, super-resolution microscopy further revealed glycan-independent dimerization of the Siglec-10 receptor on the surface of human monocytes. Ligand blockade mediated by anti-Siglec-10 mAb (clone S10A) restores CAR-T cell cytotoxicity in vitro, supporting its role as an immune checkpoint receptor. Finally, although CD24 was not identified as a Siglec-10 ligand on T cells, proximity labeling and mass spectrometry uncovered other sialylated glycoproteins that may mediate this interaction. Together, these results identify Siglec-10 as a modulatory receptor with structural and functional features distinct from other Siglec family members and highlight its potential for therapeutic targeting in cancer immunotherapy.

molecular biology↗

bioRxiv2025

sKL/mKL Transcript Ratio and Protein Localization Define a Species- and Region-Specific Klotho Signature in the CNS and AD Progression

-Klotho is a multifunctional protein widely recognized for its anti-aging and neuroprotective properties. This study investigates the expression and localization of the secreted Klotho (s-KL) isoform in the human brain and its potential role in Alzheimers disease. Using RT-qPCR, we observed that the s-KL transcript predominates over the membrane-bound KL (m-KL) in multiple brain regions, a pattern consistent in macaques and lemurs. Immunohistochemistry and immunoprecipitation assays confirmed the presence of the s-KL protein in human and mouse brain parenchyma, revealing species-specific cellular localization. In human cerebrospinal fluid (CSF), s-KL constitutes [~]28% of total KL, with levels significantly reduced in mild dementia-AD patients. These findings underscore s-KLs potential neuroprotective role and highlight its differential regulation and expression during AD progression.

neuroscience↗

bioRxiv2025

Fibrillarin shapes oncogenic protein pools and ribosomal composition in triple-negative breast cancer

Fibrillarin (FBL), a core component of the C/D box snoRNP complex, catalyzes 2-O-methylation (Nm) of ribosomal RNA (rRNA), influencing ribosome heterogeneity and oncogene translation. In triple-negative breast cancer (TNBC), FBL dysregulation creates an aberrant Nm signature. This study explores role of FBL in TNBC progression via translation-driven mechanisms. FBL knockdown impaired tumorigenic traits, induced metabolic stress, and reduced the translation efficiency of oncogenes, including metastasis-associated protein 1 (MTA1), interleukin-1 receptor-associated kinase 1 (IRAK1), and thymosin beta 10 (TMSB10). RiboMethSeq analysis revealed differential rRNA Nm site sensitivity to FBL depletion. Additionally, FBL knockdown lowered RPS28 protein levels, suggesting its misincorporation into ribosomes. Notably, silencing RPS28 also suppressed oncogenic traits and downregulated MTA1, IRAK1, and TMSB10, highlighting its role in FBL-mediated translation. These findings uncover a complex interplay between FBL, rRNA Nm modifications, and RPS28 in shaping oncogenic protein pools and ribosomal composition in TNBC. Targeting this pathway could offer novel therapeutic strategies for this aggressive cancer subtype.

molecular biology↗

bioRxiv2024

Adsorption-driven deformation and landing-footprints of the RBD proteins in SARS-CoV-2 variants onto biological and inanimate surfaces

Respiratory viruses, carried through airborne microdroplets, frequently adhere to surfaces, including plastics and metals. However, our understanding of the interactions between viruses and materials remains limited, particularly in scenarios involving polarizable surfaces. Here, we investigate the role of receptor-binding domain (RBD) mutations on the adsorption of SARS-CoV-2 to hydrophobic and hydrophilic surfaces employing molecular simulations. To contextualize our findings, we contrast the interactions on inanimate surfaces with those on native-biological interfaces, specifically the ACE2 receptor. Notably, we identify a twofold increase in structural deformations for the proteins receptor binding motif onto the inanimate surfaces, indicative of enhanced shock-absorbing mechanisms. Furthermore, the distribution of amino acids (landing-footprints) on the inanimate surface reveals a distinct regional asymmetry relative to the biological interface. In spite of the H-bonds formed at the hydrophilic substrate, the simulations consistently show a higher number of contacts and interfacial area with the hydrophobic surface, with the WT RBD adsorbed more strongly than the delta or omicron RBDs. In contrast, the adsorption of delta and omicron to hydrophilic surfaces was characterized by a distinctive hopping-pattern. The novel shock-absorbing mechanisms identified in the virus adsorption on inanimate surfaces could lead current experimental efforts in the design of virucidal surfaces.

biophysics↗

bioRxiv2023

Molecular Epidemiology of multidrug-resistant Klebsiella pneumoniae, Enterobacter cloacae, and Escherichia coli outbreak among neonates in Tembisa Hospital, South Africa

BackgroundAn outbreak of multidrug-resistant Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae infections in a neonatal ward within a tertiary hospital in South Africa resulted in the mortality of 10 patients within six months. In this work, the genomic epidemiology of and the molecular factors mediating this outbreak were investigated. MethodsBacterial cultures obtained from clinical samples collected from the infected neonates underwent phenotypic and molecular analyses to determine their species, sensitivity to antibiotics, production of carbapenemases, complete resistance genes profile, clonality, epidemiology, and evolutionary relationships. Mobile genetic elements flanking the resistance genes and facilitating their spread were also characterized. ResultsThe outbreak was centered in two major wards and affected mainly neonates between September 2019 and March 2020. Most isolates (n = 27 isolates) were K. pneumoniae while both E. coli and E. cloacae had three isolates each. Notably, 33/34 isolates were multidrug resistant (MDR), with 30 being resistant to at least four drug classes. All the isolates were carbapenemase-positive, but four blaOXA-48 isolates were susceptible to carbapenems. BlaNDM-1 (n = 13) and blaOXA-48/181 (n = 15) were respectively found on IS91 and IS6-like IS26 composite transposons in the isolates alongside several other resistance genes. The repertoire of resistance and virulence genes, insertion sequences, and plasmid replicon types in the strains explains their virulence, resistance, and quick dissemination among the neonates. ConclusionsThe outbreak of fatal MDR infections in the neonatal wards were mediated by clonal (vertical) and horizontal (plasmid-mediated) spread of resistant and virulent strains (and genes) that have been also circulating locally and globally.

microbiology↗

bioRxiv2022

Profiling cell envelope-antibiotic interactions reveals vulnerabilities to β-lactams in a multidrug-resistant bacterium

The cell envelope of the Gram-negative Burkholderia cepacia complex (Bcc) presents unique restrictions to antibiotic penetration. As a consequence, Bcc species are notorious for causing recalcitrant multidrug-resistant infections in immunocompromised individuals, such as those living with cystic fibrosis. To systematically identify cell envelope-associated resistance and susceptibility determinants at the genome level, we constructed a high-density, randomly-barcoded transposon mutant library in the clinical isolate B. cenocepacia K56-2 and exposed it to a panel of more than twenty cell envelope-targeting antibiotics. By quantifying relative mutant fitness with BarSeq, followed by validation with CRISPR-interference, we profiled over a hundred new functional associations and identified novel mediators of antibiotic susceptibility in the Bcc cell envelope. We revealed new connections between {beta}-lactam susceptibility, peptidoglycan synthesis, and blockages in undecaprenyl phosphate metabolism, which highlight a vulnerability in sharing this lipid intermediate. We then show that the clinically relevant synergy of the {beta}-lactam/{beta}-lactamase inhibitor combination ceftazidime/avibactam is primarily mediated by inhibition of the PenB carbapenemase. Importantly, we found that avibactam more strongly potentiates the activity of aztreonam and meropenem than ceftazidime in a panel of Bcc clinical isolates. Finally, we characterize for first time in the Bcc the iron and receptor-dependent activity of the novel siderophore-cephalosporin antibiotic, cefiderocol. Overall, our work has implications for antibiotic target prioritization, and for using additional combinations of {beta}-lactam/{beta}-lactamase inhibitors that can extend the utility of our current clinical arsenal of antibacterial therapies. Author SummaryWhile the Gram-negative cell envelope is a major barrier to antibiotic action, we have an incomplete picture of how each component contributes to antibiotic resistance. To answer this question from a genome-wide perspective in a model of antibiotic-resistant bacteria, we constructed a library of randomly-barcoded transposon mutants in Burkholderia cenocepacia and exposed it to a panel of diverse cell envelope-targeting antibiotics. We identified individual genes and whole pathways associated with antibiotic resistance, including several novel players. Focusing on clinically relevant antibiotics, our strategy dissected how the synergy of the important {beta}-lactam/{beta}-lactamase inhibitor combination ceftazidime/avibactam primarily depends upon inhibition of a single {beta}-lactamase. Additionally, we are the first to characterise the molecular basis of the antibiotic mechanism of cefiderocol in Burkholderia, a new and very potent siderophore-cephalosporin conjugate drug. Encouragingly, we found that cefiderocol susceptibility was greatest at physiological iron concentrations. Overall, we highlight several avenues that can be taken to enhance or develop new therapeutic strategies against Burkholderia infection.

microbiology↗

bioRxiv2021

Hypoxia reduces cell attachment of SARS-CoV-2 spike protein by modulating the expression of ACE2 and heparan sulfate

A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1-dependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signaling pathway might offer therapeutic opportunities for the treatment of COVID-19.

cell biology↗

bioRxiv2020

A high-affinity antibody against the CSP N-terminal domain lacks Plasmodium falciparum inhibitory activity

Malaria is a global health concern and research efforts are ongoing to develop a superior vaccine to RTS,S/AS01. To guide immunogen design, we seek a comprehensive understanding of the protective humoral response against Plasmodium falciparum circumsporozoite protein (PfCSP). In contrast to the well-studied responses to the repeat region and the C-terminus, the antibody response against the N-terminal domain of PfCSP (N-CSP) remains obscure. Here, we characterized the molecular recognition and functional efficacy of the N-CSP-specific monoclonal antibody 5D5. The crystal structure at 1.85 [A] resolution revealed that 5D5 binds an -helical epitope in N-CSP with high affinity through extensive shape and charge complementarity, and the unusual utilization of an N-linked glycan. Nevertheless, functional studies indicated low 5D5 binding to live Pf sporozoites, and lack of sporozoite inhibition in vitro and in mosquitoes. Overall, our data on low recognition and inhibition of sporozoites do not support the inclusion of the 5D5 epitope into the next generation of CSP-based vaccines. Summary StatementThe Plasmodium falciparum sporozoite surface protein, PfCSP, is an attractive vaccine target, but the antibody response against the CSP N-terminal domain has remained understudied. Here, to guide immunogen design, Thai et al. provide insights into the binding motif and functional efficacy of the N-terminal domain-specific monoclonal antibody, 5D5.

immunology↗

bioRxiv2019

Evolution of protective human antibodies against Plasmodium falciparum circumsporozoite protein repeat motifs

Circumsporozoite protein of the human malaria parasite Plasmodium falciparum (PfCSP) is the main target of antibodies that prevent the infection and disease. Protective antibodies recognize the central PfCSP domain, but our understanding of how parasite inhibition is associated with recognition of this domain and with the evolution of potent antibodies remains scattered. Here, we characterized the epitope specificity of 200 human monoclonal PfCSP antibodies. We show that the majority of PfCSP antibodies bind to NANP and NANP-like motifs with different preferences and define the molecular basis for recognition. Epitope cross-reactivity evolved with increasing antibody affinity around a conserved (N/D)P-NANP-N(V/A) core. High affinity to this motif, but not binding to NANP-like motifs, was associated with parasite inhibition and protection. Thus, NANP drives the development of potent PfCSP antibodies independently of their cross-reactivity profile, a finding with direct implications for the design of a second-generation PfCSP-based malaria vaccine.\n\nHIGHLIGHTSO_LIThe majority of human PfCSP antibodies recognize multiple epitopes\nC_LIO_LINANP affinity maturation drives the evolution of cross-reactive PfCSP antibodies\nC_LIO_LIPreferential PfCSP antibody binding to a conserved core motif\nC_LIO_LIHigh affinity not epitope specificity is associated with PfCSP antibody potency\nC_LI

immunology↗