Biology subjects

Tan, K. S.

Publications and source records attributed to Tan, K. S..

14 recordsLinked to original sources

bioRxiv2026

Cross-phosphorylation of RR06 by the non-cognate kinase VncS activates the adhesin CbpA in Streptococcus pneumoniae.

Streptococcus pneumoniae establishes asymptomatic nasopharyngeal colonization before progressing to invasive disease, a process that requires precise spatiotemporal regulation of surface adhesins. Using a barcoded capsule-switch mutant library and randomly barcoded transposon sequencing (RB-TnSeq), we identified determinants of pneumococcal adhesion to the human alveolar epithelial cell line A549. The capsule masked surface adhesins and reduced binding across all 84 serotypes tested. The transposon screen revealed that overexpression of vncS, the sensor kinase of the two-component system TCS10, markedly increased adhesion, whereas disruption of its cognate response regulator VncR had no detectable effect. Transcriptomic profiling and genetic epistasis established that the adhesin CbpA, known to be regulated by TCS06, is the effector of VncS-driven adhesion. VncS activated cbpA expression through the non-cognate response regulator RR06, independently of VncR. Phos-tag gel analysis demonstrated that VncS cross-phosphorylates RR06 in vivo, and that the cognate kinase HK06 limits this crosstalk by acting primarily as a phosphatase toward RR06. Substitution of the conserved S241 residue of HK06 increased RR06[~]P levels and upregulated cbpA. By contrast, deleting vncS and hk06 reduced RR06 phosphorylation to nearly undetectable levels. These findings resolve a long-standing mechanistic controversy regarding RR06 activation and uncover a potentially unrecognized role for VncS in regulating pneumococcal host cell adhesion. SIGNIFICANCEStreptococcus pneumoniae is a leading cause of pneumonia, and its ability to colonize the human airway depends on surface adhesins. Several two-component signaling systems are known to regulate adhesin expression, but how their outputs are integrated remains incompletely understood. Here, we identify determinants of pneumococcal adhesion to human alveolar epithelial cells at both the serotype and genotype levels. We show that the histidine kinase VncS cross-phosphorylates the non-cognate response regulator RR06 to activate the adhesin CbpA, whereas the cognate kinase HK06 primarily acts as a phosphatase to restrain this crosstalk. These findings resolve a long-standing controversy regarding RR06 activation and reveal a regulatory node that links two signaling systems to a single virulence output.

microbiology↗

bioRxiv2026

S3-enriched kidney proximal nephrons from stem cells facilitate tubular injury modelling

Chronic kidney disease (CKD) is a major global health challenge affecting >840 million people, with rising prevalence driven by common conditions including diabetes, hypertension, obesity, and drug toxicity. The proximal tubule (PT) is central to kidney function yet is highly injury-prone, particularly within the S3 segment which plays key roles in drug clearance and CKD pathology. Despite decades of in vitro PT model development, achieving accurate functional and structural segmentation of the PT with distinct PT sub-cell types has remained challenging. Here, we report an enrichment of S3-like cells within PT-enhanced kidney organoids (PT-EKO) that confers segment-specific functionality and injury susceptibility. This advanced platform facilitated physiologically relevant modelling of hyperglycaemia-induced damage, including rapid detection of injury biomarker Kidney Injury Molecule-1 (KIM-1). Integrating with both static and organ-on-chip culture systems, the translational potential of S3-enriched PT-EKO was underscored by its amenability to scale-up via cryopreservation of day 13 progenitors with retained differentiation capacity. PT-EKO applications were further broadened as an expandable high-yield source of isolatable PT cells, retaining PT characteristics across multiple passages and cryopreservation. Together, these findings present a high-fidelity platform for modelling tubular injury and advancing translational applications including CKD drug development, cell-specific nephrotoxicity testing and cellular therapies. SIGNIFICANCE STATEMENTAdvancing stem cell-based proximal nephron models, here we identify proximal tubule-enhanced kidney organoids (PT-EKO) as an enriched source of the nephrons most injury-prone cell type; S3 proximal tubule cells. This advanced platform provides physiologically-relevant modelling of tubular injury and a scalable source of high-quality PT cells, paving the way for more accurate modelling, screening, and cellular therapy applications.

bioengineering↗

bioRxiv2025

Bioengineered human tonsil organoids as an immuno-engineering platform for evaluating immune functions

Although animal models remain the preclinical gold standard, they are constrained by interspecies differences in adaptive immunity, including antibody class-switching, affinity maturation, and multicellular interactions. To address this gap, we established a more physiologically relevant platform, a bioengineered tonsil organoid (BTO) system that combines autologous tonsil-derived stromal cells with immune cells to generate highly uniform organoids, preserving multicellular complexity and sustained stromal-immune interactions. BTO cultures mounted robust, antigen-specific recall responses to influenza, tetanus, and COVID-19 antigens. Notably, stimulation with ovalbumin and SARS-CoV-1 spike protein expanded antigen-recognizing B cells, indicating capacity for naive responses. Digital spatial transcriptomics and proteomics analysis confirmed immune-stromal crosstalk underpinning both humoral and cellular responses of BTO after challenge. These data highlight stromal cells as active scaffolds that not only maintain 3D architecture but also facilitate immune activation, memory recall, and recognition of naive antigens. Together, BTO offers a human-relevant model for mechanistic immunology and for pre-clinical evaluation on immunotherapies and vaccines not achievable with 2D cultures or animal models.

bioengineering↗

bioRxiv2025

Crucial role for iron metabolism in mediating influenza A virus infection and associated disease

Rationale and ObjectivesIron availability and metabolism are important in the pathogenesis of bacterial infections. More recently, links have been reported between iron and the severity of viral infections. In this study, we characterize a crucial relationship between iron metabolism and IAV infection and disease. MethodsIron-related gene expression was assessed in human airway epithelial cells (AEC) infected with IAV. AECs were cultured with ferric iron, iron-loaded transferrin, or iron chelator, deferoxamine (DFO), prior to infection with IAV. Mice were placed on a high iron diet for 8 weeks prior to infection with IAV or treated with anti-transferrin receptor-1 (TFR1) antibody during IAV infection. The effects of iron modulation and depletion of TFR1-mediated responses on IAV infection were assessed. Measurements and main resultsIron-related gene expression and metabolism are altered systemically and in lung tissues and AECs during IAV infections. Increasing iron availability increases viral titer in AECs, while DFO protects against iron-induced increased susceptibility to infection. Increasing systemic iron loading, which increases iron levels in the lung, increases viral titer, proinflammatory responses, airway inflammation, and worsens IAV-induced disease in terms of lung function and weight loss in vivo. Inhibition of TFR1 protects against IAV-induced disease in vivo. ConclusionIAV infections remain a major threat to human health and global economies. Strategies that boost protective, or reduce pathogenic, host responses may provide broadly effective, long-term therapeutic options. We have identified a key role for iron metabolism in modifying host responses to IAV that can be harnessed to protect against disease. Key MessagesO_LIIron metabolism is altered systemically, and in airway epithelial cells and lung tissues, during IAV infection. C_LIO_LIIncreased iron availability increases viral titer both in vitro and in vivo C_LIO_LISystemic iron loading worsens IAV-induced inflammation and disease outcomes in vivo, highlighting iron as a crucial factor for modulating IAV infections and disease. C_LIO_LIHost epithelial cells and lung tissues reduce TFRC gene expression, whilst the number and proportion of TFR1hi expressing cells increase, in response to IAV infection. Neutralising TFR1 protects against IAV-induced disease in vivo, highlighting TFR1 as a potential therapeutic target for IAV infections. C_LI

immunology↗

bioRxiv2023

Single shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad and durable systemic and mucosal immune responses

Current COVID-19 vaccines face certain limitations, which include waning immunity, immune escape by SARS-CoV-2 variants, limited CD8+ cellular response, and poor induction of mucosal immunity. Here, we engineered a Clec9A-RBD antibody construct that delivers the Receptor Binding Domain (RBD) from SARS-CoV-2 spike protein to conventional type 1 dendritic cells (cDC1). We showed that single dose immunization with Clec9A-RBD induced high RBD-specific antibody titers with a strong T-helper 1 (TH1) isotype profile and exceptional durability, whereby antibody titers were sustained for at least 21 months post-vaccination. Uniquely, affinity maturation of the antibody response was observed over time, as evidenced by enhanced neutralization potency and breadth across the sarbecovirus family. Consistently and remarkably, RBD-specific T-follicular helper cells and germinal center B cells were still detected at 12 months post-immunization. Increased antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the immune sera was also measured over time with comparable efficacy against ancestral SARS-CoV-2 and variants, including Omicron. Furthermore, Clec9A-RBD immunization induced a durable poly-functional TH1-biased cellular response that was strongly cross-reactive against SARS-CoV-2 variants, including Omicron, and with robust CD8+ T cell signature. Lastly, Clec9A-RBD single dose systemic immunization primed effectively RBD-specific cellular and humoral mucosal immunity in lung. Taken together, Clec9A-RBD immunization has the potential to trigger robust and sustained, systemic and mucosal immune responses against rapidly evolving SARS-CoV2 variants.

immunology↗

bioRxiv2023

Chloroquine induces eryptosis in P. falciparum-infected red blood cells and the release of extracellular vesicles with a unique protein profile

Malaria is a vector-borne parasitic disease that affects millions worldwide. In order to reach the objective, set by the World Health Organization to decrease the cases by 2030, antimalarial drugs with novel modes of action are required. Previously, a novel mechanism of action of chloroquine (CQ) was reported involving features of programmed cell death in the parasite, mainly characterized by calcium efflux from the digestive vacuole (DV) permeabilization. Increased intracellular calcium induces the suicidal death of erythrocytes also known as eryptosis. This study aimed to identify the hallmarks of eryptosis due to calcium redistribution and the downstream cellular effects during CQ treatment in iRBCs. Plasmodium falciparum 3D7 at mid-late trophozoites were used for the antimalarial drug treatment. Our results revealed increased phosphatidylserine (PS) exposure, cell shrinkage and membrane blebbing, delineating an eryptotic phenotype in the host RBC. Interestingly, the blebs on the surface of the iRBCs released to the extracellular milieu become extracellular vesicles (EVs) which are essential for intercellular communication due to their cargo of proteins, nucleic acids, lipids and metabolites. The proteomic characterization displayed 2 highly enriched protein clusters in EVs from CQ-treated iRBCs, the proteasome and ribosome. We demonstrated that this unique protein cargo is not associated with the parasite growth rate. Additionally, we found that these particular EVs might activate IFN signaling pathways mediated by IL-6 in THP-1-derived macrophages. Our findings shed new insights into a novel drug-induced cell death mechanism that targets the parasite and specific components of the infected host RBC. IMPORTANCEOur previous studies have shown that chloroquine (CQ) treatment in iRBCs triggers Plasmodium falciparum digestive vacuole (DV) membrane permeabilization leading to calcium redistribution. Interestingly, increased intracellular calcium concentration is the main inducer of the suicidal death of red blood cells (RBCs) called eryptosis. The present study shed new insights into a novel CQ-induced cell death mechanism that targets the parasite and the infected host RBC by inducing key phenotypic hallmarks of eryptosis: PS exposure, cell shrinkage and membrane blebbing. Moreover, the proteomic characterization of the blebs released to the extracellular milieu also known as extracellular vesicles (EVs) revealed a cargo highly enriched in ribosomal proteins and proteasome subunits relevant for host-parasite interactions. These findings highlight CQs effect on calcium homeostasis disruption in infected red blood cells (iRBCs) with cellular and immunological consequences of great significance for malaria pathogenesis and potential clinical implications.

cell biology↗

bioRxiv2022

Helicobacter pylori vacuolating cytotoxin A exploits human endosomes for intracellular activation

Helicobacter pylori infection is the main cause of gastric cancer. Vacuolating cytotoxin A (VacA) is a H. pylori pore-forming toxin and a key determinant of gastric cancer risk. VacA is secreted as an 88-kDa polypeptide (p88) that upon interaction with host cells induces cytotoxic effects, including cell vacuolation and mitochondrial dysfunction. These effects are currently believed to be due to VacA p88 accumulating inside host cells and forming oligomeric anion-specific channels in membranes of intracellular compartments. However, the molecular nature of intracellular VacA channels in host cells remains undefined. Here we show that VacA p88 does not accumulate inside human epithelial cells, but instead is rapidly processed in endosomes into smaller p31/p28 and p37 products in a manner that precedes VacA-induced vacuolation. VacA processing requires endosomal acidification and concerted cleavage by multiple endo-lysosomal proteases including cathepsins. In situ structural mapping reveals that upon processing, the toxins central hydrophilic linker and globular C-terminus are excised, whereas oligomerization determinants are retained. Congruently, the processed products are constituents of a high-molecular-weight complex inside the host cell [boxh] which we propose is the intracellular, mature and active VacA pore. These findings suggest that VacA exploits human endosomes for proteolytic processing and intracellular activation. Significance StatementHelicobacter pylori is a cancer-causing bacterium that infects the stomach of billions of people worldwide. Vacuolating cytotoxin A (VacA) is an important H. pylori virulence factor and its activity directly correlates with gastric carcinogenesis. Yet despite decades of intense research, the mechanisms underlying VacA activity in human cells remain incompletely understood. Here, we present evidence suggesting that VacA is activated inside human cells by multi-step proteolytic processing involving endo-lysosomal proteases including cathepsins. We also track and identify the functional processed VacA isoforms in host cells. These results revolutionize our understanding of the mechanism of VacA activation in human cells, whilst expanding our knowledge of the diversity of microbial virulence factors that exploit human endo-lysosomes for pathogenesis.

molecular biology↗

bioRxiv2021

Discovery of a conserved rule behind the assembly of β-barrel membrane proteins.

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a "{beta}-signal" imprinted in the final {beta}-strand of the OMP engages the {beta}-barrel assembly machinery (BAM complex) to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule, that signals equivalent to the {beta}-signal are repeated in other, internal {beta}-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the {beta}-signal, arranging several {beta}-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults. TEASERBacterial outer membrane proteins are recognized and bound by BamD at specific signals located in multiple {beta}-strands at the C-terminus of these proteins.

biochemistry↗

bioRxiv2021

Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids

While pluripotent stem cell-derived kidney organoids are now being used to model renal disease, the proximal nephron remains immature with limited evidence for key functional solute channels. This may reflect early mispatterning of the nephrogenic mesenchyme and/or insufficient maturation. Here we show that enhanced specification to metanephric nephron progenitors results in elongated and radially aligned proximalised nephrons with distinct S1 - S3 proximal tubule cell types. Such PT-enhanced organoids possess improved albumin and organic cation uptake, appropriate KIM-1 upregulation in response to cisplatin, and improved expression of SARS-CoV-2 entry factors resulting in increased viral replication. The striking proximo-distal orientation of nephrons resulted from localized WNT antagonism originating from the organoid stromal core. PT-enhanced organoids represent an improved model to study inherited and acquired proximal tubular disease as well as drug and viral responses.

bioengineering↗

bioRxiv2020

Direct cleavage of human NLRP1 by enteroviral 3C protease triggers inflammasome activation in airway epithelium

Viruses pose a constant threat to human health. As a result our innate immune system has evolved multiple strategies to detect the presence of intracellular viral pathogen-associated molecular patterns (PAMPs) (1). The full repertoire of human immune sensors and their PAMP ligands are not completely understood. Here we report that human NLRP1 senses and is activated by 3C proteases (3Cpros) of enteroviruses. Mechanistically, 3Cpros cleave human NLRP1 at a single site immediately after its primate-specific PYRIN domain, leading to oligomerization of its C-terminal fragment. Expression of 3Cpros in primary human cells cause NLRP1-dependent ASC oligomerization, pyroptotic cell death and IL-1 secretion. Consistent with our observation that NLRP1 is the predominant endogenous inflammasome sensor in human airway epithelium, we find that its genetic deletion, or that of ASC, abrogates IL-18 secretion from rhinovirus (HRV)-infected primary human bronchial epithelial cells. Our findings identify the first cognate PAMP ligand for human NLRP1 and assign a new function for the NLRP1 inflammasome in human antiviral immunity and airway inflammation. These results challenge the widely held notion that viral proteases largely serve to disable host immune sensing, and suggest that the human NLRP1 inflammasome may be a therapeutic target to treat inflammatory airway diseases including asthma. One Sentence SummaryHuman NLRP1 is activated by enteroviral 3C proteases

immunology↗

bioRxiv2020

Calcitriol, the active form of vitamin D, is a promising candidate for COVID-19 prophylaxis

COVID-19, the disease caused by SARS-CoV-2 (1), was declared a pandemic by the World Health Organization (WHO) in March 2020 (2). While awaiting a vaccine, several antivirals are being used to manage the disease with limited success (3, 4). To expand this arsenal, we screened 4 compound libraries: a United States Food and Drug Administration (FDA) approved drug library, an angiotensin converting enzyme-2 (ACE2) targeted compound library, a flavonoid compound library as well as a natural product library. Of the 121 compounds identified with activity against SARS-CoV-2, 7 were shortlisted for validation. We show for the first time that the active form of Vitamin D, calcitriol, exhibits significant potent activity against SARS-CoV-2. This finding paves the way for consideration of host-directed therapies for ring prophylaxis of contacts of SARS-CoV-2 patients.

microbiology↗

bioRxiv2019

Reticulocyte Infection Leads to Altered Behaviour, Drug Sensitivity and Host Cell Remodelling by Plasmodium falciparum.

Plasmodia are host-specific, both at the organism and cellular levels. During asexual development, Plasmodium spp. infect cells of erythroid lineage, with an overall propensity towards reticulocytes. This applies to even Plasmodium (P.) falciparum, the most common causative agent of human malaria, implications of which remain unexplored. Herein, for the first time, we characterize the developmental stages and features of P. falciparum cultured in vitro in young reticulocytes (CD71+) in comparison to standard normocyte (CD71-) cultures. We demonstrate that there are notable differences in the patterns of invasion, development and sensitivity to potent antimalarials (such as artemisinin and dihydroartemisinin) for parasites residing in CD71+ reticulocytes. Through a transcriptomic approach, we report that P. falciparum parasites are able to sense the host cell environment, and calibrate their metabolic and host cell remodelling pathways through differential gene expression. These results form an exciting avenue on which hitherto unexplored interactions between Plasmodium spp and different stages of host red blood cells could be investigated in the broader contexts of drug resistance, host tropism and zoonosis. Author SummaryParasites causing malaria infect red blood cells for development and proliferation during asexual development. This asexual erythrocytic stage determines higher parasite densities and eventual disease manifestation. Although the most virulent species of Plasmodium infecting humans known as Plasmodium falciparum is able to infect red blood cells of all ages, these parasites show a preference for younger blood cells. Of note, the biochemical and biophysical properties of young and adult red blood cells vary significantly. Herein, we undertook a comparative profiling of invasion process, parasite development and drug response of Plasmoddium falciparum in two host cells: young red blood cells (reticulocytes) and mature red blood cells (normocytes). We demonstrate that P. falciparum infects human reticulocytes with higher affinity and demonstrate differential sensitivity to drugs such as artemisinin while they reside within reticulocytes. Furthermore, we show that P. falciparum is able to detect differences in host environment and adapt to it by changing the expression of genes required for host cell remodelling.

microbiology↗

bioRxiv2018

Bioprinted pluripotent stem cell-derived kidney organoids provide opportunities for high content screening.

Recent advances in the directed differentiation of human pluripotent stem cells to kidney brings with it the prospect of drug screening and disease modelling using patient-derived stem cell lines. Development of such an approach for high content screening will require substantial quality control and improvements in throughput. Here we demonstrate the use of the NovoGen MMX 3D bioprinter for the generation of highly reproducible kidney organoids from as few as 4,000 cells. Histological and immunohistochemical analyses confirmed the presence of renal epithelium, glomeruli, stroma and endothelium, while single cell RNAseq revealed equivalence to the cell clusters present within previously described organoids. The process is highly reproducible, rapid and transferable between cell lines, including genetically engineered reporter lines. We also demonstrate the capacity to bioprint organoids in a 96-well format and screen for response to doxorubicin toxicity as a proof of concept for high content compound screening.

cell biology↗

bioRxiv2018

Fate-mapping within human kidney organoids reveals conserved mammalian nephron progenitor lineage relationships.

While mammalian kidney morphogenesis has been well documented, human kidney development is poorly understood. Here we combine reprogramming, CRISPR/Cas9 gene-editing and organoid technologies to study human nephron lineage relationships in vitro. Early kidney organoids contained a SIX2+ population with a transcriptional profile akin to human nephron progenitors. Lineage-tracing using gene-edited induced pluripotent stem cell (iPSC) lines revealed that SIX2-expressing cells contribute to nephron formation but not to the putative collecting duct epithelium. However, Cre-mediated temporal induction of the SIX2+ lineage revealed a declining capacity for these cells to contribute to nephron formation over time. This suggests human kidney organoids, unlike the developing kidney in vivo, lack a nephron progenitor niche capable of both self-renewal and ongoing nephrogenesis. Nonetheless, human iPSC-derived kidney tissue maintains previously identified lineage relationships supporting the utility of pluripotent stem cell-derived kidney organoids for interrogating the molecular and cellular basis of early human development.

cell biology↗