Biology subjects

Manna, S.

Publications and source records attributed to Manna, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Global population structure and phase variation of serotype 12F Streptococcus pneumoniae following the introduction of pneumococcal conjugate vaccine

BackgroundAfter the global deployment of pneumococcal conjugate vaccines (PCVs), serotype 12F has become the predominant serotype responsible for invasive pneumococcal disease (IPD) worldwide. As PCVs that include serotype 12F are gradually introduced, we aim to characterise the global population structure and genetic diversity of the 12F capsule locus using whole-genome sequencing. Capsule variants with vaccine evasion potential were further investigated by functional experiments. MethodsA global collection of pneumococcal serotype 12F genomes (n=806) from 37 countries across six continents were included in this study. To characterise the serotype 12F population, Global Pneumococcal Sequence Cluster (GPSC), in silico serotype, and antimicrobial resistance profile were inferred from whole-genome data for each isolate. The capsule biosynthesis (cps) locus was analysed for gene content variations that could alter polysaccharide capsule production or structure, thereby influencing recognition by vaccine-induced antibodies. These isolates were further investigated by assessing their capsule production using immunofluorescence assays and its susceptibility to vaccine-elicited antibody killing by opsonophagocytosis assays. FindingsThe global increase in serotype 12F was driven by both distinct pneumococcal lineages across different continents, and a globally-disseminated and multidrug-resistant lineage GPSC26. We identified six capsule variants in nine isolates that had disruptive mutations in cps genes including wze, wcil, wciJ and fnlA. Most (6/9) of the disruptive mutations were a result of strand-slippage mutations. A convergent strand-slippage mutation disrupting the glycosyltransferase gene wciJ was identified in four isolates from distinct lineages and countries. Despite the truncation, three of four isolates with available Quellung typing results still identified them as 12F, indicating the production of the capsule. We then created a genetically engineered lab strain with wciJ knockout and complemented with wciJ containing the strand-slipppage mutation. The knockout strain did not produce any capsule. In contrast, the lab strain with wciJ containing the strand-slippage mutation produced a mixed population of encapsulated and non-encapsulated pneumococci, even within the same chain of pneumococcal cells. This observation indicated encapsulated subpopulation possesses a functional WciJ and rapidly reversible strand-slippage mutation during replication. Opsonophagocytosis assays indicated that the clinical 12F strain with strand-slippage mutation in wciJ exhibited reduced susceptibility to vaccine-elicited serum killing, compared to a genetically closely related 12F clinical strain with an intact wciJ. However, substantial inter-individual antisera variation limits definitive interpretation. InterpretationOur work revealed the global rise of serotype 12F pneumococci has been driven by both regional-specific lineages, and a globally-disseminated and multidrug-resistant lineage GPSC26. We demonstrated that strand-slippage mutation is one of the major drivers of serotype 12F capsule variants and represents a novel mechanism enabling reversible on-off switching of capsule production. The ability to switch off capsule expression in a subpopulation may enable evasion of antibody-mediated killing but increase susceptibility to innate immune clearance. FundingBill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control and Prevention.

genomics↗

bioRxiv2026

Modulating radical propagation in proteins by proton-coupled electron transfer and hydrogen bonding

Long-range protein electron transfer (ET) often depends on tryptophan and tyrosine residues acting as radical relay sites. For example, cytochrome c peroxidase (CcP) generates a W191^dot+ radical to increase ET from cytochrome c (Cc) to the active center. W191 substitution to Tyr reduces ET rates, but introduction of an adjacent general base (as Glu or His) at position 232 (Y191:E/H232 CcP) recovers activity. E232 fluorination lowers the pKa of the conjugate base and confirms that a hydrogen bond is critical to elevate the Y191^dot formal potential for effective ET. Photoinitiated ET between Zn-porphyrin (ZnP) CcP (ZnCcP) and Cc also depends on activating Y191 with a basic residue, but through a different mechanism than for the peroxide-driven system. In ZnCcP, pH dependencies and solvent isotope effects indicate that proton-coupled electron transfer to the basic residue and ZnP^dot+, respectively, facilitates Y191^dot formation. Replacing Cc with the irreversible oxidant [Co(NH3)5Cl]2+ isolates distinct protein radicals for characterization by Electron Paramagnetic Resonance (EPR) spectroscopy. Radical distributions and computation indicate that W191^dot+ lies close in potential to ZnP^dot+ and that the two radicals exchange on a slow time scale despite their close separation. Remarkably, Y191:E/H232 ZnCcP variants propagate radicals differently to peripheral sites depending on the nature of the 232 residue. QM/MM calculations support radical exchange between ZnP^dot+/Trp^dot+ and the importance of a hydrogen bond to Y191^dot for maintaining a high potential to oxidize peripheral donors. These resolved reactivity patterns of CcP/ZnCcP have general relevance for engineering proton management to separate and migrate charge in proteins and potentially other molecular systems.

biochemistry↗

bioRxiv2025

Advancing Knock-In Approaches for Robust Genome Editing in Zebrafish

Precise genome editing remains a major challenge in functional genomics, particularly for generating knock-in (KI) alleles in model organisms. Here, we introduce the mini-golden system, a versatile Golden Gate-based subcloning platform that enables rapid assembly of donor constructs containing homology arms and a gene of interest. This system offers a library of middle entry vectors including diverse genes, enhancing the preparation of donor minicircles for KI applications. Using the mini-golden system, we efficiently generated a foxd3CreER KI zebrafish line, allowing conditional recombination in neural crest cells. To further improve genome editing precision, we developed a synthetic exon-based donor template strategy combined with fluorescence screening. Using this approach, we successfully engineered a targeted isoleucine-to-valine substitution (Ile-to-Val) in hbaa1.2, one of the two adult hemoglobin alpha genes in zebrafish. Importantly, despite the high sequence similarity between hbaa1.2 and its paralog hbaa1.1, our strategy specifically edited hbaa1.2, demonstrating the effectiveness of the synthetic exon approach. This method minimized undesired recombination and significantly improved the identification of lines carrying the edited genome. Together, we provide a robust toolkit for efficient and precise genome engineering in zebrafish, with broad applicability to other model systems.

developmental biology↗

bioRxiv2025

Nasopharyngeal colonisation by Streptococcus pneumoniae enhances host anti-viral responses to respiratory virus infection

Viral-bacterial interactions during co-infection are often synergistic and can increase disease severity. However, emerging evidence indicates that some bacteria can antagonise viral infection, although the host responses driving this process remains unclear. Using infant mice co-infected with the nasopharyngeal inhabitant and pathogen Streptococcus pneumoniae and pneumonia virus of mice (PVM) to model antagonistic interactions, we found that prior bacterial colonisation enhances and prolongs anti-viral immune responses during co-infection, compared with viral infection alone. Transcriptomic, immunological, and histological analyses showed that pneumococcal colonisation prior to PVM infection enhanced and prolonged interferon signalling, increased anti-viral cytokine and chemokine protein levels and CD8+ cell responses. Notably, over 50% of differentially expressed host genes during co-infection were not differentially expressed in either infection alone. Our work shows that bacterial colonisation can modulate host immunity, shaping how the immune system responds to incoming viral infections, which has the potential to open novel therapeutic applications. HighlightsO_LIPneumococcal mono-infection in infant mice resulted in a delayed host transcriptomic response that was not detectable until 12 days post-infection. C_LIO_LIHost transcriptomic and immune responses to PVM were minimal except at the peak of viral replication and rapidly returned to baseline levels. C_LIO_LIPrior pneumococcal colonisation enhanced anti-viral immune responses to PVM infection, with more than half of the differentially expressed genes unique to co-infection. C_LIO_LIPneumococcal nasopharyngeal colonisation shapes the immune response, changing how the host responds to incoming viral infections with multiple anti-viral responses that were only transiently activated during PVM mono-infection being active for a longer duration during co-infection C_LI

microbiology↗

bioRxiv2025

Phase separation as a concentrating mechanism of protein/peptide hormones for the secretory granule storage

Protein/peptide hormones in the regulated secretory pathway are stored within secretory granules (SGs) as densely packed aggregates for extended periods of time until their stimulated release. These aggregated dense cores are reported as functional amyloids for several pituitary hormones. However, the molecular events initiating hormone aggregation and SG biogenesis remain poorly understood. Using a diverse set of protein/peptide hormones with different sizes and structures, we demonstrated that all these hormones undergo phase separation in trans-Golgi network (TGN)-relevant conditions (including low pH, the presence of glycosaminoglycans, etc.), which rapidly transitioned into solid-like structures and eventually form amyloid fibrils. Further, the cellular model expressing two protein hormones (GH-EGFP and PRL-EGFP) showed the highly dynamic, liquid-like condensate formation at the TGN volume, which was transported via microtubule to the cellular periphery/tips and formed a ready pool of mature SGs containing amyloid-rich hormone aggregates. Importantly, both in vitro and cellular studies confirmed that solid-like amyloid aggregates are reversible and can release bioactive monomeric hormones, recapitulating regulated exocytosis. Together, our findings suggest hormone phase separation at the TGN as a common molecular principle for concentrating cargo, driving rapid functional aggregation, and ensuring efficient storage within SGs.

biochemistry↗

bioRxiv2025

Streptococcus pneumoniae vaccine serotype persistence following 13-valent pneumococcal conjugate vaccine introduction in Mongolia: investigating changes in epidemiology, immunology and virulence

BackgroundStreptococcus pneumoniae is a leading cause of pneumonia globally. Vaccine serotypes can persist despite pneumococcal conjugate vaccine (PCV) introduction. To examine serotype persistence, we leveraged 6,545 nasopharyngeal swabs collected from children hospitalised with pneumonia before and after PCV13 introduction in Mongolia and undertook molecular, epidemiological and experimental analyses. MethodsSerotype, genetic lineage and antimicrobial resistance genes were inferred from DNA microarray. Patients carrying lineages that were predominant pre- and post-PCV introduction were examined for differences in disease severity. We also compared the pre- and post-PCV lineages by bacterial adhesion to hydrocarbon (BATH) and enzyme-linked immunosorbent (ELISA) assays. Capsule gene expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR), capsule thickness by transmission electron microscopy, and virulence using an infant mouse model. FindingsChanges in lineage composition were observed within serotypes 6A, 6B, 14, 19F and 23F over the six-year surveillance period. Children carrying pre-PCV lineages were more likely to have severe pneumonia than those carrying post-PCV lineages (serotypes 6B, 14 and 19F). Pre-PCV lineages were more likely to be multi-drug resistant than post-PCV lineages (serotypes 6A, 6B and 23F). For serotype 6B the post-PCV lineage had higher cell surface hydrophobicity, lower IgG, lower expression of capsule genes and evidence of thinner capsule than the pre-PCV lineage. Notably, the post-PCV 6B lineage was also less virulent in mice than the pre-PCV 6B lineage. InterpretationDespite persistence of vaccine serotypes in highly vaccinated populations, vaccination may select for lineages with differences in capsule and antibody binding and that are less virulent. When evaluating the true value of vaccination, it is important to consider factors beyond serotype alone. FundingNational Health and Medical Research Council, Murdoch Childrens Research Institute, GAVI, the Vaccine Alliance.

microbiology↗

bioRxiv2025

AKNA drives neural stem cell fate transition through differential localization and coordinating the modulation of chromatin from H3K27me3 to H3K27ac

Neuronal stem cells (NSCs) play pivotal role in adult neurogenesis, however, detail mechanisms of obtaining pluripotency or undergoing differentiation remain unknown. Herein, how AT-hook protein AKNA regulates pluripotency and stemness in neuroblastoma cells is demonstrated by gene knockdown, immunofluorescence, chromatin immunoprecipitation (ChIP), localization of AKNA, signaling interactions and transcriptional activity. AKNA was abundant and mostly nuclear during induction of pluripotency and its knockdown reduced stemness, even in presence of other pluripotency factors, including OCT4 and SOX2. During the course of induction of differentiation, AKNA remain localized in the cytosol, essential for regulated differentiation. Cytosolic retention of AKNA is possibly driven by FAK signaling. In the nucleus, AKNA promotes KDM6B demethylase for H3K27me3 demethylation to H3K27, and subsequently promotes CBP/p300 to acetylate H3K27 to H3K27ac deposition on promoter region of respective genes to trigger their transcription. Data obtained from knockdown and overexpression of AKNA and KDM6B further reinforce its importance that, they physically interact to drive pluripotency and stemness. These findings establish AKNA as a critical regulator for NSCs fate determination in association with epigenetic modifiers and signaling pathway, offering potential targets for neuroblastoma therapies and regenerative medicines for neurodegenerative diseases.

molecular biology↗

bioRxiv2025

Dietary fatty acids as determinants of cuticular wax profiles in Tribolium castaneum

Cuticular waxes are complex mixtures of hydrocarbons and other lipophilic compounds that form an outer protective layer over the insect cuticle playing a role in ecological interactions as well as physiological processes. While CHC profiles are largely genetically determined, several studies have linked diet to cuticular hydrocarbons and limited research indicates a potential influence of dietary fatty acids. Considering this, the current work investigates the effect of nine cereal flours, i.e., wheat, rice, maize, barley, oat, ragi, sorghum, soybean and gram, each with a distinct fatty acid composition, on the cuticular wax profile of Tribolium castaneum (Coleoptera: Tenebrionidae), a globally significant stored grain insect. Fatty acid compositions of the nine flours and the cuticular waxes from the beetle populations reared on each diet were characterized using gas chromatography mass spectrometry (GC-MS). In all the beetle populations, the most abundant compound was 1-pentadecene, followed by cis-9-tetradecen-1-ol and 1-heptadecene, irrespective of the diet. While no qualitative differences in cuticular wax composition were found, significant quantitative variation in cuticular wax profile was observed across dietary groups. Similarity percentage (SIMPER) analysis revealed a cumulative Bray- Curtis dissimilarity of 47.72 among the flour samples based on their fatty acid profiles, and a dissimilarity of 18.51 among T. castaneum populations reared on these flours based on their cuticular wax composition. Non-metric multidimensional scaling (NMDS) further indicated substantial variation in the cuticular wax profiles of beetle populations raised on different dietary sources. Pearsons correlation analysis between the concentrations of individual cuticular wax components and the fatty acid content of the respective diets demonstrated strong correlations between specific cuticular compounds and dietary fatty acids. Notably, levels of 1-pentadecene in the beetle cuticle were strongly associated with the concentrations of hexadecanoic and octadecanoic acids in the flours. These results support the hypothesis that, alongside endogenous conserved biosynthetic pathways, dietary fatty acid composition contributes to the modulation of insect cuticular chemistry. This dietary influence may have important implications for understanding insect ecological plasticity and diet-mediated changes in surface chemistry.

zoology↗

bioRxiv2025

SeroBA(v2.0) and SeroBAnk: a robust genome-based serotyping scheme and comprehensive atlas of capsular diversity in Streptococcus pneumoniae

The unprecedented number of Streptococcus pneumoniae (the pneumococcus) genomes sequenced in recent years has accelerated the discovery of novel serotypes and highlighted the genetic diversity both between and within each serotype. A novel serotype should demonstrate a distinct cps locus, capsular structure, and serological profile. In only the past four years, nine new serotypes have been identified. Accurate and timely serotyping of pneumococcal isolates is key to understanding its global distribution, evolution, and the response of the bacterial population to vaccination. However, current bioinformatics serotyping tools are infrequently updated, and struggle to accommodate the rapid discovery of new serotypes in a timely manner. To address these limitations, we built a comprehensive and curated library (SeroBAnk) encompassing all known pneumococcal serotypes; this resource is presented as an atlas on a dedicated publicly accessible webpage (https://www.pneumogen.net/gps/#/serobank). Building upon this resource, we developed SeroBA(v2.0), a tool with an easy-to-update database that can accurately identify 102 of 107 known pneumococcal serotypes (except for serotypes 24B, 24C, 24F, 7D and 6H) and 18 genetic subtypes within serotypes 6A, 6B, 11A, 19A, 19F and 33F. We validated SeroBA(v2.0) on 26,306 genomes from the Global Pneumococcal Sequencing project, reference isolates and simulated reads derived from the reference genetic sequences of capsular polysaccharide biosynthetic (cps) locus and showed that SeroBA(v2.0) can reliably detect the nine recently discovered serotypes. Additionally, we show that in silico serotypes inferred by SeroBA(v2.0) had high concordance with phenotypic serotypes determined by either Quellung or latex agglutination at the serotype level (88.9%; 15,945/17,933), and at the serogroup level (91.9%; 16,480/17,933). Finally, we propose a community-contribution based approach to ensure that SeroBA(v2.0) is maintained and updated as novel serotypes continue to be discovered. The global community can submit putative novel serotypes through our public repository on GitHub (https://github.com/GlobalPneumoSeq/seroba/issues). The submitted putative novel serotypes will be curated based on the genetic sequence of cps region, capsular structure and serological profile by people of relevant expertise in the field. SeroBA(v2.0) can be accessed at https://github.com/GlobalPneumoSeq/seroba. Data summaryGenome sequences are available in the European Nucleotide Archive (ENA) and are also available alongside metadata on the Monocle Database available at https://data.monocle.sanger.ac.uk/. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. Impact StatementThe polysaccharide capsule has been an effective vaccine antigen against diseases caused by Streptococcus pneumoniae (the pneumococcus). The pneumococcal conjugate vaccine has been estimated to have halved pneumococcal-related childhood mortality over 15 years (2000-2015). We collated the genetic locus and capsular structure of each known capsule type (serotype), alongside with pneumococcal vaccine formulation and licensure history, into a single webpage (SeroBAnk), providing a valuable resource for basic research and vaccine development. With increasing use of whole genome sequencing in clinical and public health laboratories, we also provided a fast and accurate bioinformatics tool, SeroBA(v2.0), to identify 102 pneumococcal serotypes, alongside a proposed system to expand SeroBA(v2.0) to include new serotypes as they are discovered, ensuring that the tool remains valuable to the global research community in the long-term.

bioinformatics↗

bioRxiv2025

RNA-triggered CRISPR-Cas12a2 Preferentially and Cooperatively Cleaves Collateral DNA

CRISPR-Cas systems often rely on collateral cleavage of nucleic-acid substrates to combat recognized mobile genetic elements. Of the CRISPR-associated (Cas) RNA-guided effector nucleases, Cas12a2 stands out as the only known example exhibiting rapid collateral cleavage of three distinct substrates: single-stranded (ss)RNA, ssDNA, and double-stranded (ds)DNA, after activating upon binding cognate RNA. However, little is known about the underlying mechanisms of collateral cleavage. Here, we show, using enzyme kinetics and inhibition assays, that Cas12a2 preferentially cleaves collateral DNA over RNA substrates, even when RNA substrates are more abundant. Additionally, using enzyme mutants, enzyme kinetics, and plasmid cleavage assays, we determine that the dsDNA cleavage mechanism relies on the aromatic clamp residues that stabilize unwound and distorted dsDNA in the RuvC nuclease active site. Leveraging the cleavage preference for collateral DNA, we demonstrate that RNA-activated Cas12a2 can readily cleave a ssDNA probe in the presence of high concentrations of non-target RNA, while an RNA-targeting Cas13a cannot. This work provides foundational kinetic and biochemical insights into the collateral cleavage mechanism and substrate preferences of Cas12a2, with immediate implications for understanding Cas12a2-based immunity and developing Cas12a2-based technologies.

biochemistry↗

bioRxiv2024

Functional insights on chemical communication of fishing cats: a strategic mechanism for adaptation in semi-aquatic habitat

Chemical communication system of fishing cats, focusing on volatile organic compounds present in their urine (UR) and marking fluid (MF) has been explored. Using solid-phase microextraction guided gas chromatography mass spectrometry analysis, a diverse range of functional groups from UR and MF were detected, including ketones, aldehydes, sulfur- and nitrogen-containing compounds, among which 3-Mercapto-3-methylbutanol (3MMB) and its related sulfur compounds were found to be sex-specific, predominantly present in males. The unique behavior of fishing cats to urinate frequently in water when experimentally tested with synthetic 3MMB, was aligned for its retention in aqueous medium upto 21 days. This suggests that fishing cats may employ an energy-efficient scent-marking strategy by leveraging aquatic environments for prolonging chemical signalling process. The analysis of less-volatile compounds, such as triglycerides and fatty acids, suggests that these compounds could be acted as fixatives, helping to stabilize and prolong the intensity of chemical messages coded to highly volatile molecules. The combination of highly volatile, less volatile compounds and lipids may create complex synergy to facilitate interindividual communication, potentially serving as pheromones. This research enhances our understanding of the fishing cats adaptation to semi-aquatic environments and its unique behavioural pattern in marshy landforms, thus concentrated with high population density in deltaic South Bengal.

zoology↗

bioRxiv2024

Unveiling the chemical and behavioural ecology of Tribolium castaneum (Herbst, 1797) in wheat flour: Alterations in flour metabolic content and the role of chemical cues in modulating beetles' behaviour and regulating population growth.

Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae) is a notorious secondary pest of stored grains and flours, accounting for 10-15% of global annual losses in stored products. This study first evaluated the impact of T. castaneum infestation on the nutritional parameters of stored wheat flour. Infested flour exhibited reductions in total soluble sugars, triacylglycerol levels, and unsaturated fatty acids, particularly Oleic and Linoleic acid, while secondary metabolites such as phenolics, flavonoids, and tannins were increased. Behavioural assays revealed that adult beetles were significantly more attracted towards infested flour odour due to the presence of 4,8-Dimethyldecanal (4,8-DMD), an aggregation pheromone of T. castaneum. Another key compound in infested flour, 1-Pentadecene, derived from the beetles cuticular wax, was found to influence adult beetle behaviour. At higher concentrations, 1-Pentadecene acted as a repellent for the species. Molecular docking studies suggested that 1-Pentadecene competes with 4,8-DMD for binding to the odorant receptor TcOR1, which could potentially inhibit aggregation behaviour and reduce population growth. The docking analysis revealed that 1-Pentadecene interacts with the same binding pocket on TcOR1, sharing key amino acids (MET49, PHE69, THR72, GLN73, THR134, VAL135, TYR138, LEU154, ILE177, GLY180, ALA181, TYR286) and forming similar intermolecular bonds, such as a pi-alkyl bond, with a binding affinity ({Delta}G = -5.7) comparable to 4,8-DMD ({Delta}G = -5.8). Additionally, beetles reared in wheat flour containing higher concentrations of 1-Pentadecene exhibited lower population growth compared to those reared in flour with lower concentrations of this compound.

zoology↗

bioRxiv2024

Modulation of H3K4 trimethylation by KDM5A and MLLs impacts metabolic adaptability in prostate and cervical cancer cells

AbstractChemical modifications of chromatin modulate gene expression and induce essential metabolic plasticity for tumor growth. Accumulation of H3K4me3 in the promoter of a gene activates transcription by making the promoter accessible to the polymerases. Methylation of H3K4 is catalysed by MLLs and demethylation of H3K4me3 is catalysed by KDM5 family proteins. Herein, we investigated if genes encoding the enzymes involved in glucose metabolism are dependent on KDM5A and MLL1, and if targeting the H3K4me3 would help in modulating the resilience of cancer cells. We present that KDM5A modulates most of the metabolic genes in a demethylase dependent manner as assesses by H3K4me3 occupancy on G6PD and catalase promoters. Targeting its expression would indeed help in sensitizing cancer cells to ROS dependent apoptotic cell death. We elucidated the differences in the epigenetic regulation in cancerous cells originated from cervical and prostate tissues and used a normal skin keratinocyte for comparison. In cervical and prostate cancers - KDM5A activated glycolysis but downregulates other metabolic processes. In cervical cancer, which majorly depends on PPP, changes in KDM5A did not modulate the G6PD expression. Further, we have shown that curcumin treatment enhanced KDM5A expression and downregulated MLL2 in cancer cell lines but not in keratinocyte cells. Curcumin inhibited metabolic pathways and enhanced apoptosis in cancer cells without affecting keratinocyte cells by modulating KDM5A and MLL levels. This work also strengthens the basic concept that, epigenetic modulations of genes in a tissue precisely depends on signal and sites of modification(s).

cancer biology↗

bioRxiv2023

Osteopontin in colitis-associated carcinoma

Patients with ulcerative colitis (UC) and Crohns disease (CD) face a lifelong risk of developing colitis-associated carcinoma (CAC). Current insights into CAC development originate from murine CAC models, while human CAC studies primarily focus on mutational analysis of patient samples. Although the mutational landscape reveals distinct patterns and frequencies compared to colorectal cancer, it falls short in elucidating the inflammatory mechanisms of CAC development. Consequently, we adopted a multi-omics approach to unravel CAC carcinogenesis from an immunological perspective. Our data revealed a robust upregulation of SPP1 gene in CAC at both RNA and protein levels, expressed by CD68+ macrophages. In vitro OPN stimulation demonstrated no direct effect on intestinal epithelial organoids. However, the mutually exclusive spatial location of SPP1/OPN+ macrophages and CD8+ T cells suggests a crucial indirect role of SPP1/OPN in mediating an immunosuppressive tumour microenvironment.

cancer biology↗

bioRxiv2023

Streptococcus pneumoniae serotype 33G: genetic, serological, and structural analysis of a new capsule type

Streptococcus pneumoniae (the pneumococcus) is a human pathogen responsible for a spectrum of diseases such as pneumonia, sepsis, and meningitis. The capsule is the major pneumococcal virulence factor and is encoded by the capsular polysaccharide (cps) locus, a recombination hot spot that has resulted in over 100 distinct capsular polysaccharide types (serotypes) identified to date. Recently, 33X (also known as 10X) was proposed as a putative novel serotype, but the capsule structure had not been elucidated. Here, we provide an in-depth investigation of 33X, demonstrating it is a new pneumococcal capsular serotype. In this study, we screened 12,850 nasopharyngeal swabs from both healthy children and pneumonia patients (adults and children) in Mongolia collected between 2015-2022. We identified 20 pneumococcal 33X isolates. Using whole genome sequencing, we found that the 33X cps locus is a chimera of genes from pneumococcal serogroups 35, 10 and 33, as well as other Streptococcal species. Serotyping of 33X pneumococci by the Quellung reaction revealed a unique serological profile, typing as both 10B and 33B. Competitive ELISAs confirmed that antibodies that were generated in mice directed against 33X were inhibited by 33X pneumococci but not 10B or 33B. Lastly, elucidation of the 33X capsule structure revealed that the polysaccharide is distinct from other serotypes, consisting of an O-acetylated hexasaccharide repeat unit of [->]5)-{beta}-Galf-(1[->]3)-{beta}-Glcp-(1[->]5)-{beta}-Galf 2Ac-(1[->]3)-{beta}-GalpNAc-(1[->]3)--Galp-(1[->]4)-Rib-ol-(5[->]P[->]. Therefore, 33X meets the requisite genetic, serological, and biochemical criteria to be designated as a new serotype, which we have named 33G. IMPORTANCEStreptococcus pneumoniae (the pneumococcus) is a bacterial pathogen with the greatest burden of disease in Asia and Africa. The pneumococcal capsular polysaccharide has biological relevance as a major virulence factor, as well as public health importance as it is the target for currently licensed vaccines. These vaccines have limited valency, covering up to 23 of the >100 known capsular types (serotypes) with higher valency vaccines in development. Here, we have characterized a new pneumococcal serotype, which we have named 33G. We detected serotype 33G in nasopharyngeal swabs (n=20) from children and adults hospitalized with pneumonia, as well as healthy children in Mongolia. We show that the genetic, serological, and biochemical properties of 33G differs from existing serotypes, satisfying the criteria to be designated as a new serotype. Future studies should focus on the geographical distribution of 33G and any changes in prevalence following vaccine introduction.

microbiology↗

bioRxiv2023

EGFR-to-Src family tyrosine kinase switching in proliferating-DTP TNBC cells creates a hyperphosphorylation-dependent vulnerability to EGFR TKI

Triple-Negative Breast Cancer (TNBC) is the most aggressive type of breast malignancy, with chemotherapy as the only mainstay treatment. TNBC patients have the worst prognoses as a large fraction of them do not achieve complete pathological response post-treatment and develop drug-resistant residual disease. Molecular mechanisms that trigger proliferation in drug-resistant chemo-residual TNBC cells are poorly understood due to the lack of investigations using clinically relevant cellular models. In this study, we have established TNBC subtype-specific cellular models of proliferating drug-tolerant persister (PDTP) cells using different classes of chemotherapeutic agents that recapitulate clinical residual disease with molecular heterogeneity. Analysis of total phospho-tyrosine signals in TNBC PDTPs showed an enhanced phospho-tyrosine content compared to the parental cells (PC). Interestingly, using mass-spectrometry analysis, we identified a dramatic decrease in epidermal growth factor receptor (EGFR) expression in the PDTPs, while the presence of hyper-activated tyrosine phosphorylation of EGFR compared to PC. Further, we show that EGFR has enhanced lysosomal trafficking in PDTPs with a concomitant increase in N-Myc Downstream Regulated-1 expression that co-localizes with EGFR to mediate receptor degradation. More surprisingly, we found that reduced protein levels of EGFR are coupled with a robust increase in Src family kinases, including Lyn and Fyn kinases, that creates a hyper-phosphorylation state of EGFR-Src tyrosine kinases axis in PDTPs and mediates downstream over-activation of STAT3, AKT and MAP kinases. Moreover, paclitaxel-derived PDTPs show increased sensitivity to EGFR TKI Gefitinib and its combination with paclitaxel selectively induced cell death in PDTP-P TNBC cells and 3D spheroids by strongly downregulating phosphorylation of EGFR-Src with concomitant downregulation of Lyn and Fyn tyrosine kinases. Collectively, this study identifies a unique hyper-phosphorylation cellular state of TNBC PDTPs established by switching of EGFR-Src family tyrosine kinases creating a vulnerability to EGFR TKI.

cancer biology↗

bioRxiv2023

GPX4-VIM equates a proliferating DTP state in TNBC subtypes with converged vulnerabilities to autophagy and glutathione inhibition.

Frequent metastatic relapses in Triple-Negative Breast Cancer (TNBC) patients with residual disease is a clinical challenge, largely due to tumor heterogeneity and absence of strategies that target proliferating chemo-tolerant cells. Here, we longitudinally modeled cellular state transitions from dormant drug-tolerant persister (DTP) into proliferating drug-tolerant persister (PDTP) in cells representing all TNBC subtypes. Combining subcellular imaging with phenotypic and biochemical assays, we identified distinct and converged spectrums of alterations in TNBC-PDTPs. We show that PDTPs retain acquired resistance with increased invasion potential. Moreover, Basal-Like DTPs enter into a non-reversible mesenchymal state while luminal androgen receptor-positive gain partial-Epithelial-to-Mesenchymal Transition (EMT) with vimentin upregulation. PDTP state dwells on high autophagy with reduced glutathione and GPX4 levels, rendering it vulnerable to autophagy suppression and ferroptosis. Interestingly, we find that GPX4 negatively regulates EMT and autophagy in TNBC, and an inverse correlation of GPX4-VIM expression along with autophagy genes predicts survival in TNBC patients undergoing chemotherapy.

cancer biology↗

bioRxiv2023

Therapeutic immunization with a whole cell vaccine reduces pneumococcal nasopharyngeal density, shedding, and middle ear infection in mice

Pneumococcal Conjugate Vaccines (PCVs) have substantially reduced the burden of disease caused by Streptococcus pneumoniae (the pneumococcus). However, protection is limited to vaccine serotypes, and when administered to children who are colonized with pneumococci at the time of vaccination, immune responses to the vaccine are blunted. Here, we investigate the potential of a killed whole cell pneumococcal vaccine (WCV) to reduce existing pneumococcal carriage and mucosal disease when given therapeutically to infant mice colonized with pneumococci. We show that a single dose of WCV reduced pneumococcal carriage density in an antibody-dependent manner. Therapeutic vaccination induced robust immune responses to pneumococcal surface antigens CbpA, PspA (family 1) and PiaA. In a co-infection model of otitis media, a single dose of WCV reduced pneumococcal middle ear infection. Lastly, in a two-dose model, therapeutic administration of WCV reduced nasal shedding of pneumococci. Taken together, our data demonstrate that WCV administered in colonized mice reduced pneumococcal density in the nasopharynx and the middle ear, and decreased shedding. A vaccine with similar properties in children would be beneficial in low and middle-income settings where pneumococcal carriage is high. ImportanceAlthough typically asymptomatic, pneumococcal carriage plays an essential role in transmission and the development of disease. Pneumococcal Conjugate Vaccines (PCVs) have reduced the burden of pneumococcal disease worldwide. However, their use has increased carriage and disease caused by non-vaccine serotypes, prompting investigations into serotype-independent pneumococcal vaccines. An additional limitation of PCVs is immune hypo-responsiveness to vaccines in children carrying pneumococci at the time of vaccination. Therefore, there is great interest in next generation vaccines such as whole cell vaccines. In this study we investigate a pneumococcal whole cell vaccine (WCV) for it effect on carriage in mice that are already colonized at the time of vaccination. We show that this therapeutic vaccination of mice can reduce pneumococcal carriage density, shedding and infection of the middle ear. Our study suggests that WCV could be beneficial in high burden settings where carriage at the time of vaccination is more common.

microbiology↗