Biology subjects

Spiga, L.

Publications and source records attributed to Spiga, L..

15 recordsLinked to original sources

bioRxiv2026

Spatiotemporal control of STING activation and saRNA delivery decouples humoral and cellular immunity

Self-amplifying mRNA (saRNA) vaccine formulations link the location and timing of antigen expression to innate immune stimulation by both RNA cargo and carrier. We hypothesized that targeting a polymeric STING agonist prodrug (polySTING) to antigen-presenting cells (APCs) independently of saRNA delivery could tune vaccines responses without limiting saRNA expression. By varying the time and location of STING signaling relative to saRNA delivered by muscle-restricted (polyplex, PP) and lymph node-draining (lipid nanoparticle, LNP) saRNA formulations, we studied how spatiotemporal colocalization of type I interferon (IFN-I) and antigen expression impacts innate and adaptive vaccine responses. Co-administering PP + polySTING decreased antigen-specific adaptive type 1 responses by mismatching early lymph node IFN-I and later arriving antigen; this could be reversed by delaying polySTING delivery timing. Conversely, synchronizing early LNP saRNA delivery and polySTING IFN-I in the lymph node boosted saRNA type 1 T cell responses without impacting humoral immunity, which re-directed antigen-intrinsic immune skewing and enhanced protection against pulmonary Acinetobacter baumannii challenge. Collectively, these findings define formulation-specific innate immune responses to saRNA vaccines and establish APC-targeted adjuvants as a strategy to tune saRNA vaccine immunogenicity.

immunology↗

bioRxiv2026

A nitrite-responsive regulatory RNA locus sustains commensal resilience against nitrosative stress

Commensal microbes in the gastrointestinal tract are central to host health, yet they must adapt to frequent perturbations such as intestinal inflammation that challenges microbial homeostasis. A major challenge during inflammation is exposure to host-derived reactive nitrogen species (RNS), which damage macromolecules and impair microbial fitness, but how commensals orchestrate defense against nitrosative stress remains poorly defined. Here, we show that Bacteroides thetaiotaomicron mounts a protective RNS-defense program centered on the hybrid cluster protein Hcp, which is required for fitness under nitrosative stress. We identify a nitrite-responsive SnoA locus (Stress-responsive Nitric Oxide regulator A) that promotes HcpR-dependent hcp expression. In vivo, this pathway promotes commensal resilience in both an antibiotic-perturbed, Nos2-dependent model of intestinal nitrosative stress and during Salmonella-induced gut inflammation. Together, our findings identify a regulatory pathway that enables a dominant gut commensal to withstand host-derived nitrosative stress and persist during intestinal inflammation.

microbiology↗

bioRxiv2026

Rational engineering of facultative anaerobiosis enables commensal survival in the oxygenated gut

Life originated in the absence of oxygen. Despite its substantial energetic advantages, many modern microbes remain obligate anaerobes, confined to anoxic niches such as the mammalian gut. Why these organisms cannot tolerate oxygen has remained unresolved for more than two centuries. Here, using integrated multi-omics analyses, we identify a network of interlocking vulnerabilities in central metabolism, biosynthetic pathways, and redox homeostasis that together impose an aerobic growth barrier in the obligate anaerobic commensal Bacteroides thetaiotaomicron. Rational repair of these vulnerabilities restores metabolic integrity and progressively enhances oxygen tolerance, yielding engineered strains capable of robust growth at 10% O2 and markedly improved resilience in the oxygenated, inflamed gut. These findings define a molecular basis for obligate anaerobiosis and establish a framework for engineering commensal bacteria to function in oxidative environments, expanding their ecological range and therapeutic potential.

microbiology↗

bioRxiv2026

A novel C57BL/6 mouse model for the study of severe Citrobacter rodentium infection

The study of human enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) has been limited by the inability of these pathogens to effectively colonize murine models without prior antibiotic treatment. Because it mimics key features of human EPEC and EHEC infection, Citrobacter rodentium, a natural mouse pathogen that colonizes the lower intestine, has become the primary model for investigating these organisms. C57BL/6 mice are most commonly used for C. rodentium research, however, unless they carry specific genetic mutations, they typically develop only mild disease and clear the infection within weeks. As a result, models of severe disease in genetically unmodified hosts are lacking. Here, we describe the development of a non-genetically modified C57BL/6 mouse line with an undisturbed intestinal microbiota, highly susceptible to severe C. rodentium infection. Early infection in these mice was marked by significantly elevated cecal bacterial burdens and tissue pathology. Immune profiling revealed broad reductions in multiple lymphoid subsets, indicating impaired early mucosal activation. Although overall cytokine expression patterns were similar between groups, ceca of susceptible mice exhibited elevated baseline and early post-infection IL-18, as well as increased G-CSF at day 1. Microbiota analyses showed broadly comparable communities with wildtype controls, with some altered groups, such as Lachnospiraceae, Prevotellaceae, Desulfovibrionaceae, and Erysipelotrichaceae. Together, these findings characterize a robust C57BL/6 model that reproducibly develops severe C. rodentium-induced disease. This phenotype is driven by microbiota-associated alterations and impaired early cecal immunity, providing a valuable system for studying host-microbiota interactions in enteric infections.

microbiology↗

bioRxiv2025

Le click c'est chic: a plug-and-play virus-like particle vaccination platform enabled by non-canonical amino acid incorporation and click chemistry in the tobacco BY-2 cell-free protein synthesis system

Non-canonical amino acids (ncaas) can provide recombinant proteins with novel exciting functionalities beyond the limits of nature, such as orthogonal reaction groups. Notably, ncaa introduction can be used in vaccinology to enhance the adaptability and immunogenicity of putative vaccine candidates. Cell-free protein synthesis (CFPS) represents the most promising methodology to introduce ncaa into recombinant proteins of interest. However, traditionally used prokaryotic CFPS systems show limitations to produce complex proteins requiring post-translational modifications, whilst eukaryotic CFPS systems have historically been difficult to scale and show low protein yields. In this work, we establish the site-specific introduction of ncaas into complex proteins with the high-yielding and scalable eukaryotic tobacco BY-2 CFPS system (BYL), commercialised as ALiCE(R). The tyrosine transferase from Escherichia coli (eTyrT) was tested for amber suppression-mediated ncaa incorporation in BYL. eTyrT showed high incorporation yields of up to 2mg/ml recombinant protein for the azido-tyrosine and alkyne-tyrosine ncaas, with linear scalability up to 10ml without any losses in protein yield. We applied ncaa incorporation in BYL to enable click chemistry bioconjugation of the receptor binding domain (RBD) of influenza hemagglutinin to pre-assembled hepatitis B core (HBc) virus-like particles (VLPs). BYL efficiently produced the alkyne-modified RBD and azido-modified HBc VLPs, and their conjugation via copper-catalysed azide-alkyne cycloaddition (CuAAC) led to structurally intact, RBD-coated particles. VLP-RBD conjugates could efficiently hemagglutinate chicken erythrocytes where the individual proteins could not, proving both the sialic-acid binding activity of the RBD and its multivalent presentation by the HBc VLP. Finally, when used to vaccinate mice the conjugated RBD-VLPs showed a greater protection against live influenza challenge than free RBD. This research thus enables ncaa introduction for recombinant proteins produced in BYL, constructing a novel plug-and-play vaccine platform and further expanding the capabilities of BYL to produce vaccine candidates and other proteins of interest.

molecular biology↗

bioRxiv2025

An Anaerobic Pathogen Rewires Host Metabolism to Fuel Oxidative Growth in the Inflamed Gut

To colonize their host and cause disease, enteric pathogens must deploy their virulence factors to establish distinct nutrient niches. How obligate anaerobic pathogens construct nutrient niches in the densely populated large intestine remains poorly understood. Enterotoxigenic Bacteroides fragilis (ETBF) is considered an obligate anaerobic bacterium and has been implicated in inflammation-associated diseases, including colitis and colorectal cancer. Here we show that ETBF uses its virulence factor, Bacteroides fragilis toxin, to reprogram colonic epithelial cell metabolism to colonize the inflamed gut. Bacteroides fragilis toxin activates colonic epithelial signaling and hijacks the host bile acid recycling pathway, inducing a metabolic shift in the epithelium from oxidative phosphorylation to glycolysis. This shift increases local concentrations of lactate and oxygen, nutrients that support an oxidative metabolism in ETBF. These findings reveal an unexpected strategy by which a pathogenic organism, previously considered to be an obligate anaerobic bacterium, generates and exploits an oxidative niche in the inflamed gut.

microbiology↗

bioRxiv2025

Pathobiont-triggered induction of epithelial IDO1 drives regional susceptibility to Inflammatory Bowel Disease

The structure and function of the mammalian gut vary by region, yet why inflammatory diseases manifest in specific regions and not others remains unclear. We use a TNF-overexpressing Crohns disease (CD) model (Tnf{Delta}ARE/+), which typically presents in the terminal ileum (TI), to investigate how environmental factors interact with the hosts immune susceptibility to drive region-specific disease. We identified Chlamydia muridarum, an intracellular bacterium and murine counterpart to the human sexually transmitted C. trachomatis, as necessary and sufficient to trigger disease manifestation in the ascending colon (AC), another common site of human CD. Disease manifestation in the AC depends on indoleamine 2,3-dioxygenase (IDO1) activity induced by hypersensitive surface secretory cells in genetically susceptible hosts. Single-cell and microbial analyses of human specimens also implicates this pathobiont-epithelial IDO1 pathway in patients with a history of CD in the AC. Our findings demonstrate that genetic and microbial factors can independently drive region-specific disease and provide a unique model to study CD specific to the AC.

cell biology↗

bioRxiv2024

Gene regulation of infection-associated L-tartrate metabolism in Salmonella enterica serovar Typhimurium

Enteric pathogens such as Salmonella enterica serovar Typhimurium experience spatial and temporal changes to the metabolic landscape throughout infection. Host reactive oxygen and nitrogen species non-enzymatically convert monosaccharides to alpha hydroxy acids, including L-tartrate. Salmonella utilizes L-tartrate early during infection to support fumarate respiration, while L-tartrate utilization ceases at later time points due to the increased availability of exogenous electron acceptors such as tetrathionate, nitrate, and oxygen. It remains unknown how Salmonella regulates its gene expression to metabolically adapt to changing nutritional environments. Here, we investigated how the transcriptional regulation for L-tartrate metabolism in Salmonella is influenced by infection-relevant cues. L-tartrate induces the transcription of ttdBAU, genes involved in L-tartrate utilization. L-tartrate metabolism is negatively regulated by two previously uncharacterized transcriptional regulators TtdV (STM3357) and TtdW (STM3358), and both TtdV and TtdW are required for sensing of L-tartrate. The electron acceptors nitrate, tetrathionate, and oxygen repress ttdBAU transcription via the two-component system ArcAB. Furthermore, regulation of L-tartrate metabolism is required for optimal fitness in a mouse model of Salmonella-induced colitis. TtdV, TtdW, and ArcAB allow for the integration of two cues, substrate availability and availability of exogenous electron acceptors, to control L-tartrate metabolism. Our findings provide novel insights into how Salmonella prioritizes utilization of different electron acceptors for respiration as it experiences transitional nutrient availability throughout infection. IMPORTANCEBacterial pathogens must adapt their gene expression profiles to cope with diverse environments encountered during infection. This coordinated process is carried out by the integration of cues that the pathogen senses to fine-tune gene expression in a spatiotemporal manner. Many studies have elucidated the regulatory mechanisms on how Salmonella sense metabolites in the gut to activate or repress its virulence program, however our understanding of how Salmonella coordinates its gene expression to maximize the utilization of carbon and energy sources found in transitional nutrient niches is not well understood. In this study, we discovered how Salmonella integrates two infection-relevant cues, substrate availability and exogenous electron acceptors, to control L-tartrate metabolism. From our experiments, we propose a model for how L-tartrate metabolism is regulated in response to different metabolic cues in addition to characterizing two previously unknown transcriptional regulators. This study expands our understanding of how microbes combine metabolic cues to enhance fitness during infection.

microbiology↗

bioRxiv2023

The RNA-binding protein RbpB is a central regulator of polysaccharide utilization in gut Bacteroides

Paramount to human health, symbiotic bacteria in the gastrointestinal tract rely on the breakdown of complex polysaccharides to thrive in this sugar-deprived environment. Gut Bacteroides are metabolic generalists and deploy dozens of polysaccharide utilization loci (PULs) to forage diverse dietary and host-derived glycans. The expression of the multi-protein PUL complexes is tightly regulated at the transcriptional level. However, how PULs are orchestrated at translational level in response to the fluctuating levels of their cognate substrates is unknown. Here, we identify the RNA-binding protein RbpB and a family of noncoding RNAs as key players in post-transcriptional PUL regulation. Ablation of RbpB in Bacteroides thetaiotaomicron displays compromised colonization in the mouse gut in a host diet-dependent manner. Current dogma holds that individual PULs are regulated by dedicated transcriptional regulators. We demonstrate that RbpB acts as a global RNA binder that directly interacts with several hundred cellular transcripts. This includes a paralogous noncoding RNA family comprised of 14 members, the FopS (family of paralogous sRNAs) cluster. Through a series of in-vitro and in-vivo assays, we reveal that FopS sRNAs repress the translation of a SusC-like glycan transporter when substrates are limited--an effect antagonized by RbpB. Together, this study implicates RNA-coordinated metabolic control as an important, yet previously overlooked, factor contributing to the in-vivo fitness of predominant microbiota species in dynamic nutrient landscapes.

microbiology↗

bioRxiv2023

Byproducts of inflammatory radical metabolism provide transient nutrient niches for microbes in the inflamed gut

Louis Pasteurs experiments on tartaric acid laid the foundation for our understanding of molecular chirality, but major questions remain. By comparing the optical activity of naturally-occurring tartaric acid with chemically-synthesized paratartaric acid, Pasteur realized that naturally-occurring tartaric acid contained only L-tartaric acid while paratartaric acid consisted of a racemic mixture of D- and L-tartaric acid. Curiously, D-tartaric acid has no known natural source, yet several gut bacteria specifically degrade D-tartaric acid. Here, we investigated the oxidation of monosaccharides by inflammatory reactive oxygen and nitrogen species. We found that this reaction yields an array of alpha hydroxy carboxylic acids, including tartaric acid isomers. Utilization of inflammation- derived D- and L-tartaric acid enhanced colonization by Salmonella Typhimurium and E. coli in murine models of gut inflammation. Our findings suggest that byproducts of inflammatory radical metabolism, such as tartrate and other alpha hydroxy carboxylic acids, create transient nutrient niches for enteric pathogens and other potentially harmful bacteria. Furthermore, this work illustrates that inflammatory radicals generate a zoo of molecules, some of which may erroneously presumed to be xenobiotics.

microbiology↗

bioRxiv2023

Laser Desorption Rapid Evaporative Ionisation Mass Spectrometry (LD-REIMS) demonstrates a direct impact of hypochlorous acid stress on PQS-mediated quorum sensing in Pseudomonas aeruginosa

To establish infections in human hosts, Pseudomonas aeruginosa must overcome innate immune generated oxidative stress, such as the hypochlorous acid (HOCl) produced by neutrophils. We set out to find specific biomarkers of oxidative stress through the development of a protocol for the metabolic profiling of P. aeruginosa cultures grown in the presence of different oxidants using a novel ionisation technique for mass spectrometry, laser desorption rapid evaporative ionisation mass spectrometry (LD-REIMS). We demonstrated the ability of LD-REIMS to classify samples as untreated or treated with a specific oxidant with 100 % accuracy and identified a panel of 54 metabolites with significantly altered concentrations after exposure to one or more of the oxidants. Key metabolic changes were conserved in P. aeruginosa clinical strains isolated from patients with cystic fibrosis lung infections. These data demonstrated that HOCl stress impacted the Pseudomonas Quinolone Signal (PQS) quorum sensing system. Ten 2-alkyl-4-quinolones (AHQs) associated with the PQS system were significantly lower in concentration in HOCl-stressed P. aeruginosa cultures, including 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), the most active signal molecule of the PQS system. The PQS system regulates the production of virulence factors, including pyocyanin and elastase, and their levels were markedly affected by HOCl stress. No pyocyanin was detectable and elastase concentrations were significantly reduced in cultures grown with sub-lethal concentrations of HOCl, suggesting that this neutrophil-derived oxidant may disrupt the ability of P. aeruginosa to establish infections through interference with production of PQS-associated virulence factors. IMPORTANCEThis work demonstrates that a high-throughput ambient ionisation mass spectrometry method can be used successfully to study a bacterial stress response. Its application to the opportunistic pathogen Pseudomonas aeruginosa led to the identification of specific oxidative stress biomarkers, and demonstrated that hypochlorous acid, an oxidant specifically produced by human neutrophils during infection, affects quorum sensing and markedly reduces production of the virulence factors pyocyanin and elastase in this bacterium. This approach has the potential to be widely applicable to the characterisation of the stress responses of bacteria.

microbiology↗

bioRxiv2023

Iron acquisition by a commensal bacterium modifies host nutritional immunity during Salmonella infection

During intestinal inflammation, host nutritional immunity starves microbes of essential micronutrients such as iron. Pathogens scavenge iron using siderophores, which is counteracted by the host using lipocalin-2, a protein that sequesters iron-laden siderophores, including enterobactin. Although the host and pathogens compete for iron in the presence of gut commensal bacteria, the roles of commensals in nutritional immunity involving iron remain unexplored. Here, we report that the gut commensal Bacteroides thetaiotaomicron acquires iron in the inflamed gut by utilizing siderophores produced by other bacteria including Salmonella, via a secreted siderophore-binding lipoprotein termed XusB. Notably, XusB-bound siderophores are less accessible to host sequestration by lipocalin-2 but can be "re-acquired" by Salmonella, allowing the pathogen to evade nutritional immunity. As the host and pathogen have been the focus of studies of nutritional immunity, this work adds commensal iron metabolism as a previously unrecognized mechanism modulating the interactions between pathogen and host nutritional immunity.

microbiology↗

bioRxiv2022

Taxonomic and metagenomic analyses define the development of the microbiota in the chick

Chicks are ideal to follow the development of the intestinal microbiota and to understand how a pathogen perturbs this developing population. Taxonomic/metagenomic analyses captured the development of the chick microbiota in unperturbed chicks and in chicks infected with Salmonella enterica serotype Typhimurium (STm) during development. Taxonomic analysis suggests that colonization by the chicken microbiota takes place in several waves. The cecal microbiota stabilizes at day 12 post-hatch with prominent Gammaproteobacteria and Clostridiales. Introduction of S. Typhimurium at day 4 post-hatch disrupted the expected waves of intestinal colonization. Taxonomic and metagenomic shotgun sequencing analyses allowed us to identify species present in uninfected chicks. Untargeted metabolomics suggested different metabolic activities in infected chick microbiota. This analysis, and GS-MS on ingesta confirmed that lactic acid in cecal content coincides with the stable presence of Enterococci in STm infected chicks. Unique metabolites including 2-isopropylmalic acid, an intermediate in the biosynthesis of leucine, was present only in the cecal content of STm infected chicks. Metagenomic data suggested that the microbiota in STm infected chicks contained a higher abundance of genes, from STm itself, involved in branched chain amino acid synthesis. We generated a deletion mutant in ilvC (STM3909) encoding ketol-acid-reductoisomerase, a gene required for the production of L-isoleucine and L-valine. {Delta}ilvC mutants are disadvantaged for growth during competitive infection with the wild type. Providing the ilvC gene in trans restored growth of the {Delta}ilvC mutant. Our integrative approach identified biochemical pathways used by STm to establish a colonization niche in the chick intestine during development. IMPORTANCEChicks are an ideal model to follow the development of the intestinal microbiota and to understand how a pathogen perturbs this developing population. Using taxonomic and metagenomic analyses we captured the development of the chick microbiota to 19 days post-hatch in unperturbed chicks and in chicks infected with Salmonella enterica serotype Typhimurium (STm). We show that normal development of the microbiota takes place in waves, and is altered in the presence of a pathogen. Metagenomics and metabolomics suggested that branched chain amino acid biosynthesis is especially important for Salmonella growth in the infected chick intestine. Salmonella mutants unable to make L-isoleucine and L-valine colonize the chick intestine poorly. Restoration of the pathway for biosynthesis of these amino acids restored the colonizing ability of Salmonella. Integration of multiple analyses allowed us to correctly identify biochemical pathways used by Salmonella to establish a niche for colonization in the chick intestine during development.

microbiology↗

bioRxiv2020

Novel structural components generate distinct type VI secretion system anchoring modes

The type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the assembly of the T6SS sheath structure, the contraction of which propels a payload of effector proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected diversity and exist in two major forms, a short (TssAS) and a long (TssAL) TssA. Whilst TssAL proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssAS-containing T6SSs remains unknown. Here we discover a novel class of structural components that interact with short TssA proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the baseplate. We demonstrate that the presence of these components is important for full sheath extension and optimal firing. Moreover, we show that the pairing of each form of TssA with a different class of sheath stabilization proteins results in T6SS apparatuses that either reside in the cell for a while or fire immediately after sheath extension, thus giving rise to different aggression behaviors. We propose that this functional diversity could contribute to the specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches.

microbiology↗

bioRxiv2020

Response of Pseudomonas aeruginosa to the innate immune system-derived oxidants hypochlorous acid and hypothiocyanous acid

Pseudomonas aeruginosa is a significant nosocomial pathogen and associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite the host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and survives attack from HOCl and HOSCN, and the contribution of such responses to its success as a CF pathogen. We investigated the P. aeruginosa response to these oxidants by screening 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators involved in antibiotic resistance, methionine biosynthesis and catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), that were required for HOCl survival. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress, and responds to both oxidants by upregulating expression of a putative peroxiredoxin, rclX (PA14_07355). While there was specificity in the transcriptional response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated) there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type III secretion system (T3SS), sulphur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinated the transcriptional response to HOCl and HOSCN, including upregulation of pyocyanin biosynthesis genes, and in response to HOSCN alone RclR downregulated chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role.

microbiology↗