Biology subjects

Frankel, G.

Publications and source records attributed to Frankel, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Two IncHI1 megaplasmids in Klebsiella species reveal transposable-element-mediated blaIMP-1 mobilisation

Introduction. Carbapenemase-producing Enterobacterales (CPE) represent a major threat to hospitalised patients worldwide. The dissemination of carbapenemase genes, such as blaIMP, is frequently mediated by mobile genetic elements including plasmids. During a previously described multispecies, healthcare-associated outbreak of blaIMP-positive CPE in North West London, two unusual isolates, IMP47 (Klebsiella grimontii) and IMP76 (K. pneumoniae), recovered in 2019 from routinely collected rectal swabs of inpatients, were predicted to harbour blaIMP-1-carrying IncHI1 megaplasmids. Aims. This study aimed to determine complete genomic sequences of IMP47 and IMP76, resolve the genetic context of blaIMP-1, and assess the conjugative mobility of blaIMP-1-carrying megaplasmids. Methods. Genomic sequences of both isolates were recovered through hybrid assembly of Oxford Nanopore and Illumina sequencing reads. Complete plasmid sequences were characterised to determine replicons, conjugation machinery, and genes encoding resistance to antimicrobials or other stress factors. Integrons and transposable elements (TEs) within flanking regions of blaIMP-1 were resolved through genome annotation and search against public databases. Liquid-mating experiments were performed to assess the mobility of blaIMP-1-carrying plasmids. Results. Completed genome assemblies were generated from both isolates, confirming two blaIMP-1-carrying megaplasmids, pIMP47 (391 kbp) and pIMP76_1 (519 kbp), of the replicon type IncHI1A(pNDM-CIT)/IncHI1B(pNDM-CIT). The blaIMP-1 locus was carried by nearly identical class 1 integrons in both plasmids and a closely related IncHI1 megaplasmid pEB3_IMP1 (361 kbp) previously identified in South West England. Comparative analysis revealed conserved genetic structures linking blaIMP-1 to mercury-resistance genes and TEs Tn6025, TnAs3, IS26, and IS5075, suggesting a history of recombination and potential for TE-mediated mobilisation. Conjugation experiments confirmed transfer of pIMP76_1 into a recipient K. pneumoniae strain, resulting in acquisition of ertapenem resistance, whereas transfer of pIMP47 was not observed under the tested conditions. Conclusion. Two IMP-producing IncHI1 megaplasmids in gut-colonising Klebsiella species revealed TE-mediated blaIMP-1 mobilisation. The co-localisation of blaIMP-1 and metal-resistance genes in both plasmids highlights the potential for co-selection of blaIMP-1 in environments enriched with metal ions. Our findings underscore the importance of longitudinal genomic surveillance of carbapenemase-encoding megaplasmids in healthcare settings.

microbiology↗

bioRxiv2026

Citrobacter rodentium infection reveals preserved intestinal resilience in aged intestine

Ageing is generally associated with increased susceptibility to enteric infection and is widely considered to reflect progressive deterioration of intestinal function. However, whether increased susceptibility represents complete loss of intestinal resilience or a distinct host response to infection remains unclear. Using Citrobacter rodentium (CR) infection as a model of infectious colitis, we investigated how ageing shapes epithelial responses to enteric bacterial infection. Infection of aged mice with wild type CR (CRWT) resulted greater bacterial colonisation, deeper crypt localisation, impaired fluid-ion homeostasis and more severe colitis than young mice. Conversely, infection of aged mice with CRM12, a CR strain lacking 12 type III secretion system effectors, resulted in reduced epithelial colonisation, preservation of epithelial barrier function and fluid-ion homeostasis, restrained inflammatory responses, and markedly lower disease severity than CRWT infection. Deep quantitative proteomic analysis demonstrated that these divergent outcomes were accompanied by distinct adaptive molecular programmes within the aged intestinal epithelium. Together, our findings demonstrate that although aged mice exhibit increased susceptibility to enteric infection, the aged intestine retains the ability to mount distinct epithelial, inflammatory and molecular responses to pathogens with different virulence repertoires. Thus, increased susceptibility in aged mice should not be interpreted as complete loss of intestinal resilience.

microbiology↗

bioRxiv2026

Personalized microbiotas (counter-)select for antibiotic resistant strains

Antibiotic resistant pathogens are an increasing public health threat, as development of novel therapeutics is outpaced by resistance emergence and dissemination. Approaches to slow down or even revert antibiotic resistance are necessary to maintain efficacy of both existing and new antibiotics. Such approaches exploit the fitness cost of resistance elements, but have largely relied on assessing this cost in laboratory conditions that poorly reflect the native context in which pathogens reside. Here we present a method that allows to investigate the influence of personalized human gut microbiota compositions on the competitive fitness of antibiotic resistant pathogens. Using fecal matter-derived microbiomes we identified a specific community that selected for a carbapenem-resistant Klebsiella pneumoniae strain. This selective advantage was due to mutations arising in a LacI-type transcriptional regulator, GlyR, which upregulated expression of the downstream glycoporin GlyP, causing the effect. By deconvoluting the microbiome composition, we identified a focal E. coli strain as a central driver of the selection, which was further modulated by other microbiota members. We further demonstrate that the selective advantage was due to carbohydrate competition, and in particular for glycerol-containing compounds. Importantly, glyR mutations are under strong positive but conditional selection in clinical K. pneumoniae isolates. This implies a reduced competitiveness in other environments, which we experimentally validated in vitro. Overall, this study offers a path to identify microbiome-specific interactions that modulate the competitiveness of antibiotic resistant pathogens.

microbiology↗

bioRxiv2026

The capsule of hypervirulent Klebsiella pneumoniae maintains neutrophils in neutral activation state

Hypervirulent Klebsiella pneumoniae (hvKp) cause invasive infections despite robust neutrophil recruitment, yet the mechanisms enabling persistence in neutrophil-rich environments remain poorly defined. Here, we show that hvKp do not simply resist neutrophil antimicrobial mechanisms but instead constrain neutrophils into a neutral functional state characterised by limited bactericidal activity. Using genetic dissection of the rmpADC locus, pharmacological inhibition of neutrophil effector pathways, and analysis of a diverse panel of clinical isolates, we demonstrate that rmpADC-driven capsule properties uncouple neutrophil recognition and activation from bacterial killing. Deletion of rmpADC restores phagocytosis, degranulation, and intraphagosomal killing, whereas loss of individual rmpD or rmpC permits neutrophil activation without bacterial killing. Moreover, hypermucoviscosity alone is sufficient to protect bacteria from neutrophil-mediated killing across multiple genetic backgrounds. Together, these findings identify capsule-driven immune state control as a central mechanism of hvKp neutrophil evasion and reveal distinct thresholds governing neutrophil activation and bactericidal outcome.

immunology↗

bioRxiv2025

TraN variants mediate conjugation species specificity of IncA/C, IncH and Acinetobacter baumannii plasmids

IncA/C and IncH plasmids commonly carry antimicrobial resistance genes, notably blaNDM-1. Although these plasmids disseminate among Gram-negative pathogens via conjugation, the mechanisms underlying mating pair stabilisation (MPS) and conjugation species specificity remain poorly understood. In IncF plasmids, MPS is mediated by interactions between outer membrane proteins (OMP) encoded by the plasmids in the donor (TraN) and by the chromosome in the recipient. Using the Plascad database, we extracted 1,436 TraN sequences: 62.5% (898/1,436), mainly in IncF plasmids, are 550-660aa (we renamed TraN short, TraNS); 15% (216/1,436), in IncA/C plasmids, are 880-950aa (TraN medium, TraNM); and 11% (160/1,436), in IncH plasmids, are 1,050-1,070aa (TraN long, TraNL). A group of six TraN from Acinetobacter baumannii plasmids (891aa) were designated TraN V-shaped (TraNV). Like TraNS, TraNM and TraNL contain base and distal tip domains essential for conjugation, whereas TraNV has a base and two distinct tip domains forming a V-shaped structure. TraNM, TraNL and TraNV determine conjugation species specificity, with TraNL cooperating with OmpA. Tip swapping reverses conjugation specificity, revealing how TraNM and TraNL diversity influence plasmid host range and AMR dissemination. Our new data reveal the molecular basis of plasmid host specificity and broaden our understanding of how conjugation drives the dissemination of antimicrobial resistance genes among clinically relevant bacteria.

microbiology↗

bioRxiv2025

GBP1 recruitment to actin-rich pedestals induced by extracellular Gram-negative bacteria promotes pyroptosis.

The IFN{gamma}-induced GTPase guanylate binding protein 1 (GBP1) binds to lipopolysaccharide (LPS) on cytosolic Gram-negative bacteria and promotes pyroptosis via the recruitment and activation of caspase-4 on the bacterial outer membrane. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) are extracellular pathogens that also induce LPS-and caspase-4-dependent pyroptosis. However, whether GBP1 is involved in this process remains unknown. EPEC and EHEC adhere intimately to intestinal epithelial cells via avid interactions between the bacterial adhesin Intimin and Tir (Translocated intimin receptor), a type 3 secretion system effector protein. Intimin-mediated clustering of Tir triggers actin polymerisation, leading to pedestal-like structures at bacterial attachment sites. Here we show that GBP1 is recruited to actin-rich pedestals in human cells infected with EPEC and EHEC in vitro and mouse colonocytes infected with the EPEC-like murine pathogen Citrobacter rodentium in vivo. GBP1-dependent caspase-4 trafficking to these sites leads to pyroptosis and IL-18 release. GBP1 mutants defective in LPS coatomer formation also localised to EPEC pedestals, pointing to LPS-independent mobilisation of GBP1 by actin polymerisation induced by extracellular A/E pathogens. To further dissect the underlying mechanism, we engineered a chimeric receptor (Fc{gamma}R-Tir) by combining the intracellular signalling domain of Tir and the extracellular ligand-binding domain of the Fc{gamma} receptor. Clustering of Fc{gamma}R-Tir with IgG-coated beads produced sterile actin-rich pedestals that were sufficient to recruit GBP1 independently of bacteria. Our findings reveal that cytosolic GBP1 is mobilised to sites of pathogen-induced actin remodelling independently of LPS. We establish that GBP1 not only operates as a pattern-recognition receptor but also orchestrates effector-triggered immunity against pathogens that hijack the actin cytoskeleton. 175-word AbstractThe IFN{gamma}-induced GTPase guanylate binding protein 1 (GBP1) binds to lipopolysaccharide (LPS) on cytosolic Gram-negative bacteria and promotes pyroptosis via the recruitment and activation of caspase-4 on the bacterial outer membrane. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) are extracellular pathogens that adhere to host cells and stimulate dense actin polymerisation underneath their attachment sites, generating structures called actin-rich pedestals. Here we show that GBP1 traffics to actin-rich pedestals in human cells infected with EPEC and EHEC in vitro and mouse colonocytes infected with the EPEC-like murine pathogen Citrobacter rodentium in vivo. GBP1 promotes caspase-4 recruitment to actin-rich pedestals, leading to pyroptosis and IL-18 release. GBP1 mutants defective in LPS coatomer formation also localised to EPEC pedestals. Our novel assay that mimics pathogenic effector activity revealed GBP1 recruitment to sterile actin polymerisation sites. We conclude that cytosolic GBP1 is mobilised to sites of pathogen-induced actin remodelling independently of LPS. Our study establishes that GBP1 not only operates as a pattern-recognition receptor but also orchestrates effector-triggered immunity against pathogens that hijack the actin cytoskeleton.

immunology↗

bioRxiv2025

Intestinal challenges shape the polarisation of protective dural memory CD4 T cells

The meninges house several innate and adaptive immune cell populations1-3. These predominantly localise within the dura mater and include gut-derived IgA-secreting plasma cells4. Whether T cell adaptive memory in the dura is similarly linked to the gut is currently unknown. Here we show that dural CD4 T cell polarisation to a T helper (Th) 1, Th2 and Th17 state is determined by the nature of the immunological challenge encountered in the gastrointestinal tract. We find that intestinally polarised CD4 T cells seed to the dura in a CXCR6-CXCL16-dependent manner, express tissue-residency markers and are long-lived. Functionally, these orally-primed dural CD4 T are capable of a rapid antigen-specific recall response that limits pathogen spread into the brain following intravenous re-challenge. Our work reveals how linked intestinal and dural immunity enables the central nervous system to accrue immunological memory of gut microbes, the most likely source of life-threatening bloodborne pathogens.

immunology↗

bioRxiv2025

Evaluation of Klebsiella pneumoniae antigen MrkA reveals expression-limited therapeutic potential

Klebsiella pneumoniae (KP) poses a major global health concern, particularly as hospital-acquired strains become increasingly antibiotic resistant. MrkA, the major type 3 fimbrial (T3F) subunit, is a recognised prospective vaccine and monoclonal antibody (mAb) target. We assessed T3F structural (mrkABCDF) and regulatory (mrkHIJ) gene prevalence and allelic variation across 1649 KP genomes, using 100 diverse isolates for detailed phenotypic characterisation. Despite high conservation MrkA expression itself is variable with genotype failing to predict phenotype. We constructed isogenic KP T3F mutants with absent and supraphysiological MrkA expression to investigate functional consequences and therapeutic responses during infection. In a severe pneumonia model blood dissemination inversely correlated with T3F expression. In vivo antigen profiling revealed only [~]20% of wild-type lung bacteria express MrkA, dropping to [~]5% in blood, with low MrkA expression defining bacteraemic KP irrespective of infection route. Critically, anti-MrkA mAb efficacy was contingent on antigen abundance, with therapeutic benefit restricted to supraphysiological MrkA expression. These findings reveal substantial in vivo MrkA expression variability with critical onward implications for vaccine and mAb development.

microbiology↗

bioRxiv2025

Genomic and molecular characterisation of a Klebsiella pneumoniae clinical isolate resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam

This article reports an unusual Klebsiella pneumoniae clinical isolate, KpMVR1, resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, and investigates the underlying genetic alterations using comparative genomics and molecular experiments. Resistance to carbapenems and third-generation cephalosporins is increasing in K. pneumoniae globally, restricting therapeutic options. The {beta}-lactam/{beta}-lactamase inhibitor combinations are widely used to circumvent {beta}-lactamase-mediated resistance. In 2021, isolate KpMVR1 was recovered from a hospitalised patient in England. Two additional isolates with the same variable-number tandem-repeat profile--KpMVS1, collected from the same patient 42 days before KpMVR1, and KpMVS2, from another patient in the same hospital--were susceptible to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam. Illumina and nanopore whole-genome sequencing and hybrid genome assembly were conducted for these three isolates. Annotated genome assemblies were compared to identify genetic variation, and mutagenesis experiments were performed to verify predicted functional alterations. All isolates belonged to a novel clone ST8134 and carried blaKPC-2-like alleles (KpMVR1: blaKPC-157; KpMVS1 and KpMVS2: blaKPC-2) in presumptively conjugative plasmids. ISEc68 caused a frameshift mutation in KpMVR1s ompK36 gene, reducing the meropenem-vaborbactam and imipenem-relebactam susceptibility. KPC-157 demonstrated decreased hydrolysis of imipenem and ceftazidime when compared with KPC-2. KpMVR1 also encoded a disrupted transcriptional repressor MarR and a destabilising mutation in AcrB, a component of the AcrAB-TolC multidrug efflux pump. In conclusion, KpMVR1 harboured complex resistance-associated genetic alterations, with evidence for in vivo emergence of antimicrobial resistance. Our study underlines routine screening for resistant pathogens in vulnerable patients to guide antimicrobial chemotherapy as well as the need to characterise underlying resistance mechanisms to help assess the potential for onward transmission. Data summaryIllumina and nanopore sequencing reads, hybrid genome assemblies, and anonymised metadata of isolates KpMVS1, KpMVR1, and KpMVS2 have been deposited in databases of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) under BioProject accession PRJNA1084250, with BioSample accessions SAMN46778009 (KpMVS1), SAMN46778010 (KpMVR1), and SAMN46778011 (KpMVS2). The genome assemblies of these isolates have also been deposited in Pasteur Institutes database for K. pneumoniae species complex (bigsdb.pasteur.fr/klebsiella/) under ids 75608 (KpMVS1), 75609 (KpMVR1), and 75610 (KpMVS2). Impact statementThis is the first blaKPC-positive K. pneumoniae isolate referred to the UKs national reference laboratory with resistance to three last-resort {beta}-lactam/{beta}-lactamase inhibitor combinations meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, implicating in vivo emergence of this unusual resistance profile during prolonged antimicrobial chemotherapy. This isolate belonged to a novel clone ST8134 and harboured a plasmid-borne blaKPC-2-like allele blaKPC-157. We identified complex genetic alterations in this isolate: chromosomal large deletions, point mutations, and an ISEc68-induced loss-of-function truncation of the ompK36 porin gene. We determined the impact of KPC-2, KPC-157, and the ompK36 truncation on the susceptibility of K. pneumoniae to meropenem, meropenem-vaborbactam, imipenem, imipenem-relebactam, imipenem-avibactam, aztreonam, aztreonam-avibactam, ceftazidime, ceftazidime-avibactam, and cefiderocol. Our work underscores the need to monitor emerging resistance to beta-lactam/beta-lactamase inhibitor combinations in healthcare and to understand underlying resistance mechanisms for assessing the potential of resistance transmission.

microbiology↗

bioRxiv2025

R27 pilus biogenesis is promiscuous and electrostatic perturbations impair functionality

During conjugation, plasmid DNA is transferred from donor to recipient bacteria via the plasmid-encoded mating pilus, formed as thin helical assemblies of polymerised pilin subunits. In the IncHI1 R27 plasmid-encoded pilus, the TrhA pilin undergoes cyclisation (via a peptide bond between Gly1 and Asp69), essential for conjugation. Gly1 and Asp69 are exposed on the pilus surface and conserved in all TrhA pilins in the Plascad database. Substituting Asp69 with Asn, Ala, Gly, or Arg did not prevent cyclisation or pilus formation, which remained structurally indistinguishable from the wild type. Conjugation efficiency of the Asp69 substitutions across multiple recipient species correlated with side chain size, in the order Asp69Asn > Asp69Ala > Asp69Gly. However, Asp69Arg, as well as Asp69Lys and Gly1Lys substitutions abolished conjugation, likely due to the positively charged pilus surface (opposite to the wild-type negative charge) forming unfavourable electrostatic interactions with the recipient outer membranes inner leaflet, composed solely of zwitterionic phosphatidylethanolamine (PE). Consistently, conjugation was rescued in recipients lacking PE. These findings indicate strong selective pressure to maintain Gly1 and Asp69, as efficient DNA transfer depends on precise electrostatic and steric constraints of the pilus surface.

microbiology↗

bioRxiv2025

Stable coexistence of Citrobacter rodentium with a lytic bacteriophage during in vivo murine infection

Bacteriophages are ubiquitously present in bacterial communities, yet phage-bacteria interactions in complex environments like the gut remain poorly understood. While antibiotic resistance is driving a renewed interest in phage therapy, most studies have been conducted in in vitro systems, offering limited insight into the complexity of such dynamics in physiological contexts. Here, we use Citrobacter rodentium (CR), a natural mouse-restricted enteric pathogen and well-established model for human enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) infections, to investigate phage-pathogen interactions in vivo. We isolate and characterise Eifel2, a novel lytic phage infecting CR, and generate anti-phage specific antibodies that enable the visualisation of phage infections in vitro. In a murine model of CR infection, oral administration of Eifel2 led to robust phage replication in the gut without reducing the bacterial burden or infection-associated inflammation, confirming the establishment of a stable coexistence in the gut. Despite the emergence of a sub-population of phage-resistant CR mutants in vivo, they did not undergo clonal expansion, indicating that additional selective pressures impaired their widespread dissemination in the gut. Together, our findings demonstrate that imaging approaches can capture key infection stages in vitro, while in vivo models are essential for capturing the complexity of phage-bacteria interactions. This work highlights the importance of studying phage therapy in host-pathogen contexts that include a normal microbiota and a suitable host environment, where dynamic co-existence rather than eradication may define therapeutic outcomes. ImportanceBacteriophages, or phages, are viruses which can either kill or persist inside bacteria. Current interests in phage biology are in part ignited by the fact that they could be used to treat infections caused by antibiotic-resistant bacteria. However, most of our understanding of phage-bacterial interactions comes from in vitro models and/or in vivo gut models relying on altering the endogenous microbiota. Here we report the finding of a novel phage, Eifel2, which specifically targets Citrobacter rodentium (CR), the mouse equivalent of human diarrhoeagenic E. coli pathogens. Despite effectively killing CR in vitro, CR and Eifel2 develop a coexistence relationship in mice with an intact microbiota. While CR phage-resistant mutants emerge, host and microbial factors constrain their expansion. This work highlights the importance of studying phage therapy in host-pathogen contexts that include the complete microbiota, where therapeutic outcomes may rely on dynamic co-existence and containment rather than eradication.

microbiology↗

bioRxiv2025

Phospholipid-independent biogenesis of a functional RP4 conjugation pilus

Conjugation, the process of DNA transfer between bacteria, is initiated universally by the formation of a mating pair formation (MPF) via a conjugative pilus. Conjugation of the IncP RP4 plasmid is mediated by short, non-retractable, rigid mating pili. Here, we report the cryo-EM structure of the RP4 pilus at 2.75 [A] resolution. Uniquely, and consistently with quantitative mass spectral analysis, this revealed that the cyclic TrbC pilin subunit is not lipidated. Consistently, an E. coli pgsA mutant lacking phosphatidylglycerol (PG) can serve as a donor of RP4 but not of R27, encoding the H-pilus consisting of PG-associated cyclic pilin subunits (TrhA). The RP4 is the first example of a lipid-independent functional mating pilus. This discovery not only challenges the long-held assumption that an amphipathic lipid moiety is essential for the construction of conjugative pili and for MPF, but also expanding our understanding of the diverse mechanisms that bacteria employ to transfer genetic material.

microbiology↗

bioRxiv2025

Enteric infection priming confers IL-17A dependent protection from chemically-induced Colitis

Background and AimsEnteric infections trigger mucosal immune responses. However, whether such immune imprinting influences susceptibility to sterile inflammatory diseases like colitis remains unclear. The aims of this study were to investigate whether a resolved Citrobacter rodentium (CR) infection in mouse alters host susceptibility to chemically induced colitis and to identify the underlying immune mechanisms. MethodsC57BL/6 mice were infected with wild-type CR or CR {Delta}map{Delta}espF, a mutant lacking tight junction-disrupting effectors. 3 weeks post clearance, mice were subjected to dextran sodium sulfate (DSS)-induced colitis. Disease severity was assessed by weight change, colon length, histopathology, and myeloperoxidase levels. Colonic immune cell populations were characterised by flow cytometry, and cytokine levels were measured from colon explants. Functional roles of IL-17A were evaluated using recombinant cytokine administration and neutralising antibody treatment. ResultsMice that cleared CR infection fared better following DSS treatment compared to uninfected or {Delta}map{Delta}espF-infected mice. This protective phenotype was not directly dependent on microbiota, as confirmed by co-housing experiments. Protected mice displayed elevated numbers of colonic Th1 and Th17 cells and higher levels of IL-17A, IL-22, and IL-2 cytokines. Prophylactic treatment with IL-17A conferred protection in naive mice, whereas IL-17A neutralisation in previously infected mice abrogated the benefit, identifying IL-17A as a key mediator of protection. ConclusionsResolved intestinal infection with CR confers long-term protection against colitis via persistent IL-17A-mediated immune reprogramming. These findings resonate with the "hygiene hypothesis" and highlight how prior microbial exposure can shape mucosal resilience.

microbiology↗

bioRxiv2025

The accessory type III secretion system effectors shape intestinal inflammatory infection outcomes

Injection of effectors via a type III secretion system (T3SS) is an infection strategy shared by various Gram-negative bacterial pathogens, many infecting mucosal surfaces. While individual T3SS effectors are well characterized, their network-level organization and the distinction between core and accessory effectors remain incompletely understood. Here, by systematically dissecting the T3SS effector network of the enteric mouse pathogen Citrobacter rodentium (CR) we identified a subset of 12 accessory effectors that, while dispensable for colonization, significantly alter infection outcomes. A strain lacking the accessory effectors (CRM12) remained virulent in susceptible mouse hosts yet resulted in reduced epithelial barrier damage, inflammation, and immune cell infiltration in resistant mice. Deep proteomic analysis specifically targeting CR-attached colonic epithelial cells revealed that, despite lacking 39% of its effector repertoire, infection with CRM12 results in similar changes to global protein expression as seen in mice infected with the wild-type strain, though key regulators of barrier integrity were differentially expressed. Using a host model with impaired barrier repair, we confirmed that accessory effectors shape infection outcomes without significantly impacting virulence. This study refines the concept of core and accessory effectors, providing a basis for further studies into effector-driven host adaptation.

microbiology↗

bioRxiv2025

citrOgen: a synthesis-free polysaccharide and protein antigen-presentation to antibody-induction platform

Existing technologies employed to generate antibodies against bacterial polysaccharides and proteins rely on the availability of purified or synthetic antigens. Here we present a genetics-based platform that utilises Citrobacter rodentium (CR), an enteric mouse pathogen, to both produce and present complex heterologous polysaccharides and protein antigen complexes during natural infection. As proof of concept, we use lipopolysaccharides (O), capsular polysaccharides (K) and type 3 fimbrial (T3F) antigens expressed by the WHO critical priority pathogens Klebsiella pneumoniae (KP) and Escherichia coli (EC). Following one infection cycle (28 days) CR induces specific IgG antibodies against KPO1, ECO25b, KPK2 and KPT3F. We demonstrate that the antibodies are functional in downstream applications including protection against pathogenic KP challenge, KP capsular serotyping and KP biofilm inhibition. Whilst KP and EC antigens were used as prototypical examples, this modular platform is now readily adaptable to generate antibodies against diverse polysaccharide and protein antigens, with basic science, public health and therapeutic applications.

microbiology↗

bioRxiv2025

Capsule-mediated O-antigen masking protects hypervirulent Klebsiella pneumoniae from neutrophil killing

Klebsiella pneumoniae is listed as a critical priority pathogen by the WHO due to prevalent extended-spectrum {beta}-lactamase and carbapenem resistance and high mortality rates Hypervirulent K. pneumoniae (hvKp) causes life-threatening infections in immunocompetent individuals, while classical K. pneumoniae (cKp) causes nosocomial infections in immunosuppressed patients. The convergence of hypervirulence with antibiotic resistance is now a major global health threat. Using a murine pulmonary infection model, we show that hvKp resisted clearance despite significant neutrophil recruitment. Using clinical hvKp and cKp isolates, bone marrow derived neutrophils, cell biology, pharmacology and imaging (both light and electron microscopy) in ex vivo assays revealed that the hvKp hypermucoviscous capsule prevents bacterial recognition and neutrophil activation, blocking ROS production, degranulation, and neutrophil extracellular traps (NETs) formation. In the absence of capsule ({Delta}wcaJ) or hypermucoid capsule determinants ({Delta}rmpADC), hvKp activates neutrophils, leading to degranulation, NET formation and bacterial killing. While superimposing the absence of O-antigen ({Delta}rfb) on the {Delta}wcaJ abrogates degranulation, neutrophils still elaborate NETs that eliminate the {Delta}wcaJ/{Delta}rfb mutant at high efficiency. This suggests that O-antigen is a double-edged sword, functioning as a PAMP triggering neutrophil degranulation yet assisting the pathogen to evade NET-mediated destruction. These findings shed light on the mechanism underpinning immune evasion by the hypervirulent capsule in hvKp.

microbiology↗

bioRxiv2025

IL-18 activates mucosal group 2 innate lymphoid cells following enteric bacterial infection

Group 3 innate lymphoid cells (ILC3s) play a major role in protecting against infection with the enteric mouse pathogen Citrobacter rodentium, used to model infections with enteropathogenic and enterohaemorrhagic Escherichia coli. ILC3s-secreted IL-22, shown to be indispensable for protection against C. rodentium infection, induces secretion of IL-18, antimicrobial peptides and nutritional immunity proteins as well as activation of tissue regeneration processes. While ILC2s have traditionally been associated with immune responses to helminth infection and allergic inflammation via the production of type 2 cytokines (e.g. IL-4, IL-5, IL-9 and IL-13), more recently they have been implicated in protection against Clostridium difficile and Helicobacter pylori infections. Here we show that colonic lamina propria ILC2s proliferate in response to C. rodentium infection and secrete IL-4, IL-5 and IL-13, which are involved in maintenance of the intestinal barrier function, tissue repair and mucus secretion. When stimulated with IL-18, colonic ILC2s from uninfected naive mice secreted type 2 cytokines. Injection of IL-18 binding protein (IL18BP), at 2- and 3-days post C. rodentium infection, blocked activation of ILC2s. These data show that ILC2s are activated in response to infection with an enteric Gram-negative pathogen, where stimulation with IL-18 plays a role in inducing proliferation and secretion of type 2 cytokines. Author SummaryWhile group 3 innate lymphoid cells (ILC3s) play a key role in protecting from bacterial infections, ILC2s are mainly associated with immune responses to helminth infection. Here we investigated if ILC2s are activated in responses to infection with the enteric mouse pathogen Citrobacter rodentium. We show that in infected mice, gut ILC2s expand and secreted type 2 cytokines. ILC2 isolated from uninfected mice were activated by IL-18. Consistently, administration of IL-18 binding protein into C. rodentium-infected mice inhibited ILC2 activation. These findings suggest that gut ILC2s are activated by Gram negative enteric pathogens, which is mediated in part by IL-18.

immunology↗

bioRxiv2024

The exopolysaccharide Poly-N-Acetyl-Glucosamine (PNAG) coats Klebsiella pneumoniae in vivo

The conserved bacterial polysaccharide Poly-N-Acetyl-Glucosamine (PNAG) is a potential broad-spectrum vaccine candidate. While the immunogenicity of PNAG-based vaccine candidates has been established, characterisation of PNAG production across clinically relevant bacteria remains largely unknown. In particular, PNAG production in the Gram-negative pathogen Klebsiella pneumoniae (KP) is not well understood. Here, we demonstrate that PNAG production is prevalent in clinical KP isolates, where it is secreted as extracellular networks during adherent growth conditions. However, during severe KP pulmonary infection, KP PNAG production undergoes a switch to a cell-associated phenotype, coating the bacterial cell surface. By screening a panel of isogenic KP mutants in prominent cell surface components ({Delta}wcaJ,{Delta} rmpADC,{Delta} rfb,{Delta} ompA and{Delta} ompk36), we identified KP capsular polysaccharide as a key determinant underpinning the phenotype. Deleting genes involved in capsule synthesis ({Delta}wcaJ) and regulation ({Delta}rmpADC) resulted in cell-associated PNAG during adherent growth and infection of alveolar epithelial cells in vitro. Taken together, we describe a novel interaction between KP surface polysaccharides and detect for the first time, cell-associated PNAG in KP during lung infection, highlighting PNAG as an attractive KP vaccine antigen. Author summaryThe Gram-negative pathogen Klebsiella pneumoniae (KP) is a leading cause of hospital-associated lung and bloodstream infections worldwide. As KP exhibits resistance to most frontline antibiotics, there is a growing demand for immune-based strategies to treat KP infections. Poly-N-Acetyl-Glucosamine (PNAG) is a surface sugar produced by most clinically relevant bacteria, including KP. However, relatively little is known about PNAG production in KP. Therefore, we set out to characterise PNAG production in KP during in vitro growth and following lung infection in a pulmonary mouse model. During in vitro growth, KP produces extracellular PNAG networks. In contrast, during an in vivo severe lung infection, PNAG is found cell-associated, coating the bacterial surface. We propose that the visible change in KP PNAG between in vitro and in vivo environments is due to crosstalk with capsule, another polysaccharide on the KP surface. Together, this supports PNAG as an attractive KP antigen.

microbiology↗