Biology subjects

Kujawska, M.

Publications and source records attributed to Kujawska, M..

14 recordsLinked to original sources

bioRxiv2025

PNGaseL from Flavobacterium akiainvivens targets a diverse range of N-glycan structures

PNGases are used by a wide range of organisms to remove N-glycan structures from proteins for use as either nutrients or in glycoprotein processing. PNGaseF is the most well-characterised enzyme of this family and is widely used in glycobiology to allow study of the N-glycome of a specific protein, cell and tissues, for instance. Despite this, PNGaseF has limitations in the types of N-glycan structures it can target. In this study, we explored the specificities of six uncharacterised PNGases selected from diverse parts of the PNGaseF superfamily. One of these enzymes, PNGaseL from Flavobacterium akiainvivens, is the highlight of this study due to its very broad specificity exemplified by its ability to cleave mammalian-, plant- and invertebrate-type complex N-glycans as well as high-mannose N-glycans. A detailed biochemical and structural characterisation was carried out against a variety of substrates to illustrate the advanced capability of PNGaseL in comparison to the canonical PNGaseF and PNGaseA enzymes. To determine the optimal reaction conditions, assess stability and define limitations of PNGaseL, a series of validation studies was performed. The data reveal that PNGaseL has potential utility in a range of glycobiology applications that are superior to the current commercially available options.

biochemistry↗

bioRxiv2025

Mutualistic interactions between Escherichia coli and Bifidobacterium bifidum enable degradation of human milk oligosaccharides in healthy infants

The development of the human gut microbiota during infancy is marked by frequent colonization of Enterobacteriaceae, a bacterial family notoriously associated with various diseases. Yet, despite their prominence in the absence of illness, their exact ecological role during healthy maturation of the infant gut remains poorly explored. Here, we analyse longitudinal stool samples from healthy, term-born, breastfed neonates (n=41) at two, six, and eleven months post-delivery, as well as microbiota of related mothers (n=30) with shotgun metagenomic sequencing, complemented by novel computational approaches and experimentation. Strain-resolved profiling indicates that dominant Bifidobacterium are frequently shared between infants and parenting mothers, while Escherichia coli originate from other sources, yet often persist within individuals. Despite their ecological differences, these genera co-exist, and both display evolutionary adaptations related to the utilization of human milk oligosaccharide (HMO) degradation products. We demonstrate that interactions between E. coli and Bifidobacterium bifidum are mutualistic in co-culture, where E. coli supplies cysteine to its auxotrophic partner, facilitating the cooperative degradation of 2'-fucosyllactose (2'FL), the predominant HMO. In turn, the liberated monosaccharides support E. coli proliferation and niche occupation. These findings reveal a fundamental cross-feeding interaction during development of healthy infant gut microbiota.

microbiology↗

bioRxiv2025

Diverse Defence Systems and Prophages in Human-Associated Bifidobacterium Species Reveal "Arms Race" Dynamics

Bacteria of the genus Bifidobacterium are pivotal for human health, especially in early life, where they dominate the gut microbiome in healthy infants. Bacteriophages, viruses of bacteria, are drivers of gut bacterial composition in the human gut and could affect bifidobacterial abundance. Here, we use a bioinformatics approach to explore the direct interactions occurring between human-associated Bifidobacterium spp. and prophages, as evidenced by their genomes. A total of 1,086 bifidobacterial genomes were analysed in this study, revealing complex systems to prevent viral invasion. Despite their characteristically small genomes, Bifidobacterium strains harboured more than double the number of defence systems as most bacteria. In total, 34 defence system types and 56 subtypes were detected, including several different CRISPR-Cas systems with spacers that targeted almost three-quarters of bifidobacteria-derived prophages. We identified at least one prophage which met our stringent quality control measures in [~]63% of strains, with phages exhibiting high genomic diversity and evidence of historical recombination. Additionally, prophages were found to encode various anti-defence systems, such as anti-CRISPR genes and restriction modification resistance mechanisms. In summary, our investigation reveals "arms race" dynamics drive genomic diversity in both bifidobacteria and their phages. ImportanceMembers of the Bifidobacterium genus are widely acknowledged as being highly important for human health, particularly in infants. To date, there have been a limited number of large-scale studies that have investigated the presence of prophages and anti-viral defence systems of Bifidobacterium strains from multiple human-associated species. Here, we have uncovered a complex set of anti-phage strategies encoded by Bifidobacterium strains. In addition, we have also identified a highly diverse phage mobilome present within the genomes of bifidobacteria across the genus, which also encode several unique systems for overcoming bacterial defences. Elucidating these co-evolutionary dynamics between phages and bifidobacteria may provide valuable insight into developing high-throughput methods for identifying next-generation probiotic or live biotherapeutic candidates, which may then be applied for the prevention and treatment of various diseases in humans.

microbiology↗

bioRxiv2025

Host-specific microbiome and genomic signatures in Bifidobacterium reveal co-evolutionary and functional adaptations across diverse animal hosts

Animal hosts harbour divergent microbiota, including various Bifidobacterium species and strains, yet their evolutionary relationships, and functional adaptions remain understudied. By integrating taxonomic, genomic and predicted functional annotations, we uncover how Bifidobacterium adapts to host-specific environments, shaped by vertical transmission, dietary influences, and host phylogeny. Our findings reveal that host phylogeny is a major determinant of gut microbiota composition. Distinct microbial networks in mammalian and avian hosts reflect evolutionary adaptations to dietary niches, such as carnivory, and ecological pressures. At a strain-resolved level, we identify strong co-phylogenetic associations between Bifidobacterium strains and their hosts, driven by vertical transmission and dietary selection, underscoring the intricate co-evolutionary dynamics between these microbes and their hosts. Functional analyses highlight striking host-specific metabolic adaptations in Bifidobacterium, particularly in carbohydrate metabolism and oxidative stress responses. In mammals, we observe an enrichment of glycoside hydrolases (GH) tailored to complex carbohydrate-rich diets, including multi-domain GH13_28 -amylases featuring diverse carbohydrate-binding modules (CBM25, CBM26, and the novel CBM74). These adaptations emphasise the ecological flexibility of Bifidobacterium in breaking down -linked glucose polysaccharides, such as resistant starch. Together, our study provides new insights into the evolutionary trajectories and ecological plasticity of Bifidobacterium, revealing how host phylogeny and dietary ecology drive microbial diversity and function. These findings deepen our understanding of host-microbe co-evolution and the critical role of microbiota in shaping animal health and adaptation.

microbiology↗

bioRxiv2024

Weberviruses are gut-associated phages that infect Klebsiella spp.

Weberviruses are bacteriophages (phages) that can infect and lyse clinically relevant, multidrug-resistant (MDR) strains of Klebsiella. They are an attractive therapeutic option to tackle Klebsiella infections due to their high burst sizes, long shelf life and associated depolymerases. In this study we isolated and characterized seven new lytic phages and compared their genomes with those of their closest relatives. Gene-sharing network, ViPTree proteome and terL gene-sequence-based analyses incorporating all publicly available webervirus genomes [n=258 from isolates, n=65 from metagenome-assembled genome (MAG) datasets] confirmed the seven phages as members of the genus Webervirus and identified a novel genus (Defiantjazzvirus) within the family Drexlerviridae. Using our curated database of 265 isolated phage genomes and 65 MAGs (n=330 total), we found that weberviruses are distributed globally and primarily associated with samples originating from the gut: sewage (154/330, 47 %), wastewater (83/330, 25 %) and human faeces (66/330, 20 %). We identified three distinct clusters of potential depolymerases encoded within the 330 genomes. Due to their global distribution, frequency of isolation and lytic activity against the MDR clinical Klebsiella strains used in this study, we conclude that weberviruses and their depolymerases show promise for development as therapeutic agents against Klebsiella spp.

microbiology↗

bioRxiv2024

Bifidobacterium pseudocatenulatum capsular exopolysaccharide enhances systemic anti-tumour immunity in pre-clinical breast cancer

Gut microbes have merged as powerful regulators of cancer responses, with Bifidobacterium species and strains playing a key role in promoting anti-tumour immunity. While they represent promising candidates for cancer therapeutics, the specific underlying microbial mechanisms driving their efficacy remains poorly understood. In this study, we demonstrate the broad potential of Bifidobacterium species to inhibit breast cancer progression across multiple pre-clinical mouse models. We identify a novel strain, Bifidobacterium pseudocatenulatum 210, which induces systemic anti-tumour immunity and enhances responses to standard-of-care therapies via its cell surface capsular exopolysaccharide (EPS). B. pseudocatenulatum 210 EPS promotes dendritic cell activation and increases systemic cDC1 infiltration, leading to robust CD8+ T cell-mediated anti-tumour activity. Our findings position Bifidobacterium EPS as a novel class of therapeutic compounds with significant potential for cancer treatment.

cancer biology↗

bioRxiv2023

Gut microbiota and faecal cytokine profiles of rural Cambodian infants and links to early life nutrition and diarrhoeal episodes.

The gut microbiota of infants in low-middle income countries like Cambodia remain underrepresented in microbiome research. This study aimed to explore the faecal gut microbiota composition and faecal cytokine profiles in a cohort of infants living in a rural province of Cambodia and explore the impact of sample storage conditions and infant environment on microbiota composition. Faecal samples collected at three time points from 32 infants (96 samples in total) after 7 months of age were analysed using 16S rRNA amplicon sequencing to determine the composition of the microbiota. Bacterial strains were isolated and subjected to whole genome sequencing and genomic analysis and concentrations of faecal cytokines were also measured. Initially, we compared the effects of two sample collection methods due to the challenges of faecal sample collection and storage in a rural location. Storage of faecal samples in a DNA preservation solution retained a greater abundance of Bacteroides. Analyses of microbiota composition of samples stored in DNA preservation solution indicated that Bifidobacterium was the most abundant genus with Bifidobacterium longum the most abundant species, particularly in breastfed infants. Most infants had detectable pathogenic taxa indicating frequent pathogen exposure, with Shigella and Klebsiella more abundant in infants with recent diarrheal illness. We did not detect antibiotic-associated perturbations in the gut microbiota, and no associations were found between the gut microbiota and infant growth. Genomic analysis of isolated strains indicated the presence of gene clusters encoding the ability to digest human milk oligosaccharides in B. longum and Bifidobacterium breve isolates. The presence of antibiotic-resistant genes was also identified in potentially pathogenic species, as well as in beneficial genera including Bifidobacterium. Faecal cytokine analysis showed higher concentrations of Interlukin-1alpha and vascular endothelial growth factor in breastfed infants, which may influence the infant gut mucosal immune system. This study provides insights into an underrepresented population of rural Cambodian infants, emphasising the impact of pathogen exposure and breastfeeding on gut microbiota composition and faecal immune profiles.

microbiology↗

bioRxiv2022

A novel bacteriocin produced by Bifidobacterium longum subsp. infantis has dual antimicrobial and immunomodulatory activity

Bacteriocins are ribosomally-synthesized antimicrobial peptides produced by bacteria with either narrow or broad spectrum activity. Many genome mining studies have indicated that bacteriocin gene clusters are widespread within certain gut microbiota members. In early life, Bifidobacterium comprise the dominant microbiota genus in vaginally delivered and breast-fed infants, with high levels associated with improved health. However, in many cases the mechanisms underlying these beneficial effects are unknown, although a limited number of studies have suggested that bacteriocin production by Bifidobacterium may represent a key mechanism for preventing pathogen over-growth. Here, we used BAGEL4 and antiSMASH to identify putative bacteriocin sequences in the whole genome sequences of 33 Bifidobacterium strains isolated from infants participating in two clinical studies. We identified a novel non-lantibiotic bacteriocin from Bifidobacterium longum subsp. infantis LH_664, with 40% sequence homology to Lactococcin 972 from Lactococcus lactis subsp. lactis. The putative bacteriocin (Bifidococcin_664) was chemically synthesized and studied for antimicrobial and immune-modulatory activities. We determined it has discrete activity against Clostridium perfringens and it appears to have novel immune stimulatory activities, promoting macrophage phagocytosis and specific cytokine release. These data highlight strain-specific beneficial properties in the early life genus Bifidobacterium, and suggest avenues for development of novel and highly specific dual action antimicrobials, and possible probiotic strains, that are active against clinically important bacterial pathogens. Data summarySamples LH_9 to LH_666 were previously sequenced and deposited to ENA under accession numbers ERS2658025-ERS2658043. Samples LH_986 to LH_1052 are newly sequenced and deposited to NCBI under accession numbers SAMN24838598-SAMN24838611. Additionally, previously assembled publicly available sequences (n=7) were retrieved online from NCBI Genomes database. See Supplementary Table S1 for further details.

microbiology↗

bioRxiv2021

Bifidobacterium castoris strains isolated from wild mice show evidence of frequent host switching and diverse carbohydrate metabolism potential

Members of the gut microbiota genus Bifidobacterium are widely distributed human and animal symbionts believed to exert beneficial effects on their hosts. However, in-depth genomic analyses of animal-associated species and strains are somewhat lacking, particularly in wild animal populations. Here, to examine patterns of host specificity and carbohydrate metabolism capacity, we sequenced whole genomes of Bifidobacterium isolated from wild-caught small mammals from two European countries (UK and Lithuania). Members of B. castoris, B. animalis and B. pseudolongum were detected in wild mice (Apodemus sylvaticus, A. agrarius and A. flavicollis), but not voles or shrews. B. castoris constituted the most commonly recovered Bifidobacterium (78% of all isolates), with the majority of strains only detected in a single population, although populations frequently harboured multiple co-circulating strains. Phylogenetic analysis revealed that the mouse-associated B. castoris clades were not specific to a particular location or host species, and their distribution across the host phylogeny was consistent with regular host shifts rather than host-microbe codiversification. Functional analysis suggested that mouse-derived B. castoris strains encoded an extensive arsenal of carbohydrate-active enzymes, including putative novel glycosyl hydrolases such as chitosanases that may act on chitin-derived substrates such as mushrooms or insects, along with genes encoding putative exopolysaccharides, some of which may have been acquired via horizontal gene transfer. Overall, these results provide a rare genome-level analysis of host specificity and genomic capacity among important gut symbionts of wild animals, and reveal that Bifidobacterium has a labile relationship with its host over evolutionary time scale.

microbiology↗

bioRxiv2020

The kleboxymycin biosynthetic gene cluster is encoded by several species belonging to the Klebsiella oxytoca complex

As part of ongoing studies with clinically relevant Klebsiella spp., we characterized the genomes of three clinical GES-5-positive ST138 strains originally identified as Klebsiella oxytoca. blaOXY gene, average nucleotide identity and phylogenetic analyses showed the strains to be Klebsiella michiganensis. Affiliation of the strains to ST138 led us to demonstrate that the current multi-locus sequence typing scheme for K. oxytoca can be used to speciate members of this genetically diverse complex of bacteria. The strains encoded the kleboxymycin biosynthetic gene cluster (BGC), previously only found in K. oxytoca strains and one strain of Klebsiella grimontii. The finding of this BGC, associated with antibiotic-associated haemorrhagic colitis, in K. michiganensis led us to carry out a wide-ranging study to determine the prevalence of this BGC in Klebsiella spp. Of 7,170 publicly available Klebsiella genome sequences screened, 88 encoded the kleboxymycin BGC. All BGC-positive strains belonged to the K. oxytoca complex, with strains of four (K. oxytoca, K. pasteurii, K. grimontii, K. michiganensis) of the six species of the complex found to encode the complete BGC. In addition to being found in K. grimontii strains isolated from preterm infants, the BGC was found in K. oxytoca and K. michiganensis metagenome-assembled genomes recovered from neonates. Detection of the kleboxymycin BGC across the K. oxytoca complex may be of clinical relevance and this cluster should be included in databases characterizing virulence factors, in addition to those characterizing BGCs. IMPACT STATEMENTMembers of the Klebsiella oxytoca complex are difficult to speciate using phenotypic and chemotaxonomic methods. Consequently, many genomes deposited in public databases are misclassified as K. oxytoca. Here we demonstrate that the current multi-locus sequence typing (MLST) system for the complex can be used to accurately speciate many strains, which will be of use to clinical laboratories in resource-limited settings which rely on the MLST scheme for typing and epidemiological tracking of isolates. In addition, extended analyses of the genomes of Klebsiella spp. have revealed the kleboxymycin biosynthetic gene cluster (BGC) is restricted to species of the Klebsiella oxytoca complex (K. oxytoca, K. michiganensis, K. pasteurii and K. grimontii). Species- and/or gene-specific differences in the clusters sequences may be relevant to virulence of K. oxytoca and related species. The finding of the kleboxymycin BGC in the preterm infant gut microbiota may have implications for disease presentation in a subset of neonates.

microbiology↗

bioRxiv2020

Succession of Bifidobacterium longum strains in response to the changing early-life nutritional environment reveals specific adaptations to distinct dietary substrates.

Diet-microbe interactions play a crucial role in infant development and modulation of the early-life microbiota. The genus Bifidobacterium dominates the breast-fed infant gut, with strains of B. longum subsp. longum (B. longum) and B. longum subsp. infantis (B. infantis) particularly prevalent. Although transition from milk to a more diversified diet later in infancy initiates a shift to a more complex microbiome, specific strains of B. longum may persist in individual hosts for prolonged periods of time. Here, we sought to investigate the adaptation of B. longum to the changing infant diet. Genomic characterisation of 75 strains isolated from nine either exclusively breast- or formula-fed (pre-weaning) infants in their first 18 months revealed subspecies- and strain-specific intra-individual genomic diversity with respect to glycosyl hydrolase families and enzymes, which corresponded to different dietary stages. Complementary phenotypic growth studies indicated strain-specific differences in human milk oligosaccharide and plant carbohydrate utilisation profiles of isolates between and within individual infants, while proteomic profiling identified active polysaccharide utilisation loci involved in metabolism of selected carbohydrates. Our results indicate a strong link between infant diet and B. longum subspecies/strain genomic and carbohydrate utilisation diversity, which aligns with a changing nutritional environment: i.e. moving from breast milk to a solid food diet. These data provide additional insights into possible mechanisms responsible for the competitive advantage of this Bifidobacterium species and its long-term persistence in a single host and may contribute to rational development of new dietary therapies for this important developmental window.

microbiology↗

bioRxiv2019

Infant diet promotes Bifidobacterium community cooperation within a single ecosystem

Diet-microbe interactions play an important role in modulating the early life microbiota, with Bifidobacterium strains and species dominating the gut microbiota of breast-fed infants. Here, we sought to explore how infant diet drives distinct bifidobacterial community composition and dynamics within individual infant ecosystems. Genomic characterisation of 19 strains isolated from breast-fed infants revealed a diverse genomic architecture enriched in carbohydrate metabolism genes, which was distinct to each strain, but collectively formed a pangenome across infants. Presence of gene clusters implicated in digestion of human milk oligosaccharides (HMOs) varied between species, with growth studies indicating within infant differences in the ability to utilise 2FL and LNnT HMOs between strains. We also performed cross-feeding experiments using metabolic products from growth on 2FL or LNnT for non-HMO degrading isolates, these compounds were identified to include fucose, galactose, acetate and N-acetylglucosamine. These data highlight the cooperative nature of individual bifidobacterial founder strains within an infant ecosystem, and how sharing resources maximises nutrient consumption from the diet. We propose that this social behaviour contributes to the diversity and dominance of Bifidobacterium in early life and suggests avenues for development of new diet and microbiota based therapies to promote infant health.

microbiology↗

bioRxiv2019

Microbiota supplementation with Bifidobacterium and Lactobacillus modifies the preterm infant gut microbiota and metabolome

Supplementation with members of the early-life microbiota or probiotics is becoming increasingly popular to attempt to beneficially manipulate the preterm gut microbiota. We performed a large longitudinal study comprising two preterm groups; 101 orally supplemented with Bifidobacterium and Lactobacillus (Bif/Lacto) and 133 non-supplemented (Control) matched by age, sex, birth-mode, and diet. 16S rRNA metataxonomic profiling on stool samples (n = 592) indicated a predominance of Bifidobacterium, and a reduction of pathobionts in the Bif/Lacto group. Metabolic phenotyping found a parallel increase in fecal acetate and lactate in the Bif/Lacto group compared to the Control group, which positively correlated with Bifidobacterium abundance consistent with the ability of the supplemented Bifidobacterium strain to metabolize human milk oligosaccharides and reduced gut pH. This study demonstrates that microbiota supplementation can modify the preterm microbiome and the gastrointestinal environment to more closely resemble that of a full-term infant.

microbiology↗

bioRxiv2017

Rapid MinION metagenomic profiling of the preterm infant gut microbiota to aid in pathogen diagnostics

The Oxford Nanopore MinION sequencing platform offers near real time analysis of DNA reads as they are generated, which makes the device attractive for in-field or clinical deployment, e.g. rapid diagnostics. We used the MinION platform for shotgun metagenomic sequencing and analysis of gut-associated microbial communities; firstly, we used a 20-species human microbiota mock community to demonstrate how Nanopore metagenomic sequence data can be reliably and rapidly classified. Secondly, we profiled faecal microbiomes from preterm infants at increased risk of necrotising enterocolitis and sepsis. In single patient time course, we captured the diversity of the immature gut microbiota and observed how its complexity changes over time in response to interventions, i.e. probiotic, antibiotics and episodes of suspected sepsis. Finally, we performed real-time runs from sample to analysis using faecal samples of critically ill infants and of healthy infants receiving probiotic supplementation. Real-time analysis was facilitated by our new NanoOK RT software package which analysed sequences as they were generated. We reliably identified potentially pathogenic taxa (i.e. Klebsiella pneumoniae and Enterobacter cloacae) and their corresponding antimicrobial resistance (AMR) gene profiles within as little as one hour of sequencing. Antibiotic treatment decisions may be rapidly modified in response to these AMR profiles, which we validated using pathogen isolation, whole genome sequencing and antibiotic susceptibility testing. Our results demonstrate that our pipeline can process clinical samples to a rich dataset able to inform tailored patient antimicrobial treatment in less than 5 hours.

genomics↗