Biology subjects

Hirt, R. P.

Publications and source records attributed to Hirt, R. P..

11 recordsLinked to original sources

bioRxiv2026

CAZyme Annotation of Five Parasitic Trichomonads Reveals a Conserved Repertoire of Host Glycan Targeting Proteins

Deciphering the functional potential of the unusually large and repetitive genomes of trichomonads has remained challenging since the first Trichomonas vaginalis genome assembly in 2007. These genomes are characterised by extensive gene duplication, widespread lateral gene transfer from prokaryotes, and numerous hypothetical proteins. Here, we systematically re-annotated five parasitic trichomonad genomes using the CAZy annotation pipeline, identifying 1,406 CAZyme-encoding genes (which encompass all four core CAZyme classes and carbohydrate-binding modules (CBMs)) - including 516 genes previously annotated as encoding hypothetical proteins - and generating predictions of CAZy genes originating from potential lateral gene transfer, encoding candidate secreted proteins, and their candidate metabolic pathway involvement. Comparative analyses revealed a conserved, highly duplicated repertoire of candidate mucin O-glycan-targeting CAZymes across all five species, alongside broad conservation of a candidate antibacterial CAZyme toolkit. Together, these findings substantially enrich functional annotation of these genomes and highlight in particular CAZymes and CBMs as promising targets for the future characterisation of the molecular and cellular basis of parasite-host and parasite-microbiota interactions underlying mucosal disease and dysbiosis associated with trichomonads.

evolutionary biology↗

bioRxiv2026

An enhancer-centric approach applied to human immune system epigenomes revealed the association of macrophage enhancers with cardiovascular disease

Complex diseases are influenced by both genetic and environmental factors. Immune cells are key mediating interactions with the environment, but the impact of genetic variation on the immune system and how it influences complex diseases is not fully understood. Moreover, most genome-wide analyses (GWAS) variants associated with complex diseases are non-coding and difficult to interpret. Here, we investigated the association of non-coding variants with immune cell enhancers. As part of BLUEPRINT and the International Human Epigenome (IHEC) consortia, we generated and analysed a comprehensive set of epigenomes for human primary immune cells, including 107 epigenomes derived from 749 ChIP-Seq experiments across 24 cell types. We identified multicell enhancer activity patterns across the genome and examined their links with non-coding variants from 518 GWAS traits. This analysis revealed 117 significant associations, including novel links between cardiovascular disease variants and macrophage-specific enhancers that regulate genes involved in lipid metabolism and immunity, such as the gene encoding for the nuclear receptor LXR-alpha (NR1H3) and many of its known target genes. Together, these data will help to better understand the influence of genetic variability in immune function and related diseases.

genomics↗

bioRxiv2025

BtuJ1, a novel surface-exposed B12-binding protein in Bacteroidetes, functions as an extracellular vitamin reservoir that enhances fitness.

The acquisition of vitamin B12 and related cobamides is a key determinant for the fitness of Bacteroidetes in the gut. Depending on the species, this uptake process relies on one to four transport systems centered on conserved core outer membrane (OM) complexes composed of the TonB-dependent transporter BtuB and the surface-exposed lipoprotein BtuG. Additionally, the surface-exposed lipoprotein BtuH, although not tightly associated with the BtuBG complex, contributes to cobamide uptake and provides a fitness advantage. Here, we report the functional and structural characterization of BtuJ1 from Bacteroides thetaiotaomicron (B. theta), a novel B12-binding lipoprotein. Under limiting B12 conditions, BtuJ1 is the most abundant component among the three B12-transport systems encoded by B. theta. BtuJ1 is surface exposed and binds vitamin B12 and cobinamide (an intermediate in B12 biosynthesis) with low nM affinity, conferring a fitness advantage in B12-limited environments. In vitro B12 transfer experiments suggest a role for BtuJ1 as an extracellular reservoir for B12, expanding the functionalities of the diverse group of accessory OM proteins employed by Bacteroides to scavenge this essential cofactor in the competitive environment of the human gut. IMPORTANCEUnderstanding how key molecules support bacterial colonization of the human GI tract is essential to rationalize the structure of the complex microbial community inhabiting the distal gut. The Bacteroidetes are one of the dominant phyla in this environment. Given that most Bacteroidetes cannot make vitamin B12 but depend on it for growth, the fitness of many species likely depends on the acquisition of vitamin B12. Unlike the classical model bacterium Escherichia coli, which encodes a single OM B12 transporter, the genomes of Bacteroidetes often encode multiple uptake systems comprising a heterogeneous repertoire of surface-exposed lipoproteins. These proteins may assist OM assemblies for vitamin B12 transport that provide fitness advantages in vivo.

microbiology↗

bioRxiv2025

An evolutionarily conserved laterally acquired toolkit enables microbiota targeting by Trichomonas

Trichomonas species are a diverse group of obligate extracellular symbionts associated with or attributed to various inflammatory diseases. They colonise mucosal surfaces across a wide range of hosts, all of which harbour a resident microbiota. Their evolutionary history likely involved multiple host transfers, including zoonotic events from columbiform birds to mammals. Using comparative transcriptomics, this study examines Trichomonas gallinae co-cultured with Escherichia coli, identifying a molecular toolkit that Trichomonas species may use to interact with bacterial members of the microbiota. Integrating transcriptomic data with comparative genomics and phylogenetics revealed a conserved repertoire of protein-coding genes likely acquired through multiple lateral gene transfers (LGT) in a columbiform-infecting ancestor. These LGT-derived genes encode muramidases, glucosaminidases, and antimicrobial peptides--enzymes and effectors capable of targeting bacterial cell walls, potentially affecting the bacterial microbiota composition across both avian and mammalian hosts. This molecular toolkit suggests that Trichomonas species can actively compete with and exploit their surrounding microbiota for nutrients, potentially contributing to the dysbiosis associated with Trichomonas infections. Their ability to target bacterial populations at mucosal surfaces provides insight into how Trichomonas species may have adapted to diverse hosts and how they could influence inflammatory mucosal diseases in birds and mammals.

molecular biology↗

bioRxiv2025

Discovery of a distinct BAM complex in the Bacteroidetes

The BAM ({beta}-barrel assembly machinery) complex is an evolutionarily conserved, multiprotein machine that catalyses the folding and membrane insertion of newly synthesised {beta}-barrel outer membrane (OM) proteins in Gram-negative bacteria. Based on Proteobacteria, bacterial BAM is also structurally conserved, with an essential BamAD core and up to three auxiliary periplasmic lipoproteins of poorly defined function. Here we show, using structural biology, quantitative proteomics and functional assays, that the BAM complex is radically different within the Bacteroidetes, a large and important phylum widely distributed within the environment and animal microbiomes. Cryogenic electron microscopy (cryo-EM) structures of BAM complexes from the human gut symbiont Bacteroides thetaiotaomicron and the human oral pathogen Porphyromonas gingivalis show similar, seven-component complexes of [~]325 kDa in size with most of the mass in the extracellular space. In addition to canonical BamA and BamD, the complexes contain an integral OM protein named BamF that is essential and intimately associated with BamA, as well as four surface-exposed lipoproteins (SLPs) named BamG-J. Together, BamF-J form a large, extracellular dome that likely serves as an assembly cage for the {beta}-barrel-SLP complexes that are a hallmark of the Bacteroidetes. Our data suggest that BAM functionality in Bacteroidetes is substantially expanded from that in Proteobacteria and underscores the importance of studying other phyla for a more complete understanding of fundamental biological processes.

microbiology↗

bioRxiv2024

A genetic locus in the gut microbe Bacteroides thetaiotaomicron encodes activities consistent with mucin-O-glycoprotein processing and plays a critical role in N-acetylgalactosamine metabolism

It is increasingly appreciated that members of the gut microbiota are key modulators of human health and the status of major diseases including cancer, diabetes and inflammatory bowel disease. Central to their survival is the ability to metabolise complex dietary and host-derived glycans including intestinal mucins. The latter are critical components of the gut epithelium glycocalyx and mucus barriers, essential for microbiota-gut homeostasis and protection from infections by pathogens. The prominent and model human gut microbe Bacteroides thetaiotaomicron (B. theta) is a versatile and highly efficient complex glycan degrader thanks to the expansion of gene clusters termed polysaccharide utilisation loci (PULs) in its genome. While the mechanisms for several singular dietary glycan-induced PULs have been elucidated, studies on the 16-18 mucin-induced PULs in B. theta significantly lag behind. A combination of the scale and complexity of B. theta transcriptomic response to mucins and complex glycan configurations of mucins represent major hurdles for the functional characterisation of the mucin induced PULs. As a result, there is very limited knowledge on how mucin metabolism is coordinated in B. theta and what specific PULs, genes and metabolites are critical for mucin-B. theta, and more generally mucin-microbiota interactions and their importance in microbiota-gut homeostasis. Here we show that a mucin inducible PUL BT4240-50, (i) encodes activities consistent with a machinery that couples the processing of mucin-O glycan glycoproteins with the metabolism of N-acetylgalactosamine (GalNAc), an abundant mucin O-glycan sugar; (ii) is important for competitive growth on mucins in-vitro; (iii) encodes a key kinase enzyme (BT4240) that is critical for GalNAc metabolism and (iv) has related PULs encoded by a range of prominent Bacteroides species in the human gut. Furthermore, BT4240 kinase was also critical for glycosaminoglycan metabolism, thus extending the PULs function beyond mucins. Our work advances our understanding of the vital metabolic processes that govern mucosal glycoprotein metabolism and by implication, a key aspect of host-microbiota interactions at mucosal surfaces and highlight GalNAc as a key metabolite targeted for competitive growth.

biochemistry↗

bioRxiv2022

Mammalian intestinal intracellular Microsporidia subvert host cell autophagy to promote replication

Intracellular pathogens such as Microsporidia can interact with host proteostasis pathways such as autophagy. Previous work done in Caenorhabditis elegans demonstrated involvement of autophagy in controlling Microsporidian proliferation through ubiquitin labelling of the parasite and subsequent degradation by autolysomes. However, it remains unknown if such mechanisms also play the role in mammalian models. Here we used immunochemistry assays, super-resolution fluorescence microscopy, and modulation of the hosts autophagy flux through siRNA silencing and chemical agents to elucidate how Microsporidia interact with host mammalian cell autophagy flux. We show that despite targeting by early autophagy markers (ubiquitin and p62); the host cell was not able to complete autophagy-mediated removal of Encephalitozoon cuniculi. Furthermore, instead of helping to control Microsporidia proliferation, the induction of autophagy dramatically increased proliferation of two Microsporidian species in two different mammalian cell models. Finally, we showed that the reduction of the autophagy flux using siRNA treatment or gut microbiota-derived metabolites known to be important for the intestinal epithelium homeostasis was able to reduce the parasite proliferation. Taken together, our results indicate that Microsporidia infecting mammalian cells not only developed strategies to evade the host autophagy but also evolved way to divert the autophagy flux to promote their own growth.

microbiology↗

bioRxiv2022

Mining the human gut microbiome identifies mycobacterial D-arabinan degrading enzymes

Division and degradation of bacterial cell walls requires coordinated action of a myriad of enzymes. This particularly applies to the elaborate cell walls of acid-fast organisms such as Mycobacterium tuberculosis, which consist of a multi-layered cell wall that contains an unusual glycan called arabinogalactan. Enzymes that cleave the O_SCPLOWDC_SCPLOW-arabinan core of this structure have not previously been identified in any organism. We have interrogated the diverse carbohydrate degrading enzymes expressed by the human gut microbiota and uncovered four families of glycoside hydrolases with the capability to degrade the O_SCPLOWDC_SCPLOW-arabinan or O_SCPLOWDC_SCPLOW-galactan components of arabinogalactan. Using novel exo-O_SCPLOWDC_SCPLOW-galactofuranosidases from gut bacteria we generated enriched O_SCPLOWDC_SCPLOW-arabinan and used it to identify D. gadei as a D-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave D-arabinan. We have identified new members of the DUF2961 family (GH172), and a novel family of glycoside hydrolases (DUF4185) that display endo-O_SCPLOWDC_SCPLOW-arabinofuranase activity. The DUF4185 enzymes are conserved in mycobacteria and found in many microbes, suggesting that the ability to degrade mycobacterial glycans plays an important role in the biology of diverse organisms. All mycobacteria encode two conserved endo-O_SCPLOWDC_SCPLOW-arabinanases that display different preferences for the O_SCPLOWDC_SCPLOW-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall.

biochemistry↗

bioRxiv2021

'Lose-to-gain' adaptation to genome decayin the structure of the smallest eukaryotic ribosomes

AO_SCPLOWBSTRACTC_SCPLOWThe evolution of microbial parasites involves the interplay of two opposing forces. On the one hand, the pressure to survive drives parasites to improve through Darwinian natural selection. On the other, frequent genetic drifts result in genome decay, an evolutionary process in which an ever-increasing burden of deleterious mutations leads to gene loss and gradual genome reduction. Here, seeking to understand how this interplay occurs at the scale of individual macromolecules, we describe cryo-EM and evolutionary analyses of ribosomes from Encephalitozoon cuniculi, a eukaryote with one of the most reduced genomes in nature. We show that E. cuniculi ribosomes, the smallest eukaryotic cytoplasmic ribosomes to be structurally characterized, employ unparalleled structural innovations that allow extreme rRNA reduction without loss of ribosome integrity. These innovations include the evolution of previously unknown rRNA features such as molten rRNA linkers and bulgeless rRNA. Furthermore, we show that E. cuniculi ribosomes withstand the loss of rRNA and protein segments by evolving a unique ability to effectively trap small molecules and use them as ribosomal building-blocks and structural mimics of degenerated rRNA and protein segments. Overall, our work reveals a recurrent evolutionary pattern, which we term "lose-to-gain" evolution, where it is only through the loss of rRNA and protein segments that E. cuniculi ribosomes evolve their major innovations. Our study shows that the molecular structures of intracellular parasites long viewed as reduced, degenerated, and suffering from various debilitating mutations instead possess an array of systematically overlooked and extraordinary structural features. These features allow them to not only adapt to molecular reduction but evolve new activities that parasites can possibly use to their advantage.

evolutionary biology↗

bioRxiv2018

BspA and Pmp proteins of Trichomonas vaginalis mediate adherence to host cells

Trichomonas vaginalis is one of the most widespread, sexually transmitted pathogens. The infection involves a morphological switch from a free-swimming pyriform trophozoite to an amoeboid cell upon adhesion to host epithelial cells. While details on how the switch is induced and to what proteins of the host surface the parasite adheres remain poorly characterized, several surface proteins of the parasite itself have been identified as potential candidates. Among those are two expanded protein families that harbor domains that share similarity to functionally investigated surface proteins of prokaryotic oral pathogens; these are the BspA proteins of Bacteroidales and Spirochaetales, and the Pmp proteins of Chlamydiales. We sequenced the transcriptomes of five Trichomonads and screened for the presence of BspA and Pmp domain-containing proteins and tested the ability of individual T. vaginalis candidates to mediate adhesion. Here we demonstrate that (i) BspA and Pmp domain-containing proteins are specifically expanded in T. vaginalis in comparison to other Trichomonads, and that (ii) individual proteins of both families have the ability to increase adhesion performance in a non-virulent T. vaginalis strain and Tetratrichomonas gallinarum, a parasite usually known to infect birds but not humans. Our results initiate the functional characterization of these two broadly distributed protein families, whose origin we trace back to the origin of Trichomonads themselves.

cell biology↗

bioRxiv2018

The protozoan Trichomonas vaginalis targets bacteria with laterally-acquired NlpC/P60 peptidoglycan hydrolases

Trichomonas vaginalis is a human eukaryotic pathogen and the causative agent of trichomoniasis, the most prevalent non-viral sexually transmitted infection worldwide. This extracellular protozoan parasite is intimately associated with the human vaginal mucosa and microbiota but key aspects of the complex interactions between the parasite and the vaginal bacteria remain elusive. We report that T. vaginalis has acquired, by lateral gene transfer from bacteria, genes encoding peptidoglycan hydrolases of the NlpC/P60 family. Two of the T. vaginalis enzymes were active against bacterial peptidoglycan, retaining the active site fold and specificity as DL-endopeptidases. The endogenous NlpC/P60 genes are transcriptionally up regulated in T. vaginalis when in the presence of bacteria. The over-expression of an exogenous copy produces a remarkable phenotype where the parasite is capable of competing out bacteria from mixed cultures, consistent with the biochemical activity of the enzyme in vitro. Our study highlights the relevance of the interactions of this eukaryotic pathogen with bacteria, a poorly understood aspect on the biology of this important human parasite.\n\nAuthor summaryTrichomonas vaginalis is a protozoan parasite that causes a very common sexually transmitted disease known as trichomoniasis. This extracellular parasite resides in the vagina where it is in close association with the mucosa and the local microbiota. Very little is known about the nature of the parasite-bacteria interactions. Here, we report that this parasite had acquired genes from bacteria which retained their original function producing active enzymes capable of degrading peptidoglycan, a polymer that is chemically unique to the cell envelope of bacteria. Our results indicate that these enzymes help the parasite compete out bacteria in mixed cultures. These observations suggest that these enzymes may be critical for the parasite to establish infection in the vagina, a body site that is densely colonised with bacteria. Our study further highlights the importance of understanding the interactions between pathogens and microbiota, as the outcomes of these interactions are increasingly understood to have important implications on health and disease.

microbiology↗