Biology subjects

Holland, G.

Publications and source records attributed to Holland, G..

8 recordsLinked to original sources

bioRxiv2026

Dual species interactions shield Campylobacter against multiple antibiotics

BackgroundCampylobacter is a major food-borne pathogen causing diarrhea. In severe cases, treatment typically involves fluoroquinolones (FQ) and macrolides. However, high proportions of FQ resistance (FQR) have made macrolides the remaining first-line treatment option. As part of the Campylobacter surveillance program in Germany, we investigated multidrug resistance (MDR) in clinical Campylobacter samples collected 2010 to 2022. MethodsA total of 6,980 samples was single-colony purified and analyzed for antimicrobial resistance phenotypes. Genome sequencing was performed for 2,912 samples, and antimicrobial resistance genes were mapped. Cultures showing MDR were further analyzed for contaminating DNA and studied using scanning electron microscopy (SEM). FindingsWe found that 453 (6%) Campylobacter samples were resistant to both FQ and macrolides. Two of the C. jejuni samples were resistant to antibiotics from ten different classes. Genome analysis revealed that these samples, despite being derived from single colonies, contained >10% Enterococcus DNA reads. SEM confirmed the presence of coccoid bacteria interspersed with spiral-shaped Campylobacter. Additional culture-based purification resulted in pure C. jejuni isolates that retained FQR but lost macrolide resistance. We further showed that presence of MDR Enterococcus spp. in the mixed samples protected C. jejuni from above-MIC (minimum inhibitory concentration) of several ribosome-targeting antimicrobials whereas pure Campylobacter were susceptible. These findings revealed that Campylobacter may gain antimicrobial resistance advantages through close interactions with Enterococcus spp. InterpretationMDR phenotypes in Campylobacter cultures may arise through close, dual-species interactions with other highly resistant intestinal bacteria, such as MDR Enterococcus spp. and may have practical implications for antibiotic treatment. FundingThis study was funded by the German Ministry of Health dedicated to the National Reference Centre for Salmonella and other Bacterial Enteric Pathogens.

microbiology↗

bioRxiv2026

On the role of cell chaining in the attenuation of a Listeria monocytogenes divIVA mutant

Listeria monocytogenes is a facultative intracellular human pathogen capable of invading non-phagocytic host cells, replicating within their cytosol, and spreading directly from cell to cell. These processes are mediated by specialized virulence factors but also depend on the DivIVA protein. DivIVA aids in the secretion of peptidoglycan-degrading autolysins in a process that requires the accessory secretion ATPase SecA2, thereby promoting daughter cell separation following cytokinesis. Consequently, a {Delta}divIVA mutant forms elongated chains of unseparated daughter cells, which may explain its attenuated virulence. To further explore the role of cell chaining for attenuation, we here investigated how different morphological aberrations affect the virulence of L. monocytogenes. To this end, we generated coccoid mreB and filamentous ezrA mutants, and compared them to the {Delta}divIVA mutant in different in vitro infection assays. Coccoid or filamentous morphologies did not impair host cell invasion or intracellular replication, unlike cell chaining of the {Delta}divIVA mutant. Introduction of a hyperactive allele of the PrfA virulence regulator, resulting in constitutive overexpression of virulence genes, was sufficient to restore the invasion defect of the {Delta}divIVA mutant, despite its pronounced cell chaining phenotype, but did not recover intracellular replication. We isolated suppressors of the {Delta}divIVA mutant carrying mutations in secA2, which likely enhance the SecA2 ATPase activity. In these suppressors, autolysin secretion, daughter cell separation, and invasion were fully restored, and intracellular replication was partially recovered. Thus, maintaining normal rod-shaped morphology plays a minor role in L. monocytogenes pathogenesis. Instead, virulence attenuation in the {Delta}divIVA mutant is better explained by distortions in PrfA- and SecA2-dependent processes. Author SummaryListeria monocytogenes infects humans by entering host cells, replicating intracellularly, and spreading from infected cells to neighboring cells. The bacterium is rod-shaped and normally occurs as single or double cells that can efficiently complete all stages of infection. However, a class of virulence-attenuated L. monocytogenes mutants forms long cell chains, suggesting a link between cell morphology and virulence. One such mutant lacks the cell division protein DivIVA and is severely impaired in infection, as it neither invades host cells, replicates in the cytoplasm nor spreads from cell to cell. To determine whether virulence attenuation is caused by cell chaining itself or by additional functional defects, we compared the virulence of the divIVA mutant with that of mutants exhibiting other morphological abnormalities. In addition, we isolated suppressor mutations that restore normal cell separation in the divIVA mutant and examined their effects on virulence. Our results show that cell morphology per se does not generally determine listerial virulence. Instead, they indicate that specific virulence-related functions are impaired in chain-forming L. monocytogenes mutants. These findings advance our understanding of the virulence defects associated with cell chaining and provide a basis for identifying their real underlying molecular causes.

microbiology↗

bioRxiv2025

AEBP2-Directed H3K27me2 Defines a Specific Vulnerability in EZH2-mutant Lymphoma

The catalytic subunit of Polycomb Repressive Complex 2 (PRC2), EZH2, is recurrently mutated in 25% of diffuse large B-cell lymphomas (DLBCL), causing increased H3K27me3 and decreased H3K27me2 levels. EZH2 inhibitors provide clinical benefit, but resistance frequently develops, highlighting the need for alternative therapeutic targets. Here, we identify the PRC2 accessory protein AEBP2 as a specific genetic dependency in EZH2-mutant DLBCL. While AEBP2 acts through PRC2, its essential role is surprisingly independent of canonical H3K27me3-mediated gene silencing. Instead, AEBP2 functions within a PRC2.2 complex lacking JARID2, using its zinc-finger domains to sample intergenic chromatin to sustain H3K27me2. Notably, loss of AEBP2 or NSD2 caused contrasting changes in intergenic H3K27me2 levels, driving sensitivity or resistance to PRC2 inhibitors, respectively. Our findings identify AEBP2-PRC2.2-maintained intergenic H3K27me2 as a therapeutic vulnerability in EZH2-mutant DLBCL and highlight dysregulated H3K27me2 as an underappreciated form of PRC2 dysfunction in cancer, with important therapeutic implications.

cancer biology↗

bioRxiv2024

Recruitment of the cellular lipid transport protein CERT to C. psittaci inclusions regulates the timing of bacterial egress

Egress of intracellular pathogens is highly regulated and carefully timed. For the zoonotic bacterium C. psittaci, the predominant egress pathway is Chlamydia-containing sphere (CCS) formation, a calcium-dependent sequential mechanism including protease activity, inclusion membrane destabilization, intracellular calcium increase, and plasma membrane blebbing. How egress is regulated to ensure that it takes place only after C. psittaci intracellular development is thus far unknown. Here, we show that C. psittaci recruits the cellular ceramide transporter CERT to its inclusion during intracellular development, but this recruitment is reduced at late time points prior to egress. In addition, an early loss of CERT at the inclusion membrane induced by CERT-KO induces premature egress by CCS formation. Complementation of the CERT-KO with different CERT-GFP variants prevents premature egress, except of complementation with a variant lacking the inclusion targeting PH domain, showing that the localization of CERT is critical for CCS formation. The CERT-KO induced premature CCS are formed by the sequential process described for mature CCS, but they contain mostly RBs and are predominantly non-infectious. Thus, our findings suggest that the timing of C. psittaci egress by CCS formation is regulated by the recruitment of CERT to the inclusion. We propose that CERT stabilizes the chlamydial inclusion by the formation of ER-inclusion membrane contact sites during intracellular development, and the loss of CERT recruitment facilitates inclusion membrane destabilization and CCS formation.

microbiology↗

bioRxiv2023

The formation of Chlamydia-containing spheres, a novel non-lytic egress pathway of the zoonotic pathogen Chlamydia psittaci

Egress of intracellular bacteria from host cells and cellular tissues is a critical process during the infection cycle. This egress process is essential for bacteria to spread inside the host and can influence the outcome of an infection. For the obligate intracellular Gram-negative zoonotic bacterium Chlamydia psittaci little is known about the mechanisms resulting in chlamydial egress from the infected epithelium. Here, we describe and characterize a novel non-lytic egress pathway of C. psittaci by formation of Chlamydia-containing spheres (CCS). CCS are spherical, low phase contrast structures surrounded by a phosphatidylserine exposing membrane with specific barrier functions. They contain infectious progeny and morphologically impaired cellular organelles. The formation of CCS shares characteristics of apoptotic cell death including a proteolytic cleavage of the peptide DEVD albeit independent of active caspase-3, an increase in the intracellular calcium concentration of infected cells, followed by blebbing of the plasma membrane and rupture of the inclusion membrane. Finally, infected blebbing cells detach and leave the monolayer thereby forming CCS. These results support that Chlamydia psittaci egresses the epithelial cell by a novel non-lytic egress pathway, a process beneficial for the bacterium, which might influence the outcome of the infection in organisms. ImportanceHost cell egress is essential for intracellular pathogens to spread within an organism and for host-to-host-transmission. Here, we describe CCS formation as a novel egress pathway for the intracellular, zoonotic bacterial pathogen C. psittaci. This non-lytic egress pathway is fundamentally different from previously described Chlamydia egress pathways. Interestingly, CCS formation shares several characteristics of apoptotic cell death. However, the sequence of proteolytic activity, followed by plasma membrane blebbing and the final detachment of a whole phosphatidylserine exposing former host cell is unique for C. psittaci. Thus, CCS formation represents a new egress pathway for intracellular pathogens that could possibly be linked to C. psittaci biology including host tropism, protection from host cell defense mechanisms or bacterial pathogenicity.

microbiology↗

bioRxiv2021

MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling

Gram-positive bacteria are protected by a thick mesh of peptidoglycan (PG) completely engulfing their cells. This PG network is the main component of the bacterial cell wall, it provides rigidity and acts as foundation for the attachment of other surface molecules. Biosynthesis of PG consumes a high amount of cellular resources and therefore requires careful adjustments to environmental conditions. An important switch in the control of PG biosynthesis of Listeria monocytogenes, a Gram-positive pathogen with a high infection fatality rate, is the serine/threonine protein kinase PrkA. A key substrate of this kinase is the small cytosolic protein ReoM. We have shown previously that ReoM phosphorylation regulates PG formation through control of MurA stability. MurA catalyzes the first step in PG biosynthesis and the current model suggests that phosphorylated ReoM prevents MurA degradation by the ClpCP protease. In contrast, conditions leading to ReoM dephosphorylation stimulate MurA degradation. How ReoM controls degradation of MurA and potential other substrates is not understood. Also, the individual contribution of the [~]20 other known PrkA targets to PG biosynthesis regulation is unknown. We here present murA mutants which escape proteolytic degradation. The release of MurA from ClpCP-dependent proteolysis was able to constitutively activate PG biosynthesis and further enhances the intrinsic cephalosporin resistance of L. monocytogenes. This activation required the RodA3/PBP B3 transglycosylase/transpeptidase pair as additional effectors of the PrkA signaling route. One murA escape mutation not only fully rescued an otherwise non-viable prkA mutant during growth in batch culture and inside macrophages but also overcompensated cephalosporin hypersensitivity. Our data collectively indicate that the main purpose of PrkA-mediated signaling in L. monocytogenes is control of MurA stability during extra- and intracellular growth. These findings have important implications for the understanding of PG biosynthesis regulation and {beta}-lactam resistance of L. monocytogenes and related Gram-positive bacteria. Author SummaryPeptidoglycan (PG) is the main component of the bacterial cell wall and many of the PG synthesizing enzymes are antibiotic targets. We previously have discovered a new signaling route controlling PG production in the human pathogen Listeria monocytogenes. This route also determines the intrinsic resistance of L. monocytogenes against cephalosporins, a group of {beta}-lactam antibiotics. Signaling involves PrkA, a membrane-embedded protein kinase, that is activated during cell wall stress to phosphorylate its target ReoM. Depending on its phosphorylation, ReoM activates or inactivates PG production by controlling the proteolytic stability of MurA, which catalyzes the first step in PG biosynthesis. MurA degradation depends on the ClpCP protease and we here have isolated murA mutations that escape this degradation. Using these mutants, we could show that regulation of PG biosynthesis through control of MurA stability is the primary purpose of PrkA-mediated signaling in L. monocytogenes. Further experiments identified the transglycosylase RodA and the transpeptidase PBP B3 as additional effectors of PrkA signaling. Our results suggest that both proteins act together to translate the signals received by PrkA into intensification of PG biosynthesis. These findings shed new light on the regulation of PG biosynthesis in Gram-positive bacteria with intrinsic {beta}-lactam resistance.

microbiology↗

bioRxiv2020

Dissection of barrier dysfunction in organoid-derived human intestinal epithelia induced by Giardia duodenalis

Background and aimsThe protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed to overcome these limitations and to recapitulate the authentic series of events in the primary human duodenal tissue by using the human organoid system. MethodsWe established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis-induced barrier breakdown by functional analysis of transcriptional, electrophysiological and tight junction components. ResultsOrganoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of epithelial barrier upon G. duodenalis infection. Transcriptomic analysis suggested major expression changes in genes contributing to ion transport and tight junction structure. SLC12A2/NKCC1- and CFTR-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy. ConclusionData suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection ignited by alterations of cellular ion transport followed by breakdown of the tight junctional complex and loss of epithelial integrity. The newly established organoid-derived model to study G. duodenalis infection will help enable further molecular dissection of the disease mechanism and, thus, can help to find new options treating disease and infection, in particular relevant for chronic cases of giardiasis.

microbiology↗

bioRxiv2019

A genomic dating tool for ancient genomes resolves the origins of hundreds of Eurasian genomes

Radiocarbon dating is the gold-standard in archaeology to estimate the age of skeletons, a key to studying their origins. Nearly half of all published ancient human genomes lack reliable and direct dates, which results in obscure and contradictory reports. Here, we developed the Temporal Population Structure (TPS), the first DNA-based dating method for ancient genomes ranging from the Upper Palaeolithic to modern-day samples and applied it to 1559 ancient Europeans, Asians, and Caucasus individuals and to 2117 modern worldwide individuals. We show that TPS predictions for dated skeletons align with their known dates and correctly account for kin relationships. The TPS-dating of poorly dated Eurasian samples resolves conflicts and sheds new light on disputed findings, as illustrated by four test cases. We discuss the phenotypic traits of the Time Informative Markers (TIMs) that underlie TPS. SummaryTPS is a novel method to date humans from the Upper Palaeolithic to modern time from their DNA sequences. Accurate dating is essential to the interepretation of paleogemonic data.. The gold-standard method in archaeology is radiocarbon dating1. However, a major limitation of radiocarbon dating is the high amount of collagen extraction (500 mg) involved in the process2. Consequently, half of all published ancient human genomes lack reliable and direct dates, which results in obscure and contradictory reports. Here, we present the Temporal Population Structure (TPS), the first genomic dating method for ancient genomes ranging from the Upper Palaeolithic to modern-day samples. We show that TPS predictions for 961 radiocarbon-dated Eurasian skeletons align with their known dates. We replicate these findings on 598 other Europeans, Asians and Caucasus individuals. Using kin-pairs, we demonstrate that TPS has produced more accurate results than radiocarbon and other dating. We show how our findings resolve conflicts and sheds new light on disputed findings as illustrated by four test cases. Finally, we discuss the phenotypic traits of the Time Informative Markers (TIMs) that underlie TPS. TPS is a novel dating technique, which can be used when radiocarbon dating is unfeasible or uncertain or to develop alternative hypotheses. TPS cannot be used for older (<14,000 years ago) samples, and its accuracy depends on the temporal and geographical breadth of radiocarbon-dated samples in the training dataset, though this limitation can be improved over time. Overall, TPS can improve the accuracy of archeological and paleogenomic studies.

genomics↗