Campylobacter jejuni is currently the most common cause of bacterial gastroenteritis worldwide. However, its genome provides few clues about how it interacts with the host. Moreover, infection screens have often been limited to classical cell culture or animal models. To identify C. jejuni genes involved in host cell interactions, we applied transposon sequencing in a humanized 3D intestinal infection model based on tissue engineering. This revealed key proteins required for host cell adherence and/or internalization, including an Rrf2 family transcriptional regulator as well as three so far uncharacterized genes ( pflC / Cj1643 , pflD / Cj0892c , pflE / Cj0978c ), which we demonstrate to encode factors essential for motility. Deletion mutants of pflC / D / E are non-motile but retain intact, paralysed flagella filaments. We demonstrate that two of these newly identified motility proteins, PflC and PflD, are components of the C. jejuni 's periplasmic disk structures of the high torque motor. The third gene, pflE , encodes a small protein of only 57 aa. Using CryoET imaging we uncovered that the small protein has a striking effect on motor biogenesis, leading to a complete loss of the flagellar disk and motor structures upon its deletion. While PflE does not appear to be a structural component of the motor itself, our data suggests that it is a lipoprotein and supports localization of the main basal disk protein FlgP, which is the first assembly step of the flagellar disk structure. Despite being annotated as a lipoprotein, we find that C. jejuni FlgP instead relies on PflE for its association with the outer membrane. Overall, our genome-wide screen revealed novel C. jejuni host interaction factors including a transcriptional regulator as well as two structural components and a small protein crucial for biogenesis of the C. jejuni high torque flagella motor. Since the flagella machinery is a critical virulence determining factor for C. jejuni , our work demonstrates how such a small protein can, quite literally, bring a bacterial pathogen to a halt.

The bacterial flagellar motor, which spins a helical propeller for propulsion, has undergone evolutionary diversification across bacterial species, often involving the addition of structures associated with increasing torque for motility in viscous environments. Understanding how such structures function and have evolved is hampered by challenges in visualizing motors in situ. Here we developed a Campylobacter jejuni minicell system for in situ cryogenic electron microscopy imaging and single-particle analysis of its motor, one of the most complex flagellar motors known, to subnanometre resolution. Focusing on the large periplasmic structures which are essential for increasing torque, our structural data, interpreted with molecular models, show that the basal disk comprises concentric rings of FlgP. The medial disk is a lattice of PflC with PflD, while the proximal disk is a rim of PflB attached to spokes of PflA. PflAB dimerization is essential for proximal disk assembly, recruiting FliL to scaffold more stator complexes at a wider radius which increases torque. We also acquired insights into universal principles of flagellar torque generation. This in situ approach is broadly applicable to other membrane-residing bacterial molecular machines.

One hurdle to understanding how molecular machines work, and how they evolve, is our inability to see their structures in situ. Here we describe a minicell system that enables in situ cryogenic electron microscopy imaging and single particle analysis to investigate the structure of an iconic molecular machine, the bacterial flagellar motor, which spins a helical propeller for propulsion. We determine the structure of the high-torque Campylobacter jejuni motor in situ, including the subnanometre-resolution structure of the periplasmic scaffold, an adaptation essential to high torque. Our structure enables identification of new proteins, and interpretation with molecular models highlights origins of new components, reveals modifications of the conserved motor core, and explain how these structures both template a wider ring of motor proteins, and buttress the motor during swimming reversals. We also acquire insights into universal principles of flagellar torque generation. This approach is broadly applicable to other membrane-residing bacterial molecular machines complexes.

The small, regulatory RNA RepG ( Re gulator of p olymeric G -repeats) regulates the expression of the chemotaxis receptor TlpB in Helicobacter pylori by targeting a variable G-repeat in the tlpB mRNA leader. Here, we show that RepG additionally controls lipopolysaccharide (LPS) phase variation by also modulating the expression of a gene ( hp0102 ) that is co-transcribed with tlpB . The hp0102 gene encodes a glycosyltransferase required for LPS O-chain biosynthesis and in vivo colonization of the mouse stomach. The G-repeat length defines a gradual (rather than ON/OFF) control of LPS biosynthesis by RepG, and leads to gradual resistance to a membrane-targeting antibiotic. Thus, RepG-mediated modulation of LPS structure might impact host immune recognition and antibiotic sensitivity, thereby helping H. pylori to adapt and persist in the host.

The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens. Author summary Enteric pathogens have evolved numerous strategies to successfully colonize and persist in the human gastrointestinal tract. However, especially for the research of virulence mechanisms of human pathogens, often only limited infection models are available. Here, we have applied and further advanced a tissue-engineered human intestinal tissue model based on an extracellular matrix scaffold reseeded with human cells that can faithfully mimic pathogenesis-determining processes of the zoonotic pathogen Campylobacter jejuni. Our three-dimensional (3D) intestinal infection model allows for the assessment of epithelial barrier function during infection as well as for the quantification of bacterial adherence, internalization, and transmigration. Investigation of C. jejuni mutant strains in our 3D tissue model revealed isolate-specific infection phenotypes, in-vivo relevant infection outcomes, and uncovered the involvement of a small RNA pair during C. jejuni pathogenesis. Overall, our results demonstrate the power of tissue-engineered models for studying host-pathogen interactions, and our model will also be helpful to investigate other gastrointestinal pathogens.

Many bacterial pathogens regulate their virulence genes via phase variation, whereby length-variable simple sequence repeats control the transcription or coding potential of those genes. Here, we have exploited this relationship between DNA structure and physiological function to discover a globally acting small RNA (sRNA) regulator of virulence in the gastric pathogen Helicobacter pylori, Our study reports the first sRNA whose expression is affected by a variable thymine (T) stretch in its promoter. We show the sRNA post-transcriptionally represses multiple major pathogenicity factors of H. pylori, including CagA and VacA, by base pairing to their mRNAs. We further demonstrate transcription of the sRNA is regulated by the nickel-responsive transcriptional regulator NikR (thus named NikS for nickel-regulated sRNA), thereby linking virulence factor regulation to nickel concentrations. Using in-vitro infection experiments, we demonstrate NikS affects host cell internalization and epithelial barrier disruption. Together, our results show NikS is a phase-variable, post-transcriptional global regulator of virulence properties in H. pylori.

The human gastrointestinal tract is a complex ecosystem in which epithelial cells and microorganisms of the intestinal microbiota live in symbiosis. Certain members of the microbiota, in particular Escherichia coli strains of the B2 phylotype, carry the polyketide synthase-island encoding the genotoxin colibactin. Colibactin is a nonribosomal peptide or polyketide-nonribosomal peptide hybrid of still unsolved structure, which induces DNA double strand breaks (DSBs) in eukaryotic cells. However, direct contact between live bacteria and host cell is required in order to elicit these genotoxic effects. In this study, we used a variety of cell culture models, among them, a 3D cell culture approach based on decellularised small intestinal submucosa, to investigate whether the intestinal mucus layer has the potential to interfere with colibactin activity. We demonstrate that the expression of mucins and the formation of an adherent mucus layer significantly increased with increasing complexity of cell culture. Moreover, we show that the presence of an adherent mucus layer on epithelial cells attenuates the genotoxic activity of colibactin, by preventing the induction of DNA-DSBs. Removal of the adherent mucus layer restored the occurrence of DNA-DSBs.

ABSTRACT Samples from patients at high risk for invasive aspergillosis (IA) were prospectively collected and analyzed for the presence of molecular markers of fungal infection. Serum specimens were screened for galactomannan and Aspergillus DNA, and whole-blood specimens were screened only for Aspergillus DNA. Fungal infections were categorized according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group, National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria. Forty-seven cases (proven and probable IA) and 31 controls (no evidence of IA) were selected retrospectively for this case-control study, comprising 803 samples, in order to determine the performance of whole-blood PCR, serum PCR, and serum galactomannan testing. Although no single assay was able to detect every case of IA, a combination of different assays provided the best performance. There was no significant difference between the use of whole-blood and serum specimens for PCR-based diagnosis of IA, but there was a trend for whole blood to be more sensitive (85% versus 79%) and to yield an earlier positive result (36 days versus 15 days) than for serum. However, DNA extraction from serum specimens is easier and faster than that from whole-blood specimens, and it allows the same specimen to be used for both galactomannan and PCR assays. In conclusion, the appropriate sample type for DNA extraction should be determined by the local requirements and the technical platforms available at each individual center. A combination of biomarker tests offered the best diagnostic utility for detecting IA.

Distal spinal muscular atrophy type 1 (DSMA1) is an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. In this disease, the degeneration of alpha-motoneurons is caused by mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2). This protein has been implicated in DNA replication, pre-mRNA splicing and transcription, but its precise function in all these processes has remained elusive. We have purified catalytically active recombinant IGHMBP2, which has enabled us to assess its enzymatic properties and to identify its cellular targets. Our data reveal that IGHMBP2 is an ATP-dependent 5' --> 3' helicase, which unwinds RNA and DNA duplices in vitro. Importantly, this helicase localizes predominantly to the cytoplasm of neuronal and non-neuronal cells and associates with ribosomes. DSMA1-causing amino acid substitutions in IGHMBP2 do not affect ribosome binding yet severely impair ATPase and helicase activity. We propose that IGHMBP2 is functionally linked to translation, and that mutations in its helicase domain interfere with this function in DSMA1 patients.