7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Chair

Prof. Dr.

Candida auris Transmission in Germany

Eukaryotic Pathogens

IntroductionCandida auris is an emerging fungal pathogen, which is resilient to environmental stress and rapidly develops antifungal drug resistance. Unlike most other Candida species, C. auris causes nosocomial transmissions and infections leading to severe hospital outbreaks. However, in comparison to other European countries, the number of reported C. auris infections in Germany is still considered low.GoalsWe analysed fungal isolates and patient data from C. auris cases reported to the German National Reference Center for Invasive Fungal Infections (NRZMyk) to (i) monitor C. auris epidemiology in Germany (ii) record and classify possible/ probable transmission events (iii) to gain insights into colonisation and infection dynamics.Materials &amp; MethodsStrains were genetically and phenotypically characterized and subdivided into clades. Whole genome sequencing was performed on selected isolates recording the diversity between closely related isolates from patient-to-patient transmission events. Antifungal drug susceptibility was tested with broth microdilution according to EUCAST or other commercially available assays.ResultsWhin the last two years (2022/23) the number of C. auris primary isolates increased over six-fold from 12 to 77 cases. The majority (66) belonged to Clade I. Non outbreak related clinical data were available for 28 and signs of invasive infection were suspected in 13 cases. 49 isolates were assigned to overall 3 suspected events of transmission. Of these, two events were rapidly contained affecting 5 patients in total. However, the so far biggest recorded outbreak in Germany had an impact on 44 patients and over 4 different university hospital units. Whole genome sequencing revealed close relationships between the patients strains and thus proofed transmission.SummaryWe observed a further rise of C. auris cases in the last years. Increased awareness for colonization and infection with C. auris is required for a timely development of appropriate strategies regarding containment and surveillance. This will also contribute to a better understanding of patient-to-patient transmissions.

Oral Presentation

Dr. rer. nat.

Single-cell force spectroscopy of Giardia duodenalis trophozoites revealed a unique attachment mode compared to other eukaryotic cells

Introduction: The unicellular parasite Giardia duodenalis is the causative agent of giardiasis, a gastrointestinal disease with global spread. Disease initiation is provoked by adhesion between G. duodenalis and the human intestinal epithelium. A unique microtubule-based attachment organelle, the cup-shaped ventral disc, facilitates this process. However, the detailed physical function of the ventral disc is still debated. In this study, we investigate adhesion forces involved during attachment of single G. duodenalis trophozoites, in comparison to adhesion mechanisms of the opportunistic pathogen Candida albicans and spreading human keratinocytes.Material &amp; Methods: Single cell force spectroscopy, based on fluidic force microscopy (FluidFM) was used to investigate the adhesion parameters of G. duodenalis trophozoites adhering to a flat glass surfaces. Comparison were made with the aforementioned cell types.Results: Force-distance curves displayed novel and so far undescribed characteristics for a microorganism, namely, gradual force increase on the pulled trophozoite, with localization of adhesion force shortly before cell detachment length. Adhesion forces reached 7.7 ± 4.2 nN at 1 µm/s pulling speed. Importantly, this unique force pattern was different from the saw-tooth pattern of Candida albicans and from the long cell interaction length of spreading keratinocytes, both considered for comparison in this study. Furthermore, the attachment mode of G. duodenalis trophozoites was mechanically resilient to tensile forces when pulling speeds were increased to 10 µm/s while adhesion forces rose to approximately 28.7 nN.Conclusions: Comparative force spectroscopy revealed novel and unique retract force curve characteristics for attached G. duodenalis trophozoites, suggesting a ligand-independent suction or clutching mechanism that substantially differs from other well-described microbial adhesion strategies.

Multi-reporter: fluorescent reporter for temporal detection of cell death in infection

Most microbial pathogens, including bacteria, fungi, and viruses, can lead to the death of their host cells. Host cells, however, die in a multitude of different ways. Some are inflammatory, such as pyroptosis and necroptosis, and some are not, such as apoptosis. The currently available tools for cell stress and death detection are limited and cannot easily distinguish between the diverse cellular events leading to cell death.Therefore, we are creating fluorescent reporters for cellular stress and different cell death pathways such as apoptosis, pyroptosis or necroptosis, which allow real-time detection by fluorescent microscopy. Cell lines are created via lentiviral transduction, which also allows multiple transductions into a single cell line to create multi-pathway reporters. Cell lines include THP-1 macrophages and C2BBe1 intestinal epithelial cells. We have created fluorescent reporters for redox status (stress), using a roGFP2 fluorophore, and apoptosis (cell death), using a bimolecular fluorescence complementation (BiFC) reporter for live detection of caspase 3/7 activation. Another fluorescent reporter for live detection of pyroptosis, via visualization of Gasdermin-D oligomerization, is being validated. As all reporters work with non-overlapping spectra, we aim to create a single "multi-reporter" cell line, which will enable us to detect simultaneously different types of cell stress and death pathways.This reporter system will allow to monitor cellular stress and to differentiate the different host cell death pathways that are active during infection. Importantly, it will be possible to follow the state of the host cells in a temporally and spatially resolved manner. Our new reporter will thereby help to answer many of the open questions on host-pathogen interactions during infection.

Professor

Reconstitution of an essential metabolic pathway of Plasmodium falciparum in Escherichia coli, facilitating functional and inhibitor screens

Introduction: New and highly selective drug targets for the malaria-causing parasite Plasmodium falciparum are still needed. The ferredoxin redox system (Fd-FNR), located in a plant-derived organelle called apicoplast, is essential for parasite survival due to its involvement in the methylerythritol phosphate pathway (MEP) for isoprenoid biosynthesis. However, in vitro screens for inhibitors are challenging due to the labile nature of the recombinant iron-sulfur proteins involved.Goal: To develop an in situ model in E. coli which reports essential functions of PfFd and its downstream electron acceptors (i.e. the terminal enzymes of the MEP pathway, shared by both organisms) as growth/intermediate/no growth.Materials &amp; Methods: Deletion of the two essential genes of E. coli, flavodoxin (EcFldA) and MEP enzyme EcIspH, and inducible expression of the respective P. falciparum proteins (PfFd, PfFd reductase (PfFNR) and PfIspH), followed standard procedures.Results: We report for the first time a double mutant of E. coli FldA and IspH. It is dependent on the mevalonate bypass system which provides the essential metabolite IPP. Removing mevalonate makes the strain entirely dependent on the simultaneous expression of plasmid-encoded PfFd, PfFNR, PfIspH, which documents that the three parasite proteins are required functionally active to replace the respective endogenous E. coli proteins. This system will allow initial functional tests to be performed. As a proof of concept, several amino acids of PfFd suspected to be involved in the interaction with PfIspH were individually mutated and the respective redox function could be evaluated by simple growth assays. Metabolomic studies are under way to characterize the mutants' consequences on the MEP.Summary: We describe an engineered E. coli strain dependent on three P. falciparum proteins, PfFd, PfNR and the terminal MEP enzyme PfIspH, which will be very useful for functional as well as drug screening purposes of this essential parasite pathway before findings are validated in the parasite.

Mechanisms of Candida albicans intracellular filamentation in human macrophages leading to peptide toxin secretion and escape

Phagocytes are essential for the initial immune response to infections with the opportunistic fungal pathogen Candida albicans. While the fungus is efficiently phagocytosed by macrophages, it also possesses strategies to survive inside phagosomes and to escape from these phagocytes within hours.Rapid initiation of filamentation inside the phagosome is a critical step for C. albicans escape since subsequently, filamentation leads to exertion of physical forces and drives damage to the macrophage. Filamentation also occurs in conjunction with the production of the peptide toxin candidalysin. After phagosomal escape, the toxin contributes to host cell damage, activation of caspase-1, and the NLRP3 inflammasome leading to the release of IL-1β. However, candidalysin is mostly dispensable for the induction of caspase-1-dependent pyroptosis. This undermines the importance of understanding how filamentation is initiated in the host phagosome.This key process is likely triggered by the amino acid arginine. Under phagosome-mimetic conditions such as acidic pH and nutrient deprivation, addition of arginine and the related amino acids ornithine and proline selectively induces filamentation. This is the case in both arginine-prototrophic and -auxotrophic strains. During infection with human primary monocyte-derived macrophages, the same strains exhibit similar filamentation and escape rates and induce comparable levels of damage. These data support the hypothesis that fungal arginine biosynthesis is not required for induction of intraphagosomal filamentation, but rather host-derived arginine. Our initial data suggests that an amino acid transporter expressed by macrophages localizes to the phagosome, yielding a potential source for this signal.In summary, host-cell escape requires a combination of hyphal extension and candidalysin production. In ongoing work, we are investigating the role of fungal arginine catabolism on the escape of C. albicans, especially with regard to the importance of arginine in macrophage polarization and the function of candidalysin in macrophage cell death.

Using a human airway organoid-derived monolayer co-culture model to study Cryptococcus neoformans and other fungal infections

IntroductionCryptococci are environmental fungi that cause localized or disseminated infections. The WHO considers C.neoformans to have the highest need for research among fungal pathogens. Studies on pathogenicity are mainly based on animal models or immune cells. However, to understand the pathomechanisms associated with the transition from pulmonary colonization to tissue infection, complementary models are required. We are using a human lung infection model to investigate parameters of early cryptococcal infection and epithelial transition.MethodsWe used well-characterized human airway-organoid derived monolayers (ODMs) from nasal and bronchial tissue and co-cultured them with C.neoformans (VNI) and C.gattii (VGII). Barrier function of ODMs was probed by measuring the transepithelial electrical resistance (TEER). We quantified internalization of fungi and fungal viability by immunofluorescence and colony forming unit assays. Fungal morphology was assessed using ink stainings and image analysis.ResultsHuman ODMs could be co-cultured with different cryptococcal strains and TEER measurements revealed no breakdown of barrier function, even after prolonged time intervals. Rare events of fungal internalization by airway epithelial cells were detected. Fungi remained viable during co-culture and were able to further proliferate. Quantification of cryptococcal morphology revealed a shift from uniform cells towards distinct phenotypes, including micro- and giant cells.DiscussionThe established co-culture model provides a suitable in vitro model to study host-cryptococcal interactions. Quantification of TEER indicate no impact of different cryptococcal strains on the barrier function of airway epithelial cells, despite the fact that both fungal strains show different disease phenotypes in humans. However, the model revealed that fungi undergo morphological changes, indicating that they adapt different phenotypes as a consequence of epithelial cell presence. Changes in morphology have been observed in in vivo-studies and are associated with pathogenic potential. Thus, the co-culture model can be used in the future for risk assessment of different fungal pathogens.

Oral Session
Eukaryotic Pathogens

Appointment

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Oral SessionEukaryotic Pathogens

Appointment

Chair

Topic

Programme

Oral Session
Eukaryotic Pathogens