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

Prof. Dr.

Chair

The combination of microfluidic cell infection with TraDIS reveals potential adherence factors of Streptococcus canis

Host-pathogen interactions and clinics of zoonotic Infections

Introduction: Streptococcus canis (S. canis) is an animal pathogen and causes zoonotic infections such as septicemia in humans. In previous studies, the M-protein of S. canis has been characterized as a bacterial surface receptor. S. canis is also known to produce a pore-forming toxin targeting the membrane of eukaryotic cells. Apart from these studies, little is known about other factors directly contributing to cell adherence and bacterial invasion into various host niches.Goals: The aim of this study is to identify and characterize novel adherence factors that play a role in the pathogenesis of systemic infections, particularly in bloodstream infections.Materials &amp; Methods: For bacterial infection analyses of human primary endothelial cells (EC) under physiological conditions defining bloodstream infections, a microfluidic system was used to apply a defined shear stress. Bacterial infection was quantified and visualized microscopically after differential immunofluorescence staining followed by confocal laser scanning microscopy. A transposon-mediated mutagenesis library of a clinical S. canis isolate was generated, representing a high genome coverage. During the cell culture infection process, a differential selection of adherent and non-adherent transposants enabled an efficient transposon directed insertion-site sequencing (TraDIS).Results: S. canis bacteria effectively adhere to EC even at high shear force levels during flow infection. A serial selection process of transposants successfully differentiates between a recovered pool of adherent and of non-adherent bacteria. Bioinformatical analyses of sequence pools resulted in identification of several putative factors contributing to bacterial adherence and cell damage.Summary: The combination of microfluidic infection of endothelial cells with the TraDIS technique proved to be ideally suited for the analysis of S. canis infections in the vascular system and for the search for new potential adherence factors.

Oral Presentation

Professor

"Metabolic upcycling" – insights into the metabolic underpinning of Haemophilus influenzae persistence in human airways

Haemophilus influenzae (Hi) infections are associated with recurring acute exacerbations of chronic respiratory diseases in children and adults including otitis media, pneumonia, chronic obstructive pulmonary disease and asthma. Physiological investigations show that persistence and recurrence of Hi infections are closely linked to Hi metabolic properties, where preferred growth substrates are aligned to the metabolome of human airway epithelial surfaces and include lactate, pentoses, and nucleosides, but not glucose that is typically used for studies of Hi growth in vitro. Enzymatic and physiological investigations revealed that utilization of lactate, the preferred Hi carbon source, required the LldD L-lactate dehydrogenase (conservation: 98.8% of strains), but not the two redox-balancing D-lactate dehydrogenases Dld and LdhA. Utilization of preferred substrates was directly linked to Hi infection and persistence. When unable to utilize L-lactate or forced to rely on salvaged guanine, Hi extra- and intra-cellular persistence was reduced in a murine model of lung infection and in normal human nasal epithelia, with up to 3000-fold attenuation observed in competitive infections. In contrast, D-lactate dehydrogenase mutants only showed a very slight reduction compared to the wild-type strain. Interestingly, acetate, the major Hi metabolic end-product, had anti-inflammatory effects on cultured human tissue cells in the presence of live but not heat-killed Hi, suggesting that metabolic endproducts also influence Hi-host interactions. Our work provides significant new insights into the critical role of metabolism for Hi persistence in contact with host cells and reveals for the first time the immunomodulatory potential of Hi metabolites.

Establishing a human in vitro 3D gut-on-a-chip model to study enterohemorrhagic Escherichia coli infection

IntroductionEnterohemorrhagic Escherichia coli (EHEC) are pathogenic bacteria that can cause severe intestinal infections and the hemolytic uremic syndrome. To study EHEC infections, cell culture systems or animal models can be applied. As none of the currently available animal models fully reflects EHEC pathogenesis in humans, human 3D cell culture systems mimicking in vivo conditions are a promising technology for monitoring EHEC infection.GoalsWe intend to establish and characterize an easy-to-use 3D cell culture model comprising four different cell types that mimics the human gut and is suitable for studying EHEC infections.Materials &amp; MethodsCaco-2 alone or together with HT29-MTX intestinal epithelial cells were seeded against extracellular matrix (ECM) in an OrganoPlate® 3-lane 40 (Mimetas). Cells were cultivated under flow conditions until leak-tight intestinal tubules were formed. Infection experiments with bacterial culture supernatant for 24 h or bacteria for 3 h were carried out using the 2011 outbreak strain EHEC O104:H4. Prior and after infection, barrier integrity was assessed by monitoring the translocation of fluorophore-labeled dextrans from tubule lumen to ECM channel.ResultsLeak-tight intestinal tubules were formed after six days of cultivation. Infection of tubules with culture supernatant from EHEC O104:H4 wild type, but not from its Shiga toxin 2 (Stx2) phage-cured derivative, induced epithelial barrier disruption, reflecting the known cytotoxic effect of Stx2. Additionally, infection experiments with bacteria revealed that an EHEC O104:H4 rpoS-mutant with similar Stx2 levels, but enhanced fimbriae and flagellin production compared to the wild type, caused a more severe tubule barrier disruption, indicating synergistic effects of Stx2 and these virulence factors.SummarySo far, we established an easy-to-handle 3D bi-culture model suitable for studying EHEC infections in vitro. To fully picture host defense and EHEC disease pathology, we are currently implementing macrophages and neutrophils into the 3D cell culture system.

Characterization of egress mechanisms of Coxiella burnetii

Intracellular bacteria evolved mechanisms to invade host cells, establish an intracellular niche allowing survival and replication and exit the host cell after completion of the replication cycle to infect new target cells. Coxiella burnetii (C.b.) is a Gram-negative, obligate intracellular pathogen and the causative agent of the zoonotic disease Q fever. Resident alveolar macrophages are the first target cells, but C.b. spread to other organs and cell types. While we have information about the initial step of C.b. uptake and the maturation process of the C. burnetii-containing vacuole (CCV), it is not well studied how C.b. spreads, i.e. how it exits its host cell to enter new target cells. Understanding this essential step will be crucial to develop novel strategies to prevent C.b. from spreading and, thus, disease.Here, we used human endothelial EA.hy926 cells and epithelial HeLa cells, as well as mouse fibroblasts lacking the lysosomal associated membrane proteins 1 and 2 (LAMP-1/ -2) to study the escape of intracellular C. burnetii with live cell imaging, confocal laser scanning microscopy, electron microscopy, a trans-well system, and by establishing a FACS-based spreading assay.We could demonstrate that C.b. inhibits host cell apoptosis early during infection, but induces and/or increases apoptosis at later stages of infection. Egress depends on previously established large bacteria-filled vacuoles and a functional intrinsic apoptotic cascade. The released bacteria are not enclosed by a host cell membrane and can infect and replicate in new target cells. However, in addition to cell death, lysosomal exocytosis might be involved, as egress is inhibited in LAMP-1/-2 deficient cells.In summary, our data argue that C. burnetii egress in a non-synchronous way at late stages of infection. Apoptosis-induction is important for C.b. egress, but other pathways such as lysosomal exocytosis most likely contribute.

The role of the RGD motif of streptococcal protein IdeC in Streptococcus canis infection

IntroductionStreptococcus canis is a Lancefield group-G opportunistic pathogen that colonises the mucosal surfaces and skin of the host, predominantly affecting cats and dogs. S. canis is a zoonotic pathogen with human infections being observed more recently, mainly caused by contact with companion animals.Different virulence factors, e.g. proteases, enable colonization, survival and replication during pathogenesis of streptococci. In silico analysis has shown that the IgG-specific protease of S. canis termed IdeC is a secreted protein containing a signal sequence but no membrane anchor. IdeC is suggested to act with a similar mechanism to the IdeS protein of Streptococcus pyogenes, a secreted cysteine kinase that cleaves the IgG molecule between the hinge and CH2 region. The IdeC protein also has an Arg-Gly-Asp (RGD) motif, an important integrin binding motif. Bacterial proteins containing RGD motifs have been implicated in adhesion and invasion of host cells. This along with the ability of IdeC to bind back to the bacterial surface after secretion suggest a possible second function of IdeC in adhesion.Goals This project is focused on elucidating the functional role of the S. canis protein IdeC. Specifically, what role this protein may play in mediating host-pathogen interactions.Materials &amp; MethodsFluorescent latex beads coated in recombinant IdeC protein are used to assess the interaction of IdeC with epithelial and endothelial cells. Further, a recombinant protein with RGD replaced with RGE has been produced to test the role of the motif in any interaction observed.ResultsBased on fluorescence microscopy analysis, IdeC coated latex beads displayed increased interaction with both epithelial and endothelial cells when compared to IdeC_RGE and BSA controls. Electron microscopy indicates that IdeC coated beads may be internalised however, a mechanism is yet to be determined.SummaryHere we present evidence that the streptococcal protein IdeC may have a second function, involving bacterial attachment and invasion into host cells during infection.

PD. Dr.

A natural focus of Francisella tularensis holarctica

Healthcare-associated infections and pathogens: Prevention, surveillance, outbreaks und antibiotic stewardship

Francisella tularensis, the causative agent of Tularemia, is a Gram-negative bacterium that can be transmitted by aerosol, contact with mucous membranes or by the alimentary route. Most common in Germany and Europe is the vector-borne infection. While in Scandinavia mosquitoes are the dominating vector, in Germany most infections originate from tick-borne transmission. As for other vector borne diseases, i.e. Tick-Borne Encephalitis Virus, the occurence of natural foci is known, where a single strain of the pathogen circulates over time, indicated by the high genetic stability of isolates over time.When a case of tularemia associated with a tick bite in Munich occured, we started to analyse ticks from this area for Francisella tularensis. Isolation of the bacterium was conducted with all samples positive in Francisella tularensis holarctica specific PCR. For this a selective medium comprising high cystein and iron content combined with antibiotics to eliminate companion bacteria was used. All isolates were tested for antibiotic susceptibility and showed no resistance against classes of antibiotics with therapeutical relevance for Tularemia. Moreover, NGS was performed and whole genomes compared to data from Francisella tularensis holarctica from Germany, Austria and Switzerland. All strains belong to Clade 33 of Francisella tularensis holarctica. Compared to other strains of Francisella tularensis holarctica the isolates only differ in very few single nucleotide polymorphisms. This low diversity was seen not only in isolates from the same year but for all isolates from 2022 to 2023. The sampling of the respective area will be further continued to monitor the behaviour of Francisella tularensis holarctica in a natural focus.

Oral Session
Host-Pathogen Interactions and Clinics of Zoonotic Infections

Appointment

Chair

Topic

Programme

Oral SessionHost-Pathogen Interactions and Clinics of Zoonotic Infections

Appointment

Chair

Topic

Programme

Oral Session
Host-Pathogen Interactions and Clinics of Zoonotic Infections