Escherichia coli Nissle 1917 Antagonizes Candida albicans Growth and Protects Intestinal Cells from C. albicans -Mediated Damage

Candida albicans is a pathobiont of the gastrointestinal tract. It can contribute to the diversity of the gut microbiome without causing harmful effects. When the immune system is compromised, C. albicans can damage intestinal cells and cause invasive disease. We hypothesize that a therapeutic approach against C. albicans infections can rely on the antimicrobial properties of probiotic bacteria. We investigated the impact of the probiotic strain Escherichia coli Nissle 1917 (EcN) on C. albicans growth and its ability to cause damage to intestinal cells. In co-culture kinetic assays, C. albicans abundance gradually decreased over time compared with C. albicans abundance in the absence of EcN. Quantification of C. albicans survival suggests that EcN exerts a fungicidal activity. Cell-free supernatants (CFS) collected from C. albicans -EcN co-culture mildly altered C. albicans growth, suggesting the involvement of an EcN-released compound. Using a model of co-culture in the presence of human intestinal epithelial cells, we further show that EcN prevents C. albicans from damaging enterocytes both distantly and through direct contact. Consistently, both C. albicans ’s filamentous growth and microcolony formation were altered by EcN. Taken together, our study proposes that probiotic-strain EcN can be exploited for future therapeutic approaches against C. albicans infections

Proteomic and transcriptomic experiments reveal an essential role of RNA degradosome complexes in shaping the transcriptome of Mycobacterium tuberculosis.

The phenotypic adjustments of Mycobacterium tuberculosis are commonly inferred from the analysis of transcript abundance. While mechanisms of transcriptional regulation have been extensively analysed in mycobacteria, little is known about mechanisms that shape the transcriptome by regulating RNA decay rates. The aim of the present study is to identify the core components of the RNA degradosome of M. tuberculosis and to analyse their function in RNA metabolism. Using an approach involving cross-linking to 4-thiouridine-labelled RNA, we mapped the mycobacterial RNA-bound proteome and identified degradosome-related enzymes polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlE), ribonuclease E (RNase E) and ribonuclease J (RNase J) as major components. We then carried out affinity purification of eGFP-tagged recombinant constructs to identify protein-protein interactions. This identified further interactions with cold-shock proteins and novel KH-domain proteins. Engineering and transcriptional profiling of strains with a reduced level of expression of core degradosome ribonucleases provided evidence of important pleiotropic roles of the enzymes in mycobacterial RNA metabolism highlighting their potential vulnerability as drug targets

Neutrophils versus Pathogenic Fungi : through the magnifying glass of nutritional immunity

Neutrophils are among the first white blood cells recruited to the site of infection once microbial pathogens enter the host organism. At site, they perform a well-orchestrated chain of processes that aims to kill the microbial invader. Most prominent, neutrophils engulf microbes to inactivate them intracellularly, a process called phagocytosis. Alternatively, neutrophils can release neutrophil extracellular traps (NETs). NETs consist of chromatin decorated with antimicrobial effector proteins – a structure that can entangle bacteria and fungi. Neutrophils are crucial during fungal infections. This is reflected in the increased risk of fungal infections resulting of neutropenia. The concept of nutritional immunity describes every infection as a battle for resources. Those are mostly metal trace elements. For a long time, neutrophils were seen as powerful, but “mindless”, killers with a limited set of actions and no transcriptional capacity, but this view is in the flux. In the presented thesis, it was my goal to gain new insights into the interplay of neutrophils and fungi – with special attention to metal-nutritional aspects. We compared human neutrophils lacking the ability to undergo NETosis, due to a non-functional NADPH complex, and neutrophils from the same person that were “cured” by gene therapy. We investigated those NETs and found that their inhibitory activity towards the mold A. nidulans depends on calprotectin, a known zinc-chelator. Considering the high influx of neutrophils, we wanted to unravel the neutrophils’ contribution to the metal milieu at the site of infection and trace element changes resulting from NETosis. By combining synchrotron radiation XRF and ICP-MS, we analyzed the neutrophil metallome and the spatial element distribution in activated neutrophils and NETs. Most strikingly, we found neutrophils to be exceptionally high in Fe and the process of NETosis to be reducing available Zn in the surrounding and the early phagosome, possibly by the formation of Zn-rich vesicles. Using RNA-sequencing, we analyzed the interplay of the C. albicans and neutrophils face-to-face. We dissected their transcriptional profile and revealed a manifold response in neutrophils that include cytokine induction and cellular rearrangement. We further were the firsts to explore the transcriptional response of C. albicans to NETs. Our data indicates a distinct response compared to intact neutrophils or other known stress triggers. Metal homeostasis was affected in Candida in both set-ups. In summary, this thesis provides new insights into the interaction of fungal pathogens with neutrophils and emphasizes the impact of nutritional aspects on this interplay. A deeper understanding of the nutritional immunity during fungal infection might open up new strategies to tackle fungal infections – a growing threat worldwide

Fungal-Bacterial Interactions in Health and Disease

Fungi and bacteria encounter each other in various niches of the human body. There, they interact directly with one another or indirectly via the host response. In both cases, interactions can affect host health and disease. In the present review, we summarized current knowledge on fungal-bacterial interactions during their commensal and pathogenic lifestyle. We focus on distinct mucosal niches: the oral cavity, lung, gut, and vagina. In addition, we describe interactions during bloodstream and wound infections and the possible consequences for the human host

Immune Resolution Dilemma: Host Antimicrobial Factor S100A8/A9 Modulates Inflammatory Collateral Tissue Damage During Disseminated Fungal Peritonitis

Intra-abdominal infection (peritonitis) is a leading cause of severe disease in surgical intensive care units, as over 70% of patients diagnosed with peritonitis develop septic shock. A critical role of the immune system is to return to homeostasis after combating infection. S100A8/A9 (calprotectin) is an antimicrobial and pro-inflammatory protein complex used as a biomarker for diagnosis of numerous inflammatory disorders. Here we describe the role of S100A8/A9 in inflammatory collateral tissue damage (ICTD). Using a mouse model of disseminated intra-abdominal candidiasis (IAC) in wild-type and S100A8/A9-deficient mice in the presence or absence of S100A9 inhibitor paquinimod, the role of S100A8/A9 during ICTD and fungal clearance were investigated. S100A8/A9-deficient mice developed less ICTD than wild-type mice. Restoration of S100A8/A9 in knockout mice by injection of recombinant protein resulted in increased ICTD and fungal clearance comparable to wild-type levels. Treatment with paquinimod abolished ICTD and S100A9-deficient mice showed increased survival compared to wild-type littermates. The data indicates that S100A8/A9 controls ICTD levels and antimicrobial activity during IAC and that targeting of S100A8/A9 could serve as promising adjunct therapy against this challenging disease

Trace element landscape of resting and activated human neutrophils on the sub-micrometer level

Every infection is a battle for trace elements. Neutrophils migrate first to the infection site and accumulate quickly to high numbers. They fight pathogens by phagocytosis and intracellular toxication. Additionally, neutrophils form neutrophil extracellular traps (NETs) to inhibit extracellular microbes. Yet, neutrophil trace element characteristics are largely unexplored. We investigated unstimulated and phorbol myristate acetate-stimulated neutrophils using synchrotron radiation X-ray fluorescence (SR-XRF) on the sub-micron spatial resolution level. PMA activates pinocytosis, cytoskeletal rearrangements and the release of NETs, all mechanisms deployed by neutrophils to combat infection. By analyzing Zn, Fe, Cu, Mn, P, S, and Ca, not only the nucleus but also vesicular granules were identifiable in the elemental maps. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) revealed a neutrophil-specific composition of Zn, Fe, Cu, and Mn in comparison with J774 and HeLa cells, indicating a neutrophil-specific metallome complying with their designated functions. When investigating PMA-activated neutrophils, the SR-XRF analysis depicted typical subcellular morphological changes: the transformation of nucleus and granules and the emergence of void vacuoles. Mature NETs were evenly composed of Fe, P, S, and Ca with occasional hot spots containing Zn, Fe, and Ca. An ICP-MS-based quantification of NET supernatants revealed a NETosis-induced decrease of soluble Zn, whereas Fe, Cu, and Mn concentrations were only slightly affected. In summary, we present a combination of SR-XRF and ICP-MS as a powerful tool to analyze trace elements in human neutrophils. The approach will be applicable and valuable to numerous aspects of nutritional immunity.This article is part of themed collection: Metals in infectious diseases and nutritional immunity.</p

Augmented enterocyte damage during Candida albicans and Proteus mirabilis coinfection

The human gut acts as the main reservoir of microbes and a relevant source of life-threatening infections, especially in immunocompromised patients. There, the opportunistic fungal pathogen Candida albicans adapts to the host environment and additionally interacts with residing bacteria. We investigated fungal-bacterial interactions by coinfecting enterocytes with the yeast Candida albicans and the Gram-negative bacterium Proteus mirabilis resulting in enhanced host cell damage. This synergistic effect was conserved across different P. mirabilis isolates and occurred also with non-albicans Candida species and C. albicans mutants defective in filamentation or candidalysin production. Using bacterial deletion mutants, we identified the P. mirabilis hemolysin HpmA to be the key effector for host cell destruction. Spatially separated coinfections demonstrated that synergism between Candida and Proteus is induced by contact, but also by soluble factors. Specifically, we identified Candida-mediated glucose consumption and farnesol production as potential triggers for Proteus virulence. In summary, our study demonstrates that coinfection of enterocytes with C. albicans and P. mirabilis can result in increased host cell damage which is mediated by bacterial virulence factors as a result of fungal niche modification via nutrient consumption and production of soluble factors. This supports the notion that certain fungal-bacterial combinations have the potential to result in enhanced virulence in niches such as the gut and might therefore promote translocation and dissemination