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In vitro and in vivo analyses of mono- and mixed-species biofilms formed by microbial pathogens
(2022)
Microbial biofilms can be defined as multicellular clusters of microorganisms embedded in a self-produced extracellular matrix (ECM), which is primarily composed of polymeric biomolecules. Biofilms represent one of the most severe burdens in both industry and healthcare worldwide, causing billions of dollars of treatment costs annually because biofilms are inherently difficult to prevent, treat, and eradicate. In health care settings, patients suffering from cystic fibrosis, or patients with medical implants are highly susceptible to biofilm infections. Once a biofilm is formed, it is almost impossible to quantitatively eradicate it by mechanical, enzymatical, chemical, or antimicrobial treatment. Often the only remaining option to fully eradicate the biofilm is removing of the infected implant or body part. The primary reasons for the inherent resistance of biofilms against all forms of antimicrobial treatment are (I) a reduced metabolic activity of biofilm-embedded cells climaxing in the presence of metabolic inactive persister cells, as well as (II) the protective nature of the biofilm matrix acting as a (diffusion) barrier against antimicrobials and the host immune system. Consequently, there is an urgent need to better understand microbial biofilms from a structural and (patho-) physiological point of view in order to be able to develop new treatment strategies.
Therefore, the aims of this study were to investigate fundamental physiological properties of different clinically relevant single and multi-species biofilms, both in vitro and in vivo. Furthermore, the effectiveness of a novel treatment strategy using cold atmospheric pressure plasma was evaluated in vitro to treat biofilms of the pathogenic fungus C. albicans.
In article I, the intracellular and ECM protein inventory of Staphylococcus aureus during in vitro biofilm growth in a flow reactor was analyzed by liquid-chromatography coupled to tandem mass-spectrometry (LC-MS/MS) analysis combined with metabolic footprint analysis. This analysis showed that anaerobiosis within biofilms releases organic acids lowering the ECM pH. This, in turn, leads to protonation of alkaline proteins – mostly ribosomal proteins originating from cell lysis as well as actively secreted virulence factors – resulting in a positive net charge of these proteins. As a consequence, these proteins accumulate within the ECM and form an electrostatic network with negatively charged cell surfaces, eDNA, and metabolites contributing to the overall biofilm stability.
In article II, the in vivo metaproteome of the multi-species biofilm community in cystic fibrosis sputum was investigated. To this end, an innovative protocol was developed allowing the enrichment of microbial cells, the extraction of proteins from a small amount of cystic fibrosis sputum, and subsequent metaproteome analysis. This protocol also allows 16S sequencing, metabolic footprint analysis, and microscopy of the same sample to complement the metaproteome data. Applying this protocol, we were able to significantly enhance microbial protein coverage providing first insights into important physiological pathways during CF lung infection. A key finding was that the arginine deaminase pathway as well as microbial proteases play a so far underappreciated role in CF pathophysiology.
In articles III and IV, a novel treatment strategy for biofilms formed by the important fungal pathogen Candida albicans was evaluated in vitro. Biofilms were treated with two different sources of nonthermal plasma (with the Nonthermal Plasma Jet “kINPen09” as well as with the Microwave-induced plasma torch “MiniMIP”) and the effect on growth, survival, and viability was assessed by counting colony-forming units (CFU), by cell proliferation assays, as well as by live/dead staining combined with fluorescence microscopy, confocal laser scanning microscopy, (CLSM) and atomic force microscopy (AFM). These tests revealed that biofilms were effectively inactivated mostly on the bottom side of biofilms, indicating a great potential of these two plasma sources to fight biofilms.
Staphylococcus aureus (S. aureus) endocarditis is still one of the most fatal heart diseases, with a mortality rate of 20-45%. In recent years, the importance of endothelial cells (ECs) in the context of endocarditis has become more evident. The vascular endothelium forms a selective barrier between blood and the adjacent tissue by maintaining an anti-inflammatory and anti-thrombogenic phenotype. However, in case of insertion of cardiac implants, an injury of the endothelium can occur which promotes platelet aggregation followed by S. aureus adherence to the platelets, especially in areas with low hemodynamic shear stress. This process is considered as a key event in the development of infective endocarditis (IE) and allows bacteria to colonize the heart valves. Despite extensive research, the pathogenesis of IE is still not completely understood. Therefore, further investigations are needed to enable an effective prevention of this life-threatening disease.
In order to study the infection process of S. aureus, internalization experiments with two different S. aureus strains, one control strain (HG001) and one strain isolated from an endocarditis patient (T-72949) were performed in human coronary artery endothelial cells (HCAEC). Subsequently, an extensive proteome analysis of the host cells was carried out. More specific analyses were performed using peptidoglycan (PGN), a cell wall component of Gram-positive bacteria, which causes a pro-inflammatory response in ECs. In this context, the focus remained on the analysis of cellular changes in terms of cell stiffness, wound healing, and additionally platelet aggregation.
The analysis of the HCAEC host proteome revealed a time-related difference depending on the infecting bacterial strain. Several proteins involved in host cell signaling pathways exhibited a higher abundance at earlier time points in host cells infected with endocarditis strain T-72949 compared to those infected with HG001. Further proteome analysis uncovered several adaptations on the cellular side that enable internalization and replication of both S. aureus strains as well as the activation of pathways that promote cellular recovery. Furthermore, it could be shown that PGN reduced cellular stiffness which could lead to an increased bacterial uptake and would thereby promote the development of a chronic S. aureus infection. Additionally, PGN prevented effective wound healing which promotes a pro-thrombotic and pro-inflammatory condition. This status could facilitate the bacterial infection of further cells. Apart from that, PGN induced platelet aggregation which could ease bacterial adhesion to thrombotic surfaces (e.g., dysfunctional endothelium). The following formation of a mature vegetation might protect the bacteria from the immune system and antibiotics.
The results of the present work emphasize the central role of ECs in the context of IE. It could be demonstrated that a healthy monolayer of ECs enables a beneficial cell response and may prevent the development of vascular diseases. Moreover, the comprehensive proteome dataset which was generated in this project provides a valuable source of information for future studies to unravel further molecular mechanisms of endocarditis and possible therapeutic approaches.
Influenza A Virus (IAV), Staphylococcus aureus (staphylococci), and Streptococcus pneumoniae (pneumococci) are leading viral and bacterial causes of pneumonia. Dendritic cells (DCs) are present in the lower respiratory tract. They are characterized by low expression of co-stimulatory molecules, including CD80 and CD86 and high capacity of antigen uptake. Subsequently, DCs upregulate co-stimulatory signals and cytokine secretion to effectively induce T-cell priming. Here, we investigated these processes in response to bacterial and viral single as well as coinfections using human monocyte-derived (mo)DCs. Irrespective of single or coinfections, moDCs matured in response to IAV and/or staphylococcal infections, secreted a wide range of cytokines, and activated CD4+, CD8+ as well as double-negative T cells. In contrast, pneumococcal single and coinfections impaired moDC maturation, which was characterized by low expression of CD80 and CD86, downregulated expression of CD40, and a mild cytokine release resulting in abrogated CD4+ T-cell activation. These actions were attributed to the cholesterol-dependent cytotoxin pneumolysin (Ply). Infections with a ply-deficient mutant resulted in restored moDC maturation and exclusive CD4+ T-cell activation. These findings show that Ply has important immunomodulatory functions, supporting further investigations in specific modalities of Ply-DC interplay.