Host-pathogen interactions in invasive Staphylococcus aureus infections

Staphylococcus aureus is a versatile human pathogen causing a wide range of diseases from uncomplicated skin and soft tissue infections to life-threatening invasive diseases like endocarditis, bacteremia, necrotizing pneumonia, and fasciitis. The pathogen has become increasingly resistant to -lactam antibiotics, and of special concern is the rise in community- acquired (CA)-MRSA strains, as specific CA-MRSA clones have been associated with highly aggressive infections. The ability of S. aureus to cause such a multitude of infections is linked to the production of a wide array of virulence factors. Several virulence factors have been implicated in disease pathogenesis, including the exotoxins Panton-Valentine Leukocidin (PVL), alpha-toxin (α-toxin), superantigens and phenol soluble modulins. This thesis project aimed to characterize S. aureus strains in the community as well as through use of clinical invasive isolates and human lung/skin organotypic tissue models explore the role of specific staphylococcal toxins and virulence regulation in the pathologic events leading to the destructive infections in lung and skin. In paper I, molecular characterization of Indian community S. aureus isolates to determine their lineage and to analyze their virulence and immune-evasion factors was conducted. The percentage of methicillin resistance was 26% in carrier isolates while 60% among disease isolates. 69% of the isolates were positive for PVL genes along with combinations of many other toxins. The patterns of presence and absence of virulence and immune evasion factors strictly followed the sequence type (ST). We are reporting several new STs, which have not been reported earlier, along with factors influencing virulence and host-pathogen interactions. Next, we demonstrate in paper II that community S. aureus strains displayed stable phenotypic response profiles, defined by either proliferative or cytotoxic responses. The cytotoxic supernatants contained significantly higher levels of α-toxin as compared to proliferative supernatants. Furthermore, a significant association between agr type and phenotypic response profile was found, with agr I and agr IV strains being predominantly cytotoxic whereas agr II and III strains were proliferative. This differential response profiles associated with certain S. aureus strains with varying toxin production abilities could have an impact on disease outcome and may reflect upon the existence of specific pathotypes. In paper III we focused on the pathogenesis of CA S. aureus severe pneumonia, in particular, the impact of exotoxins produced by strains isolated from varying severity of lung infections on human host cells and in human 3D organotypic lung tissue. α-toxin had a direct damaging effect on the epithelium whereas PVL contributed indirectly to the tissue pathology by triggering lysis of neutrophils. We demonstrated that severe tissue pathology is associated with a combination and high levels of both α-toxin and PVL, and fatal outcome correlated with higher toxin production in pneumonia. Notably, both α-toxin and PVL mediated cytotoxic effect and epithelial disruption was significantly abrogated by addition of polyclonal intravenous immunoglobulins. In paper IV we focused on skin and soft tissue infections caused by ST22 strains, one of the most critically expanding MRSA clones world-wide. Here we identified a mechanism for which new variants, cytotoxic vs. persistent phenotype, can emerge. We link this phenotype switch to a specific mutation of receptor histidine kinase AgrC. The phenotypic switch to a persistence phenotype is associated with upregulation of bacterial surface proteins, less severe skin tissue damage, resistance to antimicrobials, and induction of autophagy. In contrast, cytotoxic phenotype strains showed upregulated exotoxin expression and caused infections characterized by inflammasome activation and severe skin tissue pathology. This study shows a strong effect of a single amino acid substitution in AgrC as a critical factor contributing to virulence properties and infection outcome. Together, the studies in this thesis demonstrate that several different toxins will contribute to tissue pathology, but they target different cells and their impact may be tissue-specific. Also, distinct functional differences between the isolates were identified that are likely to contribute to disease outcome. Such insight should promote the development of novel diagnostics or therapeutic strategies

Single-cell patterning and characterisation of antibiotic persistent bacteria using bio-sCAPA

In microbiology, accessing single-cell information within large populations is pivotal. Here we introduce bio-sCAPA, a technique for patterning bacterial cells in defined geometric arrangements and monitoring their growth in various nutrient environments. We demonstrate bio-sCAPA with a study of subpopulations of antibiotic-tolerant bacteria, known as persister cells, which can survive exposure to high doses of antibiotics despite lacking any genetic resistance to the drug. Persister cells are associated with chronic and relapsing infections, yet are difficult to study due in part to a lack of scalable, single-cell characterisation methods. As >10$^{5}$ cells can be patterned on each template, and multiple templates can be patterned in parallel, bio-sCAPA allows for very rare population phenotypes to be monitored with single-cell precision across various environmental conditions. Using bio-sCAPA, we analysed the phenotypic characteristics of single Staphylococcus aureus cells tolerant to flucloxacillin and rifampicin killing. We find that antibiotic-tolerant S. aureus cells do not display significant heterogeneity in growth rate and are instead characterised by prolonged lag-time phenotypes alone

Pulmonary Surfactant Proteins are Inhibited by IgA Autoantibodies in Severe COVID-19

Rationale: Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome with fatal outcomes. Evidence suggests that dysregulated immune responses, including autoimmunity, are key pathogenic factors. Objectives: To assess whether IgA autoantibodies target lung-specific proteins and contribute to disease severity. Methods: We collected 147 blood, 9 lung tissue, and 36 bronchoalveolar lavage fluid samples from three tertiary hospitals in Switzerland and one in Germany. Severe COVID-19 was defined by the need to administer oxygen. We investigated the presence of IgA autoantibodies and their effects on pulmonary surfactant in COVID-19 using the following methods: immunofluorescence on tissue samples, immunoprecipitations followed by mass spectrometry on bronchoalveolar lavage fluid samples, enzyme-linked immunosorbent assays on blood samples, and surface tension measurements with medical surfactant. Measurements and main results: IgA autoantibodies targeting pulmonary surfactant proteins B and C were elevated in patients with severe COVID-19, but not in patients with influenza or bacterial pneumonia. Notably, pulmonary surfactant failed to reduce surface tension after incubation with either plasma or purified IgA from patients with severe COVID-19. Conclusions: Our data suggest that patients with severe COVID-19 harbor IgA against pulmonary surfactant proteins B and C and that these antibodies block the function of lung surfactant, potentially contributing to alveolar collapse and poor oxygenation. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Staphylococcus aureus impairs dermal fibroblast functions with deleterious effects on wound healing.

Chronic wounds are a major disease burden worldwide. The breach of the epithelial barrier facilitates transition of skin commensals to invasive facultative pathogens. Therefore, we investigated the potential effects of Staphylococcus aureus (SA) on dermal fibroblasts as key cells for tissue repair. In co-culture systems combining live or heat-killed SA with dermal fibroblasts derived from the BJ-5ta cell line, healthy individuals, and patients with systemic sclerosis, we assessed tissue repair including pro-inflammatory cytokines, matrix metalloproteases (MMPs), myofibroblast functions, and host defense responses. Only live SA induced the upregulation of IL-1β/-6/-8 and MMP1/3 as co-factors of tissue degradation. Additionally, the increased cell death reduced collagen production, proliferation, migration, and contractility, prerequisite mechanisms for wound closure. Intracellular SA triggered inflammatory and type I IFN responses via intracellular dsDNA sensor molecules and MyD88 and STING signaling pathways. In conclusion, live SA affected various key tissue repair functions of dermal fibroblasts from different sources to a similar extent. Thus, SA infection of dermal fibroblasts should be taken into account for future wound management strategies

Systemic application of bone-targeting peptidoglycan hydrolases as a novel treatment approach for staphylococcal bone infection

The rising prevalence of antimicrobial resistance in S. aureus has rendered treatment of staphylococcal infections increasingly difficult, making the discovery of alternative treatment options a high priority. Peptidoglycan hydrolases, a diverse group of bacteriolytic enzymes, show high promise as such alternatives due to their rapid and specific lysis of bacterial cells, independent of antibiotic resistance profiles. However, using these enzymes for the systemic treatment of local infections, such as osteomyelitis foci, needs improvement, as the therapeutic distributes throughout the whole host, resulting in low concentrations at the actual infection site. In addition, the occurrence of intracellularly persisting bacteria can lead to relapsing infections. Here, we describe an approach using tissue-targeting to increase the local concentration of therapeutic enzymes in the infected bone. The enzymes were modified with a short targeting moiety that mediated accumulation of the therapeutic in osteoblasts and additionally enables targeting of intracellularly surviving bacteria

Levels of alpha-toxin correlate with distinct phenotypic response profiles of blood mononuclear cells and with agr background of community-associated Staphylococcus aureus isolates

Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of α-toxin than did the proliferative supernatants. Addition of α-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, δ-toxin or phenol soluble modulin α-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes

The Role of Streptococcal and Staphylococcal Exotoxins and Proteases in Human Necrotizing Soft Tissue Infections

Necrotizing soft tissue infections (NSTIs) are critical clinical conditions characterized by extensive necrosis of any layer of the soft tissue and systemic toxicity. Group A streptococci (GAS) and Staphylococcus aureus are two major pathogens associated with monomicrobial NSTIs. In the tissue environment, both Gram-positive bacteria secrete a variety of molecules, including pore-forming exotoxins, superantigens, and proteases with cytolytic and immunomodulatory functions. The present review summarizes the current knowledge about streptococcal and staphylococcal toxins in NSTIs with a special focus on their contribution to disease progression, tissue pathology, and immune evasion strategies

Antibiotic resistance and persistence—Implications for human health and treatment perspectives

Antimicrobial resistance (AMR) and persistence are associated with an elevated risk of treatment failure and relapsing infections. They are thus important drivers of increased morbidity and mortality rates resulting in growing healthcare costs. Antibiotic resistance is readily identifiable with standard microbiological assays, and the threat imposed by antibiotic resistance has been well recognized. Measures aiming to reduce resistance development and spreading of resistant bacteria are being enforced. However, the phenomenon of bacteria surviving antibiotic exposure despite being fully susceptible, so‐called antibiotic persistence, is still largely underestimated. In contrast to antibiotic resistance, antibiotic persistence is difficult to measure and therefore often missed, potentially leading to treatment failures. In this review, we focus on bacterial mechanisms allowing evasion of antibiotic killing and discuss their implications on human health. We describe the relationship between antibiotic persistence and bacterial heterogeneity and discuss recent studies that link bacterial persistence and tolerance with the evolution of antibiotic resistance. Finally, we review persister detection methods, novel strategies aiming at eradicating bacterial persisters and the latest advances in the development of new antibiotics

Single-cell patterning and characterisation of antibiotic persistent bacteria using bio-sCAPA

In microbiology, accessing single-cell information within large populations is pivotal. Here we introduce bio-sCAPA, a technique for patterning bacterial cells in defined geometric arrangements and monitoring their growth in various nutrient environments. We demonstrate bio-sCAPA with a study of subpopulations of antibiotic-tolerant bacteria, known as persister cells, which can survive exposure to high doses of antibiotics despite lacking any genetic resistance to the drug. Persister cells are associated with chronic and relapsing infections, yet are difficult to study due in part to a lack of scalable, single-cell characterisation methods. As >105 cells can be patterned on each template, and multiple templates can be patterned in parallel, bio-sCAPA allows for very rare population phenotypes to be monitored with single-cell precision across various environmental conditions. Using bio-sCAPA, we analysed the phenotypic characteristics of single Staphylococcus aureus cells tolerant to flucloxacillin and rifampicin killing. We find that antibiotic-tolerant S. aureus cells do not display significant heterogeneity in growth rate and are instead characterised by prolonged lag-time phenotypes alone.ISSN:1473-0197ISSN:1473-018

Antibacterial Neutrophil Effector Response: Ex Vivo Quantification of Regulated Cell Death Associated with Extracellular Trap Release

Regulated cell death (RCD) and the concomitant release of extracellular traps by neutrophils (NETs) constitute an important antibacterial effector response. Usually, the dynamic processes of RCD and NETs release are assessed independently of each other by either unspecific or time-consuming methods. Here, we describe a flow cytometry-based high-throughput analysis method incorporating neutrophil RCD and NETs release with visual live-imaging conformation upon ex vivo bacterial challenge. This combined approach allows to quantify and closely follow the kinetics of the dynamic neutrophil effector response towards bacterial infection