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

Photo-oxidative tuning of individual and coupled GaAs photonic crystal cavities

We demonstrate a new photo-induced oxidation technique for tuning GaAs photonic crystal cavities using a $390~\mathrm{nm}$ pulsed laser with an average power of $10~\mathrm{\mu W}$. The laser oxidizes a small $\left(\sim 500~\mathrm{nm}\right)$ diameter spot, reducing the local index of refraction and blueshifting the cavity. The tuning progress can be actively monitored in real time. We also demonstrate tuning an individual cavity within a pair of proximity-coupled cavities, showing that this method can be used to correct undesired frequency shifts caused by fabrication imperfections in cavity arrays.Comment: 4 pages, 3 figure

Nanobeam photonic crystal cavity quantum dot laser

The lasing behavior of one dimensional GaAs nanobeam cavities with embedded InAs quantum dots is studied at room temperature. Lasing is observed throughout the quantum dot PL spectrum, and the wavelength dependence of the threshold is calculated. We study the cavity lasers under both 780 nm and 980 nm pump, finding thresholds as low as 0.3 uW and 19 uW for the two pump wavelengths, respectively. Finally, the nanobeam cavity laser wavelengths are tuned by up to 7 nm by employing a fiber taper in near proximity to the cavities. The fiber taper is used both to efficiently pump the cavity and collect the cavity emission.Comment: 8 pages; 6 figure

Optical fiber tips functionalized with semiconductor photonic crystal cavities

We demonstrate a simple and rapid epoxy-based method for transferring photonic crystal cavities to the facets of optical fibers. Passive Si cavities were measured via fiber taper coupling as well as direct transmission from the fiber facet. Active quantum dot containing GaAs cavities showed photoluminescence that was collected both in free space and back through the original fiber. Cavities maintain a high quality factor (2000-4000) in both material systems. This new design architecture provides a practical mechanically stable platform for the integration of photonic crystal cavities with macroscale optics and opens the door for novel research on fiber-coupled cavity devices.Comment: 10 pages, 5 figure

A photonic crystal cavity-optical fiber tip nanoparticle sensor for biomedical applications

We present a sensor capable of detecting solution-based nanoparticles using an optical fiber tip functionalized with a photonic crystal cavity. When sensor tips are retracted from a nanoparticle solution after being submerged, we find that a combination of convective fluid forces and optically-induced trapping cause an aggregation of nanoparticles to form directly on cavity surfaces. A simple readout of quantum dot photoluminescence coupled to the optical fiber shows that nanoparticle presence and concentration can be detected through modified cavity properties. Our sensor can detect both gold and iron oxide nanoparticles and can be utilized for molecular sensing applications in biomedicine.Comment: 13 pages, 5 figure

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

Coupled fiber taper extraction of 1.53 um photoluminescence from erbium doped silicon nitride photonic crystal cavities

Optical fiber tapers are used to collect photoluminescence emission at ~1.5 um from photonic crystal cavities fabricated in erbium doped silicon nitride on silicon. Photoluminescence collection via fiber taper is enhanced 2.5 times relative to free space, with a total taper collection efficiency of 53%. By varying the fiber taper offset from the cavity, a broad tuning range of coupling strength is obtained. This material system combined with fiber taper collection is promising for building on-chip optical amplifiers.Comment: 10 pages, 7 figure

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/)