Characterization of pathogenic modulation through bacterial-fungal interactions in the skin microbiome|RISS 상세보기

국문 초록 (Abstract)

피부 미생물군 내에서 일어나는 세균-진균 사이의 복잡한 상호작용은 피부 건강과 질병에 중추적인 역할을 한다. Staphylococcus와 Malassezia는 우점하는 피부미생물로, 병원성과 면역 반응을 조절하며 상호작용하는것으로 알려져 있지만, 상호작용 기저에 있는 구체적인 메커니즘은 아직 완전히 이해되지 않았다. 따라서 피부 건강과 질병 진행에 미치는 미생물 상호작용의 역할을 규명하기 위한 종합적인 연구가 필요하다.
본 연구는 간단한 in vitro 공동배양에서 재구된 인간 표피(RHE)모델에 이르기까지 다양한 실험 환경을 사용하여 Staphylococcus와 Malassezia 간의 미생물 역학을 탐색하였다. 통제된 액상 공동배양 시스템에서 M. restricta는 Staphylococcus 종에 따라 상이한 영향을 미쳤으며, 이러한 상호작용의 결과는 표현형적 특성으로 분석할 수 있었다. 실험실 균주인 S. aureus NCTC 8325-4는 M. restricta에 거의 반응하지 않았으나, S. epidermidis는 활발한 상호작용을 보였다.
M. restricta에 대한 S. aureus NCTC 8325-4의 최소 반응은 생물막 환경에서도 일관되게 나타났다. S. aureus 균주의 strain수준에서 병원성에 따른 공간적 상호작용을 비교하기 위해, 한천 공동배양 시스템에서 콜로니의 공간역학을 평가하였다. S. aureus NCTC 8325-4와 달리, 병원성 균주인 S. aureus USA300은 M. restricta와 공동 배양 시 향상된 운동성으로 독특한 공간 구조를 형성하였다. 이러한 행동은 S. aureus가 운동성과 생물막 형성의 이점을 활용하여 감염성을 향상시키는 방법을 강조한다.
최종 목표인 다중 미생물 상호작용이 숙주의 면역 반응에 미치는 영향을 종합적으로 이해하기 위해, S. aureus USA300과 M. restricta를 3D 재구성 인간 표피(RHE)에 동시에 감염시켰다. 그 결과, 공동 감염이 염증성 사이토카인 생성 증가 및 피부 장벽 무결성에 영향을 미친다는 것을 확인하였다. 이는 미생물 상호작용이 단순한 경쟁이나 협력을 넘어 숙주 방어 기전에 적극적으로 영향을 미친다는 것을 시사한다.
이러한 결과들을 통해 피부 세균과 곰팡이 사이의 복잡한 관계에 대한 새로운 통찰력을 얻을 수 있었다. M. restricta가 S. aureus의 병원성을 조절하고 숙주 면역 반응에 미치는 영향을 밝힘으로써, 향후 박테리아-진균 상호작용을 제어하여 피부 건강을 유지하거나 회복하는 치료 접근법의 기초를 마련하였다.

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피부 미생물군 내에서 일어나는 세균-진균 사이의 복잡한 상호작용은 피부 건강과 질병에 중추적인 역할을 한다. Staphylococcus와 Malassezia는 우점하는 피부미생물로, 병원성과 면역 반응을 조...

다국어 초록 (Multilingual Abstract)

Complex interactions between bacteria and fungi within the skin microbiome are essential for determining skin health and disease. Among the predominant microorganisms, Staphylococcus and Malassezia interact in ways that influence the pathogenicity and immune responses. The mechanisms underlying this self-organization among bacteria remain largely unknown. Comprehensive investigations are required to understand how microbial interactions affect skin health and disease progression. This study employed a diverse experimental environment to investigate the microbial dynamics of Staphylococcus and Malassezia restricta. The study ranged from primary in vitro co-cultures to advanced 3D reconstructed human epidermis (RHE) models. In a controlled liquid co-culture system, the presence of M. restricta resulted in differential effects on Staphylococcus species, and the results of this interaction could be analyzed by phenotypic characterization. S. aureus NCTC 8325-4 exhibited minimal phenotypic response to M. restricta, unlike the active interactions observed with S. epidermidis.
The minimal response of S. aureus NCTC 8325-4 to M. restricta was consistent, even in biofilm environments. To compare the spatial interactions of the S. aureus strains based on pathogenicity, we evaluated the spatial dynamics of the two colonies using an agar co-culture system. In contrast to the laboratory strain S. aureus NCTC 8325-4, the pathogenic strain S. aureus USA300 forms a unique spatial structure with enhanced motility with M. restricta. This highlights how S. aureus exploits motility and biofilm formation to enhance infectivity. The ultimate goal of this study was to comprehensively understand how polymicrobial interactions influence host immune responses. RHE was simultaneously infected with S. aureus USA300 and M. restricta. RHE demonstrated that co-infection increased inflammatory cytokine production and affected the skin barrier integrity.
This dissertation provides novel insights into the complex relationships between skin bacteria and fungi. Elucidating how M. restricta regulates S. aureus pathogenicity and affects host immune responses establishes a foundation for future therapeutic approaches to control bacterial-fungal interactions in skin health.

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목차 (Table of Contents)

TABLE OF CONTENTS i
LIST OF FIGURES vii
LIST OF TABLES xi
ABSTRACT xii
Chapter 1 Introduction 1

TABLE OF CONTENTS i
LIST OF FIGURES vii
LIST OF TABLES xi
ABSTRACT xii
Chapter 1 Introduction 1
1.1 Microbial interactions as drivers of microbiome functionality 1
1.2 Conventional approaches to microbial interaction studies and their limitations 2
1.3 Research objective and the structure of dissertation 4
Chapter 2 Literature review 6
2.1 Overview of the skin microbiome composition 6
2.1.1 Role of the skin microbiome 6
2.1.2 Skin microbiome composition 7
2.1.3 Staphylococcus: Prevalence, ecological niche, and role in health and disease 11
2.1.4 Malassezia: Prevalence, ecological niche, and role in health and disease 12
2.2 Pathogenicity behavior of skin microbe 15
2.2.1 Staphylococcus 15
2.2.2 Malassezia 16
2.3 Bacterial-fungal interaction 18
2.3.1 Review of traditional co-culture methodology 20
2.3.2 Advanced co-culture techniques 21
2.4 Omics and laser based approaches to uncovering microbial interactions 22
2.4.1 RNA Sequencing 23
2.4.2 Raman Spectroscopy 24
Chapter 3 Interactive phenotyping of single cells determines the type of spatially separated bacterial-fungal interactions 27
3.1 Introduction 27
3.2 Materials and methods 31
3.2.1 Strains and culture conditions 31
3.2.2 Experimental setup 31
3.2.3 Raman spectroscopy 33
3.2.4 Flow cytometry 33
3.2.5 Biofilm formation assay 34
3.2.6 RNA extraction and sequencing 34
3.2.7 Raman spectral processing 35
3.2.8 RNA-Seq data alignment and transcriptome profile determination 35
3.2.9 Statistics and reproducibility 36
3.3 Results 37
3.3.1 Positive effects on the growth of Staphylococcus in co-culture with Malassezia species 37
3.3.2 Effect of the presence of Malassezia restricta on the viability of Staphylococcus 38
3.3.3 Effect of Staphylococcus species on phenotypic plasticity 42
3.3.4 Effect of co-culturing Staphylococcus with Malassezia on the transcriptomic profiles of Staphylococcus 51
3.4 Discussion 62
Chapter 4 Specific Spatial Dynamics in Staphylococcus aureus Interactions with Malassezia Lead to Enhanced Pathogenicity 69
4.1 Introduction 69
4.2 Materials and methods 72
4.2.1 Strains and culture conditions 72
4.2.2 Interaction assay 74
4.2.3 Bacterial population dynamics 76
4.2.4 Confocal laser scanning microscopy 77
4.2.5 Fluorescence area calculation and bacterial growth rate analysis 77
4.2.6 Scanning electron microscopy 78
4.2.7 RNA extraction and sequencing 79
4.2.8 RNA-Seq data alignment and transcriptome profile determination 81
4.2.9 Matrix-assisted laser desorption ionization (MALDI-TOF) 82
4.2.10 Statistics and reproducibility 82
4.2.11 Data availability 83
4.3 Results 84
4.3.1 S. aureus USA300 establishes robust vertical biofilm architecture on M. restricta 84
4.3.2 Malassezia-specific cues drive exclusive vertical colonization by USA300 90
4.3.3 Spatial quantification reveals divergent interaction strategies with M. restricta 93
4.3.4 Transcriptomic analyses reveal specialized metabolic adaptations 98
4.3.5 Region-dependent metabolic reprogramming of USA300: Amino acid synthesis and mannitol utilization 104
4.3.6 Differential PSM profiles and agr-mediated δ-toxin production during vertical migration 105
4.3.7 USA300 enhances virulence factors to gain a competitive advantage 107
4.3.8 PSM-driven vertical growth and colony restructuring in clinical S. aureus 113
4.4 Discussion 118
Chapter 5 The influence of spatial co-colonization by S. aureus and Malassezia restricta on skin barrier integrity 124
5.1 Introduction 124
5.2 Materials and methods 127
5.2.1 Bacterial and fungal cultures 127
5.2.2 3D reconstructed human epidermis (RHE) culture 128
5.2.3 Colonization of the RHE with glass cylinder methodology 128
5.2.4 Multi-parameter end point analysis of organotypic cultures exposed to bacteria and fungi 129
5.2.5 Histological and immunofluorescent staining 131
5.2.6 Cell vailbility Assay 132
5.2.7 Enzyme-linked immunosorbent assay (ELISA) 132
5.2.8 Trypsin activity analysis 133
5.2.9 Fungi RNA extraction and sequencing 133
5.2.10 RNA-Seq data alignment and transcriptome profile determination 134
5.2.11 Statistics and reproducibility 134
5.3 Results 136
5.3.1 Epidermal structure and thickness are altered by microbial treatments 136
5.3.2 Filaggrin expression and distribution indicate barrier disruption 139
5.3.3 Fungal and bacterial co-treatment amplify inflammatory cytokine responses and disrupt skin barrier homeostasis 143
5.3.4 Fungal and bacterial co-treatment increases trypsin activity and suggests proteolytic imbalance 147
5.3.5 Transcriptional analysis of M. restricta co-treated with S. aureus reveals indirect evidence of S. aureus cytotoxicity and enhanced host toxicity 149
5.4 Discussion 151
Chapter 6 General conclusions 154
6.1 Main achievements of this dissertation 154
6.2 Future prospects 155
References 157
ABSTRACT in KOREAN 177

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