Bacterial Colonization of Staphylococcus Aureus on Human Skin

CHAPTER ONE

OBJECTIVES

The main objective of this study was to investigate the molecular mechanisms underlying the determinants involved in S. aureus colonization and/or infection of its host. We aimed to provide increased knowledge regarding the role of the S. aureus CWA proteins SdrD and SasG in bacterial adhesion to host cells and/or evasion of the host immune defence mechanisms.

Our specific research questions were:

1. Does S. aureus SdrD interact with specific host ligand(s) in the epithelium? What could be the role of such ligand interaction?
2. How conserved is the sdrD gene within S. aureus isolates from healthy individuals? Could variation within the sdrD gene influence SdrD function?
3. Does S. aureus SasG contribute to host cells adhesion to human keratinocytes? Under what conditions is SasG expressed? Does S. aureus SasG have immune evasion properties?

CHAPTER TWO

AUREUS COLONIZATION

Significance of Colonization

Humans are constantly exposed to S. aureus in their environment and our body provides a range of ecological niches for the S. aureus and other microbes to thrive⁵⁶. However, not every exposure to S. aureus will lead to successful colonization. S. aureus colonization of its host involves a complex interplay of factors from the bacterium and its host⁵⁷. Longitudinal studies have shown that 20-30% of the healthy adult population is persistently colonized by the S. aureus in their anterior nares^1,2,58,59. S. aureus colonization requires that the bacterium is able to adhere to the receptors present at the ecological niches. Furthermore, it must also be able to thrive and not be eradicated by the host defence mechanisms or resident microbiota^60,61.

1. aureus colonization is an important and essential risk factor for subsequent development of
2. aureus infection and hospital acquired infections^1,4,62,63. Danbolt established the first correlation between nasal carriage and furunculosis skin infection in 1932 (reviewed in²³). In addition, correlation between S. aureus nasal carriage and infections such as continuous peritoneal dialysis (CPD) related infections⁶⁴, HIV⁶⁵, post-operative infections^66,67and foot ulcer⁶⁸have been observed. Studies have shown that the infecting strains are S. aureus strain, which had colonized its carriers’ nares^62,69,70. In addition, patients colonized with MRSA before hospital admission, have a higher risk of developing MRSA infections^71–73 and serve as depots for transmission to other patients⁷⁴.

Sites of  aureus Colonization

The primary ecological niche for S. aureus colonization in human is the nose^63,75. However, aureushave been reported to colonize other sites within the human body including skin⁷⁵, perineum⁷⁶, vagina⁷⁷, axillae⁷⁸, pharynx^75,77, gastrointestinal tract^75,79,80, urinary tract and throat^81,82. Exclusive S. aureus throat, intestinal and pharynx colonization without nasal carriage have been reported^79,81–83. In addition, some studies show higher incidences of S. aureus prevalence in the throat and pharynx compared to the nasal carriage^81,84. The ability of S. aureus to survive in different ecological niches of the human body shows its versatility and diversity in colonizing its host.

Nasal Colonization by  aureus

aureus nasal carriage influences the bacterial colonization of other parts of the human body⁸⁵. This implies that S. aureus nasal carriage most likely serve as a repository for the dispersalofS. aureus into environment or colonization of other body parts⁸⁶. Habits such as nose picking could be an avenue for transfer of S. aureus carried in the nose to other areas of the human body⁸⁷. In addition, patients and healthcare workers nasally colonized by S. aureus can also spread the bacterium to non-colonized persons in hospital settings^74,88.

Based on the risk posed by S. aureus nasal carriage, calls to develop effective nasal decolonization strategies have increased^89,90. Decolonization of S. aureus in the anterior nares following courses of intranasal application of the antibiotics mupirocin has been reported^91,92. In addition, nasal decolonization treatment also eliminated S. aureus from the hands of health workers⁹³. Eradication of S. aureus in the anterior nares in the patients reduced the occurrence of S. aureus infections^94–96. Application of mupirocin has also been used to eradicate MRSA carriage⁹⁷. These observations further strengthen the notion that the nasal environment provides a very viable environment for the colonization and subsequent propagation of S. aureus. However, despite the success of mupirocin in eradicating S. aureus nasal colonization, S. aureus has developed resistance to the antibiotic⁹⁸.

In human nose, the main ecological niche of S. aureus is the moist squamous epithelium of the anterior nares of healthy adults in a general population^2,63,99. This has been further supported by in vitro cell studies, which showed an increased adherence of S. aureus to desquamated epithelial cells isolated from the anterior nares^100,101. However, S. aureus also colonize other regions nose as well, from mid region nares to the deeper regions of the nose¹⁰². Interestingly, Kaspar et al., observed within the sampling population of their study that the posterior region of the nose was consistently colonized compared to the anterior nares¹⁰³. The surface of the anterior nares is lined with a skin-like epithelium while the middle and posterior region of the nose is lined with pseudostratified columnar ciliated epithelium^102,104,105. The role of these different surface cellular constituents on nasal microbiota has been suggested¹⁰². However, in another study where the human nasal microbiome evaluated, they concluded that the epithelium constituent does not affect the nasal microbial diversity¹⁰³, but a large proportion of the participants in the study had chronic nasal inflammation.

₦5,000.00 – DOWNLOAD CHAPTERS 1-5 PROCEED TO DOWNLOAD Added to cart

 

CHAPTER THREE

METHODOLOGY

A detailed description of the experimental procedures performed in this thesis is listed and explained in the articles and manuscript herein. An overview of some of the experimental procedures performed is explained below.

Host Protein-Pathogen Protein Interaction

Protein-protein interaction detection assays have been employed to determine the interaction between host proteins and S. aureus virulence factors. Elucidating these interactions is particularly helpful in deciphering some of the molecular mechanism engaged by S. aureus in colonizing humans. It has been suggested that this might form the basis for the development of some anti-colonization strategies in future²⁹⁷. In Paper I, a yeast two-hybrid (Y2H) assay²⁹⁸ was ordered from Hybrigenics to find the potential host partners for S. aureus SdrD protein. Basically, Y2H assays are performed in yeast strains containing two proteins. One of the proteins referred to as “bait” is fused with a DNA binding domain (DBD) while the other protein referred to as “prey” is fused with an Activation domain (AD). Physical interaction of the two proteins brings these two domains in close proximity and thus forming a functional transcription factor. The transcription factor then activates the reporter gene, which can be assayed by growing the yeast strain on a selective medium or observing for colorimetric changes. In Paper I, the SdrD A region fused with GAL4DBD was the bait protein and this was used to screen the human reconstituted skin libraries fused with GAL4AD (the “prey”). We selected Y2H assay because it allowed for screening of large proteins libraries for SdrD putative host partners. It is important to note that Y2H assay has some limitations, one of which is the high false positive rates of potential candidates²⁹⁹. However, in Paper I, we have employed complementary methods to validate the putative partners as indicated by the Y2H.

CHAPTER FOUR

GENERAL DISCUSSION

 

Adequate understanding of the mechanisms of S. aureus colonization is vital in developing alternatives strategies to combat its ability to cause infections and diseases. Host colonization by S. aureus involves a complex interaction of diverse factors, which are the pathogen, host and the environment^23,316. In this thesis, mechanisms of some of the bacterial determinants involved in S. aureus colonization and infection were investigated. Particularly, we focused on the role of the S. aureus CWA proteins SdrD and SasG in adhesion and immune evasion processes.

CHAPTER FIVE

CONCLUSION

 

Though our understanding of S. aureus interaction with humans has improved greatly since the discovery of the bacteria, there are still a lot of questions regarding the molecular determinants involved. Despite this, S. aureus remains an efficient colonizer and an important human pathogen. Host colonization and/or infection by S. aureus is both complex and multifactorial. This study has characterized the role of S. aureus cell wall anchored proteins in ensuring its adhesion and/or immune evasion.

 

We showed that CWA proteins SdrD and SasG mediate bacterial adhesion to host cells (Paper I, II and III). Furthermore, we identified Dsg1 as the host ligand of SdrD and show that this interaction promotes bacterial adhesion to host cells (Paper I). In addition, we found that genetic variations within the sdrD gene is concentrated within its SdrD A domain and R domain (Paper II). Furthermore, we revealed that expression of S. aureus SasG is upregulated in human blood and in the presence of serum components (Paper III). However, we found that SasG does not promote bacterial survival in human blood ex vivo (Paper III).

 

Our findings are consistent with the multifactorial nature of S. aureus-host interaction. We have provided increased knowledge about the molecular mechanism S. aureus uses in its interaction with the host. Additional studies into the subsequent events following this interaction is needed in order to further clarify the mechanism used by S. aureus to colonize and invade host cells.

 

In the future, our findings (Paper I, II and III) should help in providing molecular mechanistic knowledge required in developing alternative therapeutics to combat S. aureus colonization and/or infection.

 

REFERENCES

 

• Kluytmans, J. et al. Nasal carriage of Staphylococcus aureus: epidemiology, underlyingmechanisms, and associated  Clinical microbiology reviews 10, 505– 20 (1997).
• vanBelkum,  et al. Reclassification of Staphylococcus aureus Nasal Carriage Types. The Journal of Infectious Diseases 199, 1820–1826 (2009).
• Wertheim, F. et al. The role of nasal carriage in Staphylococcus aureus infections. Lancet Infectious Diseases 5, 751–762 (2005).
• Wertheim, F. et al. Risk and outcome of nosocomial Staphylococcus aureus bacteraemia in nasal carriers versus non-carriers. Lancet 364, 703–705 (2004).
• Foster, J. et al. Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus. Nature reviews. Microbiology 12, 49–62 (2014).
• Geoghegan, J. A. & Foster, T. J. Cell wall-anchored surface proteins of Staphylococcusaureus: Many proteins, multiple  in Current Topics in Microbiology and Immunology 409, 95–120 (2017).
• Pendleton, N. et al. Clinical relevance of the ESKAPE pathogens. Expert Review of Anti-infective Therapy 11, 297–308 (2013).
• Rice, B. Federal Funding for the Study of Antimicrobial Resistance in Nosocomial Pathogens: No ESKAPE. The Journal of Infectious Diseases 197, 1079–1081 (2008).
• Tong, S. Y. C. et al. Staphylococcus aureus infections: epidemiology, pathophysiology,clinical manifestations, and  Clinical microbiology reviews 28, 603–61 (2015).
• Chambers, F. & DeLeo, F. R. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nature Reviews Microbiology 7, 629–641 (2009).
• Ogston, Report upon micro-organisms in surgical diseases. British Medical Journal  1, 369–377 (1881).
• Ogston, Micrococcus Poisoning. Journal of Anatomy and Physiology 17, 24–58 (1882).\
• Rosenbach, J. Mikro-organismen bei den Wund-Infections-Krankheiten des Menschen. (J.F. Bergmann, 1884).
• Marshall, H. & Wilmoth, G. J. Pigments of Staphylococcus aureus, a series of triterpenoid carotenoids. Journal of Bacteriology 147, 900–913 (1981).
• Foster, Staphylococcus. in Medical Microbiology.(4 th ed.) Galveston (TX): (ed. Baron S) 1–11 (University of Texas Medical Branch at Galveston, 1996).

₦5,000.00 – DOWNLOAD CHAPTERS 1-5 PROCEED TO DOWNLOAD Added to cart