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Maren Depke Introduction STAPHYLOCOCCUS AUREUS General Features of S. aureus Staphylococci are Gram-positive bacteria of approximately 1 µm diameter, which often aggregate in grape-like structures. The golden color of Staphylococcus aureus colonies – derived from staphyloxanthin and other C 30 triterpenoid carotenoids (Marshall/Wilmoth 1981) which function as antioxidant protection molecules (Liu GY et al. 2005) – led to its naming. Several S. aureus properties like the ability to perform hemolysis and expression of catalase and coagulase allow differentiation of the species from other bacteria. S. aureus can produce energy by aerobic respiration, but is facultatively able to survive in anaerobic environments. In situations without oxygen it can apply lactate fermentation. S. aureus seems to be naturally auxotrophic and requires e. g. amino acids as growth supplement. The staphylococcal cell wall peptidoglycan features a special crossbridge structure formed by multiple glycine residues. These are the target structures for lysostaphin, a staphylocidal enzyme from Staphylococcus simulans first described by Schindler and Schuhardt in 1964, which was taken over by scientist for several laboratory research purposes and which might even be applied as mupirocin substitute for eradication of staphylococcal nasal colonization (Recsei et al. 1987, Kokai-Kun et al. 2003). Staphylococci are resistant to lysozyme, a muramidase, which is present as antimicrobial enzyme in body fluids and at sites of infection as part of the innate immune defense. The resistance is mediated by O- acetylation in the muramic acid of peptidoglycan (position C6-OH) which is conducted by the peptidoglycan-specific O-acetyltransferase OatA. OatA mutants are susceptible to lysozyme (Bera et al. 2005). The S. aureus genome has 32 % GC content. Its chromosome is about 2.7E+06 to 2.9E+06 nucleotides in length, contains about 2600 to 3000 genes, and has been sequenced for several strains until now (Table I.1). Additionally, sequence information for several plasmids is available (http://www.ncbi.nlm.nih.gov/sites/entrez?db=Genome). For sub-division purposes, the S. aureus genome has been classified into two parts. The core genome, which occupies approximately 75 % of the complete genome, includes genes with basic functions e. g. in house-keeping and central metabolism. These genes are present in the genomes of most strains and exhibit a high degree of conservation in the individual genes’ sequences in different strains. The core part also features a high similarity to the phylogenetically related Bacillus species genomes (Hiramatsu et al. 2004). The remaining 25 % belong to the accessory part of the genome. This part consists of further genes which have been acquired by some S. aureus strains e. g. from mobile genetic elements. Here, variation in the presence of genes is observed in different strains. Many virulence genes are classified as fraction of the accessory genome (Lindsay/Holden 2004). S. aureus has many options of varying its accessory genome part by different mobile genetic elements (Plata et al. 2009). Staphyloccoccal pathogenicity islands (SaPI) are located at constant positions of the genome, contain superantigen genes and sequences with similarity to prophages (Lindsay et al. 1998, Novick RP 2003). Similarly, genes of the νSa-family of pathogenicity genomic islands, of which several types (1-4, α, β, γ) exist, code for virulence factors and toxins (Gill et al. 20
Maren Depke Introduction 2005). SaPIs and some νSa-members can be excised from the genome and are involved in horizontal gene transfer. S. aureus strains often harbor prophages, which also incorporate virulence factors and are an aid for their transfer between strains and in general influence S. aureus evolution (Lindsay/Holden 2004). The Panton-Valentine leukocidin is a phage-encoded staphylococcal toxin. S. aureus NCTC8325 carries three lysogenic phages, φ11, φ12, and φ13, of which φ13 introduces the virulence factor staphylokinase (sak) into the strain (Iandolo et al. 2002). Kwan et al. reported in 2005 the genome sequences and predicted proteins of 27 bacteriophages of S. aureus (Kwan et al. 2005). Additionally, gene transfer can be accomplished by shorter insertion sequences or by longer transposons which bring along their own transposase genes and recognition sites (Hiramatsu et al. 2004, Plata et al. 2009). Plasmids are a further place of encoding useful, although not essential information, e. g. for antibiotic resistances. S. aureus strains include different plasmids of varying size and copy number. For example, the methicillinresistant S. aureus strain COL owns the 4440 nt plasmid pT181 (accession number NC_006629; http://www.ncbi.nlm.nih.gov/sites/entrez?db=Genome), whose sequence became available in 2005. Some S. aureus strains have also acquired the vanA operon for vancomycin resistance from an Enterococcus spp. transposon via a conjugative plasmid (Péricon/Courvalin 2009). Table I.1: Complete, RefSeq genome sequences for bacterial chromosomes of Staphylococcus aureus strains from NCBI Entrez Genome database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=Genome) of August 25 th 2010. accession number strain length / nt date created genes NC_002745 Staphylococcus aureus subsp. aureus N315 2814816 2001/04/21 2664 NC_002951 Staphylococcus aureus subsp. aureus COL 2809422 2001/09/14 2723 NC_002758 Staphylococcus aureus subsp. aureus Mu50 2878529 2001/10/04 2774 NC_002952 Staphylococcus aureus subsp. aureus MRSA252 2902619 2001/11/06 2839 NC_002953 Staphylococcus aureus subsp. aureus MSSA476 2799802 2001/11/06 2715 NC_003923 Staphylococcus aureus subsp. aureus MW2 2820462 2002/05/31 2704 NC_007622 Staphylococcus aureus RF122 2742531 2005/11/24 2663 NC_007793 Staphylococcus aureus subsp. aureus USA300_FPR3757 2872769 2006/02/11 2648 NC_007795 Staphylococcus aureus subsp. aureus NCTC8325 2821361 2006/02/18 2969 NC_009487 Staphylococcus aureus subsp. aureus JH9 2906700 2007/05/23 2816 NC_009632 Staphylococcus aureus subsp. aureus JH1 2906507 2007/07/03 2870 NC_009641 Staphylococcus aureus subsp. aureus Newman 2878897 2007/07/06 2687 NC_009782 Staphylococcus aureus subsp. aureus Mu3 2880168 2007/09/06 2768 NC_010079 Staphylococcus aureus subsp. aureus USA300_TCH1516 2872915 2007/12/03 2799 NC_013450 Staphylococcus aureus subsp. aureus ED98 2824404 2009/11/19 2752 S. aureus, a Commensal and Opportunistic Pathogen Staphylococcus aureus is a persistent commensal in the anterior nares of approximately 20 % of the human population (persistent carriers). The bacterium is found intermittently in 30 % to 60 % of the population, and in non-carriers, which is the remaining fraction of the population, nasal swabs are never positive for S. aureus (Kluytmans et al. 1997, Wertheim et al. 2005). Some authors subdivide the group of intermittent carriers into intermittent and occasional carriers (Eriksen et al. 1995). More recent publications classify the carriage status only into two groups: persistent carriers and others (van Belkum et al. 2009). The occurrence of the two contrary groups of persistent and non-carriers strongly hints for an influence of the host’s immune system on the possibility for S. aureus to colonize persistently 21
Page 1 and 2: G E N O M E W I D E C H A R A C T E
Page 3 and 4: Maren Depke C O N T E N T S GENOMEW
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Page 7 and 8: Maren Depke Zusammenfassung der Dis
Page 9 and 10: Maren Depke S U M M A R Y O F D I S
Page 11 and 12: Maren Depke Summary of Dissertation
Page 13 and 14: Maren Depke I N T R O D U C T I O N
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Page 31 and 32: Maren Depke Introduction STUDIES OF
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Page 39 and 40: Maren Depke M A T E R I A L A N D M
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Maren Depke Results Liver Gene Expr
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liver weight [g] Maren Depke Result
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infection rate cfu / 10 mg tissue c
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Maren Depke Results Kidney Gene Exp
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Table R.2.1: Genes displaying stati
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Maren Depke BR1 sign DsigB vs wt BR
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Maren Depke Results GENE EXPRESSION
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Maren Depke Results Gene Expression
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log2(ratio C57BL/6 / BALB/c) at IFN
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Maren Depke Results Host Cell Gene
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Maren Depke Results Pathogen Gene E
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S. aureus RN1HG sample conditions a
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mean normalized intensity mean norm
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mean normalized intensity Maren Dep
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log 2 (ratio) log 2 (ratio) log 2 (
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Maren Depke D I S C U S S I O N A N
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Maren Depke Discussion and Conclusi
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Maren Depke References Athanasopoul
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Maren Depke References Byrne GI, Le
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Maren Depke References Demling R. T
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Maren Depke References Foss GS, Pry
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Maren Depke References Hagopian K,
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Maren Depke References Jin T, Bokar
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Maren Depke References Kwan T, Liu
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Maren Depke References Luster AD, U
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Maren Depke References Namiki S, Na
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Maren Depke References Puccetti P.
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Maren Depke References Siewert E, B
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Maren Depke References van den Akke
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Maren Depke References Yang XL, Sch
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Maren Depke A F F I D A V I T / E R
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Maren Depke Acknowledgments Kidney
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