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PhD thesis: a PhD thesis.
Ythier M.
Moreillon  P.
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Université de Lausanne, Faculté de biologie et médecine
Faculté de biologie et de médecine Université de Lausanne UNIL - Bugnon Rue du Bugnon 21 - bureau 4111 CH-1015 Lausanne SUISSE
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Staphylococcus aureus is a highly successful pathogen responsible of a wide variety of
diseases, from minor skin infection to life-threatening sepsis or infective endocarditis, as well as food
poisoning and toxic shock syndrome. This heterogeneity of infections and the ability of S. aureus to
develop antibiotic-resistance to virtually any available drugs reflect its extraordinary capacity to adapt
and survive in a great variety of environments. The pathogenesis of S. aureus infection involves a
wide range of cell wall-associated adhesins and extracellular toxins that promote host colonization and
invasion. In addition, S. aureus is extremely well equipped with regulatory systems that sense
environmental conditions and respond by fine tuning the expression of metabolic and virulence
determinants. Surface adhesins referred to MSCRAMMs - for Microbial Surface Component
Recognizing Adherence Matrix Molecules - mediate binding to the host extracellular matrix or serum
components, including fibrinogen, fibronectin, collagen and elastin, and promote tissue colonization
and invasion. Major MSCRAMMs include a family of surface-attached proteins covalently bound to the
cell wall peptidoglycan via a conserved LPXTG motif. Genomic analyses indicate that S. aureus
contain up to 22 LPXTG surface proteins, which could potentially act individually or in synergy to
promote infection.
In the first part of this study we determined the range of adherence phenotypes to fibrinogen
and fibronectin among 30 carriage isolates of S. aureus and compared it to the adherence phenotypes
of 30 infective endocarditis and 30 blood culture isolates. Overall there were great variations in in vitro
adherence, but no differences were observed between carriage and infection strains. We further
determined the relation between in vitro adherence and in vivo infectivity in a rat model of
experimental endocarditis, using 4 isolates that displayed either extremely low or high adherence
phenotypes. Unexpectedly, no differences were observed between the in vivo infectivity of isolates
that were poorly and highly adherent in vitro. We concluded that the natural variability of in vitro
adherence to fibrinogen and fibronectin did not correlate with in vivo infectivity, and thus that
pathogenic differences between various strains might only be expressed in in vivo conditions, but not
in vitro. Therefore, considering the importance of adhesins expression for infection, direct
measurement of those adhesins present on the bacterial surface were made by proteomic approach.
In the second series of experiments we assessed the physical presence of the LPXTG species
at the staphylococcal surface, as measured at various time points during growth in different culture
media. S. aureus Newman was grown in either tryptic soy broth (TSB) or in Roswell Park Memorial
Institute (RPMI) culture medium, and samples were removed from early exponential growth phase to
late stationary phase. Experiments were performed with mutants in the global accessory-gene
regulator (agr), surface protein A (Spa) and clumping factor A (ClfA). Peptides of surface proteins were
recovered by "trypsin-shaving" of live bacteria, and semi-quantitative proteomic analysis was
performed by tandem liquid-chromatography and mass-spectrometry (LC-MS). We also determined in
parallel the mRNA expression by microarrays analysis, as well as the phenotypic adherence of the
bacteria to fibrinogen in vitro. The surface proteome was highly complex and contained numerous
proteins theoretically not belonging to the bacterial envelope, including ribosomal proteins and
metabolic enzymes. Sixteen of the 21 known LPXTG species were detected, but were differentially
expressed. As expected, 9 known agr-regulated proteins (e.g. including Spa, FnBPA, ClfA, IsdA, IsdB,
SasH, SasD, SasG and FmtB) increased up to the late exponential growth phase, and were abrogated
in agr-negative mutants. However, only Spa and SasH modified their proteomic and mRNA profiles in
parallel in the parent and its agr negative mutant, while all other LPXTG proteins modified their
proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active
in in vitro fibrinogen adherence tests during late growth (24h), whereas it remained poorly detected by
proteomics. Differential expression was also detected in iron-rich TSB versus iron-poor RPMI. Proteins
from the iron-regulated surface determinant (isd) system, including IsdA, IsdB and IsdH were barely
expressed in iron-rich TSB, whereas they increased their expression by >10 time in iron-poor RPMI.
We conclude that semi-quantitative proteomic analysis of specific protein species is feasible in S.
aureus and that proteomic, transcriptomic and adherence phenotypes demonstrated differential
profiles in S. aureus. Furthermore, peptide signatures released by trypsin shaving suggested
differential protein domain exposures in various environments, which might be relevant for antiadhesins
vaccines. A comprehensive understanding of the S. aureus physiology should integrate all
these approaches.
Staphylococcus aureus, proteomic, transcriptomic, adherence, cell wall, adhesins, LPXTG
Create date
28/01/2013 15:12
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20/08/2019 14:33
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