The Staphylococcus aureus secretome - TI Pharma

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
by pseudopilin-specific SPases, like ComC, takes place at the cytoplasmic side of the
membrane and leaves the H-domain attached to the translocated protein.
While many proteins that end up in the extracellular milieu or the cell wall of Gram-positive
bacteria have signal peptides, proteins without known export signals can also be found on
these locations. The relative numbers of proteins without known signal peptides seem to vary
per organism. While these numbers are relatively low for B. subtilis and S. aureus, they are
high for group A Streptococcus and M. tuberculosis (Tjalsma et al., 2004). As indicated
above, some of the proteins without known export signals appear to be liberated from the cell
by lysis, while others are actively exported, for example via the Ess pathway. Although the
precise export signal in proteins secreted via the Ess pathway has not yet been defined, a
WxG motif is shared by many of these proteins and may serve a function in protein targeting
(Pallen, 2002). Furthermore, the signal for specific release of lysins via holins is presently not
known.
Signal peptide predictions
Several prediction programs that are accessible through the world-wide web are useful tools
to predict whether a given protein contains some sort of sorting signal or SPase cleavage site.
The programs that we have used in this and other studies were: SignalP-NN and SignalP-
HMM version 2.0 (Nielsen et al., 1997), LipoP version 1.0 (Juncker et al., 2003), PrediSi
(Hiller et al., 2004) and Phobius (Kall et al., 2004). These programs have been designed to
identify Sec type signal peptides, N-terminal membrane anchors (Phobius), or lipoprotein
signal peptides in Gram-negative bacteria (LipoP). The TMHMM-program version 2.0
(Cserzö et al., 1997) was used to exclude proteins with (predicted) multiple membrane
spanning domains. Predictions for proteins containing a signal peptide were performed with
the SignalP program, using the Neural Network (NN) and Hidden Markov Model algorithms
(HMM). Version 2.0 of the SignalP program was preferred above Version 3.0 (Bendtsen et
al., 2004) for our signal peptide predictions in S. aureus and S. epidermidis, because the best
overall prediction accuracy was obtained with Version 2.0 in a recent proteomics-based
verification of predicted export and retention signals in B. subtilis (Tjalsma and van Dijl,
2005). Specifically, the Hidden Markov Model in SignalP 2.0 assigns probability scores to
each amino acid of a potential signal peptide and indicates whether it is likely to belong to the
N-, H-, or C-domains. Proteins with no detectable N-, H-, and C-domain were excluded from
the set. Searching for transmembrane domains was performed with the TMHMM program
and proteins with more than one (predicted) transmembrane domain were excluded from the
set, because they are most likely integral membrane proteins. All proteins with a predicted Cterminal
transmembrane segment in addition to a signal peptide were screened for the
presence of a conserved motif for covalent cell wall binding. It should be noted that this
approach does not automatically result in the exclusion of potential membrane proteins with
one N-terminal transmembrane domain. The LipoP-program was used to predict lipoproteins.
The combined results of all these programs resulted in a list of proteins, which have: a) signal
peptides with distinctive N-, H-, and C-domains, b) no additional transmembrane domains,
and c) predicted extracytoplasmic localizations. These proteins were scanned for the presence
of proteomics-based consensus motifs for type I, type II or pseudopilin-specific SPase
recognition and cleavage sites, twin-arginine motifs, and known leader peptides of
bacteriocins by BLAST searches and by use of the PATTINPROT program (http://npsapbil.ibcp.fr)
as previously described (Tjalsma and van Dijl, 2005). To define the core
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