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Previous Article | Next Article 
Microbiology and Molecular Biology Reviews, September 2006, p. 755-788, Vol. 70, No. 3
1092-2172/06/$08.00+0 doi:10.1128/MMBR.00008-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Mapping the Pathways to Staphylococcal Pathogenesis by Comparative Secretomics
M. J. J. B. Sibbald,1
A. K. Ziebandt,2
S. Engelmann,2
M. Hecker,2
A. de Jong,3
H. J. M. Harmsen,1
G. C. Raangs,1
I. Stokroos,4
J. P. Arends,1
J. Y. F. Dubois,1 and
J. M. van Dijl1*
Department
of Medical Microbiology, University Medical Centre Groningen and
University of Groningen, Hanzeplein
1, P.O. Box 30001, 9700 RB
Groningen, The Netherlands,1
Institut für
Mikrobiologie, Ernst-Moritz-Arndt Universität,
Greifswald, F.-L.-Jahnstr. 15, D-17487
Greifswald, Germany,2
Department of Genetics,
Groningen Biomolecular Sciences
and Biotechnology Institute, Kerklaan 30,
9751 NN Haren, The Netherlands,3
Department
of Cell Biology and Electron Microscopy,
University Medical Centre Groningen and University
of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The
Netherlands4
The gram-positive bacterium Staphylococcus aureus is a
frequent component of the human microbial flora that can turn into a
dangerous pathogen. As such, this organism is capable of infecting
almost every tissue and organ system in the human body. It does so by
actively exporting a variety of virulence factors to the cell surface
and extracellular milieu. Upon reaching their respective destinations,
these virulence factors have pivotal roles in the colonization and
subversion of the human host. It is therefore of major importance to
obtain a clear understanding of the protein transport pathways that are
active in S. aureus. The present review aims to provide a
state-of-the-art roadmap of staphylococcal secretomes, which include
both protein transport pathways and the extracytoplasmic proteins of
these organisms. Specifically, an overview is presented of the exported
virulence factors, pathways for protein transport, signals for cellular
protein retention or secretion, and the exoproteomes of different
S. aureus isolates. The focus is on S. aureus, but
comparisons with Staphylococcus epidermidis and other
gram-positive bacteria, such as Bacillus subtilis, are
included where appropriate. Importantly, the results of genomic and
proteomic studies on S. aureus secretomes are integrated
through a comparative "secretomics" approach, resulting
in the first definition of the core and variant secretomes of this
bacterium. While the core secretome seems to be largely employed for
general housekeeping functions which are necessary to thrive in
particular niches provided by the human host, the variant secretome
seems to contain the "gadgets" that S. aureus
needs to conquer these well-protected
niches.
The gram-positive
bacterium Staphylococcus aureus is a frequent
component of the human microbial flora that can turn into a dangerous
pathogen. As such, this organism is capable of infecting almost every
tissue and organ system in the human body. It does so by exporting a
variety of virulence factors to the cell surface and extracellular
milieu of the human host. Like all living organisms
(201), S.
aureus contains several protein transport pathways, among which
the general secretory (Sec) pathway is the most well known and best
described. Proteins that need to be transported to an extracytoplasmic
location generally contain an N-terminal signal peptide that is needed
to target the newly synthesized protein from the ribosome to
the translocation machinery in the cytoplasmic membrane. Next, the
protein is threaded through the Sec translocon in an unfolded state.
During this translocation step, or shortly thereafter, the signal
peptide is removed by a so-called signal peptidase (SPase). Upon
complete membrane translocation, the protein has to fold into its
correct conformation and will then be retained in an extracytoplasmic
compartment of the cell or secreted into the extracellular milieu. In
the case of gram-positive cocci, such as S. aureus (Fig.
1), we distinguish three extracytoplasmic subcellular compartments, namely,
the membrane, the membrane-cell wall interface, and the cell wall.
Since surface-exposed and secreted proteins of S. aureus play
pivotal roles in the colonization and subversion of the human host, it
is of major importance to obtain a clear understanding of the protein
transport pathways that are active in this organism
(103). Knowledge about
the protein sorting mechanism has become all the more relevant with the
emergence of staphylococcal resistance against last-defense
antibiotics, such as vancomycin. The scope of this review is to provide
a state-of-the-art roadmap of staphylococcal secretomes, which include
both protein transport pathways and the extracytoplasmic proteins of
staphylococcal organisms. The focus is on S. aureus, but
comparisons with Staphylococcus epidermidis and the
best-characterized gram-positive bacterium, Bacillus subtilis,
are included where appropriate. Importantly, the present review aims to
integrate the results of genomic and proteomic studies on S.
aureus secretomes, representing the first documented
"comparative secretomics" study. Specifically, this
review deals with known and predicted exported virulence
factors, pathways for protein transport, signals for subcellular
protein sorting or secretion, and the exoproteomes of different S.
aureus isolates, as defined by two-dimensional polyacrylamide gel
electrophoresis (2D-PAGE) and mass spectrometry (Fig.
2 and 3). The
exoproteome is defined by all S. aureus proteins that can be
identified in the extracellular milieu of this organism and thus
includes proteins actively secreted by living cells and the remains of
dead cells. For a clear appreciation of the present review, it is
important to bear in mind that the proteins exported from the cytoplasm
could be directly involved in staphylococcal virulence, whereas the
respective protein export systems represent the "pathways to
pathogenesis."
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FIG. 1. Imaging
of S. aureus RN6390. (A) For scanning electron
microscopy, a drop of washed culture of bacteria was fixated for 30 min
with 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.38. Next, the
fixated bacteria were placed on a piece (1 cm2) of cleaved
0.1% poly-L-lysine-coated mica sheet and washed in 0.1 M
cacodylate buffer. This specimen was dehydrated in an ethanol series
consisting of 30%, 50%, 70%, 96%, and anhydrous 100% (3x)
solutions for 10 min each, critical point dried with CO2,
and sputter coated with 2 to 3 nm Au/Pd (Balzers coater). The specimen
was fixed on a scanning electron microscope stub holder and observed in
a JEOL FE-SEM 6301F microscope. (B) Micrograph of a cluster
of S. aureus cells grown in blood culture medium. The cells
were fixed with ethanol and hybridized with the fluorescein-labeled
peptide nucleic acid (PNA) probe PNA-Stau. The image was generated by
merging an epifluorescence image with the negative of a phase-contrast
image.
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FIG. 2. Extracellular
proteomes of different S. aureus strains. Proteins in the
growth medium fractions of different staphylococcal isolates, grown in
TSB medium (37°C) to an optical density at 540 nm
(OD540) of 10, were separated by 2D-PAGE using immobilized
pH gradient strips in the pH range of 3 to 10 (Amersham Pharmacia
Biotech, Piscataway, N.J.). Each gel was loaded with 350 µg
protein extracts and, after electrophoresis, stained with colloidal
Coomassie blue. Proteins were identified by matrix-assisted laser
desorption ionization-time of flight mass spectrometry. The
corresponding protein spots are labeled with protein names according to
the S. aureus N315 database or NCBI entries for proteins not
present in N315. The S. aureus strains that were used in these
experiments are RN6390 and COL and four clinical isolates from the
University Medical Center Groningen, named MRSA693331, 035699y/bm,
0440579/rmo, and
CA-MRSA021708m/rmo.
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FIG. 3. Dynamics
of the amount of extracellular proteins during growth of S.
aureus RN6390 in TSB medium. (A) Individual dual-channel
2D patterns of extracellular proteins during the different phases of
the growth curve for cells grown in TSB medium were assembled into a
movie. The protein pattern at an OD540 of 1 (labeled in
green) was compared with the protein patterns at higher optical
densities (labeled in red). As a consequence of dual channel labeling,
spots where the intensities do not differ between the compared gels are
yellow, and spots with different intensities are either green or red
(15). (B)
Growth curve for S. aureus RN6390 grown in TSB medium, as
determined by OD540 readings. The sampling points for
proteomics analyses are indicated by arrows. (C) Proteomic
signatures of selected proteins representing different regulatory
groups, as revealed by dual-channel imaging. The amounts of the
respective proteins at an OD540 of 1 (spots labeled in
green) for cells grown in TSB medium were compared with the relative
amounts of these proteins at higher optical densities (spots labeled in
red). Proteins were stained with Sypro
ruby.
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EXPORTED STAPHYLOCOCCAL VIRULENCE FACTORS
|
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S. aureus
and S. epidermidis are organisms that occur naturally in and
on the human body. While S. epidermidis is mostly present on
human skin, S. aureus can be found on mucosal surfaces. S.
aureus is carried by 30 to 40% of the population
(143) and can be
identified readily in the nose, but the organism can also be detected
in other moist regions of the human body, such as the axillae,
perineum, vagina, and rectum, which thereby form a major reservoir for
infections. Although most staphylococcal infections are nosocomial
(i.e., hospital acquired), an increase in the number of cases of
community-acquired, antibiotic (methicillin)-resistant infections is
currently being observed worldwide
(27,
67,
185). The risk of
intravascular and systemic infection by S. aureus rises when
the epithelial barrier is disrupted by intravascular catheters,
implants, mucosal damage, or trauma. Interestingly, after infection,
cells of S. aureus can persist unnoticed in the human body for
a long time (years), after which they can suddenly cause another
infection. S. aureus is primarily an extracellular pathogen
whose colonization and invasion of human tissues and organs can lead to
severe cytotoxic effects. Nevertheless, S. aureus can also be
internalized by various cells, including nonphagocytic cells, which
seems to induce apoptosis
(43,
72,
120). Although S.
aureus has the potential to form biofilms
(64), S.
epidermidis infections are particularly notorious for the
formation of thick multilayered biofilms on indwelling catheters and
other implanted devices. The formation of such a biofilm takes place in
several steps, during which the bacteria first adhere rapidly to the
surface of a polymer material that has been coated with a film of
proteinaceous and nonproteinaceous organic host molecules
(56). Bacteria that
adhere to this film produce extracellular polymeric substances, mostly
polysaccharides and proteins, in turn resulting in a strong attachment
to the polymer surface and other bacteria in the growing biofilm.
Ultimately, the biofilm is composed of multiple layers of cells,
cellular debris, polysaccharides, and proteins. S. epidermidis
factors that are essential for biofilm formation include the
polysaccharide intercellular adhesin
(107), the
accumulation-associated protein
(157), and the
biofilm-associated protein
(184). Polysaccharide
intercellular adhesin is most likely identical to the polysaccharide
adhesion protein.
Virulence of S. aureus
The pathogenicity of S. aureus is caused by
the expression of an arsenal of virulence factors (Table
1), which can lead to superficial skin lesions, such as styes,
furunculosis, and paronychia, or to more serious infections, such as
pneumonia, mastitis, urinary tract infections, osteomyelitis,
endocarditis, and even sepsis. In very rare cases, S. aureus
causes meningitis. The virulence factors that S. aureus
employs to cause these diseases are displayed at the surface of the
staphylococcal cell or secreted into the host milieu
(57). Specifically, these
virulence factors include (i) surface proteins that promote adhesion to
and colonization of host tissues, (ii) invasins that are exported to an
extracytoplasmic location and promote bacterial spread in tissues
(leukocidin, kinases, and hyaluronidase), (iii) surface factors that
inhibit phagocytic engulfment (capsule and protein A), (iv) biochemical
properties that enhance staphylococcal survival in phagocytes
(carotenoid and catalase production), (v) immunological disguises
(protein A, coagulase, and clotting factor), (vi) membrane-damaging
toxins that disrupt eukaryotic cell membranes (hemolysins and
leukotoxin), (vii) superantigens that contribute to the
symptoms of septic shock (SEA-G, toxic shock syndrome toxin [TSST], and
ET), and (viii) determinants for inherent and acquired resistance to
antimicrobial agents.
Most virulence factors are expressed in a
coordinated fashion during the growth cycle of S. aureus. The
best-characterized regulators of virulence factors are the accessory
gene regulator (agr)
(124,
144,
152) and the
staphylococcal accessory regulator (SarA)
(29,
30). Ziebandt et al.
(208) showed that
extracellular proteins can be divided into two groups based on the
timing of their expression in cells grown in tryptic soy broth (TSB),
i.e., proteins that are expressed only at low cell densities and
proteins exclusively expressed at high cell densities. agr
seems to be an important positive regulator of proteins that are
expressed at higher optical densities (e.g., proteases, hemolysins, and
lipases) and a negative regulator of proteins that are expressed during
the exponential growth phase (e.g., immunodominant antigen A, secretory
antigen precursor, and several proteins with unknown functions). In
addition, Gill et al.
(63) identified 15 other
two-component regulatory systems in the genomes of S. aureus
and S. epidermidis that are potentially involved in
staphylococcal virulence. In this respect, it is interesting that the
antibiotic cerulenin, which is known to inhibit protein secretion by
S. aureus at sub-MIC levels, was recently reported to block
the transcriptional activation of at least two regulatory determinants,
agr and sae. Thus, it seems that cerulenin inhibits
the transcription of genes for secretory proteins rather than the
secretion process of these proteins
(1). In contrast, it was
previously believed that cerulenin would interfere with membrane
function through an inhibition of normal fatty acid
synthesis.
Notably, to date, relatively little information is
available on the molecular nature of the stimuli that are perceived by
the major regulators of the expression of virulence factors. Overall,
it should be clear that strain-specific differences in gene regulation
by agr, sae, sarA, or other regulators may
dramatically influence the repertoire of produced virulence factors,
thereby having a profound impact on the disease-causing potential of
different strains. Since the interplay of different regulators and
cell-to-cell communication can impact differently on the expression of
different virulence factors, even the disease-causing potential of
individual S. aureus cells within a genetically identical
population may vary.
Resistance of S. aureus to Antibiotics
Resistance of S. aureus to
antibiotics was observed very soon after the introduction of penicillin
about 60 years ago. In the following years, the amazing ability of
staphylococci to develop resistance to antibiotics has
resulted in the emergence of methicillin-resistant
S. aureus (MRSA) and S. epidermidis strains. In fact,
methicillin resistance was observed already in 1961 in nosocomial
isolates of S. aureus, 1 year after the introduction of
methicillin (85).
Resistance towards methicillin is a result of the production of an
altered penicillin binding protein, PBP2a (or PBP2'), which has
less affinity for most ß-lactam antibiotics. The PBP2a protein,
which is located at the membrane-cell wall interface, is of major
importance for cell wall biogenesis by mediating the cross-linking of
peptidoglycans. PBP2a is encoded by the mecA gene, which is
located on a mobile genetic element known as the staphylococcal
cassette chromosome mec element (SCCmec)
(28,
84). SCCmec is a
basic mobile genetic element that serves as a vehicle for gene exchange
among staphylococcal species
(49). In addition to the
mecA gene, SCCmec carries the mecA
regulatory genes mecI and mecR, an insertion sequence
element (IS431mec), and a unique cassette of recombinase genes
(ccr), which are responsible for SCCmec chromosomal
integration and excision. Five different types of SCCmec
elements, types I to V, have been identified so far, based on the
classes of mecA gene and ccr gene complexes
(84). The type I
SCCmec contains the mecA gene as the only resistance
element, while the type II and III elements contain, besides
mecA, multiple determinants for resistance against
non-ß-lactam antibiotics. Accordingly, type II and III
SCCmec elements are responsible for multidrug resistance in
nosocomial MRSA isolates. Type IV SCCmec elements, like type I
elements, contain no resistance genes other than mecA, and
they are significantly smaller than the type II and III elements. This
might serve as an evolutionary advantage, making it easier for these
mobile genetic elements to spread across bacterial populations. Type V
SCCmec elements are also small compared to the other elements
and differ in their set of recombinase genes
(84). Whereas the type I
to IV SSCmec elements contain the two recombinase genes
ccrA and ccrB, the type V elements contain a single
copy of a gene, ccrC, homologous to a cassette chromosome
recombinase gene. In addition, two open reading frames, hsdS
and hsdM, which encode a restriction-modification system, are
unique to these elements. Phylogenetic analyses of these genes showed a
distant relationship with their homologues in other S. aureus
genomes and suggested a foreign origin for these
genes.
Vancomycin resistance was first reported for
Enterococcus faecium
(101), and transfer of
vancomycin resistance from enterococci, such as Enterococcus
faecalis, to S. aureus has been shown to occur
(137). Vancomycin has
long been a last-resort antibiotic for multiple-drug-resistant S.
aureus strains, but already in 1996 a strain was
isolated which showed reduced sensitivity towards vancomycin
(78). Shortly afterwards,
additional strains were isolated in different countries and were
designated vancomycin intermediately resistant S. aureus
(VISA). These strains show a significantly thickened cell wall, which
allows them to sequester more vancomycin than non-VISA strains, thereby
preventing the detrimental effects of this antibiotic
(42). A search for the
genetic basis of the lowered vancomycin sensitivity of the S.
aureus Mu50 strain revealed that important genes for cell wall
biosynthesis and intermediary metabolism have mutations compared to
those in MRSA strains, which might lead to altered expression of genes
involved in cell wall metabolism and a thickened cell wall
(4). The first
highly-vancomycin-resistant strain was isolated in 2002
(199). This strain was
shown to carry a plasmid which contains, among other resistance genes,
the vanA gene plus several additional genes required for
vancomycin resistance. The proteins encoded by these genes are
responsible for replacing the C-terminal
D-alanyl-D-alanine
(D-Ala-D-Ala) of the
disaccharide pentapeptide cell wall precursor with a
depsipeptide,
D-alanyl-D-lactate
(D-Ala-D-Lac), thereby lowering the cell
wall affinity for vancomycin
(24).
Export of Virulence Factors from the Cytoplasm
Since most
proteinaceous virulence factors are displayed at the surface of the
staphylococcal cell or released into the medium, it is important for
our understanding of the pathogenic potential of these organisms to map
their pathways for protein transport. While specific questions relating
to surface display or secretion of particular virulence factors have
been addressed for several years, more holistic studies on the genomics
and proteomics of these processes have been documented in the
scientific literature only very recently. Moreover, no systematic
analysis of pathways and cellular machinery for protein transport has
thus far been performed for staphylococci. This review is aimed at
filling this knowledge gap. To do so, we have taken full advantage of
the availability of six completely sequenced and annotated S.
aureus genomes and one of the two sequenced S.
epidermidis strains as well as recently published data on the
analysis of staphylococcal cell wall proteomes and exoproteomes.
Additionally, we have combined published information with
bioinformatics-derived data on all potential signals for protein export
from the cytoplasm and secretion into the extracellular milieu or
retention in the membrane or cell wall. Since polytopic membrane
proteins do not appear to have major direct roles in virulence other
than causing drug resistance, such membrane proteins remain beyond the
scope of this review. Furthermore, since the secretome of B.
subtilis has been characterized extensively, at the level of both
the protein export machinery and the exoproteome, we have compared the
staphylococcal secretomes with that of B. subtilis. To our
knowledge, this has resulted in the first "comparative
secretomics" study.
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S. AUREUS STRAINS SUITABLE FOR COMPARATIVE SECRETOMICS
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Nine sequenced and fully annotated genomes of S. aureus are
available in public databases (Table
2) (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi;
http://www.tigr.org),
and six of these genomes were used in the present study. These include
the genome of one of the first hospital-acquired MRSA isolates, S.
aureus COL (63),
which has been used widely in research on staphylococcal methicillin
and vancomycin resistance. The sequenced MRSA252 strain
(79) is a
hospital-acquired epidemic strain, which was isolated from a patient
who died as a consequence of septicemia. The sequenced MSSA476 strain
(79) is a
community-acquired invasive strain that is penicillin and fusidic acid
resistant but susceptible to most commonly used antibiotics. S.
aureus Mu50 and N315
(100) are
hospital-acquired MRSA strains isolated from Japanese patients. In
addition, the Mu50 strain displays intermediate vancomycin sensitivity.
Finally, the community-acquired isolate S. aureus MW2
(7) is a highly virulent
MRSA strain isolated from a 16-month-old girl from the United States.
Notably, the most widely used laboratory strain, NCTC8325, has been
sequenced, but the nucleotide sequence and corresponding annotation
were not available for the present analyses. This was also the case for
the community-acquired MRSA strain USA300
(46). Furthermore, the
sequence of S. aureus RF122, a strain associated with mastitis
in cattle, is now also available in the NCBI database (unpublished),
but it was not included in the present review, which is primarily
focused on staphylococcal pathogenicity towards humans. Using
multilocus sequence typing with seven housekeeping genes of the
different S. aureus strains, Holden et al.
(79) showed that the
MRSA252 strain is phylogenetically most distantly related to the other
sequenced strains, while the Mu50 and N315 strains are
indistinguishable by multilocus sequence typing, as are the MSSA476 and
MW2 strains. The COL and NCTC8325 strains are relatively closely
related to each other.
Sequenced and annotated genomes
of other staphylococcal species, such as S.
epidermidis, Staphylococcus haemolyticus,and Staphylococcus carnosus, are also publicly
available. However, with the exception of S. epidermidis
strain ATCC 12228 (207),
these are not included in the present review, which focuses primarily
on S. aureus. A comparative genomic analysis of S.
aureus COL, Mu50, MW2, and N315 and the sequenced S.
epidermidis strains RP62a and ATCC 12228 revealed that these
species and strains have a set of 1,681 genes in common
(63). In contrast, 454
genes are present in the S. aureus strains but not in S.
epidermidis, whereas 286 genes are present in S.
epidermidis but not in S. aureus. Most of the
strain-specific and species-specific genes can be related to the
presence or absence of particular prophages and genomic
islands.
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PATHWAYS FOR STAPHYLOCOCCAL PROTEIN TRANSPORT
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The
bacterial machinery for protein transport is currently best described
for Escherichia coli (gram negative) and B. subtilis
(gram positive) (for reviews, see references
44,
174, and
175). Many of the known
components that are involved in the different routes for protein export
from the cytoplasm and in posttranslocational modification of exported
proteins in these organisms are also conserved in S. aureus
and S. epidermidis (Table
3). In general, proteins that are exported are synthesized with an
N-terminal signal peptide, which directs them to a particular transport
pathway. Consequently, the presently known signal peptides are
classified according to the export pathway into which they direct the
corresponding proteins or the type of signal peptidase that is
responsible for their removal (processing) upon membrane translocation.
The staphylococcal protein export pathways that have been characterized
experimentally or that can be deduced from sequenced genomes are shown
schematically in Fig.
4 and are discussed below. Since these pathways are likely used for the
export of virulence factors to the cell surface and the milieu of the
host, Fig. 4 can be
regarded as a subcellular road map to staphylococcal
pathogenesis.
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FIG. 4. Staphylococcal
pathways to pathogenesis. The figure shows a schematic representation
of a staphylococcal cell with potential pathways for protein sorting
and secretion. (A) Proteins without signal peptides reside in
the cytoplasm. (B) Proteins with one or more
transmembrane-spanning domains can be inserted into the membrane via
the Sec, Tat, or Com pathway. (C) Lipoproteins are exported
via the Sec pathway and are anchored to the membrane after lipid
modification. (D) Proteins with cell wall retention signals
are exported via the Sec, Tat, or Com pathway and retained in the cell
wall via covalent or high-affinity binding to cell wall components.
(E) Exported proteins with a signal peptide and without a
membrane or cell wall retention signal can be secreted into the
extracellular milieu via the various indicated
pathways.
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Components of the Sec Pathway
The most commonly used pathway for bacterial protein transport is
the general secretory (Sec) pathway. Specifically, this pathway is
responsible for the secretion of the majority of the proteins found in
the exoproteome of B. subtilis, which is probably also the
case for most other gram-positive bacteria, including S.
aureus (174).
Unfortunately, there are very few published data available concerning
the Sec pathway of S. aureus, and therefore we have filled in
the current knowledge gaps with data obtained from studies of B.
subtilis or E. coli. Proteins that are exported via the
Sec pathway contain signal peptides with recognition sites for
so-called type I or type II SPases. Notably, type II SPase recognition
sites overlap with the recognition sites for the diacylglyceryl
transferase Lgt. Precursor proteins with a type II SPase recognition
sequence are lipid modified prior to being processed, and the resulting
mature proteins are retained as lipopoteins in the cytoplasmic membrane
via their diacylglyceryl moieties. Furthermore, the Sec-dependent
export of proteins can be divided into the following three stages: (i)
targeting to the membrane translocation machinery by export-specific or
general chaperones, (ii) translocation across the membrane by the Sec
machinery, and (iii) posttranslocational folding and modification. If
the translocated proteins of gram-positive bacteria lack specific
retention signals for the membrane or cell wall, they are secreted into
the growth medium.
Preprotein targeting to the membrane.
In B. subtilis, the
only known secretion-specific chaperone is the signal recognition
particle (SRP), which consists of small cytoplasmic RNA (scRNA), the
histone-like protein HBsU, and the Ffh protein. Ffh and HBsU bind to
different moieties of the scRNA. Studies with E. coli have
shown that upon emergence from the ribosome, the signal peptide of a
nascent secretory protein can be recognized by several cytoplasmic
chaperones and/or targeting factors, such as Ffh or trigger factor (TF)
(55). In contrast to Ffh,
which is required for cotranslational protein export in E.
coli, the cytoplasmic chaperone SecB has mainly been implicated in
posttranslational protein targeting. Notably, however, SecB is absent
from the sequenced gram-positive bacteria, including S. aureus
and B. subtilis. Most likely, ribosome-nascent chain complexes
of S. aureus are thus targeted to the membrane by SRP, which,
by analogy to the case in B. subtilis and E. coli,
will probably involve the SRP receptor FtsY. At the membrane, the
nascent preprotein will be directed to the translocation machinery.
This process is likely stimulated by negatively charged phospholipids
(45), the Sec translocon
(17,
45), and/or the SecA
protein (25). In this
respect, SecA may function not only as the translocation motor (see
below) but also as a chaperone for preprotein targeting
(75). While it has been
shown that Ffh is essential for growth and viability in E.
coli and B. subtilis, this does not seem to be the case
for all bacteria. For example, Ffh, FtsY, and scRNA are not essential
in Streptococcus mutans. In this organism, the SRP is merely
required for growth under stressful conditions, such as low pH
(<pH 5), high salt (3.5% NaCl), or the presence of
H2O2 (0.3 mM). This suggests that SRP has an
important role in the export of proteins to the membrane or cell wall
to protect S. mutans against environmental insults
(71).
For B.
subtilis, it has been proposed that the general chaperone CsaA may
have a role in preprotein targeting to the membrane, similar to SecB of
E. coli. This view is supported by the observation that the
B. subtilis CsaA protein has binding affinity for SecA and
preproteins (126).
However, CsaA is not conserved in S. aureus. Therefore, it
remains to be investigated whether other chaperones with a preprotein
targeting function are present in S.
aureus.
Translocation across the membrane.
As deduced from known genome
sequences, the translocation machinery of S. aureus consists
of several Sec proteins. The mode of action of these proteins has been
studied in great detail in E. coli
(44,
197). After binding of a
preprotein to a SecA dimer, the SecA molecules will bind ATP, resulting
in conformational changes that promote their insertion together with
the preprotein into the membrane-embedded translocation channel.
Subsequent hydrolysis of ATP causes SecA to release the preprotein,
return to its original conformation, and deinsert from the
translocation channel. Repeated cycles of ATP binding and hydrolysis by
SecA, together with the proton motive force, drive further
translocation of the preprotein across the membrane. The translocation
channel is essentially formed by the SecE and SecY proteins, which are
conserved in all bacteria
(189). An additional
nonessential channel component is SecG, which serves to increase the
translocation efficiency. While the SecY proteins of different bacteria
show a relatively high degree of sequence similarity, the SecE and SecG
proteins, though present in all bacteria, are less well conserved.
Specifically, the SecE and SecG proteins in B. subtilis,
S. aureus, and S. epidermidis are considerably
shorter than the equivalent proteins of E. coli. Although SecA
and SecY of S. aureus (referred to here as SecA1 and SecY1)
have not yet been characterized functionally, they are of major
importance for the growth of S. aureus. This was demonstrated
with the help of specific antisense RNAs
(86). Upon secA
antisense induction, a strong growth defect was observed, and
secY antisense induction turned out to be
lethal.
Remarkably, the genome of S. aureus contains a
second set of secA and secY genes, referred to as
secA2 and secY2, respectively. In contrast to the
SecA1 and SecY1 proteins, their homologues are not essential for growth
and viability. It is presently unknown whether SecA2 and SecY2
transport specific proteins across the membrane of S. aureus.
However, it has been shown for other pathogenic gram-positive bacteria
which also possess a second set of SecA and SecY proteins that these
proteins are required for the transport of certain proteins related to
virulence. In Streptococcus gordonii, the export of GspB, a
large cell surface glycoprotein that contributes to platelet binding,
seems to be dependent on the presence of SecA2 and SecY2
(13). This protein
contains large serine-rich repeats, an LPXTG motif for cell wall
anchoring (see below), and a very large signal peptide of 90 amino
acids. In Streptococcus parasanguis, two other proteins, FimA
and Fap1, are known to be secreted via SecA2-dependent membrane
translocation. FimA is a (predicted) lipoprotein which is a major
virulence factor implicated in streptococcal endocarditis. The FimA
homologue in S. aureus is a manganese-binding lipoprotein
(MntA) associated with an ATP-binding cassette (ABC) transporter. Fap1
of S. parasanguis is involved in adhesion to the surfaces of
teeth. Like FimA, Fap1 has a long signal peptide of 50 amino acids,
serine-rich repeats, and an LPXTG motif for cell wall anchoring. To
date, it is not known what determines the difference in specificity of
the SecA1/SecY1 and SecA2/SecY2 translocases. It is also not known
whether the SecA2/SecY2 translocase shares SecE and/or SecG with the
SecA1/SecY1 translocase, whether these translocases function completely
independently from each other, or whether mixed translocases can occur.
Clearly, the secE and secG genes are not duplicated
in S. aureus. The SecE and SecG functions in the SecA2/SecY2
translocase may, however, be performed by the S. aureus
homologues of the Asp4 and Asp5 proteins of S. gordonii, for
which SecE- and SecG-like functions have been proposed
(172).
In E.
coli, the heterotrimeric SecYEG complex is associated with another
heterotrimeric complex composed of the SecD, SecF, and YajC proteins
(138). This complex has
been shown to be involved in the cycling of SecA
(51) and the release of
the translocated protein from the translocation channel
(113). SecD and SecF are
separate but structurally related proteins in most bacteria, including
E. coli. Interestingly, in B. subtilis and S.
aureus, natural gene fusions between the secD and
secF genes are observed. Accordingly, the corresponding SecDF
proteins can be regarded as molecular "Siamese twins"
(20). Unlike SecA, SecY,
and SecE, the SecDF protein of B. subtilis is not essential
for growth and viability, and its role in protein secretion is
presently poorly understood
(20). B. subtilis
secDF mutants showed only a mild secretion defect under conditions
of high-level synthesis of secretory proteins. The known SecDF proteins
have 12 (predicted) transmembrane domains with two large
extracytoplasmic loops, between the first and second transmembrane
segments and between the seventh and eighth transmembrane segments. For
E. coli SecD, it has been shown that small deletions in the
large extracytoplasmic loop result in malfunctioning of the protein,
while the stability of the SecDF-YajC complex is not affected
(138). It has therefore
been proposed that this loop in SecD might provide a protective
structure in which translocated proteins can fold more efficiently. The
large extracytoplasmic loop in SecF has been proposed to interact with
SecY, thereby stabilizing the translocation channel formed by SecYEG.
Homologues of the E. coli YajC protein are present in many
bacteria, including S. aureus and B. subtilis (YrbF),
but their role in protein secretion has not yet been established. It is
presently not known whether the S. aureus SecDF-YajC complex
associates specifically with the SecA1/SecY1 translocase, the
SecA2/SecY2 translocase, or both.
Type I signal peptidases.
Signal peptides of
preproteins are cleaved during or shortly after translocation by an
SPase I or SPase II, depending on the nature of the signal peptide
(180,
187). The B.
subtilis chromosome encodes five type I SPases, named SipS, SipT,
SipU, SipV, and SipW
(176,
178,
186). Two of these, SipS
and SipT, are of major importance for the processing of secretory
preproteins, growth, and viability. In S. aureus, only two
SPase I homologues are present, namely, SpsA and SpsB. The
catalytically active SPase I in S. aureus is SpsB, which is
probably essential for growth and viability
(38). This SPase can be
used to complement an E. coli strain that is temperature
sensitive for preprotein processing. In general, type I SPases
recognize residues at the –1 and –3 positions relative
to the cleavage site
(187). For B.
subtilis, it has been shown that all secretory proteins identified
by proteomics have Ala at the –1 position and that 71% of these
secretory proteins have Ala at the –3 position
(174). In contrast,
various residues are tolerated at the –2 position, including
Ser, Lys, Glu, His, Tyr, Gln, Gly, Phe, Leu, Ala, Asp, Asn, Trp, and
Pro. Interestingly, Bruton et al.
(23) studied the cleavage
sites in substrates of S. aureus SpsB and showed that this
enzyme has a preference for basic residues at the –2 position
and tolerance for hydrophobic residues at this position. However, an
acidic residue at the –2 position resulted in a significantly
reduced rate of processing. The second SPase I homologue of S.
aureus (SpsA) appears to be inactive, since it lacks the catalytic
Ser and Lys residues, which are replaced with Asp and Ser residues,
respectively. The presence of an apparently catalytically inactive SpsA
homologue is a conserved feature of all staphylococci with sequenced
genomes. Notably, in addition to an inactive SpsA homologue, S.
epidermidis contains two SpsB homologues, which show the greatest
similarity to SipS and SipU of B. subtilis. To date, it is not
known whether the inactive SpsA homologues contribute somehow to
protein secretion in these
organisms.
Lipid modification of lipoproteins.
In E. coli, lipid
modification of prolipoproteins involves three sequential steps that
are catalyzed by cytoplasmic membrane-bound proteins. The first step
involves the transfer of a diacylglyceryl group from
phosphatidylglycerol to the sulfhydryl group of the invariant Cys
residue present at the +1 position of the signal peptide
cleavage site in lipoprotein precursors. This step is catalyzed by a
phosphatidyl glycerol diacylglyceryl transferase (Lgt), as shown for
E. coli by Sankaran and Wu
(161). The recognition
sequence for Lgt, which includes the Cys residue that becomes modified
with diacylglyceryl, is known as the lipobox. Lipid modification of the
lipobox Cys residue is necessary for the lipoprotein-specific type II
signal peptidase (LspA) to recognize and cleave the signal peptide of a
prolipoprotein, which represents the second step in lipoprotein
modification. The third step involves the transfer of an
N-acyl group by an N-acyl transferase (Lnt),
resulting in the formation of N-acyl diacylglycerylcysteine at
the N terminus of the mature lipoprotein. Although Lgt and LspA are
present in most, if not all, bacteria, Lnt is present only in
gram-negative bacteria
(180). Like the case for
other gram-positive bacteria, no homologue of Lnt could be detected in
the genomes of S. aureus or S. epidermidis
(169), which suggests
that the lipoproteins of these organisms are not N
acylated.
S. aureus Lgt is a protein of 279 amino acids
that contains a highly conserved HGGLIG motif (residues 97 to 102).
Although the His residue in this motif was shown to be essential for
the catalytic activity of E. coli Lgt
(160), it is not
strictly conserved in all known Lgt proteins. On the other hand, the
strictly conserved Gly at position 103 of E. coli Lgt, which
is equivalent to Gly98 of S. aureus Lgt, is required for the
activity of this protein. Stoll et al.
(169) showed that an
S. aureus lgt mutation has no effect on growth in broth, as
also observed for B. subtilis
(104). Nevertheless, the
absence of Lgt has a considerable effect on the induction of an
inflammatory response. Importantly, lipid modification serves to retain
exported proteins at the membrane-cell wall interface. This is
particularly relevant for gram-positive bacteria, which lack an outer
membrane that represents a retention barrier for exported proteins. In
the absence of Lgt, B. subtilis cells release a variety of
lipoproteins into the extracellular milieu, in the form of both
unmodified precursor proteins and alternatively processed mature
proteins that lack the N-terminal Cys residue
(3). Similarly, the S.
aureus lgt mutation resulted in the shedding of certain abundant
lipoproteins, such as OppA, PrsA, and SitC, into the broth. These
lipoproteins are normally retained in the membrane or cell wall of
S. aureus.
Type II signal peptidase.
As described above, lipoprotein
signal peptides of prolipoproteins are cleaved by type II SPases after
the Cys residue in the lipobox is modified by Lgt. Although B.
subtilis and many other bacteria contain only one copy of the
lspA gene, some organisms, such as S. epidermidis,
Bacillus licheniformis, and Listeria monocytogenes,
contain a second copy. LspA is a membrane protein that spans the
membrane four times, and both its N and C termini face the cytoplasmic
side of the membrane
(178,
188). Six amino acid
residues are important for SPase II activity, of which two Asp residues
form the active site
(178). While processing
of lipoproteins by LspA is essential for growth and viability
for E. coli and other gram-negative bacteria
(202), it is not
essential for B. subtilis
(177) and other
gram-positive bacteria, such as Lactococcus lactis
(190). This suggests
that processing of prolipoproteins is not essential for their
functionality. This view is supported by the fact that PrsA, a
lipoprotein required for correct folding of translocated proteins, is
essential for viability of B. subtilis
(99). In the absence of
LspA, some of the lipoproteins of B. subtilis are processed in
an alternative way by unidentified proteases, and the activity of
unprocessed lipoproteins in lspA mutants is reduced. Also, in
these B. subtilis mutants, secretion of the nonlipoprotein
AmyQ was severely reduced
(177). This reduction
might be the consequence of a malfunction of unmodified PrsA in AmyQ
folding. Although most lspA mutants have been studied in
gram-negative bacteria and a few nonpathogenic gram-positive bacteria
(177,
190), Sander et al.
(159) showed a severely
attenuated phenotype of lspA mutants of the pathogen
Mycobacterium tuberculosis, which implies an important role
for lipoprotein processing by LspA during infection with M.
tuberculosis. In S. aureus, both the lspA and
lgt genes are present as single copies in the genomes of all
six sequenced strains. Interestingly, one of the two LspA homologues in
S. epidermidis (125 amino acids) is considerably shorter than
other known LspA proteins, including its large paralogue (177 amino
acids). This is mainly the result of an additional N-terminal
transmembrane domain in the large LspA proteins. As a result, the short
S. epidermidis LspA protein is predicted to have three
membrane-spanning domains, with the N terminus located on the outside
of the cell, the C terminus located on the inside of the cell, and the
(putative) active-site Asp residues located on the outer surface of the
cytoplasmic membrane.
Signal peptide peptidase.
After translocation and
processing of the preproteins by signal peptidases, the signal peptides
are rapidly degraded by signal peptide peptidases (SPPases). In B.
subtilis, two SPPases, TepA and SppA, are known to be involved in
translocation and processing of preproteins
(21). While TepA is
required for translocation and processing of preproteins, SppA is
required only for efficient processing of preproteins. Remarkably, no
homologues of SppA or TepA were detectable by BLAST searches in the
sequenced genomes of S. aureus and S. epidermidis. As
reported by Meima and van Dijl
(119), L.
lactis contains a protein that shows limited similarity
to TepA of B. subtilis and ClpP of
Caenorhabditis elegans, suggesting that
this protein might be an SPPase analog in L. lactis. In S.
aureus and S. epidermidis, this protein homologue also
seems to be present and is predicted to be a cytoplasmic membrane
protein (our unpublished
observations).
Folding catalysts (PrsA and BdbD).
Proteins that are transported
across the membrane in a Sec-dependent manner emerge at the
extracytoplasmic membrane surface in an unfolded state. These proteins
need to be rapidly and correctly folded into their native and
protease-resistant conformation before they are degraded by proteases
in the cell wall or extracellular environment
(162). An important
folding catalyst in B. subtilis is PrsA, which shows homology
to peptidyl-prolyl cis/trans-isomerases. PrsA is a
lipoprotein (see "Lipoproteins" below) that is
essential for efficient protein secretion and cell viability in B.
subtilis (99,
162). Studies on the
effects of PrsA depletion showed that the relative amounts of
extracellular proteins from PrsA-depleted cells were significantly
reduced (192). No data
have been published on S. aureus mutants lacking PrsA, and it
will be interesting to investigate whether PrsA is also essential for
the viability and virulence of this organism. It has already been shown
that S. aureus lacking Lgt releases an increased amount of
PrsA into the extracellular milieu
(169), which might
indicate that (most) pre-PrsA is not fully functional but is sufficient
for viability. The observation by Stoll et al.
(169) also shows that,
like the case in B. subtilis
(3), unmodified pre-PrsA
is not effectively retained in the cytoplasmic membrane.
Other
proteins that are involved in proper folding of extracellular proteins
in B. subtilis are the membrane proteins BdbC and BdbD, which
are involved in the formation of disulfide bonds. Both proteins have
been shown to be necessary for stabilization of the
membrane- and cell wall-associated pseudopilin
ComGC (118). This
protein, which is required for DNA binding and uptake during natural
competence, contains an intramolecular disulfide bond
(31). Both BdbC and BdbD
are also important for the folding of heterologously produced E.
coli PhoA, which contains two disulfide bonds, into an active and
protease-resistant conformation
(21,
118). Although a
homologue of BdbD (named DsbA) is present in S. aureus, there
is no homologue of BdbC in this organism. The same appears to be true
for S. epidermidis. Nevertheless, measurements of the redox
potential of purified DsbA indicated that this protein can act as an
oxidase, and this view was confirmed by complementation studies with a
dsbA mutant strain of E. coli
(53). The absence of a
BdbC homologue in the staphylococci is remarkable, since B.
subtilis BdbC and BdbD are jointly required for the folding of
ComGC and E. coli PhoA. Notably, all sequenced S.
aureus genomes encode homologues of ComGC, including the Cys
residues that form the disulfide bond in B. subtilis ComGC.
This raises the question of whether ComGC of S. aureus does
indeed contain a disulfide bond and, if so, which protein(s) is
involved in the formation of this disulfide bond. Notably, S.
aureus DsbA was recently shown to be a lipoprotein that does not
seem to contribute to the virulence of this organism, as tested in
mouse and Caenorhabditis elegans models
(53). Furthermore, DsbA
was shown to be dispensable for ß-hemolysin activity, despite
the fact that this protein contains a disulfide bond, which is required
for activity (54).
Therefore, the biological function of DsbA in staphylococci remains to
be elucidated.
Tat Pathway
The twin-arginine translocation (Tat) pathway exists in many
bacteria, archaea, and chloroplasts. This pathway was named after the
consensus double (twin) Arg residues that are present in the signal
peptide. The twin Arg residues are part of a motif that directs
proteins specifically into the Tat pathway. In contrast to the Sec
machinery, where only unfolded proteins are translocated across the
membrane, the Tat machinery is capable of translocating folded
proteins. In gram-negative bacteria, streptomycetes, mycobacteria, and
chloroplasts, an active Tat pathway seems to require three core
components, named TatA, TatB, and TatC
(14,
47,
125,
154,
204). In all
gram-positive bacteria except streptomycetes and Mycobacterium
smegmatis, the Tat pathway involves only TatA and TatC
(47,
204). Recent studies
with E. coli and chloroplasts have resulted in a model that
proposes key roles for TatB-TatC complexes in signal peptide reception
and for TatA-TatB-TatC complexes in preprotein translocation
(2,
36). Interestingly,
certain mutations in E. coli TatA have been shown to allow
this protein to compensate for the absence of TatB
(18). This demonstrates
that TatA is intrinsically bifunctional, which is consistent with the
fact that most gram-positive bacteria lack TatB but have TatA
(90). In B.
subtilis, two minimal TatA-TatC translocases with distinct
specificities are active
(88). While the
constitutively expressed TatAy-TatCy translocase of B.
subtilis is required for secretion of a protein with unknown
function, YwbN, the TatAd-TatCd translocase seems to be expressed only
under conditions of phosphate starvation for secretion of the
phosphodiesterase PhoD
(175,
188). Most other
gram-positive bacteria that have tatA and tatC genes,
including S. aureus, appear to have only one TatA-TatC
translocase. The functionality of the S. aureus Tat
translocase remains to be demonstrated. In contrast to S.
aureus, S. epidermidis seems to lack a Tat
pathway.
Pseudopilin Export (Com) Pathway
In B. subtilis, four proteins, ComGC, ComGD,
ComGE, and ComGG, have been identified as having an N-terminal
pseudopilin-like signal peptide
(174,
175). All four of these
proteins are involved in DNA binding and uptake and are localized in
the membrane and cell wall. It is thought that these proteins form a
pilus-like structure in the cell wall or modify the cell wall to
provide a passage for DNA uptake. Translocation to the extracytoplasmic
membrane surface is possible only when these proteins are processed by
the pseudopilin-specific SPase ComC in B. subtilis
(52). SPases of this type
are bifunctional and catalyze not only signal peptide cleavage but also
methylation of the N terminus of the mature protein
(170). Furthermore,
export and functionality of the four ComG proteins depend on the
integral membrane protein ComGB and the traffic ATPase ComGA, which is
located at the cytoplasmic side of the membrane
(32,
69). Homologues of ComC,
ComGA, ComGB, and ComGC, but not ComGD, ComGE, and ComGG, are present
in the six sequenced S. aureus strains. This suggests that the
Com system of S. aureus is not involved in DNA uptake but is
part of another solute transport
process.
ABC Transporters
Bacteriocins are peptides or proteins that inhibit the
growth of other bacteria. Most of the characterized bacteriocins can be
divided into several classes, depending on specific posttranslational
modifications, the presence and processing of particular leader
peptides, and the machinery for export from the cytoplasm. A
well-described class of bacteriocins is formed by the lantibiotics.
Members of this class are composed of short peptides that contain
posttranslationally modified amino acids, such as lanthionine and
ß-methyllanthionine
(117). The production of
bacteriocins in S. aureus has been described for various
strains. S. aureus C55 produces the two lantibiotics
C55
and C55ß
(129). These
lantibiotics are both encoded by a 32-kb plasmid, which is readily lost
upon growth at elevated temperatures. C55 and C55ß
showed antimicrobial activity towards other S. aureus strains
and Micrococcus luteus but not towards S.
epidermidis. Furthermore, the nonlantibiotics BacR1
(40), aureocin A53
(134), and aureocin A70
(132,
133) have been
identified as bacteriocins with activity against a broad range of
bacteria. The genes for both aureocins are located on a
plasmid that is present in S. aureus strains isolated from
milk. By analogy with the well-described bacteriocin export machineries
of other organisms (73,
145), it can be
anticipated that all of the aforementioned bacteriocins are exported to
the external staphylococcal milieu by dedicated ABC transporters.
However, no experimental evidence for this assumption has been
published for S. aureus. Notably, it has been demonstrated
that secretion of the lantibiotics epidermin and gallidermin of S.
epidermidis Tü3298 and Staphylococcus gallinarum,
respectively, is facilitated by so-called one-component ABC
transporters. Specifically, the ABC transporter GdmT has been
implicated in the transport of these lantibiotics
(145).
Holins
Holins are dedicated export systems for peptidoglycan-degrading
endolysins that have been implicated in the programmed cell death of
bacteria. These exporters, which are composed of homo-oligomeric
complexes, can be subdivided into two classes, depending on the number
of transmembrane segments. While class I holin subunits have three
transmembrane segments, class II holin subunits have two transmembrane
segments (206). In
S. aureus, the lrg and cid operons are
involved in murein hydrolase activity and antibiotic tolerance
(66,
153). A disrupted
lrg operon leads to an increase in murein hydrolase activity
and a decrease in penicillin tolerance, and a disrupted cid
operon leads to a decrease in murein hydrolase activity and an increase
in penicillin tolerance. It is still unclear how the CidA and LrgA
proteins are involved in these mechanisms, but these proteins display
significant similarity to the bacteriaphage holin protein family,
suggesting that they have a role in protein export. It has therefore
been proposed that the CidA and LrgA proteins act on murein hydrolase
activity and antibiotic tolerance in a manner analogous to that of
holins and antiholins, respectively
(11,
153). Sequence
similarity searches showed that the genes for LrgA and CidA are
conserved in the six sequenced S. aureus strains as well as in
S. epidermidis and B. subtilis. Notably, none of the
three holins of B. subtilis were shown to be involved in the
secretion of proteins to the extracellular milieu
(174,
200).
ESAT-6 Pathway
The ESAT-6 secretion pathway was first described for
M. tuberculosis. It has been proposed that at least two
virulence factors, ESAT-6 (early secreted antigen target, 6 kDa) and
CFP-10 (culture filtrate protein, 10 kDa), are secreted via this
pathway in a Sec-independent manner
(16,
166). Since this pathway
was discovered in mycobacteria, it is also known as the Snm pathway
(secretion in mycobacteria)
(37). The genes for
ESAT-6 and CFP-10 are located in conserved gene clusters, which also
encode proteins with domains that are conserved in FtsK- and
SpoIIIE-like transporters. These conserved FtsK/SpoIIIE domains have
been termed FSDs (26). In
other gram-positive bacteria, including S. aureus,
B. subtilis, Bacillus anthracis,
Clostridium acetobutylicum, and L.
monocytogenes, homologues of ESAT-6 have been
identified (140). The
genes for these ESAT-6 homologues are also found in gene clusters that
contain at least one gene for a membrane protein with an FSD. In S.
aureus, two proteins, named EsxA and EsxB, have been identified
that seem to be secreted via the ESAT-6 pathway
(26). The esxA
and esxB genes are part of a cluster containing six other
genes for proteins that have been implicated in the translocation of
EsxA and EsxB. These include the EsaB and EsaC proteins, with a
predicted cytoplasmic location, as well as the predicted membrane
proteins EsaA, EssA, EssB, and EssC, among which EssC contains an FSD.
Mutations in essA, essB, or essC result in a
loss of EsxA and EsxB production, which may be related to inhibition of
the synthesis of these proteins or their folding into a
protease-resistant conformation. All sequenced S. aureus
strains contain this cluster of esa, ess, and
esx genes, but it seems to be absent from S.
epidermidis. Interestingly, the genes for EsxB and EsaC appear to
be absent from the S. aureus MRSA252 strain. This implies that
the ESAT-6 machinery of this strain may be required for the transport
of only EsxA and perhaps a few other unidentified proteins. If so, EsaC
would be dispensable for an active ESAT-6 pathway and might be
specifically involved in the export of EsxB. Alternatively, the ESAT-6
pathway could be inactive in the S. aureus MRSA252 strain due
to the absence of EsaC.
Lysis
Various studies have shown that certain proteins with typical
cytoplasmic functions and without known signals for protein secretion
can nevertheless be detected in the extracellular proteomes of
different bacteria
(174). Notably, many of
these proteins, such as catalase, elongation factor G, enolase,
glyceraldehyde-3-phosphate dehydrogenase, GroEL, and superoxide
dismutase, are among the most highly abundant cytoplasmic proteins.
This makes it likely that they are detectable in the extracellular
proteome due to cell lysis. Perhaps such proteins are more resistant to
extracytoplasmic degradation than are other proteins that are
simultaneously released by lysis. However, the possibility that the
extracellular localization of typical cytoplasmic proteins is due to
the activity of as yet unidentified export pathways cannot be excluded.
Clearly, until recently this possibility did still apply for the EsxA
and EsxB proteins, which are now known to be exported via the ESAT-6
pathway. A clear indication that the presence of certain
"cytoplasmic" proteins in the extracytoplasmic milieu
of bacteria may relate to specific export processes was provided by
Boël and coworkers
(19), who showed that
2-phosphoglycerate-dependent automodification of enolase is necessary
for its export from the
cytoplasm.
|
PROPERTIES OF STAPHYLOCOCCAL SIGNAL PEPTIDES AND CELL RETENTION SIGNALS
|
|---|
Signal Peptides
All proteins that have to be
transported from the cytoplasm across the membrane to the
extracytoplasmic compartments of the cell, or the extracellular milieu,
need to contain a specific sorting signal for their distinction from
resident proteins of the cytoplasm. The known bacterial sorting signals
for protein export from the cytoplasm are signal peptides
(195). These signal
peptides can be classified by the transport and modification pathways
into which they direct proteins. Presently, four different bacterial
signal peptides are recognized that share a common architecture but
differ in the details (Fig.
5). Two of these direct proteins into the widely used Sec pathway,
including the secretory (Sec-type) signal peptides and the lipoprotein
signal peptides. Proteins with Sec-type or lipoprotein signal peptides
are processed by different SPases (type I and type II SPases,
respectively) and are targeted to different destinations. In S.
aureus, proteins with Sec-type signal peptides are processed by
the type I SPase SpsB and are targeted to the cell wall or
extracellular milieu. Proteins with a lipoprotein signal peptide are
lipid modified by Lgt prior to being processed by the type II SPase
LspA. In principle, these lipoproteins are retained at the
membrane-cell wall interface, but they can be liberated from this
compartment by proteolytic removal of the N-terminal Cys that contains
the diacylglyceryl moiety
(3). Proteins with
twin-arginine (RR) signal peptides appear to be processed by type I
SPases, at least in B. subtilis, and are targeted to the cell
wall or extracellular milieu
(174). Proteins with a
pseudopilin signal peptide are processed by the
pseudopilin signal peptidase ComC and most likely
are localized to the cytoplasmic membrane and the cell wall. Finally,
bacteriocins contain a completely different sorting and modification
signal that is usually called the leader peptide. The known leader
peptides show no resemblance to the aforementioned signal peptides. The
export of bacteriocins via ABC transporters results in their secretion
into the extracellular milieu
(121,
164).
View larger version (20K):
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|
FIG. 5. General
properties and classification of S. aureus signal peptides.
Signal peptide properties are based on SPase cleavage sites and the
export pathways by which the preproteins are exported. Predicted signal
peptides (144) were
divided into the following five distinct classes: secretory (Sec-type)
signal peptides, twin-arginine (RR/KR) signal peptides, lipoprotein
signal peptides, pseudopilin-like signal peptides, and bacteriocin
leader peptides. Most of these signal peptides have a tripartite
structure, with a positively charged N domain (N) containing
lysine and/or arginine residues (indicated by plus signs), a
hydrophobic H domain (H, indicated by a black box), and a C domain
(C) that specifies the cleavage site for a specific SPase.
Where appropriate, the most frequently occurring amino acid residues at
particular positions in the signal peptide or mature protein are
indicated. Also, the numbers of signal peptides identified for each
class and the respective SPase are
indicated.
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Sec-type,
lipoprotein, and RR signal peptides contain three distinguishable
domains, the N, H, and C domains. The N-terminal domain contains
positively charged amino acids, which are thought to interact with the
secretion machinery and/or negatively charged phospholipids in the
membrane. The H domain is formed by a stretch of hydrophobic amino
acids which facilitate membrane insertion. Helix-breaking residues in
the middle of the H domain may facilitate H domain looping during
membrane insertion and translocation of the precursor protein. The
subsequent unlooping of the H domain would display the SPase
recognition and cleavage site at the extracytoplasmic membrane surface,
where the catalytic domains of type I and type II SPases are localized
(187). Helix-breaking
residues just before the SPase recognition and cleavage site would
facilitate precursor processing by SPase I or II. In fact, these
helix-breaking residues and the SPase cleavage site, respectively,
define the beginning and the end of the C domain. Notably, the C
domains of pseudopilin signal peptides are located between the N and H
domains (32,
33,
106,
148). Accordingly,
processing by pseudopilin-specific SPases, such as 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 walls of gram-positive bacteria
have signal peptides, proteins without known export signals can also be
found at these locations. The relative number of proteins without known
signal peptides seems to vary for each organism. While these numbers
are relatively low for B. subtilis and S. aureus,
they are high for group A streptococcus and M. tuberculosis
(174). 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 ESAT-6 pathway. Although the precise export signal
in proteins secreted via the ESAT-6 pathway has not yet been defined, a
WXG motif is shared by these proteins and may serve a function in
protein targeting (140).
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 for predicting whether a
given protein contains some type of sorting signal or SPase cleavage
site. The programs that we used in this and other studies were
SignalP-NN and SignalP-HMM, version 2.0
(136), LipoP, version
1.0 (94), PrediSi
(76), and Phobius
(95). These programs were
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
(41), 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 and hidden Markov
model algorithms. Version 2.0 of the SignalP program was preferred
above version 3.0 (12)
for our signal peptide predictions for 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
(179). Specifically, the
hidden Markov model in SignalP 2.0 assigns a probability score to each
amino acid of a potential signal peptide and indicates whether it is
likely to belong to the N, H, or C domain. Proteins with no detectable
N, H, or 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 most likely are integral membrane
proteins. All proteins with a predicted C-terminal 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
(i) signal peptides with distinctive N, H, and C domains, (ii) no
additional transmembrane domains, and (iii) 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://npsa-pbil.ibcp.fr),
as previously described
(179). To define the
core exoproteome and variant exoproteome of the S. aureus
strains, the sets of proteins with predicted signal peptides were used
in multiple BLAST searches with the freeware BLASTall from the NCBI.
The output was then filtered using Genome2D
(10).
Secretory (Sec-type) signal peptides.
Proteomics-based data sets of membrane, cell wall,
and extracellular proteins were extremely valuable for a recent
verification of signal peptide predictions for B. subtilis
(179). Such data sets
are now becoming available for S. aureus, as exemplified by
studies on the membrane and cell wall proteomes of S. aureus
Phillips (127) and the
extracellular proteomes of S. aureus strains derived from the
recently sequenced NCTC8325 and COL strains
(208,
209) (Fig.
2). Additionally, the
extracellular proteomes of several clinical S. aureus isolates
have been analyzed (Fig.
2). The membrane, cell
wall, and extracellular proteins of S. aureus that have been
identified by proteomics, involving 2D-PAGE and subsequent
mass spectrometry, are listed
in Tables
4 and
5.These tables also show the –3-to-+1 amino acid
sequences of the respective signal peptidase cleavage sites, if
present.
View this table:
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TABLE 5. Identified
proteins in extracellular proteomes of various S. aureus
strains without known signal peptides
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Based on the proteomics data for membrane and
extracellular proteins of B. subtilis, the optimized
–3-to-+1 pattern (AVSTI)-(SEKYHQFLDGPW)-A-(AQVEKDFHLNS)
for signal peptide recognition and cleavage by type I SPases of this
organism was identified
(179). SPase cleavage
occurs C-terminal of the invariant Ala residue at the –1
position. The residues in parentheses in the pattern are listed in the
order of frequency, with the most frequently identified residue at each
position being placed in the first position. By comparing the predicted
SPase recognition and cleavage sites for signal peptides of
proteomically identified extracellular proteins of S. aureus
(Table 4), we defined the
–3-to-+1 pattern (AVS)-(KHNDQSYEGLR)-A-(AESDIKL) for
productive recognition and cleavage by the type I SPase SpsB. Compared
to the equivalent pattern in B. subtilis, it is interesting
that the frequencies of certain residues at the –3, –2,
and +1 positions differ, as reflected by the order of frequency
with which they are listed in the pattern. Moreover, Asn can be present
at the –2 position, while Ile is accepted at the +1
position. These residues are found at the –2 and +1
positions of certain serine proteases, hemolysins, immunoglobulin G
(IgG) binding protein A, and aureolysin (Table
4). It should also be
noted that compared to the optimized SPase recognition pattern in
B. su
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