Excretion of cytosolic proteins (ECP) in bacteria.

G Model

ARTICLE IN PRESS

IJMM-50923; No. of Pages 8

International Journal of Medical Microbiology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Medical Microbiology journal homepage: www.elsevier.com/locate/ijmm

Mini Review

Excretion of cytosolic proteins (ECP) in bacteria Friedrich Götz ∗ , Wenqi Yu, Linda Dube, Marcel Prax, Patrick Ebner Microbial Genetics, Interfaculty Institute for Microbiology and Infection Medicine Tübingen (IMIT), University of Tübingen, 72076 Tübingen, Germany

a r t i c l e

i n f o

Keywords: Cytoplasmic membrane (CM) Excretion of cytosolic proteins (ECP) Secretome Bacteria Bacillus Staphylococcus Streptococcus

a b s t r a c t Excretion of cytosolic proteins (ECP) has been reported in bacteria and eukaryotes. As none of the classical signal peptide (SP) dependent or SP-independent pathways could be associated with ECP, it has been also referred to as ‘non-classical protein export’. When microbiologists first began to study this subject in 1990, mainly singular cytoplasmic proteins were investigated, such as GAPDH at the cell surface and in the supernatant of pathogenic streptococci or glutamine synthetase (GlnA) as a major extracellular protein in pathogenic mycobacteria. Later, with the rising popularity of proteomics, it became obvious that the secretome of most bacteria contained a copious amount of cytosolic proteins. In particular ancient proteins such as glycolytic enzymes, chaperones, translation factors or enzymes involved in detoxification of reactive oxygen were found in the supernatants. As the excreted proteins do not possess a common motive, the most widespread opinion is that ECP is due to cell lysis. Indeed, upregulation of autolysins or distortion of the murein structure increased ECP, suggesting that enhanced ECP is some sort of survival strategy to counteract osmotic stress. However, in the meantime there are mounting evidences and hints that speak against cell lysis as a primary mechanism for ECP. Very likely, ECP belongs to the normal life cycle of bacteria and involves a programmed process. This review provides a brief overview of the ‘non-classical protein export’. © 2015 Published by Elsevier GmbH.

Introduction The cytoplasmic membrane (CM) is the most dynamic structure of bacterial cells. Its main function is the formation of a selective permeability barrier that regulates the passage of substances into and out of the cell. It allows the undirected transition of water and uncharged molecules up to MW of about 100 Da, but does not allow the passage of larger molecules or any charged substances except by means of special transport systems. Proteome analysis of the Staphylococcus aureus membrane revealed that exponential growing cells contain at least 270 integral proteins (Becher et al., 2009) and approximately 30% of the encoded proteome (±2600 proteins) could be secreted (Kusch and Engelmann, 2014). Normally, proteins that are translocated over the cytoplasmic membrane are distinguished by appropriate signal peptides and are translocated by defined transport systems. However, there is

Abbreviations: ECP, excretion of cytosolic proteins; GAPDH, glyceraldehyde-3phosphate dehydrogenase; PGN, peptidoglycan; SP, signal peptide; S., Staphylococcus; Sec, major secretion system; WTA, wall teichoic acid. ∗ Corresponding author at: Mikrobielle Genetik, Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany. Tel.: +49 7071 2974636; fax: +49 7071 295937. E-mail address: [email protected] (F. Götz).

an increasing number of typical cytosolic proteins described which do not have a signal sequence and are still found extracellularly. It is hotly debated whether the release of such proteins is due to cell lysis or whether they are exported by a so far unknown mechanism (Wang et al., 2013). In eukaryotes such proteins were secreted distinct from the classical ER-Golgi route and the pathway was referred to as ‘nonclassical protein export’ (Muesch et al., 1990). At least four distinct types of nonclassical export were distinguished (Nickel, 2003): (a) for IL-1␤, En2 (transcription factor engrailed homeoprotein isoform 2) and HMGB1 (intra-nuclear factor that mediates the assembly of site-specific DNA-binding proteins within chromatin), export involves import into intracellular vesicles, which are probably endosomal sub-compartments (Rubartelli et al., 1992, 1990); (b) FGF-1 and 2 (fibroblast growth factor 1 and 2) probably reach the extracellular space by direct translocation across the plasma membrane; (c) the Leishmania cell surface protein HASPB is also translocated directly across the plasma membrane via dual acylation at the N-terminus and using a flip-flop mechanism to localize the protein in the outer leaflet of the plasma membrane; (d) the final postulated pathway of non-classical export involves exosomal vesicles formed on the outer surface of the cell in a process known as membrane blebbing. Exosomes are labile structures that release their contents into extracellular space. It has been suggested that this pathway may be used by galectins (Nickel, 2003). One possible

http://dx.doi.org/10.1016/j.ijmm.2014.12.021 1438-4221/© 2015 Published by Elsevier GmbH.

Please cite this article in press as: Götz, F., et al., Excretion of cytosolic proteins (ECP) in bacteria. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2014.12.021

G Model IJMM-50923; No. of Pages 8 2

ARTICLE IN PRESS F. Götz et al. / International Journal of Medical Microbiology xxx (2015) xxx–xxx

benefit of the ‘non-classical protein export’ in eukaryotes could be that this export system is a way to clear unfolded proteins from the cytoplasm (Sloan et al., 1994). For ECP in eukaryotes, the term ‘moonlighting proteins’ has also been coined (Jeffery, 1999). Moonlighting refers to a single protein that has multiple functions. For example the mammalian thymidine phosphorylase catalyzes the intracellular dephosphorylation of thymidine but acts outside as a platelet-derived endothelial cell growth factor, which stimulates endothelial cell growth and chemotaxis (Jeffery, 1999). Most moonlighting proteins represent evolutionarily conserved (ancient) enzymes. The glycolytic enzymes, GAPDH and enolase and the cell stress proteins chaperonin 60, Hsp70 and peptidyl prolyl isomerase, are among the most common of the bacterial moonlighting proteins. They play a role in bacterial virulence, since they are involved in adhesion and modulation of cell signaling processes. An overview of moonlighting proteins deriving from bacteria and their role in bacterial virulence is given by (Henderson and Martin, 2013).

Sibbald et al., 2006; Tjalsma et al., 2004; Trost et al., 2005; Xia et al., 2008). Proteome analyses were carried out in staphylococci to study the expression of cytosolic proteins under biofilm and anoxic growth conditions (Fuchs et al., 2007; Resch et al., 2006), of growing and non-growing cells (Kohler et al., 2005) or of global regulator mutants such as agr, sigmaB and clpC (Chatterjee et al., 2009; Ziebandt et al., 2004, 2001). Only later, secretome studies were also conducted to show that a number of typical cytosolic proteins were present in the culture supernatants of B. subtilis and S. aureus (Sibbald et al., 2010; Tjalsma et al., 2004; Ziebandt et al., 2004). A recent study compared the exoproteomes of three different S. epidermidis strains (Siljamaki et al., 2014). Approximately 80% of the proteins identified in their analysis belonged to the cytoplasmic fraction. Interestingly, strain specificity with respect to the protein composition could be made, hypothesizing a possible correlation of pathogenicity and the level of ECP. Only certain cytosolic proteins are excreted

Early observation of excretion of cytosolic proteins (ECP) in bacteria One of the first reports that typical cytosolic proteins are found on bacterial cell surface came from Vincent Fischetti’s group. They found that the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is present in large amounts on the cell surface of pathogenic streptococcal groups (Pancholi and Fischetti, 1992) and also in the supernatant of several bacteria, fungi and even protozoans (Pancholi and Chhatwal, 2003). Interestingly, this GAPDH functions also as an ADP-ribosylating enzyme catalyzing the NADdependent, auto-ADP-ribosylation at its cysteine residue via a thioglycosidic linkage (Pancholi and Fischetti, 1993), a modification that is stimulated by nitric oxide (NO). GAPDH is a very ‘sticky’ protein as it binds to various human proteins, including plasmin(ogen) (D’Costa and Boyle, 2000; Lottenberg et al., 1992; Winram and Lottenberg, 1996), lysozyme, myosin, actin, fibronectin (Pancholi and Fischetti, 1992) and PAR/CD87 on pharyngeal cells (Jin et al., 2005). GAPDH also stimulates B-lymphocytes and induces an early IL-10 production that facilitates host colonization (Madureira et al., 2007). In group B streptococci (GBS) GAPDH acts as an inducer of apoptosis of murine macrophages (Oliveira et al., 2012). Moreover, in enterohemorrhagic and enteropathogenic E. coli GAPDH was exposed on surface where it binds to human plasminogen and fibrinogen, suggesting a role in pathogenesis (Egea et al., 2007). Besides GAPDH, a number of other cytosolic proteins have been found on the surface or to be excreted, such as ␣-enolase (Pancholi and Fischetti, 1998), glucose-6-phosphate isomerase (Hughes et al., 2002), glutamine synthetase (Suvorov et al., 1997), ornithine carbamoyltransferase (Hughes et al., 2002), fibrinogenbinding protein A of Listeria monocytogenes (Dramsi et al., 2004) or Fbp54 of Streptococcus pyogenes (Courtney et al., 1996). All these proteins do not possess a traditional signal peptide and appear to be typical ‘moonlighting’ proteins with different intra- and extracellular activities.

Secretome analysis revealed much more cytosolic proteins Release of typical cytosolic proteins into the culture supernatant is not restricted to individual species; as it has been observed in Gram-positive and Gram-negative bacteria such as staphylococci, streptococci, Bacillus subtilis, Listeria monocytogenes or E. coli. In particular, glycolytic enzymes, chaperones, translation factors or enzymes involved in detoxification of reactive oxygen species were found in the supernatants by secretome analysis (Li et al., 2004;

A comparative proteomic analysis of cytosolic and culture supernatant proteins demonstrated that in mid-exponential culture of S. aureus SA113, quite a number of cytosolic proteins were found in the secretome, while many other cytosolic proteins were missing (Pasztor et al., 2010). Using a 2D-PAGE gel, cytosolic proteins of SA113 were separated and proteins found also in the secretome were labeled blue, while those solely found in the cytoplasm were labeled red (Fig. 1, adapted from Pasztor et al., 2010). Notably, highly expressed cytosolic proteins such as Fhs, GuaB, SA0802 (Ndh-2), EF-TS, GlnA, PdhD, SucC were not found in the secretome. Table 1 lists excreted and non-excreted proteins. Two conclusions can be drawn from this observation: (a) there is no correlation between the quantity and the excretion level of cytosolic proteins and (b) a specific selection procedure in the excretion of cytosolic proteins has to exist. In this context an interesting observation has been made with the glycolytic enolase of E. coli. Like in other bacteria, the enolase from E. coli is also excreted. The enzymatic reaction involves a transient covalent binding of the substrate 2-phosphoglycerate (2-PG) to the active site Lys341. Replacement of Lys341 with other amino acids not only prevented the automodification but also the export of enolase (Boel et al., 2004). One of the enolase mutants (K341E) was almost as active as the wild-type enzyme and still was not exported, suggesting that the enolase export was correlated with the loss of modification and not the loss of glycolytic activity. This is one of the strongest examples that excretion of cytosolic proteins is selective; this selectivity speaks against the indiscriminate excretion by cell lysis. The unsolved question is however, via which transport system cytosolic proteins could be excreted. For this reason the known transport systems in S. aureus are briefly addressed. Specific protein transport systems in Gram-positive bacteria The main protein/peptide transport systems can be grouped in signal peptide dependent and signal peptide independent systems (Fig. 2). The signal peptide dependent systems can be subdivided into the Sec translocation system, representing the major secretion system, and the twin-arginine translocation system (Tat). Proteins are translocated through this pathway in a more or less unfolded state (Driessen and Nouwen, 2008) and are targeted to the Sec translocon via their N-terminal signal peptide (von Heijne, 1990). The Sec translocon is also used for the translocation of lipoproteins, which are distinguished by their own signal peptide (Babu et al., 2006). To answer the question whether the Sec pathway is involved in ECP, a secA-temperature sensitive B. subtilis mutant was used to


G Model IJMM-50923; No. of Pages 8


3

Fig. 1. 2D-PAGE of cytosolic proteins of S. aureus SA113. Blue labeled protein spots represent proteins that are also excreted; red labeled protein spots mark proteins that were not found in the secretome. The intensity of the spots is an indication for the abundance in the cytoplasm; many highly abundant proteins were not found excreted, suggesting that there is no correlation between abundance and excretion. Adapted from Pasztor et al. (2010).

Fig. 2. Overview of protein/peptide export systems in Staphylococcus. The upper half shows the signal peptide (SP) dependent translocation systems. The lower part illustrates the SP-independent export systems. Cell lysis by prophage-induction and increased autolysin activity are no real transport systems; it is unlikely that they account for ECP under normal conditions.

disrupt the translocation machinery. Despite the downregulation of the Sec system, they still identified several cytosolic proteins in the supernatant of B. subtilis, e.g. Gap, SodA and KatA, which suggests that these proteins were excreted in a Sec-independent manner (Hirose et al., 2000). In M. tuberculosis, there are two secA genes (secA1 and secA2). SecA1 is the essential ‘housekeeping’ SecA protein whereas SecA2 is an accessory secretion factor. It turned out that SecA2 contributes not only to the pathogenicity of M. tuberculosis but it is also necessary for the excretion of superoxide-dismutase (SodA)

and catalase-peroxidase (KatG), both involved in the detoxification of reactive oxygen intermediates (ROI). SecA2 appears to be part of a specialized secretion system involved in signal peptide independent secretion (Braunstein et al., 2003). In Listeria monocytogenes, SecA2 is involved in secretion of murein hydrolases such as the p60 and N-acetylmuramidase (NamA) autolysins (Lenz et al., 2003). S. aureus contains an accessory Sec2 pathway too; here however, it appears that the SecA2 pathway is only required for the export of SraP, a cell wall-anchored, glycosylated, serine-rich platelet adhesin (Li et al., 2014; Siboo et al., 2008).




4

Table 1 Cytosolic proteins of Staphylococcus aureus SA113 present and not present in the secretome. Present in secretome ClpC DnaK EF-G EF-TU Eno FbaA GapA GlpD GroEL IleS LeuS PdhB PdhC PtsI PykA RpoB RpsB OdhA SA0829 TrxB

Putative ATPase/chaperone Chaperone protein DnaK (Hsp70) homolog Elongation factor EF-G Translational elongation factor TU homolog 2-Phospho-d-glycerate hydrolase (enolase) homolog Fructose-bisphosphate aldolase Glyceraldehyde-3-phosphate dehydrogenase Aerobic glycerol-3-phosphate dehydrogenase homolog GroEL protein homolog Putative isoleucyl-tRNA synthetase Putative leucyl-tRNA synthetase Pyruvate dehydrogenase E1 component beta subunit homolog Dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex E2 Phosphoryl transfer from PEP to enzyme I Pyruvate kinase homolog RNA polymerase beta chain homolog 30S ribosomal protein S2 2-Oxoglutarate dehydrogenase subunit Small hypothetical protein Thioredoxin reductase

Not present in secretome AckA AhpC AldA EF-TS Fhs GlnA GuaB KatA PdhD Pta RocD RpsA SA0224 SA0508 SA0707 SA0802 SodA SucC Tig

Acetate kinase homolog Alkyl hydroperoxide reductase subunit C Putative aldehyde dehydrogenase Putative elongation factor TS Formate-tetrahydrofolate ligase homolog Glutamine synthase Putative inositol-monophosphate dehydrogenase Catalase Dihydrolipoamide dehydrogenase component of pyruvate dehydrogenase E3 Phosphotransacetylase Ornithine aminotransferase Small ribosomal protein 3-Hydroxyacyl-CoA dehydrogenase 2-Amino-3-ketobutyrate CoA ligase Ribosomal subunit interface protein (yfiA) Ndh-2, NADH dehydrogenase Manganese superoxide dismutase Putative succinyl-CoA synthetase, beta chain Trigger factor homolog

Proteins that are translocated via the twin-arginine translocation system (Tat) carry a characteristic signal peptide, including two consecutive arginine residues that are essential for recognition by the Tat translocon (Berks et al., 2005; Jongbloed et al., 2004). The Tat pathway operates independently from the Sec pathway and translocates proteins across the cytoplasmic membrane in a fully folded conformation; many of these proteins are even complexed with cofactors (Palmer and Berks, 2012). In Staphylococcus, the Tat pathway, composed of TatA and TatC, is present only in some species (Biswas et al., 2009); TatC, an integral membrane protein, is the central component of the Tat pathway capturing substrate proteins by binding their signal peptides. Subsequently, TatC recruits TatA family proteins to form the active translocation complex. In S. aureus only the iron-dependent peroxidase (FepB) is translocated by this system (Biswas et al., 2009). It is unlikely that the Sec or Tat-secretion systems, which mainly translocate proteins with specific signal sequences at the N-terminus, play a major role in non-classical protein excretion. Beside these two secretion systems, there are a few protein/peptide translocation systems that do not require a specific signal sequence in Gram-positive bacteria. These systems could be involved in excretion of cytosolic proteins.

The Holin–Antiholin system in S. aureus There is a family of programmed cell death effectors of which S. aureus Cid and Lrg proteins are the prototypical members (Ranjit et al., 2011). This system comprises membrane-associated proteins CidA, CidB, LrgA, and LrgB encoded by the cid and lrg operons respectively (Rice and Bayles, 2003). CidA is a holin-like protein with a positive effect on murein hydrolase activity, and LrgA is an antiholin-like protein abrogating the effect of these enzymes. It is thought that their biochemical properties of hole formation and murein hydrolase activation are similar to those of the holin/antiholin system of bacteriophage ␭. Interestingly, the last gene of the cid operon, cidC, encodes for a pyruvate oxidase which is involved in generation of acetic acid that contributes to cell death and lysis of high-glucose stationary phase cultures (Patton et al., 2005). The S. aureus holin–antiholin system does not contribute to the release of cytosolic proteins, as lrgAB- and cidAB-deletion mutants showed now significant difference in release of GAPDH (Pasztor, 2011).

ESAT-6 or type seven secretion system T7SS In Mycobacterium tuberculosis and M. marinum, a novel ESAT6 secretion complex-1 (ESX-1 or type VII secretion system) has been identified which enables secretion of two small proteins, the early-secreted antigen EsxA (6 kDa) and the culture filtrate protein EsxB (10 kDa), both are suggested to be involved in host membrane lysis and cell-to-cell spread (Abdallah et al., 2007). The translocation of ESAT-6 from the phagolysosome to the cytosol depends on its C-terminal domain and is crucial for its role in virulence (Houben et al., 2012). Homologues of ESAT-6 are also present in other Gram-positive bacteria, including Bacillus, Staphylococcus or Clostridium (Pallen, 2002). S. aureus, for example, secretes EsxA, EsxB and EsaC via the transport system composed of EssA, EssB and EssC (Burts et al., 2008). Like in the mycobacterial counterpart, the C-terminal domain is important for secretion (Anderson et al., 2013). The translocation protein, EssB, a 52-kDa integral membrane protein; the 22-kDa C-terminal fragment is predicted to be on the trans-side of the cytoplasmic membrane, while the 25 kDa N-terminal domain (EssB-N) is on the inside of the cytoplasmic membrane (Sundaramoorthy et al., 2008). EssB resembles a membrane inserted pseudokinase (Zoltner et al., 2013). The translocated EsxA interferes with the host cell apoptotic pathways and together with EsxB, mediates the release of S. aureus from the host cell (Korea et al., 2014). A potential contribution of ESAT-6 in ECP has not been investigated so far.

ABC transporters in Gram-positive bacteria ATP-binding cassette (ABC) transporters are widespread transport systems in Gram-positive bacteria and are involved in several tasks, e.g. efflux of antibiotics, export of small peptides or uptake of nutrients. Some examples are described for S. aureus. Selfprotection of the epidermin-producing strain S. epidermidis Tü3298 against the Lipid II-binding lantibiotic epidermin is mediated by an ABC transporter composed of the EpiF, EpiE and EpiG proteins (Hille et al., 2001; Peschel and Götz, 1996). The EpiFEG transporter works by expelling the lantibiotic from the cytoplasmic membrane into the surrounding medium (Otto et al., 1998). vraFG of S. aureus encodes an ABC transporter that is responsible for increased vancomycin and polymyxin B resistance, most likely by pumping out the antibiotics (Meehl et al., 2007); and vraFG expression is controlled by the global regulator GraRS (Herbert et al., 2007). The interaction between these two systems, GraRS and VraFG, is




required for cationic antimicrobial peptide sensing and resistance in S. aureus (Falord et al., 2012). Phenol-soluble modulins (PSMs: PSM␣, PSM␤ and ␦-toxin) are a family of amphipathic, ␣-helical peptides found in S. aureus, S. epidermidis, and other staphylococci (Cheung et al., 2014; Joo et al., 2011; Mehlin et al., 1999; Otto et al., 2004). Several PSMs have cytolytic activities, for example, toward human neutrophils and other human cells. Based on their surfactant-like properties, PSMs have also been proposed to impact biofilm maturation and detachment (Periasamy et al., 2012; Vuong et al., 2000). PSM gene expression is positively regulated by the global accessory regulator system, Agr, by direct binding of the AgrA response regulator to the corresponding promoters (Queck et al., 2008). Recently, it has been found that PSMs are secreted by the four-component ABC transporter Pmt (PSM transporter) (Chatterjee et al., 2013). The Pmt transporter protects the PSM producer from the antimicrobial activity of the expressed PSMs that might attack the cytoplasmic membrane from inside if they are not efficiently secreted. Interestingly, PSMs are secreted with their formyl-methionine group and can therefore be sensed by the human formyl peptide receptor 2 (Kretschmer et al., 2010). A possible role of PSMs in ECP is conceivable, as they have detergent-like character and are membrane active. Furthermore, it cannot be excluded that the Pmt-ABC transporter is unspecific enough to release also certain cytosolic proteins attached to PSMs in a random manner. One should not rule out that any of the signal peptide independent protein/peptide translocation systems is at least partially involved in the excretion of cytosolic proteins. Currently, the most popular explanation for ECP is that cytosolic proteins are released by cell lysis via the action of autolysins or prophages. Therefore, the role of prophages and autolysins will be briefly discussed.

There is no evidence for the involvement of prophages in ECP under normal conditions Temperate phages (like lambda phage) can integrate into the host’s genome and are replicated indirectly with the genome as an integral element without affecting cell growth, unless the prophage is induced by mutagens or UV radiation to enter the lytic cycle (Young et al., 2000). During phage assembly, holin molecules accumulate in the cytoplasmic membrane without detectable or destructive effect on the host (Gründling et al., 2001; Wang et al., 2000). Then, at a time programmed into their primary structure, holins trigger the disruption of the cytoplasmic membrane and causing cell lysis by activating murein hydrolytic enzymes that have accumulated in inactive conformation within the membrane. In other cases murein hydrolytic enzymes are exported by the host sec system (Xu et al., 2005). As many bacteria carry one or more prophages, it is possible that during cell growth a certain percentage of the prophages becomes activated for some unknown reason. Indeed, this activation can account for the leakage of a certain amount of cytoplasmic proteins within a small subpopulation, but it does not give an explanation why some proteins with a high abundance in the cytoplasm cannot be found in the secretome. In B. subtilis various possibilities/hypothesis have been discussed focusing on the question, how cytosolic proteins could be excreted (Tjalsma et al., 2004). It was speculated that prophageencoded holins could form pores in the membrane through which cytosolic proteins can escape (Young and Bläsi, 1995). However, elimination of several prophages from a strain had no effect on the excretion level of cytosolic proteins (Westers et al., 2003). Thus, cytosolic proteins do not seem to exit the cytoplasm in B. subtilis via prophage-encoded holins, as it was proposed for L. lactis (Walker and Klaenhammer, 2001).

5

In S. aureus too prophages do not seem to play a crucial role in ECP. Release of GAPDH by S. aureus containing the three prophages ␾11, ␾12, and ␾13 (Iandolo et al., 2002) was compared with S. aureus strain 8325-4 (Novick, 1967) cured of prophages and no difference was observed (Pasztor et al., 2010). These results suggest that prophages under normal conditions do not significantly contribute to release of cytosolic proteins. The influence of major autolysin (Atl) and distortion of the cell wall structure on release of cytosolic proteins The major autolysin (Atl) is the main peptidoglycan hydrolase in staphylococci (Heilmann et al., 1997; Oshida et al., 1995). Atl represents a bifunctional protein composed of a propeptide region, an amidase and an endo-beta-N-acetylglucosaminidase domain; the major lytic activity resides in the amidase (Biswas et al., 2006; Schlag et al., 2010). Autolysis is markedly decreased in an atl mutant (Schlag et al., 2010). Atl is sec-dependently exported and is targeted to the septum region (Schlag et al., 2010; Zoll et al., 2012). In the secretome of the S. aureus atl mutant, the amount of typical cytosolic proteins was significantly decreased and alternative cell wall hydrolases were increased (Pasztor et al., 2010). GAPDH, as a cytosolic marker protein, was hardly detectable in the culture supernatant of the atl mutant. At first glance, it seemed as if less cytosolic proteins were exported in the atl mutant. However, it turned out that in the atl mutant the cytosolic proteins were still translocated through the cytoplasmic membrane but were entrapped within the huge cell clusters of this mutant and therefore decreased in the secretome (Pasztor et al., 2010). Autolysins play a role in ECP, which has been demonstrated with the wall teichoic acid deficient tagO mutant, characterized by increased autolysis activity (Schlag et al., 2010), which correlated with higher amounts of GAPDH in the supernatant (Pasztor et al., 2010). Recently, it has been shown that the integrity of peptidoglycan structure plays a role in ECP. In the Staphylococcus carnosus femB mutant, the penta-glycine interpeptide bridge is shortened by two glycine residues (Fig. 3). This structural alteration caused a decrease in peptidoglycan cross-linking, affected the cell separation and caused increased susceptibility to methicillin (Henze et al., 1993). However, the most eye-catching phenotype of the femB mutant was the massive increase of ECP, which likely stems

Fig. 3. Staphylococcal peptidoglycan structure. In the femB mutant glycine residues 4 and 5 are absent. The resulting 3-glycine interpeptide bridge causes decreased cross-linking, upregulation of autolysins and massively ECP. Shown are the enzymes involved in the interpeptide bridge formation (FemX, FemA and FemB) as well as the cleavage sites of muramidase, amidase and lysostaphin.




6

from both the altered murein structure and the accompanied up-regulation of certain autolysins (Nega et al., 2014). Upregulated autolysins might impair the murein network, which is an important protective shield for the cytoplasmic membrane in Gram-positive bacteria. A compromised murein network should increase the susceptibility against osmotic stress. For example, permeabilization of the outer membrane of E. coli with EDTA led to osmotic stress and the release of a specific subset of cytoplasmic proteins such as EF-Tu, thioredoxin and DnaK (Vazquez-Laslop et al., 2001). Discussed mechanism for ECP There are indeed some hints that increased autolytic activity or cell fragility led to an increase of ECP. This has been demonstrated in staphylococci with the wall teichoic acid-less tagO or the femB mutant. In both cases the cell wall structures were altered resulting in an increased cell fragility, autolysis activity and ECP (Nega et al., 2014; Pasztor et al., 2010). Another mechanism for excretion of cytoplasmic proteins could be the use of membrane vesicles (MVs). MVs appear to be evolutionally conserved just as in mammalians, some proteins such as IL-1␤ are carried within intracellular vesicles which are translocated (Rubartelli et al., 1990). In Salmonella, MVs are released at constricted division septa and along the cell body; protein composition of septum- and cell body-derived MVs differed with respect to quantity and spectrum (Deatherage et al., 2009). But also Grampositive bacteria, such as S. aureus release MVs together with a mixture of Sec secreted, wall- and membrane associated and cytosolic proteins into the extracellular milieu (Lee et al., 2009). How these vesicles pass the murein barrier is unknown. So far there is no evidence that MVs are responsible for ECP in Gram-positive bacteria. The alternative Sec-2 pathway in mycobacteria may account for the excretion of cytosolic proteins in this genus. However, other bacterial species either do not have this pathway or if they have it, like L. monocytogenes or S. aureus, it appears that this pathway is restricted to only certain proteins. Successful immunization with non-classical secreted proteins of S. aureus Recently, nearly 40 proteins were identified on the cell surface of S. aureus including various cytosolic proteins such as enolase, oxoacyl reductase and the hypothetical protein hp2160 (Glowalla et al., 2009). These three proteins were used to vaccinate mice which led to a decreased spreading of S. aureus. Particularly, the immunization with rhp2160 protected mice from lethal S. aureus infection. The protective effect of immunization could have two reasons: (a) the corresponding antigens are tightly associated with the cell surface leading to good opsonization – as enolase and oxoacyl reductase were also found in the secretome one would expect a restricted opsonization; (b) the selected candidates may have ‘moonlighting’ function and might represent virulence proteins, thus interaction with the antibodies might neutralize their virulence potential. Meanwhile, there is no doubt that excreted cytosolic proteins play a role in virulence: GAPDH in S. pyogenes and group B streptococci binds to human proteins and stimulates B lymphocytes, in E. coli GAPDH induces an early IL-10 production, in Streptococcus pneumoniae excreted enolase binds to human complement inhibitor C4b-binding protein and contributes to complement evasion (Agarwal et al., 2012) and finally, vaccination with certain cytosolic proteins protects mice from lethal S. aureus infection. Alone the contribution of ECP in virulence merits paying more attention to this topic.

Fig. 4. ECP and open questions. There are many questions unanswered regarding mechanism, function and benefit of ECP in environment and infection.

Conclusion The phenomenon of ‘non-classical protein excretion’ is observed in both bacteria and mammalian cells, suggesting that this is an evolutionary conserved event. Despite the many studies that document and analyze ECP in various bacteria, there is no conclusive and generally applicable pathway identified up to now. As autolysins and the cell wall integrity influence the degree of ECP it is a widespread belief that ECP is essentially caused by cell lysis. However, there are several arguments and evidences that ECP is more than the accidental release of cytosolic proteins by cell lytic processes: (1) from the energetic point of view it would be an enormous waste of energy and resources if bacteria excrete proteins without a good reason; (2) ECP has been observed in unstressed bacterial cultures that are not prone to cell lysis and during all growth phases; (3) as outlined for S. aureus, the presence or absence of prophages has no effect on ECP; (4) there is only a defined spectrum of cytosolic proteins excreted, which implies that a specific selection process has to occur; (5) a strong argument for a selective process involved in ECP is the E. coli enolase whose excretion depended on 2-PG modification and not enzyme activity – this kind of protein selection is incompatible with simple cell lysis which randomly should expel cytosolic proteins. There are many questions unsolved and some of the open questions are illustrated (Fig. 4). For example, a defined mechanism for the ‘non-classical protein excretion’ in bacteria is not yet identified. We also need to learn more about the reason and driving forces of ECP since it is associated with a loss of energy and recourses. Presumably ECP is part of the normal life cycle of bacteria, however, its benefit is unclear. Analyzing the excretion mechanism and identifying the reason of ECP is a fundamental problem in bacterial cell biology. Acknowledgement This work was supported by the Deutsche Forschungsgemeinschaft: SFB766. References Abdallah, A.M., Gey van Pittius, N.C., Champion, P.A., Cox, J., Luirink, J., Vandenbroucke-Grauls, C.M., Appelmelk, B.J., Bitter, W., 2007. Type VII secretion – mycobacteria show the way. Nat. Rev. Microbiol. 5, 883–891. Agarwal, V., Hammerschmidt, S., Malm, S., Bergmann, S., Riesbeck, K., Blom, A.M., 2012. Enolase of Streptococcus pneumoniae binds human complement inhibitor C4b-binding protein and contributes to complement evasion. J. Immunol. 189, 3575–3584. Anderson, M., Aly, K.A., Chen, Y.H., Missiakas, D., 2013. Secretion of atypical protein substrates by the ESAT-6 secretion system of Staphylococcus aureus. Mol. Microbiol. 90, 734–743. Babu, M.M., Priya, M.L., Selvan, A.T., Madera, M., Gough, J., Aravind, L., Sankaran, K., 2006. A database of bacterial lipoproteins (DOLOP) with functional assignments to predicted lipoproteins. J. Bacteriol. 188, 2761–2773. Becher, D., Hempel, K., Sievers, S., Zuhlke, D., Pane-Farre, J., Otto, A., Fuchs, S., Albrecht, D., Bernhardt, J., Engelmann, S., Volker, U., van Dijl, J.M., Hecker, M.,




2009. A proteomic view of an important human pathogen – towards the quantification of the entire Staphylococcus aureus proteome. PLoS One 4, e8176. Berks, B.C., Palmer, T., Sargent, F., 2005. Protein targeting by the bacterial twinarginine translocation (Tat) pathway. Curr. Opin. Microbiol. 8, 174–181. Biswas, L., Biswas, R., Nerz, C., Ohlsen, K., Schlag, M., Schäfer, T., Lamkemeyer, T., Ziebandt, A.K., Hantke, K., Rosenstein, R., Götz, F., 2009. Role of the twin-arginine translocation pathway in Staphylococcus. J. Bacteriol. 191, 5921–5929. Biswas, R., Voggu, L., Simon, U.K., Hentschel, P., Thumm, G., Götz, F., 2006. Activity of the major staphylococcal autolysin Atl. FEMS Microbiol. Lett. 259, 260–268. Boel, G., Pichereau, V., Mijakovic, I., Maze, A., Poncet, S., Gillet, S., Giard, J.C., Hartke, A., Auffray, Y., Deutscher, J., 2004. Is 2-phosphoglycerate-dependent automodification of bacterial enolases implicated in their export? J. Mol. Biol. 337, 485–496. Braunstein, M., Espinosa, B.J., Chan, J., Belisle, J.T., Jacobs Jr., W.R., 2003. SecA2 functions in the secretion of superoxide dismutase A and in the virulence of Mycobacterium tuberculosis. Mol. Microbiol. 48, 453–464. Burts, M.L., DeDent, A.C., Missiakas, D.M., 2008. EsaC substrate for the ESAT-6 secretion pathway and its role in persistent infections of Staphylococcus aureus. Mol. Microbiol. 69, 736–746. Chatterjee, I., Schmitt, S., Batzilla, C.F., Engelmann, S., Keller, A., Ring, M.W., Kautenburger, R., Ziebuhr, W., Hecker, M., Preissner, K.T., Bischoff, M., Proctor, R.A., Beck, H.P., Lenhof, H.P., Somerville, G.A., Herrmann, M., 2009. Staphylococcus aureus ClpC ATPase is a late growth phase effector of metabolism and persistence. Proteomics 9, 1152–1176. Chatterjee, S.S., Joo, H.S., Duong, A.C., Dieringer, T.D., Tan, V.Y., Song, Y., Fischer, E.R., Cheung, G.Y., Li, M., Otto, M., 2013. Essential Staphylococcus aureus toxin export system. Nat. Med. 19, 364–367. Cheung, G.Y., Kretschmer, D., Queck, S.Y., Joo, H.S., Wang, R., Duong, A.C., Nguyen, T.H., Bach, T.H., Porter, A.R., DeLeo, F.R., Peschel, A., Otto, M., 2014. Insight into structure–function relationship in phenol-soluble modulins using an alanine screen of the phenol-soluble modulin (PSM) alpha3 peptide. FASEB J. 28, 153–161. Courtney, H.S., Dale, J.B., Hasty, D.I., 1996. Differential effects of the streptococcal fibronectin-binding protein, FBP54, on adhesion of group A streptococci to human buccal cells and HEp-2 tissue culture cells. Infect. Immun. 64, 2415–2419. D’Costa, S.S., Boyle, M.D., 2000. Interaction of group A streptococci with human plasmin(ogen) under physiological conditions. Methods 21, 165–177. Deatherage, B.L., Lara, J.C., Bergsbaken, T., Rassoulian Barrett, S.L., Lara, S., Cookson, B.T., 2009. Biogenesis of bacterial membrane vesicles. Mol. Microbiol. 72, 1395–1407. Dramsi, S., Bourdichon, F., Cabanes, D., Lecuit, M., Fsihi, H., Cossart, P., 2004. FbpA, a novel multifunctional Listeria monocytogenes virulence factor. Mol. Microbiol. 53, 639–649. Driessen, A.J., Nouwen, N., 2008. Protein translocation across the bacterial cytoplasmic membrane. Annu. Rev. Biochem. 77, 643–667. Egea, L., Aguilera, L., Gimenez, R., Sorolla, M.A., Aguilar, J., Badia, J., Baldoma, L., 2007. Role of secreted glyceraldehyde-3-phosphate dehydrogenase in the infection mechanism of enterohemorrhagic and enteropathogenic Escherichia coli: interaction of the extracellular enzyme with human plasminogen and fibrinogen. Int. J. Biochem. Cell Biol. 39, 1190–1203. Falord, M., Karimova, G., Hiron, A., Msadek, T., 2012. GraXSR proteins interact with the VraFG ABC transporter to form a five-component system required for cationic antimicrobial peptide sensing and resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 56, 1047–1058. Fuchs, S., Pane-Farre, J., Kohler, C., Hecker, M., Engelmann, S., 2007. Anaerobic gene expression in Staphylococcus aureus. J. Bacteriol. 189, 4275–4289. Glowalla, E., Tosetti, B., Kronke, M., Krut, O., 2009. Proteomics-based identification of anchorless cell wall proteins as vaccine candidates against Staphylococcus aureus. Infect. Immun. 77, 2719–2729. Gründling, A., Manson, M.D., Young, R., 2001. Holins kill without warning. Proc. Natl. Acad. Sci. U. S. A. 98, 9348–9352. Heilmann, C., Hussain, M., Peters, G., Götz, F., 1997. Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Mol. Microbiol. 24, 1013–1024. Henderson, B., Martin, A., 2013. Bacterial moonlighting proteins and bacterial virulence. Curr. Top. Microbiol. Immunol. 358, 155–213. Henze, U., Sidow, T., Wecke, J., Labischinski, H., Berger-Bächi, B., 1993. Influence of femB on methicillin resistance and peptidoglycan metabolism in Staphylococcus aureus. J. Bacteriol. 175, 1612–1620. Herbert, S., Bera, A., Nerz, C., Kraus, D., Peschel, A., Goerke, C., Meehl, M., Cheung, A., Götz, F., 2007. Molecular basis of resistance to muramidase and cationic antimicrobial peptide activity of lysozyme in staphylococci. PLoS Pathog. 3, e102. Hille, M., Kies, S., Götz, F., Peschel, A., 2001. Dual role of GdmH in producer immunity and secretion of the Staphylococcal lantibiotics gallidermin and epidermin. Appl. Environ. Microbiol. 67, 1380–1383. Hirose, I., Sano, K., Shioda, I., Kumano, M., Nakamura, K., Yamane, K., 2000. Proteome analysis of Bacillus subtilis extracellular proteins: a two-dimensional protein electrophoretic study. Microbiology 146, 65–75. Houben, D., Demangel, C., van Ingen, J., Perez, J., Baldeon, L., Abdallah, A.M., Caleechurn, L., Bottai, D., van Zon, M., de Punder, K., van der Laan, T., Kant, A., Bossers-de Vries, R., Willemsen, P., Bitter, W., van Soolingen, D., Brosch, R., van der Wel, N., Peters, P.J., 2012. ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria. Cell Microbiol. 14, 1287–1298. Hughes, M.J., Moore, J.C., Lane, J.D., Wilson, R., Pribul, P.K., Younes, Z.N., Dobson, R.J., Everest, P., Reason, A.J., Redfern, J.M., Greer, F.M., Paxton, T., Panico, M., Morris, H.R., Feldman, R.G., Santangelo, J.D., 2002. Identification of major outer surface proteins of Streptococcus agalactiae. Infect. Immun. 70, 1254–1259.

7

Iandolo, J.J., Worrell, V., Groicher, K.H., Qian, Y., Tian, R., Kenton, S., Dorman, A., Ji, H., Lin, S., Loh, P., Qi, S., Zhu, H., Roe, B.A., 2002. Comparative analysis of the genomes of the temperate bacteriophages phi 11, phi 12 and phi 13 of Staphylococcus aureus 8325. Gene 289, 109–118. Jeffery, C.J., 1999. Moonlighting proteins. Trends Biochem. Sci. 24, 8–11. Jin, H., Song, Y.P., Boel, G., Kochar, J., Pancholi, V., 2005. Group A streptococcal surface GAPDH, SDH, recognizes uPAR/CD87 as its receptor on the human pharyngeal cell and mediates bacterial adherence to host cells. J. Mol. Biol. 350, 27–41. Jongbloed, J.D., Grieger, U., Antelmann, H., Hecker, M., Nijland, R., Bron, S., van Dijl, J.M., 2004. Two minimal Tat translocases in Bacillus. Mol. Microbiol. 54, 1319–1325. Joo, H.S., Cheung, G.Y., Otto, M., 2011. Antimicrobial activity of communityassociated methicillin-resistant Staphylococcus aureus is caused by phenolsoluble modulin derivatives. J. Biol. Chem. 286, 8933–8940. Kohler, C., Wolff, S., Albrecht, D., Fuchs, S., Becher, D., Buttner, K., Engelmann, S., Hecker, M., 2005. Proteome analyses of Staphylococcus aureus in growing and non-growing cells: a physiological approach. Int. J. Med. Microbiol. 295, 547–565. Korea, C.G., Balsamo, G., Pezzicoli, A., Merakou, C., Tavarini, S., Bagnoli, F., Serruto, D., Unnikrishnan, M., 2014. The staphylococcal Esx proteins modulate apoptosis and release of intracellular Staphylococcus aureus during infection in epithelial cells. Infect. Immun. 82, 4144–4153. Kretschmer, D., Gleske, A.K., Rautenberg, M., Wang, R., Koberle, M., Bohn, E., Schoneberg, T., Rabiet, M.J., Boulay, F., Klebanoff, S.J., van Kessel, K.A., van Strijp, J.A., Otto, M., Peschel, A., 2010. Human formyl peptide receptor 2 senses highly pathogenic Staphylococcus aureus. Cell Host Microbe 7, 463–473. Kusch, H., Engelmann, S., 2014. Secrets of the secretome in Staphylococcus aureus. Int. J. Med. Microbiol. 304, 133–141. Lee, E.Y., Choi, D.Y., Kim, D.K., Kim, J.W., Park, J.O., Kim, S., Kim, S.H., Desiderio, D.M., Kim, Y.K., Kim, K.P., Gho, Y.S., 2009. Gram-positive bacteria produce membrane vesicles: proteomics-based characterization of Staphylococcus aureus-derived membrane vesicles. Proteomics 9, 5425–5436. Lenz, L.L., Mohammadi, S., Geissler, A., Portnoy, D.A., 2003. SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis. Proc. Natl. Acad. Sci. U. S. A. 100, 12432–12440. Li, M., Rosenshine, I., Tung, S.L., Wang, X.H., Friedberg, D., Hew, C.L., Leung, K.Y., 2004. Comparative proteomic analysis of extracellular proteins of enterohemorrhagic and enteropathogenic Escherichia coli strains and their ihf and ler mutants. Appl. Environ. Microbiol. 70, 5274–5282. Li, Y., Huang, X., Li, J., Zeng, J., Zhu, F., Fan, W., Hu, L., 2014. Both GtfA and GtfB are required for SraP glycosylation in Staphylococcus aureus. Curr. Microbiol. 69, 121–126. Lottenberg, R., Broder, C.C., Boyle, M.D., Kain, S.J., Schroeder, B.L., Curtiss 3rd, R., 1992. Cloning, sequence analysis, and expression in Escherichia coli of a streptococcal plasmin receptor. J. Bacteriol. 174, 5204–5210. Madureira, P., Baptista, M., Vieira, M., Magalhaes, V., Camelo, A., Oliveira, L., Ribeiro, A., Tavares, D., Trieu-Cuot, P., Vilanova, M., Ferreira, P., 2007. Streptococcus agalactiae GAPDH is a virulence-associated immunomodulatory protein. J. Immunol. 178, 1379–1387. Meehl, M., Herbert, S., Götz, F., Cheung, A., 2007. Interaction of the GraRS two-component system with the VraFG ABC transporter to support vancomycin-intermediate resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 51, 2679–2689. Mehlin, C., Headley, C.M., Klebanoff, S.J., 1999. An inflammatory polypeptide complex from Staphylococcus epidermidis: isolation and characterization. J. Exp. Med. 189, 907–918. Muesch, A., Hartmann, E., Rohde, K., Rubartelli, A., Sitia, R., Rapoport, T.A., 1990. A novel pathway for secretory proteins? Trends Biochem. Sci. 15, 86–88. Nega, M., Dube, L., Kull, M., Ziebandt, A.K., Ebner, P., Albrecht, D., Krismer, B., Rosenstein, R., Hecker, M., Götz, F., 2014. Secretome analysis revealed adaptive and non-adaptive responses of the Staphylococcus carnosus femB mutant. Proteomics, http://www.ncbi.nlm.nih.gov/pubmed/25430637 Nickel, W., 2003. The mystery of non-classical protein secretion. A current view on cargo proteins and potential export routes. Eur. J. Biochem. 270, 2109–2119. Novick, R., 1967. Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology 33, 155–166. Oliveira, L., Madureira, P., Andrade, E.B., Bouaboud, A., Morello, E., Ferreira, P., Poyart, C., Trieu-Cuot, P., Dramsi, S., 2012. Group B streptococcus GAPDH is released upon cell lysis, associates with bacterial surface, and induces apoptosis in murine macrophages. PLoS One 7, e29963. Oshida, T., Sugai, M., Komatsuzawa, H., Hong, Y.M., Suginaka, H., Tomasz, A., 1995. A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-l-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and characterization. Proc. Natl. Acad. Sci. U. S. A. 92, 285–289. Otto, M., O’Mahoney, D.S., Guina, T., Klebanoff, S.J., 2004. Activity of Staphylococcus epidermidis phenol-soluble modulin peptides expressed in Staphylococcus carnosus. J. Infect. Dis. 190, 748–755. Otto, M., Peschel, A., Götz, F., 1998. Producer self-protection against the lantibiotic epidermin by the ABC transporter EpiFEG of Staphylococcus epidermidis Tu3298. FEMS Microbiol. Lett. 166, 203–211. Pallen, M.J., 2002. The ESAT-6/WXG100 superfamily – and a new Gram-positive secretion system? Trends Microbiol. 10, 209–212. Palmer, T., Berks, B.C., 2012. The twin-arginine translocation (Tat) protein export pathway. Nat. Rev. Microbiol. 10, 483–496. Pancholi, V., Chhatwal, G.S., 2003. Housekeeping enzymes as virulence factors for pathogens. Int. J. Med. Microbiol. 293, 391–401.


G Model IJMM-50923; No. of Pages 8 8


Pancholi, V., Fischetti, V.A., 1992. A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate-dehydrogenase with multiple binding activity. J. Exp. Med. 176, 415–426. Pancholi, V., Fischetti, V.A., 1993. Glyceraldehyde-3-phosphate dehydrogenase on the surface of group A streptococci is also an ADP-ribosylating enzyme. Proc. Natl. Acad. Sci. U. S. A. 90, 8154–8158. Pancholi, V., Fischetti, V.A., 1998. Alpha-enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J. Biol. Chem. 273, 14503–14510. Pasztor, L., (Thesis) 2011. Untersuchung der Signalpeptidunabhängigen Translokationswege in Staphylococcus aureus, Microbial Genetics. University Tübingen., pp. 198. Pasztor, L., Ziebandt, A.K., Nega, M., Schlag, M., Haase, S., Franz-Wachtel, M., Madlung, J., Nordheim, A., Heinrichs, D.E., Götz, F., 2010. Staphylococcal major autolysin (atl) is involved in excretion of cytoplasmic proteins. J. Biol. Chem. 285, 36794–36800. Patton, T.G., Rice, K.C., Foster, M.K., Bayles, K.W., 2005. The Staphylococcus aureus cidC gene encodes a pyruvate oxidase that affects acetate metabolism and cell death in stationary phase. Mol. Microbiol. 56, 1664–1674. Periasamy, S., Joo, H.S., Duong, A.C., Bach, T.H., Tan, V.Y., Chatterjee, S.S., Cheung, G.Y., Otto, M., 2012. How Staphylococcus aureus biofilms develop their characteristic structure. Proc. Natl. Acad. Sci. U. S. A. 109, 1281–1286. Peschel, A., Götz, F., 1996. Analysis of the Staphylococcus epidermidis genes epiF, -E, and -G involved in epidermin immunity. J. Bacteriol. 178, 531–536. Queck, S.Y., Jameson-Lee, M., Villaruz, A.E., Bach, T.H., Khan, B.A., Sturdevant, D.E., Ricklefs, S.M., Li, M., Otto, M., 2008. RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus. Mol. Cell 32, 150–158. Ranjit, D.K., Endres, J.L., Bayles, K.W., 2011. Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties. J. Bacteriol. 193, 2468–2476. Resch, A., Leicht, S., Saric, M., Pasztor, L., Jakob, A., Götz, F., Nordheim, A., 2006. Comparative proteome analysis of Staphylococcus aureus biofilm and planktonic cells and correlation with transcriptome profiling. Proteomics 6, 1867–1877. Rice, K.C., Bayles, K.W., 2003. Death’s toolbox: examining the molecular components of bacterial programmed cell death. Mol. Microbiol. 50, 729–738. Rubartelli, A., Bajetto, A., Allavena, G., Wollman, E., Sitia, R., 1992. Secretion of thioredoxin by normal and neoplastic cells through a leaderless secretory pathway. J. Biol. Chem. 267, 24161–24170. Rubartelli, A., Cozzolino, F., Talio, M., Sitia, R., 1990. A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence. EMBO J. 9, 1503–1510. Schlag, M., Biswas, R., Krismer, B., Köhler, T., Zoll, S., Yu, W., Schwarz, H., Peschel, A., Götz, F., 2010. Role of staphylococcal wall teichoic acid in targeting the major autolysin Atl. Mol. Microbiol. 75, 864–873. Sibbald, M.J., Winter, T., van der Kooi-Pol, M.M., Buist, G., Tsompanidou, E., Bosma, T., Schafer, T., Ohlsen, K., Hecker, M., Antelmann, H., Engelmann, S., van Dijl, J.M., 2010. Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in Staphylococcus aureus. J. Bacteriol. 192, 3788–3800. Sibbald, M.J., Ziebandt, A.K., Engelmann, S., Hecker, M., de Jong, A., Harmsen, H.J., Raangs, G.C., Stokroos, I., Arends, J.P., Dubois, J.Y., van Dijl, J.M., 2006. Mapping the pathways to staphylococcal pathogenesis by comparative secretomics. Microbiol. Mol. Biol. Rev. 70, 755–788. Siboo, I.R., Chaffin, D.O., Rubens, C.E., Sullam, P.M., 2008. Characterization of the accessory Sec system of Staphylococcus aureus. J. Bacteriol. 190, 6188–6196. Siljamaki, P., Varmanen, P., Kankainen, M., Sukura, A., Savijoki, K., Nyman, T.A., 2014. Comparative exoprotein profiling of different Staphylococcus epidermidis strains reveals potential link between non-classical protein export and virulence. J. Proteome Res. 13, 3249–3261. Sloan, I.S., Horowitz, P.M., Chirgwin, J.M., 1994. Rapid secretion by a non-classical pathway of overexpressed mammalian mitochondrial rhodanese. J. Biol. Chem. 269, 27625–27630.

Sundaramoorthy, R., Fyfe, P.K., Hunter, W.N., 2008. Structure of Staphylococcus aureus EsxA suggests a contribution to virulence by action as a transport chaperone and/or adaptor protein. J. Mol. Biol. 383, 603–614. Suvorov, A.N., Flores, A.E., Ferrieri, P., 1997. Cloning of the glutamine synthetase gene from group B streptococci. Infect. Immun. 65, 191–196. Tjalsma, H., Antelmann, H., Jongbloed, J.D., Braun, P.G., Darmon, E., Dorenbos, R., Dubois, J.Y., Westers, H., Zanen, G., Quax, W.J., Kuipers, O.P., Bron, S., Hecker, M., van Dijl, J.M., 2004. Proteomics of protein secretion by Bacillus subtilis: separating the “secrets” of the secretome. Microbiol. Mol. Biol. Rev. 68, 207–233. Trost, M., Wehmhoner, D., Karst, U., Dieterich, G., Wehland, J., Jansch, L., 2005. Comparative proteome analysis of secretory proteins from pathogenic and nonpathogenic Listeria species. Proteomics 5, 1544–1557. Vazquez-Laslop, N., Lee, H., Hu, R., Neyfakh, A.A., 2001. Molecular sieve mechanism of selective release of cytoplasmic proteins by osmotically shocked Escherichia coli. J. Bacteriol. 183, 2399–2404. von Heijne, G., 1990. The signal peptide. J. Membr. Biol. 115, 195201. Vuong, C., Saenz, H.L., Götz, F., Otto, M., 2000. Impact of the agr quorum-sensing system on adherence to polystyrene in Staphylococcus aureus. J. Infect. Dis. 182, 1688–1693. Walker, S.A., Klaenhammer, T.R., 2001. Leaky Lactococcus cultures that externalize enzymes and antigens independently of culture lysis and secretion and export pathways. Appl. Environ. Microbiol. 67, 251–259. Wang, G., Chen, H., Xia, Y., Cui, J., Gu, Z., Song, Y., Chen, Y.Q., Zhang, H., Chen, W., 2013. How are the non-classically secreted bacterial proteins released into the extracellular milieu? Curr. Microbiol. 67, 688–695. Wang, I.N., Smith, D.L., Young, R., 2000. Holins: the protein clocks of bacteriophage infections. Annu. Rev. Microbiol. 54, 799–825. Westers, H., Dorenbos, R., van Dijl, J.M., Kabel, J., Flanagan, T., Devine, K.M., Jude, F., Seror, S.J., Beekman, A.C., Darmon, E., Eschevins, C., de Jong, A., Bron, S., Kuipers, O.P., Albertini, A.M., Antelmann, H., Hecker, M., Zamboni, N., Sauer, U., Bruand, C., Ehrlich, D.S., Alonso, J.C., Salas, M., Quax, W.J., 2003. Genome engineering reveals large dispensable regions in Bacillus subtilis. Mol. Biol. Evol. 20, 2076–2090. Winram, S.B., Lottenberg, R., 1996. The plasmin-binding protein Plr of group A streptococci is identified as glyceraldehyde-3-phosphate dehydrogenase. Microbiology 142, 2311–2320. Xia, X.X., Han, M.J., Lee, S.Y., Yoo, J.S., 2008. Comparison of the extracellular proteomes of Escherichia coli B and K-12 strains during high cell density cultivation. Proteomics 8, 2089–2103. Xu, M., Arulandu, A., Struck, D.K., Swanson, S., Sacchettini, J.C., Young, R., 2005. Disulfide isomerization after membrane release of its SAR domain activates P1 lysozyme. Science 307, 113–117. Young, I., Wang, I., Roof, W.D., 2000. Phages will out: strategies of host cell lysis. Trends Microbiol. 8, 120–128. Young, R., Bläsi, U., 1995. Holins: form and function in bacteriophage lysis. FEMS Microbiol. Rev. 17, 191–205. Ziebandt, A.K., Becher, D., Ohlsen, K., Hacker, J., Hecker, M., Engelmann, S., 2004. The influence of agr and sigmaB in growth phase dependent regulation of virulence factors in Staphylococcus aureus. Proteomics 4, 3034–3047. Ziebandt, A.K., Weber, H., Rudolph, J., Schmid, R., Hoper, D., Engelmann, S., Hecker, M., 2001. Extracellular proteins of Staphylococcus aureus and the role of SarA and sigma B. Proteomics 1, 480–493. Zoll, S., Schlag, M., Shkumatov, A.V., Rautenberg, M., Svergun, D.I., Götz, F., Stehle, T., 2012. Ligand-binding properties and conformational dynamics of autolysin repeat domains in staphylococcal cell wall recognition. J. Bacteriol. 194, 3789–3802. Zoltner, M., Fyfe, P.K., Palmer, T., Hunter, W.N., 2013. Characterization of Staphylococcus aureus EssB, an integral membrane component of the Type VII secretion system: atomic resolution crystal structure of the cytoplasmic segment. Biochem. J. 449, 469–477.


Detection of cytosolic bacteria by inflammatory caspases.

EDN are regulated in a reciprocal manner.

Selective degradation of cytosolic proteins by lysosomes.

Interaction of mitochondrial porin with cytosolic proteins.

The ECP calcium fibre polyp prevention study. The ECP Colon Group.

Guanine nucleotides stimulate carboxyl methylation of kidney cytosolic proteins.

Pyruvate production and excretion by the luminous marine bacteria.

Maturation of cytosolic and nuclear iron-sulfur proteins.

Modifiable chromatophore proteins in photosynthetic bacteria.

Miscellaneous indications for extracorporeal photochemotherapy (ECP).

Eosinophil granule proteins ECP and EPX as markers for a potential early-stage inflammatory lesion in female genital schistosomiasis (FGS).

Toxoplasma gondii ingests and digests host cytosolic proteins.

Hydrolysis of milk proteins by bacteria used in cheese making.

Absorption, distribution, metabolism, and excretion of bacteria-derived hyaluronic acid in rats and rabbits.

Transport proteins in bacteria: common themes in their design.

RNA-binding proteins involved in post-transcriptional regulation in bacteria.

Diverse mechanisms for inflammasome sensing of cytosolic bacteria and bacterial virulence.

Recurrent background mutations in WHI2 impair proteostasis and degradation of misfolded cytosolic proteins in Saccharomyces cerevisiae.

Sputum ECP levels correlate with parameters of airflow obstruction.

Functional characterization of ECP-heparin interaction: a novel molecular model.

NBP35 interacts with DRE2 in the maturation of cytosolic iron-sulphur proteins in Arabidopsis thaliana.

Peptidoglycan-associated outer membrane proteins in gammegatine bacteria.

Dancing to another tune-adhesive moonlighting proteins in bacteria.

Tracking Proteins Secreted by Bacteria: What's in the Toolbox?