The Twin-Arginine Signal Peptide of Bacillus subtilis YwbN Can Direct either Tat- or Sec-Dependent Secretion of Different Cargo Proteins: Secretion of Active Subtilisin via the B. subtilis Tat Pathway

Proteins that are produced for commercial purposes in Bacillus subtilis are commonly secreted via the Sec pathway. Despite its high secretion capacity, the secretion of heterologous proteins via the Sec pathway is often unsuccessful. Alternative secretion routes, like the Tat pathway, are therefore of interest. Two parallel Tat pathways with distinct specificities have previously been discovered in B. subtilis. To explore the application potential of these Tat pathways, several commercially relevant or heterologous model proteins were fused to the signal peptides of the known B. subtilis Tat substrates YwbN and PhoD. Remarkably, the YwbN signal peptide directed secretion of active subtilisin, a typical Sec substrate, via the B. subtilis TatAyCy route. In contrast, the same signal peptide directed Tat-independent secretion of the Bacillus licheniformis α-amylase (AmyL). Moreover, the YwbN signal peptide directed secretion of SufI, an Escherichia coli Tat substrate, in a Tat-independent manner, most likely via Sec. Our results suggest that cytoplasmic protein folding prior to translocation is probably a major determinant of Tat-dependent protein secretion in B. subtilis, as is the case with E. coli. We conclude that future applications for the Tat system of B. subtilis will most likely involve commercially interesting proteins that are Sec incompatible.     

Environmental salinity determines the specificity and need for Tat-dependent secretion of the YwbN protein in Bacillus subtilis

Twin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described "minimal Tat translocase" consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate.     

Salt sensitivity of minimal twin arginine translocases

Bacterial twin arginine translocation (Tat) pathways have evolved to facilitate transport of folded proteins across membranes. Gram-negative bacteria contain a TatABC translocase composed of three subunits named TatA, TatB, and TatC. In contrast, the Tat translocases of most Gram-positive bacteria consist of only TatA and TatC subunits. In these minimal TatAC translocases, a bifunctional TatA subunit fulfils the roles of both TatA and TatB. Here we have probed the importance of conserved residues in the bifunctional TatAy subunit of Bacillus subtilis by site-specific mutagenesis. A set of engineered TatAy proteins with mutations in the cytoplasmic hinge and amphipathic helix regions were found to be inactive in protein translocation under standard growth conditions for B. subtilis or when heterologously expressed in Escherichia coli. Nevertheless, these mutated TatAy proteins did assemble into TatAy and TatAyCy complexes, and they facilitated membrane association of twin arginine precursor proteins in E. coli. Interestingly, most of the mutated TatAyCy translocases were salt-sensitive in B. subtilis. Similarly, the TatAC translocases of Bacillus cereus and Staphylococcus aureus were salt-sensitive when expressed in B. subtilis. Taken together, our present observations imply that salt-sensitive electrostatic interactions have critical roles in the preprotein translocation activity of certain TatAC type translocases from Gram-positive bacteria.     

Two minimal Tat translocases in Bacillus

Activity of the Tat machinery for protein transport across the inner membrane of Escherichia coli and the chloroplast thylakoidal membrane requires the presence of three membrane proteins: TatA, TatB and TatC. Here, we show that the Tat machinery of the Gram-positive bacterium Bacillus subtilis is very different because it contains at least two minimal Tat translocases, each composed of one specific TatA and one specific TatC component. A third, TatB-like component is apparently not required. This implies that TatA proteins of B. subtilis perform the functions of both TatA and TatB of E. coli and thylakoids. Notably, the two B. subtilis translocases named TatAdCd and TatAyCy both function as individual, substrate-specific translocases for the twin-arginine preproteins PhoD and YwbN, respectively. Importantly, these minimal TatAC translocases of B. subtilis are representative for the Tat machinery of the vast majority of Gram-positive bacteria, Streptomycetes being the only known exception with TatABC translocases.     

A mutation leading to super-assembly of twin-arginine translocase (Tat) protein complexes

The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report the analysis of the Bacillus subtilis TatAyCy system, which occurs as separate TatAyCy and TatAy complexes at steady state, using single-particle electron microscopy (EM) and advanced atomic force microscopy (AFM) approaches. We show that a P2A mutation in the TatAy subunit leads to apparent super-assembly of Tat complexes. Purification of TatCy-containing complexes leads to a large increase in the TatA:TatC ratio, suggesting that TatAy^P2A complexes may have attached to the TatAyCy complex. EM and AFM analyses show that the wild-type TatAyCy complex purifies as roughly spherical complexes of 9–16 nm diameter, whereas the P2A mutation leads to accumulation of large (up to 500 nm long) fibrils that are chains of numerous complexes. Time lapsed AFM imaging, recorded on fibrils under liquid, shows that they adopt a variety of tightly curved conformations, with radii of curvature of 10–12 nm comparable to the size of single TatAy^P2A complexes. The combined data indicate that the mutation leads to super-assembly of TatAy^P2A complexes and we propose that an individual TatAy^P2A complex assembles initially with a TatAy^P2ACy complex, after which further TatAy^P2A complexes attach to each other. The data further suggest that the N-terminal extracytoplasmic domain of TatAy plays an essential role in Tat complex interactions.     

Functional association of the stress-responsive LiaH protein and the minimal TatAyCy protein translocase in Bacillus subtilis

The bacterial twin-arginine (Tat) pathway serves in the exclusive secretion of folded proteins with bound cofactors. While Tat pathways in Gram-negative bacteria and chloroplast thylakoids consist of conserved TatA, TatB and TatC subunits, the Tat pathways of Bacillus species and many other Gram-positive bacteria stand out for their minimalist nature with the core translocase being composed of essential TatA and TatC subunits only. Here we addressed the question whether the minimal TatAyCy translocase of Bacillus subtilis recruits additional cellular components that modulate its activity. To this end, TatAyCy was purified by affinity- and size exclusion chromatography, and interacting co-purified proteins were identified by mass spectrometry. This uncovered the cell envelope stress responsive LiaH protein as an accessory subunit of the TatAyCy complex. Importantly, our functional studies show that Tat expression is tightly trailed by LiaH induction, and that LiaH itself determines the capacity and quality of TatAyCy-dependent protein translocation. In contrast, LiaH has no role in high-level protein secretion via the general secretion (Sec) pathway. Altogether, our observations show that protein translocation by the minimal Tat translocase TatAyCy is tightly intertwined with an adequate bacterial response to cell envelope stress. This is consistent with a critical need to maintain cellular homeostasis, especially when the membrane is widely opened to permit passage of large fully-folded proteins via Tat.     

Degradation of the Twin-Arginine Translocation Substrate YwbN by Extracytoplasmic Proteases of Bacillus subtilis

Bacterial twin-arginine translocases can export fully folded proteins from the cytoplasm. Such proteins are usually resistant to proteolysis. Here we show that multiple extracellular proteases degrade the B. subtilis Tat substrate YwbN. This suggests either that secreted YwbN is not fully folded or that folded YwbN exposes protease cleavage sites.     

Subcellular Localization of TatAd of Bacillus subtilis Depends on the Presence of TatCd or TatCy

The gram-positive bacterium Bacillus subtilis contains two minimal Tat translocases, TatAdCd and TatAyCy, which are each involved in the secretion of one or more specific protein substrates. We have investigated the subcellular localization of the TatA components by employing C-terminal green fluorescent protein (GFP) fusions and fluorescence microscopy. When expressed from a xylose-inducible promoter, the TatA-GFP fusion proteins displayed a dual localization pattern, being localized peripherally and showing bright foci which are predominantly located at the division sites and/or poles of the cells. Importantly, the localization of TatAd-GFP was similar when the protein was expressed from its own promoter under phosphate starvation conditions, indicating that these foci are not the result of artificial overexpression. Moreover, the TatAd-GFP fusion protein was shown to be functional in the translocation of its substrate PhoD, provided that TatCd is also present. Furthermore, we demonstrate that the localization of TatAd-GFP in foci depends on the presence of the TatCd component. Remarkably, however, the TatAd-GFP foci can also be observed in the presence of TatCy, indicating that TatAd can interact not only with TatCd but also with TatCy. These results suggest that the formation of TatAd complexes in B. subtilis is controlled by TatC.The bacterial twin-arginine translocation (Tat) machinery is able to transport folded proteins across the cytoplasmic membrane (26). Preproteins translocated by the Tat pathway are characterized by a twin-arginine (RR) motif in their signal sequences.In Escherichia coli, the Tat system consists of three components, the TatA, TatB, and TatC proteins. In the currently favored model for its mode of action, a TatB-TatC complex is involved in initial RR signal peptide recognition and binding of precursor proteins. Multiple TatA subunits then associate with this complex to form a protein-conducting channel (1). TatA, which is homologous to TatB, can be found complexed to TatBC but also forms a wide range of large, homooligomeric complexes (7, 23). In a few cases, the TatB protein can be functionally replaced by the TatA protein, indicating that TatA and TatC are able to form an active, minimal translocase (6, 10).Most gram-positive bacteria contain only two types of Tat subunit, a TatC protein and a TatA protein which has characteristics and the ability to perform the function of both TatA and TatB of E. coli (2, 13). Bacillus subtilis contains two substrate-specific Tat systems: a TatAyCy translocase that is required for translocation of the iron-dependent DyP peroxidase YwbN and a TatAdCd translocase which translocates the phosphodiesterase PhoD (12). In addition, B. subtilis contains a third TatA component, designated TatAc. This protein is dispensable for Tat-dependent translocation of YwbN or PhoD, and its function is currently unknown.TatAd is the most-studied TatA component of B. subtilis, and like TatA of E. coli, it is able to form both homooligomeric complexes and complexes with TatCd (2, 31). Despite the fact that it contains an N-terminal transmembrane segment (17), TatAd was also found in the cytosol, where it appears to interact with its substrate, pre-PhoD, via the signal sequence (24). TatCd was proposed to act as a receptor for the anchoring at and subsequent incorporation into the membrane of this TatAd-PhoD complex (28).The subcellular localization of Tat components in E. coli has been extensively investigated by fluorescence microscopy. Green fluorescent protein (GFP) fusions of TatA were localized at the periphery of the cells, but punctate regions of fluorescence were also reported (4, 25). In these studies, TatB was localized all over the membrane, with some accumulation at the cell poles. TatC was mainly distributed evenly throughout the periphery of the cells, with some small punctate regions. Recently, the oligomeric state of TatA-yellow fluorescent protein (YFP) in living E. coli cells was determined by single-molecule imaging (18). TatA complexes with a broad range of stoichiometries were observed as fluorescent foci, and TatA was also present in a dispersed state in the membrane.For B. subtilis, the subcellular localization of only one Tat component has been reported so far. Both N- and C-terminal fusions of GFP to TatCy were shown to be localized throughout the membrane, with frequent foci at the cell poles and division septa, and this localization pattern was classified as “polar” (20).In this study, we have investigated the subcellular localization of the three TatA proteins of B. subtilis by using GFP fusions, functionality assessments, and fluorescence microscopy. TatAc and TatAd showed a dual localization pattern, with fluorescence in the membrane as well as in foci which were enriched at the cell poles. Notably, the localization of TatAd-GFP in foci was shown to depend on the presence of a TatC component, suggesting that TatC drives complex formation by TatAd.     

High-Salinity Growth Conditions Promote Tat-Independent Secretion of Tat Substrates in Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis contains two Tat translocases, which can facilitate transport of folded proteins across the plasma membrane. Previous research has shown that Tat-dependent protein secretion in B. subtilis is a highly selective process and that heterologous proteins, such as the green fluorescent protein (GFP), are poor Tat substrates in this organism. Nevertheless, when expressed in Escherichia coli, both B. subtilis Tat translocases facilitated exclusively Tat-dependent export of folded GFP when the twin-arginine (RR) signal peptides of the E. coli AmiA, DmsA, or MdoD proteins were attached. Therefore, the present studies were aimed at determining whether the same RR signal peptide-GFP precursors would also be exported Tat dependently in B. subtilis. In addition, we investigated the secretion of GFP fused to the full-length YwbN protein, a strict Tat substrate in B. subtilis. Several investigated GFP fusion proteins were indeed secreted in B. subtilis, but this secretion was shown to be completely Tat independent. At high-salinity growth conditions, the Tat-independent secretion of GFP as directed by the RR signal peptides from the E. coli AmiA, DmsA, or MdoD proteins was significantly enhanced, and this effect was strongest in strains lacking the TatAy-TatCy translocase. This implies that high environmental salinity has a negative influence on the avoidance of Tat-independent secretion of AmiA-GFP, DmsA-GFP, and MdoD-GFP. We conclude that as-yet-unidentified control mechanisms reject the investigated GFP fusion proteins for translocation by the B. subtilis Tat machinery and, at the same time, set limits to their Tat-independent secretion, presumably via the Sec pathway.     

Mapping the twin‐arginine protein translocation network of Bacillus subtilis

Bacteria employ twin‐arginine translocation (Tat) pathways for the transport of folded proteins to extracytoplasmic destinations. In recent years, most studies on bacterial Tat pathways addressed the membrane‐bound TatA(B)C subunits of the Tat translocase, and the specific interactions between this translocase and its substrate proteins. In contrast, relatively few studies investigated possible coactors in the TatA(B)C‐dependent protein translocation process. The present studies were aimed at identifying interaction partners of the Tat pathway of Bacillus subtilis, which is a paradigm for studies on protein secretion by Gram‐positive bacteria. Specifically, 36 interaction partners of the TatA and TatC subunits were identified by rigorous application of the yeast two‐hybrid (Y2H) approach. Our Y2H analyses revealed that the three TatA isoforms of B. subtilis can form homo‐ and heterodimers. Subsequently, the secretion of the Tat substrates YwbN and PhoD was tested in mutant strains lacking genes for the TatAC interaction partners identified in our genome‐wide Y2H screens. Our results show that the cell wall‐bound protease WprA is important for YwbN secretion, and that the HemAT and CsbC proteins are required for PhoD secretion under phosphate starvation conditions. Taken together, our findings imply that the Bacillus Tat pathway is embedded in an intricate protein–protein interaction network.     

The Escherichia coli TatABC system and a Bacillus subtilis TatAC-type system recognise three distinct targeting determinants in twin-arginine signal peptides

The Tat system transports folded proteins across bacterial and thylakoid membranes. In Gram-negative organisms, it is encoded by tatABC genes and the system recognizes substrates bearing signal peptides with a conserved twin-arginine motif. Most Gram-positive organisms lack a tatB gene, indicating major differences in organisation and/or mechanism. Here, we have characterized the essential targeting determinants that are recognized by a Bacillus subtilis TatAC-type system, TatAdCd. Substitution by lysine of either of the twin-arginine residues in the TorA signal peptide can be tolerated, but the presence of twin-lysine residues blocks export completely. We show that additional determinants can be as important as the twin-arginine motif. Replacement of the −1 serine by alanine, in either the TorA or DmsA signal peptide, almost blocks export by either the B. subtilis TatAdCd or Escherichia coli TatABC systems, firmly establishing the importance of this −1 residue in these signal peptides. Surprisingly, the +2 leucine in the DmsA signal peptide (sequence SRRGLV) appears to play an equally important role and substitution by alanine or phenylalanine blocks export by both the B. subtilis and E. coli systems. These data identify three distinct determinants, whose importance varies depending on the signal peptide in question. The data also show that the B. subtilis TatAdCd and E. coli TatABC systems recognize very similar determinants within their target peptides, and exhibit surprisingly similar responses to mutations within these determinants.     

The Chlamydia Type III Secretion System C-ring Engages a Chaperone-Effector Protein Complex

In Gram-negative bacterial pathogens, specialized chaperones bind to secreted effector proteins and maintain them in a partially unfolded form competent for translocation by type III secretion systems/injectisomes. How diverse sets of effector-chaperone complexes are recognized by injectisomes is unclear. Here we describe a new mechanism of effector-chaperone recognition by the Chlamydia injectisome, a unique and ancestral line of these evolutionarily conserved secretion systems. By yeast two-hybrid analysis we identified networks of Chlamydia-specific proteins that interacted with the basal structure of the injectisome, including two hubs of protein-protein interactions that linked known secreted effector proteins to CdsQ, the putative cytoplasmic C-ring component of the secretion apparatus. One of these protein-interaction hubs is defined by Ct260/Mcsc (Multiple cargo secretion chaperone). Mcsc binds to and stabilizes at least two secreted hydrophobic proteins, Cap1 and Ct618, that localize to the membrane of the pathogenic vacuole (“inclusion”). The resulting complexes bind to CdsQ, suggesting that in Chlamydia, the C-ring of the injectisome mediates the recognition of a subset of inclusion membrane proteins in complex with their chaperone. The selective recognition of inclusion membrane proteins by chaperones may provide a mechanism to co-ordinate the translocation of subsets of inclusion membrane proteins at different stages in infection.     

The signal peptide as a new target for drug design

Many current and potential drug targets are membrane-bound or secreted proteins that are expressed and transported via the Sec61 secretory pathway. They are targeted to translocon channels across the membrane of the endoplasmic reticulum (ER) by signal peptides (SPs), which are temporary structures on the N-termini of their nascent chains. During translation, such proteins enter the lumen and membrane of the ER by a process known as co-translational translocation. Small molecules have been found that interfere with this process, decreasing protein expression by recognizing the unique structures of the SPs of particular proteins. The SP may thus become a validated target for designing drugs for numerous disorders, including certain hereditary diseases.     

In Vitro Interaction of the Housekeeping SecA1 with the Accessory SecA2 Protein of Mycobacterium tuberculosis

The majority of proteins that are secreted across the bacterial cytoplasmic membrane leave the cell via the Sec pathway, which in its minimal form consists of the dimeric ATP-driven motor protein SecA that associates with the protein-conducting membrane pore SecYEG. Some Gram-positive bacteria contain two homologues of SecA, termed SecA1 and SecA2. SecA1 is the essential housekeeping protein, whereas SecA2 is not essential but is involved in the translocation of a subset of proteins, including various virulence factors. Some SecA2 containing bacteria also harbor a homologous SecY2 protein that may form a separate translocase. Interestingly, mycobacteria contain only one SecY protein and thus both SecA1 and SecA2 are required to interact with SecYEG, either individually or together as a heterodimer. In order to address whether SecA1 and SecA2 cooperate during secretion of SecA2 dependent proteins, we examined the oligomeric state of SecA1 and SecA2 of Mycobacterium tuberculosis and their interactions with SecA2 and the cognate SecA1, respectively. We conclude that both SecA1 and SecA2 individually form homodimers in solution but when both proteins are present simultaneously, they form dissociable heterodimers.     

A new Escherichia coli gene,fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

Type V secretion: From biogenesis to biotechnology

The two membranes of Gram-negative bacteria contain protein machines that have a general function in their assembly. To interact with the extra-cellular milieu, Gram-negatives target proteins to their cell surface and beyond. Many specialized secretion systems have evolved with dedicated translocation machines that either span the entire cell envelope or localize to the outer membrane. The latter act in concert with inner-membrane transport systems (i.e. Sec or Tat). Secretion via the Type V secretion system follows a two-step mechanism that appears relatively simple. Proteins secreted via this pathway are important for the Gram-negative life-style, either as virulence factors for pathogens or by contributing to the survival of non-invasive environmental species. Furthermore, this system appears well suited for the secretion of biotechnologically relevant proteins. In this review we focus on the biogenesis and application of two Type V subtypes, the autotransporters and two-partner secretion (TPS) systems. For translocation across the outer membrane the autotransporters require the assistance of the Bam complex that also plays a generic role in the assembly of outer membrane proteins. The TPS systems do use a dedicated translocator, but this protein shows resemblance to BamA, the major component of the Bam complex. Interestingly, both the mechanistic and more applied studies on these systems have provided a better understanding of the secretion mechanism and the biogenesis of outer membrane proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

The synthesis of hemolymph proteins by the larval epidermis of an insect Calpodes ethlius (Lepidoptera: Hesperiidae)

Sheets of the dorsal abdominal integument from fifth instar larvae of Calpodes ethlius (Lepidoptera: Hesperiidae) were incubated in artificial hemolymph in the presence of [³⁵S]methionine to investigate protein synthesis and vectorial secretion. The epidermis synthesizes and secretes at least 13 polypeptides basally and 15 apically. Two dimensional analysis of proteins labeled in vitro and in vivo showed that (a) most of the polypeptides secreted on apical and basal surfaces are different, (b) in vitro apical secretions are the same as in vivo cuticular proteins, (c) at least four of the basal secretions can be demonstrated in hemolymph labeled in vivo.Antibodies made against whole hemolymph recognized five basally secreted polypeptides and one apically secreted polypeptide both on fluorograms of immunoprecipitates and immunoblots. Arylphorin is secreted from both surfaces. Arylphorin synthesized in vitro has been identified through its precipitation by antibodies to hemolymph arylphorin in epidermis, cuticle and medium. We conclude that insect epidermis has bi-directional secretion. Cuticular proteins are carried to the apical face. A different set of proteins are carried basally to the hemolymph.     

Analysis of the Secretomes of Paracoccidioides Mycelia and Yeast Cells

Paracoccidioides, a complex of several phylogenetic species, is the causative agent of paracoccidioidomycosis. The ability of pathogenic fungi to develop a multifaceted response to the wide variety of stressors found in the host environment is important for virulence and pathogenesis. Extracellular proteins represent key mediators of the host-parasite interaction. To analyze the expression profile of the proteins secreted by Paracoccidioides, Pb01 mycelia and yeast cells, we used a proteomics approach combining two-dimensional electrophoresis with matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF MS/MS). From three biological replicates, 356 and 388 spots were detected, in mycelium and yeast cell secretomes, respectively. In this study, 160 non-redundant proteins/isoforms were indentified, including 30 and 24 proteins preferentially secreted in mycelia and yeast cells, respectively. In silico analyses revealed that 65% of the identified proteins/isoforms were secreted primarily via non-conventional pathways. We also investigated the influence of protein export inhibition in the phagocytosis of Paracoccidioides by macrophages. The addition of Brefeldin A to the culture medium significantly decreased the production of secreted proteins by both Paracoccidioides and internalized yeast cells by macrophages. In contrast, the addition of concentrated culture supernatant to the co-cultivation significantly increased the number of internalized yeast cells by macrophages. Importantly, the proteins detected in the fungal secretome were also identified within macrophages. These results indicate that Paracoccidioides extracellular proteins are important for the fungal interaction with the host.     

Specificity of Streptococcus pyogenes NAD(+) glycohydrolase in cytolysin-mediated translocation

The mechanism by which the cytolysin-mediated translocation (CMT) pathway of the Gram-positive pathogen Streptococcus pyogenes injects effector proteins into the cytosol of an infected host cell via the pore-forming protein streptolysin O is unknown. Key questions include whether the pathway can discriminate between different substrates for translocation, and whether the effector protein plays an active or passive role in the translocation process. Here we show that CMT can discriminate between a known effector of the pathway, the S. pyogenes NAD(+) glycohydrolase (SPN), and a second secreted protein, the mitogenic factor (MF), routing the former into the host cell cytosol and the latter into the extracellular milieu. Residues within the amino-terminal 190 residues of SPN were essential for discrimination, as deletions within this domain produced proteins that retained full enzymatic activity, but were completely uncoupled from the translocation pathway. The enzymatic domain itself played a pivotal role in the discrimination as deletions within this domain also produced translocation incompetent proteins and the conversion of MF to a translocation-competent form required fusion with both SPN domains in a contiguous orientation. These data establish that CMT is discriminatory, and that SPN is a multidomain protein that plays an active role in its translocation.