Signal peptides direct surface proteins to two distinct envelope locations of Staphylococcus aureus

Surface proteins of Gram-positive bacteria are covalently linked to the cell wall envelope by a mechanism requiring an N-terminal signal peptide and a C-terminal LPXTG motif sorting signal. We show here that surface proteins of Staphylococcus aureus arrive at two distinct destinations in the bacterial envelope, either distributed as a ring surrounding each cell or as discrete assembly sites. Proteins with ring-like distribution (clumping factor A (ClfA), Spa, fibronectin-binding protein B (FnbpB), serine-aspartate repeat protein C (SdrC) and SdrD) harbour signal peptides with a YSIRK/GS motif, whereas proteins directed to discrete assembly sites (S. aureus surface protein A (SasA), SasD, SasF and SasK) do not. Reciprocal exchange of signal peptides between surface proteins with (ClfA) or without the YSIRK/GS motif (SasF) directed recombinant products to the alternate destination, whereas mutations that altered only the YSIRK sequence had no effect. Our observations suggest that S. aureus distinguishes between signal peptides to address proteins to either the cell pole (signal peptides without YSIRK/GS) or the cross wall, the peptidoglycan layer that forms during cell division to separate new daughter cells (signal peptides with YISRK/GS motif).     

Transfer functions of the conjugative integrating element pSAM2 from Streptomyces ambofaciens: characterization of a kil-kor system associated with transfer.

pSAM2 is an 11-kb integrating element from Streptomyces ambofaciens. During matings, pSAM2 can be transferred at high frequency, forming pocks, which are zones of growth inhibition of the recipient strain. The nucleotide sequences of the regions involved in pSAM2 transfer, pock formation, and maintenance have been determined. Seven putative open reading frames with the codon usage typical of Streptomyces genes have been identified: traSA (306 amino acids [aa]), orf84 (84 aa), spdA (224 aa), spdB (58 aa), spdC (51 aa), spdD (104 aa), and korSA (259 aa). traSA is essential for pSAM2 intermycelial transfer and pock formation. It could encode a protein with similarities to the major transfer protein, Tra, of pIJ101. TraSA protein contains a possible nucleotide-binding sequence and a transmembrane segment. spdA, spdB, spdC, and spdD influence pock size and transfer efficiency and may be required for intramycelial transfer. A kil-kor system similar to that of pIJ101 is associated with pSAM2 transfer: the korSA (kil-override) gene product could control the expression of the traSA gene, which has lethal effects when unregulated (Kil phenotype). The KorSA protein resembles KorA of pIJ101 and repressor proteins belonging to the GntR family. Thus, the integrating element pSAM2 possesses for transfer general features of nonintegrating Streptomyces plasmids: different genes are involved in the different steps of the intermycelial and intramycelial transfer, and a kil-kor system is associated with transfer. However, some differences in the functional properties, organization, and sizes of the transfer genes compared with those of other Streptomyces plasmids have been found.     

The YSIRK-G/S motif of staphylococcal protein A and its role in efficiency of signal peptide processing

Many surface proteins of pathogenic gram-positive bacteria are linked to the cell wall envelope by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins of Streptococcus pneumoniae harbor another motif, YSIRK-G/S, which is positioned within signal peptides. The signal peptides of some, but not all, of the 20 surface proteins of Staphylococcus aureus carry a YSIRK-G/S motif, whereas those of surface proteins of Listeria monocytogenes and Bacillus anthracis do not. To determine whether the YSIRK-G/S motif is required for the secretion or cell wall anchoring of surface proteins, we analyzed variants of staphylococcal protein A, an immunoglobulin binding protein with an LPXTG sorting signal. Deletion of the YSIR sequence or replacement of G or S significantly reduced the rate of signal peptide processing of protein A precursors. In contrast, cell wall anchoring or the functional display of protein A was not affected. The fusion of cell wall sorting signals to reporter proteins bearing N-terminal signal peptides with or without the YSIRK-G/S motif resulted in hybrid proteins that were anchored in a manner similar to that of wild-type protein A. The requirement of the YSIRK-G/S motif for efficient secretion implies the existence of a specialized mode of substrate recognition by the secretion pathway of gram-positive cocci. It seems, however, that this mechanism is not essential for surface protein anchoring to the cell wall envelope.     

Unexpected link between lipooligosaccharide biosynthesis and surface protein release in Mycobacterium marinum

The mycobacterial cell envelope is characterized by the presence of a highly impermeable second membrane, which is composed of mycolic acids intercalated with different unusual free lipids, such as lipooligosaccharides (LOS). Transport across this cell envelope requires a dedicated secretion system for extracellular proteins, such as PE_PGRS proteins, which are specific mycobacterial proteins with polymorphic GC-rich sequence (PGRS). In this study, we set out to identify novel components involved in the secretion of PE_PGRS proteins by screening Mycobacterium marinum transposon mutants for secretion defects. Interestingly, most mutants were not affected in secretion but in the release of PE_PGRS proteins from the cell surface. These mutants had insertions in a gene cluster associated with LOS biosynthesis. Lipid analysis of these mutants revealed a role at different stages of LOS biosynthesis for 10 novel genes. Furthermore, we show that regulatory protein WhiB4 is involved in LOS biosynthesis. The absence of the most extended LOS molecule, i.e. LOS-IV, and a concomitant accumulation of LOS-III was already sufficient to reduce the release of PE_PGRS proteins from the mycobacterial cell surface. A similar effect was observed for major surface protein EspE. These results show that the attachment of surface proteins is strongly influenced by the glycolipid composition of the mycobacterial cell envelope. Finally, we tested the virulence of a LOS-IV-deficient mutant in our zebrafish embryo infection model. This mutant showed a marked increase in virulence as compared with the wild-type strain, suggesting that LOS-IV plays a role in the modulation of mycobacterial virulence.     

Staphylococcus aureus protein A mediates invasion across airway epithelial cells through activation of RhoA GTPase signaling and proteolytic activity

Staphyococcus aureus and especially the epidemic methicillin-resistant S. aureus strains cause severe necrotizing pneumonia. The mechanisms whereby these organisms invade across the mucosal epithelial barrier to initiate invasive infection are not well understood. Protein A (SpA), a highly conserved and abundant surface protein of S. aureus, activates TNF receptor 1 and EGF receptor (EGFR) signaling cascades that can perturb the cytoskeleton. We demonstrate that wild-type S. aureus, but not spa mutants, invade across polarized airway epithelial cell monolayers via the paracellular junctions. SpA stimulated a RhoA/ROCK/MLC cascade, resulting in the contraction of the cytoskeleton. SpA(+) but not SpA(-) mutants stimulated activation of EGFR and along with subsequent calpain activity cleaved the membrane-spanning junctional proteins occludin and E-cadherin, facilitating staphylococcal transmigration through the cell-cell junctions. Treatment of polarized human airway epithelial monolayers with inhibitors of ROCK, EGFR, MAPKs, or calpain prevented staphylococcal penetration through the monolayers. In vivo, blocking calpain activity impeded bacterial invasion into the lung parenchyma. Thus, S. aureus exploits multiple receptors available on the airway mucosal surface to facilitate invasion across epithelial barriers.     

Isolation and properties of minB, a complex genetic locus involved in correct placement of the division site in Escherichia coli.

Mutation of Escherichia coli minicell locus (minB) results in aberrant placement of the division septum. In this paper we report the isolation and characterization of the minB locus. Replacement of the chromosomal minB+ allele by cloned minB sequences containing transposon insertions resulted in the minicell phenotype, indicating that minB+ function is required to maintain the normal division pattern. Paradoxically, overexpression of the locus also resulted in the minicell phenotype. The locus codes for several peptides whose expression is coordinately affected by transposon mutations that also eliminate minB+ function. A subset of the minB peptides is sufficient to prevent minicell formation in minB1 mutants or to induce minicell formation when overproduced in wild-type strains, implicating these peptides in the normal process of localization of the division site. The results indicate that minB is a complex locus whose expression must be maintained within certain limits to maintain the normal pattern of localization of the division septum.     

The use of Tn phoA in Erwinia amylovora to generate random fusions of alkaline phosphatase to extracytoplasmic proteins

Abstract: Tn phoA has been introduced with high efficiency into the chromosome of the phytopathogenic bacterium Erwinia amylovora . Transposition occurred with apparent randomness and single insertions were predominant. Fusion proteins were detected in cell envelope fractions from PhoA⁺ mutants and a range of alkaline phosphatase activities was onserved. The results provide the first evidence that sequences encoding signal peptides are present in E. amylovora and that Tn phoA may be a valuable tool for the study of the translocation, regulation and function of E. amylovora extracytoplasmic proteins. In particular, Tn phoA mutagenesis should be applicable to the identification of cell envelope proteins involved in the virulence of this organism.     

Characterization of novel Mycobacterium tuberculosis and Mycobacterium smegmatis mutants hypersusceptible to beta-lactam antibiotics

Our laboratory previously constructed mutants of Mycobacterium tuberculosis and Mycobacterium smegmatis with deletions in the genes for their major beta-lactamases, BlaC and BlaS, respectively, and showed that the mutants have increased susceptibilities to most beta-lactam antibiotics, particularly the penicillins. However, there is still a basal level of resistance in the mutants to certain penicillins, and the susceptibilities of the mutants to some cephalosporin-based beta-lactams are essentially the same as those of the wild types. We hypothesized that characterizing additional mutants (derived from beta-lactamase deletion mutants) that are hypersusceptible to beta-lactam antibiotics might reveal novel genes involved with other mechanisms of beta-lactam resistance, peptidoglycan assembly, and cell envelope physiology. We report here the isolation and characterization of nine beta-lactam antibiotic-hypersusceptible transposon mutants, two of which have insertions in genes known to be involved with peptidoglycan biosynthesis (ponA2 and dapB); the other seven mutants have insertions which affect novel genes. These genes can be classified into three groups: those involved with peptidoglycan biosynthesis, cell division, and other cell envelope processes. Two of the peptidoglycan-biosynthetic genes (ponA2 and pbpX) may encode beta-lactam antibiotic-resistant enzymes proposed to be involved with the synthesis of the unusual diaminopimelyl linkages within the mycobacterial peptidoglycan.     

Cell wall antibiotics provoke accumulation of anchored mCherry in the cross wall of Staphylococcus aureus

A fluorescence microscopy method to directly follow the localization of defined proteins in Staphylococcus was hampered by the unstable fluorescence of fluorescent proteins. Here, we constructed plasmid (pCX) encoded red fluorescence (RF) mCherry (mCh) hybrids, namely mCh-cyto (no signal peptide and no sorting sequence), mCh-sec (with signal peptide), and mCh-cw (with signal peptide and cell wall sorting sequence). The S. aureus clones targeted mCh-fusion proteins into the cytosol, the supernatant and the cell envelope respectively; in all cases mCherry exhibited bright fluorescence. In staphylococci two types of signal peptides (SP) can be distinguished: the +YSIRK motif SP(lip) and the -YSIRK motif SP(sasF). mCh-hybrids supplied with the +YSIRK motif SP(lip) were always expressed higher than those with -YSIRK motif SP(sasF). To study the location of the anchoring process and also the influence of SP type, mCh-cw was supplied on the one hand with +YSIRK motif (mCh-cw1) and the other hand with -YSIRK motif (mCh-cw2). MCh-cw1 preferentially localized at the cross wall, while mCh-cw2 preferentially localized at the peripheral wall. Interestingly, when treated with sub-lethal concentrations of penicillin or moenomycin, both mCh-cw1 and mCh-cw2 were concentrated at the cross wall. The shift from the peripheral wall to the cross wall required Sortase A (SrtA), as in the srtA mutant this effect was blunted. The effect is most likely due to antibiotic mediated increase of free anchoring sites (Lipid II) at the cross wall, the substrate of SrtA, leading to a preferential incorporation of anchored proteins at the cross wall.     

Comprehensive Mutational Analysis of the Moloney Murine Leukemia Virus Envelope Protein

The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the amino-terminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the amino-terminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.     

Transposon insertions in the Flavobacterium johnsoniae ftsX gene disrupt gliding motility and cell division

Flavobacterium johnsoniae is a gram-negative bacterium that exhibits gliding motility. To determine the mechanism of flavobacterial gliding motility, we isolated 33 nongliding mutants by Tn4351 mutagenesis. Seventeen of these mutants exhibited filamentous cell morphology. The region of DNA surrounding the transposon insertion in the filamentous mutant CJ101-207 was cloned and sequenced. The transposon was inserted in a gene that was similar to Escherichia coli ftsX. Two of the remaining 16 filamentous mutants also carried insertions in ftsX. Introduction of the wild-type F. johnsoniae ftsX gene restored motility and normal cell morphology to each of the three ftsX mutants. CJ101-207 appears to be blocked at a late stage of cell division, since the filaments produced cross walls but cells failed to separate. In E. coli, FtsX is thought to function with FtsE in translocating proteins involved in potassium transport, and perhaps proteins involved in cell division, into the cytoplasmic membrane. Mutations in F. johnsoniae ftsX may prevent translocation of proteins involved in cell division and proteins involved in gliding motility into the cytoplasmic membrane, thus resulting in defects in both processes. Alternatively, the loss of gliding motility may be an indirect result of the defect in cell division. The inability to complete cell division may alter the cell architecture and disrupt gliding motility by preventing the synthesis, assembly, or functioning of the motility apparatus.     

Staphylococcus aureus mutants with increased lysostaphin resistance

Staphylococcus simulans secretes lysostaphin, a bacteriolytic enzyme that specifically binds to the cell wall envelope of Staphylococcus aureus and cleaves the pentaglycine cross bridges of peptidoglycan, thereby killing staphylococci. The study of S. aureus mutants with resistance to lysostaphin-mediated killing has revealed biosynthetic pathways for cell wall assembly. To identify additional genes involved in cell wall envelope biosynthesis, we have screened a collection of S. aureus strain Newman transposon mutants for lysostaphin resistance. Bursa aurealis insertion in SAV2335, encoding a polytopic membrane protein with predicted protease domain, caused a high degree of lysostaphin resistance, similar to the case for a previously described femAB promoter mutant. In contrast to the case for this femAB mutant, transposon insertion in SAV2335, herein named lyrA (lysostaphin resistance A), did not cause gross alterations of cell wall cross bridges such as truncations of pentaglycine to tri- or monoglycine. Also, inactivation of LyrA in a methicillin-resistant S. aureus strain did not precipitate a decrease in beta-lactam resistance as observed for fem (factor essential for methicillin resistance) mutants. Lysostaphin bound to the cell wall envelopes of lyrA mutants in a manner similar to that for wild-type staphylococci. Lysostaphin resistance of lyrA mutants is attributable to altered cell wall envelope properties and may in part be due to increased abundance of altered cross bridges. Other lyr mutants with intermediate lysostaphin resistance carried bursa aurealis insertions in genes specifying GTP pyrophosphokinase or enzymes of the purine biosynthetic pathway.     

Is there evidence for a common amino acid sequence in proteins with membrane attaching ability?

A comparison between the primary sequence of staphylococcal protein A (SpA) and a wide range of published protein sequences revealed a limited, but striking homology in approximately two-thirds of them. The region in the SpA sequence with the common homology was identified as the octapeptide repeats comprising the cell wall peptidoglycan-binding domain. Available structural information and the known location of the proteins within their host cells suggests that this common octapeptide may be important in interaction of the protein with the cell surface (either membrane or wall).     

Genome-wide identification of Mycobacterium tuberculosis exported proteins with roles in intracellular growth

The exported proteins of Mycobacterium tuberculosis that are localized at the bacterial cell surface or secreted into the environment are ideally situated to interact with host factors and to function in virulence. In this study, we constructed a novel β-lactamase reporter transposon and used it directly in M. tuberculosis for genome-wide identification of exported proteins. From 177 β-lactam-resistant transposon mutants, we identified 111 different exported proteins. The majority of these proteins have no known function, and for nearly half of the proteins, our demonstration that they are exported when fused to a β-lactamase reporter is the first experimental proof of their extracytoplasmic localization. The transposon mutants in our banked library were of further value as a collection of mutants lacking individual exported proteins. By individually testing each of 111 mutants for growth in macrophages, six attenuated mutants with insertions in mce1A, mce1B, mce2F, rv0199, ctaC, and lppX were identified. Given that much of the M. tuberculosis genome encodes proteins of unknown function, our library of mapped transposon mutants is a valuable resource for efforts in functional genomics. This work also demonstrates the power of a β-lactamase reporter transposon that could be applied similarly to other bacterial pathogens.     

Redirecting retroviral tropism by insertion of short, nondisruptive peptide ligands into envelope

A potentially powerful approach for in vivo gene delivery is to target retrovirus to specific cells through interactions between cell surface receptors and appropriately modified viral envelope proteins. Previously, relatively large (>100 residues) protein ligands to cell surface receptors have been inserted at or near the N terminus of retroviral envelope proteins. Although viral tropism could be altered, the chimeric envelope proteins lacked full activity, and coexpression of wild-type envelope was required for production of transducing virus. Here we analyze more than 40 derivatives of ecotropic Moloney murine leukemia virus (MLV) envelope, containing insertions of short RGD-containing peptides, which are ligands for integrin receptors. In many cases pseudotyped viruses containing only the chimeric envelope protein could transduce human cells. The precise location, size, and flanking sequences of the ligand affected transduction specificity and efficiency. We conclude that retroviral tropism can be rationally reengineered by insertion of short peptide ligands and without the need to coexpress wild-type envelope.     

Mutational Analysis of the Fusion Peptide of Moloney Murine Leukemia Virus Transmembrane Protein p15E

Fusion peptides are hydrophobic sequences located at the N terminus of the transmembrane (TM) envelope proteins of the orthomyxoviruses and paramyxoviruses and several retroviruses. The Moloney murine leukemia virus TM envelope protein, p15E, contains a hydrophobic stretch of amino acids at its N terminus followed by a region rich in glycine and threonine residues. A series of single amino acid substitutions were introduced into this region, and the resulting proteins were examined for their abilities to be properly processed and transported to the cell surface and to induce syncytia in cells expressing the ecotropic receptor. One substitution in the hydrophobic core and several substitutions in the glycine/threonine-rich region that prevented both cell-cell fusion and the transduction of NIH 3T3 cells when incorporated into retroviral vector particles were identified. In addition, one mutation that enhanced the fusogenicity of the resulting envelope protein was identified. The fusion-defective mutants trans dominantly interfered with the ability of the wild-type envelope protein to cause syncytium formation in a cell-cell fusion assay, although no trans-dominant inhibition of transduction was observed. Certain substitutions in the hydrophobic core that prevented envelope protein processing were also found. These data indicate that the N-terminal region of p15E is important both for viral fusion and for the correct processing and cell surface expression of the viral envelope protein.     

Distribution of protein A on the surface of Staphylococcus aureus

Surface proteins of Staphylococcus aureus fulfill many important roles during the pathogenesis of human infections and are anchored to the cell wall envelope by sortases. Although the chemical linkage of proteins to cell wall cross bridges is known, the mechanisms whereby polypeptides are distributed on the staphylococcal surface have not been revealed. We show here that protein A, the ligand of immunoglobulin, is unevenly distributed over the staphylococcal surface. Upon removal with trypsin, newly synthesized polypeptide is deposited at two to four discrete foci. During subsequent growth, protein A appears to be slowly distributed from these sites. When viewed through multiple focal planes by laser scanning microscopy, protein A foci are arranged in a circle surrounding the bacterial cell. This pattern of distribution requires the LPXTG sorting signal of protein A as well as sortase A, the transpeptidase that anchors polypeptides to cell wall cross bridges. A model is presented whereby protein A deposition at discrete sites coupled with cell wall synthesis enables distribution of protein A on the staphylococcal surface.     

Bap, a Staphylococcus aureus surface protein involved in biofilm formation

Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing Staphylococcus aureus isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (bap [for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (Pseudomonas aeruginosa and Salmonella enterica serovar Typhi) and gram-positive (Enteroccocus faecalis) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids. bap was present in a small fraction of bovine mastitis isolates (5% of the 350 S. aureus isolates tested), but it was absent from the 75 clinical human S. aureus isolates analyzed. All staphylococcal isolates harboring bap were highly adherent and strong biofilm producers. In a mouse infection model bap was involved in pathogenesis, causing a persistent infection.