Src kinase has a central role in in vitro cellular internalization of Staphylococcus aureus

Traditionally recognized as an extracellular pathogen, the Gram-positive bacterium Staphylococcus aureus can also be internalized by a variety of cell types in vitro. Internalization is known to involve binding of the host extracellular protein fibronectin to the bacterium, recognition of the fibronectin-coated bacterium by the fibronectin-binding integrin alpha5beta1 on the host cell surface, and integrin-mediated internalization. Here we examine elements of mammalian cell signalling pathways involved in S. aureus internalization. The mouse fibroblast cell line GD25, in which the gene encoding the beta1 integrin subunit is inactivated, has been complemented with a beta1 integrin cDNA encoding a tyrosine (Y) to phenylalanine (F) mutation in each of the two beta1 integrin intracellular NPXY motifs. This cell line, GD25beta1 A Y783/795F, is defective in migration on fibronectin coated surfaces and intracellular signalling activities involving the tyrosine kinase Src. GD25beta1 A Y783/795F cells have a decreased ability to internalize S. aureus compared to GD25beta1 A cells expressing wild-type beta1 integrins. Furthermore, using mouse embryo fibroblasts in which different members of the Src family kinases are genetically inactivated, we demonstrate that optimal internalization is dependent on expression of Src kinase. Interferon, which has been implicated in repression of the effects of the viral homologue of Src inhibits internalization of S. aureus indicating that internalization may be blocked by inhibitors of Src kinase function. We then demonstrate that Src family kinase specific inhibitors effectively block S. aureus internalization into HeLa cells leading to the conclusion that a function unique to Src is required for optimal internalization of S. aureus in vitro.     

Polymeric Immunoglobulin Receptor-mediated Invasion of Streptococcus pneumoniae into Host Cells Requires a Coordinate Signaling of SRC Family of Protein-tyrosine Kinases,ERK, and c-Jun N-terminal Kinase

Streptococcus pneumoniae are commensals of the human nasopharynx with the capacity to invade mucosal respiratory cells. PspC, a pneumococcal surface protein, interacts with the human polymeric immunoglobulin receptor (pIgR) to promote bacterial adherence to and invasion into epithelial cells. Internalization of pneumococci requires the coordinated action of actin cytoskeleton rearrangements and the retrograde machinery of pIgR. Here, we demonstrate the involvement of Src protein-tyrosine kinases (PTKs), focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinases (MAPK) in pneumococcal invasion via pIgR. Pharmacological inhibitors of PTKs and MAPKs and genetic interference with Src PTK and FAK functions caused a significant reduction of pIgR-mediated pneumococcal invasion but did not influence bacterial adhesion to host cells. Furthermore, pneumococcal ingestion by host cells induces activation of ERK1/2 and JNK. In agreement with activated JNK, its target molecule and DNA-binding protein c-Jun was phosphorylated. We also show that functionally active Src PTK is essential for activation of ERK1/2 upon pneumococcal infections. In conclusion, these data illustrate the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells.     

Sticky connections: extracellular matrix protein recognition and integrin-mediated cellular invasion by Staphylococcus aureus

Staphylococcus aureus is a leading cause of hospital-acquired and often persistent infections. A key feature of pathogenic S. aureus is the expression of an array of extracellular matrix-binding proteins. In particular, the fibronectin-binding proteins FnBP-A and FnBP-B afford the pathogen the ability to connect to cellular integrins and to trigger internalization into host cells. Recent work has highlighted the role of host cell invasion in the pathogenesis of S. aureus, the structure-function relationship of FnBPs, and the host factors required to allow bacterial uptake. Understanding the invasive capacity of S. aureus should open up new avenues to control this microorganism in diverse disease settings.     

Staphylococcus saprophyticus ATCC 15305 is internalized into human urinary bladder carcinoma cell line 5637

Invasion of bacteria into nonphagocytic host cells is an important pathogenicity factor for escaping the host defence system. Gram-positive organisms, for example Staphylococcus aureus and Listeria monocytogenes, are invasive in nonphagocytic cells, and this mechanism is discussed as an important part of the infection process. Uropathogenic Escherichia coli and Staphylococcus saprophyticus can cause acute and recurrent urinary tract infections as well as bloodstream infections. Staphylococcus saprophyticus shows strong adhesion to human urinary bladder carcinoma and Hep2 cells and expresses the 'Microbial Surface Components Recognizing Adhesive Matrix molecule' (MSCRAMM)-protein SdrI with collagen-binding activity. MSCRAMMs are responsible for adhesion and collagen binding in S. aureus and are discussed as an important pathogenicity factor for invasion. To investigate internalization in S. aureus, several fluorescence activated cell sorting (FACS) assays have been described recently. We used a previously described FACS assay, with slight modifications, in addition to an antibiotic protection assay and transmission electron microscopy to show that S. saprophyticus ATCC 15305 and the wild-type strain 7108 were internalized into the human urinary bladder carcinoma cell line 5637. The discovery of the internalization of S. saprophyticus may be an important step for understanding the pathogenicity of recurrent infections caused by this organism.     

Factors influencing the internalization of Staphylococcus aureus and impacts on the course of infections in humans

Staphylococcus aureus is the primary etiological agent of several human diseases. S. aureus has classically been considered an extracellular pathogen; however, recent evidence indicates that S. aureus invades and persists in non-professional phagocytes. Experiments demonstrate that actin microfilaments, microtubules, receptor-mediated endocytosis, and protein tyrosine kinases play important roles in the uptake of S. aureus. Fibronectin-binding proteins and beta-integrins are implicated as critical cell surface molecules associated with internalization of S. aureus by non-phagocytic cells. Following invasion of eukaryotic cells, S. aureus induces the release of cytokines that have the potential to exacerbate disease and induce apoptosis. Finally, S. aureus has the ability to persist inside host cells as small colony variants, a phenotype associated with persistent and recurrent infections.     

Alpha-toxin interferes with integrin-mediated adhesion and internalization of Staphylococcus aureus by epithelial cells

Staphylococcus aureus is an important human and animal pathogen. During infection, this bacterium is able to attach to and enter host cells by using its cell surface-associated factors to bind to the host's extracellular matrix (ECM) proteins. In this study, we determined that a protein exported by S. aureus, alpha-toxin, can interfere with the integrin-mediated adhesion and internalization of S. aureus by human lung epithelial cells (A549). The downregulation of alpha-toxin production significantly increased bacterial adhesion and invasion into the epithelial cells. In contrast, bacterial adhesion and invasion was inhibited by both overproduction of alpha-toxin and the addition of alpha-toxin to the culture medium. Moreover, our results showed that the quantitative effects on invasion closely parallel those of adherence. This suggests that the effect on invasion is probably secondary to, and a consequence of, the reduced adherence caused by alpha-toxin exposure. Specifically, we demonstrated that alpha-toxin interacts with the hosts' ECM protein's receptor, beta1-integrin, which indicates that beta1-integrin may be a potential receptor of alpha-toxin on epithelial cells. Taken together, our results indicate that exported alpha-toxin inhibits the adhesion and internalization of S. aureus by interfering with integrin-mediated pathogen-host cell interactions.     

The B lymphocyte adaptor molecule of 32 kilodaltons (Bam32) regulates B cell antigen receptor internalization

The B lymphocyte adaptor molecule of 32 kDa (Bam32) is an adaptor that plays an indispensable role in BCR signaling. In this study, we found that upon BCR ligation, Bam32 is recruited to the plasma membrane where it associates with BCR complexes and redistributes and internalizes with BCRs. BCR ligation induced colocalization of Bam32 with lipid rafts, clathrin, and actin filaments. An inhibitor of Src family protein tyrosine kinases (PTKs) blocked both BCR-induced tyrosine phosphorylation of Bam32 and BCR internalization. Moreover, BCR internalization is impaired in Bam32-/- and Lyn-/- cells, and expression of Bam32 with a mutation of its tyrosine phosphorylation site (Y139F) inhibited BCR internalization. These data suggest that Bam32 functions downstream of Src family PTKs to regulate BCR internalization. Bam32 deficiency does not affect tyrosine phosphorylation of clathrin or the association of clathrin with lipid rafts upon BCR cross-linking. However, BCR-induced actin polymerization is impaired in Bam32-/- cells. Collectively, these findings indicate a novel role of Bam32 in connecting Src family PTKs to BCR internalization by an actin-dependent mechanism.     

Construction and characterization of plasmid vectors for cloning in Staphylococcus aureus and Staphylococcus carnosus

Several plasmid vectors for cloning in Staphylococcus aureus and S. carnosus have been constructed and characterized. The chimeric plasmids are composed of parts of the following parental plasmids: The chloramphenicol-resistance plasmid, pC194, the tetracycline-resistance plasmid, pMK148, and the erythromycin-resistance plasmid, pE12. All the chimeric plasmids confer two selectable antibiotic-resistance markers on host cells. Insertional inactivation of the various antibiotic-resistance markers occurred at the BclI site of pE12, and the Sau96- or AvaII-site of pMK148; only a slight inactivation of the chloramphenicol-resistance marker occurred at the HaeIII-site of pC194. The chimeric plasmids pCT20 and pCE10 are both stable in S. aureus and S. carnosus. In addition, the hybrid plasmids of pCT20 and pCE10, containing lambda-DNA fragments in various restriction sites between 0.4 and 1.2 kb, are stably maintained. The inserted lambda-DNA fragments appear unchanged.     

Invasion of bone cells by Staphylococcus epidermidis

Bone implants infected with Staphylococcus epidermidis often require surgical intervention because of the failure of antibiotic treatment. The reasons why such infections are resistant to therapy are poorly understood. We have previously reported that another bacterium, Staphylococcus aureus, can invade bone cells and thereby evade antimicrobial therapy. In this study we have investigated the hypothesis that S. epidermidis can also invade bone cells and may therefore explain the difficulties of treating infections with this organism. We found that S. epidermidis was capable of invading bone cells but that there were significant strain dependent differences in this capacity. A recombinant protein encompassing the D1-D4 repeat region of S. aureus fibronectin-binding protein B completely inhibited internalization of S. aureus but failed to block internalization of S. epidermidis. Similarly a blocking antibody to alpha5beta1 integrin inhibited internalization of S. aureus by bone cells but had no effect on the uptake of S. epidermidis. Therefore unlike S. aureus, S. epidermidis does not gain entrance into bone cells through a fibronectin bridge between the alpha5beta1 integrin and a bacterial adhesin.     

Staphylococcus aureus alpha-toxin induces apoptosis in endothelial cells

The internalization of Staphylococcus aureus by cultured human umbilical vein endothelial cells was recently shown to induce apoptosis. We examined the role of alpha-toxin, a major pore-forming toxin secreted by S. aureus, in causing apoptosis in vitro. Purified alpha-toxin, at sublytic concentrations, induced apoptosis in endothelial cell monolayers. Comparisons of two alpha-toxin (hla)-positive S. aureus strains and their isogenic hla-deficient mutants in the invasion assay of endothelial cells demonstrated that the capacity to produce alpha-toxin was associated with a greater propensity for apoptosis in endothelial cells. These results demonstrate for the first time that expression of alpha-toxin during endothelial cell invasion by S. aureus enhances apoptosis.     

Expression of the S. aureus hysA gene in S. carnosus from a modified E. coli-staphylococcal shuttle vector

We have modified an E. coli-staphylococcal shuttle vector for use in the general cloning and expression of genes from pathogenic staphylococci in Staphylococcus carnosus. As S. carnosus is non-pathogenic, this expression system will facilitate the study of the roles of individual gene products in the disease process. To evaluate the use of this expression system, a DNA fragment containing the Staphylococcus aureus hyaluronate lyase (hysA) gene was cloned into the modified vector, pNW21, and introduced into S. carnosus. Hyaluronate lyase was both produced and secreted by S. carnosus. In addition, the secreted HysA protein was enzymatically active, as determined using a zymographic assay.     

Incorporation of carbon from decomposing litter of two pioneer plant species into microbial communities of the detritusphere

For several Staphylococci, such as Staphylococcus aureus, Staphylococcus saprophyticus, and Staphylococcus epidermidis, invasion of eukaryotic cells has been described and this mechanism has been considered an important part of the infection process. The fibrinogen-binding protein (Fbl) of Staphylococcus lugdunensis, a homolog of the clumping factor A of S. aureus, has been described as fibrinogen-binding adhesin and might promote invasion of cells. We therefore characterized several clinical strains of S. lugdunensis in terms of whole cell fibrinogen and fibronectin binding and correlated these results with the invasion of epithelial and endothelial cells by S. lugdunensis. We described for the first time invasion of cells by S. lugdunensis. As invasion of cells by S. lugdunensis was only partly inhibited by cytochalasin D in contrast to a complete inhibition of invasion of cells by S. aureus, further invasion mechanisms are likely to be present in S. lugdunensis. In addition, the Fbl of S. lugdunensis is not involved in the invasion of cells as ruled out by an isogenic fbl mutant.     

Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin alpha5beta1

The ability of Staphylococcus aureus to invade mammalian cells may explain its capacity to colonize mucosa and to persist in tissues after bacteraemia. To date, the underlying molecular mechanisms of cellular invasion by S. aureus are unknown, despite its high prevalence and difficulties in treatment. Here, we show cellular invasion as a novel function for an S. aureus adhesin, previously implicated solely in attachment. S. aureus , but not S. epidermidis , invaded epithelial 293 cells in a temperature- and F-actin-dependent manner. Formaldehyde-fixed and live bacteria were equally invasive, suggesting that no active bacterial process was involved. All clinical S. aureus isolates analysed, but only a subset of laboratory strains, were invasive. Fibronectin-binding proteins (FnBPs) acted as S. aureus invasins, because: (i) FnBP deletion mutants of invasive laboratory strains lost invasiveness; (ii) expression of FnBPs in non-invasive strains conferred invasiveness; and (iii) the soluble isolated fibronectin-binding domain of FnBP (D1–D4) completely blocked invasion. Integrin α₅β₁ served as host cell receptor, which interacted with staphylococcal FnBPs through cellular or soluble fibronectin. FnBP-deficient mutants lost invasiveness for epithelial cells, endothelial cells and fibroblasts. Thus, fibronectin-dependent bridging between S. aureus FnBPs and host cell integrin α₅β₁ is a conserved mechanism for S. aureus invasion of human cells. This may prove useful in developing new therapeutic and vaccine strategies for S. aureus infections.     

Staphylococcal surface display of immunoglobulin A (IgA)- and IgE-specific in vitro-selected binding proteins (affibodies) based on Staphylococcus aureus protein A.

An expression system designed for cell surface display of hybrid proteins on Staphylococcus carnosus has been evaluated for the display of Staphylococcus aureus protein A (SpA) domains, normally binding to immunoglobulin G (IgG) Fc but here engineered by combinatorial protein chemistry to yield SpA domains, denoted affibodies, with new binding specificities. Such affibodies, with human IgA or IgE binding activity, have previously been selected from a phage library, based on an SpA domain. In this study, these affibodies have been genetically introduced in monomeric or dimeric forms into chimeric proteins expressed on the surface of S. carnosus by using translocation signals from a Staphylococcus hyicus lipase construct together with surface-anchoring regions of SpA. The recombinant surface proteins, containing the IgA- or IgE-specific affibodies, were demonstrated to be expressed as full-length proteins, localized and properly exposed at the cell surface of S. carnosus. Furthermore, these chimeric receptors were found to be functional, since recombinant S. carnosus cells were shown to have gained IgA and IgE binding capacity, respectively. In addition, a positive effect in terms of IgA and IgE reactivity was observed when dimeric versions of the affibodies were present. Potential applications for recombinant bacteria with redirected binding specificity in their surface proteins are discussed.     

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system. Purification and protein sequencing of the Staphylococcus carnosus histidine-containing protein, and cloning and DNA sequencing of the ptsH gene

The histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Staphylococcus carnosus and purified to homogeneity. The protein sequence was determined by Edman degradation of peptides obtained by proteolytic digestion with proteases V8, trypsin and chemical cleavage with BrCN. Furthermore, immunological screening of a chromosomal S. carnosus DNA gene library in pUC19 vector enabled us to isolate S. carnosus HPr-expressing colonies. The nucleotide sequence of this ptsH gene and its flanking regions was determined by the dideoxy-chain-termination technique. Upstream, the 264-bp open reading frame of the ptsH gene is flanked by a putative S. carnosus promoter structure and a putative ptsI gene downstream suggesting that ptsH gene is the first gene in the PTS operon of S. carnosus. Comparison of the amino acid sequence of S. carnosus HPr with the HPr sequence of Staphylococcus aureus (derived from peptide sequencing) showed a high degree of similarity.     

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli

Enzyme IIImtl is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, we report the isolation of IIImtl from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of IIImtl with [32P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase Glu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which we assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the IIImtl proteins was found to be 15,000. We have also determined the N-terminal sequence of both proteins. Comparison of the IIImtl peptide sequences and the C-terminal part of the enzyme IImtl of Escherichia coli reveals considerable sequence homology, which supports the suggestion that IImtl of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II. In particular, the homology of the active-center peptide of IIImtl of S. aureus and S. carnosus with the enzyme IImtl of E. coli allows one to predict the N-3 histidine phosphorylation site within the E. coli enzyme.     

Staphylococcus aureus keratinocyte invasion is dependent upon multiple high-affinity fibronectin-binding repeats within FnBPA

Staphylococcus aureus is a commensal organism and a frequent cause of skin and soft tissue infections, which can progress to serious invasive disease. This bacterium uses its fibronectin binding proteins (FnBPs) to invade host cells and it has been hypothesised that this provides a protected niche from host antimicrobial defences, allows access to deeper tissues and provides a reservoir for persistent or recurring infections. FnBPs contain multiple tandem fibronectin-binding repeats (FnBRs) which bind fibronectin with varying affinity but it is unclear what selects for this configuration. Since both colonisation and skin infection are dependent upon the interaction of S. aureus with keratinocytes we hypothesised that this might select for FnBP function and thus composition of the FnBR region. Initial experiments revealed that S. aureus attachment to keratinocytes is rapid but does not require FnBRs. By contrast, invasion of keratinocytes was dependent upon the FnBR region and occurred via similar cellular processes to those described for endothelial cells. Despite this, keratinocyte invasion was relatively inefficient and appeared to include a lag phase, most likely due to very weak expression of α(5)β(1) integrins. Molecular dissection of the role of the FnBR region revealed that efficient invasion of keratinocytes was dependent on the presence of at least three high-affinity (but not low-affinity) FnBRs. Over-expression of a single high-affinity or three low-affinity repeats promoted invasion but not to the same levels as S. aureus expressing an FnBPA variant containing three high-affinity repeats. In summary, invasion of keratinocytes by S. aureus requires multiple high-affinity FnBRs within FnBPA, and given the importance of the interaction between these cell types and S. aureus for both colonisation and infection, may have provided the selective pressure for the multiple binding repeats within FnBPA.     

Host-parasite interactions in Staphylococcus aureus keratitis

Ulcerative keratitis is among the leading ocular bacterial infections, and Streptococcus aureus accounts for approximately 25% of cases in some surveys. Although S. aureus expresses numerous virulence factors, many of which are under the control of staphylococcal global regulatory genes, their pathophysiologic roles in keratitis are largely unknown. Similarly, the nature of the host response during S. aureus keratitis is unclear. Following a review of previously published research on the pathophysiology of S. aureus ocular infection, we present the results of a study designed to assess the host-parasite relationship between S. aureus and human corneal epithelial cells (HCECs) in vitro. In this model system, a wild-type S. aureus strain and its isogenic mutants harboring mutations in agr and sar global regulatory genes or fibronectin-binding proteins A and B (fnbAB) were tested for their ability to bind and invade confluent HCEC monolayers. The contribution of host cell factors was assessed by preincubating HCECs with various inhibitory agents. These studies demonstrated that S. aureus not only adhered to the surface of HCECs but was also internalized, as has been previously observed in other nonocular cell lines. Adherence and invasion of HCECs was saturable at 1 h of incubation in the presence of approximately 10(7) CFU per HCEC monolayer (multiplicity of infection approximately 10). A mutant defective in both agr and sar global regulators was not significantly different in invasive capacity compared to its isogenic wild-type parent strain. In contrast, mutations in fibronectin-binding proteins A and B (fnbAB) reduced the invasiveness of S. aureus by 99% compared to the wild-type strain. Pretreatment of HCECs with colchicine had little effect on S. aureus invasion. In sharp contrast, cytochalasin D and genistein were each capable of inhibiting invasion by >99%. In summary, the results of this study point to fibronectin-binding protein as a key S. aureus surface adhesin facilitating invasion of HCECs in vitro. Furthermore, these results suggest an active mechanism for S. aureus internalization by HCECs, likely involving actin polymerization and tyrosine kinase activity. Additional studies are warranted to determine the applicability of these findings in vivo, and to facilitate the rational design of therapeutic agents aimed at blocking the establishment and progression of S. aureus keratitis.     

Staphylococcus aureus subvert autophagy for induction of caspase-independent host cell death

Staphylococcus aureus is a common bacterial etiology of serious infectious diseases. S. aureus can invade various types of non-professional phagocytes to produce host cell death. We show here that shortly after invasion of HeLa cells S. aureus transit to autophagosomes was characterized by double membranes and co-localization with LC3. S. aureus were not able to replicate and produce cell death in autophagy-deficient atg5-/- mouse embryonic fibroblasts. S. aureus-containing autophagosomes do not acidify nor do they acquire lysosome-associated membrane protein-2, indicating that S. aureus inhibits autophagosome maturation and fusion with lysosomes. Eventually, S. aureus escape from autophagosomes into the cytoplasm, which results in caspase-independent host cell death. S. aureus strains deficient for agr, a global regulator of S. aureus virulence, were not targeted by autophagy and did not produce host-cell death. Autophagy induction by rapamycin restored both replication and cytotoxicity of agr-deficient S. aureus strains, indicating that an agr-regulated factor(s) is required for autophagy-mediated cytotoxicity. The results of this study suggest that rapid induction of autophagy is essential for S. aureus replication, escape into the cytoplasm, and host cell killing.