Staphylococcal Adhesion and Host Cell Invasion: Fibronectin-Binding and Other Mechanisms

1Staphylococci are commensal bacteria that make up a large part of the microbiota of skin and mucous membranes. In pathogenic conditions, they cause opportunistic and life-threatening infections such as pneumonia, endocarditis, bone and joint infections, and sepsis. So far, 47 species and 23 sub-species of staphylococci have been identified, of which a few are coagulase-positive, such as S. aureus or S. pseudintermedius, but most are coagulase-negative species (CoNS), e.g., S. epidermidis, S. lugdunensis, S. saprophyticus, or S. haemolyticus. Like most bacteria, staphylococci express a broad range of surface proteins involved in their adhesion to extracellular matrix (ECM), plasma proteins or directly to host cells. The most prevalent of these proteins are the microbial surface component recognizing adhesive matrix molecules (MSCRAMMs), also found in enterococci and streptococci. All MSCRAMMs share a similar structure, with two adjacent IgG-folded domains mediating their attachment to components of the host ECM such as collagen, fibrinogen, or Fn. This binding capacity is closely related to the pathogenicity of staphylococci since their adherence to ECM or plasma proteins is a crucial step in the formation of biofilm and in the invasion of host cells. In this review, we discuss the mechanisms of cell adherence and internalization of coagulase-positive and -negative staphylococci. The main focus will be on the role of the ECM protein Fn and staphylococcal Fn-binding proteins (FnBPs) in the adhesion to and invasion of non-professional phagocytic cells (NPPCs) such as epithelial cells, endothelial cells, fibroblasts, and osteoblasts.

2The capacity of S. aureus to adhere to cells has been known since the early 1980s and has been demonstrated for both primary cells and cell lines from various tissues. Host cell adhesion mainly involves Fn forming a bridge between α5β1 integrin on the cellular side and Fn binding proteins (FnBPs, which are MSCRAMMs) on the bacteria. This step is a prerequisite for any internalization into NPPCs; indeed, DU5883, an isogenic mutant of S. aureus NCTC 8325-4 defective in FnBP expression, cannot invade NPPCs.

3There are two FnBP isoforms in S. aureus, FnBPA and FnBPB, encoded respectively by the fnbA and fnbB loci, with very similar domain organizations and sequences. However, their presence varies across the population. They consist of an amino-terminal secretion signal sequence followed by an A domain that is closely related to fibrinogen-binding protein clumping factor A (ClfA, Figure 1A). This domain can bind fibrinogen and elastin and is involved in Fn binding. The A domains of FnBPA and FnBPB only share 40% sequence identity. The A domain is followed by tandem repeats of Fn-binding regions (FnBRs, 95% identity between FnBPA and FnBPB), 11 in FnBPA versus 10 in FnBPB (Figure 1A). This additional FnBR, along with the higher Fn affinity of certain FnBRs, might explain FnBPA’s higher overall affinity for Fn and the fact that FnBA alone is sufficient for adhesion and cell invasion. Finally, the C-terminal peptidoglycan-binding motif (LPXTG) and the wall and membrane spanning domains anchor FnBPs to the cell wall.

4In vivo, the deletion of either FnBP strongly attenuates the ability of strain SH1000 to colonize the kidney and multiply, and to cause fatal sepsis in mice. Although FnBPA contributes more to these symptoms than FnBPB does, both FnBPs appear necessary for the development of severe infections. This observation suggests that cooperation between the two FnBPs may be required for strong cell adhesion or to efficiently trigger the internalization pathway in vivo.

5The high affinity and specificity of FnBPs for Fn is conferred by the tandem β-zipper structure they form together. Fn bridges S. aureus and the host cell through its binding to cellular α5β1 integrin. The efficiency of this adhesion varies both between cell types—one high-affinity FnBR is sufficient for adhesion and invasion in endothelial cells but three are needed in keratinocytes—and S. aureus strains. Indeed, whereas studies have shown that isolates from infections of cardiovascular devices have amino acid changes in the FnBR domain that increase the affinity for Fn, this is not the case for isolates from prosthetic joint infections. Likewise, certain methicillin-resistant S. aureus strains involved in endovascular infections harbor an additional FnBR that, together with a substitution in FnBR 11, reduces its affinity for Fn but promotes cell invasion. Although this fits with the hypothesis that the pathogenesis of endovascular infections involves the invasion of endothelial cells, it seems to go against the idea that better adhesion promotes internalization. However, it is estimated that one FnBP can bind 6–9 Fn molecules and thus could cluster α5β1 integrins on the cell surface to trigger the efficient intracellular signaling required for internalization. One hypothesis is that when affinity for Fn decreases, more interactions are needed to achieve sufficient cell adhesion, mobilizing more Fn and α5β1 integrins and thus leading to their clustering.

6The signaling pathway of staphylococci internalization involves focal adhesion kinases (FAKs) and activated Src that subsequently recruit cortactin to promote actin polymerization and mobilize the endocytic machinery (Figure 1B). Downstream of the FAK-Src pathway, the activation of PI3K and Akt is also important for the internalization of S. aureus, although the molecular pathway is still far from being fully understood. Previous studies have shown that internalization is inhibited by cytochalasin D and is temperature dependent (, specifically, inhibited at 4 and 14°C and facilitated at 37°C versus room temperature). These results respectively show that the biological prerequisites for internalization are (1) a dynamic actin cytoskeleton and (2) a fluid host cell membrane. Since bacterial uptake can occur with heat-killed or fixed bacteria, this mechanism appears to be an active process on the cellular side only. However, it has also been shown that S. aureus can stimulate its own uptake by upregulating β1 integrin expression in the host cell by secreting α-hemolysin. In this case, the bacteria must be viable as the process requires both cellular and bacterial activity.

7In summary, S. aureus adhere to cells via interactions between FnBPs, Fn, and α5β1 integrins. The resulting clustering of integrins may then be sufficient to trigger the signaling cascade involving FAK, Src, PI3K, and Akt. Finally, S. aureus mobilizes the actin cytoskeleton and possibly the endocytosis machinery to enter host cells (Figure 1).

8The FnBP-Fn-α5β1 integrin pathway is widely acknowledged to be the main internalization process. However, other factors have been shown to affect the efficiency of this internalization. In epithelial cells indeed, the internalization efficiency is maximal when the bacterial FnBPs interact directly with Hsp60 at the cell surface. Whether Hsp60 acts as a co-receptor to strengthen the binding of FnBPs-Fn to α5β1 integrin or is involved in signal transduction has still to be investigated (Figure 1C). Another clue that the FnBP-Fn-α5β1 integrin pathway is not the only internalization mechanism is that blocking α5β1 integrin or the binding of Fn by FnBPs with an antibody does not fully prevent internalization by MG63 osteoblast cells and by primary keratinocytes. The latter even internalize the FnBP-defective strain DU5883. Together, these results suggest that alternative mechanisms are involved in the adhesion and internalization of S. aureus (Table 1).

9These mechanisms mainly involve bacterial serine aspartate repeat-containing protein D (SdrD), clumping factor A (ClfA), autolysin (Atl), and serine-rich adhesin for platelets (SraP). These proteins are MSCRAMMs and (except for Atl) have the cell-wall anchoring sequence LPXTG located in their C-terminal portion. Their N-terminal part contains a signal peptide for their secretion, followed by a ligand binding region mainly consisting of repeated sequences often rich in Serine. The mechanisms involving these proteins are Fn-independent: SdrD binds directly to Desmoglein 1 on the cell surface of keratinocytes and desquamated nasal cells; ClfA interacts directly with host cells or through fibrinogen bridges; Atl seems to mediate S. aureus internalization via direct interactions with Hsc70; and SraP adheres to A549 cells through the salivary scavenger protein gp340 (Figure 1C). Note that the SdrD and Atl mechanisms require both bacterial and cellular activity as their efficiency depends on the expression of the bacterial and of the cellular interactor. Indeed, SdrD expression is upregulated following contact with neutrophils and Hsc70 production is stimulated by S. aureus infection of EA.hy 926 cells. Moreover, Atl has also been shown to be involved in the secretion of several S. aureus proteins, among which SdrD. Atl could thus have an “active” role as a secondary mechanism of internalization in some cells and act as a regulator of those secondary mechanisms in other cell types. More generally, these observations might reflect the fact that the bacteria can adapt their internalization strategy to the environmental conditions, i.e., an absence or scarcity of Fn, by finding alternative binding partners and/or by upregulating one side or the other of the adhesion machinery (e.g., SdrD and Atl). For instance, ClfA binds to annexin 2 on the surface of bovine mammary epithelial cells (MAC-T cells) but has two receptors on the surface of endothelial cells, namely αvβ3 integrin using fibrinogen as a bridge and von Willebrand factor using self-secreted von Willebrand factor binding protein as a bridge (Table 1 and Figure 1C). This might increase its capacity to adhere to and possibly enter endothelial cells, thereby supporting its role in the pathogenesis of endocarditis. Fibrinogen binding also primes platelets aggregation and abscess formation and can lead to thromboembolic lesions in the heart during sepsis.

10In other cases, the alternative mechanisms may support the FnBP-Fn-α5β1 integrin-mediated uptake of S. aureus instead. This is notably illustrated by S. aureus extracellular adherence protein (Eap) that plays a role in the adherence to fibroblasts and epithelial cells independently of any binding to Fn or fibrinogen (Figure 1C). Recently, its deletion has been shown not to affect the adhesion and internalization steps, contradicting the early idea that its role was to compensate for FnBP function loss. Rather, Eap seems to promote the adhesion and internalization of S. aureus and other pathogenic bacteria encountered in the context of polymicrobial skin infection. As for all the previously cited mechanisms, the molecular and signaling pathways underlying this activity are not understood yet and more work is needed to clarify whether Eap (1) is secreted and binds to α5β1 integrin via Fn, triggering the internalization cascade pathway and enhancing the uptake of bacteria bound to molecules other than α5β1 integrin in cells that express this integrin poorly; or (2) triggers actin-dependent phagocytosis, as its effect is fully blocked by cytochalasin D.

11The need to elucidate the molecular and signaling pathways of these alternative mechanisms is highlighted by their phenotypical relevance. ClfA and SraP are involved in the pathogenesis of endocarditis. SdrD-mediated binding appears to help the bacteria survive in vivo—whether this is because of an increased ability to kill neutrophils and/or to invade cells is an open question. Also, unraveling whether internalization occurs via the same FAK/Src/PI3K/Akt cascade as for the FnBP-Fn-β1 integrin pathway is essential as this may offer a potential avenue to treat chronic infections.

12The minor mechanism involving GapC during the adhesion to and invasion of MAC-T cells is also noteworthy as it has been involved in the development of mastitis. Finally, another point to bear in mind is that in addition to adhesins, other (probably less expected) molecules are involved in the adhesion to and invasion of NPPCs by S. aureus. In particular, the Tet38 efflux pump extrudes tetracycline and unsaturated free fatty acids but also interacts with CD36, a cellular transporter of long chain fatty acids, to trigger S. aureus adhesion and entry into A549 cells. Tet38 is also involved in phagosomal escape, facilitating the replication and persistence of S. aureus.

13Staphylococcus pseudintermedius is a coagulase-positive species mostly responsible for infections in dogs, notably necrotic skin lesions and bone and joint infections. However, human infections with S. pseudintermedius have also been described, revealing a risk of zoonotic transmission. Recently, performed a comparative study examining various CoNS species and reported that S. pseudintermedius was the only species that significantly adheres to human Fn and invades MG-63 cells with higher rates than S. aureus. This internalization does not occur in β1 integrin-deficient murine osteoblasts and is mediated through SpsD and SpsL, two cell wall-anchored proteins that share homologies with S. aureus FnBPA and FnBPB.

14The ability of S. epidermidis and other CoNS to bind fibronectin and to be internalized has also been investigated. The results of some of these studies and their interpretation are controversial however.

15The first study of Fn binding in S. epidermidis and other CoNS species found that S. epidermidis was a good Fn binder. However, this study also revealed huge variations in binding activity between S. epidermidis strains and between CoNS species. Later, reported the presence of FnBP genes in S. epidermidis and other CoNS using PCR inside a repeat unit region (D1–D4) found in both fnbA and fnbB from S. aureus (Figure 1A). Still, no FnBP-like protein has clearly been identified in S. epidermidis so far and no other study has found FnBPs at genomic, transcriptomic, or protein levels in CoNS. In S. epidermidis, the giant extracellular matrix binding protein (Embp) has been shown to bind Fn. Embp harbors 59 “Found In Various Architectures” (FIVAR) domains, involved in Fn binding, and 38 protein G-related albumin-binding (GA). A recombinant protein containing the Fn-binding domain of Embp blocks S. epidermidis binding to Fn but not that of S. aureus. Conversely, the competitive use of recombinant FnBPB (D1–D4 units) has been shown to block the binding of S. aureus, but not of S. epidermidis, to Fn. S. epidermidis Embp and S. aureus FnBPs must therefore have distinct Fn interaction sites.

16The internalization of S. epidermidis (and that of other CoNS) by NPPCs is a more controversial issue. While several studies have reported that S. epidermidis is internalized by different types of NPPCs, namely endothelial cells, MAC-T cells and human osteoblast-like MG-63 cells, others have minimized its ability to invade MG-63 cells. Indeed, using appropriate infection conditions, and found that even with a multiplicity of infection (MOI) exceeding 500:1, the rate of S. epidermidis internalization 2 h after infection was very low. (The rate of S. epidermidis internalization at an MOI 500:1 was about 100 times lower than that of S. aureus at an MOI of 100:1.) Although the significance of such a low level of invasion by S. epidermidis remains unclear, the uptake can happen and the following mechanisms have been proposed and identified to explain this.

17One tempting hypothesis is that the internalization of S. epidermidis by NPPCs occurs through a tripartite Embp-Fn-α5β1 system analogous to the FnBP-Fn-α5β1 integrin process for S. aureus. Using a recombinant protein that blocks the Fn-binding domain of S. aureus FnBP, completely inhibited the internalization of S. aureus but failed to block that of S. epidermidis. This fits with there being no FnBP in S. epidermidis, as discussed below, but does not refute the “Embp/Fn/α5β1 integrin” hypothesis. However, the use of an anti-α5β1 integrin antibody was found to block the internalization of S. aureus but not that of S. epidermidis by MG-63 cells. The uptake of S. epidermidis is therefore independent of the α5β1 integrin, which rules out the “Embp-Fn-α5β1 integrin” system and supports the intervention of secondary mechanisms.

18In CoNS, the only internalization mechanism that has been described to date involves Atl. As mentioned above for Atl in S. aureus, S. epidermidis is internalized through direct interactions between AtlE and Hsc70. This would seem to be the logical alternative mechanism for internalization as the other CoNS also have autolysins. However, existing studies only describe an ability to bind to Fn through autolysin and/or become internalized but do not offer a mechanistic explanation (Table 1). It is also noteworthy that the involvement in the internalization process of the fibrinogen-binding protein Fbl produced in S. lugdunensis, a homolog of S. aureus ClfA, has also been investigated, with negative results.

19As this short review shows, adhesion to fibronectin is a major explanation for the virulence of staphylococci. In S. aureus and S. pseudintermedius, adhesion involves fibronectin and α5β1 integrin and leads to internalization in host cells, which favors intracellular persistence and chronic infections. There are alternative mechanisms through which staphylococci become internalized and these may explain why in the absence of a major Fn-related internalization mechanism, S. epidermidis still invades cells, albeit at a low level. It is not yet clear whether these secondary mechanisms are completely independent of the FnBP-Fn-α5β1 integrin pathway (i.e., substitute it) or if they support it by strengthening binding or by triggering internalization more effectively. Identifying (1) the detailed molecular mechanisms of the FnBP-Fn-α5β1 pathway, (2) the surface proteins involved in alternative mechanisms, and (3) the role of these mechanisms, would be major steps toward more efficient anti-bacterial treatments for chronic staphylococci infections.

20JJ and AD prepared the draft of the papers with the help of FL. FL revised the version of the text.

21The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.