Integrin inactivators: balancing cellular functions in vitro and in vivo.

Integrins mediate cell-matrix and cell-cell interactions and integrate extracellular cues to the cytoskeleton and cellular signalling pathways. Integrin function on the cell surface is regulated by their activity switching such that intracellular proteins interacting with the integrin cytoplasmic domains increase or decrease integrin-ligand binding affinity. It is widely accepted that integrin activation by specific proteins is essential for cell adhesion and integrin linkage to the actin cytoskeleton. However, there is also increasing evidence that integrin-inactivating proteins are crucial for appropriate integrin function in vitro and in vivo and that the regulation of integrin-ligand interactions is a fine-tuned balancing act between inactivation and activation.

ICAP-1 loss impairs CD8+ thymocyte development and leads to reduced marginal zone B cells in mice.

ICAP-1 regulates ß1-integrin activation and cell adhesion. Here, we used ICAP-1-null mice to study ICAP-1 potential involvement during immune cell development and function. Integrin α4ß1-dependent adhesion was comparable between ICAP-1-null and control thymocytes, but lack of ICAP-1 caused a defective single-positive (SP) CD8+ cell generation, thus, unveiling an ICAP-1 involvement in SP thymocyte development. ICAP-1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP-1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP-1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP-1-/- spleen T and B cells displayed upregulation of α4ß1-mediated adhesion, indicating that ICAP-1 negatively controls their attachment. Furthermore, CD3+ - and CD19+ -selected spleen cells from ICAP-1-null mice showed reduced proliferation in response to T- and B-cell stimuli, respectively. Finally, loss of ICAP-1 caused a remarkable decrease in marginal zone B- cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP-1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B-cell numbers.

The mechano-sensitive response of ß1 integrin promotes SRC-positive late endosome recycling and activation of Yes-associated protein.

Yes-associated protein (YAP) signaling has emerged as a crucial pathway in several normal and pathological processes. Although the main upstream effectors that regulate its activity have been extensively studied, the role of the endosomal system has been far less characterized. Here, we identified the late endosomal/lysosomal adaptor MAPK and mTOR activator (LAMTOR) complex as an important regulator of YAP signaling in a preosteoblast cell line. We found that p18/LAMTOR1-mediated peripheral positioning of late endosomes allows delivery of SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) to the plasma membrane and promotes activation of an SRC-dependent signaling cascade that controls YAP nuclear shuttling. Moreover, ß1 integrin engagement and mechano-sensitive cues, such as external stiffness and related cell contractility, controlled LAMTOR targeting to the cell periphery and thereby late endosome recycling and had a major impact on YAP signaling. Our findings identify the late endosome recycling pathway as a key mechanism that controls YAP activity and explains YAP mechano-sensitivity.

ß1D integrin splice variant stabilizes integrin dynamics and reduces integrin signaling by limiting paxillin recruitment.

Heterodimeric integrin receptors control cell adhesion, migration and extracellular matrix assembly. While the α integrin subunit determines extracellular ligand specificity, the ß integrin chain binds to an acidic residue of the ligand, and cytoplasmic adapter protein families such as talins, kindlins and paxillin, to form mechanosensing cell matrix adhesions. Alternative splicing of the ß1 integrin cytoplasmic tail creates ubiquitously expressed ß1A, and the heart and skeletal muscle-specific ß1D form. To study the physiological difference between these forms, we developed fluorescent ß1 integrins and analyzed their dynamics, localization, and cytoplasmic adapter recruitment and effects on cell proliferation. On fibronectin, GFP-tagged ß1A integrin showed dynamic exchange in peripheral focal adhesions, and long, central fibrillar adhesions. In contrast, GFP-ß1D integrins exchanged slowly, forming immobile and short central adhesions. While adhesion recruitment of GFP-ß1A integrin was sensitive to C-terminal tail mutagenesis, GFP-ß1D integrin was recruited independently of the distal NPXY motif. In addition, a P786A mutation in the proximal, talin-binding NPXY783 motif switched ß1D to a highly dynamic integrin. In contrast, the inverse A786P mutation in ß1A integrin interfered with paxillin recruitment and proliferation. Thus, differential ß1 integrin splicing controls integrin-dependent adhesion signaling, to adapt to the specific physiological needs of differentiated muscle cells.

Phosphorylation of Merlin by Aurora A kinase appears necessary for mitotic progression.

Although Merlin's function as a tumor suppressor and regulator of mitogenic signaling networks such as the Ras/rac, Akt, and Hippo pathways is well-documented, in mammals as well as in insects, its role during cell cycle progression remains unclear. In this study, using a combination of approaches, including FACS analysis, time-lapse imaging, immunofluorescence microscopy, and co-immunoprecipitation, we show that Ser-518 of Merlin is a substrate of the Aurora protein kinase A during mitosis and that its phosphorylation facilitates the phosphorylation of a newly discovered site, Thr-581. We found that the expression in HeLa cells of a Merlin variant that is phosphorylation-defective on both sites leads to a defect in centrosomes and mitotic spindles positioning during metaphase and delays the transition from metaphase to anaphase. We also show that the dual mitotic phosphorylation not only reduces Merlin binding to microtubules but also timely modulates ezrin interaction with the cytoskeleton. Finally, we identify several point mutants of Merlin associated with neurofibromatosis type 2 that display an aberrant phosphorylation profile along with defective α-tubulin-binding properties. Altogether, our findings of an Aurora A-mediated interaction of Merlin with α-tubulin and ezrin suggest a potential role for Merlin in cell cycle progression.

Identification and Characterization of Staphylococcus delphini Internalization Pathway in Nonprofessional Phagocytic Cells.

The intracellular lifestyle of bacteria is widely acknowledged to be an important mechanism in chronic and recurring infection. Among the Staphylococcus genus, only Staphylococcus aureus and Staphylococcus pseudintermedius have been clearly identified as intracellular in nonprofessional phagocytic cells (NPPCs), for which the mechanism is mainly fibronectin-binding dependent. Here, we used bioinformatics tools to search for possible new fibronectin-binding proteins (FnBP-like) in other Staphylococcus species. We found a protein in Staphylococcus delphini called Staphylococcus delphini surface protein Y (SdsY). This protein shares 68% identity with the Staphylococcus pseudintermedius surface protein D (SpsD), 36% identity with S. aureus FnBPA, and 39% identity with S. aureus FnBPB. The SdsY protein possesses the typical structure of FnBP-like proteins, including an N-terminal signal sequence, an A domain, a characteristic repeated pattern, and an LPXTG cell wall anchor motif. The level of adhesion to immobilized fibronectin was significantly higher in all S. delphini strains tested than in the fibronectin-binding-deficient S. aureus DU5883 strain. By using a model of human osteoblast infection, the level of internalization of all strains tested was significantly higher than with the invasive-incompetent S. aureus DU5883. These findings were confirmed by phenotype restoration after transformation of DU5883 by a plasmid expression vector encoding the SdsY repeats. Additionally, using fibronectin-depleted serum and murine osteoblast cell lines deficient for the ß1 integrin, the involvement of fibronectin and ß1 integrin was demonstrated in S. delphini internalization. The present study demonstrates that additional staphylococcal species are able to invade NPPCs and proposes a method to identify FnBP-like proteins.

ß1 integrin-dependent Rac/group I PAK signaling mediates YAP activation of Yes-associated protein 1 (YAP1) via NF2/merlin.

Cell adhesion to the extracellular matrix or to surrounding cells plays a key role in cell proliferation and differentiation and is critical for proper tissue homeostasis. An important pathway in adhesion-dependent cell proliferation is the Hippo signaling cascade, which is coregulated by the transcription factors Yes-associated protein 1 (YAP1) and transcriptional coactivator with PDZ-binding motif (TAZ). However, how cells integrate extracellular information at the molecular level to regulate YAP1's nuclear localization is still puzzling. Herein, we investigated the role of ß1 integrins in regulating this process. We found that ß1 integrin-dependent cell adhesion is critical for supporting cell proliferation in mesenchymal cells both in vivo and in vitro ß1 integrin-dependent cell adhesion relied on the relocation of YAP1 to the nucleus after the down-regulation of its phosphorylated state mediated by large tumor suppressor gene 1 and 2 (LATS1/2). We also found that this phenotype relies on ß1 integrin-dependent local activation of the small GTPase RAC1 at the plasma membrane to control the activity of P21 (RAC1)-activated kinase (PAK) of group 1. We further report that the regulatory protein merlin (neurofibromin 2, NF2) interacts with both YAP1 and LATS1/2 via its C-terminal moiety and FERM domain, respectively. PAK1-mediated merlin phosphorylation on Ser-518 reduced merlin's interactions with both LATS1/2 and YAP1, resulting in YAP1 dephosphorylation and nuclear shuttling. Our results highlight RAC/PAK1 as major players in YAP1 regulation triggered by cell adhesion.

LKB1 signaling in cephalic neural crest cells is essential for vertebrate head development.

Head development in vertebrates proceeds through a series of elaborate patterning mechanisms and cell-cell interactions involving cephalic neural crest cells (CNCC). These cells undergo extensive migration along stereotypical paths after their separation from the dorsal margins of the neural tube and they give rise to most of the craniofacial skeleton. Here, we report that the silencing of the LKB1 tumor suppressor affects the delamination of pre-migratory CNCC from the neural primordium as well as their polarization and survival, thus resulting in severe facial and brain defects. We further show that LKB1-mediated effects on the development of CNCC involve the sequential activation of the AMP-activated protein kinase (AMPK), the Rho-dependent kinase (ROCK) and the actin-based motor protein myosin II. Collectively, these results establish that the complex morphogenetic processes governing head formation critically depends on the activation of the LKB1 signaling network in CNCC.

The RNA-binding protein Mex3b regulates the spatial organization of the Rap1 pathway.

The four related mammalian MEX-3 RNA-binding proteins are evolutionarily conserved molecules for which the in vivo functions have not yet been fully characterized. Here, we report that male mice deficient for the gene encoding Mex3b are subfertile. Seminiferous tubules of Mex3b-deficient mice are obstructed as a consequence of the disrupted phagocytic capacity of somatic Sertoli cells. In addition, both the formation and the integrity of the blood-testis barrier are compromised owing to mislocalization of N-cadherin and connexin 43 at the surface of Sertoli cells. We further establish that Mex3b acts to regulate the cortical level of activated Rap1, a small G protein controlling phagocytosis and cell-cell interaction, through the activation and transport of Rap1GAP. The active form of Rap1 (Rap1-GTP) is abnormally increased at the membrane cortex and chemically restoring Rap1-GTP to physiological levels rescues the phagocytic and adhesion abilities of Sertoli cells. Overall, these findings implicate Mex3b in the spatial organization of the Rap1 pathway that orchestrates Sertoli cell functions.

Calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII)-mediated intramolecular opening of integrin cytoplasmic domain-associated protein-1 (ICAP-1α) negatively regulates ß1 integrins.

Focal adhesion turnover during cell migration is an integrated cyclic process requiring tight regulation of integrin function. Interaction of integrin with its ligand depends on its activation state, which is regulated by the direct recruitment of proteins onto the ß integrin chain cytoplasmic domain. We previously reported that ICAP-1α, a specific cytoplasmic partner of ß1A integrins, limits both talin and kindlin interaction with ß1 integrin, thereby restraining focal adhesion assembly. Here we provide evidence that the calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII) is an important regulator of ICAP-1α for controlling focal adhesion dynamics. CaMKII directly phosphorylates ICAP-1α and disrupts an intramolecular interaction between the N- and the C-terminal domains of ICAP-1α, unmasking the PTB domain, thereby permitting ICAP-1α binding onto the ß1 integrin tail. ICAP-1α direct interaction with the ß1 integrin tail and the modulation of ß1 integrin affinity state are required for down-regulating focal adhesion assembly. Our results point to a molecular mechanism for the phosphorylation-dependent control of ICAP-1α function by CaMKII, allowing the dynamic control of ß1 integrin activation and cell adhesion.

Escherichia coli producing CNF1 toxin hijacks Tollip to trigger Rac1-dependent cell invasion.

Rho GTPases, which are master regulators of both the actin cytoskeleton and membrane trafficking, are often hijacked by pathogens to enable their invasion of host cells. Here we report that the cytotoxic necrotizing factor-1 (CNF1) toxin of uropathogenic Escherichia coli (UPEC) promotes Rac1-dependent entry of bacteria into host cells. Our screen for proteins involved in Rac1-dependent UPEC entry identifies the Toll-interacting protein (Tollip) as a new interacting protein of Rac1 and its ubiquitinated forms. We show that knockdown of Tollip reduces CNF1-induced Rac1-dependent UPEC entry. Tollip depletion also reduces the Rac1-dependent entry of Listeria monocytogenes expressing InlB invasion protein. Moreover, knockdown of Tollip, Tom1 and clathrin, decreases CNF1 and Rac1-dependent internalization of UPEC. Finally, we show that Tollip, Tom1 and clathrin associate with Rac1 and localize at the site of bacterial entry. Collectively, these findings reveal a new link between Rac1 and Tollip, Tom1 and clathrin membrane trafficking components hijacked by pathogenic bacteria to allow their efficient invasion of host cells.

Protein dynamics at invadopodia control invasion-migration transitions in melanoma cells.

Cell invasion is a highly complex process that requires the coordination of cell migration and degradation of the extracellular matrix. In melanoma cells, as in many highly invasive cancer cell types these processes are driven by the regulated formation of adhesives structures such as focal adhesions and invasive structures like invadopodia. Structurally, focal adhesion and invadopodia are quite distinct, yet they share many protein constituents. However, quantitative understanding of the interaction of invadopodia with focal adhesion is lacking, and how invadopodia turn-over is associated with invasion-migration transition cycles remains unknown. In this study, we investigated the role of Pyk2, cortactin and Tks5 in invadopodia turnover and their relation with focal adhesions. We found that active Pyk2 and cortactin are localised at both focal adhesions and invadopodia. At invadopodia, localisation of active Pyk2 is correlated with ECM degradation. During invadopodia disassembly, Pyk2 and cortactin but not Tks5 are often relocated at nearby nascent adhesions. We also show that during ECM degradation, cell migration is reduced which is likely related to the sharing of common molecules within the two structures. Finally, we found that the dual FAK/Pyk2 inhibitor PF-431396 inhibits both focal adhesion and invadopodia activities thereby reducing both migration and ECM degradation.

Structural and functional analysis of LIM domain-dependent recruitment of paxillin to αvß3 integrin-positive focal adhesions.

The LIM domain-dependent localization of the adapter protein paxillin to ß3 integrin-positive focal adhesions (FAs) is not mechanistically understood. Here, by combining molecular biology, photoactivation and FA-isolation experiments, we demonstrate specific contributions of each LIM domain of paxillin and reveal multiple paxillin interactions in adhesion-complexes. Mutation of ß3 integrin at a putative paxillin binding site (ß3VE/YA) leads to rapidly inward-sliding FAs, correlating with actin retrograde flow and enhanced paxillin dissociation kinetics. Induced mechanical coupling of paxillin to ß3VE/YA integrin arrests the FA-sliding, thereby disclosing an essential structural function of paxillin for the maturation of ß3 integrin/talin clusters. Moreover, bimolecular fluorescence complementation unveils the spatial orientation of the paxillin LIM-array, juxtaposing the positive LIM4 to the plasma membrane and the ß3 integrin-tail, while in vitro binding assays point to LIM1 and/or LIM2 interaction with talin-head domain. These data provide structural insights into the molecular organization of ß3 integrin-FAs.

Expression of the type 1 lysophosphatidic acid receptor in osteoblastic cell lineage controls both bone mineralization and osteocyte specification.

Lysphosphatidic acid (LPA) is a major natural bioactive lipid mediator whose biological functions affect multiple organs. These include bone as demonstrated by global Lpar1-knockout mice (Lpar1-/-) which present a bone growth defect. LPA acts on all bone cells including osteoblasts, that are responsible for bone formation, and osteoclasts, which are specialized cells that resorb bone. LPA appears as a potential new coupling molecule during bone remodeling. LPA1 is the most ubiquitous LPA receptor among the six LPA receptor family members (LPA1-6). To better understand the specific role of LPA via its receptor LPA1 in osteoblastic cell lineage we generated osteoblast-specific Lpar1 knockout mice (Lpar1-∆Ob) by crossing Lpar1flox/flox and Osx:Cre+ mouse lines. Lpar1-∆Ob mice do not recapitulate the bone defects of Lpar1-/- mice but revealed reduced bone mineralization and decreased cortical thickness, as well as increased bone porosity associated with an augmentation in the lacunae areas of osteocyte and their apoptotic yield. In vitro, primary Lpar1-∆Ob and immortalized cl1-Ob-Lpar1-/- osteoblasts revealed a remarkable premature expression of alkaline phosphatase, reduced cell proliferation associated with decreased YAP-P nuclear accumulation, and reduced mineralization activity. Osteocyte specification is markedly impaired as demonstrated by reduced expression of early (E11) and late (DMP1, DKK1, SOST) osteocyte markers ex vivo in enriched osteocytic fractions of Lpar1-∆Ob mouse bone explants. In addition, E11 expression and dendrite formation induced by FGF2 are markedly impaired in both primary Lpar1-∆Ob and immortalized cl1-Ob-Lpar1-/- osteoblasts. Taken together these results suggest a new role for LPA in bone mass control via bone mineralization and osteocyte function.

Podosome-type adhesions and focal adhesions, so alike yet so different.

Cell-matrix adhesions are essential for cell migration, tissue organization and differentiation, therefore playing central roles in embryonic development, remodeling and homeostasis of tissues and organs. Matrix adhesion-dependent signals cooperate with other pathways to regulate biological functions such as cell survival, cell proliferation, wound healing, and tumorigenesis. Cell migration and invasion are integrated processes requiring the continuous, coordinated assembly and disassembly of integrin-mediated adhesions. An understanding of how integrins regulate cell migration and invasiveness through the dynamic regulation of adhesions is fundamental to both physiological and pathological situations. A variety of cell-matrix adhesions has been identified, namely, focal complexes, focal adhesions, fibrillar adhesions, podosomes, and invadopodia (podosome-type adhesions). These adhesion sites contain integrin clusters able to develop specialized structures, which are different in their architecture and dynamics although they share almost the same proteins. Here we compare recent advances and developments in the elucidation of the organization and dynamics of focal adhesions and podosome-type adhesions, in order to understand how such subcellular sites - though closely related in their composition - can be structurally and functionally different. The underlying question is how their respective physiological or pathological roles are related to their distinct organization.

FGFR2-Cbl interaction in lipid rafts triggers attenuation of PI3K/Akt signaling and osteoblast survival.

Fibroblast growth factor receptor (FGFR) signaling plays an important role in skeletogenesis. The molecular mechanisms triggered by activated FGFR in bone forming cells are however not fully understood. In this study, we identify a role for phosphatidylinositol 3-kinase (PI3K) signaling in cell apoptosis induced by FGFR2 activation in osteoblasts. We show that FGFR2 activation leads to decrease PI3K protein levels, resulting in attenuation of PI3K signaling in human osteoblasts. Biochemical and molecular analyses revealed that the attenuated PI3K signaling induced by FGFR2 activation is due to increased Cbl-PI3K molecular interaction mediated by the Cbl Y731 residue, which results in increased PI3K ubiquitination and proteasome degradation. Biochemical and immunocytochemical analyses showed that FGFR2 and Cbl interact in raft micro-domains at the plasma membrane. FGFR2 activation increases FGFR2 and Cbl recruitment in micro-domains, resulting in increased molecular interactions. Consistently, functional analyses showed that the attenuation of PI3K/Akt signaling triggered by FGFR2 activation results in increased osteoblast apoptosis. These results identify a functional molecular mechanism by which activated FGFR2 recruits Cbl in raft micro-domains to trigger PI3K ubiquitination and proteasome degradation, and reveal a novel role for PI3K/Akt attenuation in the control of osteoblast survival by FGFR2 signaling.

Nuclear translocation of integrin cytoplasmic domain-associated protein 1 stimulates cellular proliferation.

Integrin cytoplasmic domain-associated protein 1 (ICAP-1) has been shown to interact specifically with the beta1 integrin cytoplasmic domain and to control cell spreading on fibronectin. Interestingly, ICAP-1 also is observed in the nucleus, by immunocytochemical staining, and after biochemical cell fractionation, suggesting that it has additional roles that have yet to be determined. We show that the nucleocytoplasmic shuttling capability of ICAP-1 is dependent on a functional nuclear localization signal. In addition, overexpression of beta1 integrin strongly reduced this nuclear localization, suggesting that integrin activity could modulate ICAP-1 shuttling by sequestering it in the cytoplasm. Indeed, the nuclear localization of ICAP-1 is dependent on the stage of cell spreading on fibronectin, and we also show that ICAP-1 expression stimulates cellular proliferation in a fibronectin-dependent manner. This function is dependent on its nuclear localization. Moreover, ICAP-1 is able to activate the c-myc promoter in vitro. Together, these results demonstrate that ICAP-1 shuttles between the nucleus and cytoplasm in a beta1 integrin-dependent manner. It could act as a messenger that relays information from sites of integrin-dependent cell adhesion to the nucleus for controlling gene expression and cell proliferation.

ß1 integrins mediate the BMP2 dependent transcriptional control of osteoblast differentiation and osteogenesis.

Osteoblast differentiation is a highly regulated process that requires coordinated information from both soluble factors and the extracellular matrix. Among these extracellular stimuli, chemical and physical properties of the matrix are sensed through cell surface receptors such as integrins and transmitted into the nucleus to drive specific gene expression. Here, we showed that the conditional deletion of ß1 integrins in the osteo-precursor population severely impacts bone formation and homeostasis both in vivo and in vitro. Mutant mice displayed a severe bone deficit characterized by bone fragility and reduced bone mass. We showed that ß1 integrins are required for proper BMP2 dependent signaling at the pre-osteoblastic stage, by positively modulating Smad1/5-dependent transcriptional activity at the nuclear level. The lack of ß1 integrins results in a transcription modulation that relies on a cooperative defect with other transcription factors rather than a plain blunted BMP2 response. Our results point to a nuclear modulation of Smad1/5 transcriptional activity by ß1 integrins, allowing a tight control of osteoblast differentiation.

Unraveling ICAP-1 function: toward a new direction?

Cell adhesion to either the extracellular matrix (ECM) or to neighboring cells is of critical importance during both physiological and pathological situations. Integrins are a large family of cell adhesion receptors composed of two non-covalently linked alpha and beta subunits. They have a well-identified dual function of mediating both firm adhesion and signaling. The short cytoplasmic domain of integrin can interact with cytoplasmic proteins that are either shared by several different integrins or specific for one type of integrin. Integrin cytoplasmic domain-associated protein-1 (ICAP-1) is a small cytoplasmic protein that specifically interacts with the beta1 integrin subunit. In this review we will discuss recent findings on ICAP-1, not only at the structural and functional level, but also its possible interconnection in other signaling pathways such as those that control cell proliferation.

Pathophysiological Mechanisms of Staphylococcus Non-aureus Bone and Joint Infection: Interspecies Homogeneity and Specific Behavior of S. pseudintermedius.

Implicated in more than 60% of bone and joint infections (BJIs), Staphylococci have a particular tropism for osteoarticular tissue and lead to difficult-to-treat clinical infections. To date, Staphylococcus aureus internalization in non-professional phagocytic cells (NPPCs) is a well-explored virulence mechanism involved in BJI chronicity. Conversely, the pathophysiological pathways associated with Staphylococcus non-aureus (SNA) BJIs have scarcely been studied despite their high prevalence. In this study, 15 reference strains from 15 different SNA species were compared in terms of (i) adhesion to human fibronectin based on adhesion microplate assays and (ii) internalization ability, intracellular persistence and cytotoxicity based on an in vitro infection model using human osteoblasts. Compared to S. aureus, S. pseudintermedius was the only species that significantly adhered to human fibronectin. This species was also associated with high (even superior to S. aureus) internalization ability, intracellular persistence and cytotoxicity. These findings were confirmed using a panel of 17 different S. pseudintermedius isolates. Additionally, S. pseudintermedius internalization by osteoblasts was completely abolished in ß1 integrin-deficient murine osteoblasts. These results suggest the involvement of ß1 integrin in the invasion process, although this mechanism was previously restricted to S. aureus. In summary, our results suggest that internalization into NPPCs is not a classical pathophysiologic mechanism of SNA BJIs. S. pseudintermedius appears to be an exception, and its ability to invade and subsequently induce cytotoxicity in NPPCs could explain its severe and necrotic forms of infection, notably in dogs, which exhibit a high prevalence of S. pseudintermedius infection.