ReviewHost-Pathogen Interactions

Targeting of host cell receptor tyrosine kinases by intracellular pathogens

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Science Signaling  17 Sep 2019:
Vol. 12, Issue 599, eaau9894
DOI: 10.1126/scisignal.aau9894

Abstract

Intracellular pathogens use complex and tightly regulated processes to enter host cells. Upon initial interactions with signaling proteins at the surface of target cells, intracellular microbes activate and co-opt specific host signaling pathways that mediate cell surface–cytosol communications to facilitate pathogen internalization. Here, we discuss the roles of host receptor tyrosine kinases (RTKs) in the establishment of productive infections by major intracellular pathogens. We evaluate the gaps in the current understanding of this process and propose a comprehensive approach for assessing the role of host cell signaling in the biology of intracellular microorganisms and viruses. We also discuss RTK-targeting strategies for the treatment of various infections.

INTRODUCTION

Receptor tyrosine kinases (RTKs) constitute one of the major families of plasma membrane–associated proteins (1) and play a key role in cellular responses to external stimuli, such as changes in cell survival, proliferation, differentiation, and migration. The 58 RTKs that have been identified in humans have similar topologies, with each containing a ligand-binding extracellular region and a cytosolic kinase domain that are linked by a single transmembrane α helix. With the exception of the insulin receptor, which is dimeric in the inactive state, RTKs are monomeric when not bound to ligand; binding of their cognate ligand to the ectodomain causes dimerization, which leads to activation by transautophosphorylation of tyrosine residues, a crucial step in the transmission process of external signals to the cytoplasm (2). The phosphorylated tyrosine residues provide docking sites for cytoplasmic signaling proteins containing Src homology 2 and phosphotyrosine binding domains, which transmit the signal to downstream signaling elements such as non-RTKs (i.e., tryrosine kinases that lack a receptor domain and are usually cytosolic), serine-threonine kinases, and guanosine triphosphatases (GTPases) (2). Upon interaction with their ligands, RTKs are endocytosed and, depending on the ligand concentration, targeted for either recycling or degradation (3). The internalization and subsequent presence of activated RTKs on endosomes facilitates the activation of signaling proteins that do not reside at the plasma membrane, such as protein kinase B (PKB; also known as Akt), the mechanistic target of rapamycin complex (mTORC), and extracellular signal–regulated kinases 1 and 2 (ERK1/2) (3).

The endocytic process itself requires RTK-mediated actin cytoskeleton remodeling. Actin cytoskeleton dynamics is tightly controlled by phosphatidylinositol 3-kinase (PI3K) and its effector GTPases, the activated forms of which are bound to guanosine 5′-triphosphate (GTP). GTPases mediate multiple physiological functions, including plasma membrane protrusion and invagination during exocytosis and endocytosis, respectively, and trafficking and fission of exocytic and endocytic vesicles. Five major GTPases have been implicated in mediating the internalization of intracellular pathogens: three members of the Rho subfamily [Ras-related C3 botulinum toxin substrate 1 (Rac1), Cdc42, and RhoA], dynamin, and the Ras family adenosine diphosphate–ribosylation factor 6. Dynamin is essential for the entry of several pathogens and mediates various physiological functions, including endocytic vesicle fission, actin polymerization, and removal of the capping protein gelsolin from actin, which enables filament elongation. Upon activation, Rho members initiate actin nucleation and polymerization through the neural Wiskott-Aldrich syndrome (N-WASP) proteins or WASP family verprolin-homologous proteins, which mediate regulation of the key players such as actin-related proteins 2 and 3 (Arp2/3) [reviewed in (4)].

CRUCIAL ROLES OF RTKs IN THE LIFE CYCLE OF INTRACELLULAR PATHOGENS

Most intracellular pathogens enter their host cells by hijacking cell signaling proteins that promote endocytosis and macropinocytosis, the major mechanisms through which nonphagocytic cells internalize membrane-associated receptors and extracellular materials [reviewed in (58)]. Other entry strategies also exist, such as those used by Legionella pneumophila [reviewed in (8)], which enters cells through phagocytosis, and microsporidia, which transfer spores into host cells through a specialized organelle that penetrates the host cell membrane (9), but these mechanisms will not be discussed here. Given the accessibility of RTKs at the cell surface and their role in triggering endocytic mechanisms, it is not unexpected that many intracellular pathogens have evolved to exploit them for their own benefit. Several RTKs have indeed been implicated in the life cycles of various pathogens (Figs. 1 to 3 and Table 1). Two of these RTKs (Axl and Tyro3) are members of the Tyro3-Axl-Mer (TAM) family, and seven [erythropoietin-producing hepatocellular receptor A2 (EphA2), epidermal growth factor receptor (EGFR), avian erythroblastosis oncogene B (ErbB2), fibroblast growth factor receptor 1 (FGFR1), platelet-derived growth factor receptor (PDGFR), hepatocyte growth factor receptor (HGFR; also known as c-Met), and tropomyosin receptor kinase C (TrkC)] are members of the growth factor receptor (GFR) family. A 10th RTK, the insulin receptor, is an outlier that plays a role in the replication of dengue and Zika viruses (DENV and ZIKV) in cells of both the human host and the mosquito vector (10, 11); in both cases, the underlying mechanisms remain to be elucidated.

Fig. 1 RTKs and downstream signaling elements hijacked by intracellular pathogens.

The interaction of some intracellular pathogens with RTKs or RTK-interacting receptors at the host cell surface is critical for the pathogens to enter the cells. Whereas some pathogens interact with RTKs directly, others do so through adaptor proteins. These interactions may trigger pathogen entry independently of receptor kinase activity or mediate the activation of RTKs, which results in signaling to downstream components that enable entry. Some intracellular pathogens also target RTK-mediated signaling events necessary for proper trafficking of the pathogen or for suppressing innate immune responses during the early stages of infection.

CREDIT: A. KITTERMAN/SCIENCE SIGNALING
Fig. 2 Interactions of viruses with RTKs and mechanisms triggered by these interactions.

The interaction of viruses with RTKs, directly or through adaptors such as Gas6, triggers several mechanisms that facilitate entry, including receptor clustering in lipid rafts, lateral movement of viral particles to sites of internalization near tight junctions, clathrin-mediated endocytosis, and actin cytoskeleton dynamics. AcMAPV, autographica californica multicapsid nucleopolyhedrovirus; AMAV, Amapari virus; CVB, coxsackievirus B; CHIKV, chikungunya virus; DENV, dengue virus; EEEV, eastern equine encephalitis virus; HCMV, human cytomegalovirus; HCV, hepatitis C virus; HIV-1, human immunodeficiency virus 1; HPV, human papillomavirus; HSV-1, herpes simplex virus type 1; IAV, influenza A virus; KSHV, Kaposi’s sarcoma-associated herpesvirus; LCMV, lymphocytic choriomeningitis virus; MARV, Marburg virus; NiV, Nipah virus; PICV, Pichinde virus; RRV, Rose River virus; SINV, Sindbis virus; SV40, simian virus 40; TCRV, Tacaribe virus; TGEV, porcine transmissible gastroenteritis virus; VACV, vaccinia virus; VSV, vesiculovirus; WNV, West Nile virus; YFV, yellow fever virus; ZIKV, Zika virus; CAR, coxsackievirus and adenovirus receptor; EBOV (Ebola virus); LASV (Lassa virus).

CREDIT: A. KITTERMAN/SCIENCE SIGNALING
Fig. 3 Interactions of prokaryotic and eukaryotic microorganisms with RTKs.

Several intracellular bacterial pathogens target RTKs for entry into the host cell. These bacteria stimulate signaling pathways to control actin cytoskeleton dynamics that mediate their entry into the host cell. C. trachomatis can bind to FGFRs indirectly through its interaction with FGF2. C. pneumoniae, Salmonella, and Listeria bind directly to RTKs through the bacterial cell surface proteins Pmp21, Rck, and InlB, respectively. Listeria can also interact with host cells after it has been packaged and secreted from infected macrophages within host cell membrane–derived vesicles that have exposed phosphatidylserine (PS). It is possible that the PS on these vesicles interacts with TAM through Gas6. Cbl, casitas B-lineage lymphoma.

CREDIT: A. KITTERMAN/SCIENCE SIGNALING
Table 1 RTKs that mediate pathogen entry.

AAV2 and AAV3, adeno-associated viruses 2 and 3; AcMAPV, autographica californica multicapsid nucleopolyhedrovirus; AMAV, Amapari virus; CHIKV, chikungunya virus; DENV, dengue virus; EBOV, Ebola virus; EEEV, eastern equine encephalitis virus; HCMV, human cytomegalovirus; HCV, hepatitis C virus; HPV, human papillomavirus; IAV, influenza A virus; HSV-1, herpes simplex virus 1; JEV, Japanese encephalitis virus; KSHV, Kaposi’s sarcoma-associated herpesvirus; LASV, Lassa virus; LCMV, lymphocytic choriomeningitis virus; MARV, Marburg virus; PICV, Pichinde virus; RRV, Rose River virus; RSV, respiratory syncytial virus; SARSV, severe acute respiratory syndrome virus; SINV, Sindbis virus; SV40, simian virus 40; TCRV, Tacaribe virus; TGEV, porcine transmissible gastroenteritis virus; PRRSV, porcine reproductive and respiratory syndrome virus; VACV, vaccinia virus; VSV, vesicular stomatitis virus; WNV, West Nile virus; YFV, yellow fever virus; ZIKV, Zika virus.

View this table:

Here, we review and discuss (i) the initial interaction of pathogens with RTKs, (ii) mechanistic roles of RTKs in lateral movement of pathogens at the plasma membrane, (iii) RTK-mediated alterations in plasma membrane lipid composition, (iv) recruitment of the endocytic machinery to mediate pathogen entry, (v) RTK-induced cytoskeleton dynamics leading to pathogen internalization, and (vi) suppression of RTK-mediated innate immune responses.

INITIAL INTERACTIONS OF INTRACELLULAR MICROBES WITH RTKs

RTKs are endocytosed upon binding to their cognate ligands (12). This mechanism is exploited by many intracellular microbes through physical interactions with RTKs that enable them to enter the target cells.

The TAM family of RTKs

Members of the TAM family mediate clearance of apoptotic cells through recognition of phosphatidylserine (PS), which flips from the cytoplasmic surface of the plasma membrane onto the external surface in apoptotic cells. Two serum proteins that share 42% amino acid similarity, growth arrest–specific gene 6 (Gas6) and protein S, have been implicated as TAM ligands. They bridge the PS molecules on the surface of apoptotic cells to TAM receptors on phagocytes and promote efferocytosis, a noninflammatory uptake mechanism (13). The role of PS in the biology of viruses, bacteria, and protists has been reviewed elsewhere (14). Many enveloped viruses acquire PS on their surface during secretion from various cellular compartments and, as a consequence, mimic apoptotic cells (15, 16). The PS on the surface of these viruses indirectly interacts with TAM-displaying target cells, thus expanding the repertoire of cells susceptible to infection (Fig. 2 and Table 1) [reviewed in (15, 17)].

The role of TAM receptors in the entry of the mosquito-borne, enveloped flaviviruses DENV, ZIKV, and West Nile virus (WNV) has been extensively studied. DENV and ZIKV are transmitted to the mammalian host through a bite by an infected mosquito. The viruses infect various Axl-displaying skin cell types such as keratinocytes and fibroblasts (1719). DENV and WNV bind to Gas6 only when virions are produced in an insect cell culture system (19), suggesting that insufficient quantities of PS are present on the surface of virions generated in mammalian cells. This is not true for the mammalian cell–derived ZIKV, which, unlike DENV or WNV, can exploit Axl to infect the endothelial cells of umbilical veins, a likely prerequisite for the infection of fetuses that leads to the microcephaly syndrome (19). Axl and Tyro3 are essential for the infectivity of DENV and WNV, but their kinase activities are dispensable for virus endocytosis into human embryonic kidney (HEK) 293T cells (20), indicating that these viruses exploit alternative signaling pathways for internalization. In contrast, the kinase activities of Axl and Tyro3 are essential for ZIKV entry into glial cells; however, Axl is dispensable for entry into Tyro3-enriched neural progenitor cells, infection of which underlies the pathogenesis of microcephaly (16, 17). This suggests that Axl and Tyro3 likely play a redundant role in ZIKV entry, highlighting the need for innovative therapeutic strategies using broad-acting TAM inhibitors, rather than selective Axl inhibitors (16, 21, 22)) for the treatment of ZIKV infection.

Murine models have been developed for DENV and ZIKV infection (23, 24). TAM kinases are not essential for the development of mouse embryos, providing an opportunity to generate TAM knockout mice to examine the role of these enzymes in virus replication in animal models (13). TAM knockout mice are susceptible to ZIKV infection (25), which raises the question of the relevance of this animal model for the study of ZIKV entry in human infections. It would be of interest to investigate whether DENV requires TAM for replication in the aforementioned mouse model.

Some nonenveloped viruses have developed PS-independent mechanisms to benefit from apoptotic mimicry. Simian virus 40 (SV40) is a nonenveloped polyomavirus that does not display PS on its surface. The SV40 capsid protein viral protein 1 has evolved to directly bind to Gas6, which thus bridges the viral particle to Axl, leading to virus uptake despite the absence of a PS-carrying envelope (26). Enteroviruses such as poliovirus and coxsackievirus B3 also lack a cell-derived lipid-containing envelope. However, these virions are secreted from host cells within PS-containing vesicles, and treatment with annexin V, which binds to and masks PS, reduces infectivity of the virus-containing vesicles (27). Hepatitis A virus, another enterovirus, is also cloaked and secreted within multivesicular bodies (2830), but in this case, the role of PS and TAM receptors in the infectivity of the packaged virus remains to be investigated.

Bacteria rapidly convert PS into phosphatidylethanolamine, and, consequently, the former molecule is not normally found in the bacterial membrane (31). Listeria monocytogenes, similar to the enteroviruses mentioned above, is packaged and secreted from macrophages within host cell membrane–derived vesicles that carry exposed PS [reviewed in (14)]. However, there are no reports indicating a role for the TAM receptors in the entry of this bacterium into host cells through interaction with these PS-displaying vesicles. Because activation of host cell Src, a non-RTK, is required for L. monocytogenes entry, it would be of interest to investigate whether Axl, a known activator of Src, is required for this process. The importance of PS-mediated immunosuppression during infections with protists has been described [reviewed in (14)], but the role of PS and TAM receptors in parasite entry has not been explored.

The GFR family of RTKs

Several GFRs have been implicated in the entry of intracellular pathogens (Table 1). The ErbB family of proteins contains four RTKs structurally related to the EGFR. In humans, the family includes human EGFR-related 1 (Her1; also known as EGFR or ErbB1), Her2 (also called Neu or ErbB2), Her3 (also called ErbB3), and Her4 (also called ErbB4). The contribution of Her1 and Her2 to the biology of medically important viruses, bacteria, and fungi has been reviewed elsewhere (32). It is established that GFRs physically interact with some intracellular pathogens (Table 1), but there is limited information on the pathogen-encoded proteins that interact with them. Initial studies with human embryonic lung fibroblasts indicated that Her1 and PDGFR physically interact with the envelope glycoprotein B (gB) of human cytomegalovirus (HCMV) (33, 34). The viral gL-gH-gO envelope glycoprotein complex binds to PDGFRα and then recruits gB to the complex, leading to the virus entry. This process does not require the enzymatic activity of PDGFRα (35).

GFRs are also exploited by prokaryotic pathogens and at least two bacterial surface proteins, the Rck protein of Salmonella enterica and the Pmp21 (polymorphic membrane protein 21) surface protein of Chlamydia pneumoniae, physically interact with and activate EGFR, the kinase activity of which is essential for bacterial entry (36). In L. monocytogenes infection, the bacterial surface protein InlB directly binds to and activates the RTK c-Met, which triggers a cascade of signaling regulating actin cytoskeleton dynamics required for bacterial entry [reviewed in (37)]. The crystal structure of the InlB–c-Met complex has mapped the interaction site outside of the binding site of c-Met with its physiological ligand, the HGF (38). The above findings suggest that GFRs, in contrast to members of the TAM family, directly interact with infectious particles; therefore, pathogen-encoded RTK binding domains represent potential candidates for the development of vaccines targeting RTK binding sites in microbial proteins.

There is some evidence that pathogens may also interact indirectly with GFRs. The FGFR ligand FGF2 has been implicated in enhancing the replication of Chlamydia trachomatis and ZIKV. Infection with either of these pathogens stimulates FGF2 transcription (3941). The secreted FGF2 binds to and promotes C. trachomatis entry during subsequent rounds of infection. The mechanistic role of FGF2 in ZIKV replication remains to be understood.

Additional signaling proteins may facilitate the entry process after the interaction of pathogens with GFRs. In influenza A virus (IAV) infection, protein kinase C–βII (PKC-βII), a host serine-threonine kinase, promotes virus entry by stimulating EGFR internalization (42), which is a crucial step in hepatitis C virus (HCV) infection as well (43). The role of this kinase in internalization of other EGFR-dependent pathogens remains uncharacterized.

RTKs IN LATERAL MOVEMENT OF PATHOGENS AT THE PLASMA MEMBRANE

After attachment to their primary receptors on the cell surface, some viruses are translocated to a defined site within the plasma membrane to reach the receptors that ultimately mediate entry into the target cell. The signaling mechanisms mediating lateral diffusion of viral particles is the best documented for HCV (44). Binding of HCV to its primary receptor tetraspanin CD81 on the hepatocyte surface initiates the translocation of viral particles to a tight junction, where the co-receptors claudin-1 and occludin mediate entry. Interaction of CD81 with a cognate antibody triggers trafficking of the complex to the tight junction, indicating that ligand binding to CD81 is sufficient to initiate translocation, a process that also requires EGFR kinase activity (43). HCV-mediated stimulation of EGFR activates the Ras-BRaf pathway, which, in turn, mediates lateral diffusion of CD81 (45).

EGFR is also required for efficient interaction between CD81 and claudin-1 during fusion of the HCV envelope with the plasma membrane, the step that follows translocation to the tight junction (46). However, stimulation of the pathway by the binding of EGF to EGFR promotes neither translocation nor CD81-claudin interaction, suggesting that the kinase activity of EGFR is not sufficient for trafficking the CD81-virion complex to a tight junction and that as-yet unidentified factors are required for this phenomenon to occur. EGFR-dependent trafficking to the tight junction is a target of innate immune responses: The antiviral factor interferon (IFN)-α–induced 6 protein (IRI6), which cells produce upon being invaded by various viruses, interferes with EGFR activation and subsequent CD81–claudin-1 interactions, thereby impairing virus entry (47). Tight junction proteins are essential for the entry of DENV, rotaviruses, and porcine epidemic diarrhea virus, but the mechanistic roles of signaling elements in the entry processes for these viruses remain to be clarified (48). In cultured cells, herpes simplex virus type 1 (HSV-1) induces the formation of filopodia, binds to them, and is subsequently translocated to the cell body, where internalization occurs through activation of Cdc42 (49), but the role of RTKs in this process is unknown.

L. monocytogenes recruits CD81 to the entry site, which, in turn, recruits phosphatidylinositol 4-kinase type II α (PI4KIIα); this plays a key promoting role in bacterial entry (50). In addition, the bacterium disrupts the tight junction to translocate across the intestinal barrier (51), but whether the CD81-EGFR pathway, as described for HCV, mediates trafficking of the bacterium to the tight junction remains unclear. CD81 is also co-opted for invasion of hepatocytes by the sporozoites of the malaria parasite Plasmodium falciparum. In the initial stage of the infection of the human host, the sporozoites injected during a mosquito bite reach the bloodstream and invade hepatocytes, a process for which CD81 is crucial; pretreatment of cells with antibodies specific for CD81 blocks parasite entry (52). As mentioned above in the context of HCV entry, interaction of CD81 with its ligand results in the activation of the EGFR-Ras-BRaf pathway; it would be of interest to investigate whether these pathways are also essential for Plasmodium invasion of hepatocytes. The susceptibility of the liver and blood stages of the Plasmodium life cycle to the broad-spectrum protein tyrosine kinase (PTK) inhibitor genistein (53) supports the notion that PTKs play an important role in P. falciparum infection; it is likely that this is mediated by host cell PTKs because the parasite’s kinome does not include members of this family (54). In fact, Plasmodium sporozoites elicit HGF secretion and, therefore, activation of c-Met, an RTK whose enzymatic activity is required for early steps in parasite entry (55), as it is for the uptake of some viruses and bacteria (see above and Table 1).

RTK-STIMULATED LIPID RAFT CLUSTERING

The stimulation of RTKs by pathogens can lead to major alterations in plasma membrane composition. Activation of EGFR by several viruses including IAV (56), porcine transmissible gastroenteritis virus (TGEV) (57), and HSV-1 (58) leads to the clustering of lipid rafts in the vicinity of the virus binding site. Lipid rafts may facilitate accumulation of—and cross-talk between—various signaling molecules, resulting in signal enhancement (59). In L. monocytogenes infection, the integrity of lipid rafts is critical for correct localization of the phosphoinositides, intermediates of downstream signaling, produced by c-Met–activated PI3K during bacterial entry (60). In addition, the accumulation of lipid rafts at viral binding sites causes a pre-endosome curvature of the membrane (61). The mechanism underlying this lipid raft clustering and the exact mechanistic role of RTK-dependent lipid rafts formation remain to be fully understood.

RTK-MEDIATED RECRUITMENT OF ENDOCYTIC MACHINERY TO THE ENTRY SITE

Clathrin-mediated endocytosis [reviewed in (61, 62)] is the major means for internalization of ligand-RTK complexes (12). After the engagement of the RTK with ligand or cargo, more than 50 proteins are recruited to the site. Some of these proteins act as adaptors for the recruitment of critical signaling factors such as Src and PI3K. Clathrin is phosphorylated by Src and recruited to the membrane through activating protein 2 (AP-2), an initial event in the formation of clathrin-coated pits (62). The Hip1R and Ent1 adaptors then link clathrin to the actin network, a crucial step in membrane invagination and vesicle formation. After endocytosis of cargo-containing clathrin-coated vesicles, EGFR pathway substrate 15 mediates targeting of the endosomal contents for degradation or recycling (63).

Many viruses and bacteria enter their host cells through the clathrin-mediated pathway (6). Clathrin-coated vesicles allow viruses to move to their site of replication, which is often distant from the plasma membrane. Some pathogens such as DENV and canine parvovirus hijack preexisting clathrin-coated pits, but many stimulate de novo clathrin recruitment upon attachment to the plasma membrane (64).

The role of the endocytic machinery components in the entry of L. monocytogenes has been studied extensively. This bacterium exploits clathrin to promote its entry as wrapped in plasma membrane-derived vacuoles, which are subsequently lysed in the cytosol, allowing bacterial escape and replication (Fig. 3) [reviewed in (8)]. The interaction of L. monocytogenes InlB with c-Met (see above) triggers the recruitment of the endocytic machinery, a critical step in pulling the bacterium into the target cell (8). In general, activation of RTKs leads to PI3K-mediated phosphatidylinositol 3,4,5-trisphosphate production, which, in turn, binds to the membrane-bound adaptor AP-2, eventually recruiting clathrin to the entry site. In the case ofL. monocytogenes infection, disabled homolog 2 directly recruits clathrin, and AP-2 is therefore dispensable for bacterial entry (65).

RTK REGULATION OF ACTIN CYTOSKELETAL REARRANGEMENT DURING PATHOGEN ENTRY

Members of both the TAM and GFR families stimulate cytoskeletal remodeling. PI3K is a key effector of RTKs and stimulates downstream signaling proteins such as Rho-associated protein kinase (PK) (Rock), p21-activated kinases (PAK), Akt, and LIM kinase 1 (LIMK), as well as various GTPase intermediates such as Rho, Rac1, and Cdc42. This eventually results in the phosphorylation and deactivation of cofilin, an actin-severing protein, thus disrupting the actin depolymerization dynamics that are required for plasma membrane protrusion and invagination.

After it has translocated along filopodia to the cell body, HSV-1 activates the EGFR-PI3K-MEK1/2-ERK1/2-Rock-LIMK cascade, which leads to dynamic oscillation between inactivation and activation of cofilin (58). Binding of C. pneumoniae Pmp21 to EGFR triggers activation of the MEK1 (mitogen-activated protein kinase kinase 1)–Erk1/2 pathway, through which EGFR mediates entry (36). It would be of interest to investigate whether Rock-LIMK function downstream of Erk1/2 in the context of Chlamydia infection as they do in HSV-1 infection. The MEK1-Erk1/2 pathway is also activated by Mycobacterium leprae binding to another member of the ErbB family, Her2, although the bacterial binding protein(s) that mediate this interaction have not yet been identified (32). The EGFR-LIMK–dependent phosphorylation of cofilin, and thus the regulation of actin filaments stability, has been reported to mediate the entry of TGEV (57) and porcine reproductive and respiratory syndrome virus (PRRSV), albeit through different intermediates (66).

The detailed mechanisms of L. monocytogenes entry into its host cells have been reviewed elsewhere (8, 37). In this context, Grb2-associated binder 1 (Gab1) mediates the recruitment of PI3K which, in turn, activates Rho GTPases involved in actin polymerization. After Gab1-mediated or direct interaction with c-Met, activated PI3K mediates Akt activation through phosphorylation of mTORC (67, 68), which, as described above for PRRSV, regulates cytoskeleton dynamics through activation of LIMK-dependent phosphorylation of cofilin (69). PKC-α is also activated by mTORC, which is important for L. monocytogenes entry (68). It has been established that some components of the exocytosis machinery play a role in the uptake of L. monocytogenes. Activation of PKC-α, mTORC, and the PKC-α substrate scaffold protein filamin A promote c-Met–induced exocytosis during bacterial entry and is essential for the recruitment of exocyst component Exo70 to bacterial entry sites (70, 71). This has provided new insights into pathogen entry mechanisms; it would be of interest to investigate whether the exocytosis proteins are crucial for the entry of other pathogens.

The Rck outer membrane protein of Salmonella binds to and activates EGFR (72) but does so by engaging a binding site on EGFR that is distinct from the binding site for EGF. EGFR kinase activity is critical for activation of the Src-PI3K pathway that facilitates Salmonella entry into fibroblasts and epithelial cells (73), likely through reorganization of the cytoskeleton as described for L. monocytogenes.

In addition to the TAM receptors, some flaviviruses exploit the GFR pathways to manipulate cytoskeletal dynamics at entry sites. For example, the Japanese encephalitis virus (JEV) activates the EGFR-PI3K-Rac1/RhoA-PAK1/Rock pathway to stimulate phosphorylation of cofilin, thus facilitating endocytosis through actin reorganization (66). After activation of the EGFR-PI3K pathway, JEV stimulates RhoA-Rock to phosphorylate caveolin-1, which is required for activation of Rac1-PAK and promotion of caveolin-mediated endocytosis (66).

Parasitic protists also manipulate the host cell cytoskeleton. For example, a study of sporozoites of the rodent malaria parasite Plasmodium berghei and tachyzoites of the Plasmodium-related apicomplexan parasite Toxoplasma gondii demonstrated that de novo polymerization of host cell actin is important for the zoite to pass through the moving junction established between the parasite and host cell membranes during the invasion process. The host Arp2/3 complex was shown to be recruited near the moving junction and important for parasite entry. The authors proposed that zoites activate the host Arp2/3 complex, leading to reorganization of host actin for anchoring the moving junction to the cytoskeleton, thereby providing traction for the “glideosome,” a molecular motor located under the zoite’s membrane (74). Although the signaling events regulating this process have yet to be elucidated, it is likely that they will implicate some of the same factors that mediate cytoskeleton dynamics during the entry of other pathogens. Consistent with this hypothesis, phosphoproteomics analysis of host cell proteins during invasion of erythrocytes by Plasmodium has revealed that the cytoskeletal protein β-spectrin and the cytoskeleton-linked piezo-type mechanosensitive ion channel component 1 are phosphorylated during the process (75).

RTK-MEDIATED MODULATION OF HOST CELL INNATE IMMUNE RESPONSES

After entry into target cells, intracellular pathogens need to suppress host defenses to survive and replicate. The involvement of RTKs in pathogen-mediated suppression of the innate immune response is an emerging and rapidly growing area of research. Viruses have evolved many mechanisms to escape the major component of the innate immune response, IFN, but existing information suggests that RTKs play a limited role in this arms race (76). Suppression of the type I IFN and Toll-like receptor innate immune responses through TAM RTKs has been established in the case of enveloped viruses that infect dendritic cells (77). In this context, the kinase activity of TAM family members enhances postentry virus replication through inhibition of IFN antiviral activity (77). Similar TAM-mediated IFN pathway inhibition has been reported for ZIKV infection of glial cells (16), but the exact mechanism of TAM-mediated IFN inhibition remains to be explored.

IAV and rhinovirus activate the EGFR-PI3K pathway through which they suppress production of the antiviral cytokines IFN-λ, IFN-γ, interferon-inducible protein-10 (IP-10), and macrophage inflammatory protein-1 α (MIP-1α) by infected cells (56, 78, 79). This is clinically relevant because patients suffering from chronic obstructive pulmonary disease have increased abundance of PI3K-p110α in primary bronchial epithelial cells, leading to the enhancement of viral replication and severe symptoms in the event of an infection with IAV (78). Suppressor of cytokine signaling 5, which is suppressed by the highly pathogenic IAV strain H5N1, enhances viral infection by negatively regulating of EGFR activity (80). The above-described mechanisms might also play a role in the suppression of cellular defenses in the context of other infections in which the EGFR-PI3K pathway is triggered.

Cytoplasmic cellular and viral DNA triggers signaling through cyclic guanosine 3´,5´-monophosphate–adenosine monophosphate synthase and stimulator of IFN genes (STING), leading to IFN production (81). Her2, a ligand-independent RTK, is strongly associated with STING and plays an inhibiting role in this process (82, 83). Activated Her2 stimulates PI3K-AKT1 signaling in HEK293T cells; AKT1 phosphorylates the TANK-binding kinase 1 (TBK1), thereby impeding TBK1-STING signaling and, consequently, induction of type 1 IFN production (83). Infection of cells with DNA viruses, such as HSV-1 or vaccinia virus (VACV), activates this Her2-AKT1 pathway, ultimately suppressing STING signaling and hence host antiviral defense mechanisms (83). The bacteria M. leprae and Neisseria meningitidis and the fungus Candida albicans (Table 1 and references therein) also interact with Her2, but whether this leads to suppression of innate immune responses is unknown.

CONCLUDING REMARKS AND FUTURE DIRECTIONS

As obligate parasites, intracellular pathogens have evolved strategies to maximize the exploitation of resources from their host cells; this includes the hijacking of signaling components. RTKs are upstream regulators of cell signaling cascades and are accessible to the incoming pathogens on the cell surface. It is therefore not surprising that many intracellular microbes have evolved to interact with RTKs as primary receptors and triggers of downstream mechanisms, such as cytoskeletal dynamics, that are then exploited by the pathogen for entering its host cell. In addition to initial recognition, RTKs play a diverse range of roles in the subsequent steps of the life cycles of intracellular pathogens, including transport of viral particles to defined plasma membrane microdomains, alteration of plasma membrane composition and topology, and triggering of signaling pathways that modulate the host defense system. A wide range of phylogenetically unrelated pathogens, including viruses, bacteria, protists, and fungi, displays substantial overlap in the signaling pathways that they mobilize for entry (Figs. 2 and 3 and Table 1), highlighting the convergent evolutionary paths that these organisms have followed to solve the problem of invasion of their host cells.

After internalization, pathogens adopt various strategies to hijack the cellular translation machinery and promote cell survival. It is well known that viruses encode specific proteins that influence translation (84). However, it is unclear whether stimulation of RTKs by intracellular pathogens plays a role in the process. As discussed above, RTKs of both the TAM and GFR groups stimulate the PI3K-Akt and the MEK-Erk pathways (85), both of which are known to play a critical role in protein translation and survival (84, 86). It is therefore reasonable to propose that the stimulation of RTKs by intracellular pathogens during entry affects these processes to establish intracellular conditions that are favorable for the completion of the life cycle. Cross-talk between members of these two RTK families leads to modular diversification of downstream signaling pathways (87). For example, EGFR-Axl heterodimerization leads to the phosphorylation of Axl by EGFR and activates distinct pathways in cancer cells (88). It would be of great interest to investigate whether the same association exists between these two RTKs in the context of infectious diseases.

The role of TAM family members in cytoskeletal regulation is an emerging area of human functional kinomics. In the context of breast cancer signaling, the interaction of Axl with Gas6 promotes phosphorylation of the scaffolding protein engulfment and cell motility (ELMO) by Axl; this promotes or stabilizes the interaction between dedicator of cytokinesis 180 (Dock180) and Rac1, a GTPase that is a key regulator of cytoskeletal dynamics (89). The ELMO-Dock180-Rac1 complex stabilizes Rac in a nucleotide-free state, enabling the exchange of guanosine-5′-diphosphate and GTP and hence activation of downstream signaling events. In addition, ELMO-independent binding of CT10 regulator of kinase II to Dock180 is essential for Rac1 activation [reviewed in (90)]. As yet, there is no report indicating a role for the above proteins in the entry of intracellular pathogens known to activate Axl during entry (Fig. 2).

Global phosphoproteomics, RNA sequencing, and RNA microarray analyses have been used to profile the responses of host cells to pathogens. However, an integrated and comprehensive picture of signaling mobilization during infection and of cross-talk dynamics between different signaling cascades is lacking. We have successfully used an antibody microarray to investigate the signaling response of host hepatocytes to HCV infection (91) and mosquito cells to the endosymbiotic bacterium Wolbachia pipientis (10). The microarray comprises ~900 antibodies recognizing human signaling proteins and their regulatory phosphosites. Implementation of such host cell–targeted approaches provides a comprehensive picture of cell signaling during internalization of any pathogen. As illustrated by the aforementioned studies (10, 91), this platform can reveal new host-encoded targets for anti-infective intervention, notably among PKs. PKs are prominent targets in cancer chemotherapy, with 48 small-molecule inhibitors on the market (25 of which target RTKs) as of 2019 and many more in development (92). As a consequence, inhibitors for a large number of human kinases are available, including some that target RTKs discussed herein (Table 2). These molecules can potentially be repurposed as anti-infectives. Host-directed therapy is indeed gaining momentum as a strategy to tackle infectious diseases in the current era of drug resistance (93). Targeting host cell enzymes presents the considerable advantage that mutant alleles of the gene encoding the target in pathogens that reduce susceptibility to the drug cannot be selected under treatment pressure, thereby limiting the pace of emergence of drug resistance. As highlighted here, some intracellular pathogens interact directly with RTKs, and this interaction is critical for pathogen entry. This establishes that pathogen-encoded RTK-interacting epitopes represent promising candidates for the development of novel therapeutic and prophylactic vaccines and of small-molecule interaction disruptors.

Table 2 RTKs that mediate entry of pathogens and some of their chemical inhibitors.

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REFERENCES AND NOTES

Acknowledgments: This research project was supported by grants from the Australian Centre for HIV and Hepatitis Virology Research (ACH2) and the Australian National Health and Medical Research Council. Competing interests: The authors declare that they have no competing interests.
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