The Salmonella pathogenicity island 1 secretion system directs cellular cholesterol redistribution during mammalian cell entry and intracellular trafficking

The bacterial pathogen Salmonella triggers its own uptake into non-phagocytic mammalian cells. Entry is induced by the delivery of bacterial effector pro-teins that subvert signalling and promote cytoskeletal rearrangement, although the molecular mechanisms that co-ordinate initial pathogen-host cell recognition remain poorly characterized. Here we show that cholesterol is essential for Salmonella uptake. Depletion and chelation of plasma membrane cholesterol specifically inhibited bacterial internalization but not adherence. Cholesterol accumulated at bacterial entry sites in cultured cells, and was retained by Salmonella -containing vacuoles following pathogen internalization. Cellular cholesterol redistribution required bacterial effector protein delivery mediated by the Salmonella pathogenicity island (SPI) 1 type III secretion system, but was independent of the SPI2-encoded system.     

Abl tyrosine kinases are required for infection by Shigella flexneri

Infection by the opportunistic bacterial pathogen Shigella flexneri stimulates tyrosine phosphorylation of host cell proteins, but the kinases involved and their effects on the regulation of cell signaling pathways during bacterial entry remain largely undefined. Here, we demonstrate a requirement for the Abl family of tyrosine kinases during Shigella internalization. Family members Abl and Arg are catalytically activated upon Shigella infection, accumulate at the site of bacterial entry, and are required for efficient bacterial uptake, as internalization is blocked upon targeted deletion of these kinases or treatment with a specific pharmacological inhibitor. We identify the adapter protein Crk as a target for Abl kinases during Shigella uptake, and show that a phosphorylation-deficient Crk mutant significantly inhibits bacterial uptake. Moreover, we define a novel signaling pathway activated during Shigella entry that links Abl kinase phosphorylation of Crk to activation of the Rho family GTPases Rac and Cdc42. Together, these findings reveal a new role for the Abl kinases, and suggest a novel approach to treatment of Shigella infections through inhibition of host cell signaling pathways.     

A region of the Yersinia pseudotuberculosis invasin protein enhances integrin-mediated uptake into mammalian cells and promotes self-association

Invasin allows efficient entry into mammalian cells by Yersinia pseudotuberculosis. It has been shown that the C-terminal 192 amino acids of invasin are essential for binding of beta1 integrin receptors and subsequent uptake. By analyzing the internalization of latex beads coated with invasin derivatives, an additional domain of invasin was shown to be required for efficient bacterial internalization. A monomeric derivative encompassing the C-terminal 197 amino acids was inefficient at promoting entry of latex beads, whereas dimerization of this derivative by antibody significantly increased uptake. By using the DNA-binding domain of lambda repressor as a reporter for invasin self-interaction, we have demonstrated that a region of the invasin protein located N-terminal to the cell adhesion domain of invasin is able to self-associate. Chemical cross-linking studies of purified and surface-exposed invasin proteins, and the dominant-interfering effect of a non-functional invasin derivative are consistent with the presence of a self-association domain that is located within the region of invasin that enhances bacterial uptake. We conclude that interaction of homomultimeric invasin with multiple integrins establishes tight adherence and receptor clustering, thus providing a signal for internalization.     

Bacterial internalization mediated by beta 1 chain integrins is determined by ligand affinity and receptor density.

Binding of bacteria to beta 1 chain integrin receptors results in either bacterial adherence or uptake by cultured cells (Isberg, 1991). In this report we show that Staphylococcus aureus coated with high affinity ligands for the beta 1 chain integrin family can be internalized efficiently, whereas bacteria coated with low affinity ligands are poorly internalized. Overproduction of the alpha 5 beta 1 integrin increased the efficiency of bacterial internalization, indicating that the uptake efficiency is directly related to the level of expression of the receptor. By using latex beads or S. aureus coated with mAbs directed against the alpha 5 beta 1 integrin, a roughly semi-logarithmic correlation was observed between the affinity of the receptor-ligand interaction and the rate of bacterial internalization. Evidence is presented that high affinity binding of the bacterium allows the microorganism to compete efficiently with receptor-ligand interactions at the basolateral surface of the cell.     

The CEACAM1 transmembrane domain, but not the cytoplasmic domain, directs internalization of human pathogens via membrane microdomains

Several bacterial pathogens exploit carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to promote attachment and uptake into eukaryotic host cells. The widely expressed isoform CEACAM1 is involved in cell–cell adhesion, regulation of cell proliferation, insulin homeostasis, and neo-angiogenesis, processes that depend on the cytoplasmic domain of CEACAM1. By analysing the molecular requirements for CEACAM1-mediated internalization of bacteria, we surprisingly find that the CEACAM1 cytoplasmic domain is completely obsolete for bacterial uptake. Accordingly, CEACAM1-4L as well as a CEACAM1 mutant with a complete deletion of the cytoplasmic domain (CEACAM1 ΔCT) promote equivalent internalization of several human pathogens. CEACAM1-4L- and CEACAM1 ΔCT-mediated uptake proceeds in the presence of inhibitors of actin microfilament dynamics, which is in contrast to CEACAM3-mediated internalization. Bacteria-engaged CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3, localize to a gangliosid GM1- and GPI-anchored protein-containing portion of the plasma membrane. In addition, interference with cholesterol-rich membrane microdomains severely blocks bacterial uptake via CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3. Similar to GPI-anchored CEACAM6, both CEACAM1-4L as well as CEACAM1 ΔCT partition into a low-density, Triton-insoluble membrane fraction upon receptor clustering, whereas CEACAM3 is not detected in this fraction. Bacterial uptake by truncated CEACAM1 or chimeric CEACAM1/CEACAM3 molecules reveals that the transmembrane domain of CEACAM1 is responsible for its association with membrane microdomains. Together, these data argue for a functional role of lipid rafts in CEACAM1-mediated endocytosis that is promoted by the transmembrane domain of the receptor and that might be relevant for CEACAM1 function in physiologic settings.     

The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.     

A novel receptor – ligand pathway for entry of <Emphasis Type="Italic">Francisella tularensis</Emphasis> in monocyte-like THP-1 cells: interaction between surface nucleolin and bacterial elongation factor Tu

Background  

Francisella tularensis, the causative agent of tularemia, is one of the most infectious human bacterial pathogens. It is phagocytosed by immune cells, such as monocytes and macrophages. The precise mechanisms that initiate bacterial uptake have not yet been elucidated. Participation of C3, CR3, class A scavenger receptors and mannose receptor in bacterial uptake have been already reported. However, contribution of an additional, as-yet-unidentified receptor for F. tularensis internalization has been suggested.     

A translocated protein of Bartonella henselae interferes with endocytic uptake of individual bacteria and triggers uptake of large bacterial aggregates via the invasome

Bartonella henselae enters human endothelial cells (ECs) by two alternative routes: either by endocytosis, giving rise to Bartonella‐containing vacuoles or by invasome‐mediated internalization. Only the latter process depends on the type IV secretion system VirB/VirD4 and involves the formation of cell surface‐associated bacterial aggregates, which get engulfed by EC membranes in an F‐actin‐dependent manner, eventually resulting in their complete internalization. Here, we report that among the VirB/VirD4‐translocated effector proteins BepA‐BepG only BepG is required for triggering invasome‐mediated internalization. Expression of BepG in the Bep‐deficient ΔbepA–G mutant restored invasome‐mediated internalization. Likewise, ectopic expression of BepG in ECs also restored invasome‐mediated internalization of the ΔbepA–G mutant, while no discernable cytoskeletal rearrangements were triggered in uninfected cells. Rather, BepG inhibited endocytic uptake of B. henselae into Bartonella‐containing vacuoles and other endocytic processes, that is, invasin‐mediated uptake of Yersinia enterocolitica and uptake of inert microspheres. BepG thus triggers invasome‐mediated internalization primarily by inhibiting bacterial endocytosis. Bacteria accumulating on the cell surface then induce locally the F‐actin rearrangements characteristic for the invasome. These cytoskeletal changes encompass both the rearrangement of pre‐existing F‐actin fibres and the de novo polymerization of cortical F‐actin in the periphery of the invasome by Rac1/Scar1/WAVE‐ and Cdc42/WASP‐dependent pathways that involve the recruitment of the Arp2/3 complex.     

CD46 (membrane cofactor protein) acts as a human epithelial cell receptor for internalization of opsonized uropathogenic Escherichia coli

Escherichia coli is a common urinary pathogen whose uptake into epithelial cells is mediated by attachment through type 1 fimbriae. In this study, we show by using using human urinary tract epithelial cells that maximal internalization of E. coli is achieved only when bacteria are opsonized with complement. The concentrations of complement proteins in the urine rise sufficiently during infection to allow bacterial opsonization. The complement regulatory protein, CD46 (membrane cofactor protein), acts in cohort with fimbrial adhesion to promote the uptake of pathogenic E. coli. This uptake is inhibited by RNA interference to lower the expression of CD46 and by soluble CD46 that will competitively inhibit opsonized bacteria binding to cell surface CD46. We propose that efficient internalization of uropathogenic E. coli by the human urinary tract depends on cooperation between fimbrial-mediated adhesion and C3 receptor (CD46)-ligand interaction. Complement receptor-ligand interaction could pose a new target for interrupting the cycle of reinfection due to intracellular bacteria.     

Salmonella typhimurium induces an inositol phosphate flux in infected epithelial cells

Salmonella typhimurium, like many other intracellular pathogens, is capable of inducing its own uptake into non-phagocytic cells by a process termed invasion, and residing within a membrane-bound inclusion. During invasion it causes significant rearrangement of the host cytoskeleton, indicating that signals are transduced between the bacterium and the host cell cytoplasm, across the eukaryotic cell membrane. We found that intracellular inositol phosphate concentrations in HeLa cells increased during S. typhimurium entry and returned to normal levels after bacterial internalization. A chelator of intracellular calcium (BAPTA/AM) blocked S. typhimurium uptake into HeLa epithelial cells, but extracellular calcium chelators (BAPTA, EGTA, EDTA) had no effect on bacterial invasion. These results indicate that S. typhimurium may activate host cell phospholipase C activity to form inositol phosphates which in turn stimulate release of intracellular calcium stores to facilitate bacterial uptake.     

Rho GTPase activity modulates Pseudomonas aeruginosa internalization by epithelial cells

The Gram-negative pathogen Pseudomonas aeruginosa invades epithelial cells in vivo and in vitro . We have examined the pathway(s) by which epithelial cells internalize P. aeruginosa strain PA103 using Madin-Darby canine kidney (MDCK) cells. We have recently demonstrated that P. aeruginosa internalization occurs by an actin-dependent Toxin B-inhibited pathway which becomes downregulated as epithelial cells become polarized, suggesting that one or more of the Rho family GTPases is involved in bacterial internalization. Here, we demonstrate that activation of the Rho family GTPases by cytotoxic necrotizing factor 1 (CNF-1) stimulates P. aeruginosa internalization. Examination of the roles of the individual Rho family GTPases in internalization shows that expression of a constitutively active allele of RhoA (RhoAV14), but not of constitutively active Rac1 (Rac1V12) or Cdc42 (Cdc42V12), is sufficient to increase uptake of PA103 pscJ . This relative increase persists when bacterial infection is established at the basolateral surface of polarized cells, suggesting that the effect of RhoAV14 is not simply due to its known ability to disrupt tight junction integrity in polarized cells. RhoAV14-mediated stimulation of bacterial uptake is actin dependent as it is abrogated by exposure to latrunculin A. We also find that endogenous Rho GTP levels in epithelial cells are increased by infection with an internalized strain of P. aeruginosa; conversely, a poorly internalized isogenic strain expressing the bacterial anti-internalization protein ExoT causes decreased Rho GTP levels. Experimental inhibition of Rho, either by expressing dominant negative RhoAN19 or by inhibiting native Rho using a membrane permeable fusion construct of a Rho-specific inhibitor, C3 ADP-ribosyltransferase, does not inhibit PA103 pscJ internalization in MDCK or HeLa cells. Models consistent with these data are presented.     

Quantification of bacterial invasion into adherent cells by flow cytometry

Quantification of invasive, intracellular bacteria is critical in many areas of cellular microbiology and immunology. We describe a novel and fast approach to determine invasion of bacterial pathogens in adherent cell types such as epithelial cells or fibroblasts based on flow cytometry. Using the CEACAM-mediated uptake of Opa-expressing Neisseria gonorrhoeae as a well-characterized model of bacterial invasion, we demonstrate that the flow cytometry-based method yields results comparable to a standard antibiotic protection assay. Furthermore, the quantification of intracellular bacteria by the novel approach is not biased by intracellular killing of the microbes and correctly discriminates between cell-associated extracellular and bona fide intracellular bacteria. As flow cytometry-based quantification is also applicable to other pathogen-host interactions such as the integrin-mediated internalization of Staphylococcus aureus, this approach provides a fast and convenient alternative for the quantification of bacterial uptake and should be particularly useful in elucidating the molecular mechanisms of pathogen-triggered host cell invasion.     

Host adaptor proteins Gab1 and CrkII promote InlB-dependent entry of Listeria monocytogenes

The bacterial surface protein InlB mediates internalization of Listeria monocytogenes into mammalian cells through interaction with the host receptor tyrosine kinase, Met. InlB/Met interaction results in activation of the host phosphoinositide (PI) 3-kinase p85-p110, an event required for bacterial entry. p85-p110 activation coincides with tyrosine phosphorylation of the host adaptor Gab1, and formation of complexes between Gab1 and the p85 regulatory subunit of PI 3-kinase. When phosphorylated in response to agonists, Gab1 is known to recruit several Src-homology 2 (SH2) domain-containing proteins including p85, the tyrosine phosphatase Shp2 and the adaptor CrkII. Here, we demonstrate that Gab1.p85 and Gab1.CrkII complexes promote entry of Listeria. Overexpression of wild-type Gab1 stimulated entry, whereas Gab1 alleles unable to recruit all SH2 proteins known to bind wild-type Gab1 inhibited internalization. Further analysis with Gab1 alleles defective in binding individual effectors suggested that recruitment of p85 and CrkII are critical for entry. Consistent with this data, overexpression of wild-type CrkII stimulated bacterial uptake. Experiments with mutant CrkII alleles indicated that both the first and second SH3 domains of this adaptor participate in entry, with the second domain playing the most critical role. Taken together, these findings demonstrate novel roles for Gab1 and CrkII in Listeria internalization.     

The phosphoinositol-3-kinase-protein kinase B/Akt pathway is critical for Pseudomonas aeruginosa strain PAK internalization

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3 accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.     

Phosphatidylinositol 3-kinases in carcinoembryonic antigen-related cellular adhesion molecule-mediated internalization of Neisseria gonorrhoeae

Neisseria gonorrhoeae can be internalized by mammalian cells through interactions between bacterial opacity-associated (Opa) adhesins and members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. We examined the role of phosphatidylinositol 3-kinases (PI3Ks) in gonococcal invasion of epithelial cell lines expressing either CEACAM1 or CEACAM3. CEACAM3-mediated internalization, but not that mediated by CEACAM1, was accompanied by localized and transient accumulation of the class I PI3K product phosphatidylinositol 3,4,5-trisphosphate at sites of bacterial engulfment. Inhibition of phosphatidylinositol 3-kinases reduced CEACAM3-mediated uptake but, paradoxically, led to an increase in intracellular survival of bacteria internalized via either CEACAM1 or CEACAM3, suggesting additional roles for PI3K products. Consistent with this finding, the class III PI3K product phosphatidylinositol 3-phosphate accumulated and persisted in the membrane of gonococcal phagosomes after internalization. Inhibition of PI3K blocked phagosomal acquisition of the late endosomal marker lysosome-associated membrane protein 2 and reduced phagosomal acidification. Inhibiting phagosomal acidification with concanamycin A also increased survival of intracellular gonococci. These results suggest two modes of action of phosphatidylinositol 3-kinases during internalization of gonococci: synthesis of phosphatidylinositol 3,4,5-trisphosphate is important for CEACAM3-mediated uptake, while phosphatidylinositol 3-phosphate is needed for phagosomal maturation and acidification, which are required for optimal bacterial killing.     

Entry of OpaA+ gonococci into HEp-2 cells requires concerted action of glycosaminoglycans, fibronectin and integrin receptors

Heparan sulphate proteoglycans are increasingly implicated as eukaryotic cell surface receptors for bacterial pathogens. Here, we report that Neisseria gonorrhoeae adheres to proteoglycan receptors on HEp-2 epithelial cells but that internalization of the bacterium by this cell type requires the serum glycoprotein fibronectin. Fibronectin was shown to bind specifically to gonococci producing the OpaA adhesin. Binding assays with fibronectin fragments located the bacterial binding site near the N-terminal end of the molecule. However, none of the tested fibronectin fragments supported gonococcal entry into the eukaryotic cells; a 120 kDa fragment carrying the cell adhesion domain with the amino acid sequence RGD even inhibited the fibronectin-mediated uptake of MS11-OpaA. This inhibition could be mimicked by an RGD-containing hexapeptide and by α5β1 integrin-specific antibodies, suggesting that interaction of the central region of fibronectin with integrin receptors facilitated bacterial uptake. Fibronectin was unable to promote gonococcal entry into HEp-2 cells that had been treated with the enzyme heparinase III, which degrades the glycosaminoglycan side-chains of proteoglycan receptors. On the basis of these results, we propose a novel cellular uptake pathway for bacteria, which involves the binding of the pathogen to glycosaminoglycans that, in turn, act as co-receptors facilitating fibronectin-mediated bacterial uptake through integrin receptors. In this scenario, fibronectin would act as a molecular bridge linking the Opa–proteoglycan complex with host cell integrin receptors.     

The Mig protein of Streptococcus dysgalactiae inhibits bacterial internalization into bovine mammary gland epithelial cells

The role of the Mig protein of Streptococcus dysgalactiae in bacterial adhesion and internalization of bovine mammary gland epithelial cells (MAC-T) was investigated with the wild-type and isogenic mig mutant strains. While there was no difference in adhesion between the strains, the wild-type strain exhibited a significantly lower level of invasion than the mutants. The lower level of internalization of the Mig(+) strain is likely due to Mig-mediated interference with uptake of the microorganisms rather than the host protein binding properties of Mig. Avoidance of intimate interactions with the host cells might be an alternative strategy for S. dysgalactiae to survive and persist in the bovine mammary glands.     

Ligation of Cell Surface Heparan Sulfate Proteoglycans by Antibody-Coated Beads Stimulates Phagocytic Uptake into Epithelial Cells: A Model for Cellular Invasion byNeisseria gonorrhoeae

Binding of a particular opacity outer membrane protein (Opa) ofNeisseria gonorrhoeaeto cell surface heparan sulfate proteoglycans (HSPGs) of epithelial cells results in tight bacterial adherence; however, the role of this ligand–receptor interaction in triggering the subsequent bacterial internalization step is uncertain. Here we have used latex beads coated with HSPG-ligating antibodies as anin vitromodel to study the role of HSPGs in gonococcal uptake into epithelial cells. Beads and gonococci showed the same cell line-specified adherence patterns and increase in phagocytic uptake mediated by serum or purified vitronectin (Vn). Heparitinase digestion as well as antibody competition experiments indicate that a critical level of HSPG ligation is necessary and sufficient to trigger phagocytic uptake into epithelial cells. Vn was found to specifically enhance HSPG-dependent phagocytic uptake while phagocytosis resulting from the ligation of other cell surface receptors was unaffected in the presence of Vn. Pharmacologicial studies with PKC inhibitors suggest a role for PKC in phagocytic uptake of HSPG-ligating beads. The use of drugs impairing cytoskeletal functions indicates that HSPG-dependent phagocytosis requires actin polymerization by a process distinct from receptor-mediated endocytosis.     

The antibacterial action of cupric ions in Pseudomonas syringae

It has been demonstrated that under iron-restricted conditions Bordetella pertussis can take up iron from human transferrin within 30 min of exposure. B. pertussis utilizes two mechanisms for acquiring iron from human transferrin, a direct contact method and a siderophore mediated system. Both systems are shown to result in bacterial internalization of iron from transferrin. However, direct contact between B. pertussis and transferrin provides far more effective iron uptake than siderophore activity alone.     

WAVE2 signaling mediates invasion of polarized epithelial cells by Salmonella typhimurium

The bacterial pathogen Salmonella penetrates the intestinal epithelium by inducing its own phagocytosis into epithelial cells. The dramatic reorganization of the actin cytoskeleton required for internalization is driven by bacterial manipulation of host signaling pathways, including activation of the Rho family GTPase Rac1 and subsequent activation of the Arp2/3 complex. However, the mechanisms linking these two events remain poorly understood. Rac1 is thought to promote activation of the Arp2/3 complex through its interaction with suppressor of cAMP receptor/WASP family verprolin-homologous (SCAR/WAVE) family proteins, but this interaction is apparently indirect. Two different Rac1 effectors have been shown to bind WAVE2: IRSp53, the SH3 domain of which binds the WAVE2 proline-rich domain, and PIR121/Sra-1, which forms a pentameric complex containing WAVE, Abi1, Nap1, and HSPC300. However, the extent to which each of these complexes contributes to Arp2/3 complex activation in the context of Salmonella infection is unclear. Here, we show that WAVE2 is necessary for efficient invasion of epithelial cells by Salmonella typhimurium. We found that although Salmonella infection strongly promotes the formation of an IRSp53/WAVE2 complex, IRSp53 is not necessary for bacterial internalization. In contrast, disruption of the PIR121/Nap1/Abi1/WAVE2/HSPC300 complex potently inhibits bacterial uptake. These results indicate that WAVE2 is an important component in signaling pathways leading to Salmonella invasion. Although infection leads to the formation of an IRSp53/WAVE2 complex, it is the association of WAVE2 with the Abi1/Nap1/PIR121/HSPC300 complex that regulates bacterial internalization.