Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy.

Xenophagy, a type of selective autophagy, is a bactericidal membrane trafficking that targets cytosolic bacterial pathogens, but the membrane homeostatic system to cope with bacterial infection in xenophagy is not known. Here, we show that the endosomal sorting complexes required for transport (ESCRT) machinery is needed to maintain homeostasis of xenophagolysosomes damaged by a bacterial toxin, which is regulated through the TOM1L2-Rab41 pathway that recruits AAA-ATPase VPS4. We screened Rab GTPases and identified Rab41 as critical for maintaining the acidification of xenophagolysosomes. Confocal microscopy revealed that ESCRT components were recruited to the entire xenophagolysosome, and this recruitment was inhibited by intrabody expression against bacterial cytolysin, indicating that ESCRT targets xenophagolysosomes in response to a bacterial toxin. Rab41 translocates to damaged autophagic membranes via adaptor protein TOM1L2 and recruits VPS4 to complete ESCRT-mediated membrane repair in a unique GTPase-independent manner. Finally, we demonstrate that the TOM1L2-Rab41 pathway-mediated ESCRT is critical for the efficient clearance of bacteria through xenophagy.

Anti-InlA single-domain antibodies that inhibit the cell invasion of Listeria monocytogenes.

Listeriosis, caused by infection with Listeria monocytogenes, is a severe disease with a high mortality rate. The L. monocytogenes virulence factor, internalin family protein InlA, which binds to the host receptor E-cadherin, is necessary to invade host cells. Here, we isolated two single-domain antibodies (VHHs) that bind to InlA with picomolar affinities from an alpaca immune library using the phage display method. These InlA-specific VHHs inhibited the binding of InlA to the extracellular domains of E-cadherin in vitro as shown by biophysical interaction analysis. Furthermore, we determined that the VHHs inhibited the invasion of L. monocytogenes into host cells in culture. High-resolution X-ray structure analyses of the complexes of VHHs with InlA revealed that the VHHs bind to the same binding site as E-cadherin against InlA. We conclude that these VHHs have the potential for use as drugs to treat listeriosis.

Group A Streptococcus cation diffusion facilitator proteins contribute to immune evasion by regulating intracellular metal concentrations.

Cation diffusion facilitators (CDFs) are a large family of divalent metal transporters with broad specificities that contribute to intracellular metal homeostasis and toxicity in bacterial pathogens. Streptococcus pyogenes (Group A Streptococcus [GAS]) expresses two homologous CDF efflux transporters, MntE and CzcD, which selectively transport Mn and Zn, respectively. We discovered that the MntE- and CzcD-deficient strains exhibited a marked decrease in the viability of macrophage-differentiated THP-1 cells and neutrophils. In addition, the viability of mice infected with both deficient strains markedly increased. Consistent with a previous study, our results suggest that MntE regulates the PerR-dependent oxidative stress response by maintaining intracellular Mn levels and contributing to the growth of GAS. The maturation and proteolytic activity of streptococcal cysteine protease (SpeB), an important virulence factor in GAS, has been reported to be abrogated by zinc and copper. Zn inhibited the maturation and proteolytic activity of SpeB in the culture supernatant of the CzcD-deficient strain. Furthermore, Mn inhibited SpeB maturation and proteolytic activity in a MntE-deficient strain. Since the host pathogenicity of the SpeB-deficient strain was significantly reduced, maintenance of intracellular manganese and zinc levels in the GAS via MntE and CzcD may not only confer metal resistance to the bacterium, but may also play an essential role in its virulence. These findings provide new insights into the molecular mechanisms of pathogenicity, which allow pathogens to survive under stressful conditions associated with elevated metal ion concentrations during host infection.

In vivo tracking of plasmid DNA/mRNA: A novel labelling precursor for 89Zr positron emission tomography.

Herein, we developed a novel labelling precursor Fe-DFO-5 for plasmid DNA (pDNA) utilizing 89Zr as a radioisotope for PET imaging. 89Zr-labelled pDNA showed comparable gene expression to non-labelled pDNA. The biodistribution of 89Zr-labelled pDNA after local or systemic administration in mice was evaluated. Furthermore, this labelling method was also applied to mRNA.

FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan.

Xenophagy, also known as antibacterial selective autophagy, degrades invading bacterial pathogens such as group A Streptococcus (GAS) to defend cells. Although invading bacteria are known to be marked with ubiquitin and selectively targeted by xenophagy, how ubiquitin ligases recognize invading bacteria is poorly understood. Here, we show that FBXO2, a glycoprotein-specific receptor for substrate in the SKP1/CUL1/F-box protein (SCF) ubiquitin ligase complex, mediates recognition of GlcNAc side chains of the GAS surface carbohydrate structure and promotes ubiquitin-mediated xenophagy against GAS. FBXO2 targets cytosolic GAS through its sugar-binding motif and GlcNAc expression on the GAS surface. FBXO2 knockout resulted in decreased ubiquitin accumulation on intracellular GAS and xenophagic degradation of bacteria. Furthermore, SCF components such as SKP1, CUL1, and ROC1 are required for ubiquitin-mediated xenophagy against GAS. Thus, SCFFBXO2 recognizes GlcNAc residues of GAS surface carbohydrates and functions in ubiquitination during xenophagy.

Biological Effect of Streptococcus pyogenes-Released Extracellular Vesicles on Human Monocytic Cells, Induction of Cytotoxicity, and Inflammatory Response.

Most bacteria naturally release spherical lipid-bilayered extracellular vesicles (EVs) containing proteins, nucleic acids, and virulence-related molecules, thus contributing to diverse biological functions including transport of virulence factors. The group A streptococcus, Streptococcus pyogenes (GAS), a major human pathogen, also releases EVs; however, it remains unclear how GAS EVs interact physiologically and pathologically with host cells, and what the differences are between invasive and non-invasive strains. The proteome profile in this study revealed that GAS EVs enclosed many virulence-related proteins such as streptolysin O and NAD-glycohydrolase, facilitating their pathogenicity, and invasive GAS EVs were more abundant than non-invasive counterparts. In terms of biological effects, invasive GAS EVs showed slo-dependent cytotoxic activity and the induction of cytokine expression, contributing to GAS pathogenicity directly. Although non-invasive GAS EVs did not show cytotoxic activity, they may be utilized as a means to prevent antibacterial mechanisms such as autophagy, leading to enhancement of their own survival in the intracellular environment after the infection. These results suggest that invasive and non-invasive GAS EVs play different roles in GAS infection strategy and pathogenicity. Our findings also indicate that EVs could be a key factor for GAS pathogenicity in GAS-host interactions.

Single-chain variable fragment (scFv) targeting streptolysin O controls group A Streptococcus infection.

Streptococcus pyogenes (Group A Streptococcus, GAS) causes a range of human diseases, including life-threatening and severe invasive GAS infections, such as streptococcal toxic shock syndrome (STSS). Several antibiotics, including penicillin, are effective against GAS. Still, invasive GAS diseases have a high mortality rate (>30%). Clinical isolates from STSS patients show higher expression of pore-forming streptolysin O (SLO). Thus, SLO is an important pathogenic factor for GAS and may be an effective target for treatment of GAS disease. We succeeded in obtaining a single-chain variable fragment (scFv) SLO-I4 capable of recognizing SLO, which significantly inhibited GAS-induced cell lytic activity in erythrocytes, macrophages, and epithelial cells. In epithelial cells, SLO-I4 significantly reduced SLO-mediated endosomal membrane damage, which consequently prevented bacterial escape from the endosome. The effectiveness of anti-SLO scFv in counteracting SLO function suggests that it might be beneficial against GAS infections.

Intracellular Group A Streptococcus Induces Golgi Fragmentation To Impair Host Defenses through Streptolysin O and NAD-Glycohydrolase.

Group A Streptococcus (GAS; Streptococcus pyogenes) is a major human pathogen that causes streptococcal pharyngitis, skin and soft tissue infections, and life-threatening conditions such as streptococcal toxic-shock syndrome. During infection, GAS not only invades diverse host cells but also injects effector proteins such as NAD-glycohydrolase (Nga) into the host cells through a streptolysin O (SLO)-dependent mechanism without invading the cells; Nga and SLO are two major virulence factors that are associated with increased bacterial virulence. Here, we have shown that the invading GAS induces fragmentation of the Golgi complex and inhibits anterograde transport in the infected host cells through the secreted toxins SLO and Nga. GAS infection-induced Golgi fragmentation required both bacterial invasion and SLO-mediated Nga translocation into the host cytosol. The cellular Golgi network is critical for the sorting of surface molecules and is thus essential for the integrity of the epithelial barrier and for the immune response of macrophages to pathogens. In epithelial cells, inhibition of anterograde trafficking by invading GAS and Nga resulted in the redistribution of E-cadherin to the cytosol and an increase in bacterial translocation across the epithelial barrier. Moreover, in macrophages, interleukin-8 secretion in response to GAS infection was found to be suppressed by intracellular GAS and Nga. Our findings reveal a previously undescribed bacterial invasion-dependent function of Nga as well as a previously unrecognized GAS-host interaction that is associated with GAS pathogenesis.IMPORTANCE Two prominent virulence factors of group A Streptococcus (GAS), streptolysin O (SLO) and NAD-glycohydrolase (Nga), are linked to enhanced pathogenicity of the prevalent GAS strains. Recent advances show that SLO and Nga are important for intracellular survival of GAS in epithelial cells and macrophages. Here, we found that invading GAS disrupts the Golgi complex in host cells through SLO and Nga. We show that GAS-induced Golgi fragmentation requires bacterial invasion into host cells, SLO pore formation activity, and Nga NADase activity. GAS-induced Golgi fragmentation results in the impairment of the epithelial barrier and chemokine secretion in macrophages. This immune inhibition property of SLO and Nga by intracellular GAS indicates that the invasion of GAS is associated with virulence exerted by SLO and Nga.

Complete Genome Sequences of Two Streptococcus suis Strains Isolated from Asymptomatic Pigs.

Streptococcus suis is an important zoonotic pathogen that causes major economic problems in the pig industry worldwide and serious infections in humans, including meningitis and septicemia. Here, we report the complete genome sequences of two strains isolated from asymptomatic pigs.

Complete Genome Sequences of Streptococcus pyogenes Serotype M3, M28, and M89 Strains Isolated from Human Patients in Japan, 1994 to 2009.

Streptococcus pyogenes (group A Streptococcus [GAS]) is a major human pathogen that occasionally causes severe and life-threatening invasive diseases. Here, we report the complete genome sequences of four GAS strains of three M types, which were isolated from patients with severe invasive disease in Japan.

Identification of Group A Streptococcus-Containing Autophagosome-Like Vacuoles.

Group A Streptococcus (GAS) is one of the major human pathogens that can invade nonphagocytic cells. GAS internalized through endocytosis secretes the pore-forming toxin Streptolysin O (SLO) to escape into the cytoplasm. The cytosolic GAS is selectively captured by autophagic membranes (GAS-containing autophagosome-like vacuoles, GcAVs) and delivered to lysosomes for degradation. Macroautophagy (referred to as autophagy hereafter) is a highly conserved lysosome-mediated catabolic process, which is critical for cellular homeostasis. Autophagy also acts as an intracellular immune system. In this section, we describe how to identify GcAVs in infected cells using fluorescent microscopy.

TBC1D9 regulates TBK1 activation through Ca2+ signaling in selective autophagy.

Invading microbial pathogens can be eliminated selectively by xenophagy. Ubiquitin-mediated autophagy receptors are phosphorylated by TANK-binding kinase 1 (TBK1) and recruited to ubiquitinated bacteria to facilitate autophagosome formation during xenophagy, but the molecular mechanism underlying TBK1 activation in response to microbial infection is not clear. Here, we show that bacterial infection increases Ca2+ levels to activate TBK1 for xenophagy via the Ca2+-binding protein TBC1 domain family member 9 (TBC1D9). Mechanistically, the ubiquitin-binding region (UBR) and Ca2+-binding motif of TBC1D9 mediate its binding with ubiquitin-positive bacteria, and TBC1D9 knockout suppresses TBK1 activation and subsequent recruitment of the ULK1 complex. Treatment with a Ca2+ chelator impairs TBC1D9-ubiquitin interactions and TBK1 activation during xenophagy. TBC1D9 is also recruited to damaged mitochondria through its UBR and Ca2+-binding motif, and is required for TBK1 activation during mitophagy. These results indicate that TBC1D9 controls TBK1 activation during xenophagy and mitophagy through Ca2+-dependent ubiquitin-recognition.

Group A Streptococcus modulates RAB1- and PIK3C3 complex-dependent autophagy.

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2.

Autophagy Receptor Tollip Facilitates Bacterial Autophagy by Recruiting Galectin-7 in Response to Group A Streptococcus Infection.

Bacterial autophagy-a type of macroautophagy that is also termed xenophagy-selectively targets intracellular bacteria such as group A Streptococcus (GAS), a ubiquitous pathogen that causes numerous serious diseases, including pharyngitis, skin infections, and invasive life-threatening infections. Although bacterial autophagy is known to eliminate invading bacteria via the action of autophagy receptors, the underlying mechanism remains unclear. Herein, we elucidated that Tollip functions as a bacterial-autophagy receptor in addition to participating involved in the intracellular immunity mechanism that defends against bacterial infection. Tollip was recruited to GAS-containing endosomal vacuoles prior to the escape of GAS into the cytosol; additionally, Tollip knockout disrupted the recruitment of other autophagy receptors, such as NBR1, TAX1BP1, and NDP52, to GAS-containing autophagosomes and led to prolonged intracellular survival of GAS. Furthermore, Tollip was found to be required for the recruitment of galectin-1 and -7 to GAS-containing autophagosomes, and immunoprecipitation results indicated that Tollip interacts with galectin-7. Lastly, our data also revealed that galectin-1 and -7 are involved in the restriction of GAS replication in cells. These results demonstrated that Tollip modulates bacterial autophagy by recruiting other autophagy receptors and galectins.

Group A Streptococcus NAD-Glycohydrolase Inhibits Caveolin 1-Mediated Internalization Into Human Epithelial Cells.

Group A Streptococcus (GAS) invades epithelial cells causing persistent infection. GAS has a variety of effector proteins that modulate host systems to affect their survival in host environments. The main effector proteins of GAS are NAD-glycohydrolase (Nga) and streptolysin O (SLO). Although Nga has NADase activity and shows SLO-dependent cytotoxicity, some clinical isolates harbor NADase-inactive subtypes of Nga, and the function of NADase-inactive Nga is still unclear. In this study, we found that deletion of nga enhanced the internalization of GAS into HeLa and Ca9-22 cells. Amino acid substitution of Nga R289K/G330D (NADase-inactive) does not enhance GAS invasion, suggesting that Nga may inhibit the internalization of GAS into host cells in an NADase-independent manner. Moreover, double deletion of slo and nga showed similar invasion percentages compared with wild-type GAS, indicating the important role of SLO in the inhibition of GAS invasion by Nga. Furthermore, enhanced internalization of the nga deletion mutant was not observed in Cav1-knockout HeLa cells. Altogether, these findings demonstrate an unrecognized NADase-independent function of Nga as a negative regulator of CAV1-mediated internalization into epithelial cells.

Complete Genome Sequences of Eight Methicillin-Resistant Staphylococcus aureus Strains Isolated from Patients in Japan.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen causing nosocomial infections, and the clinical manifestations of MRSA range from asymptomatic colonization of the nasal mucosa to soft tissue infection to fulminant invasive disease. Here, we report the complete genome sequences of eight MRSA strains isolated from patients in Japan.

Phylogenetic relationship of prophages is affected by CRISPR selection in Group A Streptococcus.

BACKGROUND: Group A Streptococcus (GAS) is a major human pathogen, which is associated with a wide spectrum of invasive diseases, such as pharyngitis, scarlet fever, rheumatic fever, and streptococcal toxic shock syndrome (STSS). It is hypothesized that differences in GAS pathogenicity are related to the acquisition of diverse bacteriophages (phages). Nevertheless, the GAS genome also harbors clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (cas) genes, which play an important role in eliminating foreign DNA, including those of phages. However, the structure of prophages in GAS strains is mosaic, and the phylogenetic relationship between prophages and CRISPR is not clear. In this study, we analyzed CRISPR and prophage structure using 118 complete genome sequences of GAS strains to elucidate the relationship between two genomic elements. Additionally, phylogenetic and M-type analyses were performed. RESULTS: Of the 118 GAS strains, 80 harbored type I-C and/or II-A CRISPR/cas loci. A total of 553 spacer sequences were identified from CRISPR/cas loci and sorted into 229 patterns. We identified and classified 373 prophages into 14 groups. Some prophage groups shared a common integration site, and were related to M-type. We further investigated the correlation between spacer sequences and prophages. Of the 229 spacer sequence patterns, 203 were similar to that of other GAS prophages. No spacer showed similarity with that of a specific prophage group with mutL integration site. Moreover, the average number of prophages in strains with type II-A CRISPR was significantly less than that in type I-C CRISPR and non-CRISPR strains. However, there was no statistical difference between the average number of prophages in type I-C strains and that in non-CRISPR strains. CONCLUSIONS: Our results indicated that type II-A CRISPR may play an important role in eliminating phages and that the prophage integration site may be an important criterion for the acceptance of foreign DNA by GAS. M type, spacer sequence, and prophage group data were correlated with the phylogenetic relationships of GAS. Therefore, we hypothesize that genetic characteristics and/or phylogenetic relationships of GAS may be estimated by analyzing its spacer sequences.

RAB30 regulates PI4KB (phosphatidylinositol 4-kinase beta)-dependent autophagy against group A Streptococcus.

Macroautophagy/autophagy plays an important role in the immune response to invasion by intracellular pathogens such as group A Streptococcus (GAS; Streptococcus pyogenes). We previously identified RAB30, a Golgi-resident GTPase, as a novel anti-bacterial autophagic regulator in the formation of GAS-containing autophagosome-like vacuoles (GcAVs); however, the precise mechanism underlying this process remains elusive. Here, we elucidate a novel property of RAB30: the ability to recruit PI4KB (phosphatidylinositol 4-kinase beta) to the Golgi apparatus and GcAVs. We found that trans-Golgi network (TGN) vesicles were incorporated into GcAVs via RAB30 to promote GcAV formation. Moreover, depletion of phosphatidylinositol-4-phosphate (PtdIns4P), a phosphatidylinositol enriched in the TGN, by wortmannin and phenylarsine oxide, followed by subsequent repletion with exogenous PtdIns4P revealed that PtdIns4P is crucial for GcAV formation. Furthermore, we identify an interaction between RAB30 and PI4KB, in which the knockdown of RAB30 decreased the localization of PI4KB to the TGN and GcAVs. Finally, PI4KB knockout suppressed autophagy by inhibiting GcAV formation, resulting in the increased survival of GAS. Our results demonstrate a novel autophagosomal formation mechanism involving coordinative functions of RAB30 and PI4KB distinct from those utilized in canonical autophagy. Abbreviations: GAS: group A Streptococcus; GcAVs: GAS-containing autophagosome-like vacuoles; PI4KB: phosphatidylinositol 4-kinase beta; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; PtdIns5P: phosphatidylinositol-5-phosphate; SLO: streptolysin O; TGN: trans-Golgi network; TGOLN2: trans-golgi network protein 2; PH: plekstrin homology; OSBP: oxysterol binding protein.

LAMTOR2/LAMTOR1 complex is required for TAX1BP1-mediated xenophagy.

Xenophagy, also known as antibacterial autophagy, plays a role in host defence against invading pathogens such as Group A Streptococcus (GAS) and Salmonella. In xenophagy, autophagy receptors are used in the recognition of invading pathogens and in autophagosome maturation and autolysosome formation. However, the mechanism by which autophagy receptors are regulated during bacterial infection remains poorly elucidated. In this study, we identified LAMTOR2 and LAMTOR1, also named p14 and p18, respectively, as previously unrecognised xenophagy regulators that modulate the autophagy receptor TAX1BP1 in response to GAS and Salmonella invasion. LAMTOR1 was localized to bacterium-containing endosomes, and LAMTOR2 was recruited to bacterium-containing damaged endosomes in a LAMTOR1-dependent manner. LAMTOR2 was dispensable for the formation of autophagosomes targeting damaged membrane debris surrounding cytosolic bacteria, but it was critical for autolysosome formation, and LAMTOR2 interacted with the autophagy receptors NBR1, TAX1BP1, and p62 and was necessary for TAX1BP1 recruitment to pathogen-containing autophagosomes. Notably, knockout of TAX1BP1 caused a reduction in autolysosome formation and subsequent bacterial degradation. Collectively, our findings demonstrated that the LAMTOR1/2 complex is required for recruiting TAX1BP1 to autophagosomes and thereby facilitating autolysosome formation during bacterial infection.

The Impact of Endogenous Breast Cancer Resistance Protein on Human P-Glycoprotein-Mediated Transport Assays Using LLC-PK1 Cells Transfected With Human P-Glycoprotein.

Lilly Laboratories cell porcine kidney 1 (LLC-PK1) cells transfected with human P-glycoprotein (LLC-PK1-P-gp) are widely used in transport assays to identify drug candidates that function as substrates of this efflux transporter. Endogenous transporters expressed in LLC-PK1 cells may complicate the interpretation of findings from P-gp-mediated transport assays. We investigated the impact of porcine breast cancer resistance protein (Bcrp) in P-gp-mediated transport assays in LLC-PK1 cells. Porcine Bcrp mRNA was detected in both LLC-PK1 wildtype (WT) and LLC-PK1-P-gp cells by quantitative RT-PCR. To investigate the activity and impact of porcine Bcrp, we conducted transport assays using 6 typical BCRP substrates in LLC-PK1 cells. Efflux ratios (ER) of the 6 BCRP substrates in LLC-PK1 WT cells were >2, and were reduced in the presence of the BCRP inhibitor Ko143. The efflux activities of the 6 BCRP substrates were confirmed using MDCKII cells transfected with human BCRP. Net ERs of prazosin and fluvastatin, dual substrates of P-gp and BCRP, determined by dividing ERs in LLC-PK1-P-gp cells by those in LLC-PK1 WT cells, were <2, but increased to >2 in the presence of Ko143. These results indicated that endogenous Bcrp in LLC-PK1 cells was involved in the transport of BCRP substrates and may interfere with the identification of P-gp substrates.