Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids.

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.

BRAFV600E-mutated serrated colorectal neoplasia drives transcriptional activation of cholesterol metabolism.

BRAF mutations occur early in serrated colorectal cancers, but their long-term influence on tissue homeostasis is poorly characterized. We investigated the impact of short-term (3 days) and long-term (6 months) expression of BrafV600E in the intestinal tissue of an inducible mouse model. We show that BrafV600E perturbs the homeostasis of intestinal epithelial cells, with impaired differentiation of enterocytes emerging after prolonged expression of the oncogene. Moreover, BrafV600E leads to a persistent transcriptional reprogramming with enrichment of numerous gene signatures indicative of proliferation and tumorigenesis, and signatures suggestive of metabolic rewiring. We focused on the top-ranking cholesterol biosynthesis signature and confirmed its increased expression in human serrated lesions. Functionally, the cholesterol lowering drug atorvastatin prevents the establishment of intestinal crypt hyperplasia in BrafV600E-mutant mice. Overall, our work unveils the long-term impact of BrafV600E expression in intestinal tissue and suggests that colorectal cancers with mutations in BRAF might be prevented by statins.

Bypassing mitochondrial defects rescues Huntington's phenotypes in Drosophila.

Huntington's disease (HD) is a fatal neurodegenerative disease with limited treatment options. Human and animal studies have suggested that metabolic and mitochondrial dysfunctions contribute to HD pathogenesis. Here, we use high-resolution respirometry to uncover defective mitochondrial oxidative phosphorylation and electron transfer capacity when a mutant huntingtin fragment is targeted to neurons or muscles in Drosophila and find that enhancing mitochondrial function can ameliorate these defects. In particular, we find that co-expression of parkin, an E3 ubiquitin ligase critical for mitochondrial dynamics and homeostasis, produces significant enhancement of mitochondrial respiration when expressed either in neurons or muscles, resulting in significant rescue of neurodegeneration, viability and longevity in HD model flies. Targeting mutant HTT to muscles results in larger mitochondria and higher mitochondrial mass, while co-expression of parkin increases mitochondrial fission and decreases mass. Furthermore, directly addressing HD-mediated defects in the fly's mitochondrial electron transport system, by rerouting electrons to either bypass mitochondrial complex I or complexes III-IV, significantly increases mitochondrial respiration and results in a striking rescue of all phenotypes arising from neuronal mutant huntingtin expression. These observations suggest that bypassing impaired mitochondrial respiratory complexes in HD may have therapeutic potential for the treatment of this devastating disorder.

Generation of Distinct Differentially Culturable Forms of Burkholderia following Starvation at Low Temperature.

Bacteria have developed unique mechanisms to adapt to environmental stresses and challenges of the immune system. Here, we report that Burkholderia pseudomallei, the causative agent of melioidosis, and its laboratory surrogate, Burkholderia thailandensis, utilize distinct mechanisms for surviving starvation at different incubation temperatures. At 21°C, Burkholderia are present as short rods which can rapidly reactivate and form colonies on solid media. At 4°C, Burkholderia convert into coccoid forms that cannot be cultured on solid agar but can be resuscitated in liquid media supplemented with supernatant obtained from logarithmic phase cultures of B. thailandensis, or catalase and Tween 80, thus displaying characteristics of differentially culturable bacteria (DCB). These DCB have low intensity fluorescence when stained with SYTO 9, have an intact cell membrane (propidium iodide negative), and contain 16S rRNA at levels comparable with growing cells. We also present evidence that lytic transglycosylases, a family of peptidoglycan-remodeling enzymes, are involved in the generation of coccoid forms and their resuscitation to actively growing cells. A B. pseudomallei ΔltgGCFD mutant with four ltg genes deleted did not produce coccoid forms at 4°C and could not be resuscitated in the liquid media evaluated. Our findings provide insights into the adaptation of Burkholderia to nutrient limitation and the generation of differentially culturable bacteria. IMPORTANCE Bacterial pathogens exhibit physiologically distinct forms that enable their survival in an infected host, the environment and following exposure to antimicrobial agents. B. pseudomallei causes the disease melioidosis, which has a high mortality rate and is difficult to treat with antibiotics. The bacterium is endemic to several countries and detected in high abundance in the environment. Here, we report that during starvation at low temperature, B. pseudomallei produces coccoid forms that cannot grow in standard media and which, therefore, can be challenging to detect using common tools. We provide evidence that the formation of these cocci is mediated by cell wall-specialized enzymes and lytic transglycosylases, and that resuscitation of these forms occurs following the addition of catalase and Tween 80. Our findings have important implications for the disease control and detection of B. pseudomallei, an agent of both public health and defense interest.

A novel role for kynurenine 3-monooxygenase in mitochondrial dynamics.

The enzyme kynurenine 3-monooxygenase (KMO) operates at a critical branch-point in the kynurenine pathway (KP), the major route of tryptophan metabolism. As the KP has been implicated in the pathogenesis of several human diseases, KMO and other enzymes that control metabolic flux through the pathway are potential therapeutic targets for these disorders. While KMO is localized to the outer mitochondrial membrane in eukaryotic organisms, no mitochondrial role for KMO has been described. In this study, KMO deficient Drosophila melanogaster were investigated for mitochondrial phenotypes in vitro and in vivo. We find that a loss of function allele or RNAi knockdown of the Drosophila KMO ortholog (cinnabar) causes a range of morphological and functional alterations to mitochondria, which are independent of changes to levels of KP metabolites. Notably, cinnabar genetically interacts with the Parkinson's disease associated genes Pink1 and parkin, as well as the mitochondrial fission gene Drp1, implicating KMO in mitochondrial dynamics and mitophagy, mechanisms which govern the maintenance of a healthy mitochondrial network. Overexpression of human KMO in mammalian cells finds that KMO plays a role in the post-translational regulation of DRP1. These findings reveal a novel mitochondrial role for KMO, independent from its enzymatic role in the kynurenine pathway.

Mycobacterial phosphatase PstP regulates global serine threonine phosphorylation and cell division.

Protein phosphatase PstP is conserved throughout the Actinobacteria in a genetic locus related to cell wall synthesis and cell division. In many Actinobacteria it is the sole annotated serine threonine protein phosphatase to counter the activity of multiple serine threonine protein kinases. We used transcriptional knockdown, electron microscopy and comparative phosphoproteomics to investigate the putative dual functions of PstP as a specific regulator of cell division and as a global regulator of protein phosphorylation. Comparative phosphoproteomics in the early stages of PstP depletion showed hyperphosphorylation of protein kinases and their substrates, confirming PstP as a negative regulator of kinase activity and global serine and threonine phosphorylation. Analysis of the 838 phosphorylation sites that changed significantly, suggested that PstP may regulate diverse phosphoproteins, preferentially at phosphothreonine near acidic residues, near the protein termini, and within membrane associated proteins. Increased phosphorylation of the activation loop of protein kinase B (PknB) and of the essential PknB substrate CwlM offer possible explanations for the requirement for pstP for growth and for cell wall defects when PstP was depleted.

Two Faces of CwlM, an Essential PknB Substrate, in Mycobacterium tuberculosis.

Tuberculosis claims >1 million lives annually, and its causative agent Mycobacterium tuberculosis is a highly successful pathogen. Protein kinase B (PknB) is reported to be critical for mycobacterial growth. Here, we demonstrate that PknB-depleted M. tuberculosis can replicate normally and can synthesize peptidoglycan in an osmoprotective medium. Comparative phosphoproteomics of PknB-producing and PknB-depleted mycobacteria identify CwlM, an essential regulator of peptidoglycan synthesis, as a major PknB substrate. Our complementation studies of a cwlM mutant of M. tuberculosis support CwlM phosphorylation as a likely molecular basis for PknB being essential for mycobacterial growth. We demonstrate that growing mycobacteria produce two forms of CwlM: a non-phosphorylated membrane-associated form and a PknB-phosphorylated cytoplasmic form. Furthermore, we show that the partner proteins for the phosphorylated and non-phosphorylated forms of CwlM are FhaA, a fork head-associated domain protein, and MurJ, a proposed lipid II flippase, respectively. From our results, we propose a model in which CwlM potentially regulates both the biosynthesis of peptidoglycan precursors and their transport across the cytoplasmic membrane.

Vps33b is crucial for structural and functional hepatocyte polarity.

BACKGROUND & AIMS: In the normal liver, hepatocytes form a uniquely polarised cell layer that enables movement of solutes from sinusoidal blood to canalicular bile. Whilst several cholestatic liver diseases with defects of hepatocyte polarity have been identified, the molecular mechanisms of pathogenesis are not well defined. One example is arthrogryposis, renal dysfunction and cholestasis syndrome, which in most patients is caused by VPS33B mutations. VPS33B is a protein involved in membrane trafficking that interacts with RAB11A at recycling endosomes. To understand the pathways that regulate hepatocyte polarity better, we investigated VPS33B deficiency using a novel mouse model with a liver-specific Vps33b deletion. METHODS: To assess functional polarity, plasma and bile samples were collected from Vps33b liver knockout (Vps33bfl/fl-AlfpCre) and control (Vps33bfl/fl) mice; bile components or injected substrates were quantitated by mass spectrometry or fluorometry. For structural analysis, livers underwent light and transmission electron microscopy. Apical membrane and tight junction protein localisation was assessed by immunostaining. Adeno-associated virus vectors were used for in vivo gene rescue experiments. RESULTS: Like patients, Vps33bfl/fl-AlfpCre mice showed mislocalisation of ATP-binding cassette proteins that are specifically trafficked to the apical membrane via Rab11a-positive recycling endosomes. This was associated with retention of bile components in blood. Loss of functional tight junction integrity and depletion of apical microvilli were seen in knockout animals. Gene transfer partially rescued these defects. CONCLUSIONS: Vps33b has a key role in establishing structural and functional aspects of hepatocyte polarity and may be a target for gene replacement therapy. LAY SUMMARY: Hepatocytes are liver cells with tops and bottoms; that is, they are polarised. At their bottoms they absorb substances from blood. They then, at their tops, secrete these substances and their metabolites into bile. When polarity is lost, this directional flow of substances from blood to bile is disrupted and liver disease follows. In this study, using a new mouse model with a liver-specific mutation of Vps33b, the mouse version of a gene that is mutated in most patients with arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome, we investigated how the Vps33b gene product contributes to establishing hepatocyte polarity. We identified in these mice abnormalities similar to those in children with ARC syndrome. Gene transfer could partly reverse the mouse abnormalities. Our work contributes to the understanding of VPS33B disease and hepatocyte polarity in general, and may point towards gene transfer mediated treatment of ARC liver disease.

Autosomal Recessive Keratoderma-Ichthyosis-Deafness (ARKID) Syndrome Is Caused by VPS33B Mutations Affecting Rab Protein Interaction and Collagen Modification.

In this paper, we report three patients with severe palmoplantar keratoderma associated with ichthyosis and sensorineural deafness. Biallelic mutations were found in VPS33B, encoding VPS33B, a Sec1/Munc18 family protein that interacts with Rab11a and Rab25 proteins and is involved in trafficking of the collagen-modifying enzyme LH3. Two patients were homozygous for the missense variant p.Gly131Glu, whereas one patient was compound heterozygous for p.Gly131Glu and the splice site mutation c.240-1G>C, previously reported in patients with arthrogryposis renal dysfunction and cholestasis syndrome. We demonstrated the pathogenicity of variant p.Gly131Glu by assessing the interactions of the mutant VPS33B construct and its ability to traffic LH3. Compared with wild-type VPS33B, the p.Gly131Glu mutant VPS33B had reduced coimmunoprecipitation and colocalization with Rab11a and Rab25 and did not rescue LH3 trafficking. Confirming the cell-based experiments, we found deficient LH3-specific collagen lysine modifications in patients' urine and skin fibroblasts. Additionally, the epidermal ultrastructure of the p.Gly131Glu patients mirrored defects in tamoxifen-inducible VPS33B-deficient Vps33bfl/fl-ERT2 mice. Both patients and murine models revealed an impaired epidermal structure, ascribed to aberrant secretion of lamellar bodies, which are essential for epidermal barrier formation. Our results demonstrate that p.Gly131Glu mutant VPS33B causes an autosomal recessive keratoderma-ichthyosis-deafness syndrome.

Regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis.

Post-translational modifications are necessary for collagen precursor molecules (procollagens) to acquire final shape and function. However, the mechanism and contribution of collagen modifications that occur outside the endoplasmic reticulum and Golgi are not understood. We discovered that VIPAR, with its partner proteins, regulate sorting of lysyl hydroxylase 3 (LH3, also known as PLOD3) into newly identified post-Golgi collagen IV carriers and that VIPAR-dependent sorting is essential for modification of lysines in multiple collagen types. Identification of structural and functional collagen abnormalities in cells and tissues from patients and murine models of the autosomal recessive multisystem disorder Arthrogryposis, Renal dysfunction and Cholestasis syndrome caused by VIPAR and VPS33B deficiencies confirmed our findings. Thus, regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis and for the development and function of multiple organs and tissues.

Novel VIPAS39 mutation in a syndromic patient with arthrogryposis, renal tubular dysfunction and intrahepatic cholestasis.

ARC syndrome is a rare autosomal recessive disease, characterized by arthrogryposis, renal tubular dysfunction and cholestasis. Herein a 2.5 month old infant with dysmorphic features, including small anterior fontanel, low set ears, beaked nose and high arched palate is presented who was referred because of icterus. He also suffered from some additional anomalies, including unilateral choanal atresia, club foot, and bilateral developmental dislocation of hip, while further studies showed renal tubular acidosis and hearing impairment in addition to cholestasis. Genetic studies showed a homozygous mutation in the VIPAS39 gene. Making the definite diagnosis of the syndrome is important, while increased risk of mutation in other siblings highlights the importance of prenatal diagnosis.

Glomerular involvement in the arthrogryposis, renal dysfunction and cholestasis syndrome.

BACKGROUND: Arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome is a multisystem autosomal-recessive disorder caused by defects in the VPS33B and VIPAR genes, involved in localization of apical membrane proteins. Affected children usually die by 1 year of age, often secondary to infective complications. The classic renal manifestation previously described in ARC syndrome is proximal-tubular dysfunction. The aim of this study is to gain further insight into the renal manifestations of this syndrome. METHODS: Clinical review of three cases of ARC syndrome presenting to a tertiary centre. Together with measurement of VPS33B and VIPAR protein expression in the human glomerulus. RESULTS: The cases demonstrated severe failure to thrive and in addition to commonly described features profound proteinuria and albuminuria, together with hypoalbuminaemia, suggesting glomerular involvement of this syndrome. Western blotting of conditionally immortalized human glomerular cells and ex vivo immunofluorescent analysis of the human glomerulus revealed that VPS33B and VIPAR were highly expressed in glomerular endothelium, and podocytes, but not in the mesangium. CONCLUSIONS: ARC syndrome affects the glomerulus as well as the proximal tubule in the kidney. Our molecular studies suggest that both cell types that constitute the glomerular filtration barrier are affected in this condition, providing an explanation for the albuminuria that we have observed in our cases.

Associations among genotype, clinical phenotype, and intracellular localization of trafficking proteins in ARC syndrome.

Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in vacuolar protein sorting 33 homologue B (VPS33B) and VPS33B interacting protein, apical-basolateral polarity regulator (VIPAR). Cardinal features of ARC include congenital joint contractures, renal tubular dysfunction, cholestasis, severe failure to thrive, ichthyosis, and a defect in platelet alpha-granule biogenesis. Most patients with ARC do not survive past the first year of life. We report two patients presenting with a mild ARC phenotype, now 5.5 and 3.5 years old. Both patients were compound heterozygotes with the novel VPS33B donor splice-site mutation c.1225+5G>C in common. Immunoblotting and complementary DNA analysis suggest expression of a shorter VPS33B transcript, and cell-based assays show that c.1225+5G>C VPS33B mutant retains some ability to interact with VIPAR (and thus partial wild-type function). This study provides the first evidence of genotype-phenotype correlation in ARC and suggests that VPS33B c.1225+5G>C mutation predicts a mild ARC phenotype. We have established an interactive online database for ARC (https://grenada.lumc.nl/LOVD2/ARC) comprising all known variants in VPS33B and VIPAR. Also included in the database are 15 novel pathogenic variants in VPS33B and five in VIPAR.

Mutation in the TCRα subunit constant gene (TRAC) leads to a human immunodeficiency disorder characterized by a lack of TCRαß+ T cells.

Inherited immunodeficiency disorders can be caused by mutations in any one of a large number of genes involved in the function of immune cells. Here, we describe two families with an autosomal recessive inherited immunodeficiency disorder characterized by increased susceptibility to infection and autoimmunity. Genetic linkage studies mapped the disorder to chromosomal region 14q11.2, and a homozygous guanine-to-adenine substitution was identified at the last base of exon 3 immediately following the translational termination codon in the TCRα subunit constant gene (TRAC). RT-PCR analysis in the two affected individuals revealed impaired splicing of the mRNA, as exon 3 was lost from the TRAC transcript. The mutant TCRα chain protein was predicted to lack part of the connecting peptide domain and all of the transmembrane and cytoplasmic domains, which have a critical role in the regulation of the assembly and/or intracellular transport of TCR complexes. We found that T cells from affected individuals were profoundly impaired for surface expression of the TCRαß complex. We believe this to be the first report of a disease-causing human TRAC mutation. Although the absence of TCRαß+ T cells in the affected individuals was associated with immune dysregulation and autoimmunity, they had a surprising level of protection against infection.

Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization.

Arthrogryposis, renal dysfunction and cholestasis syndrome (ARC) is a multisystem disorder associated with abnormalities in polarized liver and kidney cells. Mutations in VPS33B account for most cases of ARC. We identified mutations in VIPAR (also called C14ORF133) in individuals with ARC without VPS33B defects. We show that VIPAR forms a functional complex with VPS33B that interacts with RAB11A. Knockdown of vipar in zebrafish resulted in biliary excretion and E-cadherin defects similar to those in individuals with ARC. Vipar- and Vps33b-deficient mouse inner medullary collecting duct (mIMDC-3) cells expressed membrane proteins abnormally and had structural and functional tight junction defects. Abnormal Ceacam5 expression was due to mis-sorting toward lysosomal degradation, but reduced E-cadherin levels were associated with transcriptional downregulation. The VPS33B-VIPAR complex thus has diverse functions in the pathways regulating apical-basolateral polarity in the liver and kidney.

Mutations in SLC29A3, encoding an equilibrative nucleoside transporter ENT3, cause a familial histiocytosis syndrome (Faisalabad histiocytosis) and familial Rosai-Dorfman disease.

The histiocytoses are a heterogeneous group of disorders characterised by an excessive number of histiocytes. In most cases the pathophysiology is unclear and treatment is nonspecific. Faisalabad histiocytosis (FHC) (MIM 602782) has been classed as an autosomal recessively inherited form of histiocytosis with similarities to Rosai-Dorfman disease (RDD) (also known as sinus histiocytosis with massive lymphadenopathy (SHML)). To elucidate the molecular basis of FHC, we performed autozygosity mapping studies in a large consanguineous family and identified a novel locus at chromosome 10q22.1. Mutation analysis of candidate genes within the target interval identified biallelic germline mutations in SLC29A3 in the FHC kindred and in two families reported to have familial RDD. Analysis of SLC29A3 expression during mouse embryogenesis revealed widespread expression by e14.5 with prominent expression in the central nervous system, eye, inner ear, and epithelial tissues including the gastrointestinal tract. SLC29A3 encodes an intracellular equilibrative nucleoside transporter (hENT3) with affinity for adenosine. Recently germline mutations in SLC29A3 were also described in two rare autosomal recessive disorders with overlapping phenotypes: (a) H syndrome (MIM 612391) that is characterised by cutaneous hyperpigmentation and hypertrichosis, hepatomegaly, heart anomalies, hearing loss, and hypogonadism; and (b) PHID (pigmented hypertrichosis with insulin-dependent diabetes mellitus) syndrome. Our findings suggest that a variety of clinical diagnoses (H and PHID syndromes, FHC, and familial RDD) can be included in a new diagnostic category of SLC29A3 spectrum disorder.

Molecular investigations to improve diagnostic accuracy in patients with ARC syndrome.

Arthrogryposis, Renal dysfunction and Cholestasis (ARC) syndrome is a multi-system autosomal recessive disorder caused by germline mutations in VPS33B. The detection of germline VPS33B mutations removes the need for diagnostic organ biopsies (these carry a>50% risk of life-threatening haemorrhage due to platelet dysfunction); however, VPS33B mutations are not detectable in approximately 25% of patients. In order further to define the molecular basis of ARC we performed mutation analysis and mRNA and protein studies in patients with a clinical diagnosis of ARC. Here we report novel mutations in VPS33B in patients from Eastern Europe and South East Asia. One of the mutations was present in 7 unrelated Korean patients. Reduced expression of VPS33B and cellular phenotype was detected in fibroblasts from patients clinically diagnosed with ARC with and without known VPS33B mutations. One mutation-negative patient was found to have normal mRNA and protein levels. This patient's clinical condition improved and he is alive at the age of 2.5 years. Thus we show that all patients with a classical clinical course of ARC had decreased expression of VPS33B whereas normal VPS33B expression was associated with good prognosis despite initial diagnosis of ARC.

Enteropathogenic Escherichia coli (EPEC) adhesion to intestinal epithelial cells: role of bundle-forming pili (BFP), EspA filaments and intimin.

Enteropathogenic Escherichia coli (EPEC), an important paediatric diarrhoeal pathogen, employs multiple adhesins to colonize the small bowel and produces characteristic 'attaching and effacing' (A/E) lesions on small intestinal enterocytes. EPEC adhesins that have been associated with A/E adhesion and intestinal colonization include bundle-forming pili (BFP), EspA filaments and intimin. BFP are involved in bacteria-bacteria interaction and microcolony formation but their role in cell adhesion remains unclear; EspA filaments are components of the EPEC type III secretion system but since they interact directly with host cells they may also function as adhesins; intimin is the well characterized intimate EPEC adhesin which binds the translocated intimin receptor, Tir. However, other uncharacterized host cell receptors have been implicated in intimin-mediated adhesion. In this study, the role of BFP, EspA filaments and intimin in EPEC adhesion to intestinal brush border cells was assessed by observing adhesion of wild-type EPEC strain E2348/69 and a set of isogenic single, double and triple mutants in bfpA, espA and eae (intimin gene) to differentiated human intestinal Caco-2 cells. E2348/69 (bfpA(+) espA(+) eae(+)) adhered rapidly (<10 min) to the brush border of Caco-2 cells and subsequently produced microcolonies and typical A/E lesions. Non-intimate brush border adhesion of double mutant strain UMD880 (bfpA(+) espA(-) eae(-)) also occurred rapidly, whereas adhesion of strain UMD886 (bfpA(-) espA(+) eae(-)) occurred later in the infection (>1 h) and with much lower efficiency; confocal microscopy indicated BFP and EspA-mediated adhesion, respectively. Strain UMD883 (bfpA(-) espA(-) eae(+)), which is unable to translocate Tir, was non-adherent although this strain was able to form intimate attachment and A/E lesions when co-cultured with strain CVD206 (bfpA(+) espA(+) eae(-)) which supplied Tir to the membrane. Single mutant strains CVD206 (bfpA(+) espA(+) eae(-)) and UMD872 (bfpA(+) espA(-) eae(+)) showed adherence characteristics of strain UMD880 (bfpA(+) espA(-) eae(-)), whilst triple mutant strain UMD888 (bfpA(-) espA(-) eae(-)) was totally non-adherent. These results support an adhesive role for BFP and EspA in initial brush border cell attachment, and in typical EPEC which express both BFP and EspA filaments suggest a predominant role for BFP; EspA filaments, however, could serve as initial attachment factors in atypical EPEC which lacks BFP. The study found no evidence for an independent host cell intimin receptor or for other adhesive factors able to support bacterial adherence.