Thus, after purified outer membranes were subjected to a cross-linking reaction, BfpG could be copurified with the BfpB complex, demonstrating that BfpG interacts with the BfpB multimer

Thus, after purified outer membranes were subjected to a cross-linking reaction, BfpG could be copurified with the BfpB complex, demonstrating that BfpG interacts with the BfpB multimer. == The BfpB multimer requires BfpG for stability and/or formation. is also associated with accumulation of EPEC proteins in growth medium, suggesting that it may QX77 support both pilus biogenesis and protein secretion. EnteropathogenicEscherichia coli(EPEC) is an important cause of protracted diarrheal illness among children living in developing countries (11). Endoscopically directed biopsies of tissue from naturally infected children show that EPEC mainly infects the small intestine, adhering to epithelial cell surfaces where it induces actin condensation and effacement of microvilli (22,46). Studies of EPEC-infected epithelial cell monolayers revealed similar cytopathic changes and led to the identification of two unique but coordinated processes: the formation of adherent microcoloniesthe localized adherence (LA) phenotype (12,51)and characteristic changes of the cytoskeleton beneath attached Ednra bacteriathe attaching and effacing phenotype (31). The genes coding for the attaching and effacing phenotype are located around the chromosome within a large pathogenicity island, termed the locus of enterocyte effacement (LEE) (37). Among the proteins specified by LEE are intimin, required for close adherence between bacteria and the epithelial cell (25); Tir, which serves as an intimin receptor that is inserted into the epithelial cell plasma membrane (29); EspB, a protein required for the attaching and effacing effect (30); and components of the cognate type III secretion system, including SepC, a member of the secretin protein QX77 superfamily (24). Proof that LEE-encoded functions are required for virulence comes from human challenge studies showing that intimin or EspB mutants are significantly attenuated (13,55). Genes required for the LA phenotype are found around the 69-kb EPEC adherence factor (EAF) plasmid (38,53,54) in a region that specifies the bundle-forming pilus (BFP) of the organism (18). Disruption of this locus abrogates the LA phenotype (45) and significantly reduces the virulence of the mutant in orally challenged human volunteers (3). Examination of this mutant revealed that bundle-forming pili also mediate the formation of transient bacterial aggregates during growth in tissue culture medium (2,3). Time course, phase-contrast microscopy of this phenomenon, termed the autoaggregation (AA) phenotype, showed that after an overnight culture of dispersed, individual EPEC bacteria was diluted into tissue culture media, the cells reenter the exponential phase of growth. Forty-five to 60 min later, the bacteria begin to coalesce into dynamic, spherical assemblies. These autoaggregates continue to form and enlarge until late exponential phase and then, over an 20-min interval, they disaggregate, yielding a suspension of individual bacteria (3). BFP biogenesis and function are encoded by an operon made up of 14 genes, designatedbfpAtobfpL(53,54). All butbfpHare required for BFP filament production and for the LA and AA phenotypes (S. W. Ramer, unpublished data) (1,45), but the functions of only 3 of the 14 open reading frames (ORFs) have been reported.bfpAencodes the principal repeating subunit of the pilus filament; amino acid sequence analysis QX77 showed it to be a member of the type IV QX77 family of pilus proteins (53).bfpPcodes for the pre-pilin transmission peptidase (59), proven to be required for the maturation of BfpA and likely to be required for the processing of three other pilin-like proteins encoded by the operon and denoted BfpI, -J, and -K (53).bfpFis required for the AA phenotype, andbfpFmutants are hyperpiliated, form aggregates that do not disperse, and are less virulent for human volunteers (2,3). The study reported here focuses on the biochemical characterization and functional functions ofbfpGandbfpB, two genes previously shown to be required for pilus biogenesis (1,45).bfpGandbfpBare the second and third ORFs of the operon (53,54), respectively, and RNase protection assays showed that this 3 end ofbfpGand the 5 end ofbfpBare located on a continuous RNA transcript (45). BfpB is an outer-membrane lipoprotein that shares sequence similarity with theE. colibacteriophage f1 morphogenic protein pIV (45), a member of the secretin protein superfamily (17,49). Studies of pIV and several other secretin family members show them to be essential outer-membrane components of the main terminal branch of the general secretory pathway (GSP; also termed the type II secretion pathway) (4,20,44). The GSP is required.