Cloning of the genes for AF/R1 pili from rabbit enteroadherent Escherichia coli RDEC-1 and DNA sequence of the major structural subunit.

AF/R1 pili on the surface of Escherichia coli RDEC-1 promote attachment of the bacteria to rabbit intestinal brush borders. In order to characterize AF/R1 pili and manipulate their expression, we cloned the genes necessary for AF/R1 expression; determined the size of proteins produced in minicells; located the gene encoding the major structural subunit, named AfrA; and determined the DNA sequence of afrA as well as the sequence of 700 additional nucleotides upstream of afrA. Two contiguous EcoRI fragments spanning 7.9 kilobases were cloned from the 86-megadalton plasmid of RDEC-1 into vector pUC19 to make plasmid pW1. Bacteria carrying pW1 produced AF/R1 pili that were recognized by AF/R1-specific antiserum and promoted adherence of bacteria to brush borders prepared from rabbit intestine. Proteins with a molecular weight of 17,000 (17K proteins), which was the size of AfrA, as well as 15K, 15.5K, 26K, 28K, and 80K proteins were detected in minicells carrying pW1. The gene afrA was located by using an oligonucleotide probe, and its DNA sequence was determined. The DNA sequence of 700 additional nucleotides upstream was determined because this sequence may be important in the regulation of AF/R1 expression.     

Peyer's patch adherence of enteropathogenic Escherichia coli strains in rabbits.

RDEC-1 (serotype O15) is an attaching and effacing strain of rabbit enteropathogenic Escherichia coli (REPEC) that causes diarrhea in postweanling rabbits. It expresses AF/R1 pili that mediate Peyer's patch M-cell adherence. We investigated Peyer's patch adherence, the presence of virulence genes, ileal brush border aggregation, and pilus expression in 9 strains representing several serotypes of REPEC as well as in two commensal strains. Postweanling rabbits were inoculated with 10(6) organisms and sacrificed at 24 h, and tissues were prepared for examination by light microscopy. Strains B10 and RDEC-1 were also studied at 12 and 72 h postinoculation. All REPEC strains were eaeA positive, expressed pili, and adhered to ileal brush borders. Both commensal strains expressed pili, and one strain adhered to brush borders. All REPEC strains demonstrated some degree of Peyer's patch lymphoid follicle adherence, ranging from diffuse coverage to small patches covering two to three dome epithelial cells. Strains C102 and C110 had genes homologous with the structural subunit gene of the AF/R1 pilus (afrA) of RDEC-1, which correlated with greater degrees of lymphoid follicle adherence and lesser degrees of ileal villus adherence. The observation that all REPEC strains adhere to Peyer's patch epithelium suggests the possibility that human strains of enteropathogenic E. coli (EPEC) might do likewise. EPEC strains might thus serve as mucosal vaccine vectors in humans. Better understanding of the molecular mechanism of REPEC adherence should provide a model for the targeting of the Peyer's patch in humans.     

Characterization of the plasmid from Escherichia coli RDEC-1 that mediates expression of adhesin AF/R1 and evidence that AF/R1 pili promote but are not essential for enteropathogenic disease.

RDEC-1, an Escherichia coli strain that adheres to rabbit mucosa and causes an attaching, effacing lesion, expresses the pilus adhesin AF/R1 which determines in vitro attachment to rabbit intestinal brush borders. In order to determine the role of AF/R1 pili in the pathogenesis of enteropathogenic diarrhea in rabbits, we localized the genes for AF/R1 expression, constructed an AF/R1- strain, and compared the virulence of the AF/R1+ and AF/R1- strains with particular attention to the development of attaching, effacing lesions. We introduced Tn5 into the 86-megadalton (MDa) conjugative plasmid known to mediate expression of AF/R1 pili and transferred the derivative plasmids into laboratory strain HB101. Transconjugant M5 was found to contain the 86-MDa plasmid from RDEC-1 and to express AF/R1 pili. Pilus expression on M5 was confirmed by reaction with antiserum raised against purified AF/R1 pili and allowed the bacteria to adhere to the rabbit ileum in an in vitro assay. Three Tn5 insertions in the 86-MDa plasmid were obtained which resulted in loss of AF/R1 expression. Part of the plasmid was mapped, including a region necessary for AF/R1 pilus expression. AF/R1- mutant strain M34 was constructed, and its pathogenesis was investigated. M34 produced disease in rabbits but was less virulent than the parent. The characteristic effacing lesions of RDEC-1 and enteropathogenic E. coli developed in the intestine of rabbits infected with either M34 or RDEC-1, although with M34 they were much less frequent and did not involve the small bowel. We conclude that AF/R1 pilus expression is not essential for the attaching, effacing lesion but serves as an accessory virulence factor which promotes an initial interaction of RDEC-1 with normal epithelial cells.     

Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate.

Isolates of Escherichia coli from human urinary tract infections frequently express adherence properties found less often among normal intestinal isolates. These properties include adherence to human uroepithelial cells and primary monkey kidney cells, as well as D-mannose-resistant hemagglutination of human erythrocytes, and they are mediated by a pilus type different from type 1. The genes encoding this pilus type (pyelonephritis-associated pili, pap) and those encoding type 1 pili have been cloned from a urinary tract infection isolate of E. coli and transferred to an E. coli K-12 derivative. The recombinant plasmids were found to express functional pili and to endow the new host with all of the adherence properties of the urinary tract infection isolate. Both pilus types were found to be genetically distinct, and unlike the adherence genes from bovine, porcine, and human diarrheal isolates, both were found to be chromosomally encoded.     

Identification of a gene essential for piliation in Haemophilus influenzae type b with homology to the pilus assembly platform genes of gram-negative bacteria.

Haemophilus influenzae type b (Hib) pili are complex filamentous surface structures consisting predominantly of pilin protein subunits. The gene encoding the major pilin protein subunit of Hib adherence pili has been cloned and its nucleotide sequence has been determined. In order to identify specific accessory genes involved in pilus expression and assembly, we constructed isogenic Hib mutants containing insertional chromosomal mutations in the DNA flanking the pilin structural gene. These mutants were screened for pilin production, pilus expression, and hemagglutination. Pili and pilin production were assessed by immunoassays with polyclonal antisera specific for pilin and pili of Hib strain Eagan. Hemagglutination was semiquantitatively evaluated in a microtiter plate assay. Six Hib mutants produced proteins immunoreactive with antipilin antiserum but no longer produced structures reactive with antipilus antiserum. In addition, the mutants were unable to agglutinate human erythrocytes. Nucleotide sequence analysis localized the insertion sites in the six mutants to 2.5-kb open reading frame upstream of the pilin structural gene and immediately downstream of an Hib pilin chaperone gene. The amino acid sequence encoded by this open reading frame has significant homology to members of the pilus assembly platform protein family, including FhaA of Bordetella pertussis, MrkC of Klebsiella pneumoniae, and the Escherichia coli assembly platform proteins FimD and PapC. This open reading frame, designated hifC, appears to represent a gene essential to Hib pilus biogenesis that has genetic and functional similarity to the pilus platform assembly genes of other gram-negative rods.     

Characteristics of adherence of enteroaggregative Escherichia coli to human and animal mucosa.

An Escherichia coli strain (serotype O127a:H2) that had been isolated from a child with diarrhea in Thailand and that was negative for the virulence factors of the four categories of diarrheagenic E. coli (enterotoxigenic, enteropathogenic, enteroinvasive, and enterohemorrhagic) and that showed an aggregative pattern of adherence to HeLa cells was investigated for adherence to native or Formalin-fixed human and animal mucosa. The hemagglutinating activity and adherence ability of the bacteria were resistant to D-mannose and were strictly regulated by environmental conditions. Genetic data supported the close relation between the hemagglutinating activity and adherence ability. In accordance with the adherence pattern on tissue-cultured cells, the bacteria adhered to human and animal mucosa, as evidenced by a direct gold-labeling analysis. In human intestines, Formalin-fixed mucous coatings, epithelial cells of colonic mucosa, epithelial cells of ileal single lymphoid follicles and Peyer's patches, and the absorptive cells of jejunal or ileal villi provided adherence targets. Adherence to M cells in the Peyer's patch-associated epithelium was also confirmed. The adherence levels to native jejunal or ileal human villi were low, as was the case with the corresponding Formalin-fixed villi. In human urinary tract, the superficial epithelial cells of both native and Formalin-fixed ureter provided striking adherence targets. In animal (porcine and rabbit) small intestines, the bacteria adhered to the native villi to a lesser extent than to the Formalin-fixed villi. The adherence levels were compared with those of enterotoxigenic E. coli with colonization factor antigen (CFA)/I pili or CFA/II pili. The data suggested unique mucosa adherence characteristics of the enteroaggregative E. coli strain. The possibility of the adherence ability as a virulence factor was discussed.     

Functional heterogeneity of intestinal Escherichia coli strains expressing type 1 somatic pili (fimbriae): assessment of bacterial adherence to intestinal membranes and surface hydrophobicity.

Although the role of host-specific, nonmannose-sensitive pilus adhesins in the intestinal adherence of pathogenic Escherichia coli is well established, a similar role for mannose-sensitive type 1 or common pili is less clear, since these structures can be expressed by most E. coli, even nonpathogens. We first examined whether type 1 pili, expressed by the rabbit-effacing, adherent, enteropathogenic E. coli strain RDEC-1, mediated interactions with intestinal membranes of several species and compared these interactions with those mediated by the nonmannose-sensitive adhesin of RDEC-1. We next grew a series of E. coli intestinal strains in static broth to promote type 1 pilus expression and determined whether E. coli expressing type 1 pili differed in their affinity for intestinal membranes (as measured by phase-contrast microscopy and aggregometry), hydrophobic surface properties, net negative surface charge (as measured by hydrophobic interaction chromatography and salt aggregation), and hemagglutination patterns. In contrast to the species-specific attachment to rabbit brush borders of RDEC-1 expressing its nonmannose-sensitive adhesin, type 1 pili on RDEC-1 mediated mannose-sensitive attachment to intestinal membranes of all four species tested. Expression of type 1 pili on other E. coli strains resulted in varying degrees of nonspecies-specific, mannose-sensitive attachment to intestinal membranes. This attachment correlated with increasing surface hydrophobicity rather than with hemagglutination patterns. These results indicate that various E. coli strains expressing type 1 pili are functionally heterogeneous and suggest that some type 1 pili might contribute to in vivo enteroadherence.     

Characterization of the CsfC and CsfD proteins involved in the biogenesis of CS5 pili from enterotoxigenic Escherichia coli

The region required for biosynthesis of CS5 pili consists of six csf genes, with csfA encoding the major subunit. In this study, we describe the characterization of two of the genes constituting the region, csfC and csfD, but also identify the true morphology of the CS5 pilus by high resolution electron microscopy. CsfD was shown to be essential in the initiation of CS5 pilus biogenesis, did not possess any chaperone-like activity for the major subunit, and was an integral minor component of the pilus structure. Studies on CsfD translocation across the outer membrane in Escherichia coli K-12 using a csfA mutant also showed that CsfD is likely to be the first pilin subunit assembled. A specific in-frame deletion in the csfC gene resulted in the complete absence of cell surface CS5 pili and prevented the translocation of CsfA and CsfD pilins across the outer membrane. Specific cell localization studies showed an accumulation of CsfC in the outer membranes of E. coli K-12, while complementation experiments with homologous outer membrane assembly genes from CS1 and CFA/I pili systems were unable to restore assembly of CS5 pili. The CS5 pilus was shown to be a 2 nm flexible fibrillar structure, which adopted a predominantly open helical conformation under the electron microscope.     

In vitro adhesion of Escherichia coli to porcine small intestinal epithelial cells: pili as adhesive factors.

Escherichia coli strains with pili (K99 or 987P) known to facilitate intestinal colonization adhered in vitro to porcine intestinal epithelial cells. These strains adhered equally to both ileal and jejunal epithelial cells. A laboratory E. coli strain that has type 1 pili also adhered to porcine intestinal epithelial cells. When nonpiliated cells derived from 987P+, K99+, or type 1 pilus+ strains were used for in vitro adhesion assays, they failed to adhere. The attachment of piliated bacteria to epithelial cells was a saturable process that plateaued at 30 to 40 bacterial cells attached per epithelial cell. Competitive inhibition of bacterial cell attachment to epithelial cells with purified pili showed that only purified 987P competed against the 987P+ strain and only purified type 1 pili competed against the type 1 pilus+ strain. Competition between a K99+ strain and K99 was not consistently achieved. K99+, 987P+, and type 1 pilus+ bacteria could be prevented from adhering to epithelial cells by Fab fragments specific for K99, 987P, or type 1 pili, respectively. Fab fragments specific for non-K99 bacterial surface antigens did not inhibit adhesion of the K99+ strain. It is concluded that adhesion of E. coli to porcine intestinal epithelial cells in vitro is mediated by pili and that the epithelial cells used apparently had different receptors for different pili.     

Role of Escherichia coli type 1 pilus in colonization of porcine ileum and its protective nature as a vaccine antigen in controlling colibacillosis.

This study was designed to evaluate the role of Escherichia coli type 1 pili in adherence of the organism to porcine small intestines and the efficacy of pili as a vaccine antigen in controlling neonatal colibacillosis. Our results demonstrated that an E. coli phase cloned to express type 1 pili readily attached to the small intestines of colostrum-deprived newborn pigs. Immunofluorescent staining of intestine sections revealed the presence of E. coli expressing type 1 pili only on the brush border, suggesting involvement of type 1 pili in the colonization process. Administration of anti-type 1 serum to newborn pigs prior to challenge reduced the level of gut-associated E. coli sixfold compared with controls. Purified type 1 pilus vaccine induced significant protection against colibacillosis in newborn pigs following challenge with E. coli expressing type 1 pili. Pigs born to vaccinated gilts scoured less and gained more weight than pigs born to control gilts. Our results demonstrate that type 1 pili are a virulence factor, as well as an effective vaccine antigen.     

Mucosal immune response to RDEC-1 infection: study of lamina propria antibody-producing cells and biliary antibody.

Infection of rabbits with Escherichia coli RDEC-1 is a useful model for diarrheal disease caused by mucosally attaching E. coli. Understanding of the protective immunity induced by RDEC-1 infection in rabbits should provide information useful in the design of vaccines for protection against this infection and other mucosally attaching organisms as well. Thus, to define the time course and location of specific immunoglobulin A secretion in relation to bacterial colonization during primary RDEC-1 infection, we infected rabbits with RDEC-1, which express AF/R1 adherence pili, and compared sites of anti-AF/R1 antibody-containing cells in the intestinal mucosa with the sites of luminal colonization and mucosal attachment of RDEC-1. Also, anti-AF/R1 antibodies in intestinal fluids and bile were measured by enzyme-linked immunosorbent assay, and attachment sites of RDEC-1 to the intestinal epithelium were determined by immunohistochemical examination. Anti-AF/R1 pilus antibody-containing cells were most numerous in the proximal intestine (duodenum and jejunum). In contrast, both luminal colonization and attachment of RDEC-1 to epithelial cells were densest in the distal intestine (cecum and colon). Anti-AF/R1 antibodies were present in approximately equal amounts in fluids collected from all levels of the gut after week 1 postinfection. Anti-AF/R1 antibody levels in undiluted bile exceeded those in gut flushes by at least 2 orders of magnitude. Loss of RDEC-1 attachment to epithelial cells preceded resolution of diarrheal illness despite the presence of large numbers of organisms in the intestinal lumen. Our studies indicate that during RDEC-1 infection (i) sites of greatest mucosal anti-AF/R1 antibody secretion are proximal to sites of maximal RDEC-1 luminal colonization and attachment, (ii) bile is a major source of specific antibodies in the intestinal lumen, and (iii) interference with RDEC-1 attachment to epithelial cells may permit resolution of disease.     

Adherence of an enterotoxigenic Escherichia coli strain, serotype O78:H11, to purified human intestinal brush borders.

The human enterotoxigenic Escherichia coli pathogen designated H10407 expresses two different types of surface pili, one designated type 1 pili and the other designated colonization factor antigen I (CFA/I), CFA/I pili are thought to promote the adherence of H10407 to the mucosa of the human small bowel. H10407 was grown under conditions which promoted the expression of either type 1 pili or CFA/I pili, and in each case, the adherence of H10407 to purified human intestinal brush borders was quantitated. The adherence assays revealed that H10407 adhered to human brush borders only when it expressed CFA/I pili. It appears that in vitro adherence of H10407 to human intestinal epithelial cells is dependent on the expression of CFA/I.     

Utilization of anion-exchange chromatography and monoclonal antibodies to characterize multiple pilus types on a uropathogenic Escherichia coli O6 isolate.

Multiple pilus types from a uropathogenic strain of Escherichia coli O6, strain 6260, were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high-pressure liquid chromatography, binding assays, and erythrocyte adsorption. In addition, monoclonal antibodies were raised against purified pili of E. coli 6260 and used for immunological characterization. SDS-PAGE analysis of the purified pili showed at least three different subunits with molecular weights of 15,700, 17,800, and 19,300. SDS-PAGE analysis of four protein peaks from anion-exchange chromatography of intact pili showed polypeptides with molecular weights of 19,300 (fraction 1), 15,700 (fraction 2), and 17,800 and 15,700 (both fractions 3 and 4). Erythrocyte adsorption of the whole-pilus preparation removed the 17,800-molecular-weight subunit (17.8K subunit) and reduced the 15.7K subunit. Pili from an isogenic hemagglutination-negative variant of E. coli 6260, showing only the 15.7K and 19.3K subunits by SDS-PAGE, lacked the 17.8K subunit of fractions 3 and 4 present in the parent high-pressure liquid chromatography profile. Our data suggest that two of the pilus subunits, the 15.7K and 17.8K subunits, mediate mannose-resistant agglutination of human erythrocytes. Pili in fractions 1 and 2 from the parent strain bound specifically to mannose residues, while pili in fraction 4 bound to P-coated horse erythrocytes; no receptor specificity was identified for pili in fraction 3. Immunological analysis by the immunoblot technique showed that monoclonal antibody 11-2 reacted with the 19.3K subunit, monoclonal antibodies 34-3 and 73-3 reacted with the 15.7K subunit, and monoclonal antibodies 81-1, 82-1, and 91-1 reacted with polymers of subunits retained in the stacking gel. Intact pili precipitated by any of the six monoclonal antibodies showed two polypeptides by SDS-PAGE: 15.7K and 19.3K polypeptides for monoclonal antibody 11-2, and 15.7K and 17.8K polypeptides for monoclonal antibodies 34-3, 73-3, 81-1, 82-1, and 91-1. The cross-reactivity of the monoclonal antibodies with purified pili from other E. coli strains was determined by enzyme-linked immunosorbent assay. Monoclonal antibody 11-2 showed no significant cross-reactivity with heterogeneous pili. In contrast, the other monoclonal antibodies showed equivalent or greater reactivity with P pili from heterologous strains as compared with reactivity with E. coli 6260 pili.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetics of digalactoside-binding adhesin from a uropathogenic Escherichia coli strain.

The uropathogenic strain Escherichia coli J96 mediates mannose-resistant hemagglutination owing to production of a digalactoside-binding adhesin. A cosmid clone from this strain has been isolated that, when harbored in E. coli K-12, expressed Pap pili and this adhesin (R. Hull et al., Infect. Immun. 33:933-938, 1981). By transposon mutagenesis and by the construction of a number of hybrid plasmid derivatives, we have demonstrated that about 8.5 kilobases of DNA is required to generate a mannose-resistant hemagglutination-positive phenotype in E. coli K-12 strain P678-54. The structural gene for the Pap pili monomer, papA, has been identified and mapped close to the promotor-proximal end of the Pap operon. Although strain P678-54 that harbored a Tn5 insertion within papA showed a mannose-resistant hemagglutination-positive phenotype, it was negative in a competitive enzyme-linked immunosorbent assay with anti-Pap pilus serum. This could mean that a Pap adhesin is encoded by a region on the Pap operon that is distinct from papA.     

Characterization of a novel type IV pilus locus encoded on the large plasmid of locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains that are virulent for humans

The majority of Shiga-toxigenic Escherichia coli (STEC) strains isolated from humans with gastrointestinal disease carry large (approximately 90-kb) plasmids. We have been analyzing the megaplasmid (designated pO113) from an O113:H21 STEC strain (98NK2). This strain lacks the locus for enterocyte effacement (LEE) and yet was responsible for an outbreak of hemolytic uremic syndrome. In the present study, we demonstrate that pO113 carries a novel type IV pilus biosynthesis locus (pil) related to those of the IncI plasmids R721, R64, and ColIb9. The pO113 pil locus consists of 11 closely linked genes (pilL through pilV) with an additional separately transcribed upstream gene (pilI). It directs the expression of long thin pili on the 98NK2 surface and the hemagglutination of guinea pig erythrocytes. We also demonstrate that pO113 can be transferred by conjugation. However, the type IV pilus encoded by pO113 does not appear to be involved in the adherence of 98NK2 to HEp-2 or Hct-8 cells in vitro. Homologues of the pO113 pil locus were present in several other LEE-negative STEC strains but not in LEE-positive STEC strains belonging to serogroup O26, O111, or O157.     

A new fimbrial antigen harbored by CAZ-5/SHV-4-producing Klebsiella pneumoniae strains involved in nosocomial infections.

We purified and characterized a new fimbria termed KPF-28 (Klebsiella pneumoniae fimbria with a fimbrin molecular mass of 28 kDa) involved in K. pneumoniae adherence to the human carcinoma cell line Caco-2. Electron microscopy of bacterial surface protein preparations and immunogold labeling of bacterial cells showed that KPF-28 was a long, thin, and flexible fimbria about 4 to 5 nm in diameter and 0.5 to 2 microm long. The N-terminal amino acid sequence of the KPF-28 major fimbrial subunit showed no homology with type 1 and type 3 pili of K. pneumoniae but showed 61.7% identity with residues 6 to 19 of the N-terminal amino acid sequence of PapA, the Pap major pilus subunit expressed by uropathogenic Escherichia coli strains. Total amino acid content determination showed that the KPF-28 major subunit composition was close to that of the GVVPQ fimbrial family major subunits expressed by pathogenic E. coli strains. The study of the prevalence of KPF-28 among K. pneumoniae strains involved in nosocomial infections revealed that KPF-28 was found in the great majority of the K. pneumoniae strains producing the CAZ-5/SHV-4 extended-spectrum beta-lactamase. As shown by curing and mating experiments, the R plasmid encoding the CAZ-5/SHV-4 enzyme was found to be involved in but not solely responsible for KPF-28 expression. Hybridization experiments using an oligonucleotide probe corresponding to the N-terminal part of the 28-kDa protein revealed that the structural gene encoding the KPF-28 major subunit was localized on this R plasmid. KPF-28 is a putative colonization factor of the human gut, since the ceftazidine-sensitive derivative strain CF914-1C no longer adhered and since the Fab fragments of antibodies raised against KPF-28 inhibited adhesion of K. pneumoniae CF914-1 to the Caco-2 cell line.     

Gene cluster for assembly of pilus colonization factor antigen III of enterotoxigenic Escherichia coli

The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems. CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane. In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, including cofA and cofP. Several proteins encoded by cof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E. coli. The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E. coli genome (50%). The introduction of a recombinant plasmid containing the cof gene cluster into the E. coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2. This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.     

Type 1 pili (F1) of porcine enterotoxigenic Escherichia coli: vaccine trial and tests for production in the small intestine during disease.

This study was performed to determine whether the F1 (type 1) pili of a porcine strain of enterotoxigenic Escherichia coli are protective antigens and whether they are produced in the pig small intestine during disease caused by an enterotoxigenic E. coli. Reciprocal cross-absorption experiments with antisera prepared against F1 pili purified from enterotoxigenic E. coli 431 (O101:K30,99:H-:F1) and P14 (O149:K91,88ac:8H+:F1) demonstrated that the F1 antigens of the two strains were closely related or identical. Pregnant swine vaccinated with a vaccine prepared from strain P14 (F1+) responded with a significant increase in antibody against F1 in their serum and colostrum. However, the vaccinated dams did not significantly protect their suckling pigs against fatal challenge with strain 431. There was no evidence of F1 pilus production in the strain 431-infected pigs, as determined by immunofluorescent staining of ileal sections, direct electron microscopic examination of bacteria from ilea, and titration of serum agglutinins in convalescent pigs. It was concluded that strain 431 did not produce F1 in the small intestine during disease and that F1 was not a protective antigen in this system.     

Sortase A utilizes an ancillary protein anchor for efficient cell wall anchoring of pili in Streptococcus agalactiae

Pili are putative virulence factors and promising vaccine candidates in Streptococcus agalactiae (group B Streptococcus [GBS]) infection, a leading cause of neonatal sepsis and meningitis. The genes necessary for pilus synthesis and assembly are clustered in pilus islands (PI). Each gene encodes three structural subunits (a backbone and two ancillary proteins) bearing a C-terminal LPXTG motif and two subfamily C sortases (SrtC) involved in covalent polymerization of the subunits. GBS strains also possess the conserved “housekeeping” sortase A (SrtA), but its role in pilus assembly is unclear. To address this issue, pilus expression and cell wall anchoring were analyzed in srtA deletion mutants. Loss of SrtA did not affect pilus polymerization. However, pilus expression on the cell surface was reduced, and pili accumulated in the culture supernatant. Furthermore, cell-associated pili could be readily released by detergent treatment, indicating that SrtA is involved in covalent anchoring of pili to the cell wall. When each of the genes comprising PI-2a was systematically deleted, only the absence of ancillary subunit GBS150 or the SrtC required for incorporation of GBS150 into pili mimicked the srtA mutant phenotype. Thus, from these data a model for GBS pilus assembly can be proposed in which PI sortases are responsible for polymerization of the pilus structure, while SrtA is required to covalently attach it to the cell wall, utilizing ancillary pilus subunit GBS150 as the anchor protein.