TnphoA Mutants Research Articles

Enteropathogenic Providencia alcalifaciens: A Subgroup of P. alcalifaciens That Causes Diarrhea.

Despite being considered a normal flora, Providencia alcalifaciens can cause diarrhea. In a previous study, strain 2939/90, obtained from a diarrheal patient, caused invasion and actin condensation in mammalian cells, and diarrhea in a rabbit model. Four TnphoA mutants of 2939/90 produced negligible invasion and actin condensation in mammalian cells. Now, the parent strain and the mutants have been sequenced to locate TnphoA insertion sites and determine the effect on virulence. A TnphoA insertion was detected in the type three secretion system (T3SS) locus on a large plasmid and not in a T3SS locus on the chromosome. In 52 genomes of P. alcalifaciens surveyed, the chromosomal T3SS locus was present in all strains, including both P. alcalifaciens genomic clades, which we classified as group A and group B. Plasmid T3SS was present in 21 of 52 genomes, mostly in group A genomes, which included isolates from an outbreak of hemorrhagic diarrhea in dogs. The TnphoA insertion only in the plasmid T3SS locus affected the invasion phenotype, suggested that this locus is critical for causation of diarrhea. We conclude that a subgroup of P. alcalifaciens that possesses this plasmid-mediated T3SS is an enteric pathogen that can cause diarrheal disease.

CD48 and α7 Nicotinic Acetylcholine Receptor Synergistically Regulate FimH-Mediated Escherichia coli K1 Penetration and Neutrophil Transmigration Across Human Brain Microvascular Endothelial Cells.

FimH-mediated bacterial invasion and polymorphonuclear neutrophil (PMN) transmigration across human brain microvascular endothelial cells (HBMECs) are required for the pathogenesis of Escherichia coli meningitis. However, the underlying mechanism remains unclear. This study demonstrated that the TnphoA mutant (22A33) and FimH-knockout mutant (ΔFimH) of E coli strain E44, which resulted in inactivation of FimH, were less invasive and less effective in promoting PMN transmigration than their wild-type strain. FimH protein induced PMN transmigration, whereas calmodulin inhibitor significantly blocked this effect. Moreover, immunofluorescence and co-immunoprecipitation analysis indicated that colocalized CD48 and α7 nAChR formed a complex on the surface of HBMECs that is associated with increased cofilin dephosphorylation, which could be remarkably enhanced by FimH+ E44. Our study concluded that FimH-induced E coli K1 invasion and PMN migration across HBMECs may be mediated by the CD48-α7nAChR complex in lipid rafts of HBMEC via Ca2+ signaling and cofilin dephosphorylation.

Precise excision and secondary transposition of Tn phoA in non-motile mutants of a Salmonella enterica serovar Enteritidis clinical isolate

Mutagenesis with Tn phoA has been widely used in many bacteria. Here, we report the excision and secondary transposition of this transposon in three non-motile ( fliC, fliF and motB) mutants of Salmonella enterica serovar Enteritidis ( S. Enteritidis). Isolation of motile revertants showed that they were kanamycin resistant and conserved a copy of Tn phoA in their genome in an insertion site different from the initial one. They also expressed an intact flagella. Characterization of the motile revertant derived from the fliC mutant showed that Tn phoA excised precisely from the fliC gene, resulting in an equivalent amount of FliC secreted protein in the revertant compared to that of the wild-type strain. These results show that Tn phoA mutants should be used with care and underline the value of using transposon derivatives lacking the transposase gene.

Growth and colonization suppression of Salmonella enterica serovar Hadar in vitro and in vivo

Growth suppression in Salmonella enterica serovar Hadar (S. Hadar) was investigated, in vitro under strict anaerobiosis and in vivo in the intestine of the day-old chicken. Stationary-phase cultures of 20 S. Hadar field strains were tested against each other for growth suppression activity by their ability to suppress the multiplication of low counts of minority cultures inoculated into them as nalidixic acid-resistant mutants. All strains showed profound growth suppression. Four S. Hadar strains were selected and further tested for their ability to suppress growth of S. Enteritidis, S. Typhimurium, S. Virchow and S. Saintpaul. One of the four strains (S. Hadar 18) was randomly selected for further studies. Precolonization of chicken with S. Hadar 18 prevented superinfection with any of the serovars mentioned above. From more than 1000 TnphoA mutants of S. Hadar 18 screened against the parent strain anaerobically in vitro, four were non-suppressive with TnphoA insertions in dapF, aroD, sgaT or tatA. Only the dapF mutant was also non-suppressive in the chicken intestine.

Growth and colonization suppression of Salmonella enterica serovar Hadar in vitro and in vivo.

Growth suppression in Salmonella enterica serovar Hadar (S. Hadar) was investigated, in vitro under strict anaerobiosis and in vivo in the intestine of the day-old chicken. Stationary-phase cultures of 20 S. Hadar field strains were tested against each other for growth suppression activity by their ability to suppress the multiplication of low counts of minority cultures inoculated into them as nalidixic acid-resistant mutants. All strains showed profound growth suppression. Four S. Hadar strains were selected and further tested for their ability to suppress growth of S. Enteritidis, S. Typhimurium, S. Virchow and S. Saintpaul. One of the four strains (S. Hadar 18) was randomly selected for further studies. Precolonization of chicken with S. Hadar 18 prevented superinfection with any of the serovars mentioned above. From more than 1000 TnphoA mutants of S. Hadar 18 screened against the parent strain anaerobically in vitro, four were non-suppressive with TnphoA insertions in dapF, aroD, sgaT or tatA. Only the dapF mutant was also non-suppressive in the chicken intestine.

TnphoA mutants of Providencia alcalifaciens with altered invasiveness of HEp-2 cells.

Recent studies have shown that Providencia alcalifaciens is a diarrhoeal pathogen. It may cause diarrhoea by an invasive mechanism, as it invades cultured mammalian cells in vitro and intestinal epithelial cells of experimentally inoculated rabbits in vivo. To locate the gene(s) involved in invasion, TnphoA mutants of a diarrhoeal isolate of P. alcalifaciens were generated. Compared with the parent strain, these mutants exhibited negligible invasion and actin condensation in HEp-2 cells. TnphoA insertion was located in fragments of 4.9 kb and 11.1 kb of the bacterial chromosome by Southern blot. These mutants did not secrete a 28-kDa protein, which may be involved in invasion. It should be possible now to study the gene(s) involved in invasion of P. alcalifaciens with these mutants. This investigation is another example of the usefulness of TnphoA mutagenesis in the study of bacterial virulence genes.

Tir tyrosine phosphorylation and pedestal formation are delayed in enteropathogenic Escherichia coli sepZ::TnphoA mutant 30-5-1(3).

Enteropathogenic Escherichia coli (EPEC) strain 30-5-1(3) has been reported to form attaching and effacing (A/E) lesions without Tir tyrosine phosphorylation. In this study, we show that 30-5-1(3), which has a transposon insertion within the sepZ gene, forms wild-type A/E lesions including Tir tyrosine phosphorylation, but at a slower rate. A/E lesion formation by 30-5-1(3) occurs without detectable secretion of Tir or other EPEC Esp secreted proteins.

Escherichia coli K1 aslA contributes to invasion of brain microvascular endothelial cells in vitro and in vivo.

Neonatal Escherichia coli meningitis remains a devastating disease, with unacceptably high morbidity and mortality despite advances in supportive care measures and bactericidal antibiotics. To further our ability to improve the outcome of affected neonates, a better understanding of the pathogenesis of the disease is necessary. To identify potential bacterial genes which contribute to E. coli invasion of the blood-brain barrier, a cerebrospinal fluid isolate of E. coli K1 was mutagenized with TnphoA. TnphoA mutant 27A-6 was found to have a significantly decreased ability to invade brain microvascular endothelial cells compared to the wild type. In vivo, 32% of the animals infected with mutant 27A-6 developed meningitis, compared to 82% of those infected with the parent strain, despite similar levels of bacteremia. The DNA flanking the TnphoA insertion in 27A-6 was cloned and sequenced and determined to be homologous to E. coli K-12 aslA (arylsulfatase-like gene). The deduced amino acid sequence of the E. coli K1 aslA gene product shows homology to a well-characterized arylsulfatase family of enzymes found in eukaryotes, as well as prokaryotes. Two additional aslA mutants were constructed by targeted gene disruption and internal gene deletion. Both of these mutants demonstrated decreased invasion phenotypes, similar to that of TnphoA mutant 27A-6. Complementation of the decreased-invasion phenotypes of these mutants was achieved when aslA was supplied in trans. This is the first demonstration that this locus contributes to invasion of the blood-brain barrier by E. coli K1.

The gene locus yijP contributes to Escherichia coli K1 invasion of brain microvascular endothelial cells.

Most cases of Escherichia coli meningitis develop as a result of hematogenous spread, but it is not clear how circulating E. coli crosses the blood-brain barrier. A TnphoA mutant of E. coli K1 RS218 was shown to be significantly less invasive than its parent strain in bovine and human brain microvascular endothelial cells (BMEC), which constitute the blood-brain barrier. More importantly, traversal of the blood-brain barrier was significantly less with this mutant than with the parent strain in newborn rats with experimental hematogenous meningitis. A DNA segment containing the TnphoA insertion site was cloned from RS218, and the cloned DNA complemented the TnphoA mutant in invasion of BMEC. Nucleotide sequence revealed a near identity to that of a hypothetical yijP gene (also called f577) in the E. coli K-12 genome. Sequence analysis indicated that the E. coli K1 yijP gene likely encodes a 66. 6-kDa membrane protein. Deletion and complementation experiments indicated that the yijP gene was involved in E. coli K1 invasion of BMEC, i.e., the invasive ability of E. coli K1 was significantly reduced after yijP was deleted and was restored by complementation with a plasmid containing the yijP open reading frame. This is the first demonstration that the yijP gene locus plays a role in the pathogenesis of E. coli K1 meningitis.

A swarming-defective mutant of Proteus mirabilis lacking a putative cation-transporting membrane P-type ATPase.

The motile TnphoA mutant IC24 of Proteus mirabilis U6450 generates an aberrant swarming colony, and was shown to be impaired in swarm cell differentiation, i.e. cell elongation and hyperflagellation, causing delayed and slower population migration across a solid growth medium. Levels of transcript from the flagellin filament gene fliC, the flagellar master operon flhDC, and the leucine-responsive regulatory protein gene lrp, a regulator of swarming differentiation, were reduced in IC24 mutant swarm cells. The transposon had inserted into a gene encoding a putative P-type ATPase closely related to those transporting cations across bacterial membranes. This ppa gene (Proteus P-type ATPase) was maximally expressed in differentiated swarm cells. The data suggest an effect of ion homeostasis on swarm cell differentiation, possibly mediated via the lrp-flhDC pathway.

INVASION OF INTESTINAL EPITHELIAL CELLS IN VITRO BYESCHERICHIA COLI IS INFLUENCED BY MICROAEROPHILIC CONDITIONS AND THE PRESENCE OF ibe GENES.

96 Enteric bacteria are an important cause of sepsis in neonates, children with gastrointestinal diseases (such as short bowel syndrome), immunocompromised hosts, and children with central venous catheters. It is unclear what microbial factors may mediate the translocation of enteric bacteria across the intestinal epithelial barrier. E. coli RS218 is a K1 encapsulated clinical isolate from the CSF of a newborn infant with meningitis, which has been used to elucidate mechanisms of invasion of brain microvascular endothelial cells. The aims of our study were to evaluate (1) the ability of RS218 and TnphoA transposon mutants of RS218 to invade two intestinal epithelial cell lines and (2) the influence of growth conditions on RS218 invasion. Materials and Methods: Monolayers of Caco-2 and C2BBe1 cells were allowed to fully differentiate, expressing dome formation characteristic for transporting epithelia, and were infected at a minimum of 15 days post-confluency. Bacterial strains were grown overnight under either aerobic or microaerophilic conditions, and approximately 107 bacteria were added to each monolayer. Cells were incubated at 37 °C for 1.5 hours, washed, and then incubated with a gentamicin containing medium for one hour to eliminate extracellular bacteria. Cells were lysed and viable bacteria were enumerated by plating onto sheep blood agar plates. Results: RS218 was invasive in both Caco-2 and C2BBe1 cell lines. RS218 was 3 to 6.5-fold more invasive than HB101, a known noninvasive E. coli, in both cells lines. RS218 grown under microaerophilic conditions showed a 10-fold increase in invasion in both cell lines. TnphoA mutants, which have been shown to be noninvasive in brain microvascular endothelial cells, were at least 16 to 300-fold less invasive than RS218 in both cell lines. Conclusions: (1) The E. coli genes involved in the invasion of brain microvascular endothelial cells (such as ibe10) also play a role in the in vitro invasion of intestinal epithelial cells. (2) The microaerophilic environment enhances E. coli invasion of gut epithelial cells in vitro.

Invasion of Intestinal Epithelial Cells In Vitro by Escherichia coli Is Influenced by Microaerophilic Conditions and the Presence of ibe Genes † 890

Enteric bacteria are an important cause of sepsis in neonates, children with chronic gastrointestinal diseases (such as short bowel syndrome), immunocompromised hosts, and children with central venous catheters. It is unclear what microbial factors may mediate the translocation of enteric bacteria across the intestinal epithelial barrier. E. coli RS218 is a K1 encapsulated clinical isolate from the CSF of a newborn infant with meningitis, which has been used to elucidate mechanisms of invasion of brain microvascular endothelial cells. The aims of our study were to evaluate (1) the ability of RS218 and Tn phoA transposon mutants of RS218 to invade two intestinal epithelial cell lines, and (2) the influence of growth conditions on RS218 invasion. Materials and Methods: Monolayers of Caco-2 and C2BBe1 cells were allowed to fully differentiate, expressing dome formation characteristic for transporting epithelia, and were infected at a minimum of 15 days post-confluency. Bacterial strains were grown overnight under either aerobic or microaerophilic conditions, and approximately 107 bacteria were added to each monolayer. Cells were incubated at 37°C for 1.5 hours, washed, and then incubated with a gentamicin containing medium for one hour to eliminate extracellular bacteria. Cells were lysed and viable bacteria were enumerated by plating onto sheep blood agar plates. Results: RS218 was invasive in both Caco-2 and C2BBe1 cell lines. RS218 was 3 to 6.5-fold more invasive than HB101, a known noninvasive E. coli, in both cell lines. RS218 grown under microaerophilic conditions showed a 10-fold increase in invasion in both cell lines. Tn phoA mutants, which have been shown to be noninvasive in brain microvascular endothelial cells, were at least 16 to 300-fold less invasive than RS218 in both cell lines.Conclusions: (1) The E. coli genes involved in the invasion of brain microvascular endothelial cells (such as ibe10) also play a role in the in vitro invasion of intestinal epithelial cells. (2) The microaerophilic environment enhances E. coli invasion of gut epithelial cells in vitro.

Environmental regulation of Salmonella typhi invasion-defective mutants.

Salmonella typhi is the etiologic agent of human typhoid. During infection, S. typhi adheres to and invades epithelial and M cells that line the distal ileum. To survive in the human host, S. typhi must overcome numerous complex extracellular and intracellular environments. Since relatively little is known about S. typhi pathogenesis, studies were initiated to identify S. typhi genes involved in the early steps of interaction with the host and to evaluate the environmental regulation of these genes. In the present study, TnphoA mutagenesis was used to study these early steps. We isolated 16 Salmonella typhi TnphoA mutants that were defective for both adherence and invasion of the human small intestinal epithelial cell line Int407. Twelve of sixteen mutations were identified in genes homologous to the S. typhimurium invG and prgH genes, which are known to be involved in the type III secretion pathway of virulence proteins. Two additional insertions were identified in genes sharing homology with the cpxA and damX genes from Escherichia coli K-12, and two uncharacterized invasion-deficient mutants were nonmotile. Gene expression of TnphoA fusions was examined in response to environmental stimuli. We found that the cpxA, invG, and prgH genes were induced when grown under conditions of high osmolarity (0.3 M NaCl). Expression of invG and prgH genes was optimal at pH 6.5 and strongly reduced at low pH (5.0). Transcription of both invG and prgH TnphoA gene fusions was initiated during the late logarithmic growth phase and was induced under anaerobic conditions. Finally, we show that both invG and prgH genes appear to be regulated by DNA supercoiling, a mechanism influenced by environmental factors. These results are the first to demonstrate that in S. typhi, (i) the prgH and cpxA genes are osmoregulated, (ii) the invG gene is induced under low oxygen conditions, (iii) the invG gene is pH regulated and growth phase dependent, and (iv) the prgH gene appears to be regulated by DNA supercoiling. Since our experimental conditions were designed to mimic the in vivo environmental milieu, our results suggest that specific environmental conditions act as signals to induce the expression of S. typhi invasion genes.

Screening of TnphoA mutants of Vibrio cholerae O139 for identification of antigens involved in colonisation.

A new serogroup of Vibrio cholerae non-O1, designated as O139, has emerged causing cholera-like disease among adults. Laboratory and field studies clearly show that there is no cross-protection between O1 and O139 pathogenic strains. Since colonisation of the intestine is a most important step in the pathogenesis of cholera caused by O1 strains and colonising antigens are known to be protective, investigation of the colonising antigens of O139 strain was initiated. By TnphoA mutagenesis, mutants were generated with insertions in the genome encoding membrane spanning or secretory proteins. Screening of the mutants for adherence to rabbit intestinal surface and colonisation in 5-day-old mice resulted in the identification of mutant clones, which were less adhesive than was the wild-type parent strain and which could not efficiently colonise the gut. Such non-colonising strains were attenuated in virulence. Analysis of the proteins by SDS-PAGE revealed that the non-colonising mutants did not express a 40-kDa outer-membrane protein.

Identification and characterization of a K88- and CS31A-like operon of a rabbit enteropathogenic Escherichia coli strain which encodes fimbriae involved in the colonization of rabbit intestine.

Initiation of attaching-effacing lesions, which characterize infections with rabbit enteropathogenic Escherichia coli (REPEC), requires bacteria to adhere to the intestinal epithelium. This adherence is reflected in vitro by the affinity of these E. coli strains for various types of eukaryotic cells. TnphoA mutants of REPEC 83/39 (O15:H-) which had lost the ability to adhere to HEp-2 epithelial cells, guinea pig ileal brush borders, and mouse erythrocytes were generated. DNA sequencing of the region surrounding the inactivating transposon insertions within a 95-kb plasmid, designated pRAP for REPEC adherence plasmid, revealed extensive homology between that region and the structural genes of enterotoxigenic E. coli operons encoding the K88 and CS31A fimbrial adhesins and the genes for the afr2 adhesin from REPEC B10 (O103:H2). Seven genes of the ral operon (for REPEC adherence locus), including three putative minor fimbrial subunit genes (ralC, ralF, and ralH), a major fimbrial subunit gene (ralG), a gene of unknown function (ralI), and genes for two fimbrial subunit chaperones (ralD and ralE), were sequenced. When inoculated perorally into weanling rabbits, a mutant with a TnphoA insertion in the ralE gene showed a 10-fold reduction in colonizing ability, with only 1 of 10 rabbits excreting bacteria compared to all 5 of those infected with the wild-type parent strain (P = 0.002). The severity of the diarrheal illness caused by the mutant strain was also reduced. Western blotting of surface protein extracts of strain 83/39 with hyperimmune anti-83/39 antiserum, adsorbed with the ralE mutant, revealed a 32-kDa protein which was absent from protein extracts of two nonadherent mutants. The adsorbed antiserum also bound to the surface of strain 83/39 but not to nonadherent mutants, as detected by immunogold labeling. These results indicate that the ral operon of REPEC 83/39 contains genes necessary for the biosynthesis of fine fimbriae which are responsible for in vitro adherence of the bacteria and play a role in their colonization of, and hence virulence for, rabbits. The putative major fimbrial subunit is a protein with an observed molecular size of approximately 32 kDa which, when assembled, appears to form a capsule of fimbriae surrounding the bacterium similar to that described for CS31A.

A motile but non-swarming mutant of Proteus mirabilis lacks FlgN, a facilitator of flagella filament assembly.

A TnphoA mutant of Proteus mirabilis was isolated, which had lost the ability to swarm, yet was still motile. The transposon had inserted into flgN, a flagella gene encoding a 147-amino-acid protein of undefined function. Proteus flgN is arranged in an operon with the class III anti-sigma28 gene, flgM, flanked by the class II genes, flgA, flgBCD and flhBA, and a novel putative virulence-related gene. The flgN mutation caused a substantial reduction in cell surface-associated flagellin, particularly during differentiation to the normally hyperflagellated swarm cell. This was not due to an effect on flagella gene expression or a typical defect in the flagella export apparatus as there was no class III gene downregulation by FlgM feedback, or intracellular flagellin accumulation. Loss of FlgN nevertheless caused a severe reduction in the incorporation of pulse-labelled flagellin into the membrane/flagellum fraction of differentiating cells. Substantial amounts of both non-oligomeric flagellin and flagellin degradation products appeared in the extracellular medium, although the few mature filaments made by the mutant were no more sensitive to proteolysis than those of the wild type. FlgN appeared soluble and active in the cytosol. The data suggest that the function of FlgN is to facilitate the initiation of flagella filament assembly, a role that may be especially critical in attaining the much higher concentration of surface flagellin required for swarming. Proteus FlgN has leucine zipper-like motifs arranged on potential amphipathic helices, a feature conserved in cytosolic chaperones for the exported substrates of flagella-related type III virulence systems. While gel filtration of FlgN from the soluble cell fraction did not establish an interaction with flagellin, it indicated that FlgN may associate with an unknown component and/or form an oligomer.

Adhesive factor/rabbit 2, a new fimbrial adhesin and a virulence factor from Escherichia coli O103, a serogroup enteropathogenic for rabbits.

Enteropathogenic Escherichia coli-like E. coli strains belonging to serovar O103:K-:H2 and rhamnose-negative biotypes are highly pathogenic diarrhea-inducing strains for weaned European rabbits. We describe here the cloning and sequencing of the major subunit gene of a new fimbrial adhesin, adhesive factor/rabbit 2 (AF/R2), which confers on these strains the ability to attach to rabbit enterocytes and to HeLa cells in a diffuse manner and which is associated with in vivo virulence. The chromosomal operon that encodes functional AF/R2 has been cloned from strain B10. The major subunit gene afr2G, as well as an adjacent open reading frame, afr2H, has been sequenced. The Afr2G protein shows homologies with FaeG and ClpG, which are the respective major subunits of fimbrial adhesin K88 (F4) and afimbrial adhesin CS31A. Plasmid carrying the operon transcomplements an AF/R2-negative TnphoA mutant for its ability to express AF/R2. As a whole, AF/R2 is a new member of the E. coli K88 adhesin family which is associated with virulence and which may serve in the design of vaccines.

Polysaccharide side chains are not required for attaching and effacing adhesion of Escherichia coli O157:H7.

Escherichia coli of the serotype O157:H7 is an enterohemorrhagic human pathogen which demonstrates attaching and effacing adhesion to colonocytes in vivo and to epithelial cells grown in tissue culture. Transposon TnphoA mutants of E. coli O157:H7 strain CL-8 were produced. Two of 300 alkaline phosphatase positive mutants, designated JB6 and JB27, did not express O157 side chains as assessed by agglutination with specific polyclonal O157 antiserum, silver staining of lipopolysaccharide extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western immunoblots with polyclonal O157-specific antiserum. Both O157-negative mutants and the parent strain demonstrated localized adherence to HEp-2 cells when examined by Giemsa staining and bright-field microscopy. Furthermore, both O157-negative mutants showed enhanced adherence to HEp-2 cells compared with the parent strain when assessed by quantification of adherent bacterial CFUs. The parent strain, CL-8, and both of the mutants produced fluorescent foci when adherent bacteria and HEp-2 cells were stained with fluorescein isothiocyanate-labelled phalloidin. Transmission electron microscopy confirmed attaching and effacing adherence of strain CL-8 and the OO7-negative mutants to HEp-2 cells. These findings indicate that mutants deficient in O157 polysaccharide repeats exhibit adherence to tissue culture cells in vitro and that O157 polysaccharide repeats are not required to produce the attaching and effacing lesion.

Role of catechol siderophore synthesis in Vibrio vulnificus virulence.

We isolated a Vibrio vulnificus TnphoA mutant that was unable to produce catechol siderophores or to acquire iron from transferrin. This mutant showed reduced virulence in an infant mouse model. The TnphoA insertion was in an open reading frame designated venB. The venB gene cloned on a plasmid restored catechol production to the mutant. The deduced amino acid sequence of venB is 41% identical to the enzyme isochorismatase of Escherichia coli (EntB), an enzyme involved in the biosynthesis of the catechol siderophore enterobactin.

Role of Adhesive Factor/Rabbit 2 in experimental enteropathogenic Escherichia coli O103 diarrhea of weaned rabbit

The Adhesive Factor/Rabbit 2 (AF/R2) is found in Escherichia coli strains of serovar O103:K −:H2 and rhamnose-negative biovars isolated from weaned rabbits with diarrhea. This adhesin allows the colonization of the distal parts of the digestive tract, a first step leading to severe inflammatory diarrhea and death of the animals. In vitro, AF/R2 expression mediates diffuse adhesion of E. coli on HeLa cells, adhesion to ileal villi of newborn and weaned rabbits and the presence of a major 32 kDa subunit in bacterial surface extracts. In this work, we constructed Tn phoA mutants of the prototype strain B10 and selected an isogenic clone that did not express the AF/R2 32 kDa subunit when grown in permissive conditions in vitro. The pathogenicity of the wild type strain and of the isogenic mutant was compared by oral inoculation to 35-day-old weaned rabbits. The mutant showed impaired colonization and a highly significant loss of pathogenicity. However, the occurrence of residual weight losses, and of diarrheas and mortalities in some inoculated rabbits suggest that pathogenicity of rabbit O103 enteropathogenic-like E. coli (EPEC-like) strains is due to multiple virulence factors and that other virulence traits remain to be found.