Amino acid residue Ala-62 in the FimH fimbrial adhesin is critical for the adhesiveness of meningitis-associated Escherichia coli to collagens

Adhesion of meningitis-associated Escherichia coli O18acK1H7 to collagens was characterized. The E. coli strain IHE 3034 adhered to type IV and type I collagens but not to type III collagen immobilized on glass. Collagens lack terminal mannosyl units, yet the bacterial adhesion was completely abolished in the presence of alpha-methyl-D-mannoside. A cat cassette was introduced into the filmA gene of IHE 3034, and the resulting mutant strain IHE 3034-2 failed to adhere to collagens. In contrast, insertion of a Gm cassette into the sfaA gene of IHE 3034, encoding the S-fimbrillin, had no significant effect on the adhesiveness. The fim cluster from IHE 3034 was cloned and expressed in trans in the fimA::cat mutant strain IHE 3034-2. The complemented strain IHE 3034-2(pRPO-1) exhibited adhesiveness to type IV and type I collagens, confirming the function of the type 1 fimbria in the adhesion. We have previously shown that the type 1 fimbria from E. coli K-12 strain PC31 does not confer bacterial adhesiveness to collagens. The fimH genes from E. coli IHE 3034 as well as from PC31 were expressed in the fimH-null strain MS4. The FimH from IHE 3034 potentiated collagen adherence, whereas the FimH from PC31 was inactive. Sequence comparison of fimH from IHE 3034 and PC31 revealed five amino-acid differences in the predicted mature FimH proteins: at residues 27, 62, 70, 78 and 201. Each of these residues in the IHE 3034-FimH were individually substituted to the corresponding amino acid in the PC31-FimH. The substitution S62-->A completely abolished collagen adhesiveness. The reverse substitution A62-->S in the PC31-FimH as well as in the FimH from another E. coli strain induced collagen adhesiveness to the level seen with IHE 3034-FimH. Out of nine fimH genes analysed from isolates of E. coli, collagen adhesiveness as well as alanine at position 62 in FimH were found only in two O18acK1H7 isolates with the isoenzyme profile ET type 1. Our results demonstrate that the amino-acid residue Ala-62 in the FimH lectin is critical for the adhesion to collagens by a highly virulent clonal group of E. coli.     

Characterization of the rcsB gene from Erwinia amylovora and its influence on exoploysaccharide synthesis and virulence of the fire blight pathogen.

RcsB belongs to a family of positive regulators of exopolysaccharide synthesis in various enterobacteria. The rcsB gene of the fire blight pathogen Erwinia amylovora was cloned by PCR amplification with consensus primers, and its role in exopolysaccharide (EPS) synthesis was investigated. Its overexpression from high-copy-number plasmids stimulated the synthesis of the acidic EPS amylovoran and suppressed expression of the levan-forming enzyme levansucrase. Inactivation of rcsB by site-directed mutagenesis created mutants that were deficient in amylovoran synthesis and avirulent on host plants. In addition, a cosmid which complemented rcsB mutants was selected from a genomic library. The spontaneous E. amylovora mutant E8 has a similar phenotype and was complemented by the cloned rcsB gene. The rcsB region of strain E8 was also amplified by PCR, and the mutation was characterized as a nine-nucleotide deletion at the start of the rcsB gene. Nucleotide sequence analysis of the E. amylovora rcsB region and the predicted amino acid sequence of RcsB revealed extensive homology to rcsB and the encoded protein of other bacteria such as Escherichia coli and Erwinia stewartii. In all three organisms, rcsB is localized adjacent to the rcsC gene, which is transcribed in the opposite direction of rcsB. The E. amylovora rcsB gene has now been shown to strongly affect the formation of disease symptoms of a plant pathogen.     

Effect of rpoS mutations on stress-resistance and invasion of brain microvascular endothelial cells in Escherichia coli K1

Escherichia coli K1 strains are predominant in causing neonatal meningitis. We have shown that invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for E. coli K1 crossing of the blood-brain barrier. BMEC invasion by E. coli K1 strain RS218, however, has been shown to be significantly greater with stationary-phase cultures than with exponential-phase cultures. Since RpoS participates in regulating stationary-phase gene expression, the present study examined a possible involvement of RpoS in E. coli K1 invasion of BMEC. We found that the cerebrospinal fluid isolates of E. coli K1 strains RS218 and IHE3034 have a nonsense mutation in their rpoS gene. Complementation with the E. coli K12 rpoS gene significantly increased the BMEC invasion of E. coli K1 strain IHE3034, but failed to significantly increase the invasion of another E. coli K1 strain RS218. Of interest, the recovery of E. coli K1 strains following environmental insults was 10-100-fold greater on Columbia blood agar than on LB agar, indicating that growing medium is important for viability of rpoS mutants after environmental insults. Taken together, our data suggest that the growth-phase-dependent E. coli K1 invasion of BMEC is affected by RpoS and other growth-phase-dependent regulatory mechanisms.     

Construction of a novel suicide vector: selection for Escherichia coli HB101 recombinants carrying the DNA insert.

We constructed a new type of cloning vector, pERISH2, that transforms Escherichia coli HB101 only when a foreign DNA fragment is ligated into the cloning site of the plasmid vector. Plasmid pERISH2 carries the rcsB gene which is derived from the chromosome of E. coli HB101 and is involved in the regulation of colanic acid production. When E. coli HB101 is transformed by this vector carrying the intact rcsB gene, the gene product RcsB blocks bacterial growth. However, if the rcsB gene is inactivated by the insertion of a foreign DNA fragment, this recombinant plasmid no longer inhibits the growth of E. coli HB101. Although E. coli HB101 is not stably transformed by pERISH2, E. coli K-12 strains such as JM109 and C600 can harbor this vector. Therefore, pERISH2 can be amplified in JM109 and be prepared from this strain in a large quantity using conventional methods. A chromosomal gene library of Klebsiella pneumoniae is constructed easily and efficiently by the utilization of this new cloning vector.     

Isolation of an Escherichia coil strain mutant unable to form biofilm on polystyrene and to adhere to human pneumocyte cells: involvement of tryptophanase

Escherichia coli adherence to biotic and abiotic surfaces constitutes the first step of infection by promoting colonization and biofilm formation. The aim of this study was to gain a better understanding of the relationship between E. coli adherence to different biotic surfaces and biofilm formation on abiotic surfaces. We isolated mutants defective in A549 pneumocyte cells adherence, fibronectin adherence, and biofilm formation by random transposition mutagenesis and sequential passages over A549 cell monolayers. Among the 97 mutants tested, 80 were decreased in biofilm formation, 8 were decreased in A549 cells adherence, 7 were decreased in their adherence to fibronectin, and 17 had no perturbations in either of the three phenotypes. We observed a correlation between adherence to fibronectin or A549 cells and biofilm formation, indicating that biotic adhesive factors are involved in biofilm formation by E. coli. Molecular analysis of the mutants revealed that a transposon insertion in the tnaA gene encoding for tryptophanase was associated with a decrease in both A549 cells adherence and biofilm formation by E. coli. The complementation of the tnaA mutant with plasmid-located wild-type tnaA restored the tryptophanase activity, epithelial cells adherence, and biofilm formation on polystyrene. The possible mechanism of tryptophanase involvement in E. coli adherence and biofilm formation is discussed.     

Coexpression of colanic acid and serotype-specific capsular polysaccharides in Escherichia coli strains with group II K antigens.

In Escherichia coli K-12, the rcsA and rcsB gene products are positive regulators in expression of the slime polysaccharide colanic acid. We have previously demonstrated the presence of rcsA sequences in E. coli K1 and K5, strains with group II capsular K antigens, and shown that introduction of multicopy rcsA into these strains results in the expression of colanic acid. We report here the presence of rcsB sequences in E. coli K1 and K5 and demonstrate that RcsB also plays a role in the biosynthesis of colanic acid in strains with group II K antigens. In E. coli K1 and K5 grown at 37 degrees C, multicopy rcsB and the resulting induction of colanic acid synthesis had no significant effect on synthesis of the group II K antigens. K-antigen-specific sugar transferase activities were not significantly different in the presence or absence of multicopy rcsB, and introduction of a cps mutation to eliminate colanic acid biosynthesis in a K1-derivative strain did not influence the activity of the polysialyltransferase enzyme responsible for synthesis of the K1 polymer. Furthermore, immunoelectron microscopy showed no detectable difference in the size or distribution of the group II K-antigen capsular layer in cells which produced colanic acid. Colanic acid expression therefore does not appear to significantly affect synthesis of the group II K-antigen capsule and, unlike for group I K antigens, expression of group II K antigens is not positively regulated by the rcs system.     

Acetyl phosphate-sensitive regulation of flagellar biogenesis and capsular biosynthesis depends on the Rcs phosphorelay

As part of our attempt to map the impact of acetyl phosphate (acetyl approximately P) on the entire network of two-component signal transduction pathways in Escherichia coli, we asked whether the influence of acetyl approximately P on capsular biosynthesis and flagellar biogenesis depends on the Rcs phosphorelay. To do so, we performed a series of epistasis experiments: mutations in the components of the pathway that controls acetyl approximately P levels were combined with mutations in components of the Rcs phosphorelay. Cells that did not synthesize acetyl approximately P produced no capsule under normally permissive conditions, while those that accumulated acetyl approximately P synthesized capsule under conditions previously considered to be non-permissive. Acetyl approximately P-dependent capsular biosynthesis required both RcsB and RcsA, while the lack of RcsC restored capsular biosynthesis to acetyl approximately P-deficient cells. Similarly, acetyl approximately P-sensitive repression of flagellar biogenesis was suppressed by the loss of RcsB (but not of RcsA), while it was enhanced by the lack of RcsC. Taken together, these results show that both acetyl approximately P-sensitive activation of capsular biosynthesis and acetyl approximately P-sensitive repression of flagellar biogenesis require the Rcs phosphorelay. Moreover, they provide strong genetic support for the hypothesis that RcsC can function as either a kinase or a phosphatase dependent on environmental conditions. Finally, we learned that RcsB and RcsC inversely regulated the timing of flagellar biogenesis: rcsB mutants elaborated flagella prematurely, while rcsC mutants delayed their display of flagella. Temporal control of flagella biogenesis implicates the Rcs phosphorelay (and, by extension, acetyl approximately P) in the transition of motile, planktonic individuals into sessile biofilm communities.     

Interaction of the regulator proteins RcsA and RcsB with the promoter of the operon for amylovoran biosynthesis in Erwinia amylovora

The RcsA and RcsB proteins of Erwinia amylovora and Escherichia coli were expressed in E. coli and purified. Their DNA-binding activity was examined using a 1-kb DNA region containing the putative promoter of the ams operon of Ew. amylovora, which is responsible for the biosynthesis of the exopolysaccharide amylovoran. Mobility shift assays indicated specific binding of RcsA and RcsB to a region of 78?bp spanning nucleotide positions ?578 to ?501 relative to the translational start of the first open reading frame of the operon. This region includes stretches of homology to E. coliσ ⁷⁰ promoter consensus sequences and to the E. coli cps promoter region. Binding of the Rcs proteins was not found at a JUMPstart consensus, typical for various promoters of polysaccharide gene clusters. DNA-binding activity was not detected for RcsA alone and only high concentrations of RcsB were able to interact with the ams promoter in our assay. The two proteins bind cooperatively at the indicated region of the ams promoter and further evidence is provided showing that the DNA-protein complex formed involves a heterodimer of RcsA and RcsB. The specific activity of RcsA, but not of RcsB, was enhanced when the protein was expressed in E. coli at 28°?C, relative to expression at 37°?C. In addition, DNA-protein complex formation is affected by temperature. The E. coli RcsA/RcsB proteins bind to the same region of the ams promoter and are able to interact with the Rcs proteins from Ew. amylovora.     

SslE Elicits Functional Antibodies That Impair In Vitro Mucinase Activity and In Vivo Colonization by Both Intestinal and Extraintestinal Escherichia coli Strains

SslE, the Secreted and surface-associated lipoprotein from Escherichia coli, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of in vitro bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired E. coli mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslE_IHE3034), are able to inhibit translocation of E. coli strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslE_IHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in E. coli colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic E. coli species.     

Identification, cloning, and characterization of rcsF, a new regulator gene for exopolysaccharide synthesis that suppresses the division mutation ftsZ84 in Escherichia coli K-12.

A new gene, designated rcsF, was located adjacent to drpA at the 5.2-min position of the genetic map of Escherichia coli. The deduced amino acid sequence encoded by the rcsF gene indicates a small protein of 133 amino acid residues with a calculated pI of 10.8 that is rich in proline, serine, alanine, and cysteine residues. When overexpressed as a result of its presence on a multicopy plasmid, rcsF confers a mucoid phenotype and restores colony formation to ftsZ84 mutant cells on L agar medium containing no added NaCl. These two phenotypes are not observed in rcsB mutant cells. Ion mutant cells harboring an rcsF mutation accumulate considerably lower levels of exopolysaccharides, whereas the presence of a multicopy rcsF plasmid not only increases capsule synthesis but also confers a mucoid phenotype at 37 degrees C, a temperature at which ion mutant cells are known not to form mucoid colonies. RcsF does not stimulate the expression of rcsB, indicating that it exerts its action through the RcsB protein, possibly by phosphorylation. It is also shown that RcsF stimulation of capsule synthesis is RcsA-dependent, whereas colony formation of ftsZ84 mutant cells can be restored by RcsF in the absence of RcsA.     

Differential binding of Escherichia coli O157:H7 to alfalfa, human epithelial cells, and plastic is mediated by a variety of surface structures

Escherichia coli O157:H7 carried on plant surfaces, including alfalfa sprouts, has been implicated in food poisoning and outbreaks of disease in the United States. Adhesion to cell surfaces is a key component for bacterial establishment and colonization on many types of surfaces. Several E. coli O157:H7 surface proteins are thought to be important for adhesion and/or biofilm formation. Therefore, we examined whether mutations in several genes encoding potential adhesins and regulators of adherence have an effect on bacterial binding to plants and also examined the role of these genes during adhesion to Caco-2 cells and during biofilm formation on plastic in vitro. The genes tested included those encoding adhesins (cah, aidA1, and ompA) and mediators of hyperadherence (tdcA, yidE, waaI, and cadA) and those associated with fimbria formation (csgA, csgD, and lpfD2). The introduction of some of these genes (cah, aidA1, and csg loci) into an E. coli K-12 strain markedly increased its ability to bind to alfalfa sprouts and seed coats. The addition of more than one of these genes did not show an additive effect. In contrast, deletion of one or more of these genes in a strain of E. coli O157:H7 did not affect its ability to bind to alfalfa. Only the absence of the ompA gene had a significant effect on binding, and the plant-bacterium interaction was markedly reduced in a tdcA ompA double mutant. In contrast, the E. coli O157:H7 ompA and tdcA ompA mutant strains were only slightly affected in adhesion to Caco-2 cells and during biofilm formation. These findings suggest that some adhesins alone are sufficient to promote binding to alfalfa and that they may exist in E. coli O157:H7 as redundant systems, allowing it to compensate for the loss of one or more of these systems. Binding to the three types of surfaces appeared to be mediated by overlapping but distinct sets of genes. The only gene which appeared to be irreplaceable for binding to plant surfaces was ompA.     

Transcriptional regulation through RfaH contributes to intestinal colonization by Escherichia coli

The Escherichia coli regulatory protein RfaH contributes to efficient colonization of the mouse gut. Extraintestinal pathogenic (ExPEC) as well as non-pathogenic probiotic E. coli strains rapidly outcompeted their isogenic rfaH mutants following oral mixed infections. LPS-core and O-antigen side-chain as well as capsular polysaccharide synthesis are among the E. coli virulence factors affected by RfaH. In respect of colonization, deep-rough LPS mutants (waaG) but not capsular (kps) mutants were shown to behave similarly to rfaH mutants. Furthermore, alteration in the length of O-antigen side-chains did not modify colonization ability either indicating that it was the regulatory effect of RfaH on LPS-core synthesis, which affected intestinal colonization. Loss of RfaH did not significantly influence adhesion of bacteria to cultured colon epithelial cells. Increased susceptibility of rfaH mutants to bile salts, on the other hand, suggested that impaired in vivo survival could be responsible for the reduced colonization capacity.     

The pathogenicity island encoded PvrSR/RcsCB regulatory network controls biofilm formation and dispersal in Pseudomonas aeruginosa PA14

Pseudomonas aeruginosa biofilm formation is linked to persistent infections in humans. Biofilm formation is facilitated by extracellular appendages, some of which are assembled by the Chaperone Usher Pathway (Cup). The cupD gene cluster is located on the PAPI‐1 pathogenicity island of strain PA14 and has probably been acquired together with four genes encoding two‐component signal transduction proteins. We have previously showed that the RcsB response regulator activates expression of the cupD genes, which leads to the production of CupD fimbriae and increased attachment. Here we show that RcsB activity is tightly modulated by two sensors, RcsC and PvrS. While PvrS acts as a kinase that enhances RcsB activity, RcsC has a dual function, first as a phosphorelay, and second as a phosphatase. We found that, under certain growth conditions, overexpression of RcsB readily induces biofilm dispersal. Microarray analysis shows that RcsB positively controls expression of pvrR that encodes the phosphodiesterase required for this dispersal process. Finally, in addition to the PAPI‐1 encoded cupD genes, RcsB controls several genes on the core genome, some of which encode orphan response regulators. We thus discovered that RcsB is central to a large regulatory network that fine‐tunes the switch between biofilm formation and dispersal.