Nonadhesive Mutants Research Articles

Critical roles of a housekeeping sortase of probiotic Bifidobacterium bifidum in bacterium–host cell crosstalk

SummaryBifidobacterium bifidum YIT 10347 (BF-1) is adhesive in vitro. Here we studied the molecular aspects of the BF-1 adhesion process. We identified and characterized non-adhesive mutants and found that a class E housekeeping sortase was critical for the adhesion to mucin. These mutants were significantly less adhesive to GCIY cells than was the wild type (WT), which protected GCIY cells against acid treatment more than did a non-adhesive mutant. The non-adhesive mutants aberrantly accumulated precursors of putative sortase-dependent proteins (SDPs). Recombinant SDPs bound to mucin. Disruption of the housekeeping sortase influenced expression of SDPs and pilus components. Mutants defective in a pilin or in an SDP showed the same adhesion properties as WT. Therefore, multiple SDPs and pili seem to work cooperatively to achieve adhesion, and the housekeeping sortase is responsible for cell wall anchoring of its substrates to ensure their proper biological function.

Functional characterization of VC1929 of Vibrio cholerae El Tor: role in mannose-sensitive haemagglutination, virulence and utilization of sialic acid

The nonadhesive mutant CD11 of Vibrio cholerae El Tor, defective in expression of mannose-sensitive haemagglutinin, lacks a protein when compared with its parent strain. Determination of the amino acid sequence revealed the identity of the protein as the product of VC1929, which is annotated to encode a protein, DctP, involved in the transport of C₄-dicarboxylates. We cloned the dctP gene in pUC19 vector and expressed it in mutant CD11. Expression of DctP in the resulting complemented strain restored virulence, adhesive and colonizing capabilities, mannose-sensitive haemagglutination (MSHA) and ability to grow in medium containing sialic acid as a sole carbon source. The mutation in CD11 was caused by insertion of an adenine nucleotide in the reading frame of dctP. Recombinant purified DctP protein showed MSHA of human red blood cells, and protected rabbits against infection by V. cholerae. The protein was localized in membrane and cell wall fractions. The mutant, recombinant CD11 expressing DctP and parent strains were grown in M9 minimal medium in the presence of various carbohydrates (glucose, malate, fumarate, succinate or N-acetylneuraminic acid). The mutant was unable to grow in minimal medium containing N-acetylneuraminic acid (sialic acid) as the sole carbon source whereas the recombinant and parent strains utilized all the sugars tested. It is concluded that DctP is a mannose-sensitive haemagglutinin and a virulence factor and is involved in the utilization of sialic acid.

Type 1 fimbriae of Salmonella enterica serovar Typhimurium bind to enterocytes and contribute to colonization of swine in vivo.

Salmonella enterica serovar Typhimurium strain 798 is a clinical isolate from a pig and is known to be able to cause persistent, asymptomatic infections. This strain also is known to exist in two phenotypes (adhesive and nonadhesive to enterocytes) and can switch between the two phenotypes at a rate consistent with phase variation. Cells in the adhesive phenotype are more readily phagocytosed by leukocytes than nonadhesive cells. Once in a leukocyte, adhesive-phase cells survive while nonadhesive-phase cells die. In the present study, nonadhesive mutants were obtained with the transposon TnphoA. A nonadhesive mutant was selected for study and was shown by electron microscopy not to produce fimbriae. The gene encoding the adhesin was cloned and sequenced. Based on its sequence, the adhesin was shown to be FimA, the major subunit of type 1 fimbriae. The nonadhesive mutant was attenuated in its ability to colonize both mouse and pig intestines, but remained capable of systemic spread in mice. The nonadhesive mutant was phagocytosed to the same extent as parental cells in the adhesive phase and then survived intracellularly. These results demonstrated that type 1 fimbriae were important for attachment to enterocytes and promoted intestinal colonization. However, they were not important in promoting phagocytosis or intracellular survival.

Construction and characterization of mutations within the Klebsiella mrkD1P gene that affect binding to collagen type V.

The fimbria-associated MrkD1P protein mediates adherence of type 3 fimbriate strains of Klebsiella pneumoniae to collagen type V. Currently, three different MrkD adhesins have been described in Klebsiella species, and each possesses a distinctive binding pattern. Therefore, the binding abilities of mutants possessing defined mutations within the mrkD1P gene were examined in order to determine whether specific regions of the adhesin molecule were responsible for collagen binding. Both site-directed and chemically induced mutations were constructed within mrkD1P, and the ability of the gene products to be incorporated into fimbrial appendages or bind to collagen was determined. Binding to type V collagen was not associated solely with one particular region of the MrkD1P protein, and two classes of nonadhesive mutants were isolated. In one class of mutants, the MrkD adhesin was not assembled into the fimbrial shaft, whereas in the second class of mutants, the adhesin was associated with fimbriae but did not bind to collagen. Both hemagglutinating and collagen-binding activities were associated with the MrkD1P molecule, since P pili and type 3 fimbriae carrying adhesive MrkD proteins exhibited identical binding properties.

Construction and Characterization of Mutations within theKlebsiella mrkD1P Gene That Affect Binding to Collagen Type V

The fimbria-associated MrkD1P protein mediates adherence of type 3 fimbriate strains of Klebsiella pneumoniae to collagen type V. Currently, three different MrkD adhesins have been described in Klebsiella species, and each possesses a distinctive binding pattern. Therefore, the binding abilities of mutants possessing defined mutations within themrkD1P gene were examined in order to determine whether specific regions of the adhesin molecule were responsible for collagen binding. Both site-directed and chemically induced mutations were constructed within mrkD1P, and the ability of the gene products to be incorporated into fimbrial appendages or bind to collagen was determined. Binding to type V collagen was not associated solely with one particular region of the MrkD1Pprotein, and two classes of nonadhesive mutants were isolated. In one class of mutants, the MrkD adhesin was not assembled into the fimbrial shaft, whereas in the second class of mutants, the adhesin was associated with fimbriae but did not bind to collagen. Both hemagglutinating and collagen-binding activities were associated with the MrkD1P molecule, since P pili and type 3 fimbriae carrying adhesive MrkD proteins exhibited identical binding properties.

Construction and characterization of type 1 non-fimbriate and non-adhesive mutants of Salmonella typhimurium

Mutations in the fimH gene of Salmonella typhimurium result in a non-fimbriate, non-adhesive phenotype. This phenotype was shown to be due to the lack of both fimH and fimF expression since disruption of the fimH gene by insertion of a DNA cassette into this determinant results in mutants that are complemented by plasmids carrying both fimH and fimF. Deletion mutations within the S. typhimurium fimH gene carried on a recombinant plasmid can be used to complement the mutant, and these transformants are non-adhesive but fully fimbriate, consistent with the role of FimH as being necessary for fimbrial adhesin expression. Adherence to erythrocytes, HeLa, and Hep-2 cells is associated with expression of the FimH polypeptide, and fimbriate strains that cannot synthesize FimH are non-adhesive. Discrete differences in the amino acid sequences of the adhesive type 1 and the non-hemagglutinating type 2 FimH polypeptides were detected, and are most likely responsible for the differences in hemagglutinating activity.

21] Use of TnphoA and T7 RNA polymerase to study fimbrial proteins

Publisher Summary This chapter focuses on two tools that have proved to be particularly useful for studying cloned-gene clusters responsible for fimbriation and adhesion of enteric bacteria. Pertinent techniques, concepts, strategies, precautions, and advantages of (1) region-directed Tn phoA (Tn5IS 50 L :: phoA ) mutagenesis and (2) in vivo expression of cloned polycistronic genes with a T7 RNA polymerase/promoter system are presented. Because of their versatile properties, ease in handling, and disposition to molecular manipulations, transposons are among the most frequently used mutagenic tools. Two types of studies, in particular, can benefit from the use of transposon mutagenesis. In the first, the method is used to identify fimbrial or adhesion genes by random mutagenesis of a pathogenic strain followed by screening (if possible, selecting) for nonfimbriated or nonadhesive mutants. This approach is then followed by cloning and further analysis of the mutated locus. In a second type of study, a gene cluster of interest is already cloned on a multicopy number plasmid to be analyzed in Escherichia coli in more detail by region-directed mutagenesis, as discussed in the chapter. Mutated alleles can then be introduced into the original pathogenic strain by marker-exchange experiments. In both types of studies, Tn phoA has demonstrated advantages over other transposons, as it can be used to identify exported proteins. Tn phoA is particularly well suited for the study of bacterial fimbrial proteins and adhesins, because such proteins typically cross bacterial membranes to interact with host molecules.

Identification of a 33 kDa antigen associated with an adhesive and colonizing strain of Vibrio cholerae El Tor and its role in protection

Proteins from the cell-free lysates of the wild-type strain KB207 of Vibrio cholerae El Tor and the isogenic non-adhesive mutant CD11 were analysed by native and denaturing polyacrylamide gel electrophoresis. A protein of 33 kDa present in KB207 was absent from CD11. Antiserum to the surface antigens of KB207 was absorbed with CD11. Antibodies remaining in the serum after absorption reacted to KB207 but not to CD11 as judged by slide agglutination, double gel diffusion and dot blot ELISA. Antibodies in the absorbed serum inhibited adherence of KB207 to rabbit intestinal mucosa and colonization in an infant mice model. The 33 kDa protein was isolated from KB207 by immunoaffinity chromatography. Antibodies present in the absorbed serum were used as ligand. The 33 kDa antigen was immunogenic and conferred protection in the rabbit ileal loop model. Combined administration of 33 kDa protein and B-subunit of cholera toxin offered full protection.

Expression of the surface properties of the fibrillar Streptococcus salivarius HB and its adhesion deficient mutants grown in continuous culture under glucose limitation.

Streptococcus salivarius HB and four adhesion deficient mutants, HB-7, HB-V5, HB-V51 and HB-B, were grown in continuous culture in a defined medium under glucose limitation over a range of growth rates from 0.1 to 1.1 h-1. The ability to coaggregate with Veillonella parvula V1 cells and the ability to adhere to buccal epithelial cells did not alter with increasing growth rate. Cell surface hydrophobicity decreased markedly with increasing growth rate for the non-fibrillar non-adhesive mutant HB-B but not for the other four strains which all carry different combinations of fibril classes. The thickness of the ruthenium red staining layer (RRL) also varied with growth rate for strain HB-B, ranging from 19.5 +/- 3.8 nm at high growth rate to a minimum of 12.3 +/- 4.8 nm at low growth rate. Low cell surface hydrophobicity correlated with a thicker RRL for strain HB-B. Strains HB-V5 and HB-7 also showed a significant increase in RRL thickness at high growth rates although to a lesser degree than HB-B. SDS-PAGE revealed a large number of protein bands common to all strains at all growth rates, with the major common protein occurring at 15.6 kDa. Protein bands at 70, 56, 40.5 and 39 kDa appeared stronger at high growth rates than at low. A protein band at 82 kDa showed strongly only at low growth rates. Therefore, adhesion and coaggregation are not phenotypically variable with increasing growth rate but RRL thickness, hydrophobicity and cell surface proteins may be phenotypically variable depending on the strain.

Characterization of monoclonal antibodies specific for adhesion: isolation of an adhesin of Streptococcus sanguis FW213.

Monoclonal antibodies reactive to an adhesive strain of Streptococcus sanguis (FW213) and nonreactive to a nonadhesive mutant (JL7) were derived from the fusion of myeloma line X63Ag8.653 and spleen cells from BALB/c mice immunized with live S. sanguis cells. Five cell lines, belonging to subclasses of immunoglobulin G, produced monoclonal antibodies specifically directed against the adhesive strain. All five antibodies also failed to react with five additional, independently isolated, nonadhesive mutants. A spontaneous mutant of FW213 (VT508) that no longer reacted with monoclonal antibody F51 (MAbF51) was isolated by serial agglutination with the antibody. Langmuir adsorption isotherms of VT508 indicated that this mutant also had altered ability to adhere to saliva-coated hydroxyapatite further confirming the specificity of MAbF51 for adhesion. Electron microscopy revealed that VT508 had lost the peritrichous fimbriae associated with the adhesion of FW213. MAbF51 was used to purify the adhesin from lysozyme cell extracts by using an affinity column of MAbF51 linked to Sephacryl S1000. Purity was suggested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and crossed immunoelectrophoresis (CIEP). The adhesin had a molecular weight greater than 150,000 and was not denatured in sodium dodecyl sulfate reducing gels. Two peaks of near electrophoretic mobility were detected in CIEP when the purified material was run against polyclonal antibody to the whole cell. Tandem CIEP analysis and immunoprecipitation provided evidence that the two peaks represented the same antigen in two different forms.

Structural properties of fibrillar proteins isolated from the cell surface and cytoplasm of Streptococcus salivarius (K+) cells and nonadhesive mutants.

Most Streptococcus salivarius (K+) cells contain two protein antigens with different adhesive functions. The subcellular distribution and some structural properties of purified proteins were studied. Antigen B (AgB), a protein involved in interbacterial coaggregation with gram-negative bacteria, was present in the cell wall fraction only of the wild-type strain and was absent from the cells of a nonadhesive mutant. Antigen C (AgC), a glycoprotein involved in host-associated adhesive functions, was predominantly associated with the cell wall of the wild-type strain (AgCw), but accumulated in high amounts in the cytoplasmic fraction (AgCin) of mutants lacking the wall-associated form. AgB, AgCw, and AgCin had molecular weights of 380,000, 250,000 to 320,000, and 488,000, respectively, upon gel electrophoresis under nondenaturing conditions. In the presence of sodium dodecyl sulfate and beta-mercaptoethanol the molecular weights were only slightly lower, suggesting that the free, isolated molecules exist as monomers under native conditions. AgCin readily stained with periodate-Schiff reagent, indicating a significant content of carbohydrate, similar to AgCw. Circular dichroism spectra showed that about 45% of the amino acids of AgCw were involved in alpha-helical coiled structures. AgB had a significantly lower proportion of ordered coiled structure. Electron microscopic observations of low-angle-shadowed preparations of purified antigens showed that they were flexible, thin rods with thickened or globular ends. Measurements corrected for shadow thickness showed lengths of 184 nm (AgB), 112 nm (AgCin), and 87 nm (AgCw). Treatment of AgCw with protease destroyed the fibrillar core, but seemed not to affect the globular ends. Comparison of the results with the localization of the antigens in wild-type and specific mutant strains suggested that each antigen molecule may represent a single, characteristic surface fibril with a specific adhesive capacity.

Antibodies that bind to fimbriae block adhesion of Streptococcus sanguis to saliva-coated hydroxyapatite.

Antibodies raised against a fimbriated, adhesive strain of Streptococcus sanguis (FW213) were found to block the adhesion of this organism to saliva-coated hydroxyapatite. Antibodies were made specific for adhesion antigens by adsorption with isogenic, nonadhesive mutants (for rabbit polyclonal adsorbed antibody) or selection based on nonreactivity with two nonadhesive mutants (for monoclonal antibody). Rabbit antibody raised against isogenic, nonfimbriated nonadhesive mutants served as a control for antibodies present, but not related to fimbriation. Adsorbed antibody and monoclonal antibody were shown to be specific for fimbriae (antigen 1), since both antibodies could be seen by immune electron microscopy to bind 3.6-nm fimbriae, reacted only with the fimbriated parent and not the mutants in a whole bacterial cell enzyme-linked immunosorbent assay, and could immunoprecipitate fimbriae from fimbrial extracts of FW213. Antibodies isolated from preimmune and mutant sera did not react with fimbriae in any of the above assays. Only adsorbed antibody and monoclonal antibody were capable of blocking the adhesion of FW213 to saliva-coated hydroxyapatite. Adsorbed antibody, purified to immunoglobulin G (IgG), was an effective inhibitor of adhesion without causing interfering cellular aggregation. Monoclonal IgG, papain-cleaved to Fab fragments to prohibit cell-to-cell cross-linking, was also a potent inhibitor of S. sanguis FW213 adhesion. Both IgG from mutant sera and Fab fragments from normal mouse IgG could not be shown to block adhesion. These data further support the hypothesis that S. sanguis fimbriae are involved in adhesion.

Isolation of a non-adhesive mutant of Vibrio cholerae and chromosomal localization of the gene controlling mannose-sensitive adherence.

A mutant (CD11) of Vibrio cholerae was isolated after exposure of a wild-type pathogenic strain (KB207) to N-methyl-N'-nitro-N-nitrosoguanidine. Although the mutant was motile and chemotactic, it adhered poorly to rabbit intestinal mucosa. Pretreatment of vibrios with 10 mg D-mannose ml-1 and of intestinal mucosa with 100 mmol sodium metaperiodate inhibited adherence of KB207 but not of CD11. The gene (ams) controlling mannose-sensitive adherence was mapped on the bacterial chromosome closely linked to the pur locus.