Identification of the genetic determinants of Salmonella enterica serotype Typhimurium that may regulate the expression of the type 1 fimbriae in response to solid agar and static broth culture conditions

Background

Type 1 fimbriae are the most commonly found fimbrial appendages on the outer membrane of Salmonella enterica serotype Typhimurium. Previous investigations indicate that static broth culture favours S. Typhimurium to produce type 1 fimbriae, while non-fimbriate bacteria are obtained by growth on solid agar media. The phenotypic expression of type 1 fimbriae in S. Typhimurium is the result of the interaction and cooperation of several genes in the fim gene cluster. Other gene products that may also participate in the regulation of type 1 fimbrial expression remain uncharacterized.

Results

In the present study, transposon insertion mutagenesis was performed on S. Typhimurium to generate a library to screen for those mutants that would exhibit different type 1 fimbrial phenotypes than the parental strain. Eight-two mutants were obtained from 7,239 clones screened using the yeast agglutination test. Forty-four mutants produced type 1 fimbriae on both solid agar and static broth media, while none of the other 38 mutants formed type 1 fimbriae in either culture condition. The flanking sequences of the transposons from 54 mutants were cloned and sequenced. These mutants can be classified according to the functions or putative functions of the open reading frames disrupted by the transposon. Our current results indicate that the genetic determinants such as those involved in the fimbrial biogenesis and regulation, global regulators, transporter proteins, prophage-derived proteins, and enzymes of different functions, to name a few, may play a role in the regulation of type 1 fimbrial expression in response to solid agar and static broth culture conditions. A complementation test revealed that transforming a recombinant plasmid possessing the coding sequence of a NAD(P)H-flavin reductase gene ubiB restored an ubiB mutant to exhibit the type 1 fimbrial phenotype as its parental strain.

Conclusion

Genetic determinants other than the fim genes may involve in the regulation of type 1 fimbrial expression in S. Typhimurium. How each gene product may influence type 1 fimbrial expression is an interesting research topic which warrants further investigation.     

Genetic analysis of 987P adhesion and fimbriation of Escherichia coli: the fas genes link both phenotypes.

The 987P fimbrial gene cluster has recently been shown to contain eight genes (fasA to fasH) clustered on large plasmids of enterotoxigenic Escherichia coli and adjacent to a Tn1681-like transposon encoding the heat-stable enterotoxin STIa. Different genetic approaches were used to study the relationship between 987P fimbriation and adhesion. TnphoA mutagenesis, complementation assays, and T7 RNA polymerase-promoted gene expression indicated that all of the fas genes were involved in fimbrial expression and adhesion. In contrast to other fimbrial systems, the lack of expression of any single fas gene never resulted in the dissociation of fimbriation and adhesion, indicating that the adhesin is required for fimbrial expression and suggesting that FasA, the fimbrial structural subunit itself, is the adhesin. In addition, fimbrial length was shown to be modulated by the levels of expression of different fas genes.     

Characterization of a Bordetella pertussis fimbrial gene cluster which is located directly downstream of the filamentous haemagglutinin gene

The biosynthesis of fimbriae is a complex process requiring multiple genes which are generally found clustered on the chromosome. In Bordetella pertussis, only major fimbrial subunit genes have been identified, and no evidence has yet been found that they are located in a fimbrial gene cluster. To locate additional genes involved in the biosynthesis of B. pertussis fimbriae, we used TnphoA mutagenesis. A PhoA+ mutant (designated B176) was isolated which was affected in the production of both serotype 2 and 3 fimbriae. Cloning and sequencing of the DNA region harbouring the transposon insertion revealed the presence of at least three additional fimbrial genes, designated fimB, fimC and fimD. The transposon was found to be located in fimD. Analysis of PhoA activity indicated that the fimbrial gene cluster was positively regulated by the bvg locus. A potential binding site for BvgA was observed upstream of fimB. FimB showed homology with the so-called chaperone-like fimbrial proteins, while FimC was homologous with a class of fimbrial proteins located in the outer membrane and presumed to be involved in transport and anchorage of fimbrial subunits. An insertion mutation in fimB abolished the expression of fimbrial subunits, implicating this gene in the biosynthesis of both serotype 2 and 3 fimbriae. Upstream of fimB a pseudogene (fimA) was observed which showed homology with the three major fimbrial subunit genes, fim2, fim3 and fimX. The construction of a phylogenetic tree suggested that fimA may be the primordial major fimbrial subunit gene from which the other three were derived by gene duplication. Interestingly, the fimbrial gene cluster was found to be located directly downstream from the gene coding for the filamentous haemagglutinin, an important B. pertussis adhesin, possibly suggesting co-operation between the two loci in the pathogenesis of pertussis.     

Type V collagen as the target for type-3 fimbriae, enterobacterial adherence organelles

Tissue-binding specificity of the type-3 fimbriae of pathogenic enteric bacteria was determined using frozen sections of human kidney. A wild-type Klebsiella sp. strain and the recombinant strain Escherichia coli HB101(pFK12), both expressing type-3 fimbriae, as well as the purified type-3 fimbriae effectively bound to sites at or adjacent to tubular basement membranes, Bowman's capsule, arterial walls, and the interstitial connective tissue. Bacterial adherence to kidney was decreased after collagenase treatment of the tissue sections. Recombinant strains expressing type-3 fimbriae specifically adhered to type V collagen immobilized on glass slides, whereas other collagens, fibronectin or laminin did not support bacterial adherence. In accordance with these findings, specific binding of purified type-3 fimbriae to immobilized type V collagen was demonstrated. Specific adhesion to type V collagen was also seen with the recombinant strain HB101(pFK52/pDC17), which expresses the mrkD gene of the type-3 fimbrial gene cluster in association with the pap-encoded fimbrial filament of E. coli, showing that the observed binding was mediated by the minor lectin (MrkD) protein of the type-3 fimbrial filament. The interaction is highly dependent on the conformation of type V collagen molecules since type V collagen in solution did not react with the fimbriae. Specific binding to type V collagen was also exhibited by type-3 fimbriate strains of Yersinia and Salmonella, showing that the ability to use type V collagen as tissue target is widespread among enteric bacteria.     

Fimbrial biogenesis genes of Pseudomonas aeruginosa: pilW and pilX increase the similarity of type 4 fimbriae to the GSP protein-secretion systems and pilY1 encodes a gonococcal PilC homologue

Type 4 fimbriae of Pseudomonas aeruginosa are surface filaments involved in host colonization. They mediate both attachment to host epithelial cells and flagella-independent twitching motility. Four additional genes, pilW, pilX, pilY1 and pilY2, are located on Spel fragment E in the 5 kb intergenic region between the previously characterized genes pilV and pilE, which encode prepilin-like proteins involved in type 4 fimbrial biogenesis. The phenotypes of a transposon insertion and other mutations constructed by allelic exchange show that these genes are involved in the assembly of type 4 fimbriae. The PilW and PilX proteins are membrane located, possess the hydrophobic N-terminus characteristic of prepilin-like proteins, and appear to belong to the GspJ and GspK group of proteins that are required for protein secretion in a wide range of Gram-negative bacteria. These findings increase the similarities between the fimbrial biogenesis and the Gsp-based protein-secretion super-systems. PilY1 is a large protein with C-terminal homology to the PilC2 protein of Neisseria gonorrhoeae, thought to be a fimbrial tip-associated adhesin, and which, like PilY1, is involved in fimbrial assembly. PilY1 appears to be located in both the membrane and the external fimbrial fractions. PilY2 is a small protein that appears to play a subtle role In fimbrial biogenesis and represents a new class of protein.     

Structure of the Bordetella pertussis gene coding for the serotype 3 fimbrial subunit

Bordetella pertussis strains contain at least three distinct genes coding for fimbrial subunits, designated fim2, fim3, and fimX. The sequences of the fim2 and fimX genes have been published. Here we present the sequence of the fim3 gene. Proximal and distal to the fim3 gene, regions were observed that could function as rho-independent terminators, suggesting that the gene is not part of a larger operon. Comparison of the putative promoter regions of the fim2 and fim3 genes revealed a conserved region containing a stretch of approximately 13 C's. This region may be involved in fimbrial phase variation. A comparison of the deduced amino acid sequences of the three fimbrial subunits revealed conserved, variable, and hypervariable regions. The hypervariable regions coincided with predicted antigenic determinants. Peptides derived from the conserved regions may be incorporated into a future pertussis vaccine to induce antibodies which confer protection against strains producing different fimbrial serotypes.     

complex interplay between type 1 fimbrial expression and flagellum-mediated motility of uropathogenic Escherichia coli

Type 1 fimbriae and flagella have been previously shown to contribute to the virulence of uropathogenic Escherichia coli (UPEC) within the urinary tract. In this study, the relationship between motility and type 1 fimbrial expression was tested for UPEC strain CFT073 by examining the phenotypic effect of fimbrial expression on motility and the effect that induction of motility has on type 1 fimbrial expression. While constitutive expression of type 1 fimbriae resulted in a significant decrease in motility and flagellin expression (P < 0.0001), a loss of type 1 fimbrial expression did not result in increased motility. Additionally, hypermotility and flagellar gene over- and underexpression were not observed to affect the expression of type 1 fimbriae. Hence, it appeared that the relationship between type 1 fimbrial expression and motility is unidirectional, where the overexpression of type 1 fimbriae dramatically affects motility and flagellum expression but not vice versa. Moreover, the constitutive expression of type 1 fimbriae in UPEC cystitis isolate F11 and the laboratory strain E. coli K-12 MG1655 also resulted in decreased motility, suggesting that this phenomenon is not specific to CFT073 or UPEC in general. Lastly, by analyzing the repression of motility caused by constitutive type 1 fimbrial expression, it was concluded that the synthesis and presence of type 1 fimbriae at the bacterial surface is only partially responsible for the repression of motility, as evidenced by the partial restoration of motility in the CFT073 fim L-ON DeltafimAICDFGH mutant. Altogether, these data provide further insight into the complex interplay between type 1 fimbrial expression and flagellum-mediated motility.     

Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract

Escherichia coli is the leading cause of urinary tract infections (UTIs). Despite the association of numerous bacterial factors with uropathogenic E. coli (UPEC), few such factors have been proved to be required for UTI in animal models. Previous investigations of urovirulence factors have relied on prior identification of phenotypic characteristics. We used signature-tagged mutagenesis (STM) in an unbiased effort to identify genes that are essential for UPEC survival within the murine urinary tract. A library of 2049 transposon mutants of the prototypic UPEC strain CFT073 was constructed using mini-Tn5km2 carrying 92 unique tags and screened in a murine model of ascending UTI. After initial screening followed by confirmation in co-infection experiments, 19 survival-defective mutants were identified. These mutants were recovered in numbers 101- to 106-fold less than the wild type in the bladder, kidneys or urine or at more than one site. The transposon junctions from each attenuated mutant were sequenced and analysed. Mutations were found in: (i) the type 1 fimbrial operon; (ii) genes involved in the biosyn-thesis of extracellular polysaccharides including group I capsule, group II capsule and enterobacterial common antigen; (iii) genes involved in metabolic pathways; and (iv) genes with unknown function. Five of the genes identified are absent from the genome of the E. coli K-12 strain. Mutations in type 1 fimbrial genes resulted in severely attenuated colonization, even in the case of a mutant with an insertion upstream of the fim operon that affected the rate of fimbrial switching from the 'off' to the 'on' phase. Three mutants had insertions in a new type II capsule biosynthesis locus on a pathogenicity island and were impaired in the production of capsule in vivo. An additional mutant with an insertion in wecE was unable to synthesize enterobacterial common antigen. These results confirm the pre-eminence of type 1 fimbriae, establish the importance of extracellular polysaccharides in the pathogenesis of UTI and identify new urovirulence determinants.     

Identification and characterization of precursors in the biosynthesis of the K88ab fimbria of Escherichia coli

Four genes encoding for polypeptides with apparent molecular weights of 17,000, 26,000 (the fimbrial subunit), 27,000, and 81,000 have been implicated in the biosynthesis of the K88ab fimbria (Mooi et al., J. Bacteriol. 150:512-521, 1982). Escherichia coli mutants with defects in these genes were examined for the presence of fimbrial precursors. An analysis of these mutants revealed that fimbrial subunits accumulated transiently in the periplasmic space before being translocated across the outer membrane. The 81,000-dalton (d) polypeptide is probably involved in translocating fimbrial subunits across the outer membrane, because in the absence of this polypeptide the fimbrial subunits remained in the periplasmic space, where they were found to be associated with the 17,000- and 27,000-d polypeptides. In mutants with a deletion in the gene for the 27,000-d polypeptide, fimbrial precursors were not detected, because the fimbrial subunits were degraded. The 27,000-d polypeptide might be involved in stabilizing a conformation of the fimbrial subunit required to translocate it across the outer membrane. In the absence of the 17,000-d polypeptide, most fimbrial subunits were found in the periplasmic space associated with the 27,000-d polypeptide. However, small amounts of subunits were also translocated across the outer membrane. These extracellular subunits did not adhere to brushborders, suggesting that fimbrial subunits must be modified by the 17,000-d polypeptide to be assembled into functional fimbriae. A model for the biosynthesis of the K88ab fimbria is proposed.     

Identification of two genes with prepilin-like leader sequences involved in type 4 fimbrial biogenesis in Pseudomonas aeruginosa.

Type 4 fimbriae are surface filaments produced by a range of bacterial pathogens for colonization of host epithelial surfaces. In Pseudomonas aeruginosa, they are involved in adhesion as well as in a form of surface translocation called twitching motility, and sensitivity to infection by fimbria-specific bacteriophage. Analysis of the 2.5-kb intergenic region between the previously defined pilR and pilV genes on P. aeruginosa genomic SpeI fragment E has identified three new genes, fimT, fimU, and dadA*. The predicted 18.5-kDa products of the fimT and fimU genes contain prepilin-like leader sequences, whereas the third gene, dadA*, encodes a protein similar to the D-amino acid dehydrogenase of Escherichia coli. Isogenic mutants constructed by allelic exchange demonstrated that the fimU gene was required for fimbrial biogenesis and twitching motility, whereas the fimT and dada* mutants retained wild-type phenotypes. However, overexpression of the fimT gene was found to be able to functionally replace the lack of a fimU gene product, suggesting a subtle role in fimbrial biogenesis. The identification of these proteins increases the similarity between type 4 fimbrial biogenesis and the supersystems involved in macromolecular traffic, such as extracellular protein secretion and DNA uptake, all of which now possess multiple protein species that possess prepilin-like leader sequences.