Inability of encapsulated Klebsiella pneumoniae to assemble functional type 1 fimbriae on their surface.

We screened phase variants of Klebsiella pneumoniae isolates for the expression of capsule and type 1 fimbriae and found that all of the 22 blood isolates were encapsulated and did not express type 1 fimbriae while 10 of 11 urinary tract isolates expressed type 1 fimbriae but were unencapsulated. Phase variants from selected isolates were found to be either unencapsulated and fimbriated or lacked both structures. Variants expressing both structures were not detected. Fimbrial subunits FimH and FimA were localized in the periplasmic space of the parent strain and on the surface of the unencapsulated variants. The results suggest that capsule formation impedes assembly of pre-formed fimbrial subunits on the bacterial surface.

The mast cell tumor necrosis factor alpha response to FimH-expressing Escherichia coli is mediated by the glycosylphosphatidylinositol-anchored molecule CD48.

Mast cells are well known for their harmful role in IgE-mediated hypersensitivity reactions, but their physiological role remains a mystery. Several recent studies have reported that mast cells play a critical role in innate immunity in mice by releasing tumor necrosis factor alpha (TNF-alpha) to recruit neutrophils to sites of enterobacterial infection. In some cases, the mast cell TNF-alpha response was triggered when these cells directly bound FimH on the surface of Escherichia coli. We have identified CD48, a glycosylphosphatidylinositol-anchored molecule, to be the complementary FimH-binding moiety in rodent mast cell membrane fractions. We showed that (i) pretreatment of mast cell membranes with antibodies to CD48 or phospholipase C inhibited binding of FimH+ E. coli, (ii) FimH+ E. coli but not a FimH- derivative bound isolated CD48 in a mannose-inhibitable manner, (iii) binding of FimH+ bacteria to Chinese hamster ovary (CHO) cells was markedly increased when these cells were transfected with CD48 cDNA, and (iv) antibodies to CD48 specifically blocked the mast cell TNF-alpha response to FimH+ E. coli. Thus, CD48 is a functionally relevant microbial receptor on mast cells that plays a role in triggering inflammation.

Molecular basis for the enterocyte tropism exhibited by Salmonella typhimurium type 1 fimbriae.

Salmonella typhimurium exhibits a distinct tropism for mouse enterocytes that is linked to their expression of type 1 fimbriae. The distinct binding traits of Salmonella type 1 fimbriae is also reflected in their binding to selected mannosylated proteins and in their ability to promote secondary bacterial aggregation on enterocyte surfaces. The determinant of binding in Salmonella type 1 fimbriae is a 35-kDa structurally distinct fimbrial subunit, FimHS, because inactivation of fimHS abolished binding activity in the resulting mutant without any apparent effect on fimbrial expression. Surprisingly, when expressed in the absence of other fimbrial components and as a translational fusion protein with MalE, FimHS failed to demonstrate any specific binding tropism and bound equally to all cells and mannosylated proteins tested. To determine if the binding specificity of Salmonella type 1 fimbriae was determined by the fimbrial shaft that is intimately associated with FimHS, we replaced the amino-terminal half of FimHS with the corresponding sequence from Escherichia coli FimH (FimHE) that contains the receptor binding domain of FimHE. The resulting hybrid fimbriae bearing FimHES on a Salmonella fimbrial shaft exhibited binding traits that resembled that of Salmonella rather than E. coli fimbriae. Apparently, the quaternary constraints imposed by the fimbrial shaft on the adhesin determine the distinct binding traits of S. typhimurium type 1 fimbriae.

Localization of a domain in the FimH adhesin of Escherichia coli type 1 fimbriae capable of receptor recognition and use of a domain-specific antibody to confer protection against experimental urinary tract infection.

The FimH subunit of type 1-fimbriated Escherichia coli has been implicated as an important determinant of bacterial adherence and colonization of the urinary tract. Here, we sought to localize the functionally important domain(s) within the FimH molecule and to determine if antibodies against this domain would block adherence of type 1-fimbriated E. coli to the bladder mucosa in situ and in vivo in an established mouse model of cystitis. We generated translational fusion proteins of disparate regions of the FimH molecule with an affinity tag MalE, and tested each of the fusion products in vitro for functional activity. The minimum region responsible for binding mouse bladder epithelial cells and a soluble mannoprotein, horseradish peroxidase, was contained within residues 1-100 of the FimH molecule. We validated and extended these findings by demonstrating that antibodies directed at the putative binding region of FimH or at synthetic peptides corresponding to epitopes within the binding domain could specifically block type 1 fimbriae-mediated bacterial adherence to bladder epithelial cells in situ and yeast cells in vitro. Next, we compared the ability of mice passively immunized intraperitoneally with antisera raised against residues 1-25 and 253-264 of FimH or 1-13 of FimA to resist bladder colonization in vivo after intravesicular challenge with type 1-fimbriated E. coli. Only the antibody directed at the putative binding region of FimH (anti- s-FimH1-25) significantly reduced E. coli bladder infections in the experimental mouse model of urinary tract infections. Similar results were obtained when the mice were actively immunized with synthetic peptides corresponding to residues 1-25 and 253-264 of FimH or 1-13 of FimA. The mechanism of protection was attributed, at least in part, to inhibition of bacterial adherence to the bladder surface by s-FimH1-25-specific antibody molecules that had filtered through the kidneys into the urine. The level of FimH antibodies entering the bladder from the circulatory system of the immunized mice was found to be markedly enhanced upon bacterial challenge. The potential broad spectrum activity of the protective FimH antibody was indicated from its serologic cross-reactivity with various urinary tract bacterial isolates bearing type 1 fimbriae. These findings could be relevant in the design of an efficacious and broadly reactive FimH vaccine against urinary tract infections.

Mast cells as modulators of host defense in the lung.

Mast cells display a distinct spatial distribution in the lung where they are found preferentially in intraepithelial locations or in deeper tissue around blood vessels, bronchioles and mucus secreting glands. Yet the physiological role of these granule-laden cells is unknown. There are now intriguing signs that their distinctive distribution together with their intrinsic capacity to release large amounts of inflammatory mediators serve a critical role in immune surveillance. Mast cells have now been shown to be capable of recognizing and aggressively reacting to a wide range of bacteria. The mast cell responses involve ingesting and killing of adherent bacteria, in a manner not unlike that of traditional phagocytic cells. Concomitant with this endocytic activity, a large variety of potent inflammatory mediators are released by the mast cell. One such mast cell-derived mediator, TNF-alpha, was recently shown to be a critical signal for initiating neutrophil influx to sites of bacterial infection in the lung as well as the peritoneum of mice. This capacity of mast cells to recruit neutrophils, together with its recently reported participation in processing and presenting bacterial antigens to immune cells and in mediating proliferation of epithelial cells and mucosal mucus secretion, indicate that mast cells have an extraordinary ability to modulate the innate as well as adaptive immune responses to infectious microorganisms.