Commonly used antibiotics induce expression of Hsp 27 and Hsp 60 and protect human lymphocytes from apoptosis.

Heat shock proteins (Hsps) are abundant molecular chaperones participating in the cytoprotection. The kinetics of synthesis of Hsps closely correlates with the kinetics of development of resistance to cell death. In this study, we analysed the probable involvement of Hsp 27 and Hsp 60 in the protection of cells undergoing apoptosis. Human lymphocytes cultured in the presence of ampicillin or ceftriaxone produced Hsp 60 and Hsp 27, estimated by immunoblotting in a time-dependent manner and the increased levels of Hsp 60 and Hsp 27 correlated with enhanced resistance of the lymphocytes to apoptosis, as determined by flow cytometry. Cultures treated with ampicillin or ceftriaxone also exhibited smaller numbers of apoptotic cells than untreated cultures when exposed to the apoptosis-inducing agent staurosporine (1 mM). In contrast, cloramphenicol induced the production of only small amounts of Hsp 60, and no resistance apoptosis. Further studies are needed to clarify the potential role of Hsp 27 and Hsp 60 in the resistance of human cells to apoptosis and the effects of antibiotics on this phenomenon.

Chlamydia pneumoniae infections prevent the programmed cell death on THP-1 cell line.

Chlamydia pneumoniae is an obligate intracellular bacterium which frequently causes airway infection in humans and has been implicated in chronic inflammatory disease and atherosclerosis. Here we show that infection with C. pneumoniae protects THP-1 cells against the apoptosis which spontaneously occurs in macrophages in the absence of an activation signal. Analysis by flow cytometry at different post-infection times revealed that 50+/-7% of THP-1 cells were apoptotic at 48 h after onset of the experiments, whereas C. pneumoniae-infected cultures (multiplicity of infection, MOI=30) displayed only 18+/-4% of cells in apoptosis. At MOI=20 and MOI=10 the cells susceptible to apoptosis at 48 h were 28+/-5% and 35+/-6% respectively. Moreover, the results show that heat-inactivated bacteria do not give significant protection against apoptosis, even at higher MOI (MOI=30), while UV-treated Chlamydia did provide a degree of protection against apoptosis. These data suggest that the anti-apoptotic effect of C. pneumoniae requires a heat-labile component released during infection, and that the effect is not lipopolysaccharide-dependent.

Cytokine induction in murine bladder tissue by type 1 fimbriated Escherichia coli.

The local cytokine response to uropathogenic phenotype Escherichia coli KBC211 infection exhibits characteristics of both TH1 and TH2 profiles. Interleukin (IL)-6, MIP-2, IL-12, IL-18, and tumor necrosis factor-alpha (TNF-alpha) are expressed, but IL-4, IL-5, and IL-10 are also present at low levels. This is clearly a complex response that should be explored more fully. The relative contributions of the bladder epithelium and other cells of the bladder wall should also be determined. Epithelial cytokine responses may be considerable, and because these cells are the first to encounter the pathogen, they will be of great importance in the immune response to pathogenic E. coli.

p53 and anti-p53 antibodies as possible markers of a switch towards a neoplastic phenotype in patients infected by Helicobacter pylori.

Helicobacter pylori is a definite carcinogen whose mechanism of action is still unknown. The aim of this work was (1) to determine the presence of p53 protein and related antibodies in patients affected by various gastric pathologies and chronically infected with H. pylori, and (2) to try to discover a test to be used as a marker of a possible switch towards a neoplastic phenotype.

Valency conversion in the type 1 fimbrial adhesin of Escherichia coli.

FimH protein is a lectin-like adhesive subunit of type 1, or mannose-sensitive, fimbriae that are found on the surface of most Escherichia coli strains. All naturally occurring FimH variants demonstrate a conserved mannotriose-specific (i.e. multivalent) binding. Here, we demonstrate that replacement of residues 185-279 within the FimH pilin domain with a corresponding segment of the type 1C fimbrial adhesin FocH leads to a loss of the multivalent mannotriose-specific binding property accompanied by the acquisition of a distinct monomannose-specific (i.e. monovalent) binding capability. Bacteria expressing the monovalent hybrid adhesins were capable of binding strongly to uroepithelial tissue culture cells and guinea pig erythrocytes. They could not, however, agglutinate yeast or bind human buccal cells -- functions readily accomplished by the E. coli-expressing mannotriose-specific FimH variants. Based on the relative potency of inhibiting compounds of different structures, the receptor binding site within monovalent FimH-FocH adhesin has an extended structure with an overall configuration similar to that within the multivalent FimH of natural origin. The monomannose-only specific phenotype could also be invoked by a single point mutation, E89K, located within the lectin domain of FimH, but distant from the receptor binding site. The structural alterations influence the receptor-binding valency of the FimH adhesin via distal effects on the combining pocket, obviously by affecting the FimH quaternary structure.

Spa contributes to the virulence of type 18 group A streptococci.

Streptococcal protective antigen (Spa) is a newly described surface protein of group A streptococci that was recently shown to evoke protective antibodies (J. B. Dale, E. Y. Chiang, S. Liu, H. S. Courtney, and D. L. Hasty, J. Clin. Investig. 103:1261--1268, 1999). In this study, we have determined the complete sequence of the spa gene from type 18 streptococci. Purified, recombinant Spa protein evoked antibodies that were bactericidal against type 18 streptococci, confirming the presence of protective epitopes. Sera from patients with acute rheumatic fever contained antibodies against recombinant Spa, indicating that the Spa protein is expressed in vivo and is immunogenic in humans. To determine the role of Spa in the virulence of group A streptococci, we created a series of insertional mutants that were (i) Spa negative and M18 positive, (ii) Spa positive and M18 negative, and (iii) Spa negative and M18 negative. The mutants and the parent M18 strain (18-282) were used in assays to determine resistance to phagocytosis, growth in human blood, and mouse virulence. The results show that Spa is a virulence determinant of group A streptococci and that expression of both Spa and M18 is required for optimal virulence of type 18 streptococci.

Tamm-Horsfall protein binds to type 1 fimbriated Escherichia coli and prevents E. coli from binding to uroplakin Ia and Ib receptors.

The adherence of uropathogenic Escherichia coli to the urothelial surface, a critical first step in the pathogenesis of urinary tract infection (UTI), is controlled by three key elements: E. coli adhesins, host receptors, and host defense mechanisms. Although much has been learned about E. coli adhesins and their urothelial receptors, little is known about the role of host defense in the adherence process. Here we show that Tamm-Horsfall protein (THP) is the principal urinary protein that binds specifically to type 1 fimbriated E. coli, the main cause of UTI. The binding was highly specific and saturable and could be inhibited by d-mannose and abolished by endoglycosidase H treatment of THP, suggesting that the binding is mediated by the high-mannose moieties of THP. It is species-conserved, occurring in both human and mouse THPs. In addition, the binding to THP was much greater with an E. coli strain bearing a phenotypic variant of the type 1 fimbrial FimH adhesin characteristic of those prevalent in UTI isolates compared with the one prevalent in isolates from the large intestine of healthy individuals. Finally, a physiological concentration of THP completely abolished the binding of type 1 fimbriated E. coli to uroplakins Ia and Ib, two putative urothelial receptors for type 1 fimbriae. These results establish, on a functional level, that THP contains conserved high-mannose moieties capable of specific interaction with type 1 fimbriae and strongly suggest that this major urinary glycoprotein is a key urinary anti-adherence factor serving to prevent type 1 fimbriated E. coli from binding to the urothelial receptors.

Pathoadaptive mutations: gene loss and variation in bacterial pathogens.

Pathogenicity-adaptive, or pathoadaptive, mutations represent a genetic mechanism for enhancing bacterial virulence without horizontal transfer of specific virulence factors. Pathoadaptive evolution can be important within single infections and for defining the population structure of a pathogenic species.

New protective antigen of group A streptococci.

It is widely believed that the surface M protein of group A streptococci is the predominant surface protein of these organisms containing opsonic epitopes. In the present study, we identified a new surface protein, distinct from M protein, that evokes protective antibodies. A type 18 M-negative mutant was found to be both resistant to phagocytosis in human blood and virulent in mice. The wild-type strain, but not the M-negative mutant, was opsonized by antisera against purified recombinant M18 protein or a synthetic peptide copying the NH2-terminus of M18. However, antisera raised against a crude pepsin extract of the M-negative mutant opsonized both strains, indicating the presence of a protective antigen in addition to type 18 M protein. This antiserum was used to identify and purify a 24-kDa protein fragment (Spa, streptococcal protective antigen) that evoked antibodies that opsonized the M18 parent and the M-negative mutant. The results of passive mouse protection tests confirmed the presence of protective epitopes within Spa. The deduced amino acid sequence of a 636-bp 5' fragment of the spa18 gene showed no homology with sequences in GenBank. These studies reveal the presence of a new protective antigen of certain strains of group A streptococci that may prove to be an important component of vaccines to prevent streptococcal infections.

Serum opacity factor is a major fibronectin-binding protein and a virulence determinant of M type 2 Streptococcus pyogenes.

Serum opacity factor (SOF) is a fibronectin-binding protein of group A streptococci that opacifies mammalian sera and is expressed by some strains that cause impetigo, pharyngitis and acute glomerulonephritis. Although SOF is expressed by approximately 35% of known serotypes, its role in the pathogenesis of group A streptococcal infections has not been previously investigated. The sof genes from M types 2, 28 and 49 Streptococcus pyogenes were cloned, sequenced, and their deduced amino acid sequences were compared. The gene for FnBA, a fibronectin-binding protein from Streptococcus dysgalactiae, was also cloned and found to express an opacity factor. The leader sequences, the fibronectin-binding domains, and the membrane anchor regions of these proteins were highly conserved. Short spans of conserved sequences were interspersed throughout the remaining parts of the proteins. The sof2 gene was insertionally inactivated in an M type 2 S. pyogenes strain, T2MR. The resultant SOF-negative mutant (YL3) did not express SOF or opacify serum, and exhibited a 71% reduction in binding fibronectin. Complementation of the SOF-negative defect with sof28 in the recombinant strain YL3(pNZ28) fully restored fibronectin-binding activity and the ability to opacify serum. To determine whether sof plays a role in virulence, mice were challenged intraperitoneally with these strains. None of the 10 mice infected with YL3(pNZ28) survived and only 1 out of 15 mice challenged with T2MR survived, whereas 12 out of 15 mice infected with YL3 survived. These data clearly indicate that SOF is a virulence factor, and they provide the first direct evidence that a fibronectin-binding protein contributes to the pathogenesis of group A streptococcal infections in vivo.

Pathogenic adaptation of Escherichia coli by natural variation of the FimH adhesin.

Conventional wisdom regarding mechanisms of bacterial pathogenesis holds that pathogens arise by external acquisition of distinct virulence factors, whereas determinants shared by pathogens and commensals are considered to be functionally equivalent and have been ignored as genes that could become adapted specifically for virulence. It is shown here, however, that genetic variation in an originally commensal trait, the FimH lectin of type 1 fimbriae, can change the tropism of Escherichia coli, shifting it toward a urovirulent phenotype. Random point mutations in fimH genes that increase binding of the adhesin to mono-mannose residues, structures abundant in the oligosaccharide moieties of urothelial glycoproteins, confer increased virulence in the mouse urinary tract. These mutant FimH variants, however, are characterized by increased sensitivity to soluble inhibitors bathing the oropharyngeal mucosa, the physiological portal of E. coli. This functional trade-off seems to be detrimental for the intestinal ecology of the urovirulent E. coli. Thus, bacterial virulence can be increased by random functional mutations in a commensal trait that are adaptive for a pathologic environment, even at the cost of reduced physiological fitness in the nonpathologic habitat.

Diversity of the Escherichia coli type 1 fimbrial lectin. Differential binding to mannosides and uroepithelial cells.

Type 1 fimbriae are the most common adhesive organelles of Escherichia coli. Because of their virtual ubiquity, previous epidemiological studies have not found a correlation between the presence of type 1 fimbriae and urinary tract infections (UTIs). Recently it has become clear that type 1 fimbriae exhibit several different phenotypes, due to allelic variation of the gene for the lectin subunit, FimH, and that these phenotypes are differentially distributed among fecal and UTI isolates. In this study, we have analyzed in more detail the ability of isogenic, recombinant strains of E. coli expressing fimH genes of the predominant fecal and UTI phenotypes to adhere to glycoproteins and to uroepithelial cells. Evidence was obtained to indicate that type 1 fimbriae differ in their ability to recognize various mannosides, utilizing at least two different mechanisms. All FimH subunits studied to date are capable of mediating adhesion via trimannosyl residues, but only certain variants are capable of mediating high levels of adhesion via monomannosyl residues. The ability of the FimH lectins to interact with monomannosyl residues strongly correlates with their ability to mediate E. coli adhesion to uroepithelial cells. In this way, it would be possible for certain phenotypic variants of type 1 fimbriae to contribute more than others to virulence of E. coli in the urinary tract.

Conversion of M serotype 24 of Streptococcus pyogenes to M serotypes 5 and 18: effect on resistance to phagocytosis and adhesion to host cells.

In this study, we utilized recombinant strains expressing the M5 and M18 proteins and M- mutants of group A streptococci to compare the abilities of these M proteins to confer resistance to phagocytosis and to mediate adhesion to host cells. The data indicate that the M5 and M18 proteins can confer resistance to phagocytosis, that fibrinogen is required for this resistance, and that these M proteins can mediate adhesion to HEp-2 cells.

M protein mediated adhesion of M type 24 Streptococcus pyogenes stimulates release of interleukin-6 by HEp-2 tissue culture cells.

We investigated the contributions of lipoteichoic acid and M protein to reversible and irreversible adhesion of group A streptococci and the effects of such adhesion on release of interleukin-6. Streptococci in which lipoteichoic acid was masked by the hyaluronate capsule were readily washed from HEp-2 cells, indicating no attachment. Unencapsulated, M-negative streptococci in which lipoteichoic acid was exposed were removed more slowly, indicating loose attachment. Only unencapsulated streptococci that expressed both lipoteichoic acid and M protein remained stably adherent to HEp-2 cells throughout multiple washes. Streptococci expressing both M protein and lipoteichoic acid induced release of interleukin-6 from HEp-2 cells, whereas an isogenic, M-negative mutant failed to induce release of interleukin-6. These data suggest that lipoteichoic acid mediates reversible adhesion and that M protein is required for irreversible adhesion and for inducing release of interleukin-6 from HEp-2 cells.

Linker insertion analysis of the FimH adhesin of type 1 fimbriae in an Escherichia coli fimH-null background.

The gene encoding the Escherichia coli FimH adhesin of type 1 fimbriae has been subjected to linker insertion mutagenesis. Amino acid changes were introduced at a number of positions spanning the entire sequence in order to probe the structure-function relationship of the FimH protein. The effect of these mutations on the ability of bacteria to express a D-mannose binding phenotype was assessed in a fimH null mutant (MS4) constructed by allelic exchange in the E. coli K-12 strain PC31. Mutations mapping at amino acid residues 36, 58 and 279 of the mature FimH protein were shown to completely abolish binding to D-mannose receptors. Differences in the level of fimbriation were also observed as a result of some of the mutations in the fimH gene. These mutants may prove useful in dissecting receptor-ligand interactions by defining regions of the FimH protein that are important in erythrocyte binding.