Search PubMed⌕ Search

Biomedical subjects

S Normark

Publications and source records attributed to S Normark.

At least 19 recordsLinked to original sources
PubMed2001

Dynamics of penicillin-susceptible clones in invasive pneumococcal disease.

In a 10-year period, 1987-1997, there was a >4-fold increase in the rate of pneumococcal bacteremia in Sweden. Invasive pneumococcal isolates (n=1136), which were obtained from 18 Swedish clinical microbiology laboratories from 1987 through 1997, and other national and international isolates were serotyped, and their clonal relationships were determined by molecular typing. The increase in invasive pneumococcal disease in Sweden during this period was associated particularly with an increase in isolates of serotypes 1 and 14. A 3-fold increase of type 14 was seen from 1987 through 1992, and a 10-fold increase of type 1 occurred from 1992 through 1997. One dominating penicillin-susceptible clone of type 14 was responsible for the increase of type 14 during the first 5 years. This clone also was found in Canada and the United States and was shown by multilocus sequence typing to correspond to a previously identified hyper-virulent clone. A novel penicillin-susceptible clone of type 1, which was not found among invasive isolates from 1987 or 1992, was responsible for the increase of serotype 1 during the last 5 years. These results illustrate the ability of virulent penicillin-susceptible pneumococcal clones to emerge and spread rapidly within a country.

Alleles↗
PubMed2001

Clinical isolates of Streptococcus pneumoniae that exhibit tolerance of vancomycin.

The ability of Streptococcus pneumoniae to escape lysis and killing by vancomycin, a property termed "tolerance," has recently been noted in a laboratory strain of the species. Vancomycin tolerance in clinical isolates represents a potential new health risk. We determined the prevalence of vancomycin and penicillin tolerance among 116 clinical isolates of pneumococci by monitoring lysis and viability after exposure to the respective antibiotic for 4 hours. Eight percent of the strains were tolerant to penicillin and 3% were tolerant to vancomycin. The 3 vancomycin-tolerant isolates also had a high ratio of minimum bactericidal concentration to minimum inhibitory concentration, in contrast to nontolerant strains. They were of serotype 9V and had reduced susceptibility to penicillin. Only 1 was also tolerant to penicillin. Growth rate and ability to divide were not affected in the 3 vancomycin-tolerant strains, and they all lysed with deoxycholate, which indicates autolysin production. Vancomycin tolerance among clinical isolates of pneumococci will necessitate tracking to determine the magnitude of the evolving health risk, since tolerance may contribute to treatment failure (in particular, cases of meningitis, in which bactericidal activity is critical for eradication) and since it may also be a favored background for acquisition of resistance of vancomycin.

Amino Acid Sequence↗
PubMed2001

Activation of inducible nitric oxide synthase/nitric oxide by curli fibers leads to a fall in blood pressure during systemic Escherichia coli infection in mice.

Septic shock, a major cause of death, is characterized by a pathophysiologic increased production of nitric oxide (NO), which leads to vasodilation and myocardial toxicity. Septic Escherichia coli frequently express proteinaceous curli fibers. In this study, curliated E. coli induced high levels of NO by directly inducing type 2 nitric oxide synthase (NOS2) both in vitro and in vivo. More severe hypotension and higher plasma nitrite/nitrate levels were seen in wild type mice systemically infected with curliated E. coli than in animals infected with E. coli mutants that lacked curli proteins. Blood pressure remained stable in NOS2-deficient mice with curliated bacteria. Increased heart rates, transient hypothermia, and loss of gross activity were seen in all mice, regardless of curli expression. Study results suggest that expression of curli fibers by E. coli activates the NO/NOS2 arm of the innate immune system, which leads to a significant fall in blood pressure.

Animals↗
PubMed2001

Induction of innate immune responses by Escherichia coli and purified lipopolysaccharide correlate with organ- and cell-specific expression of Toll-like receptors within the human urinary tract.

Mucosal epithelial linings function as physical barriers against microbes. In addition, they participate in the first line of host defence by production of a variety of proinflammatory mediators when exposed to microbes and microbial agents. Here, we use a human urinary tract infection model to demonstrate that organ- and cell-specific innate responses induced by lipopolysaccharides (LPS) present on Gram-negative bacteria correlates with the expression of Toll-like receptor 4 (TLR4). The presence of TLR4 on human bladder epithelial cells enables them to rapidly respond to bacterial infections in vitro and in vivo. In contrast, TLR4 is not expressed on human proximal tubule cells isolated from the renal cortex, which may explain the cortical localization of bacteria in pyelonephritis. TLR4-negative renal epithelial cells, A498, are non-responsive to purified LPS, however, they respond to viable bacteria via a mannose-sensitive attachment-mediated pathway. To identify LPS components recognised by bladder epithelial cells, a bacterial lipid A mutant and LPS of different chemotypes were tested. Full interleukin 8 induction required hexa-acylated lipid A and was decreased by between 50% and 70% in the presence of O-antigen. Taken together, we propose that multiple independent pathways, which are organ- and cell-specifically expressed, mediate bacterial recognition and determine the outcome of innate responses to infection.

Cell Line↗
PubMed2000

Germ-free and colonized mice generate the same products from enteric prodefensins.

The use of germ-free mice offers the possibility to study antibacterial components in a gut uncolonized by bacteria. We have developed a method to extract and high pressure liquid chromatography-fractionate the antibacterial factors present in the small intestine of a single mouse. By mass spectrometry and sequence analyses of fractions exhibiting antimicrobial activity, we identified and characterized the defensin region in germ-free mice as well as in colonized mice. Defensins made up around 15% of the total antibacterial activity both in germ-free and colonized mice. The intestine of germ-free mice exhibited the same set of mature enteric defensins (defensins 1, 2, 3, 4, and 6) as mice colonized by a normal microflora. Mature defensins are generated through processing of larger precursors by enzymatic removal of a signal peptide and a propiece. We found that all prodefensins were cleaved at a Ser/Ala-Leu bond, giving 34-residue propiece peptides and only trace amounts of the predicted 39-residue peptide. This first step must be followed by the removal of a residual peptide to render the mature defensins, indicating that the processing is more complex than previously anticipated. The same propieces were found in both germ-free and colonized mice, suggesting that the same processing operates independent of bacterial presence in the intestine.

Amino Acid Sequence↗
PubMed2000

Alpha-haemolysin of uropathogenic E. coli induces Ca2+ oscillations in renal epithelial cells.

Pyelonephritis is one of the most common febrile diseases in children. If not treated appropriately, it causes irreversible renal damage and accounts for a large proportion of end stage renal failures. Renal scarring can occur in the absence of inflammatory cells, indicating that bacteria may have a direct signalling effect on renal cells. Intracellular calcium ([Ca2+]i) oscillations can protect cells from the cytotoxic effects of prolonged increases in intracellular calcium. However, no pathophysiologically relevant protein that induces such oscillations has been identified. Here we show that infection by uropathogenic Escherichia coli induces a constant, low-frequency oscillatory [Ca2+]i response in target primary rat renal epithelial cells induced by the secreted RTX (repeats-in-toxin) toxin alpha-haemolysin. The response depends on calcium influx through L-type calcium channels as well as from internal stores gated by inositol triphosphate. Internal calcium oscillations induced by alpha-haemolysin in a renal epithelial cell line stimulated production of cytokines interleukin (IL)-6 and IL-8. Our findings indicate a novel role for alpha-haemolysin in pyelonephritis: as an inducer of an oscillating second messenger response in target cells, which fine-tunes gene expression during the inflammatory response.

Animals↗
PubMed2000

AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways.

The regulatory programme of multicellular behaviour in Salmonella typhimurium is determined by mutations in the agfD promoter. AgfD has already been identified to regulate the extracellular matrix associated with the multicellular morphotype composed of thin aggregative fimbriae (agf). To detect additional components contributing to the multicellular morphotype in S. typhimurium, we constructed a mutant in agfD, the positive transcriptional regulator of the agfBA(C) operon encoding for fimbrial subunit proteins. The agfD mutant lacked any form of multicellular behaviour as shown by analysis at the macroscopic and microscopic level. In contrast, the agfBA mutant unable to form thin aggregative fimbriae still maintained long-range intercellular adhesion. Promoter and expression analysis revealed that the genes downstream of agfD agfEFG most likely did not contribute to the remaining aggregative behaviour. Screening of transcriptional fusions for agfD dependency uncovered adrA, a homologue of yaiC in Escherichia coli. Environmental factors regulating adrA correspond to the regulation of thin aggregative fimbriae. AdrA is a putative transmembrane protein with a C-terminal GGDEF domain of unknown function although it is present in over 50 bacterial proteins. AdrA mutant cells, which still formed thin aggregative fimbriae with all binding characteristics, exhibited community behaviour but, unlike the wild type, lacked long-range intercellular adhesion. An agfBA adrA double mutant behaved like the agfD mutant. Therefore, it was concluded that agfD regulates at least two independent pathways contributing to the multicellular morphotype in S. typhimurium.

Amino Acid Motifs↗
PubMed2000

Expression of and cytokine activation by Escherichia coli curli fibers in human sepsis.

Curli organelles are expressed by commensal Escherichia coli K12 and by Salmonella typhimurium at temperatures <37 degrees C, which bind serum proteins and activate the contact-phase system in vitro. This study demonstrates, by means of an anti-CsgA (curli major subunit) antibody, that a significant fraction of E. coli isolates (24 of 46) from human blood cultures produce curli at 37 degrees C in vitro. Serum samples from 12 convalescent patients with sepsis, but not serum from healthy controls, contained antibodies against CsgA (n=12). This study further demonstrates that a curli-expressing E. coli strain and a noncurliated mutant secreting soluble CsgA induce significantly (P<.05) higher levels of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin [IL]-6, and IL-8) in human macrophages differentiated from THP-1 cells. These data, therefore, provide direct evidence that curli are expressed in vivo in human sepsis and suggest a possible role for curli and CsgA in the induction of proinflammatory cytokines during E. coli sepsis.

Adult↗
PubMed2000

Vaccination with FimH adhesin protects cynomolgus monkeys from colonization and infection by uropathogenic Escherichia coli.

Escherichia coli FimH adhesin mediates binding to the bladder mucosa. In mice, a FimH vaccine protects against bacterial challenge. In this study, 4 monkeys were inoculated with 100 microgram of FimCH adhesin-chaperone complex mixed with MF59 adjuvant, and 4 monkeys were given adjuvant only intramuscularly. After 2 doses (day 0 and week 4), a booster at 48 weeks elicited a strong IgG antibody response to FimH in the vaccinated monkeys. All 8 monkeys were challenged with 1 mL of 108 E. coli cystitis isolate NU14. Three of the 4 vaccinated monkeys were protected from bacteruria and pyuria; all control monkeys were infected. These findings suggest that a vaccine based on the FimH adhesin of E. coli type 1 pili may have utility in preventing cystitis in humans.

Adhesins, Bacterial↗
PubMed1999

Emergence of vancomycin tolerance in Streptococcus pneumoniae.

Streptococcus pneumoniae, the pneumococcus, is the most common cause of sepsis and meningitis. Multiple-antibiotic-resistant strains are widespread, and vancomycin is the antibiotic of last resort. Emergence of vancomycin resistance in this community-acquired bacterium would be catastrophic. Antibiotic tolerance, the ability of bacteria to survive but not grow in the presence of antibiotics, is a precursor phenotype to resistance. Here we show that loss of function of the VncS histidine kinase of a two-component sensor-regulator system in S. pneumoniae produced tolerance to vancomycin and other classes of antibiotic. Bacterial two-component systems monitor environmental parameters through a sensor histidine-kinase/phosphatase, which phosphorylates/dephosphorylates a response regulator that in turn mediates changes in gene expression. These results indicate that signal transduction is critical for the bactericidal activity of antibiotics. Experimental meningitis caused by the vncS mutant failed to respond to vancomycin. Clinical isolates tolerant to vancomycin were identified and DNA sequencing revealed nucleotide alterations in vncS. We conclude that broad antibiotic tolerance of S. pneumoniae has emerged in the community by a molecular mechanism that eliminates sensitivity to the current cornerstone of therapy, vancomycin.

Animals↗
PubMed1999

The origin of cecropins; implications from synthetic peptides derived from ribosomal protein L1.

We recently showed that Helicobacter pylori grown on plates produce cecropin-like antibacterial peptides to which H. pylori is resistant. This antibacterial activity was traced to fragments from the N-terminus of ribosomal protein L1 (Pütsep et al., Nature, April 22, 1999). The evolutionary suggestion from this finding has now been extended by the synthesis of eight peptides with sequences taken from the N-terminus of ribosomal protein L1 (RpL1) of five different species. Two peptides of different length derived from H. pylori RpL1 showed a potent antibacterial activity, while a peptide with the sequence from Escherichia coli was 20 times less active. Like cecropins the H. pylori peptides were not cytolytic. We suggest that the cecropins have evolved from ribosomal protein L1 of an ancestral intracellular pathogen that developed to a symbiont ending as an organelle. When the R1 gene moved into the host nucleus, a duplication provided a copy from which today cecropins could have evolved.

Amino Acid Sequence↗
PubMed1999

Signal transduction-mediated adherence and entry of Helicobacter pylori into cultured cells.

BACKGROUND & AIMS: An ability to invade host cells could be a means for Helicobacter pylori to achieve resistance to antibiotic therapy. The aim of this study was to investigate the mechanisms involved in adherence and entry of H. pylori into cultured cells. METHODS: Coinfection with Yersinia expressing mutant or wild-type YopH tyrosine phosphatase was used. Genistein and cytochalasin D were used as inhibitors of adherence and entry; entry was monitored by a gentamicin-protection assay. Target cells were AGS cells and a beta1-integrin-deficient cell line with its corresponding beta1-integrin-expressing transfectant. RESULTS: H. pylori induced phosphorylation of 125-130-kilodalton proteins, similar in size to the target proteins of Yersinia YopH. Adherence of H. pylori was inhibited by Yersinia organisms expressing enzymatically active YopH but not by inactive YopH. Adherence and entry of H. pylori was considerably higher with beta1-integrin-transfected cells than with beta1-integrin-deficient cells. Antibodies directed against alpha5- and beta1-integrin chains reduced adherence to the alpha5beta1-integrin-expressing gastric cell line AGS. Entry was inhibited by both cytochalasin D and genistein. Entry, but not adherence, was higher for 2 type I strains than for a type II isolate. CONCLUSIONS: Invasion of gastric epithelium via an integrin-mediated pathway could contribute to the ability of H. pylori to establish persistent infection.

Bacterial Adhesion↗
PubMed1999

Multiple insertions of fimbrial operons correlate with the evolution of Salmonella serovars responsible for human disease.

On centisome 7, Salmonella spp. contain a large region not present in the corresponding region of Escherichia coli. This region is flanked by sequences with significant homology to the E. coli tRNA gene aspV and the hypothetical E. coli open reading frame yafV. The locus consists of a mosaic of differentially acquired inserts forming a dynamic cs7 region of horizontally transferred inserts. Salmonella enterica subspecies I, responsible for most Salmonella infections in warm-blooded animals, carries a fimbrial gene cluster (saf) in this region as well as a regulatory gene (sinR). These genes are flanked by inverted repeats and are inserted in another laterally transferred region present in most members of Salmonella spp. encoding a putative invasin (pagN ). S. enterica subspecies I serovar Typhi, the Salmonella serovar that causes the most severe form of human salmonellosis, contains an additional insert of at least 8 kb in the sinR-pagN intergenic region harbouring a novel fimbrial operon (tcf ) similar to the coo operon encoding the CS1 fimbrial adhesin expressed by human-specific enterotoxigenic E. coli. It is suggested that the multiple insertions of fimbrial genes that have occurred in the cs7 region have contributed to phylogenetic diversity and host adaptation of Salmonella spp.

Chromosome Mapping↗
PubMed1999

The acid-inducible asr gene in Escherichia coli: transcriptional control by the phoBR operon.

Escherichia coli responds to external acidification (pH 4.0 to 5.0) by synthesizing a newly identified, approximately 450-nucleotide RNA component. At maximal levels of induction it is one of the most abundant small RNAs in the cell and is relatively stable bacterial RNA. The acid-inducible RNA was purified, and the gene encoding it, designated asr (for acid shock RNA), mapped at 35.98 min on the E. coli chromosome. Analysis of the asr DNA sequence revealed an open reading frame coding for a 111-amino-acid polypeptide with a deduced molecular mass of approximately 11.6 kDa. According to computer-assisted analysis, the predicted polypeptide contains a typical signal sequence of 30 amino acids and might represent either a periplasmic or an outer membrane protein. The asr gene cloned downstream from a T7 promoter was translated in vivo after transcription using a T7 RNA polymerase transcription system. Expression of a plasmid-encoded asr::lacZ fusion under a native asr promoter was reduced approximately 15-fold in a complex medium, such as Luria-Bertani medium, versus the minimal medium. Transcription of the chromosomal asr was abolished in the presence of a phoB-phoR (a two-component regulatory system, controlling the pho regulon inducible by phosphate starvation) deletion mutant. Acid-mediated induction of the asr gene in the Delta(phoB-phoR) mutant strain was restored by introduction of the plasmid with cloned phoB-phoR genes. Primer extension analysis of the asr transcript revealed a region similar to the Pho box (the consensus sequence found in promoters transcriptionally activated by the PhoB protein) upstream from the determined transcription start. The asr promoter DNA region was demonstrated to bind PhoB protein in vitro. We discuss our results in terms of how bacteria might employ the phoB-phoR regulatory system to sense an external acidity and regulate transcription of the asr gene.

Acids↗
PubMed1998

Fimbriae-mediated host-pathogen cross-talk.

Recent studies show that the coupling of fimbrial adhesins of uropathogenic Escherichia coli and pathogenic Neisseria species to their complementary receptors on host cells is a dynamic event, involving specific signaling to the bacteria as well as to the host cell. These studies have unveiled intriguing and novel mechanisms by which bacteria utilize their fimbriae to promote virulence at the mucosal surface and in deeper tissue.

Adhesins, Bacterial↗
PubMed1998

Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter.

A colony morphology type is described in which cells of Salmonella typhimurium form a rigid multicellular network with expression of thin aggregative fimbriae that mediate tight intercellular bonds. Surface translocation of cells on plates and adherence to glass and polystyrene surfaces in biofilm assays are further characteristics of the morphotype. This morphotype (rdar) is normally expressed only at low temperature. However, in two unrelated S. typhimurium strains, spontaneous mutants were found forming rdar colonies independent of temperature. Allelic replacement proved a single point mutation in the promoter region of PagfD in each of the two mutants to be responsible for the constitutive phenotype of a multicellular behaviour. Transcription levels of the two divergently transcribed agf operons required for biogenesis of thin aggregative fimbriae by Northern blot analysis with gene probes for agfA and agfD as well as expression levels of AgfA by Western blotting were compared in the wild type, the constitutive mutants and their respective ompR and rpoS- derivatives. In the wild type the rdar morphotype and expression of thin aggregative fimbriae are restricted to low temperature on plates containing rich medium of low osmolarity, but biogenesis of thin aggregative fimbriae occurs upon iron starvation at 37 degrees C. In the upregulated mutants biogenesis of thin aggregative fimbriae is only abolished at high osmolarity at 37 degrees C and in the exponential phase in broth culture. Control of expression of thin aggregative fimbriae and rdar morphology takes place at the transcriptional level at the agfD promoter. A functional ompR allele is required, however an rpoS mutation abolishes transcription only in the wild type, but has no influence on expression of thin aggregative fimbriae in the constitutive mutants.

Bacterial Adhesion↗