| 1. |
- Aspholm, Marina, et al.
(författare)
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SabA is the H. pylori hemagglutinin and is polymorphic in binding to sialylated glycans.
- 2006
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Ingår i: PLoS pathogens. - : Public Library of Science (PLoS). - 1553-7374 .- 1553-7366. ; 2:10
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Tidskriftsartikel (refereegranskat)abstract
- Adherence of Helicobacter pylori to inflamed gastric mucosa is dependent on the sialic acid-binding adhesin (SabA) and cognate sialylated/fucosylated glycans on the host cell surface. By in situ hybridization, H. pylori bacteria were observed in close association with erythrocytes in capillaries and post-capillary venules of the lamina propria of gastric mucosa in both infected humans and Rhesus monkeys. In vivo adherence of H. pylori to erythrocytes may require molecular mechanisms similar to the sialic acid-dependent in vitro agglutination of erythrocytes (i.e., sialic acid-dependent hemagglutination). In this context, the SabA adhesin was identified as the sialic acid-dependent hemagglutinin based on sialidase-sensitive hemagglutination, binding assays with sialylated glycoconjugates, and analysis of a series of isogenic sabA deletion mutants. The topographic presentation of binding sites for SabA on the erythrocyte membrane was mapped to gangliosides with extended core chains. However, receptor mapping revealed that the NeuAcalpha2-3Gal-disaccharide constitutes the minimal sialylated binding epitope required for SabA binding. Furthermore, clinical isolates demonstrated polymorphism in sialyl binding and complementation analysis of sabA mutants demonstrated that polymorphism in sialyl binding is an inherent property of the SabA protein itself. Gastric inflammation is associated with periodic changes in the composition of mucosal sialylation patterns. We suggest that dynamic adaptation in sialyl-binding properties during persistent infection specializes H. pylori both for individual variation in mucosal glycosylation and tropism for local areas of inflamed and/or dysplastic tissue.
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| 2. |
- Lindberg, Stina, et al.
(författare)
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Regulatory Interactions among adhesin gene systems of uropathogenic Escherichia coli
- 2008
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 76:2, s. 771-780
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Tidskriftsartikel (refereegranskat)abstract
- Uropathogenic Escherichia coli strain J96 carries multiple determinants for fimbrial adhesins. The regulatory protein PapB of P fimbriae has previously been implicated in potential coregulatory events. The focB gene of the F1C fimbria determinant is highly homologous to papB; the translated sequences share 81% identity. In this study we investigated the role of PapB and FocB in regulation of the F1C fimbriae. By using gel mobility shift assays, we showed that FocB binds to sequences in both the pap and foc operons in a somewhat different manner than PapB. The results of both in vitro cross-linking and in vivo oligomerization tests indicated that FocB could function in an oligomeric fashion. Furthermore, our results suggest that PapB and FocB can form heterodimers and that these complexes can repress expression of the foc operon. The effect of FocB on expression of type 1 fimbriae was also tested. Taken together, the results that we present expand our knowledge about a regulatory network for different adhesin gene systems in uropathogenic E. coli and suggest a hierarchy for expression of the fimbrial adhesins.
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| 3. |
- Loh, Edmund, et al.
(författare)
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An unstructured 5'-coding region of the prfA mRNA is required for efficient translation
- 2012
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Ingår i: Nucleic Acids Research. - Oxford, England : Oxford University Press. - 0305-1048 .- 1362-4962. ; 40:4, s. 1818-1827
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Tidskriftsartikel (refereegranskat)abstract
- Expression of virulence factors in the human bacterial pathogen Listeria monocytogenes is almost exclusively regulated by the transcriptional activator PrfA. The translation of prfA is controlled by a thermosensor located in the 5'-untranslated RNA (UTR), and is high at 37 degrees C and low at temperatures < 30 degrees C. In order to develop a thermoregulated translational expression system, the 5'-UTR and different lengths of the prfA-coding sequences were placed in front of lacZ. When expressed in Escherichia coli, the beta-galactosidase expression was directly correlated to the length of the prfA-coding mRNA lying in front of lacZ. A similar effect was detected with gfp as a reporter gene in both L. monocytogenes and E. coli, emphasizing the requirement of the prfA-coding RNA for maximal expression. In vitro transcription/translation and mutational analysis suggests a role for the first 20 codons of the native prfA-mRNA for maximal expression. By toe-print and RNA-probing analysis, a flexible hairpin-loop located immediately downstream of the start-codon was shown to be important for ribosomal binding. The present work determines the importance of an unstructured part of the 5'-coding region of the prfA-mRNA for efficient translation.
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| 4. |
- Loh, Edmund, 1981-, et al.
(författare)
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The first 20 codons of the prfA-mRNA are required for efficient translation in Listeria monocytogenes
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Expression of virulence factors in the human pathogen Listeria monocytogenes is almost exclusively regulated by the transcriptional activator PrfA. The translation of prfA is controlled by a thermosensor located in the 5´-untranslated RNA (UTR), which is high at 37°C and low at temperatures below 30°C. Also, translation of the prfA transcript is inhibited by a trans-acting riboswitch RNA, SreA, which binds to the 5´-end of the thermosensor. In order to develop a thermoregulated translational expression system in Mycobacterial species, the 5´-UTR and different lengths of the prfA-coding sequences were placed in front of lacZ. When expressed in Escherichia coli, the constructs retained their thermoregulation. However, the β-galactosidase expression was directly correlated to the length of the prfA-coding mRNA fused in front of lacZ. A similar regulation was also detected when gfp was used as a reporter gene. Transcriptional stability experiments indicated that the observed difference in expression was not due to a decreased stability of transcripts lacking more of the prfA-coding RNA. The gfp constructs behaved similarly in L. monocytogenes as in E. coli, emphasizing the requirement of the prfA-coding RNA for maximal expression, also in its natural genetic background. Moreover, the different PrfA-LacZ fusion proteins showed the same proteolytic stability, ruling out post-translational mechanisms. Instead, in vitro transcription/translation experiments suggest a role of the first 20 codons of the native prfA-mRNA for maximal expression. Our data indicated that the difference in expression was not due to rare codons, stretches of certain bases or a putative downstream box. We observed an inverse correlation between the stability of the RNA secondary structure and protein expression. The first 12 codons of prfA displayed a very weak RNA secondary structure. Similar weak stabilities were detected also for thermosensors in other species, indicating a common strategy of regulation. In summary, the present work determines the importance of an unstructured 5´-coding region of the prfA-RNA for efficient translation.
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| 5. |
- Mahdavi, Jafar, et al.
(författare)
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The blood group antigen binding activity of the Helicobacter pylori baba adhesin is regulated by local ph and redox potential
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The blood group antigen hinding adhesin, BabA, which binds to fucosylated blood group antigens, such as the Lewis b (Leb) and H1 antigens constitutes one of the best recognized adhesin-receptor interactions that mediate adherence of Helicobacter pylori to the gastric epithelium. BabA belongs to a family of H. pylori outer membrane, proteins (HOPs), a group of some 30 proteins with most similar N- and C-terminal domains.We previously identified the babA1 and babA2 genes, where babA2 was found to encode the BabA adhesin in strain CCUG17875. Here, we confirmed the identity of the BabA protein by immunoblot-analysis, followed by MALDIT-OF MS analysis, which also provided molecular weight of the BabA polypeptide and the unique peptide sequences for BabA. Surprisingly, the BabA protein was found to be expressed 50-fold higher compared to the number of calculated bacterial Leb-binding sites.Furthermore, surface scan of the bacterial membrane by freeze fracture immuno-EM technique localized the BabA by immunogold labeling to the bacterial surface in numbers similar to the predicted binding sites. To help explain the binding results, crosslinker-analyses were performed which revealed that BabA form supra-molecular complexes on the bacterial surfaces. In addition, binding to the Lewis b antigen was shown to be pH dependent and took place over a broad pH range but binding activity was reversibly lost when approaching pH 3, i.e. conditions similar to the acidic gastric juice.The binding activity of the BabA adhesin was shown to be sensitive for reducing conditions, which suggests the presence of disulfide bond(s) close to the carbohydrate-binding domain. The dynamics of BabA in Leb-binding suggest that the bacterial adhesin is regulated by local variations in pH and redox potential, such as the pH gradient in the slimy mucus lining of the epithelium, and the reduced conditions of the inflamed gastric mucosa.
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| 6. |
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| 7. |
- Sjöström, Annika E., 1963-, et al.
(författare)
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Analysis of the sfaXII locus in the Escherichia coli meningitis isolate IHE3034 reveals two novel regulatory genes within the promoter-distal region of the main S fimbrial operon
- 2009
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Ingår i: Microbial Pathogenesis. - : Elsevier Ltd. - 0882-4010 .- 1096-1208. ; 46:3, s. 150-158
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Tidskriftsartikel (refereegranskat)abstract
- We describe the expression and regulation of the gene sfaXII located near the SfaII fimbrial determinant in the newborn meningitis Escherichia coli (NMEC) isolate IHE3034. sfaXII belongs to a gene family, the 17kDa genes, typically located downstream (300 – 3000 bp) of different fimbrial operons found in E. coli isolates of uropathogenic and newborn meningitis origin. Using transcriptional sfaXII reporter gene fusions we found that different environmental conditions commonly affecting expression of fimbrial genes also affected sfaXII expression. Analysis of the sfaXII transcripts showed that the gene is part of the main fimbrial operon as it is transcribed together with the rest of the fimbrial genes. In addition, the sfaXII gene can be expressed from a more proximal promoter and is found to be subject to strong down-regulation by the nucleoid protein H-NS. Studies with an sfaXII mutant derivative of IHE3034 did not reveal effects on SfaII fimbrial biogenesis as monitored by e.g. immunofluorescence microscopy. Nevertheless, a mutation in sfaXII resulted in altered expression of other surface components. Moreover, we define a new gene, sfaYII, coding for a putative phosphodiesterase that is located in between the sfaXII gene and the fimbrial biogenesis genes. Our studies by ectopic expression of sfaYII in Vibrio cholerae showed that the gene product caused reduced biofilm formation and it is proposed that sfaYII can influence cyclic-di-GMP turnover in the bacteria. Our findings demonstrate that the operons typical for S-fimbriae of extraintestinal pathogenic E. coli include previously unrecognized novel regulatory genes.
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| 8. |
- Sjöström, Annika E., 1963-, et al.
(författare)
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Analysis of the sfaXII locus in the Escherichia coli meningitis isolate IHE3034 reveals two novel regulatory genes within the promoter-distal region ofthe main S fimbrial operon
- 2009
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Ingår i: Microbial Pathogenesis. - : Elsevier Ltd. - 0882-4010 .- 1096-1208. ; 46:3, s. 150-158
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Tidskriftsartikel (refereegranskat)abstract
- We describe the expression and regulation of the gene sfaXII located near the SfaII fimbrial determinant inthe newborn meningitis Escherichia coli (NMEC) isolate IHE3034. sfaXII belongs to a gene family, the 17-kDa genes, typically located downstream (300–3000 bp) of different fimbrial operons found in E. coli isolates of uropathogenic and newborn meningitis origin. Using transcriptional sfaXII reporter genefusions we found that different environmental conditions commonly affecting expression of fimbrialgenes also affected sfaXII expression. Analysis of the sfaXII transcripts showed that the gene is part of themain fimbrial operon as it is transcribed together with the rest of the fimbrial genes. In addition, the sfaXII gene can be expressed from a more proximal promoter and is found to be subject to strong downregulationby the nucleoid protein H-NS. Studies with an sfaXII mutant derivative of IHE3034 did notreveal effects on SfaII fimbrial biogenesis as monitored by e.g. immunofluorescence microscopy. Nevertheless,a mutation in sfaXII resulted in altered expression of other surface components. Moreover, we define a new gene, sfaYII, coding for a putative phosphodiesterase that is located in between the sfaXII gene and the fimbrial biogenesis genes. Our studies by ectopic expression of sfaYII in Vibrio cholerae showed that the gene product caused reduced biofilm formation and it is proposed that sfaYII caninfluence cyclic-di-GMP turnover in the bacteria. Our findings demonstrate that the operons typical for S-fimbriae of extraintestinal pathogenic E. coli include previously unrecognized novel regulatory genes.
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| 9. |
- Sondén, Berit, et al.
(författare)
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Gap, a mycobacterial specific integral membrane protein, is required for glycolipid transport to the cell surface.
- 2005
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 58:2, s. 426-440
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Tidskriftsartikel (refereegranskat)abstract
- The cell envelope of mycobacteria is a complex multilaminar structure that protects the cell from stresses encountered in the environment, and plays an important role against the bactericidal activity of immune system cells. The outermost layer of the mycobacterial envelope typically contains species-specific glycolipids. Depending on the mycobacterial species, the major glycolipid localized at the surface can be either a phenolglycolipid or a peptidoglycolipid (GPL). Currently, the mechanism of how these glycolipids are addressed to the cell surface is not understood. In this study, by using a transposon library of Mycobacterium smegmatis and a simple dye assay, six genes involved in GPLs synthesis have been characterized. All of these genes are clustered in a single genomic region of approximately 60 kb. We show by biochemical analyses that two non-ribosomal peptide synthetases, a polyketide synthase, a methyltransferase and a member of the MmpL family are required for the biosynthesis of the GPLs backbone. Furthermore, we demonstrate that a small integral membrane protein of 272 amino acids named Gap (gap: GPL addressing protein) is specifically required for the transport of the GPLs to the cell surface. This protein is predicted to contain six transmembrane segments and possesses homologues across the mycobacterial genus, thus delineating a new protein family. This Gap family represents a new paradigm for the transport of small molecules across the mycobacterial envelope, a critical determinant of mycobacterial virulence.
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