| 1. |
- Lind, Peter A, et al.
(författare)
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Compensatory gene amplification restores fitness after inter-species gene replacements
- 2010
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 75:5, s. 1078-1089
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Tidskriftsartikel (refereegranskat)abstract
- Genes introduced by gene replacements and other types of horizontal gene transfer (HGT) represent a significant presence in many archaeal and eubacterial genomes. Most alien genes are likely to be neutral or deleterious upon arrival and their long-term persistence may require a mechanism that improves their selective contribution. To examine the fate of inter-species gene replacements, we exchanged three native S. typhimurium genes encoding ribosomal proteins with orthologues from various other microbes. The results show that replacement of each of these three genes reduces fitness to such an extent that it would provide an effective barrier against inter-species gene replacements in eubacterial populations. However, these fitness defects could be partially ameliorated by gene amplification that augmented the dosage of the heterologous proteins. This suggests that suboptimal expression is a common fitness constraint for inter-species gene replacements, with fitness costs conferred by either a lower expression level of the alien protein compared with the native protein or a requirement for an increased amount of the alien protein to maintain proper function. Our findings can explain the observation that duplicated genes are over-represented among horizontally transferred genes, and suggest a potential coupling between compensatory gene amplification after HGT and the evolution of new genes.
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| 2. |
- Liu, Yiming, et al.
(författare)
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Penicillin-binding protein SpoVD disulfide is a target for StoA in Bacillus subtilis forespores.
- 2010
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 75:1, s. 46-60
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Tidskriftsartikel (refereegranskat)abstract
- Summary The bacterial endospore is a dormant and heat-resistant form of life. StoA (SpoIVH) in Bacillus subtilis is a membrane-bound thioredoxin-like protein involved in endospore cortex synthesis. It is proposed to reduce disulfide bonds in hitherto unknown proteins in the inter-membrane compartment of developing forespores. Starting with a bioinformatic analysis combined with mutant studies we identified the sporulation-specific, high molecular weight, class B penicillin-binding protein SpoVD as a putative target for StoA. We then demonstrate that SpoVD is a membrane-bound protein with two exposed redox-active cysteine residues. Structural modelling of SpoVD, based on the well characterized orthologue PBP2x of Streptococcus pneumoniae, confirmed that a disulfide bond can form close to the active site of the penicillin-binding domain restricting access of enzyme substrate or functional association with other cortex biogenic proteins. Finally, by exploiting combinations of mutations in the spoVD, stoA and ccdA genes in B. subtilis cells, we present strong in vivo evidence that supports the conclusion that StoA functions to specifically break the disulfide bond in the SpoVD protein in the forespore envelope. The findings contribute to our understanding of endospore biogenesis and open a new angle to regulation of cell wall synthesis and penicillin-binding protein activity.
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| 3. |
- Singh, Birendra, et al.
(författare)
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Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement-inhibitory activity.
- 2010
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 75, s. 1426-1444
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Tidskriftsartikel (refereegranskat)abstract
- Summary The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (K(D)) for vitronectin binding to UspA2 was 2.3 x 10(-8) M, and the N-terminal region encompassing residues UspA2 30-170 bound vitronectin with a K(D) of 7.9 x 10(-8) M. Electron microscopy verified that the active binding domain (UspA2(30-177)) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA2(30-177) bound to the C-terminal region of vitronectin residues 312-396. Finally, when human serum was pre-incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N-terminal domain of UspA2 and the C-terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis-dependent serum resistance.
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| 4. |
- Thorsen, Michael, 1974, et al.
(författare)
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Glutathione serves an extracellular defence function to decrease arsenite accumulation and toxicity in yeast.
- 2012
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Ingår i: Molecular microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 84:6, s. 1177-88
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Tidskriftsartikel (refereegranskat)abstract
- Arsenic is an environmental toxin and a worldwide health hazard. Since this metalloid is ubiquitous in nature, virtually all living organisms require systems for detoxification and tolerance acquisition. Here, we show that during chronic exposure to arsenite [As(III)], Saccharomyces cerevisiae (budding yeast) exports and accumulates the low-molecular-weight thiol molecule glutathione (GSH) outside of cells. Extracellular accumulation of the arsenite triglutathione complex As(GS)₃ was also detected and direct transport assays demonstrate that As(GS)₃ does not readily enter cells. Yeast cells with increased extracellular GSH levels accumulate less arsenic and display improved growth when challenged with As(III). Conversely, cells defective in export and extracellular accumulation of GSH are As(III) sensitive. Taken together, our data are consistent with a novel detoxification mechanism in which GSH is exported to protect yeast cells from arsenite toxicity by preventing its uptake.
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| 5. |
- Brady, L. Jeannine, et al.
(författare)
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The changing faces of Streptococcus antigen I/II polypeptide family adhesins
- 2010
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 77:2, s. 276-286
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus mutans antigen I/II (AgI/II) protein was one of the first cell wall-anchored adhesins identified in Gram-positive bacteria. It mediates attachment of S. mutans to tooth surfaces and has been a focus for immunization studies against dental caries. The AgI/II family polypeptides recognize salivary glycoproteins, and are also involved in biofilm formation, platelet aggregation, tissue invasion and immune modulation. The genes encoding AgI/II family polypeptides are found among Streptococcus species indigenous to the human mouth, as well as in Streptococcus pyogenes, S. agalactiae and S. suis. Evidence of functionalities for different regions of the AgI/II proteins has emerged. A sequence motif within the C-terminal portion of Streptococcus gordonii SspB (AgI/II) is bound by Porphyromonas gingivalis, thus promoting oral colonization by this anaerobic pathogen. The significance of other epitopes is now clearer following resolution of regional crystal structures. A new picture emerges of the central V (variable) region, predicted to contain a carbohydrate-binding trench, being projected from the cell surface by a stalk formed by an unusual association between an N-terminal α-helix and a C-terminal polyproline helix. This presentation mode might be important in determining functional conformations of other Gram-positive surface proteins that have adhesin domains flanked by α-helical and proline-rich regions. Ever since dental caries (tooth decay) was first shown to be caused by bacteria, there has been continued interest in developing vaccine or passive immunization protocols for its control or prevention (Lehner et al., 1980). Although dental caries is not fatal, and in developed countries caries is now considered to be largely avoidable through controlled diet and good oral hygiene, there remain significant problems with childhood disease, especially among indigent populations. Consequently, caries is one of the most common worldwide infectious diseases. Therefore, research continues towards employing vaccine formulations comprised of peptide components derived from surface proteins of Streptococcus mutans, a major agent associated with dental caries (Lehner et al., 1975). One of the most promising strategies seems to be delivery of peptides, derived from glucan-binding protein B (GbpB) and antigen I/II (AgI/II) protein, via a mucosal (nasal) route. The GbpB polypeptide binds extracellular glucans, thus promoting co-adhesion of S. mutans cells in the development of dental plaque (Taubman and Nash, 2006). The AgI/II protein (also named P1, SpaP, AgB or PAc) is a major surface protein that functions as an adhesin, attaching S. mutans to the saliva-coated tooth enamel surface (Koga et al., 1990; Kelly et al., 1995). Antibodies against SpaP and GbpB block adherence and co-adhesion, respectively, thus disrupting colonization of the oral cavity by S. mutans (Ma et al., 1990; 1998; Taubman and Nash, 2006). The terminology AgI/II derives from the identification of two major cell wall antigens I and II in S. mutans by Russell et al. (1980), and the subsequent recognition that AgII was a component of AgI. Following the discovery of AgI/II, it became apparent that genes encoding orthologous proteins were widely dispersed among the streptococci (Jenkinson and Demuth, 1997). The viridans Streptococcus AgI/II adhesins range in composition from 1310 to 1653 amino acid (aa) residues, while the Streptococcus agalactiae AgI/II proteins are smaller (826–932 aa residues) (Tettelin et al., 2005). The widespread distribution of these AgI/II protein genes across the streptococci is perhaps not surprising, given the complex streptococcal communities that exist on surfaces of the oro- and naso-pharynx and within the bacterial soup of saliva. It is interesting, though, that the AgI/II family polypeptide genes have not yet been discovered in Streptococcus pneumoniae, which might be by the fact that S. pneumoniae forms a distinct evolutionary cluster (Kilian et al., 2008).
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| 6. |
- Collins, James, et al.
(författare)
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Fibrinogen-binding and platelet-aggregation activities of a Lactobacillus salivarius septicaemia isolate are mediated by a novel fibrinogen-binding protein.
- 2012
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 85:5, s. 862-877
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Tidskriftsartikel (refereegranskat)abstract
- The marketplace for probiotic foods is burgeoning, measured in billions of euro per annum. It is imperative, however, that all bacterial strains are fully assessed for human safety. The ability to bind fibrinogen is considered a potential pathogenicity trait that can lead to platelet aggregation, serious medical complications, and in some instances, death. Here we examined strains from species frequently used as probiotics for their ability to bind human fibrinogen. Only one strain (CCUG 47825), a Lactobacillus salivarius isolate from a case of septicaemia, was found to strongly adhere to fibrinogen. Furthermore, this strain was found to aggregate human platelets at a level comparable to the human pathogen Staphylococcus aureus. By sequencing the genome of CCUG 47825, we were able to identify candidate genes responsible for fibrinogen binding. Complementing the genetic analysis with traditional molecular microbiological techniques enabled the identification of the novel fibrinogen receptor, CCUG_2371. Although only strain CCUG 47825 bound fibrinogen under laboratory conditions, homologues of the novel fibrinogen binding gene CCUG_2371 are widespread among L. salivarius strains, maintaining their potential to bind fibrinogen if expressed. We highlight the fact that without a full genetic analysis of strains for human consumption, potential pathogenicity traits may go undetected. © 2012 Blackwell Publishing Ltd.
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| 7. |
- Pagels, Martin, et al.
(författare)
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Redox sensing by a Rex-family repressor is involved in the regulation of anaerobic gene expression in Staphylococcus aureus
- 2010
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 76:5, s. 1142-1161
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Tidskriftsartikel (refereegranskat)abstract
- P>An alignment of upstream regions of anaerobically induced genes in Staphylococcus aureus revealed the presence of an inverted repeat, corresponding to Rex binding sites in Streptomyces coelicolor. Gel shift experiments of selected upstream regions demonstrated that the redox-sensing regulator Rex of S. aureus binds to this inverted repeat. The binding sequence - TTGTGAAW(4)TTCACAA - is highly conserved in S. aureus. Rex binding to this sequence leads to the repression of genes located downstream. The binding activity of Rex is enhanced by NAD+ while NADH, which competes with NAD+ for Rex binding, decreases the activity of Rex. The impact of Rex on global protein synthesis and on the activity of fermentation pathways under aerobic and anaerobic conditions was analysed by using a rex-deficient strain. A direct regulatory effect of Rex on the expression of pathways that lead to anaerobic NAD+ regeneration, such as lactate, formate and ethanol formation, nitrate respiration, and ATP synthesis, is verified. Rex can be considered a central regulator of anaerobic metabolism in S. aureus. Since the activity of lactate dehydrogenase enables S. aureus to resist NO stress and thus the innate immune response, our data suggest that deactivation of Rex is a prerequisite for this phenomenon.
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| 8. |
- Singh, Birendra, et al.
(författare)
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Haemophilus influenzae protein E recognizes the C-terminal domain of vitronectin and modulates the membrane attack complex.
- 2011
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 81, s. 80-98
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Tidskriftsartikel (refereegranskat)abstract
- Haemophilus influenzae protein E (PE) is a 16 kDa adhesin that induces a pro-inflammatory immune response in lung epithelial cells. The active epithelial binding region comprising amino acids PE 84-108 also interferes with complement-mediated bacterial killing by capturing vitronectin (Vn) that prevents complement deposition and formation of the membrane attack complex (MAC). Here, the interaction between PE and Vn was characterized using site-directed mutagenesis. Protein E variants were produced both in soluble forms and in surface-expressed molecules on Escherichia coli. Mutations within PE(84-108) in the full-length molecule revealed that K85 and R86 residues were important for the Vn binding. Bactericidal activity against H. influenzae was higher in human serum pre-treated with full-length PE as compared with serum incubated with PE(K85E, R86D) , suggesting that PE quenched Vn. A series of truncated Vn molecules revealed that the C-terminal domain comprising Vn(353-363) harboured the major binding region for PE. Interestingly, MAC deposition was significantly higher on mutants devoid of PE due to a decreased Vn-binding capacity when compared with wild-type H. influenzae. Our results define a fine-tuned interaction between H. influenzae and the innate immune system, and identify the mode of control of the MAC that is important for pathogen complement evasion.
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| 9. |
- Singh, Birendra, et al.
(författare)
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Vitronectin in bacterial pathogenesis: A host protein used in complement escape and cellular invasion.
- 2010
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 78:3, s. 545-560
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Tidskriftsartikel (refereegranskat)abstract
- The multifunctional human glycoprotein vitronectin (Vn) plays a significant role in cell migration, tissue repair and regulation of membrane attack complex (MAC) formation. It also promotes neutrophil infiltration and, thus, enhances the inflammatory process during infection. In the host, a balanced homeostasis is maintained by Vn due to neutralization of the self-reactivity of the MAC. On the other hand, Vn bound to the bacterial surface protects from MAC mediated lysis and enhances adhesion. Gram-negative bacterial pathogens including Moraxella catarrhalis, Haemophilus influenzae, and Neisseria gonorrhoeae use Vn recruitment to prevent MAC deposition at their surface. Moreover, Gram-positive bacterial pathogens such as Streptococcus pneumoniae and S. pyogenes utilize Vn for effective adhesion to host cells and subsequent internalization. Vitronectin has an Arg-Gly-Asp (RGD) sequence for binding the host cell integrin receptors and a separate bacterial binding domain for pathogens, and thus more likely functions to cross-link bacteria and epithelial cells. Once bacteria are attached to the vitronectin-integrin complex, various host cell-signalling events are activated and promote internalization. In this review, we focus on the important roles of vitronectin in bacterial pathogenesis and describe different strategies used by pathogens to evade the host response by the help of this intriguing molecule.
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| 10. |
- Andersson, Dan I., et al.
(författare)
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Biological roles of translesion synthesis DNA polymerases in eubacteria
- 2010
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 77:3, s. 540-548
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Forskningsöversikt (refereegranskat)abstract
- Biological systems are strongly selected to maintain the integrity of their genomes by prevention and repair of external and internal DNA damages. However, some types of DNA lesions persist and might block the replication apparatus. The universal existence of specialized translesion synthesis DNA polymerases (TLS polymerases) that can bypass such lesions in DNA implies that replication blockage is a general biological problem. We suggest that the primary function for which translesion synthesis polymerases are selected is to rescue cells from replication arrest at lesions in DNA, a situation that, if not amended, is likely to cause an immediate and severe reduction in cell fitness and survival. We will argue that the mutagenesis observed during translesion synthesis is an unavoidable secondary consequence of this primary function and not, as has been suggested, an evolved mechanism to increase mutation rates in response to various stresses. Finally, we will discuss recent data on additional roles for translesion synthesis polymerases in the formation of spontaneous deletions and in transcription-coupled TLS, where the coupling of transcription to TLS is proposed to allow the rescue of the transcription machinery arrested at DNA lesions.
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