| 1. |
- Dryselius, Rikard
(författare)
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Bacterial gene expression inhibition with antisense peptide nucleic acids
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Regulated gene expression is widely useful for gene function studies and as a means to control genes important in disease. There are many methods for regulating the expression of genes, and one approach is to use antisense oligonucleotides that bind complementary mRNAs to inhibit translation. A number of nucleic acid analogues and mimics have been developed to improve antisense efficiency. Peptide nucleic acid (PNA) is a DNA mimic with many interesting properties. PNA has proven effective in a variety of applications and mRNA has been a frequent target with the aim to silence gene expression in a sequence specific manner. However, mRNA targeting antisense PNA has almost exclusively been used on eukaryotic cells and relatively little is known about inhibition of bacterial gene expression. In this thesis antisense PNA is used to target bacterial genes. The focus is on design features such as antisense length and uptake and also on selection of appropriate target sites and target genes. Moreover, practical applications for antisense PNAs are examined including their use as inhibitors of essential bacterial genes. In terms of design features, the results show that antisense effects are much improved when a cell penetrating peptide is conjugated to the PNA. Also, relatively short molecules show higher efficiency. It is further demonstrated that the ribosome binding site is most likely to be susceptible to antisense inhibition and that antisense PNA directed to genes within operons may strongly affect the expression of cotranscribed genes. In terms of practical applications it was observed that an antisense PNA directed to an essential bacterial gene inhibited growth and was bactericidal. Furthermore, the PNA was able to rescue a human cell culture from infection and select for PNA resistant bacteria in a mixed population, indicating that efficient killing effects can be both cell type and strain specific. Also, growth inhibitory properties of antisense PNAs were examined in combination with conventional antimicrobial drugs. Combinations of mRNA specific antisense PNAs and protein specific drugs with shared genetic targets commonly displayed antimicrobial synergy, suggesting that antisense agents can be used to potentiate conventional antimicrobial drugs. In summary, the results described here provide guidelines for the design of sequence specific antisense PNAs and demonstrate practical uses for antisense agents as inhibitors of bacterial genes.
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| 2. |
- Dryselius, Rikard, et al.
(författare)
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Variable coordination of cotranscribed genes in Escherichia coli following antisense repression
- 2006
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Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 6, s. 97-
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Tidskriftsartikel (refereegranskat)abstract
- Background: A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target. Results: To examine the effects of antisense inhibition on cotranscribed genes, we constructed a plasmid expressing the two reporter genes gfp and DsRed as one transcriptional unit. Incubation with antisense peptide nucleic acid (PNA) targeted to the mRNA start codon region of either the upstream gfp or the downstream DsRed gene resulted in a complete expression discoordination from this artificial construct. The same approach was applied to the three cotranscribed genes in the endogenously expressed lac-operon (lacZ, Y and A) and partial downstream expression coordination was seen when the lacZ start codon was targeted with antisense PNA. Targeting the lacY mRNA start codon region showed no effect on the upstream lacZ gene expression whereas expression from the downstream lacA gene was affected as strongly as the lacY gene. Determination of lacZ and lacY mRNA levels revealed a pattern of reduction that was similar to the Lac-proteins, indicating a relation between translation inhibition and mRNA degradation as a response to antisense PNA treatment. Conclusion: The results show that antisense mediated repression of genes within operons affect cotranscribed genes to a variable degree. Target transcript stability appears to be closely related to inhibition of translation and presumably depends on translating ribosomes protecting the mRNA from intrinsic decay mechanisms. Therefore, for genes within operons and clusters it is likely that the nature of the target transcript will determine the inhibitory effects on cotranscribed genes. Consequently, no simple and specific methods for expression control of a single gene within polycistronic operons are available, and a thorough understanding of mRNA regulation and stability is required to understand the results from both knock-down and knock-out methods used in bacteria.
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| 3. |
- Fischerström, Karolina, et al.
(författare)
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Outbreak of Salmonella Typhimurium linked to Swedish pre-washed rocket salad, Sweden, September to November 2022
- 2024
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Ingår i: Eurosurveillance. - : European Centre for Disease Prevention and Control. - 1025-496X .- 1560-7917. ; 29:10
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Tidskriftsartikel (refereegranskat)abstract
- In September 2022, the Public Health Agency of Sweden observed an increase in domestic Salmonella Typhimurium cases through the Swedish electronic notification system, and an outbreak strain was identified with whole genome sequencing. Overall, 109 cases with symptom onset between 17 September and 24 November 2022 were reported from 20 of 21 Swedish regions. The median age of cases was 52 years (range 4-87 years) and 62% were female. A case-control study found cases to be associated with consumption of rocket salad (adjusted odds ratio (aOR) = 4.9; 95% confidence interval (CI): 2.4-10, p value < 0.001) and bagged mixed salad (aOR = 4.0; 95% CI: 1.9-8.1, p value < 0.001). Trace-back, supported by Finnish authorities who identified the Swedish outbreak strain in a Finnish cluster during the same time period, identified rocket salad, cultivated, pre-washed and pre-packed in Sweden as the likely source of the outbreak. No microbiological analyses of rocket salad were performed. Our investigation indicates that bagged leafy greens such as rocket salad, regardless of pre-washing procedures in the production chain, may contain Salmonella and cause outbreaks, posing a health risk to consumers. We emphasise the need for primary producers of leafy greens to identify possible contamination points to prevent outbreaks.
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| 4. |
- Frisan, Teresa, 1967-, et al.
(författare)
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Ubiquitin C-terminal hydrolase-L1 interacts with adhesion complexes and promotes cell migration, survival, and anchorage independent growth
- 2012
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Ingår i: The FASEB Journal. - : The Federation of American Societies for Experimental Biology. - 0892-6638 .- 1530-6860. ; 26:12, s. 5060-5070
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Tidskriftsartikel (refereegranskat)abstract
- Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme of unknown function that is highly expressed in neurons and overexpressed in several human cancers. UCH-L1 has been implicated in the regulation of phenotypic properties associated with malignant cell growth but the underlying mechanisms have not been elucidated. By comparing cells expressing catalytically active or inactive versions of UCH-L1, we found that the active enzyme enhances cell adhesion, spreading, and migration; inhibits anoikis; and promotes anchorage independent growth. UCH-L1 accumulates at the motile edge of the cell membrane during the initial phases of adhesion, colocalizes with focal adhesion kinase (FAK), p120-catenin, and vinculin, and enhances the formation of focal adhesions, which correlates with enhanced FAK activation. The involvement of UCH-L1 in the regulation of focal adhesions and adherens junctions is supported by coimmunoprecipitation with key components of these complexes, including FAK, paxillin, p120-catenin, β-catenin, and vinculin. UCH-L1 stabilizes focal adhesion signaling in the absence of adhesion, as assessed by reduced caspase-dependent cleavage of FAK following cell detachment and sustained activity of the AKT signaling pathway. These findings offer new insights on the molecular interactions through which the deubiquitinating enzyme regulates the survival, proliferation, and metastatic potential of malignant cells.
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| 5. |
- Hägglund, Moa, et al.
(författare)
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Accounting for bacterial overlap between raw water communities and contaminating sources improves the accuracy of signature-based microbial source tracking
- 2018
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Microbial source tracking (MST) analysis is essential to identifying and mitigating the fecal pollution of water resources. The signature-based MST method uses a library of sequences to identify contaminants based on operational taxonomic units (OTUs) that are unique to a certain source. However, no clear guidelines for how to incorporate OTU overlap or natural variation in the raw water bacterial community into MST analyses exist. We investigated how the inclusion of bacterial overlap between sources in the library affects source prediction accuracy. To achieve this, large-scale sampling-including feces from seven species, raw sewage, and raw water samples from water treatment plants - was followed by 16S rRNA amplicon sequencing. The MST library was defined using three settings: (i) no raw water communities represented; (ii) raw water communities selected through clustering analysis; and (iii) local water communities collected across consecutive years. The results suggest that incorporating either the local background or representative bacterial composition improves MST analyses, as the results were positively correlated to measured levels of fecal indicator bacteria and the accuracy at which OTUs were assigned to the correct contamination source increased fourfold. Using the proportion of OTUs with high source origin probability, underpinning a contaminating signal, is a solid foundation in a framework for further deciphering and comparing contaminating signals derived in signature-based MST approaches. In conclusion, incorporating background bacterial composition of water in MST can improve mitigation efforts for minimizing the spread of pathogenic and antibiotic resistant bacteria into essential freshwater resources.
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| 6. |
- Larkin, Lesley, et al.
(författare)
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Investigation of an international outbreak of multidrug-resistant monophasic Salmonella Typhimurium associated with chocolate products, EU/EEA and United Kingdom, February to April 2022
- 2022
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Ingår i: Eurosurveillance. - : European Centre for Disease Prevention and Control. - 1025-496X .- 1560-7917. ; 27:15
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Tidskriftsartikel (refereegranskat)abstract
- An extensive multi-country outbreak of multidrug-resistant monophasic Salmonella Typhimurium infection in 10 countries with 150 reported cases, predominantly affecting young children, has been linked to chocolate products produced by a large multinational company. Extensive withdrawals and recalls of multiple product lines have been undertaken. With Easter approaching, widespread product distribution and the vulnerability of the affected population, early and effective real-time sharing of microbiological and epidemiological information has been of critical importance in effectively managing this serious food-borne incident.
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| 7. |
- Nikravesh, Abbas, et al.
(författare)
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Antisense PNA Accumulates in Escheria coli and mediates a Long Post-antibiotic Effect
- 2008
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Ingår i: Molecular Therapy. - : Elsevier BV. - 1525-0016 .- 1525-0024. ; 15:8, s. 1537-1542
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Tidskriftsartikel (refereegranskat)abstract
- Antisense agents that target growth-essential genes display surprisingly potent bactericidal properties. In particular, peptide nucleic acid (PNA) and phosphorodiamidate morpholino oligomers linked to cationic carrier peptides are effective in time kill assays and as inhibitors of bacterial peritonitis in mice. It is unclear how these relatively large antimicrobials overcome stringent bacterial barriers and mediate killing. Here we determined the transit kinetics of peptide–PNAs and observed an accumulation of cell-associated PNA in Escherichia coli and slow efflux. An inhibitor of drug efflux pumps did not alter peptide–PNA potency, indicating a lack of active efflux from cells. Consistent with cell retention, the post-antibiotic effect (PAE) of the anti-acyl carrier protein (acpP) peptide–PNA was greater than 11 hours. Bacterial cell accumulation and a long PAE are properties of significant interest for antimicrobial development.
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| 8. |
- Ottosson, Jakob, et al.
(författare)
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Bevattningsvatten : kunskapsunderlag
- 2019
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Livsmedel från växtriket kan i vissa fall sprida mikrobiologisk smitta. Bär, bladgrönt och groddarmedför en särskild risk. Det beror på att de ofta äts råa eller efter minimal tillredning. Vi värmer deminte innan vi äter dem.Om livsmedlen är kontaminerade är det med stor sannolikhet orsakat av bevattningen. Vattnet kan havarit påverkat av avföring. Det kan också bero på spillning från vilda djur. Även naturgödsel kanförorena produkterna vid stänk från jord.Syftet med detta projekt var att se över kvaliteten på vatten för bevattning i Sverige. Vi ville koppladet till mikrobiologiska risker och utifrån det ta fram underlag till nationella råd och riktlinjer. Måletär ”en säkrare produktion av ätfärdiga bär och grönsaker som bättre förebygger sjukdomsutbrott”.Det visade sig att kvaliteten på vattnet generellt sett var god hos odlarna i projektet. Det samma gällergrödor. I vatten hittade vi enstaka sjukdomsframkallande mikroorganismer och ESBL-bildande E. coli.Dessa hittade vi dock endast i ytvatten som var mer förorenade av avföring och spillning. Men endastett fåtal prov på gröda visade på högre fekal förorening. Majoriteten av dessa kom från en och sammaodlare, som bevattnade med vatten från en damm. Vi hittade inga patogener på grödor. Däremot var ettprov på vitkål positivt för ESBL-bildande E. coli.Vi gjorde en kvantitativ riskvärdering av Shigatoxinproducerande E. coli (STEC) från isbergssallat.Den visade att även en liten förorening orsakad av avföring och utan påvisade STEC-bakterier ibevattningsvattnet kan utgöra en risk för mag-och tarminfektion. Riskvärderingen kan användas somunderlag för att föreslå mikrobiologiska kriterier, till exempel på vattenkvalitet. Vi behöver dockbeakta osäkerheterna med värderingen. De viktigaste osäkerheterna var utsöndringen av ochpatogeniciteten hos STEC från infekterade nötkreatur. Hur mycket av dessa fastnar på den bevattnadegrödan? Hur stor är sannolikheten att infekteras vid låga doser (enstaka STEC-bakterier)? Och hur storär risken av stänk från jord?Det finns olika åtgärder att ta till för att minska sannolikheten för utbrott och sjukdomsfall. Dessa är 1.Kriterier för vattenkvalitet. 2: Rening av vatten som inte uppnår dessa kriterier. 3. Införa enuppehållstid mellan sista bevattning och skörd. 4. Dessutom kan konsumenterna skölja produkternainnan de konsumerar dem. Det är också viktigt att se till att hålla utrustningen ren, exempelvis rör ochmunstycken. På så vis undviks föroreningar och tillväxt av bakterier.Om ytvatten måste användas för bevattning under de sista två veckorna innan skörd bör ettprovtagningsprogram utformas. Proven bör då visa på låg fekal förorening samt avsaknad avSalmonella. I bästa fall bör ytvatten renas före bevattning. Detta kan göras till exempel med hjälp avfotokatalys, UV-ljus eller filtrering. De sista två dagarna före skörd bör dock endast vatten avdricksvattenkvalitet användas.I en framtid med sjunkande grundvattennivåer, torrare somrar och blötare vintrar finns ett behov av attsamla in vatten för bevattning. Det bör göras under höstregn och vårfloder, samt vintertid. Vattnet fårsedan lagras i magasin. Vatten från bevattningsmagasin kan behöva renas innan det används förbevattning beroende på hur skyddat magasinet är från föroreningar.
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| 9. |
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| 10. |
- Pullerits, Kristjan, et al.
(författare)
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Impact of UV irradiation at full scale on bacterial communities in drinking water
- 2020
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Ingår i: npj Clean Water. - : Springer Science and Business Media LLC. - 2059-7037. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Water in a full-scale drinking water treatment plant was irradiated with ultraviolet (UV) doses of 250, 400, and 600 J/m2, and the effect on bacterial communities investigated using 16s rRNA gene amplicon sequencing, heterotrophic plate counts (HPCs), coliform, and Escherichia coli counts. The bacteria in the irradiated water were also analyzed following storage for 6 days at 7 °C, to approximate the conditions in the distribution system. The log10 reduction of HPCs at 400 J/m2 was 0.43 ± 0.12. Phylogenetic examination, including DESeq2 analysis, showed that Actinobacteria was more resistant to UV irradiation, whereas Bacteroidetes was sensitive to UV. Phylum Proteobacteria contained monophyletic groups that were either sensitive or resistant to UV exposure. The amplicon sequence variants (ASVs) resistant to UV irradiation had a greater average GC content than the ASVs sensitive to UV, at 55% ± 1.7 (n = 19) and 49% ± 2.5 (n = 16), respectively. Families Chitinophagaceae, Pelagibacteraceae, Holophagaceae, Methylophilaceae, and Cytophagaceae decreased linearly in relative abundance, with increasing UV dose (P < 0.05, Pearson’s correlation). When irradiated water was stored, Chitinophagaceae, Comamonadaceae, and Flavobacteriaceae families decreased in relative abundance, whereas ACK-M1, Mycobacteriaceae, and Nitrosomonadaceae were increasing in relative abundance. This suggests that the impact of UV irradiation cannot only be considered directly after application but that this treatment step likely continues to influence microbial dynamics throughout the distribution system.
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