| 1. |
- Engman, Jakob, et al.
(författare)
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Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence
- 2016
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 84:5, s. 1501-1513
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Tidskriftsartikel (refereegranskat)abstract
- Neisseria meningitidis autoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase ( PNPase) is a 3'-5' exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is the N. meningitidis PNPase, and we characterize its role in N. meningitidis pathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in an in vivo model of N. meningitidis infection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence in N. meningitidis.
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| 2. |
- Negrea, Aurel
(författare)
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Pharmaceutical and mutational interference with virulence of Salmonella enterica serovar typhimurium
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Within the species Salmonella enterica are a diverse range of bacteria that can cause illness in humans and many animals. Salmonellae are extremely versatile and can adapt to a variety of environments and hosts. Typhoid and paratyphoid fever, caused by human-restricted S. enterica serovars Typhi and Paratyphi are common in the developing world. An increased resistance to the first line antibiotics has been recorded amongst salmonellae, which makes the basic and applied research aiming to farther the understanding of the disease and developing new regimes of treatment even more important. We showed that salicylidene acylhydrazides are able to inhibit the activity of virulence-associated type III secretion systems SPI1 and SPI2 in Salmonella enterica serovar Typhimurium. The compounds strongly affected Salmonella pathogenicity island (SPI) 1 activity and also SPI2-mediated intracellular bacterial replication in murine macrophage-like cells. In addition, two of the compounds significantly inhibited bacterial motility and expression of extracellular flagellin. We also found that the proton pump inhibitor omeprazole had a bacteriostatic effect on intracellular replication of S. Typhimurium mediated by virulence-associated SPI2 T3SS. We could demonstrate that S. Typhimurium rapidly acquires mutations in the putative transport protein SbmA, reducing the susceptibility of the bacteria to antimicrobial peptide PR-39 without an obvious fitness cost. We report for the first time that expression of thioredoxin 1 in the facultative intracellular pathogen S. Typhimurium is induced at conditions that prevail during intracellular infection and that thioredoxin 1 is necessary for proper T3SS expression, protein secretion and virulence. Our findings define an entirely new functional niche for thioredoxin 1 and demonstrate a new level of inter-connection between core genome functions and horizontally-acquired virulence genes in bacteria.
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| 3. |
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| 4. |
- Negrea, Aurel, et al.
(författare)
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Salicylidene Acylhydrazides That Affect Type III Protein Secretion in Salmonella enterica Serovar Typhimurium
- 2007
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804. ; 51:8, s. 2867-76
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Tidskriftsartikel (refereegranskat)abstract
- A collection of nine salicylidene acylhydrazide compounds were tested for their ability to inhibit the activity of virulence-associated type III secretion systems (T3SSs) in Salmonella enterica serovar Typhimurium. The compounds strongly affected Salmonella pathogenicity island 1 (SPI1) T3SS-mediated invasion of epithelial cells and in vitro secretion of SPI1 invasion-associated effector proteins. The use of a SPI1 effector ß-lactamase fusion protein implicated intracellular entrapment of the protein construct upon application of a salicylidene acylhydrazide, whereas the use of chromosomal transcriptional gene fusions revealed a compound-mediated transcriptional silencing of SPI1. Salicylidene acylhydrazides also affected intracellular bacterial replication in murine macrophage-like cells and blocked the transport of an epitope-tagged SPI2 effector protein. Two of the compounds significantly inhibited bacterial motility and expression of extracellular flagellin. We conclude that salicylidene acylhydrazides affect bacterial T3SS activity in S. enterica and hence could be used as lead substances when designing specific inhibitors of bacterial T3SSs in order to pharmaceutically intervene with bacterial virulence.
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| 5. |
- Pränting, Maria, et al.
(författare)
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Mechanism and fitness costs of PR-39 resistance in Salmonella enterica serovar Typhimurium LT2
- 2008
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 52:8, s. 2734-2741
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Tidskriftsartikel (refereegranskat)abstract
- PR-39 is a porcine antimicrobial peptide that kills bacteria with a mechanism that does not involve cell lysis. Here, we demonstrate that Salmonella enterica serovar Typhimurium can rapidly acquire mutations that reduce susceptibility to PR-39. Resistant mutants appeared at a rate of 0.4 x 10(-6) per cell per generation. These mutants were about four times more resistant than the wild type and showed a greatly reduced rate of killing. Genetic analysis revealed mutations in the putative transport protein SbmA as being responsible for the observed resistance. These sbmA mutants were as fit as the wild-type parental strain as measured by growth rates in culture medium and mice and by long-term survival in stationary phase. These results suggest that resistance to certain antimicrobial peptides can rapidly develop without an obvious fitness cost for the bacteria and that resistance development could become a threat to the efficacy of antimicrobial peptides if used in a clinical setting.
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| 6. |
- Puiac, Speranta, et al.
(författare)
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Small-molecular virulence inhibitors show divergent and immunomodulatory effects in infection models of Salmonella enterica serovar Typhimurium
- 2011
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Ingår i: International Journal of Antimicrobial Agents. - : Elsevier BV. - 0924-8579 .- 1872-7913. ; 38:5, s. 409-416
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Tidskriftsartikel (refereegranskat)abstract
- The virulence-associated Salmonella pathogenicity island 2 (SPI2) type III secretion system supports intracellular replication of Salmonella enterica serovar Typhimurium in macrophage-like RAW264.7 cells. In contrast, the salicylidene acylhydrazide INP0010 and the benzimidazole omeprazole prevent virulence factor-mediated replication of S. Typhimurium in these cells. Here we show that INP0010 enhances expression of inducible nitric oxide synthase (iNOS), nitric oxide (NO) production, the oxidative burst and tumour necrosis factor-alpha (TNF alpha) release in infected RAW264.7 cells. INP0010 also inhibited SPI2 activity in RAW264.7 cells. The ability of INP0010 to suppress bacterial intracellular replication correlated with NO production. The iNOS inhibitor N-monomethyl-L-arginine restored SPI2 activity and antagonised the bacteriostatic effect of INP0010. Omeprazole, which inhibited iNOS expression in RAW264.7 cells, likewise antagonised INP0010. In infected epithelioid MDCK cells that did not express NO upon infection, INP0010 enhanced bacterial intracellular replication. In Caenorhabditis elegans, INP0010 significantly attenuated the virulence of S. Typhimurium. In this infection model, the attenuating effect of INP0010 was further enhanced by omeprazole. These results demonstrate that chemically unrelated virulence inhibitors may act in an antagonistic or additive manner, that their effect depends on the infection model applied, and that the attenuating effects of INP0010 in part relate to its ability to promote the SPI2 antagonist NO.
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| 7. |
- Sun, Song, et al.
(författare)
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Genetic analysis of colistin resistance in Salmonella enterica serovar Typhimurium
- 2009
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 53:6, s. 2298-2305
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Tidskriftsartikel (refereegranskat)abstract
- Colistin is a cyclic cationic peptide that kills gram-negative bacteria by interacting with and disrupting the outer membrane. We isolated 44 independent mutants in Salmonella enterica serovar Typhimurium with reduced susceptibility to colistin and identified 27 different missense mutations located in the pmrA and pmrB genes (encoding the regulator and sensor of a two-component regulatory system) that conferred increased resistance. By comparison of the two homologous sensor kinases, PmrB and EnvZ, the 22 missense mutations identified in pmrB were shown to be located in four different structural domains of the protein. All five pmrA mutations were located in the phosphate receiver domain of the regulator protein. The mutants appeared at a mutation rate of 0.6 x 10(-6) per cell per generation. The MICs of colistin for the mutants increased 2- to 35-fold, and the extent of killing was reduced several orders of magnitude compared to the susceptible strain. The growth rates of the mutants were slightly reduced in both rich medium and M9-glycerol minimal medium, whereas growth in mice appeared unaffected by the pmrA and pmrB mutations. The low fitness costs and the high mutation rate suggest that mutants with reduced susceptibility to colistin could emerge in clinical settings.
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