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- Andersson, Christopher, 1987-
(författare)
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Regulatory pathways and virulence inhibition in Listeria monocytogenes
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Listeria monocytogenes is a rod-shaped Gram positive bacterium. It generally exist ubiquitously in nature, where it lives as a saprophyte. Occasionally it however enters the food chain, from where it can be ingested by humans and cause gastro-intestinal distress. In immunocompetent individuals L. monocytogenes is generally cleared within a couple of weeks, but in immunocompromised patients it can progress to listeriosis, a potentially life-threatening infection in the central nervous system. If the infected individual is pregnant, the bacteria can cross the placental barrier and infect the fetus, possibly leading to spontaneous abortion.The infectivity of L. monocytogenes requires a certain set of genes, and the majority of them is dependent on the transcriptional regulator PrfA. The expression and activity of PrfA is controlled at several levels, and has traditionally been viewed to be active at 37 °C (virulence conditions) where it bind as a homodimer to a “PrfA-box” and induces the expression of the downstream gene.One of these genes is ActA, which enables intracellular movement by recruiting an actin polymerizing protein complex. When studying the effects of a blue light receptor we surprisingly found an effect of ActA at non-virulent conditions, where it is required for the bacteria to properly react to light exposure.To further study the PrfA regulon we tested deletion mutants of several PrfA-regulated virulence genes in chicken embryo infection studies. Based on these studies we could conclude that the chicken embryo model is a viable complement to traditional murine models, especially when investigating non-traditional internalin pathogenicity pathways. We have also studied the effects of small molecule virulence inhibitors that, by acting on PrfA, can inhibit L. monocytogenes infectivity in cell cultures with concentrations in the low micro-molar range.
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| 2. |
- Dzhygyr, Ievgen, 1985-
(författare)
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Functional studies of Escherichia coli stringent response factor RelA
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- RelA is a ribosome associated multi-domain enzyme, which plays a crucial role in adaptation ofEscherichia coli to nutritional stress as such as amino acid deficiency. It detects the deficiency of aminoacids in the cell by monitoring whether a tRNA at the acceptor site (A-site) of the ribosome is chargedwith amino acid or not. When RelA detects uncharged, i.e. deacylated tRNA, it starts to producealarmone guanosine penta- or tetraphosphate, collectively referred to as (p)ppGpp. (p)ppGpp is aglobal metabolism regulator in bacteria. Increase in (p)ppGpp concentration alters crucial metabolicprocesses, such as DNA replication, gene expression, cell wall synthesis and translation. Thesechanges also include activation of different virulence factors and are proposed to drive formation of abacterial sub-population that is highly resilient to antibiotic treatment, the so-called persisters.For a long time the molecular mechanism of RelA’s activation by and interaction with the ribosomedeacylatedtRNA complex was unknown. Only recently several cryo-EM structures of RelA-ribosomecomplex have shed light on how C-terminal domains of RelA interact with ribosome-deacylated tRNAcomplex. Guided by these structures we investigated the role of RelA’s domains in this interaction byconstructing a set of RelA C-terminal truncates and subjecting these to biochemical and microbiologicalexperimentation. These experiments were complemented with mutations in ribosomal RNA atpositions that interact with RelA, namely A-site finger and sarcin-ricin loop.We have shown that only the full-length wild type RelA can be activated by ribosome-tRNA complex,whereas, the set of truncated proteins missing either one, two or three C-terminal domains do notrespond to the presence of uncharged tRNA in the A-site of the ribosome. However, these truncatedversions can still be activated by vacant 70S ribosome as well as pppGpp, suggesting that N-terminaldomain of RelA has an allosteric regulation site for (p)ppGpp and is able to interact with the ribosome.The mechanism of this interaction is yet to be elucidated.We have shown that A-site finger of the ribosome is required for RelA activation and recruitment tothe ribosome. Using EMSA assays, we have shown that RelA and deacylated tRNA do not form a stablecomplex off the ribosome. His432 located in TGS domain of RelA is crucial for recognition of deacylatedtRNA and a mutation of this histidine to glycine abolishes RelA activation by deacylated tRNA.Since (p)ppGpp plays an important role in bacterial survival and pathogenicity we have also testedseveral strategies for RelA inhibition by antibiotics, which target ribosomes and the interactionbetween RelA and ribosome-deacylated tRNA complex. We have shown that antibiotic thiostreptoninhibits (p)ppGpp synthesis by preventing RelA-tRNA interaction on the ribosome. (p)ppGppproduction is also inhibited by chloramphenicol and tetracycline.
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| 3. |
- Farag, Salah I., 1959-
(författare)
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Biogenesis, function and regulation of the type III secretion translocon of Yersinia pseudotuberculosis
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many Gram negative bacteria use type III secretion systems to cross-talk with eukaryotic cells. Type III secretion system assembly and function is tightly regulated. It initiates with assembly of a basal body-like structure, and is followed by a cytoplasmic-located substrate sorting and export platform that first engages with early substrates required for needle assembly. At the needle tip, a translocon is formed upon eukaryotic cell contact to allow the translocation of effector proteins to the host cell. The focus of this thesis is on understanding aspects of biogenesis, regulation and function of the translocon and its interaction with the host cell. Research questions are addressed in enteropathogenic Yersinia pseudotuberculosis model.Prioritising the secretion of translocon components before effector proteins is a task given partly to the InvE/MxiC/HrpJ family of proteins. In Yersinia, homology to this protein family is partitioned over two proteins; YopN and TyeA. Certain Yersinia strains naturally produce a single YopN/TyeA polypeptide hybrid. To understand the implications of hybrid formation towards type III secretion control, a series of mutants were engineered to produce only a single hybrid peptide. Using in vitro assays revealed no difference in substrate secretion profiles between parent and mutants. Moreover, no obvious prioritisation of secretion between translocator and effector substrates was observed. Although these in vitro studies indicate that the YopN-TyeA single polypeptide is fully functionally competent, these mutants were attenuated in the mouse infection model. Hence, natural production of YopN and TyeA as a single polypeptide alone is unlikely to confer a fitness advantage to the infecting bacteria and is unlikely to orchestrate hierarchal substrate secretion.The YopB and YopD translocon components form a pore in the host cell plasma membrane to deliver the effectors into the host cell. To better understand how YopD contributes to the biogenesis, function and regulation of the translocon pore, a series of mutants were constructed to disrupt two predicted α-helix motifs, one lying at the N-terminus and the other at the C-terminus. Based upon phenotypes associated with environmental control of Yop synthesis and secretion, effector translocation, evasion of phagocytosis, killing of immune cells and virulence in a mouse infection model, the mutants were grouped into three phenotypic classes. A particularly interesting mutant class maintained full T3SS function in vitro, but were attenuated for virulence in a murine oral-infection model. To better understand the molecular basis for these phenotypic differences, the effectiveness of RAW 264.7 cells to respond to infection by these mutants was scrutinised. Sixteen individual cytokines were profiled with mouse cytokine screen multiplex analysis. Signature cytokine profiles were observed that could again separate the different YopD mutants into distinct categories. The activation and supression of certain cytokines that function as central innate immune response modulators correlated well with the ability of mutant bacteria to modulate programmed cell death and antiphagocytosis pathways. Hence, the biogenesis of sub-optimal translocon pores alters host cell responsiveness and limits the ability of Yersinia to fortify against attack by both early and late arms of the host innate immune response.The amount of bacteria now resistant to multiple antibiotics is alarming. By providing insights into a common virulence process, this work may ultimately facilitate the design of novel broad-acting inhibitors of type III secretion, and thereby be useful to treat an array of bacterial infections.
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| 4. |
- Sabharwal, Dharmesh, 1986-
(författare)
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Regulatory roles of sRNAs in pathogenesis of Vibrio cholerae
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Gram-negative pathogen Vibrio cholerae uses variety of regulatory molecules to modulate expression of virulence factors. One important regulatory element of microorganisms is small non-coding RNAs (sRNAs), which control various cell functions such as expression of cell membrane proteins, mRNA decay and riboswitches. In this thesis studies, we demonstrated the roles of the sRNAs VrrA in regulation of outer membrane protein expression, biofilm formation and expression of ribosome binding proteins. In addition, we showed that VrrB, a newly discovered sRNA, played a role in amino acid dependent starvation survival of V. cholerae and might functioned as a riboswitch.VrrA, a 140-nt sRNAs in V. cholerae, was controlled by the alternative sigma factor σE. The outer membrane protein, OmpT is known to be regulated by environmental signals such as pH and temperature via the ToxR regulon and carbon source signals via the cAMP–CRP complex. Our studies provide new insight into the regulation of OmpT by signals received via the σE regulon through VrrA. We demonstrated that VrrA down-regulate ompT translation by base-pairing with the 5′ region of the ompT mRNA in a Hfq (RNA chaperone protein) dependent manner.V. cholerae biofilms contain three matrix proteins—RbmA, RbmC and Bap1—and exopolysaccharide. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. In our studies, we demonstrated that VrrA negatively regulated rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation was not stringently dependent on Hfq.In V. cholerae, VC0706 (Vrp) and VC2530 proteins are homologous to ribosome-associated inhibitor A (RaiA) and hibernation promoting factor (HPF) of Escherichia coli, respectively. HPF facilitates stationary phase survival through ribosome hibernation. We showed that VrrA repressed Vrp protein expression by base-pairing to the 5´ region of vrp mRNA and that this regulation required Hfq. We also showed that Vrp was highly expressed during stationary phase growth and associated with the ribosomes of V. cholerae. We further demonstrated that Vrp and VC2530 were important for V. cholerae starvation survival under nutrient-deficient conditions. While VC2530 was down-regulated in bacterial cells lacking vrrA, mutation of vrp resulted in increased expression of VC2530.Riboswitches are an important class of regulators in bacteria, which are most often located in the 5' untranslated region (5´ UTR) of bacterial mRNA. In this study, we discovered the novel non-coding sRNA, VrrB located at the 5´ UTR of a downstream gene encoding Vibrio auxotropic factor A (VafA) for phenylalanine. In V. cholerae, reduced production of VafA was observed in the presence of phenylalanine and phenylpyruvate in the culture media. Some analogs of phenylalanine and phenylpyruvate could also modulate the expression of VafA. Furthermore, bacterial cells lacking the vrrB gene exhibited high production of VafA, suggesting that VrrB might function as a riboswitch that controls VafA expression.
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