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Nordfelth, Roland
Publications (10 of 15) Show all publications
Bamyaci, S., Nordfelth, R. & Forsberg, Å. (2019). Identification of specific sequence motif of YopN of Yersinia pseudotuberculosis required for systemic infection. Virulence, 10(1), 10-25
Open this publication in new window or tab >>Identification of specific sequence motif of YopN of Yersinia pseudotuberculosis required for systemic infection
2019 (English)In: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 10, no 1, p. 10-25Article in journal (Refereed) Published
Abstract [en]

Type III secretion systems (T3SSs) are tightly regulated key virulence mechanisms shared by many Gram-negative pathogens. YopN, one of the substrates, is also crucial in regulation of expression, secretion and activation of the T3SS of pathogenic Yersinia species. Interestingly, YopN itself is also targeted into host cells but so far no activity or direct role for YopN inside host cells has been described. Recently, we were able show that the central region of YopN is required for efficient translocation of YopH and YopE into host cells. This was also shown to impact the ability of Yersinia to block phagocytosis. One difficulty in studying YopN is to generate mutants that are not impaired in regulation of the T3SS. In this study we extended our previous work and were able to generate specific mutants within the central region of YopN. These mutants were predicted to be crucial for formation of a putative coiled-coil domain (CCD). Similar to the previously described deletion mutant of the central region, these mutants were all impaired in translocation of YopE and YopH. Interestingly, these YopN variants were not translocated into host cells. Importantly, when these mutants were introduced in cis on the virulence plasmid, they retained full regulatory function of T3SS expression and secretion. This allowed us to evaluate one of the mutants, yopNGAGA, in the systemic mouse infection model. Using in vivo imaging technology we could verify that the mutant was also attenuated in vivo and highly impaired to establish systemic infection.

Place, publisher, year, edition, pages
Taylor & Francis, 2019
Keywords
Yersinia, T3SS, YopN, mouse infection, virulence
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-154803 (URN)10.1080/21505594.2018.1551709 (DOI)000453038200001 ()30488778 (PubMedID)2-s2.0-85058611719 (Scopus ID)
Funder
The Kempe Foundations, 161121Swedish Research Council, 2011-3439
Available from: 2019-01-07 Created: 2019-01-07 Last updated: 2019-01-09Bibliographically approved
Bamyaci, S., Ekestubbe, S., Nordfelth, R., Erttmann, S. F., Edgren, T. & Forsberg, Å. (2018). YopN Is Required for Efficient Effector Translocation and Virulence in Yersinia pseudotuberculosis. Infection and Immunity, 86(8), Article ID e00957-17.
Open this publication in new window or tab >>YopN Is Required for Efficient Effector Translocation and Virulence in Yersinia pseudotuberculosis
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2018 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 86, no 8, article id e00957-17Article in journal (Refereed) Published
Abstract [en]

Type III secretion systems (T3SSs) are used by various Gram-negative pathogens to subvert the host defense by a host cell contact-dependent mechanism to secrete and translocate virulence effectors. While the effectors differ between pathogens and determine the pathogenic life style, the overall mechanism of secretion and translocation is conserved. T3SSs are regulated at multiple levels, and some secreted substrates have also been shown to function in regulation. In Yersinia, one of the substrates, YopN, has long been known to function in the host cell contact-dependent regulation of the T3SS. Prior to contact, through its interaction with TyeA, YopN blocks secretion. Upon cell contact, TyeA dissociates from YopN, which is secreted by the T3SS, resulting in the induction of the system. YopN has also been shown to be translocated into target cells by a T3SS-dependent mechanism. However, no intracellular function has yet been assigned to YopN. The regulatory role of YopN involves the N-terminal and C-terminal parts, while less is known about the role of the central region of YopN. Here, we constructed different in-frame deletion mutants within the central region. The deletion of amino acids 76 to 181 resulted in an unaltered regulation of Yop expression and secretion but triggered reduced YopE and YopH translocation within the first 30 min after infection. As a consequence, this deletion mutant lost its ability to block phagocytosis by macrophages. In conclusion, we were able to differentiate the function of YopN in translocation and virulence from its function in regulation.

Keywords
phagocytosis, type III secretion, Yersinia, YopN, virulence
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-150354 (URN)10.1128/IAI.00957-17 (DOI)000439474900006 ()29760214 (PubMedID)
Available from: 2018-08-13 Created: 2018-08-13 Last updated: 2019-01-09Bibliographically approved
Fahlgren, A., Avican, K., Westermark, L., Nordfelth, R. & Fällman, M. (2014). Colonization of cecum is important for development of persistent infection by Yersinia pseudotuberculosis. Infection and Immunity, 82(8), 3471-3482
Open this publication in new window or tab >>Colonization of cecum is important for development of persistent infection by Yersinia pseudotuberculosis
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2014 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 82, no 8, p. 3471-3482Article in journal (Refereed) Published
Abstract [en]

Yersiniosis is a human disease caused by the bacterium Yersinia pseudotuberculosis or Yersinia enterocolitica. The infection is usually resolved but can lead to postinfectious sequelae, including reactive arthritis and erythema nodosum. The commonly used Yersinia mouse infection model mimics acute infection in humans to some extent but leads to systemic infection and eventual death. Here, we analyzed sublethal infection doses of Y. pseudotuberculosis in mice in real time using bioluminescent imaging and found that infections using these lower doses result in extended periods of asymptomatic infections in a fraction of mice. In a search for the site for bacterial persistence, we found that the cecum was the primary colonization site and was the site where the organism resided during a 115-day infection period. Persistent infection was accompanied by sustained fecal shedding of cultivable bacteria. Cecal patches were identified as the primary site for cecal colonization during persistence. Y. pseudotuberculosis bacteria were present in inflammatory lesions, in localized foci, or as single cells and also in neutrophil exudates in the cecal lumen. The chronically colonized cecum may serve as a reservoir for dissemination of infection to extraintestinal sites, and a chronic inflammatory state may trigger the onset of postinfectious sequelae. This novel mouse model for bacterial persistence in cecum has potential as an investigative tool to unveil a deeper understanding of bacterial adaptation and host immune defense mechanisms during persistent infection.

Place, publisher, year, edition, pages
American Society for Microbiology, 2014
National Category
Immunology in the medical area Infectious Medicine
Identifiers
urn:nbn:se:umu:diva-91821 (URN)10.1128/IAI.01793-14 (DOI)000339161400035 ()
Available from: 2014-09-01 Created: 2014-08-18 Last updated: 2018-06-07Bibliographically approved
Vonkavaara, M., Pavel, S. T., Hölzl, K., Nordfelth, R., Sjöstedt, A. & Stöven, S. (2013). Francisella is sensitive to insect antimicrobial peptides. Journal of Innate Immunity, 5(1), 50-59
Open this publication in new window or tab >>Francisella is sensitive to insect antimicrobial peptides
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2013 (English)In: Journal of Innate Immunity, ISSN 1662-811X, E-ISSN 1662-8128, Vol. 5, no 1, p. 50-59Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis causes the zoonotic disease tularemia. Arthropod vectors are important transmission routes for the disease, although it is not known how Francisella survives the efficient arthropod immune response. Here, we used Drosophila melanogaster as a model host for Francisella infections and investigated whether the bacteria are resistant to insect humoral immune responses, in particular to the antimicrobial peptides (AMPs) secreted into the insect hemolymph. Moreover, we asked to which extent such resistance might depend on LPS structure and surface characteristics of the bacteria. We analyzed F. novicida mutant strains in genes, directly or indirectly involved in specific steps of LPS biosynthesis, for virulence in wildtype and Relish E20 immune deficient flies, and tested selected mutants for sensitivity to AMPs in vitro. We demonstrate that Francisella is sensitive to specific fly AMPs, i.e. Attacin, Cecropin, Drosocin and Drosomycin. Furthermore, six bacterial genes, kpsF, manB, lpxF, slt, tolA and pal, were found to be required for resistance to Relish-dependent immune responses, illustrating the importance of structural details of Francisella lipid A and Kdo core for interactions with AMPs. Interestingly, a more negative surface charge and lack of O-antigen did not render mutant bacteria more sensitive to cationic AMPs and attenuated virulence in flies.

Place, publisher, year, edition, pages
Basel: Karger, 2013
Keywords
Francisella tularensis, Drosophila melanogaster, Antimicrobial peptides, Lipopolysaccharide, Host-pathogen interactions
National Category
Microbiology in the medical area
Research subject
Clinical Bacteriology
Identifiers
urn:nbn:se:umu:diva-54412 (URN)10.1159/000342468 (DOI)000313541600006 ()23037919 (PubMedID)
Note

Originally published in thesis in manuscript form.

Available from: 2012-04-26 Created: 2012-04-26 Last updated: 2018-06-08Bibliographically approved
Ur-Rehman, T., Nordfelth, R., Blomgren, A., Zetterström, C. E., Elofsson, M. & Gylfe, Å. (2012). Preliminary pharmacokinetics of the bacterial virulence inhibitor N'-(3,5-dibromo-2-hydroxy-benzylidenene)-nicotinic acid hydrazide. In: Advances in Yersinia Research: (pp. 349-356). Springer
Open this publication in new window or tab >>Preliminary pharmacokinetics of the bacterial virulence inhibitor N'-(3,5-dibromo-2-hydroxy-benzylidenene)-nicotinic acid hydrazide
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2012 (English)In: Advances in Yersinia Research, Springer, 2012, p. 349-356Chapter in book (Other academic)
Abstract [en]

Bacterial virulence inhibitors are potential novel drugs that may be used to treat infections. N′-(3,5-dibromo-2-hydroxy-benzylidene)-nicotinic acid hydrazide, ME0052, has been shown to inhibit type III secretion (T3S) and virulence in several Gram-negative enteric pathogens including Yersinia pseudotuberculosis. In vitro data suggest that ME0052 may be developed into drugs against bacterial gastroenteritis. Here we describe preliminary pharmacokinetics of ME0052 after intraperitoneal and subcutaneous administration in mice. The aim of this work was to identify suitable formulations and to determine pharmacokinetic parameters prior to testing in animal infection models. Peak plasma concentrations above the IC50 for virulence inhibition were achieved with high dose formulations and the elimination half-life was prolonged from 0.5 to 3.4 h using a poloxamer 407-based slow-release formulation.

Place, publisher, year, edition, pages
Springer, 2012
Series
Advances in Experimental Medicine and Biology, ISSN 0065-2598 ; 954
Keywords
salicylidene acylhydrazide, pharmacokinetics, preclinical testing, sustained release formulations, anti-chlamydial activity
National Category
Medicinal Chemistry Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Pharmaceutical Microbiology; Pharmaceutics
Identifiers
urn:nbn:se:umu:diva-40486 (URN)10.1007/978-1-4614-3561-7_42 (DOI)000333327900043 ()978-1-4614-3560-0 (ISBN)978-1-4614-3561-7 (ISBN)
Note

Originally published in thesis in manuscript form with the title: "Preliminary pharmacokinetics of the bacterial virulence inhibitor 3,5-dibromo-2-hydroxy-benzylidene nicotinic acid hydrazide"

Available from: 2011-02-24 Created: 2011-02-23 Last updated: 2018-06-08Bibliographically approved
Thorslund, S. E., Edgren, T., Pettersson, J., Nordfelth, R., Sellin, M. E., Ivanova, E., . . . Fällman, M. (2011). The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function. PLoS ONE, 6(2), e16784
Open this publication in new window or tab >>The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 2, p. e16784-Article in journal (Refereed) Published
Abstract [en]

Many Gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm. This is called antiphagocytosis, and constitutes an important virulence feature of this pathogen since it allows survival in immune cell rich lymphoid organs. We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis. We present data showing that YopK influences Yop effector translocation by modulating the ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, we show that YopK that can interact with the translocators, is exposed inside target cells and binds to the eukaryotic signaling protein RACK1. This protein is engaged upon Y. pseudotuberculosis-mediated beta1-integrin activation and localizes to phagocytic cups. Cells with downregulated RACK1 levels are protected from antiphagocytosis. This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors. Further, a yopK mutant unable to bind RACK1 shows an avirulent phenotype during mouse infection, suggesting that RACK1 targeting by YopK is a requirement for virulence. Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.

Place, publisher, year, edition, pages
San Francisco: Public Library of Science, 2011
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-43212 (URN)10.1371/journal.pone.0016784 (DOI)21347310 (PubMedID)
Available from: 2011-04-22 Created: 2011-04-22 Last updated: 2018-06-08Bibliographically approved
Obi, I., Nordfelth, R. & Francis, M. (2011). Varying dependency of periplasmic peptidylprolyl cis-trans isomerases in promoting Yersinia pseudotuberculosis stress tolerance and pathogenicity. Biochemical Journal, 439(2), 321-332
Open this publication in new window or tab >>Varying dependency of periplasmic peptidylprolyl cis-trans isomerases in promoting Yersinia pseudotuberculosis stress tolerance and pathogenicity
2011 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 439, no 2, p. 321-332Article in journal (Refereed) Published
Abstract [en]

Periplasmic PPIases (peptidylprolyl cis-trans isomerases) catalyse the cis-trans isomerization of peptidyl-prolyl bonds, which is a rate-limiting step during protein folding. We demonstrate that the surA, ppiA, ppiD, fkpA and fklB alleles each encode a periplasmic PPIase in the bacterial pathogen Yersinia pseudotuberculosis. Of these, four were purified to homogeneity. Purified SurA, FkpA and FklB, but not PpiD, displayed detectable PPIase activity in vitro. Significantly, only Y. pseudotuberculosis lacking surA caused drastic alterations to the outer membrane protein profile and FA (fatty acid) composition. They also exhibited aberrant cellular morphology, leaking LPS (lipopolysaccharide) into the extracellular environment. The SurA PPIase is therefore most critical for maintaining Y. pseudotuberculosis envelope integrity during routine culturing. On the other hand, bacteria lacking either surA or all of the genes ppiA, ppiD, fkpA and fklB were sensitive to hydrogen peroxide and were attenuated in mice infections. Thus Y. pseudotuberculosis exhibits both SurA-dependent and -independent requirements for periplasmic PPIase activity to ensure in vivo survival and a full virulence effect in a mammalian host.

Place, publisher, year, edition, pages
United Kingdom: Portland Press Limited, 2011
Keywords
chaperone, immunosuppressant, infection, membrane biogenesis, periplasmic peptidylprolyl cis–trans isomerase, protein folding, survival
National Category
Microbiology
Research subject
Microbiology; Molecular Biology; Biochemistry
Identifiers
urn:nbn:se:umu:diva-48067 (URN)10.1042/BJ20110767 (DOI)21726196 (PubMedID)
Funder
Swedish Research Council, 2006-3869Swedish Research Council, 2009-3660
Available from: 2011-10-10 Created: 2011-10-07 Last updated: 2018-06-08Bibliographically approved
Navarro, L., Koller, A., Nordfelth, R., Wolf-Watz, H., Taylor, S. & Dixon, J. E. (2007). Identification of a molecular target for the Yersinia protein kinase A.. Mol Cell, 26(4), 465-77
Open this publication in new window or tab >>Identification of a molecular target for the Yersinia protein kinase A.
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2007 (English)In: Mol Cell, ISSN 1097-2765, Vol. 26, no 4, p. 465-77Article in journal (Refereed) Published
Abstract [en]

Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject bacterial effector proteins directly into the host cytosol. One of these effectors, the Yersinia serine/threonine protein kinase YpkA, is an essential virulence determinant involved in host actin cytoskeletal rearrangements and in inhibition of phagocytosis. Here we report that YpkA inhibits multiple Galphaq signaling pathways. The kinase activity of YpkA is required for Galphaq inhibition. YpkA phosphorylates Ser47, a key residue located in the highly conserved diphosphate binding loop of the GTPase fold of Galphaq. YpkA-mediated phosphorylation of Ser47 impairs guanine nucleotide binding by Galphaq. Y. pseudotuberculosis expressing wild-type YpkA, but not a catalytically inactive YpkA mutant, interferes with Galphaq-mediated signaling pathways. Identification of a YpkA-mediated phosphorylation site in Galphaq sheds light on the contribution of the kinase activity of YpkA to Yersinia pathogenesis.

Keywords
Actins/physiology, Bacterial Proteins/metabolism, Cell Line, Cyclic AMP-Dependent Protein Kinases/genetics/*metabolism, GTP-Binding Protein alpha Subunits; Gq-G11/metabolism/*physiology, Humans, Kidney, Phosphorylation, Phosphoserine/metabolism, Protein Binding, Recombinant Fusion Proteins/metabolism, Signal Transduction/*physiology, Stress; Mechanical, Transfection, Yersinia enterocolitica/enzymology/*physiology, Yersinia pestis/enzymology/*physiology, rhoA GTP-Binding Protein/metabolism
Identifiers
urn:nbn:se:umu:diva-16674 (URN)17531806 (PubMedID)
Available from: 2007-10-08 Created: 2007-10-08 Last updated: 2018-06-09Bibliographically approved
Kauppi, A., Nordfelth, R., Hägglund, U., Wolf-Watz, H. & Elofsson, M. (2003). Salicylanilides are potent inhibitors of type III secretion in Yersinia. Advances in Experimental Medicine and Biology, 529, 97-100
Open this publication in new window or tab >>Salicylanilides are potent inhibitors of type III secretion in Yersinia
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2003 (English)In: Advances in Experimental Medicine and Biology, Vol. 529, p. 97-100Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-10054 (URN)
Available from: 2008-06-12 Created: 2008-06-12 Last updated: 2018-06-09Bibliographically approved
Kauppi, A., Nordfelth, R., Uvell, H., Wolf-Watz, H. & Elofsson, M. (2003). Targeting bacterial Virulence:  Inhibitors of type III secretion in Yersinia. Chemistry and Biology, 10(3), 241-249
Open this publication in new window or tab >>Targeting bacterial Virulence:  Inhibitors of type III secretion in Yersinia
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2003 (English)In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 10, no 3, p. 241-249Article in journal (Refereed) Published
Abstract [en]

Agents that target bacterial virulence without detrimental effect on bacterial growth are useful chemical probes for studies of virulence and potential candidates for drug development. Several gram-negative pathogens employ type III secretion to evade the innate immune response of the host. Screening of a chemical library with a luciferase reporter gene assay in viable Yersinia pseudotuberculosis furnished several compounds that inhibit the reporter gene signal expressed from the yopE promoter and effector protein secretion at concentrations with no or modest effect on bacterial growth. The selectivity patterns observed for inhibition of various reporter gene strains indicate that the compounds target the type III secretion machinery at different levels. Identification of this set of inhibitors illustrates the approach of utilizing cell-based assays to identify compounds that affect complex bacterial virulence systems.

Identifiers
urn:nbn:se:umu:diva-10053 (URN)doi:10.1016/S1074-5521(03)00046-2 (DOI)
Available from: 2008-06-11 Created: 2008-06-11 Last updated: 2018-06-09Bibliographically approved
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