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MglA and Igl proteins contribute to the modulation of Francisella tularensis live vaccine strain-containing phagosomes in murine macrophages
Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. (Sjöstedt)
Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. (Sjöstedt)
Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. (Sjöstedt)
(Sjöstedt)
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2008 (Engelska)Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 76, nr 8, s. 3502-3510Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The Francisella tularensis live vaccine strain (LVS), in contrast to its iglC mutant, replicates in the cytoplasm of macrophages. We studied the outcome of infection of the murine macrophagelike cell line J774A.1 with LVS and with iglC, iglD, and mglA mutants, the latter of which is deficient in a global regulator. Compared to LVS, all of the mutants showed impaired intracellular replication up to 72 h, and the number of the mglA mutant bacteria even decreased. Colocalization with LAMP-1 was significantly increased for all mutants compared to LVS, indicating an impaired ability to escape into the cytoplasm. A lysosomal acidity-dependent dye accumulated in approximately 40% of the vacuoles containing mutant bacteria but not at all in vacuoles containing LVS. Preactivation of the macrophages with gamma interferon inhibited the intracellular growth of all strains and significantly increased acidification of phagosomes containing the mutants, but it only slightly increased the LAMP-1 colocalization. The intracellular replication and phagosomal escape of the iglC and iglD mutants were restored by complementation in trans. In conclusion, the IglC, IglD, and MglA proteins each directly or indirectly critically contribute to the virulence of F. tularensis LVS, including its intracellular replication, cytoplasmic escape, and inhibition of acidification of the phagosomes.

Ort, förlag, år, upplaga, sidor
2008. Vol. 76, nr 8, s. 3502-3510
Nationell ämneskategori
Cell- och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-20948DOI: 10.1128/IAI.00226-08PubMedID: 18474647OAI: oai:DiVA.org:umu-20948DiVA, id: diva2:210077
Tillgänglig från: 2009-03-30 Skapad: 2009-03-30 Senast uppdaterad: 2019-03-26Bibliografiskt granskad
Ingår i avhandling
1. Mechanisms of the intracellular survival of Francisella tularensis
Öppna denna publikation i ny flik eller fönster >>Mechanisms of the intracellular survival of Francisella tularensis
2011 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Francisella tularensis is a gram-negative, highly virulent, intracellular bacterium which causes the zoonotic disease tularemia. The subspecies tularensis and holarctica are clinically important, and the former is the more virulent. The intracellular lifestyle of F. tularensis is not completely understood, but after uptake in monocytes, the bacterium escapes from the phagosome within hours and replicates massively in the cytosol. The escape is dependent on factors encoded by the Intracellular Growth Locus (igl) operon, located in the Francisella Pathogenicity Island, FPI. The thesis was aimed to clarify and understand the interaction of F. tularensis strains with the endosomal pathway of monocytic cells in general and the roles of the Igl proteins and the global regulator MglA for this interaction in particular. A focus has also been to elucidate the roles of reactive oxygen and nitrogen species for the intracellular host-parasite interaction.

We show that mutants in the IglB, IglC, or IglD proteins or their regulator MglA of the live vaccine strain, LVS (subspecies holarctica), all demonstrated reduced replication rates and lowered cytopathogenicity compared to the wild type in a J774 mouse macrophage cell model. Colocalization with LAMP-1 was significantly increased for the IglC, IglD and MglA mutants compared to LVS. This indicated an impaired ability to escape into the cytoplasm, while at the same time they, like LVS, partly prevented fusion with lysosomes. IFN-γ activation of the J774 host cells prior to infection had a bactericidal effect on LVS and all of the mutants, though the cidal effect was significantly more pronounced for the mutants. Following IFN-γ activation, a majority of the mutant-containing phagosomesfused with lysosomeswhile LVS remained localized in the cytosol without significantly increased interactions with the endosomal pathway.

Previous studies have revealed that IFN-γ activation of F. tularensis-infected macrophages leads to control of infection but conclusions about the importance of reactive nitrogen and oxygen species on bacterial killing are inconsistent. We found that the growth inhibition resulting from IFN-γ activation could not be attributed to an increased oxidative burst since PMA-induced superoxide production was still inhibited by LVS to the same extent as in non-activated macrophages. On the other hand, reactive nitrogen species may in part have contributed to the cidal effect. To further assess the role of reactive nitrogen species to the killing of F. tularensis, nitric oxide was administrated exogenously to J774 cells infected with LVS. This led to significant killing of intracellular LVS with a concomitant increased phagosomal localization and downregulation of the virulence gene regulator mglA. These effects were reversed by addition of a peroxynitrite decomposition catalyst.

A spontaneous avirulent mutant of subspecies tularensis, strain FSC043, was previously demonstrated to provide protective immunity in mice. Here, microscopic analyses of the strain revealed an unusual intracellular localization with a delayed phagosomal escape. This may account for the low virulence, while at the same time FSC043 remains immunogenic and thereby confers protection. The igl operon is intact in strain FCS043 and we hypothesize that a defect in the FPI gene pdpC contributed to the observed phenotype.

Altogether, this thesis work demonstrates the importance of the mglA and igl genes for the virulence of F. tularensis and specifically their important roles for a functional phagosomal escape and inhibition of the host cell oxidative burst. Also, addition of exogenous nitric oxide likely leads to formation of peroxynitrite intracellularly, a reactive molecule which confines the bacterium to the phagosome and confers a significant bactericidal effect on intracellular F. tularensis.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå universitet, 2011. s. 51
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1435
Nyckelord
Francisella tularensis, Igl, MglA, J774, IFN-γ, RNS, ROS, phagosome
Nationell ämneskategori
Mikrobiologi inom det medicinska området
Forskningsämne
mikrobiologi
Identifikatorer
urn:nbn:se:umu:diva-45869 (URN)978-91-7459-248-1 (ISBN)
Disputation
2011-09-16, E04, Umeå University Hospital, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2011-08-26 Skapad: 2011-08-19 Senast uppdaterad: 2018-06-08Bibliografiskt granskad

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