5 research outputs found
Mechanisms of the intracellular survival of Francisella tularensis
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-Îł 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-Îł 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-Îł 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-Îł 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
Identification of mechanisms for attenuation of the FSC043 mutant of Francisella tularensis SCHU S4
Previously, we identified a spontaneous, essentially avirulent mutant, FSC043, of the highly virulent strain SCHU S4 of Francisella tularensis subsp. tularensis. We have now characterized the phenotype of the mutant and the mechanisms of its attenuation in more detail. Genetic and proteomic analyses revealed that the pdpE gene and most of the pdpC gene were very markedly downregulated and, as previously demonstrated, that the strain expressed partially deleted and fused fupA and fupB genes. FSC043 showed minimal intracellular replication and induced no cell cytotoxicity. The mutant showed delayed phagosomal escape; at 18 h, colocalization with LAMP-1 was 80%, indicating phagosomal localization, whereas the corresponding percentages for SCHU S4 and the \u394fupA mutant were <10%. However, a small subset of the FSC043-infected cells contained up to 100 bacteria with LAMP-1 colocalization of around 30%. The unusual intracellular phenotype was similar to that of the \u394pdpC and \u394pdpC \u394pdpE mutants. Complementation of FSC043 with the intact fupA and fupB genes did not affect the phenotype, whereas complementation with the pdpC and pdpE genes restored intracellular replication and led to marked virulence. Even higher virulence was observed after complementation with both double-gene constructs. After immunization with the FSC043 strain, moderate protection against respiratory challenge with the SCHU S4 strain was observed. In summary, FSC043 showed a highly unusual intracellular phenotype, and based on our findings, we hypothesize that the mutation in the pdpC gene makes an essential contribution to the phenotype.Peer reviewed: YesNRC publication: Ye
SpojenĂ proteomu amniotickĂ© tekutiny v polovinÄ› trimestru se spontánnĂm pĹ™edÄŤasnĂ˝m porodem a dĂ©lkou tÄ›hotenstvĂ
Background Amniotic fluid is clinically accessible via amniocentesis and its protein composition may correspond to birth timing. Early changes in the amniotic fluid proteome could therefore be associated with the subsequent development of spontaneous preterm delivery. Objective The main objective of this study was to perform unbiased proteomics analysis of the association between mid-trimester amniotic fluid proteome and spontaneous preterm delivery and gestational duration, respectively. A secondary objective was to validate and replicate the findings by enzyme-linked immunosorbent assay using a second independent cohort. Methods Women undergoing a mid-trimester genetic amniocentesis at Sahlgrenska University Hospital/Ostra between September 2008 and September 2011 were enrolled in this study, designed in three analytical stages; 1) an unbiased proteomic discovery phase using LC-MS analysis of 22 women with subsequent spontaneous preterm delivery (cases) and 37 women who delivered at term (controls), 2) a validation phase of proteins of interest identified in stage 1, and 3) a replication phase of the proteins that passed validation using a second independent cohort consisting of 20 cases and 40 matched controls. Results Nine proteins were nominally significantly associated with both spontaneous preterm delivery and gestational duration, after adjustment for gestational age at sampling, but none of the proteins were significant after correction for multiple testing. Several of these proteins have previously been described as being associated with spontaneous PTD etiology and six of them were thus validated using enzyme linked immunosorbent assay. Two of the proteins passed validation; Neutrophil gelatinase-associated lipocalin and plasminogen activator inhibitor 1, but the results could not be replicated in a second cohort. Conclusions Neutrophil gelatinase-associated lipocalin and Plasminogen activator inhibitor 1 are potential biomarkers of spontaneous preterm delivery and gestational duration but the findings could not be replicated. The negative findings are supported by the fact that none of the nine proteins from the exploratory phase were significant after correction for multiple testing.DosavadnĂ vĂ˝zkum Plodová voda je klinicky pĹ™Ăstupná prostĹ™ednictvĂm amniocentĂ©zy a jejĂ proteinovĂ© sloĹľenĂ mĹŻĹľe odpovĂdat naÄŤasovánĂ porodu. ÄŚasnĂ© zmÄ›ny v proteomu plodovĂ© vody by proto mohly bĂ˝t spojeny s následnĂ˝m vĂ˝vojem spontánnĂho pĹ™edÄŤasnĂ©ho porodu. CĂl HlavnĂm cĂlem tĂ©to studie bylo provĂ©st objektivnĂ proteomickou analĂ˝zu asociace mezi proteomem plodovĂ© vody ve stĹ™ednĂm trimestru a spontánnĂm pĹ™edÄŤasnĂ˝m porodem, respektive gestaÄŤnĂ dobou. SekundárnĂm cĂlem bylo ověřit a replikovat nálezy pomocĂ enzymovĂ©ho imunosorbentnĂho testu s pouĹľitĂm druhĂ© nezávislĂ© kohorty. Metody Do tĂ©to studie, která byla koncipována ve tĹ™ech analytickĂ˝ch fázĂch, byly zaĹ™azeny Ĺľeny podstupujĂcĂ genetickou amniocentĂ©zu v polovinÄ› trimestru ve FakultnĂ nemocnici Sahlgrenska / Ostra v obdobĂ od zářà 2008 do zářà 2011; 1) nezaujatá fáze proteomickĂ©ho objevu pomocĂ LC-MS analĂ˝zy 22 Ĺľen s následnĂ˝m spontánnĂm pĹ™edÄŤasnĂ˝m porodem (pĹ™Ăpady) a 37 Ĺľen, kterĂ© porodily v termĂnu (kontroly), 2) ověřovacĂ fáze sledovanĂ˝ch proteinĹŻ identifikovanĂ˝ch ve stadiu 1 a 3 ) replikaÄŤnĂ fáze proteinĹŻ, která prošla validacĂ pomocĂ druhĂ© nezávislĂ© kohorty skládajĂcĂ se z 20 pĹ™ĂpadĹŻ a 40 shodnĂ˝ch kontrol. VĂ˝sledky DevÄ›t proteinĹŻ bylo nominálnÄ› vĂ˝znamnÄ› spojeno jak se spontánnĂm pĹ™edÄŤasnĂ˝m porodem, tak s gestaÄŤnĂm trvánĂm po ĂşpravÄ› na gestaÄŤnĂ vÄ›k pĹ™i odbÄ›ru vzorkĹŻ, ale žádnĂ˝ z proteinĹŻ nebyl vĂ˝znamnĂ˝ po korekci na vĂcenásobnĂ© testovánĂ. NÄ›kterĂ© z tÄ›chto proteinĹŻ byly dĹ™Ăve popsány jako asociovanĂ© se spontánnĂ etiologiĂ PTD a šest z nich bylo tedy validováno pomocĂ enzymovĂ©ho imunosorbentnĂho testu. Dva z proteinĹŻ prošly validacĂ; Lipokalin spojenĂ˝ s neutrofilnĂ gelatinázou a inhibitor aktivátoru plazminogenu 1, ale vĂ˝sledky nemohly bĂ˝t replikovány ve druhĂ© kohortÄ›. ZávÄ›ry Lipokalin spojenĂ˝ s neutrofilnĂ gelatinázou a inhibitor aktivátoru plazminogenu 1 jsou potenciálnĂmi biomarkery spontánnĂho pĹ™edÄŤasnĂ©ho porodu a gestaÄŤnĂho trvánĂ, ale nálezy nelze replikovat. NegativnĂ zjištÄ›nĂ podporuje skuteÄŤnost, Ĺľe žádnĂ˝ z devĂti proteinĹŻ z prĹŻzkumnĂ© fáze nebyl vĂ˝znamnĂ˝ po korekci na vĂcenásobnĂ© testovánĂ
Altered Expression of Fibronectin and Collagens I and IV in Multiple Myeloma and Monoclonal Gammopathy of Undetermined Significance
Multiple myeloma (MM) is an incurable B-cell malignancy that arises in the bone marrow (BM). The malignant cells within the BM have extensive interaction with the structural components of their microenvironment. It has been previously shown that the interactions between MM cells and the BM extracellular matrix (ECM) proteins contribute to drug resistance. To understand the underlying causes of adhesion-mediated drug resistance in MM, the components of human BM ECM available for interactions with MM cells must be characterized. We analyzed the expression and localization of fibronectin, laminin, and collagens I and IV in the core biopsies of normal donors and patients with monoclonal gammopathy of undetermined significance (MGUS) or MM. In addition, we compared the patterns of ECM expression in MM patients with low-, mid-, and high-level plasmacytosis of the BM. Although expression of laminin was the same for all groups tested, levels of fibronectin and collagen I were reduced in MM patients with high-level plasmacytosis. Expression of collagen IV in the BM of MGUS and MM patients was higher than in the BM from normal donors. Compared with the plasma cells isolated from the patients with low- and mid-level plasmacytosis, sorted CD138+ plasma cells from MM patients with high-level plasmacytosis overexpressed collagen IV. Our findings show that, compared with normal controls, the ECM composition of the bone, endosteum, and BM is aberrant in patients with MM, further establishing ECM as a key player in the MM disease process. (J Histochem Cytochem 57:239–247, 2009