Research personnel at the Division of General Microbioloy, Unviersity of Helsinki:
Kaarina Lähteenmäki, General Microbiology, UH
Ritva Virkola, General Microbiology, UH
Maini Kukkonen, General Microbiology, UH, and EU project
Päivi Kyllönen, Academy of Finland
Marjo Suomalainen, General Microbiology, UH
Leandro Lobo, Academy of Finland
Jenni Antikainen, General Microbiology, UH
Sanna Edelman, General Microbiology, UH
Veera Kuparinen, General Microbiology, UH
Ulla Hynönen, General Microbiology, UH
Johanna Haiko, General Microbiology, UH, and Academy of Finland
Key words: human, macrophages, dendritic cells, streptococci, salmonella, cytokines
Presentation of the results/JULKUNEN'S GROUP:
Results
This project aims at defining microbial and host cell factors that initiate the inflammatory response and activation of host innate immunity during bacterial-host cell interactions. As experimental systems we have used pathogenic Gram-negative bacteria Salmonella typhimurium and its PgtE virulence factor negative mutant and Gram-positive pathogenic Streptococcus pyogenes and nonpathogenic Lactobacillus rhamnosus bacterial strains. Host cell systems include primary human macrophages and dendritic cells. In addition, we have analyzed the role of microbe-induced macrophage or DC-specific cytokines in the activation of innate immune responses.
We have shown that in human primary macrophages the studied bacteria induce a very strong proinflammatory cytokine and chemokine response. In human monocyte-derived DCs pathogenic (S. typhimurium and S. pyogenes) and nonpathogenic bacteria (L. rhamnosus) induce a considerably different response. While S. typhimurium and S. pyogenes stimulate DC maturation very well and induce a strong cytokine/chemokine response, L. rhamnosus is a very weak activator of human DCs. Of interest is the observation that both salmonella and streptococci stimulate very strong Th1 type cytokine (IL-2, IL-12, IL-23, IL-27 and IFN-g), which is likely to contribute to an efficient T cell proliferation and polarization towards Th1 type cells. In addition we have analyzed the role T cell-specific cytokine IL-21 in regulating IFN-g gene expression in NK and T cells.
More recently we have characterized functional differencies between human blood-derived myeloid and plasmacytoid dendritic cells. It appears that these different cell types have a significantly different response to viral and bacterial pathogens. As analyzed by oligonucleotide chips we observed that the differentiation of human monocytes to macrophages or myeloid dendritic cells leads to a significantly different host gene expression profiles. It is likely that the differential gene expression profiles in these cells may also explain, why these cells have functionally different properties.
Our research project will give detailed functional information of human macrophages and dendritic cells, which form the most important antigen-presenting cells of the host. It is likely that the information on the virulence mechanisms of certain microbes will give us better possibilities to understand the host cell responses to different microbes. This may lead to novel modalities of treatment and prevention of microbial diseases.
Selected publications:
Veckman V., Miettinen M., Pirhonen J., Sirén J., Matikainen S. and Julkunen I. Streptococci and lactobacilli differentially induce maturation and production of cytokines and chemokines in human monocyte-derived dendritic cells. J. Leukocyte Biol. 75, 764-771, 2004
Sirén J., Pirhonen J., Julkunen I. and Matikainen S. IFN-a regulates TLR-dependent gene expression of IFN-a , IFN-b, IL-28 and IL-29. J. Immunol. 174, 1932-1937, 2005.
Österlund P., Veckman V., Sirén J., Klucher K.M., Matikainen S. and Julkunen I. Gene expression and antiviral activity of type I IFNs and IL-29 in virus-infected human monocyte-derived dendritic cells. J. Virol., 79, 9608-9617, 2005.
Pietilä T.E., Veckman V., Kyllönen P., Lähteenmäki K., Korhonen T. K. and Julkunen I. Activation, cytokine production and intracellular survival of bacteria in Salmonella-infected human monocyte-derived macrophages and dendritic cells. J. Leukocyte Biol. 78, 909-920, 2005.
Lehtonen A., Veckman V., Nikula T., Lahesmaa R., Kinnunen L., Matikainen S. and Julkunen I. Regulated expression of interferon regulatory factor (IRF) 4 during differentiation of human dendritic cells and macrophages. J. Immunol., 175, 6570-6579, 2005.
Veckman V., Österlund P., Fagerlund R., Melén K., Matikainen S. and Julkunen I. TNF-a and IFN-a pretreatment enhances influenza A virus-induced chemokine gene expression in lung epithelial cells. Virology, 345, 96-104, 2006.
Strengell M., Lehtonen A., Matikainen S. and Julkunen I. IL-21 enhances SOCS gene expression and inhibits LPS-induced cutokine production in human monocyte-derived dendritic cells. J. Leukoc. Biol. In press, 2006.
Presentation of the results/KORHONEN'S GROUP:
A Subproject: Interaction of bacterial pathogens and commensals with the plasminogen systemKeywords: adhesion, invasion, plasminogen, basement membranes, Salmonella, Yersinia, Lactobacillus
Results
We initially characterized the multifunctional plasminogen activators (PAs) of Yersinia pestis and Salmonella enterica. These PAs are 73% identical in sequence and belong to the omptin family of transmembrane, beta-barrel outer membrane proteases detected in Gram-negative bacteria. The studies characterized their substate specificity in proteolysis, virulence-associated functions, domain structure of the molecules, as well as evolution.
Pla and PgtE have a dual interaction with LPS. Their functions are inhibited by the O-side chains of LPS. Y. pestis is genetically rough, which allows full function of Pla, whereas Salmonella overcomes this problem inside and as released from mouse macrophages, where the bacteria express short O-chains and expression of PgtE is upregulated. On the other hand, both PAs reguire rough LPS to be proteolytically active. Analysis of defined LPS molecules showed that rough LPS, representing various variants, is optimal for Pla proteolysis and that degree of acylation (affecting LPS polymer structure) as well as presence of unsubstituted phosphates in lipid A are important. Mutagenesis of the conserved lipid A phosphate-binding motif in Pla greatly reduced proteolysis. These finding indicate that both PAs have adapted to the life style of their species (intracellular, extracellular) and that surface proteolysis is dependent on coordinated control of bacterial surface architecture. Purified Pla was also shown to be adhesive to laminin and basement membranes, which confirms its multifunctional nature.
The closest homolog of Pla is the ortholog Epo in a plant-pathogenic bacterium, Erwinia. Epo is not a PA but cleaves plasminogen uncorrectly. The two proteins are 75% identical in sequence, and extensive substitution of surface-associated residues in the two molecules revealed that change of 10 residues is enough to make Epo into a PA. A collaborative foreign biophysics laboratory modeled the docking of Pla onto plasminogen, and the residues in Epo we identified are among those that make the first contact with plasminogen. Omptin genes have spread horizontally in Gram-negative bacteria, and our directed evolution to change substrate specificity of Epo exemplifies how a powerful virulence factor may have evolved from a harmless soil bacterium. Search for Pla domains important for its adhesive/invasive functions was begun.
S. enterica taken from SCVs in mouse macrophages express high PgtE activity. Deletion of pgtE in S. enterica strain 14028 attenuates the strain by tenfold in orally infected mice and decreases by threefold the growth in mouse macrophages. These are first demonstrations for an in vivo virulence role of PgtE. In human monocyte and dendritic cell lines, however, pgtE deletion did not affect bacterial growth, indicating possible host specificify against killing by phagocytes. PgtE is a modest PA compared to Pla, and antiprotease inactivation seems to be one of its major virulence functions. PgtE also activates mouse and human pro-MMP9 into gelatinase, and this takes place also in vivo in the liver and the spleen of infected mice. PgtE also has gelatinase activity of its own, which is not seen with Pla, and by extensive substitutions we turned Pla into a gelatinase. This is the first demonstration of gelatinase activity and mimicry of eukaryotic MMPs in enteric bacteria, and the data also give insight into evolution and domain structure of PgtE.
An omptin ortholog is evident in the published genomic sequences of Legionella pneumophila. In collaboration with a foreign genomics laboratory and for comparison with Pla and PgtE, we expressed and purified Leo, which is a modest PA but active in antiprotease inactivation.
Unexpectedly, secreted plasminogen activation cofactors were detected in several strains of lactobacilli, including probiotic ones. Lactobacilli cause an extremely rapid enhancement of tPA-mediated activation, and this involves secreted forms of enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which we cloned from L. crispatus and expressed for functional studies. This is the first report on plasminogen interaction by lactobacilli, and an intruiging finding was that binding of plasminogen onto lactobacillar GAPDH, in absence of eukaryotic PAs, results in cleavage (seemingly by plasmin itself) into angiostatin-like molecules. In an anlogy to streptokinase and stafylokinase, we termed the novel activity as lactokinase and begun the analysis of its generation. Also, possible biological and probiotic functions of lactokinase have been adressed.
We take part in a collaborative project aiming at use of E. coli type III flagellar secretion machinery to secrete heterologous, virulence-associated peptides into the culture medium or to secrete them on cell surface at different locations on the flagellar filament. The basic methods have been developed.
Selected publications:
Kukkonen M, M Suomalainen, P Kyllönen, K Lähteenmäki, H Lång, R Virkola, IH Helander, O Holst, TK Korhonen 2004. Lack of O-antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica. Mol. Microbiol. 51:215-225.
Kukkonen M, Korhonen TK 2004. The omptin family of enterobacterial surface proteases/adhesins: from Housekeeping in Escherichia coli to systemic spread of Yersinia pestis. Int. J. Med. Microbiol. 294:7-14.
Lähteenmäki K, Edelman S, Korhonen TK 2005 Bacterial metastasis: the host plasminogen system in bacterial invasion. Trends Microbiol. 13:79-85.
Lähteenmäki K, Kyllönen P, Partanen L, Korhonen TK 2005. Antiprotease inactivation by Salmonella enterica released from infected macrophages. Cell. Microbiol. 7:529-538.
Majander K, Anton L, Antikainen J, Lång H, Brummer M, Korhonen TK, Westerlund-Wikström B 2005. Extracellular secretion of polypeptides using a modified Escherichia coli flagellar secretion apparatus.Nature Biotechnol. 23:475-481.
Majander K, Korhonen TK, Westerlund-Wikström B 2005. Simultaneous display of multiple foreign peptides in the FliD capping and the FliC filament proteins of the Escherichia coli flagellum. Appl. Environm. Microbiol. 71:4263-4268.
Pietilä TE, Veckman V, Kyllönen P, Lähteenmäki K, Korhonen TK, Julkunen I 2005. Activation, cytokine production, and intracellular survival of bacteria in Salmonella-infected human monocyte-derived macrophages and dendritic cells. Leukoc. Biol. 78:909-920.
Pouillot F, Derbise A, Kukkonen M, Foulon J, Korhonen TK, Carniel E 2005. Evaluation of O-antigen inactivation on Pla activity and virulence of Yersinia pseudotuberculosis harbouring the pPla plasmid. Microbiology 151:3759-3768.
Mann B, Orihuela C, Antikainen J, Gao G, Sublett J, Korhonen TK, Tuomanen E 2006. Multifunctional role of choline binding protein G in pneumococcal pathogenesis. Infect. Immun. 74:821-829.
An abstract of the research plan (January 2003)