M Sarvas

National Institute for Health and Welfare, Finland, Helsinki, Southern Finland Province, Finland

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Publications (96)317.86 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: A gram-negative obligate intracellular bacterium, Chlamydia pneumoniae, is a common respiratory pathogen. Here, we examined the invasion and attachment of C. pneumoniae K6 into nonphagocytic HL epithelial cell line by manipulating host plasma membranes by using cholesterol-depleting methyl-beta-cyclodextrin (MβCD) and cholesterol-loading MβCD complexed cholesterol (chol-MβCD). The invasion was attenuated by MβCD-treatment while chol-MβCD augmented the attachment and invasion. In addition, the invasion was inhibited by cholesterol sequestering reagents, nystatin and filipin. Furthermore, exposure of host cells to sphingomyelinase inhibited the invasion. RNA interference was used to assay the role of clathrin and human scavenger receptor B, type I (SR-BI) in the entry of C. pneumoniae into A549 lung epithelial adenocarcinoma cells. In contrast to Chlamydia trachomatis L2, the entry of C. pneumoniae was found to be independent of clathrin. In addition, the entry was found to be SR-BI-independent, but interestingly, the chlamydial growth was attenuated in the SR-BI-silenced cells. These findings suggest that the attachment and invasion of C. pneumoniae into nonphagocytic epithelial cells is dependent on the formation of cholesterol- and sphingomyelin-rich plasma membrane microdomains, and the entry is a clathrin-independent process. In addition, our data indicate that SR-BI supports the growth of C. pneumoniae in epithelial cells.
    Microbial Pathogenesis 12/2011; 52(3):157-64. · 1.97 Impact Factor
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    ABSTRACT: The Dlt system modulates the density of negative charge in the cell wall of Gram-positive bacteria by substituting anionic polymers (wall and lipoteichoic acids) with d-alanine. The htrA and htrB genes, regulated by the CssRS two-component system (TCS) and encoding membrane-associated protein quality control proteases, were expressed at strongly decreased levels in a mutant with defective Dlt (dltD : : miniTn10) as compared to the dlt(+) wild-type strain under a secretion stress condition (hypersecretion of AmyQ alpha-amylase). The level of HtrA protein in the extracellular proteome of the dltD mutant was decreased consistently. Expression from the promoter of the liaIHGFSR (yvqIHGFEC) operon (P(liaI)) is dependent on the LiaRS TCS. The Dlt defect increased the expression from P(liaI) under two stress conditions, AmyQ hypersecretion and treatment with a cationic antimicrobial peptide (LL-37), but decreased the expression in vancomycin-treated cells. Furthermore, Dlt inactivation enhanced the expression of the YxdJK-regulated yxdL gene in LL-37-treated cells. The increased net negative charge of the cell wall seems to cause varied and opposite effects on the expression of CssRS-, LiaRS- and YxdJK-regulated genes under different stress conditions. The results suggest that TCSs which sense misfolded proteins or peptides are modulated by the density of negative charge in the cell wall. The density of negative charge on the outer surface of the cell membrane did not have a similar effect on TCSs.
    Microbiology 08/2007; 153(Pt 7):2126-36. · 2.85 Impact Factor
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    ABSTRACT: Chlamydia pneumoniae is an intracellular pathogen that grows inside a vacuole, referred to as an inclusion. C. pneumoniae possess a type III secretion system (TTSS), which allows them to secrete effector molecules into the inclusion membrane and to the host cell cytosol. Proteins such as chlamydial outer protein N (CopN) that associate with the inclusion membrane are potential targets for the host's MHC-dependent antigen presentation, thereby representing ideal antigen candidates for T cell-based vaccination. The results of this study showed that intranasal immunization of BALB/c mice with heat-aggregated CopN protein and an Escherichia coli heat-labile toxin (LT) induced a strong immune response, detected as antigen-specific antibody production, lymphocyte proliferation and IFN-gamma production. Furthermore, the immunization induced statistically significant protection against intranasal C. pneumoniae challenge, the level of which correlated with the magnitude of CopN-specific lymphocyte proliferation. Both heat-aggregation of the antigen and the presence of LT adjuvant were required for maximal protective effect.
    Vaccine 02/2007; 25(2):283-90. · 3.49 Impact Factor
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    ABSTRACT: Chlamydia pneumoniae is a common human respiratory pathogen, and sera from infected individuals recognize several proteins of C. pneumoniae. We produced C. pneumoniae-specific proteins in a Bacillus subtilis expression system. We then used these recombinant C. pneumoniae proteins and purified C. pneumoniae elementary bodies as antigens in enzyme immunoassays to assess the kinetics and protein specificity of the systemic and mucosal antibody responses induced by C. pneumoniae intranasal infection in BALB/c mice. The systemic antibodies in mice recognized strong 'key' immunogens of Chlamydia, Omp2 and Hsp60, but weakly targeted the MOMP protein, the major immunogen in chlamydial species other than C. pneumoniae. The IgA antibodies in bronchial secretions specifically recognized the putative surface protein of C. pneumoniae, Omp4. Our preliminary observations point to the necessity of further characterization of the mucosal antibody response during C. pneumoniae infection.
    Comparative medicine 09/2006; 56(4):272-8. · 1.12 Impact Factor
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    ABSTRACT: PrsA is a peptidyl-prolyl isomerase (PPIase) from Bacillus subtilis belonging to the parvulin family of PPIases. It is a membrane bound lipoprotein at the membrane-wall interface, involved in folding of exported proteins. We present the NMR solution structure of the PPIase domain of PrsA, the first from a Gram-positive bacterium. In addition we mapped out the active site with NMR titration experiments. A high degree of conservation with other members of the parvulin family was revealed in the structure and binding site. Interactions with substrate peptides were also characterized by mutated domains revealing that H122 is indispensable for overall correct folding.
    FEBS Letters 04/2006; 580(7):1822-6. · 3.58 Impact Factor
  • FEMS Microbiology Letters 03/2006; 3(6):323 - 326. · 2.05 Impact Factor
  • Mervi Sibakov, Matti Sarvas, Ilkka Palva
    FEMS Microbiology Letters 03/2006; 17(1‐3):81 - 85. · 2.05 Impact Factor
  • FEMS Microbiology Letters 03/2006; 19(1):119 - 123. · 2.05 Impact Factor
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    ABSTRACT: When Bacillus subtilis-amylase was expressed under the control of sacR in a degU32(Hy) strain, the production of exoenzyme occurred during both the exponential and stationary phases of growth. In each phase, pulse-chase experiments showed that the rate-limiting step of the secretion process was the release of the processed form of the protein in each physiological context. The rate of this event was slightly slower (t1/2=3.2 min) during the stationary phase than during the exponential phase (t1/2=2 min). The effectors which possibly control the efficiency of the release stage, the level of PrsA or the calcium binding properties of the cell wall, remained unchanged throughout growth phases.
    FEMS Microbiology Letters 01/2006; 173(1):127 - 131. · 2.05 Impact Factor
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    Microbial Cell Factories 01/2006; · 3.31 Impact Factor
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    ABSTRACT: CD8+ T cells have been suggested to play an important role in protective immunity against pulmonary Chlamydia pneumoniae infection in mice. Moreover, several classical major histocompatibility complex class I - restricted cytotoxic CD8+ T lymphocytes (CTL) specific for C. pneumoniae- derived peptides have been identified. Here, we studied the outcome of C. pneumoniae infection in human leucocyte antigen (HLA)-A2.1 transgenic mice (HHD mice) that are only able to express a classical human class I molecule (HLA-A2.1). C. pneumoniae infection was self-restricted in HHD mice which were able to develop specific immune responses and a protective immunity against a subsequent rechallenge in a manner comparable to wildtype mice. Furthermore, accumulation of functional and C. pneumoniae-specific T cells to the site of infection was detected after challenge. Antigen processing and HLA-A2.1-dependent presentation was studied by immunizing the HHD mice with chlamydial outer protein N (CopN). Isolation of a peptide-specific CTL line from the CopN-immunized mice suggests that the HLA-A2.1 molecule can support the development of CTL response against a chlamydial protein in mice. These findings suggest that the transgenic mouse model can be used for further characterization of the HLA-A2.1-restricted CD8+ T-cell response during C. pneumoniae infection and for identification of CD8 epitopes from chlamydial antigens.
    Scandinavian Journal of Immunology 09/2005; 62(2):131-9. · 2.20 Impact Factor
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    ABSTRACT: Transcription profiling of all protein-encoding genes of Bacillus subtilis was carried out under several secretion stress conditions in the exponential growth phase. Cells that secreted AmyQ alpha-amylase at a high level were stressed only moderately: seven genes were induced, most significantly htrA and htrB, encoding quality control proteases, and yqxL, encoding a putative CorA-type Mg(2+) transporter. These three genes were induced more strongly by severe secretion stress (prsA3 mutant secreting AmyQ), suggesting that their expression responds to protein misfolding. In addition, 17 other genes were induced, including the liaIHGFSR (yvqIHGFEC) operon, csaA and ffh, encoding chaperones involved in the pretranslocational phase of secretion, and genes involved in cell wall synthesis/modification. Severe secretion stress caused downregulation of 23 genes, including the prsA paralogue yacD. Analysis of a cssS knockout mutant indicated that the absence of the CssRS two-component system, and consequently the absence of the HtrA and HtrB proteases, caused secretion stress. The results also suggest that the htrA and htrB genes comprise the CssRS regulon. B. subtilis cells respond to secretion/folding stress by various changes in gene expression, which can be seen as an attempt to combat the stress condition.
    Applied Microbiology and Biotechnology 06/2005; 67(3):389-96. · 3.69 Impact Factor
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    ABSTRACT: Stress responses of Bacillus subtilis to membrane-active cationic antimicrobial peptides were studied. Global analysis of gene expression by DNA macroarray showed that peptides at a subinhibitory concentration activated numerous genes. A prominent pattern was the activation of two extracytoplasmic function sigma factor regulons, SigW and SigM. Two natural antimicrobial peptides, LL-37 and PG-1, were weak activators of SigW regulon genes, whereas their synthetic analogue poly-L-lysine was clearly a stronger activator of SigW. It was demonstrated for the first time that LL-37 is a strong and specific activator of the YxdJK two-component systems, one of the three highly homologous two-component systems sensing antimicrobial compounds. YxdJK regulates the expression of the YxdLM ABC transporter. The LiaRS (YvqCE) TCS was also strongly activated by LL-37, but its activation is not LL-37 specific, as was demonstrated by its activation with PG-1 and Triton X-100. Other strongly LL-37-induced genes included yrhH and yhcGHI. Taken together, the responses to cationic antimicrobial peptides revealed highly complex regulatory patterns and induction of several signal transduction pathways. The results suggest significant overlap between different stress regulons and interdependence of signal transduction pathways mediating stress responses.
    Microbiology 06/2005; 151(Pt 5):1577-92. · 2.85 Impact Factor
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    ABSTRACT: To explore the potential to enhance secretion of heterologous proteins in Bacillus subtilis by engineering cell factors affecting extracytoplasmic protein folding and degradation. Bottleneck components affecting the extracytoplasmic phase of protein secretion were genetically engineered and their effects on the secretion of 11 industrially interesting heterologous proteins were studied by Western blotting and enzymatic assays. Overproduction of PrsA lipoprotein enhanced the secretion of alpha-amylase of Bacillus stearothermophilus (fourfold) and pneumolysin (1.5-fold). Increasing the net negative charge of the cell wall because of lack of the d-alanine substitution of anionic cell wall polymers enhanced the secretion of pneumolysin c. 1.5-fold. Decreasing the level of HtrA-type quality control proteases caused harmful effects on growth and did not enhance secretion. Pertussis toxin subunit, S1 was found to be a substrate for HtrA-type proteases and its secretion was dependent on these proteases. Secretion of heterologous proteins can be enhanced by engineering components involved in late stages of secretion in a protein-dependent manner. The study revealed both possibilities and limitations of modulating the post-translocational phase of secretion as a means to improve the yield of heterologous proteins.
    Journal of Applied Microbiology 02/2005; 99(2):363-75. · 2.20 Impact Factor
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    ABSTRACT: The transport of proteins from their site of synthesis in the cytoplasm to their functional location is an essential characteristic of all living cells. In Gram-positive bacteria the majority of proteins that are translocated across the cytoplasmic membrane are delivered to the membrane-cell wall interface in an essentially unfolded form. They must then be folded into their native configuration in an environment that is dominated by a high density of immobilised negative charge-in essence an ion exchange resin. It is essential to the viability of the cell that these proteins do not block the translocation machinery in the membrane, form illegitimate interactions with the cell wall or, through intermolecular interactions, form insoluble aggregates. Native Gram-positive proteins therefore have intrinsic folding characteristics that facilitate their rapid folding, and this is assisted by a variety of folding factors, including enzymes, peptides and metal ions. Despite these intrinsic and extrinsic factors, secretory proteins do misfold, particularly if the cell is subjected to certain types of stress. Consequently, Gram-positive bacteria such as Bacillus subtilis encode membrane- and cell wall-associated proteases that act as a quality control machine, clearing misfolded or otherwise aberrant proteins from the translocase and the cell wall.
    Biochimica et Biophysica Acta 12/2004; 1694(1-3):311-27. · 4.66 Impact Factor
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    ABSTRACT: Vaccination against Chlamydia pneumoniae would be a beneficial strategy for either preventing or controlling infection by this human respiratory pathogen that also causes persistent infections. In the present study, we used recombinant Semliki Forest virus (rSFV) particles for delivering C. pneumoniae antigens major outer membrane protein (MOMP) or outer membrane protein 2 (Omp2) to the mice or applied the prime-boost technique, where mice were first primed with naked DNA and then boosted with the viral vector coding for the same proteins. Partial protection suggested by the reduced number of cultivable bacteria from the lungs of the challenged mice was seen in mice immunized by either method with MOMP expressing constructs. A significant protection was also achieved after DNA/rSFV immunization with Omp2. DNA priming followed by rSFV boosting induced a more prominent IFN-gamma production after challenge at the site of the infection in pulmonary and mediastinal cells.
    Vaccine 10/2004; 22(25-26):3386-94. · 3.49 Impact Factor
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    ABSTRACT: The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.
    Journal of Biological Chemistry 05/2004; 279(18):19302-14. · 4.65 Impact Factor
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    ABSTRACT: Bacteriophage PRD1 is an icosahedral dsDNA virus with a diameter of 740 A and an outer protein shell composed of 720 copies of major coat protein P3. Spike complexes at the vertices are composed of a pentameric base (protein P31) and a spike structure (proteins P5 and P2) where the N-terminal region of the trimeric P5 is associated with the base and the C-terminal region of P5 is associated with receptor-binding protein P2. The functionality of proteins P3 and P5 was investigated using insertions and deletions. It was observed that P3 did not tolerate changes whereas P5 tolerated changes much more freely. These properties support the hypothesis that viruses have core structures and functions, which remain stable over time, as well as other elements, responsible for host interactions, which are evolutionally more fluid. The insertional probe used was the apex of exposed loop 4 of group B meningococcal outer membrane protein PorA, a medically important subunit vaccine candidate. It was demonstrated that the epitope could be displayed on the virus surface as part of spike protein P5.
    Virology 07/2003; 310(2):267-79. · 3.37 Impact Factor
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    ABSTRACT: Due to intracellular growth requirements, large-scale cultures of chlamydiae and purification of its proteins are difficult and laborious. To overcome these problems we produced chlamydial proteins in a heterologous host, Bacillus subtilis, a gram-positive nonpathogenic bacterium. The genes of Chlamydia pneumoniae major outer membrane protein (MOMP), the cysteine-rich outer membrane protein (Omp2), and the heat shock protein (Hsp60) were amplified by PCR, and the PCR products were cloned into expression vectors containing a promoter, a ribosome binding site, and a truncated signal sequence of the alpha-amylase gene from Bacillus amyloliquefaciens. C. pneumoniae genes were readily expressed in B. subtilis under the control of the alpha-amylase promoter. The recombinant proteins MOMP and Hsp60 were purified from the bacterial lysate with the aid of the carboxy-terminal histidine hexamer tag by affinity chromatography. The Omp2 was separated as an insoluble fraction after 8 M urea treatment. The purified proteins were successfully used as immunogens and as antigens in serological assays and in a lymphoproliferation test. The Omp2 and Hsp60 antigens were readily recognized by the antibodies appearing after pulmonary infection following intranasal inoculation of C. pneumoniae in mice. Also, splenocytes collected from mice immunized with MOMP or Hsp60 proteins proliferated in response to in vitro stimulation with the corresponding proteins.
    Clinical and Diagnostic Laboratory Immunology 06/2003; 10(3):367-75. · 2.51 Impact Factor
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    ABSTRACT: To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.
    Proceedings of the National Academy of Sciences 05/2003; 100(8):4678-83. · 9.74 Impact Factor

Publication Stats

3k Citations
317.86 Total Impact Points


  • 2011
    • National Institute for Health and Welfare, Finland
      Helsinki, Southern Finland Province, Finland
  • 1986–2007
    • National Public Health Institute
      Helsinki, Southern Finland Province, Finland
  • 1974–2006
    • University of Helsinki
      • Department of Virology
      Helsinki, Province of Southern Finland, Finland
  • 1999
    • Institut Jacques Monod
      Lutetia Parisorum, Île-de-France, France
  • 1993
    • Technical University of Denmark
      • Division of Food Microbiology
      Copenhagen, Capital Region, Denmark
  • 1971
    • University of California, Berkeley
      Berkeley, California, United States
  • 1970
    • Statens kriminaltekniska laboratorium
      Linköping, Östergötland, Sweden