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1.
mBio ; 4(1): e00535-12, 2012 Dec 26.
Article in English | MEDLINE | ID: mdl-23269830

ABSTRACT

UNLABELLED: Pneumococcal pili have been shown to influence pneumococcal colonization, disease development, and the inflammatory response in mice. The role of the pilus-associated RrgA adhesin in pneumococcal interactions with murine and human macrophages was investigated. Expression of pili with RrgA enhanced the uptake of pneumococci by murine and human macrophages that was abolished by antibodies to complement receptor 3 (CR3) and not seen in CR3-deficient macrophages. Recombinant RrgA, but not pilus subunit RrgC, promoted CR3-mediated phagocytosis of coated beads by murine and human macrophages. Flow cytometry showed that purified CR3 binds pneumococcal cells expressing RrgA, and purified RrgA was shown to interact with CR3 and its I domain. In vivo, RrgA facilitated spread of pneumococci from the upper airways and peritoneal cavity to the bloodstream. Earlier onset of septicemia and more rapidly progressing disease was observed in wild-type mice compared to CR3-deficient mice challenged intranasally or intraperitoneally with pneumococci. Motility assays and time-lapse video microscopy showed that pneumococcal stimulation of macrophage motility required RrgA and CR3. These findings, together with the observed RrgA-dependent increase of intracellular survivors up to 10 h following macrophage infection, suggest that RrgA-CR3-mediated phagocytosis promotes systemic pneumococcal spread from local sites. IMPORTANCE: Streptococcus pneumoniae is a major contributor to morbidity and mortality in infectious diseases globally. Symptomatology is mainly due to pneumococcal interactions with host cells leading to an inflammatory response. However, we still need more knowledge on how pneumococci talk to immune cells and the importance of this interaction. Recently, a novel structure was identified on the pneumococcal surface, an adhesive pilus found in about 30% of clinical pneumococcal isolates. The pilus has been suggested to be important for successful spread of antibiotic-resistant pneumococcal clones globally. Here we sought to identify mechanisms for how the pneumococcal pilin subunit RrgA contributes to disease development by interacting with host immune cells. Our data suggest a new way for how pneumococci may cross talk with phagocytic cells and affect disease progression. An increased understanding of these processes may lead to better strategies for how to treat these common infections.


Subject(s)
Fimbriae Proteins/immunology , Fimbriae Proteins/metabolism , Macrophage-1 Antigen/immunology , Macrophage-1 Antigen/metabolism , Macrophages/immunology , Streptococcus pneumoniae/immunology , Virulence Factors/immunology , Virulence Factors/metabolism , Adhesins, Bacterial/immunology , Adhesins, Bacterial/metabolism , Animals , Blood/microbiology , Cell Movement , Female , Flow Cytometry , Humans , Macrophages/microbiology , Male , Mice , Peritoneal Cavity/microbiology , Phagocytosis , Pneumococcal Infections/immunology , Pneumococcal Infections/microbiology , Protein Binding , Protein Interaction Domains and Motifs , Respiratory System/microbiology , Streptococcus pneumoniae/pathogenicity , Survival Analysis , Time-Lapse Imaging
2.
J Mol Biol ; 393(3): 704-16, 2009 Oct 30.
Article in English | MEDLINE | ID: mdl-19729023

ABSTRACT

The respiratory tract pathogen Streptococcus pneumoniae is a primary cause of morbidity and mortality worldwide. Pili enhance initial adhesion as well as the capacity of pneumococci to cause pneumonia and bacteremia. Pilus-associated sortases (SrtB, SrtC, and SrtD) are involved in the biogenesis of pneumococcal pili, composed of repeating units of RrgB that create the stalk to which the RrgA adhesin and the preferential pilus tip subunit RrgC are covalently associated. Using single sortase-expressing strains, we demonstrate that both pilin-polymerizing sortases SrtB and SrtC can covalently link pili to the peptidoglycan cell wall, a property shared with the non-pilus-polymerizing enzyme SrtD and the housekeeping sortase SrtA. Comparative analysis of the crystal structures of S. pneumoniae SrtC and SrtB revealed structural differences explaining the incapacity of SrtC, but not of SrtB, to incorporate RrgC into the pilus. Accordingly, site-directed mutagenesis of Thr(160) in SrtB to an arginine as in SrtC (Arg(160)) partially converted its substrate specificity into that of SrtC. Solving two crystal structures for SrtC suggests that an opening of a flexible lid and a concomitant cysteine rotation are important for catalysis and the activation of the catalytic cysteine of pilus-associated sortases.


Subject(s)
Aminoacyltransferases/chemistry , Bacterial Proteins/chemistry , Biocatalysis , Cysteine Endopeptidases/chemistry , Fimbriae, Bacterial/enzymology , Streptococcus pneumoniae/enzymology , Amino Acid Sequence , Amino Acid Substitution , Catalytic Domain , Cell Wall/enzymology , Crystallography, X-Ray , Cysteine , Fimbriae Proteins/chemistry , Molecular Sequence Data , Peptidoglycan/chemistry , Sequence Alignment , Substrate Specificity
3.
Cell Host Microbe ; 5(6): 580-92, 2009 Jun 18.
Article in English | MEDLINE | ID: mdl-19527885

ABSTRACT

Most commensal and pathogenic bacteria interacting with eukaryotic hosts express adhesive molecules on their surfaces that promote interaction with host cell receptors or with soluble macromolecules. Even though bacterial attachment to epithelial cells may be beneficial for bacterial colonization, adhesion may come at a cost because bacterial attachment to immune cells can facilitate phagocytosis and clearing. Many pathogenic bacteria have solved this dilemma by producing an antiphagocytic surface layer usually consisting of polysaccharide and by expressing their adhesins on polymeric structures that extend out from the cell surface. In this review, we will focus on the interaction between bacterial adhesins and the host, with an emphasis on pilus-like structures.


Subject(s)
Adhesins, Bacterial/metabolism , Bacteria/pathogenicity , Fimbriae, Bacterial/physiology , Host-Pathogen Interactions , Virulence Factors/metabolism , Models, Biological
4.
J Bacteriol ; 191(5): 1666-76, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19114497

ABSTRACT

The human enteropathogen Yersinia enterocolitica survives and replicates in the lymphoid tissues of its host. Previous in vivo analyses of gene expression revealed that various chromosomal genes are expressed at this stage of infection, but not in vitro. One of these, termed hreP, encodes a protease that is necessary for full virulence of Y. enterocolitica. Using transposon mutagenesis, we identified three genes, pypA, pypB, and pypC, as positive regulators of hreP transcription. PypA is an inner membrane protein with no significant similarity to any known proteins; PypB is a ToxR-like transmembrane transcriptional regulator; and PypC is a cytoplasmic transcriptional regulator with an OmpR-like winged helix-turn-helix DNA binding motif. We show that all Pyp proteins are able to activate hreP independently of each other and that PypB and PypC interact directly with the hreP promoter region. Furthermore, pypB and pypC are autoregulated and regulate each other. Additional data indicate that transcription of hreP is repressed by the histone-like nucleoid-structuring protein H-NS in a temperature-dependent manner. Our data reveal a new regulatory network that might have implications for the controlled expression of further virulence-associated functions in Yersinia.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Subtilisins/metabolism , Bacterial Proteins/genetics , DNA Transposable Elements , DNA-Binding Proteins , Gene Deletion , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutagenesis, Insertional , Promoter Regions, Genetic , Subtilisins/genetics , Yersinia enterocolitica
5.
Mol Microbiol ; 70(3): 595-607, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18761697

ABSTRACT

The rlrA genetic islet encodes an extracellular pilus in the Gram-positive pathogen Streptococcus pneumoniae. Of the three genes for structural subunits, rrgB encodes the major pilin, while rrgA and rrgC encode ancillary pilin subunits decorating the pilus shaft and tip. Deletion of all three pilus-associated sortase genes, srtB, srtC and srtD, completely prevents pilus biogenesis. Expression of srtB alone is sufficient to covalently associate RrgB subunits to one another as well as linking the RrgA adhesin and the RrgC subunit into the polymer. The active-site cysteine residue of SrtB (Cys 177) is crucial for incorporating RrgC, even when the two other sortase genes are expressed. SrtC is redundant to SrtB in permitting RrgB polymerization, and in linking RrgA to the RrgB filament, but SrtC is insufficient to incorporate RrgC. In contrast, expression of srtD alone fails to mediate RrgB polymerization, and a srtD mutant assembles heterotrimeric pilus indistinguishable from wild type. Topological studies demonstrate that pilus antigens are localized to symmetric foci at the cell surface in the presence of all three sortases. This symmetric focal presentation is abrogated in the absence of either srtB or srtD, while deletion of srtC had no effect. In addition, strains expressing srtB alone or srtC alone also displayed disrupted antigen localization, despite polymerizing subunits. Our data suggest that both SrtB and SrtC act as pilus subunit polymerases, with SrtB processing all three pilus subunit proteins, while SrtC only RrgB and RrgA. In contrast, SrtD does not act as a pilus subunit polymerase, but instead is required for wild-type focal presentation of the pilus at the cell surface.


Subject(s)
Aminoacyltransferases/metabolism , Bacterial Proteins/metabolism , Cysteine Endopeptidases/metabolism , Fimbriae Proteins/metabolism , Fimbriae, Bacterial/ultrastructure , Streptococcus pneumoniae/metabolism , Adhesins, Bacterial/genetics , Adhesins, Bacterial/metabolism , Aminoacyltransferases/genetics , Bacterial Proteins/genetics , Cysteine Endopeptidases/genetics , Fimbriae Proteins/genetics , Fimbriae, Bacterial/genetics , Fimbriae, Bacterial/metabolism , Genes, Bacterial , INDEL Mutation , Microscopy, Atomic Force , Microscopy, Fluorescence , Microscopy, Immunoelectron , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/ultrastructure , Transformation, Bacterial
6.
Infect Immun ; 75(10): 4990-7, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17682042

ABSTRACT

DNA adenine methyltransferase (Dam) not only regulates basic cellular functions but also interferes with the proper expression of virulence factors in various pathogens. We showed previously that for the human pathogen Yersinia enterocolitica, overproduction of Dam results in increased invasion of epithelial cells. Since invasion and motility are coordinately regulated in Y. enterocolitica, we analyzed the motility of a Dam-overproducing (Dam(OP)) strain and found it to be highly motile. In Dam(OP) strains, the operon encoding the master regulator of flagellum biosynthesis, flhDC, is upregulated. We show that the increased invasion is not due to enhanced expression of known and putative Y. enterocolitica invasion and adhesion factors, such as Inv, YadA, Ail, Myf fibrils, Pil, or Flp pili. However, overproduction of Dam no longer results in increased invasion for an inv mutant strain, indicating that Inv is necessary for increased invasion after overproduction of Dam. Since we show that overproduction of Dam results in an increased amount of rough lipopolysaccharide (LPS) molecules lacking O-antigen side chains, this implies that reduced steric hindrance by LPS might contribute to increased invasion by a Y. enterocolitica Dam(OP) strain. Our data add an important new aspect to the various virulence-associated phenotypes influenced by DNA methylation in Y. enterocolitica and indicate that Dam targets regulatory processes modulating the composition and function of the bacterial surface.


Subject(s)
Gene Expression Regulation, Bacterial , Locomotion/physiology , O Antigens/biosynthesis , Site-Specific DNA-Methyltransferase (Adenine-Specific)/physiology , Yersinia enterocolitica/enzymology , Yersinia enterocolitica/pathogenicity , Adhesins, Bacterial/genetics , Adhesins, Bacterial/physiology , Animals , Bacterial Proteins/biosynthesis , CHO Cells , Cricetinae , Cricetulus , DNA, Bacterial/metabolism , Flagella/genetics , Gene Deletion , Humans , Locomotion/genetics , Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics , Transcription, Genetic , Up-Regulation , Yersinia enterocolitica/genetics , Yersinia enterocolitica/physiology
7.
Int J Med Microbiol ; 297(1): 1-7, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17126598

ABSTRACT

Methylation of DNA by the DNA adenine methyltransferase (Dam) provides an epigenetic signal that influences and regulates numerous physiological processes in the bacterial cell including chromosome replication, mismatch repair, transposition, and transcription. A growing number of reports describe a role for DNA adenine methylation in regulating the expression of various bacterial genes related to virulence in diverse pathogens, suggesting that DNA methylation may be a widespread and versatile regulator of virulence gene expression. Here, we summarize the current knowledge about the influence of DNA methylation on virulence functions and discuss perspectives for future research.


Subject(s)
Bacteria/pathogenicity , DNA Methylation , DNA, Bacterial/metabolism , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Adaptation, Physiological , Adenine , Adhesins, Bacterial , Animals , Bacteria/genetics , Bacteria/metabolism , Bacterial Infections/microbiology , Bacterial Proteins/metabolism , DNA , Gene Expression Regulation, Bacterial , Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics , Virulence
8.
J Bacteriol ; 188(20): 7072-81, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17015646

ABSTRACT

DNA methylation by the DNA adenine methyltransferase (Dam) interferes with the coordinated expression of virulence functions in an increasing number of pathogens. While analyzing the effect of Dam on the virulence of the human pathogen Yersinia enterocolitica, we observed type III secretion of Yop effector proteins under nonpermissive conditions. Dam alters the Ca(2+) regulation of Yop secretion but does not affect the temperature regulation of Yop/Ysc expression. The phenotype is different from that of classical "Ca(2+)-blind" mutants of Yersinia, as Dam-overproducing (Dam(OP)) strains still translocate Yops polarly into eukaryotic cells. Although transcription of the lcrGV and yopN-tyeA operons is slightly upregulated, LcrG is absent from lysates of Dam(OP) bacteria, while the amounts of YopN and TyeA are not changed. We present evidence that clpXP expression increases after Dam overproduction and that the ClpP protease then degrades LcrG, thereby releasing a block in type III secretion. This is the first example of posttranslational regulation of type III secretion by the Clp protease and adds a new flavor to the complex regulatory mechanisms underlying the controlled release of effector proteins from bacterial cells.


Subject(s)
Bacterial Proteins/biosynthesis , Endopeptidase Clp/biosynthesis , Gene Expression Regulation, Bacterial , Site-Specific DNA-Methyltransferase (Adenine-Specific)/biosynthesis , Yersinia enterocolitica/metabolism , Bacterial Outer Membrane Proteins/biosynthesis , Bacterial Proteins/metabolism , Blotting, Western , Calcium/physiology , Cysteine Endopeptidases/biosynthesis , Electrophoresis, Polyacrylamide Gel , Gene Expression , Genes, Reporter , Pore Forming Cytotoxic Proteins , Protein Tyrosine Phosphatases/biosynthesis , Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics , Virulence Factors/biosynthesis , Yersinia enterocolitica/genetics , beta-Galactosidase/analysis , beta-Galactosidase/genetics
9.
Microbiology (Reading) ; 152(Pt 5): 1327-1335, 2006 May.
Article in English | MEDLINE | ID: mdl-16622050

ABSTRACT

It was previously shown that alpha1-antitrypsin (AAT) interacts with the type III secreted (T3S) EspB and EspD proteins of enteropathogenic Escherichia coli (EPEC), resulting in reduced functionality of the proteins. To determine if AAT is also able to interact with T3S proteins of other pathogens, the binding of AAT to Yop proteins of Yersinia enterocolitica was analysed. AAT did not interact with YopB or YopD, which have functions in type III translocation similar to EspB and EspD in EPEC, but specifically interacts with YopM, a member of the leucine-rich repeat (LRR) family of proteins, in overlay and pull-down assays. To determine regions of YopM involved in AAT binding, various N- and C-terminally truncated versions of YopM were recombinantly expressed, and their ability to interact with AAT analysed. All versions tested were able to bind AAT, indicating that at least eight LRR of YopM are sufficient for AAT interaction. The main physiological role of AAT is to inhibit neutrophil elastase; however, elastase was efficiently inhibited by AAT in the presence and absence of YopM, indicating that YopM does not interfere with the anti-protease inhibition activity of AAT, and that the domain of AAT interacting with YopM is not identical to AAT's protease interaction domain. Furthermore, it was shown that elastase efficiently degrades YopM and other Yop proteins. The data suggest that AAT has additional functions in the host response against bacterial infections that are not related to its anti-protease activity.


Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Yersinia enterocolitica/metabolism , alpha 1-Antitrypsin/metabolism , Amino Acid Motifs , Bacterial Outer Membrane Proteins/genetics , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Pancreatic Elastase/antagonists & inhibitors , Pancreatic Elastase/metabolism , Protein Interaction Mapping , Sequence Deletion
10.
Microbiology (Reading) ; 151(Pt 7): 2291-2299, 2005 Jul.
Article in English | MEDLINE | ID: mdl-16000719

ABSTRACT

DNA adenine methyltransferase (Dam) plays an important role in physiological processes of Gram-negative bacteria such as mismatch repair and replication. In addition, Dam regulates the expression of virulence genes in various species. The authors cloned the dam gene of Yersinia enterocolitica and showed that Dam is essential for viability. Dam overproduction in Y. enterocolitica resulted in an increased frequency of spontaneous mutation and decreased resistance to 2-aminopurine; however, these effects were only marginal compared to the effect of overproduction of Escherichia coli-derived Dam in Y. enterocolitica, implying different roles or activities of Dam in mismatch repair of the two species. These differences in Dam function are not the cause for the essentiality of Dam in Y. enterocolitica, as Dam of E. coli can complement a dam defect in Y. enterocolitica. Instead, Dam seems to interfere with expression of essential genes. Furthermore, Dam mediates virulence of Y. enterocolitica. Dam overproduction results in increased tissue culture invasion of Y. enterocolitica, while the expression of specifically in vivo-expressed genes is not altered.


Subject(s)
DNA, Bacterial/metabolism , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Yersinia enterocolitica/metabolism , Animals , CHO Cells , Cricetinae , DNA Repair , Escherichia coli/enzymology , Escherichia coli/genetics , Methylation , Substrate Specificity , Virulence , Yersinia Infections/microbiology , Yersinia enterocolitica/enzymology , Yersinia enterocolitica/pathogenicity
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