Spatial localization of bacteria controls coagulation of human blood by 'quorum acting'

Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
Nature Chemical Biology (Impact Factor: 13). 01/2009; 4(12):742-50. DOI: 10.1038/nchembio.124
Source: PubMed


Blood coagulation often accompanies bacterial infections and sepsis and is generally accepted as a consequence of immune responses. Though many bacterial species can directly activate individual coagulation factors, they have not been shown to directly initiate the coagulation cascade that precedes clot formation. Here we demonstrated, using microfluidics and surface patterning, that the spatial localization of bacteria substantially affects coagulation of human and mouse blood and plasma. Bacillus cereus and Bacillus anthracis, the anthrax-causing pathogen, directly initiated coagulation of blood in minutes when bacterial cells were clustered. Coagulation of human blood by B. anthracis required secreted zinc metalloprotease InhA1, which activated prothrombin and factor X directly (not via factor XII or tissue factor pathways). We refer to this mechanism as 'quorum acting' to distinguish it from quorum sensing--it does not require a change in gene expression, it can be rapid and it can be independent of bacterium-to-bacterium communication.

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Available from: Andrei P Pomerantsev,
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    • "Also the host’s capacity to form fibrin clots is crucial to contain bacterial spread after blood stream invasion [4,5]. On the other hand increased clot formation was shown to boost bacterial dissemination [6]. Recently, studies exploring the bacteria-host interaction from the host’s side showed that a clotting enzyme receptor, the protease activated receptor (PAR)-1, impairs survival in a mouse pneumococcal pneumonia model [5] further underlining the interplay between bacteria and the host’s clotting components in the modulation of bacterial virulence. "
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    ABSTRACT: The family of 4 related protease-activated receptors (PAR-1, 2, 3 & 4) expressed by mammalian cells allow to sense for and react to extracellular proteolytic activity. Since major human bacterial pathogens secret a wide array of protease(-s) we investigated whether they interfere with human PAR function. Supernatants from cultures of major human bacterial pathogens were assayed for the presence of protease(-s) capable to cleave overexpressed human PAR-1, 2, 3 and 4 reporter constructs. Group A streptococcus (GAS) was found to secret a PAR-1-cleaving protease. Experiments involving genetical and pharmacological gain and loss of function identified streptococcal pyrogenic exotoxin B SpeB as the protease responsible. On the host's side analysis of overexpressed PAR-1 carrying alanine substitutions and deletions showed the amino acid residue leucine44 on PAR-1's extracellular N-terminus to be the only cleavage site. Complementary studies on endogenously expressed PAR-1 using PAR-1 blocking antibodies further supported our conclusion. Through PAR-1 cleavage SpeB efficiently blunted thrombin-induced induction of the ERK-pathway in endothelial cells and prevented platelets aggregation in response to thrombin. Our results identify a novel function of the streptococcal virulence factor SpeB. By cleaving human PAR-1 at the N-terminal amino acid residue leucine44 SpeB rendered endothelial cells unresponsive to thrombin and prevented human platelets from thrombin-induced aggregation. These results suggest that by blunting PAR-1 signaling, SpeB modulates various innate host responses directed against invasive GAS potentially helping the invasive bacteria to escape. This may allow to tailor additional treatments in the future since upon invasion of the blood stream endothelial cells as well as platelets and mononuclear cells respond to PAR-1 agonists aiming to prevent further bacterial dissemination.
    PLoS ONE 11/2013; 8(11):e81298. DOI:10.1371/journal.pone.0081298 · 3.23 Impact Factor
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    • "In addition, this bacterium has increasingly been recognized as an opportunistic pathogen that triggers local and systemic infections such as endophthalmitis, meningitis or septicaemia, which are particularly harmful for immunocompromised patients. Extraintestinal infections arise from the non-specific activities of an array of extracellular, tissue-destructive and cytolytic proteins (for reviews see Stenfors Arnesen et al., 2008; Bottone, 2010), and it has been suggested that the combined activities of haemolysins, neutral proteases and immune inhibitor metalloproteases might counteract host immune responses (Kastrup et al., 2008; Cadot et al., 2010; Guillemet et al., 2010). In contrast, food-borne illnesses , resulting in the diarrheal syndrome or in emetic intoxications, are attributed to specific agents: the poreforming enterotoxins Nhe, CytK and Hbl that act on epithelial cells in the intestine (Beecher and Macmillan, 1991; Lund and Granum, 1996; Lund et al., 2000), and cereulide, a cyclic peptide toxin (Agata et al., 1994). "
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    ABSTRACT: Bacillus cereus causes gastrointestinal diseases and local and systemic infections elicited by the depsipeptide cereulide, enterotoxins, phospholipases, cytolysins and proteases. The PlcR-PapR quorum sensing system activates the expression of several virulence factors, whereas the Spo0A-AbrB regulatory circuit partially controls the plasmid-borne cereulide synthetase (ces) operon. Here, we show that CodY, a nutrient-responsive regulator of Gram-positive bacteria, has a profound effect on both regulatory systems, which have been assumed to operate independently of each other. Deletion of codY resulted in downregulation of virulence genes belonging to the PlcR regulon and a concomitant upregulation of the ces genes. CodY was found to be a repressor of the ces operon, but did not interact with the promoter regions of PlcR-dependent virulence genes in vitro, suggesting an indirect regulation of the latter. Furthermore, CodY binds to the promoter of the immune inhibitor metalloprotease InhA1, demonstrating that CodY directly links B. cereus metabolism to virulence. In vivo studies using a Galleria mellonella infection model, showed that the codY mutant was substantially attenuated, highlighting the importance of CodY as a key regulator of pathogenicity. Our results demonstrate that CodY profoundly modulates the virulence of B. cereus, possibly controlling the development of pathogenic traits in suitable host environments.
    Molecular Microbiology 05/2012; 85(1):67-88. DOI:10.1111/j.1365-2958.2012.08090.x · 4.42 Impact Factor
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    • "B. anthracis InhA, however, has also been suggested to play a role in anthrax virulence through cleavage of numerous host substrates (Chung et al., 2006). More specifically, InhA cleaves host proteins involved in the coagulation cascade potentially facilitating circulatory dissemination of bacteria (Chung et al., 2006; Kastrup et al., 2008). In an anthrax mouse model DinhA (background Ames 35) bacterial infection was less lethal than wild-type Ames 35 bacterial infection (Mukherjee et al., 2011). "
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    ABSTRACT: To achieve widespread dissemination in the host, Bacillus anthracis cells regulate their attachment to host endothelium during infection. Previous studies identified BslA (Bacillus anthracis S-layer Protein A), a virulence factor of B. anthracis, as necessary and sufficient for adhesion of vegetative cells to human endothelial cells. While some factors have been identified, bacteria-specific contributions to BslA mediated adhesion remain unclear. Using the attenuated vaccine Sterne 7702 strain of B. anthracis, we tested the hypothesis that InhA (immune inhibitor A), a B. anthracis protease, regulates BslA levels affecting the bacteria's ability to bind to endothelium. To test this, a combination of inhA mutant and complementation analysis in adhesion and invasion assays, Western blot and InhA inhibitor assays were employed. Results show InhA downregulates BslA activity reducing B. anthracis adhesion and invasion in human brain endothelial cells. BslA protein levels in ΔinhA bacteria were significantly higher than wild-type and complemented strains showing InhA levels and BslA expression are inversely related. BslA was sensitive to purified InhA degradation in a concentration- and time-dependent manner. Taken together these data support the role of InhA regulation of BslA-mediated vegetative cell adhesion and invasion.
    Cellular Microbiology 03/2012; 14(8):1219-30. DOI:10.1111/j.1462-5822.2012.01791.x · 4.92 Impact Factor
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