Production of Fibronectin Binding Protein A at the Surface of Lactococcus lactis Increases Plasmid Transfer In Vitro and In Vivo

ABSTRACT Since Staphylococcus aureus expresses multiple pathogenic factors, studying their individual roles in single-gene-knockout mutants is difficult. To circumvent this problem,S. aureus clumping factor A (clfA) and fibronectin-binding protein A (fnbA) genes were constitutively expressed in poorly pathogenic Lactococcus lactis using the recently described pOri23 vector. The recombinant organisms were tested in vitro for their adherence to immobilized fibrinogen and fibronectin and in vivo for their ability to infect rats with catheter-induced aortic vegetations. In vitro, bothclfA and fnbA increased the adherence of lactococci to their specific ligands to a similar extent as theS. aureus gene donor. In vivo, the minimum inoculum size producing endocarditis in ≥80% of the rats (80% infective dose [ID80]) with the parent lactococcus was ≥107CFU. In contrast, clfA-expressing andfnbA-expressing lactococci required only 105CFU to infect the majority of the animals (P < 0.00005). This was comparable to the infectivities of classical endocarditis pathogens such as S. aureus and streptococci (ID80 = 104 to 105 CFU) in this model. The results confirmed the role ofclfA in endovascular infection, but with a much higher degree of confidence than with single-gene-inactivated staphylococci. Moreover, they identified fnbA as a critical virulence factor of equivalent importance. This was in contrast to previous studies that produced controversial results regarding this very determinant. Taken together, the present observations suggest that if antiadhesin therapy were to be developed, at least both of theclfA and fnbA products should be blocked for the therapy to be effective.

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The Fibrinogen- and Fibronectin-Binding Domains of Staphylococcus aureus Fibronectin-Binding Protein A Synergistically Promote Endothelial Invasion and Experimental Endocarditis

Infection and Immunity ◽

10.1128/iai.00405-08 ◽

2008 ◽

Vol 76 (8) ◽

pp. 3824-3831 ◽

Cited By ~ 63

Author(s):

Lionel Piroth ◽

Yok-Ai Que ◽

Eleonora Widmer ◽

Alexandre Panchaud ◽

Stéphane Piu ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Amino Acid ◽

Binding Protein ◽

Protein A ◽

Binding Domains ◽

Fibrinogen Binding ◽

Fibronectin Binding Protein ◽

Fibronectin Binding

ABSTRACT Staphylococcus aureus experimental endocarditis relies on sequential fibrinogen binding (for valve colonization) and fibronectin binding (for endothelial invasion) conferred by peptidoglycan-attached adhesins. Fibronectin-binding protein A (FnBPA) reconciles these two properties—as well as elastin binding—and promotes experimental endocarditis by itself. Here we attempted to delineate the minimal subdomain of FnBPA responsible for fibrinogen and fibronectin binding, cell invasion, and in vivo endocarditis. A large library of truncated constructs of FnBPA was expressed in Lactococcus lactis and tested in vitro and in animals. A 127-amino-acid subdomain spanning the hinge of the FnBPA fibrinogen-binding and fibronectin-binding regions appeared necessary and sufficient to confer the sum of these properties. Competition with synthetic peptides could not delineate specific fibrinogen- and fibronectin-binding sites, suggesting that dual binding arose from protein folding, irrespective of clearly defined binding domains. Moreover, coexpressing the 127-amino-acid subdomain with remote domains of FnBPA further increased fibrinogen binding by ≥10 times, confirming the importance of domain interactions for binding efficacy. In animals, fibrinogen binding (but not fibronectin binding) was significantly associated with endocarditis induction, whereas both fibrinogen binding and fibronectin binding were associated with disease severity. Moreover, fibrinogen binding also combined with fibronectin binding to synergize the invasion of cultured cell lines significantly, a feature correlating with endocarditis severity. Thus, while fibrinogen binding and fibronectin binding were believed to act sequentially in colonization and invasion, they appeared unexpectedly intertwined in terms of both functional anatomy and pathogenicity (in endocarditis). This unforeseen FnBPA subtlety might bear importance for the development of antiadhesin strategies.

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Impacts of sarA and agr in Staphylococcus aureus Strain Newman on Fibronectin-Binding Protein A Gene Expression and Fibronectin Adherence Capacity In Vitro and in Experimental Infective Endocarditis

Infection and Immunity ◽

10.1128/iai.72.3.1832-1836.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1832-1836 ◽

Cited By ~ 45

Author(s):

Yan-Qiong Xiong ◽

Arnold S. Bayer ◽

Michael R. Yeaman ◽

Willem van Wamel ◽

Adhar C. Manna ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Binding Capacity ◽

Protein A ◽

Aureus Strain ◽

Global Regulators ◽

Fibronectin Binding Protein ◽

Regulatory Loci ◽

Fibronectin Binding

ABSTRACT We investigated the impacts of sarA and agr on fnbA expression and fibronectin-binding capacity in Staphylococcus aureus in vitro and in experimental endocarditis. Although sarA up-regulated and agr down-regulated both fnbA expression and fibronectin binding in vitro and in vivo, fnbA expression was positively regulated in the absence of both global regulators. Thus, additional regulatory loci contribute to fnbA regulation and fibronectin-binding capacities in S. aureus.

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Expression of fibronectin binding protein A (FnBPA) from Staphylococcus aureus at the cell surface of Lactococcus lactis improves its immunomodulatory properties when used as protein delivery vector

Vaccine ◽

10.1016/j.vaccine.2016.01.022 ◽

2016 ◽

Vol 34 (10) ◽

pp. 1312-1318 ◽

Cited By ~ 9

Author(s):

Juliana F. Almeida ◽

Natalia M. Breyner ◽

Miloud Mahi ◽

Bensoltane Ahmed ◽

Bouasria Benbouziane ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Surface ◽

Lactococcus Lactis ◽

Protein Delivery ◽

Binding Protein ◽

Protein A ◽

Delivery Vector ◽

Fibronectin Binding Protein ◽

Immunomodulatory Properties ◽

Fibronectin Binding

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Lactococcus lactis Expressing either Staphylococcus aureus Fibronectin-Binding Protein A or Listeria monocytogenes Internalin A Can Efficiently Internalize and Deliver DNA in Human Epithelial Cells

Applied and Environmental Microbiology ◽

10.1128/aem.00825-09 ◽

2009 ◽

Vol 75 (14) ◽

pp. 4870-4878 ◽

Cited By ~ 68

Author(s):

Silvia Innocentin ◽

Valeria Guimarães ◽

Anderson Miyoshi ◽

Vasco Azevedo ◽

Philippe Langella ◽

...

Keyword(s):

Binding Protein ◽

Protein A ◽

Egfp Expression ◽

Expression Plasmid ◽

Fibronectin Binding Protein ◽

Delivery Vehicles ◽

Fibronectin Binding ◽

Green Fluorescent ◽

Internalin A

ABSTRACT Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA+), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA+ and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA+) or its fibronectin binding domains C and D (L. lactis CD+). L. lactis FnBPA+ and L. lactis InlA+ showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD+ was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA+, L. lactis CD+, and L. lactis InlA+ were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA+, L. lactis CD+, and L. lactis InlA+ were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA+ or L. lactis InlA + . Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles.

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Functional Analysis of Putative Adhesion Factors in Lactobacillus acidophilus NCFM

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.8344-8351.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 8344-8351 ◽

Cited By ~ 249

Author(s):

B. Logan Buck ◽

Eric Altermann ◽

Tina Svingerud ◽

Todd R. Klaenhammer

Keyword(s):

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Binding Protein ◽

Protein A ◽

Surface Proteins ◽

Intestinal Epithelial ◽

Cell Surface Proteins ◽

Fibronectin Binding Protein ◽

Fibronectin Binding

ABSTRACT Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro.

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Increased Virulence of a Fibronectin-Binding Protein Mutant of Staphylococcus aureus in a Rat Model of Pneumonia

Infection and Immunity ◽

10.1128/iai.70.7.3865-3873.2002 ◽

2002 ◽

Vol 70 (7) ◽

pp. 3865-3873 ◽

Cited By ~ 42

Author(s):

Mary C. McElroy ◽

David J. Cain ◽

Christine Tyrrell ◽

Timothy J. Foster ◽

Christopher Haslett

Keyword(s):

Staphylococcus Aureus ◽

Epithelial Cells ◽

Rat Model ◽

Binding Proteins ◽

Binding Protein ◽

Alveolar Epithelial Cells ◽

Alveolar Epithelial ◽

Fibronectin Binding Protein ◽

Fibronectin Binding

ABSTRACT Fibronectin-binding proteins mediate Staphylococcus aureus internalization into nonphagocytic cells in vitro. We have investigated whether fibronectin-binding proteins are virulence factors in the pathogenesis of pneumonia by using S. aureus strain 8325-4 and isogenic mutants in which fibronectin-binding proteins were either deleted (DU5883) or overexpressed [DU5883(pFnBPA4)]. We first demonstrated that fibronectin-binding proteins mediate S. aureus internalization into alveolar epithelial cells in vitro and that S. aureus internalization into alveolar epithelial cells requires actin rearrangement and protein kinase activity. Second, we established a rat model of S. aureus-induced pneumonia and measured lung injury and bacterial survival at 24 and 96 h postinoculation. S. aureus growth and the extent of lung injury were both increased in rats inoculated with the deletion mutant (DU5883) in comparison with rats inoculated with the wild-type (8325-4) and the fibronectin-binding protein-overexpressing strain DU5883(pFnBPA4) at 24 h postinfection. Morphological evaluation of infected lungs at the light and electron microscopic levels demonstrated that S. aureus was present within neutrophils from both 8325-4- and DU5883-inoculated lungs. Our data suggest that fibronectin-binding protein-mediated internalization into alveolar epithelial cells is not a virulence mechanism in a rat model of pneumonia. Instead, our data suggest that fibronectin-binding proteins decrease the virulence of S. aureus in pneumonia.

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