Bioluminescent imaging of Borrelia burgdorferi in vivo demonstrates that the fibronectin-binding protein BBK32 is required for optimal infectivity

ABSTRACT BBK32, a fibronectin-binding protein of Borrelia burgdorferi, is one of many surface lipoproteins that are differentially expressed by the Lyme disease spirochete at various stages of its life cycle. The level of BBK32 expression in B. burgdorferi is highest during infection of the mammalian host and lowest in flat ticks. This temporal expression profile, along with its fibronectin-binding activity, strongly suggests that BBK32 may play an important role in Lyme pathogenesis in the host. To test this hypothesis, we constructed an isogenic BBK32 deletion mutant from wild-type B. burgdorferi B31 by replacing the BBK32 gene with a kanamycin resistance cassette through homologous recombination. We examined both the wild-type strain and the BBK32 deletion mutant extensively in the experimental mouse-tick model of the Borrelia life cycle. Our data indicated that B. burgdorferi lacking BBK32 retained full pathogenicity in mice, regardless of whether mice were infected artificially by syringe inoculation or naturally by tick bite. The loss of BBK32 expression in the mutant had no adverse effect on spirochete acquisition (mouse-to-tick) and transmission (tick-to-mouse) processes. These results suggest that additional B. burgdorferi proteins can complement the function of BBK32, fibronectin binding or otherwise, during the natural spirochete life cycle.

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A novel fibronectin-binding protein of Streptococcus suis serotype 2 contributes to epithelial cell invasion and in vivo dissemination

Veterinary Microbiology ◽

10.1016/j.vetmic.2012.09.004 ◽

2013 ◽

Vol 162 (1) ◽

pp. 186-194 ◽

Cited By ~ 25

Author(s):

Wei Li ◽

Yun Wan ◽

Zui Tao ◽

Huanchun Chen ◽

Rui Zhou

Keyword(s):

Epithelial Cell ◽

Cell Invasion ◽

Binding Protein ◽

Streptococcus Suis ◽

Fibronectin Binding Protein ◽

Fibronectin Binding ◽

Suis Serotype ◽

Streptococcus Suis Serotype 2 ◽

Epithelial Cell Invasion

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Correlation between fibronectin binding protein A expression level at the surface of recombinant lactococcus lactis and plasmid transfer in vitro and in vivo

BMC Microbiology ◽

10.1186/s12866-014-0248-9 ◽

2014 ◽

Vol 14 (1) ◽

Cited By ~ 12

Author(s):

Juliana F Almeida ◽

Denis Mariat ◽

Vasco Azevedo ◽

Anderson Miyoshi ◽

Alejandra de Moreno de LeBlanc ◽

...

Keyword(s):

Lactococcus Lactis ◽

Binding Protein ◽

Protein A ◽

Plasmid Transfer ◽

Expression Level ◽

Fibronectin Binding Protein ◽

Fibronectin Binding ◽

Recombinant Lactococcus Lactis

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Regulation of Expression of the Fibronectin-Binding Protein BBK32 in Borrelia burgdorferi

Journal of Bacteriology ◽

10.1128/jb.01199-07 ◽

2007 ◽

Vol 189 (22) ◽

pp. 8377-8380 ◽

Cited By ~ 19

Author(s):

Ming He ◽

Bethany K. Boardman ◽

Dalai Yan ◽

X. Frank Yang

Keyword(s):

Environmental Factors ◽

Borrelia Burgdorferi ◽

Response Regulator ◽

Binding Protein ◽

Regulation Of Expression ◽

Fibronectin Binding Protein ◽

Fibronectin Binding

ABSTRACT The BBK32 protein binds to host extracellular ligand fibronectin and contributes to the pathogenesis of Borrelia burgdorferi. Here we showed that expression of the BBK32 gene is influenced by multiple environmental factors and that its regulation is governed by the response regulator Rrp2 and RpoN-RpoS (σ54-σS) sigma cascade in B. burgdorferi.

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Reassessing the Role of Staphylococcus aureus Clumping Factor and Fibronectin-Binding Protein by Expression in Lactococcus lactis

Infection and Immunity ◽

10.1128/iai.69.10.6296-6302.2001 ◽

2001 ◽

Vol 69 (10) ◽

pp. 6296-6302 ◽

Cited By ~ 115

Author(s):

Yok-Ai Que ◽

Patrice François ◽

Jacques-Antoine Haefliger ◽

José-Manuel Entenza ◽

Pierre Vaudaux ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Lactococcus Lactis ◽

Gene Knockout ◽

Binding Protein ◽

Single Gene ◽

Fibronectin Binding Protein ◽

Fibronectin Binding

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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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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Identification of a 47 kDa fibronectin-binding protein expressed by Borrelia burgdorferi isolate B31

Molecular Microbiology ◽

10.1046/j.1365-2958.1998.01127.x ◽

1998 ◽

Vol 30 (5) ◽

pp. 1003-1015 ◽

Cited By ~ 199

Author(s):

William S. Probert ◽

Barbara J. B. Johnson

Keyword(s):

Borrelia Burgdorferi ◽

Binding Protein ◽

Fibronectin Binding Protein ◽

Fibronectin Binding

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Mapping the Ligand-Binding Region of Borrelia burgdorferi Fibronectin-Binding Protein BBK32

Infection and Immunity ◽

10.1128/iai.69.6.4129-4133.2001 ◽

2001 ◽

Vol 69 (6) ◽

pp. 4129-4133 ◽

Cited By ~ 57

Author(s):

William S. Probert ◽

Jung Hwa Kim ◽

Magnus Höök ◽

Barbara J. B. Johnson

Keyword(s):

Borrelia Burgdorferi ◽

Ligand Binding ◽

Binding Protein ◽

Cellular Adhesion ◽

Binding Activity ◽

Common Mechanism ◽

Binding Region ◽

Cellular Attachment ◽

Fibronectin Binding Protein ◽

Fibronectin Binding

ABSTRACT The cellular attachment and entry of pathogenic microorganisms can be facilitated by the expression of microbial adhesins that bind fibronectin. We have previously described a Borrelia burgdorferi gene, bbk32, that encodes a 47-kDa fibronectin-binding protein. In this study, the ligand-binding region of BBK32 from B. burgdorferi isolate B31 was localized to 32 amino acids. The bbk32 gene was cloned and sequenced from three additional B. burgdorferi isolates representing different genospecies of B. burgdorferi sensu lato. All four bbk32 genes encoded proteins having fibronectin-binding activity when expressed in Escherichia coli, and the deduced proteins shared 81 to 91% amino acid sequence identity within the ligand-binding domain. In addition, the ligand-binding region of BBK32 was found to share sequence homology with a fibronectin-binding peptide defined for protein F1 ofStreptococcus pyogenes. The structural and functional similarity between the ligand-binding region of BBK32 and the UR region of protein F1 suggests a common mechanism of cellular adhesion and entry for B. burgdorferi and S. pyogenes.

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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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