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1.
Ticks Tick Borne Dis ; 10(1): 207-212, 2019 01.
Article in English | MEDLINE | ID: mdl-30391124

ABSTRACT

Spirochetes have developed sophisticated means to successfully colonize host tissues and survive in unfavorable environments. Attachment to human cells is thought to be a key step for the establishment of an infection that causes multiple clinical symptoms. Infection of host tissues largely depends on the ability of spirochetes to attach to different cell types. In this study, we examine the ability of spirochetes belonging to seven distinct genospecies (Borrelia (B.) burgdorferi sensu stricto (s.s.), B. afzelii, B. garinii, B. spielmanii, B. bavariensis, B. lusitaniae, and B. valaisiana) to adhere to human keratinocytes. Among the genospecies analyzed, B. valaisiana and B. spielmanii showed the strongest adherence while B. bavariensis, B. garinii and B. afzelii displayed moderate binding activity. By contrast, only a few cells of B. burgdorferi s.s. and B. lusitaniae bound to keratinocytes. Furthermore, intra-species differences have also been observed among B. garinii, B. bavariensis, B. afzelii, and in particular B. valaisiana. To further assess the role of infection-associated borrelial outer surface proteins for mediating interaction to human cells, a non-adherent and non-infectious B. garinii strains producing distinct complement regulator-acquiring surface proteins (CRASP) were employed. Interestingly, binding capacity to human keratinocytes increased up to four-fold in B. garinii cells producing ErpC but not CspA, CspZ or ErpP compared to wild-type B. garinii cells lacking CRASPs. Taken together, these data provide evidence that distinct borrelial genospecies differ in their ability to bind to human keratinocytes and, in addition, support a role of ErpC as a potential adhesin of spirochetes.


Subject(s)
Adhesins, Bacterial/metabolism , Borrelia/physiology , Host-Pathogen Interactions , Keratinocytes/microbiology , Lyme Disease/microbiology , Membrane Proteins/metabolism , Adhesins, Bacterial/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Line , Genotype , Humans , Membrane Proteins/genetics
2.
Article in English | MEDLINE | ID: mdl-29456970

ABSTRACT

The emerging relapsing fever spirochete Borrelia (B.) miyamotoi is transmitted by ixodid ticks and causes the so-called hard tick-borne relapsing fever or B. miyamotoi disease (BMD). More recently, we identified a surface-exposed molecule, CbiA exhibiting complement binding and inhibitory capacity and rendering spirochetes resistant to complement-mediated lysis. To gain deeper insight into the molecular principles of B. miyamotoi-host interaction, we examined CbiA as a plasmin(ogen) receptor that enables B. miyamotoi to interact with the serine protease plasmin(ogen). Recombinant CbiA was able to bind plasminogen in a dose-dependent fashion. Moreover, lysine residues appear to play a crucial role in the protein-protein interaction as binding of plasminogen was inhibited by the lysine analog tranexamic acid as well as increasing ionic strength. Of relevance, plasminogen bound to CbiA can be converted by urokinase-type plasminogen activator (uPa) to active plasmin which cleaved both, the chromogenic substrate S-2251 and its physiologic substrate fibrinogen. Concerning the involvement of specific amino acids in the interaction with plasminogen, lysine residues located at the C-terminus are frequently involved in the binding as reported for various other plasminogen-interacting proteins of Lyme disease spirochetes. Lysine residues located within the C-terminal domain were substituted with alanine to generate single, double, triple, and quadruple point mutants. However, binding of plasminogen to the mutated CbiA proteins was not affected, suggesting that lysine residues distant from the C-terminus might be involved in the interaction.


Subject(s)
Bacterial Proteins/metabolism , Borrelia/physiology , Complement System Proteins/metabolism , Extracellular Matrix/metabolism , Lyme Disease/metabolism , Lyme Disease/microbiology , Plasminogen/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Complement System Proteins/immunology , Humans , Lyme Disease/immunology , Lysine/chemistry , Lysine/metabolism , Mutagenesis, Site-Directed , Mutation , Osmolar Concentration , Protein Binding , Protein Interaction Domains and Motifs , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
3.
Mol Microbiol ; 99(2): 407-24, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26434356

ABSTRACT

Borrelia (B.) bavariensis exhibits a marked tropism for nervous tissues and frequently causes neurological manifestations in humans. The molecular mechanism by which B. bavariensis overcomes innate immunity, in particular, complement remains elusive. In contrast to other serum-resistant spirochetes, none of the B. bavariensis isolates investigated bound complement regulators of the alternative (AP) and classical pathway (CP) or proteolytically inactivated complement components. Focusing on outer surface proteins BGA66 and BGA71, we demonstrated that both molecules either inhibit AP, CP and terminal pathway (TP) activation, or block activation of the CP and TP respectively. Both molecules bind complement components C7, C8 and C9, and thereby prevent assembly of the terminal complement complex. This inhibitory activity was confirmed by the introduction of the BGA66 and BGA71 encoding genes into a serum-sensitive B. garinii strain. Transformed spirochetes producing either BGA66 or BGA71 overcome complement-mediated killing, thus indicating that both proteins independently facilitate serum resistance of B. bavariensis. The generation of C-terminally truncated proteins as well as a chimeric BGA71 protein lead to the localization of the complement-interacting binding site within the N-terminus. Collectively, our data reveal a novel immune evasion strategy of B. bavariensis that is directed against the activation of the TP.


Subject(s)
Bacterial Proteins/immunology , Borrelia burgdorferi/immunology , Complement System Proteins/immunology , Lyme Disease/immunology , Animals , Bacterial Proteins/genetics , Borrelia burgdorferi/genetics , Complement Membrane Attack Complex/genetics , Complement Membrane Attack Complex/immunology , Humans , Lyme Disease/microbiology , Mice
4.
J Infect Dis ; 213(9): 1388-99, 2016 May 01.
Article in English | MEDLINE | ID: mdl-26681776

ABSTRACT

Acinetobacter baumannii is an emerging opportunistic pathogen, responsible for up to 10% of gram-negative, nosocomial infections. The global increase of multidrug-resistant and pan-resistant Acinetobacter isolates presents clinicians with formidable challenges. To establish a persistent infection,A. baumannii must overcome the detrimental effects of complement as the first line of defense against invading microorganisms. However, the immune evasion principles underlying serum resistance inA. baumannii remain elusive. Here, we identified a novel plasminogen-binding protein, termed CipA. Bound plasminogen, upon conversion to active plasmin, degraded fibrinogen and complement C3b and contributed to serum resistance. Furthermore, CipA directly inhibited the alternative pathway of complement in vitro, irrespective of its ability to bind plasminogen. A CipA-deficient mutant was efficiently killed by human serum and showed a defect in the penetration of endothelial monolayers, demonstrating that CipA is a novel multifunctional protein that contributes to the pathogenesis ofA. baumannii.


Subject(s)
Acinetobacter baumannii/metabolism , Acinetobacter baumannii/pathogenicity , Bacterial Proteins/metabolism , Complement System Proteins/metabolism , Plasminogen/metabolism , Acinetobacter Infections/microbiology , Acinetobacter baumannii/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cell Membrane/chemistry , Complement System Proteins/chemistry , Humans , Plasminogen/chemistry , Protein Binding
6.
PLoS One ; 10(7): e0134418, 2015.
Article in English | MEDLINE | ID: mdl-26230848

ABSTRACT

Acinetobacter baumannii is an important nosocomial pathogen, causing a variety of opportunistic infections of the skin, soft tissues and wounds, urinary tract infections, secondary meningitis, pneumonia and bacteremia. Over 63% of A. baumannii infections occurring in the United States are caused by multidrug resistant isolates, and pan-resistant isolates have begun to emerge that are resistant to all clinically relevant antibiotics. The complement system represents the first line of defense against invading pathogens. However, many A. baumannii isolates, especially those causing severe bacteremia are resistant to complement-mediated killing, though the underlying mechanisms remain poorly understood. Here we show for the first time that A. baumannii binds host-derived plasminogen and we identify the translation elongation factor Tuf as a moonlighting plasminogen-binding protein that is exposed on the outer surface of A. baumannii. Binding of plasminogen to Tuf is at least partly dependent on lysine residues and ionic interactions. Plasminogen, once bound to Tuf can be converted to active plasmin and proteolytically degrade fibrinogen as well as the key complement component C3b. Thus, Tuf acts as a multifunctional protein that may contribute to virulence of A. baumannii by aiding in dissemination and evasion of the complement system.


Subject(s)
Acinetobacter baumannii/metabolism , Bacterial Proteins/metabolism , Peptide Elongation Factors/metabolism , Plasminogen/metabolism , Fibrinogen/metabolism , Humans , Lysine/metabolism , Osmolar Concentration , Peptide Elongation Factors/chemistry , Protein Binding
8.
J Med Chem ; 58(8): 3626-30, 2015 Apr 23.
Article in English | MEDLINE | ID: mdl-25815530

ABSTRACT

Resistance to ß-lactam antibiotics can be mediated by metallo-ß-lactamase enzymes (MBLs). An MBL inhibitor could restore the effectiveness of ß-lactams. We report on the evaluation of approved thiol-containing drugs as inhibitors of NDM-1, VIM-1, and IMP-7. Drugs were assessed by a novel assay using a purchasable fluorescent substrate and thermal shift. Best compounds were tested in antimicrobial susceptibility assay. Using these orthogonal screening methods, we identified drugs that restored the activity of imipenem.


Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Imipenem/pharmacology , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/pharmacology , beta-Lactamase Inhibitors/chemistry , beta-Lactamase Inhibitors/pharmacology , Crystallography, X-Ray , Escherichia coli/drug effects , Escherichia coli Infections/drug therapy , Humans , Klebsiella Infections/drug therapy , Klebsiella pneumoniae/drug effects , Models, Molecular , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/drug effects , beta-Lactam Resistance/drug effects , beta-Lactamases/chemistry , beta-Lactamases/metabolism , beta-Lactams/pharmacology
9.
Infect Immun ; 82(1): 380-92, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24191298

ABSTRACT

CspA of the Lyme disease spirochete Borrelia burgdorferi represents a key molecule in immune evasion, protecting borrelial cells from complement-mediated killing. As previous studies focused almost exclusively on CspA of B. burgdorferi, here we investigate the different binding capacities of CspA orthologs of Borrelia burgdorferi, B. afzelii, and B. spielmanii for complement regulator factor H and plasminogen and their ability to inhibit complement activation by either binding these host-derived plasma proteins or independently by direct interaction with components involved in formation of the lethal, pore-like terminal complement complex. To further examine their function in serum resistance in vivo, a serum-sensitive B. garinii strain was used to generate spirochetes, ectopically producing functional CspA orthologs. Irrespective of their species origin, all three CspA orthologs impart resistance to complement-mediated killing when produced in a serum-sensitive B. garinii surrogate strain. To analyze the inhibitory effect on complement activation and to assess the potential to inactivate C3b by binding of factor H and plasminogen, recombinant CspA orthologs were also investigated. All three CspA orthologs simultaneously bound factor H and plasminogen but differed in regard to their capacity to inactivate C3b via bound plasmin(ogen) and inhibit formation of the terminal complement complex. CspA of B. afzelii binds plasmin(ogen) and inhibits the terminal complement complex more efficiently than CspA of B. burgdorferi and B. spielmanii. Taken together, CspA orthologs of serum-resistant Lyme disease spirochetes act as multifunctional evasion molecules that inhibit complement on two central activation levels, C3b generation and assembly of the terminal complement complex.


Subject(s)
Bacterial Proteins/physiology , Borrelia burgdorferi/physiology , Complement System Proteins/metabolism , Lyme Disease/microbiology , Analysis of Variance , Bacteriolysis/physiology , Blood Bactericidal Activity , Borrelia/physiology , Cells, Cultured , Complement C3b/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Lyme Disease/immunology , Plasminogen/metabolism , Protein Binding/physiology
10.
J Biol Chem ; 288(35): 25229-25243, 2013 Aug 30.
Article in English | MEDLINE | ID: mdl-23861404

ABSTRACT

The Lyme disease spirochete Borrelia burgdorferi lacks endogenous, surface-exposed proteases. In order to efficiently disseminate throughout the host and penetrate tissue barriers, borreliae rely on recruitment of host proteases, such as plasmin(ogen). Here we report the identification of a novel plasminogen-binding protein, BBA70. Binding of plasminogen is dose-dependent and is affected by ionic strength. The BBA70-plasminogen interaction is mediated by lysine residues, primarily located in a putative C-terminal α-helix of BBA70. These lysine residues appear to interact with the lysine-binding sites in plasminogen kringle domain 4 because a deletion mutant of plasminogen lacking that domain was unable to bind to BBA70. Bound to BBA70, plasminogen activated by urokinase-type plasminogen activator was able to degrade both a synthetic chromogenic substrate and the natural substrate fibrinogen. Furthermore, BBA70-bound plasmin was able to degrade the central complement proteins C3b and C5 and inhibited the bacteriolytic effects of complement. Consistent with these functional activities, BBA70 is located on the borrelial outer surface. Additionally, serological evidence demonstrated that BBA70 is produced during mammalian infection. Taken together, recruitment and activation of plasminogen could play a beneficial role in dissemination of B. burgdorferi in the human host and may possibly aid the spirochete in escaping the defense mechanisms of innate immunity.


Subject(s)
Bacterial Proteins/metabolism , Borrelia burgdorferi/metabolism , Plasminogen/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Borrelia burgdorferi/chemistry , Borrelia burgdorferi/genetics , Borrelia burgdorferi/immunology , Complement C3b/chemistry , Complement C3b/genetics , Complement C3b/immunology , Complement C3b/metabolism , Complement C5/chemistry , Complement C5/genetics , Complement C5/immunology , Complement C5/metabolism , Fibrinolysin/chemistry , Fibrinolysin/genetics , Fibrinolysin/immunology , Fibrinolysin/metabolism , Humans , Immunity, Innate , Lyme Disease/genetics , Lyme Disease/immunology , Lyme Disease/metabolism , Plasminogen/chemistry , Plasminogen/genetics , Plasminogen/immunology , Protein Binding , Protein Structure, Tertiary , Urokinase-Type Plasminogen Activator/chemistry , Urokinase-Type Plasminogen Activator/genetics , Urokinase-Type Plasminogen Activator/immunology , Urokinase-Type Plasminogen Activator/metabolism
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