Mechanism of Borrelia immune evasion by FhbA-related proteins

Author summaryRelapsing fever and Lyme Disease are infectious diseases caused by borrelia bacteria. Relapsing fever occurs sporadically worldwide, whereas distribution of Lyme Disease is restricted to the Northern Hemisphere. Both infections are transmitted to humans by blood eating ticks or lice. These infections are often difficult to diagnose due to nonspecific symptoms. To be able to cause infection, borrelia must circumvent the human immune responses. Here we describe a mechanism, how borrelia bacteria protect themselves in the human host by utilizing host proteins. By using X-ray crystallography, we solved the structure of an outer membrane protein FhbA from a relapsing fever causing borreliae, Borrelia hermsii, in complex with human complement regulator factor H. FhbA has a unique alpha-helical fold that has not been reported earlier. The structure of the complex revealed how FhbA binds factor H in a very specific manner. Factor H bound to FhbA on the surface of borrelia protects bacteria from the complement system and lysis. Based on the structure, we performed structure-guided sequence database analysis, which suggests that similar proteins are present in all relapsing fever causing borrelia and possibly in some Lyme disease agents. Immune evasion facilitates survival of Borrelia, leading to infections like relapsing fever and Lyme disease. Important mechanism for complement evasion is acquisition of the main host complement inhibitor, factor H (FH). By determining the 2.2 angstrom crystal structure of Factor H binding protein A (FhbA) from Borrelia hermsii in complex with FH domains 19-20, combined with extensive mutagenesis, we identified the structural mechanism by which B. hermsii utilizes FhbA in immune evasion. Moreover, structure-guided sequence database analysis identified a new family of FhbA-related immune evasion molecules from Lyme disease and relapsing fever Borrelia. Conserved FH-binding mechanism within the FhbA-family was verified by analysis of a novel FH-binding protein from B. duttonii. By sequence analysis, we were able to group FH-binding proteins of Borrelia into four distinct phyletic types and identified novel putative FH-binding proteins. The conserved FH-binding mechanism of the FhbA-related proteins could aid in developing new approaches to inhibit virulence and complement resistance in Borrelia.Peer reviewe

Dientamoeba fragilis - the most common intestinal protozoan in the Helsinki Metropolitan Area, Finland, 2007 to 2017

Despite the global distribution of the intestinal protozoan Dientamoeba fragilis, its clinical picture remains unclear. This results from underdiagnosis: microscopic screening methods either lack sensitivity (wet preparation) or fail to reveal Dientamoeba (formalin-fixed sample). Aim: In a retrospective study setting, we characterised the clinical picture of dientamoebiasis and compared it with giardiasis. In addition, we evaluated an improved approach to formalin-fixed samples for suitability in Dientamoeba diagnostics. Methods: This study comprised four parts: (i) a descriptive part scrutinising rates of Dientamoeba findings; (ii) a methodological part analysing an approach to detect Dientamoebalike structures in formalin samples; (iii) a clinical part corn paring demographics and symptoms between patients with dientamoebiasis (n = 352) and giardiasis (n = 272), and (iv) a therapeutic part (n = 89 patients) investigating correlation between faecal eradication and clinical improvement. Results: The rate of Dientamoeba findings increased 20-fold after introducing criteria for Dientamoeba-like structures in formalin-fixed samples (88.9% sensitivity and 83.3% specificity). A further increase was seen after implementing faecal PCR. Compared with patients with giardiasis, the symptoms in the Dientamoeba group lasted longer and more often included abdominal pain, cramping, faecal urgency and loose rather than watery stools. Resolved symptoms correlated with successful faecal eradication (p<0.001). Conclusions: Previously underdiagnosed, Dientamoeba has become the most frequently recorded pathogenic enteroparasite in Finland. This presumably results from improved diagnostics with either PCR or detection of Dientamoeba-like structures in formalin-fixed samples, an approach applicable also in resource-poor settings. Symptoms of dientamoebiasis differ slightly from those of giardiasis; patients with distressing symptoms require treatment.Peer reviewe

Staphylococcal protein Ecb impairs complement receptor-1 mediated recognition of opsonized bacteria

Staphyloccus aureus is a major human pathogen leading frequently to sepsis and soft tissue infections with abscesses. Multiple virulence factors including several immune modulating molecules contribute to its survival in the host. When S. aureus invades the human body, one of the first line defenses is the complement system, which opsonizes the bacteria with C3b and attract neutrophils by release of chemotactic peptides. Neutrophils express Complement receptor-1 [CR1, CD35) that interacts with the C3b-opsonized particles and thereby plays an important role in pathogen recognition by phagocytic cells. In this study we observed that a fraction of S. aureus culture supernatant prevented binding of C3b to neutrophils. This fraction consisted of S. aureus leukocidins and Efb. The C-terminus of Efb is known to bind C3b and shares significant sequence homology to the extracellular complement binding protein [Ecb). Here we show that S. aureus Ecb displays various mechanisms to block bacterial recognition by neutrophils. The presence of Ecb blocked direct interaction between soluble CR1 and C3b and reduced the cofactor activity of CR1 in proteolytic inactivation of C3b. Furthermore, Ecb could dose-dependently prevent recognition of C3b by cell-bound CR1 that lead to impaired phagocytosis of NHS-opsonized S. aureus. Phagocytosis was furthermore reduced in the presence of soluble CR1 [sCR1). These data indicate that the staphylococcal protein Ecb prevents recognition of C3b opsonized bacteria by neutrophil CR1 leading to impaired killing by phagocytosis and thereby contribute to immune evasion of S. aureus.Peer reviewe

Crystal structure of a tripartite complex between C3dg, C-terminal domains of factor H and OspE of Borrelia burgdorferi

Complement is an important part of innate immunity. The alternative pathway of complement is activated when the main opsonin, C3b coats non-protected surfaces leading to opsonisation, phagocytosis and cell lysis. The alternative pathway is tightly controlled to prevent autoactivation towards host cells. The main regulator of the alternative pathway is factor H (FH), a soluble glycoprotein that terminates complement activation in multiple ways. FH recognizes host cell surfaces via domains 19–20 (FH19-20). All microbes including Borrelia burgdorferi, the causative agent of Lyme borreliosis, must evade complement activation to allow the infectious agent to survive in its host. One major mechanism that Borrelia uses is to recruit FH from host. Several outer surface proteins (Osp) have been described to bind FH via the C-terminus, and OspE is one of them. Here we report the structure of the tripartite complex formed by OspE, FH19-20 and C3dg at 3.18 Å, showing that OspE and C3dg can bind simultaneously to FH19-20. This verifies that FH19-20 interacts via the “common microbial binding site” on domain 20 with OspE and simultaneously and independently via domain 19 with C3dg. The spatial organization of the tripartite complex explains how OspE on the bacterial surface binds FH19-20, leaving FH fully available to protect the bacteria against complement. Additionally, formation of tripartite complex between FH, microbial protein and C3dg might enable enhanced protection, particularly on those regions on the bacteria where previous complement activation led to deposition of C3d. This might be especially important for slow-growing bacteria that cause chronic disease like Borrelia burgdorferi.Peer reviewe

Binding of the complement inhibitor C4b-binding protein to Lyme disease borreliae

The Lyme disease spirochetes, Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. are tick-borne pathogens that can cause chronic disseminated infections. To survive in the human host borreliae need to evade the immune system. It is already well known that B. burgdorferi ss. and B. afzelii bind the complement (C) alternative pathway inhibitor factor H from serum using OspE and CRASP-1/Bba68 proteins to escape C attack. In the presence of natural antibodies and in chronic infections, when specific antibodies develop, borreliae have to protect themselves from antibody-induced classical pathway C attack. In this study we demonstrate binding of the classical pathway inhibitor, C4b-binding protein (C4bp), to three genospecies of B. burgdorferi sensu lato. Binding was strongest to B. garinii, which has been found to be the weakest factor H binder. The bacteria bound both purified I-125-labeled C4bp and C4bp from serum. Unlabeled C4bp competed for binding with I-125-C4bp, whereas BSA had no effect. Binding was salt-sensitive and inhibited by C4b and partially by heparin. C4bp maintained its cofactor activity for factor I in cleaving C4b when bound to the bacterial surface. Ligand blotting analysis of whole cell lysates and fractionated outer cell membranes of the bacteria suggested one major receptor of approximately 43 kDa (P43) for C4bp in B. garinii and B. burgdorferi sensu stricto. Binding of C4bp may thus allow Lyme disease borreliae to escape activation of the classical C pathway and allow chronic infections in humans even in the presence of specific antibodies. (C) 2009 Elsevier Ltd. All rights reserved

Loa loa Microfilariae Evade Complement Attack In Vivo by Acquiring Regulatory Proteins from Host Plasma ▿

Loa loa is a filarial nematode that infects humans. The adults live in subcutaneous tissues and produce microfilariae that live for several weeks in the blood circulation in order to be transmitted to another person via blood meals of a dipterian vector. As microfilariae live in continuous contact with plasma, it is obvious that they evade the complement system. We studied markers of complement activation and signs of complement regulation on Loa loa microfilariae in vivo. The microfilariae were isolated from anticoagulated blood samples of a Loa loa-infected Caucasian patient. C1q and some mannose-binding lectin but only a limited amount of C3b or C4b fragments and practically no C5 or C5b-9 were present on the microfilariae. The covalently microfilaria-bound C3 and C4 depositions were mainly inactive iC3b, C3c, and iC4b fragments indicating that microfilariae had regulated complement activation in vivo. Also, in vitro deposition of C3b onto the microfilariae upon serum exposure was limited. The patient-isolated microfilariae were found to carry the host complement regulators factor H and C4b-binding protein on the outermost layer, so called sheath. The microfilaria-bound factor H was functionally active. Binding of the complement regulators to the microfilariae was confirmed in vitro using 125I-labeled factor H and C4b-binding protein. In conclusion, our study shows that Loa loa microfilariae block complement activation and acquire the host complement regulators factor H and C4b-binding protein in blood circulation. This is the first time that binding of complement regulators onto nonviral pathogens has been demonstrated to occur in humans in vivo