Figure - available from: Infection and Immunity
This content is subject to copyright. Terms and conditions apply.
Analysis of C3b cleavage. Cells were incubated with or without FH (as indicated above the lanes), and then purified human FI and C3b were added. As a control for C3b cleavage, FH, FI, and C3b were coincubated in solution in the absence of cells. The samples were solubilized, subjected to SDS-PAGE, transferred to membranes, and screened with anti-C3 antiserum. Expected sizes of the C3b α and β chains, as well as the factor I-mediated C3b cleavage products α′43 and α′68, are indicated to the right of the panel, with molecular mass standards indicated on the left.
Source publication
The primary causative agent of tick-borne relapsing fever in North America is Borrelia hermsii. It has been hypothesized that B. hermsii evades complement-mediated destruction by binding factor H (FH), a host-derived negative regulator of complement. In vitro, B. hermsii produces a single FH binding protein designated FhbA (FH binding protein A). T...
Citations
... Microbes bind domains other than N-terminal domains FH1-4 so that this region can bind to and downregulate C3b. It has been previously shown that FH bound to BhFhbA retains its cofactor-activity in cleaving C3b both using purified proteins [30] and when BhFhbA is expressed on the bacterial cell surface [55]. We confirmed that FH bound to BdFhbA retains its cofactor-activity ( Fig 5E). ...
... In addition, B. miyamotoi CbiA established serum resistance when expressed in serum sensitive Bo. garinii strain G1 [34]. Surprisingly, an fhbA knockout strain created from B. hermsii strain YOR retained resistance to complement in vitro and in mice, even though the strain did not express FhbA nor bound FH [55]. ...
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 Å 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 .
... Similarly, CspA (CRASP-1) variants are not required for spirochete survival at the mouse tick bite site, though it is expressed at this location (203). Additionally, FH binding protein, FhbA of B. hermsii is not required for murine infection or human serum resistance (204). B. hermsii does not express additional FH binding proteins, suggesting the presence of additional complement evasion strategies (204). ...
... Additionally, FH binding protein, FhbA of B. hermsii is not required for murine infection or human serum resistance (204). B. hermsii does not express additional FH binding proteins, suggesting the presence of additional complement evasion strategies (204). While in vitro studies demonstrate BclA, a FH binding protein of Bacillus anthracis, downregulates complement activation and protects against cell lysis, inoculation of mice with a lethal dose of B. anthracis spores lacking BclA, did not affect animal survival or bacterial burden compared to inoculation with an isogenic wild type strain (72). ...
The complement system is an essential player in innate and adaptive immunity. It consists of three pathways (alternative, classical, and lectin) that initiate either spontaneously (alternative) or in response to danger (all pathways). Complement leads to numerous outcomes detrimental to invaders, including direct killing by formation of the pore-forming membrane attack complex, recruitment of immune cells to sites of invasion, facilitation of phagocytosis, and enhancement of cellular immune responses. Pathogens must overcome the complement system to survive in the host. A common strategy used by pathogens to evade complement is hijacking host complement regulators. Complement regulators prevent attack of host cells and include a collection of membrane-bound and fluid phase proteins. Factor H (FH), a fluid phase complement regulatory protein, controls the alternative pathway (AP) both in the fluid phase of the human body and on cell surfaces. In order to prevent complement activation and amplification on host cells and tissues, FH recognizes host cell-specific polyanionic markers in combination with complement C3 fragments. FH suppresses AP complement-mediated attack by accelerating decay of convertases and by helping to inactivate C3 fragments on host cells. Pathogens, most of which do not have polyanionic markers, are not recognized by FH. Numerous pathogens, including certain bacteria, viruses, protozoa, helminths, and fungi, can recruit FH to protect themselves against host-mediated complement attack, using either specific receptors and/or molecular mimicry to appear more like a host cell. This review will explore pathogen complement evasion mechanisms involving FH recruitment with an emphasis on: (a) characterizing the structural properties and expression patterns of pathogen FH binding proteins, as well as other strategies used by pathogens to capture FH; (b) classifying domains of FH important in pathogen interaction; and (c) discussing existing and potential treatment strategies that target FH interactions with pathogens. Overall, many pathogens use FH to avoid complement attack and appreciating the commonalities across these diverse microorganisms deepens the understanding of complement in microbiology.
... Several subsequent studies have identified additional complement factors, including FhbA, HcpA and CihC, which affect different regulators of complement activation. These factors make RF Borrelia resistant to killing by complement and provide other important virulence properties (Brenner et al., 2013;Fine et al., 2014;Grosskinsky et al., 2009;Grosskinsky et al., 2010;Schott et al., 2010;Woodman et al., 2009) ...
Relapsing fever (RF) is caused by several species of Borrelia; all, except two species, are transmitted to humans by soft (argasid) ticks. The species B. recurrentis is transmitted from one human to another by the body louse, while B. miyamotoi is vectored by hard-bodied ixodid tick species. RF Borrelia have several pathogenic features that facilitate invasion and dissemination in the infected host. In this article we discuss the dynamics of vector acquisition and subsequent transmission of RF Borrelia to their vertebrate hosts. We also review taxonomic challenges for RF Borrelia as new species have been isolated throughout the globe. Moreover, aspects of pathogenesis including symptomology, neurotropism, erythrocyte and platelet adhesion are discussed. We expound on RF Borrelia evasion strategies for innate and adaptive immunity, focusing on the most fundamental pathogenetic attributes, multiphasic antigenic variation. Lastly, we review new and emerging species of RF Borrelia and discuss future directions for this global disease.
... A significant advance in the Borrelia field has been the development of a genetic system for the relapsing fever (RF) spirochetes (Battisti et al., 2008;Fine et al., 2011;Guyard et al., 2013;Lopez et al., 2013;Fine et al., 2014;Raffel et al., 2014;James et al., 2016;Krishnavajhala et al., 2017;James et al., 2018;Jackson-Litteken et al., 2019). The availability of genetic tools and complete genomic sequences for multiple RF species (Lescot et al., 2008;Elbir et al., 2012;Miller et al., 2013;Barbour and Campeau Miller, 2014;Barbour, 2016;Wilder et al., 2016;Elbir et al., 2017;Kuleshov et al., 2020) has permitted investigation of genetically modified RF spirochetes in their respective tick vectors or rodent hosts, and informative comparisons with other Borrelia, as detailed in Radolf and Samuels (2021). ...
Genetic studies in Borrelia require special consideration of the highly segmented genome, complex growth requirements and evolutionary distance of spirochetes from other genetically tractable bacteria. Despite these challenges, a robust molecular genetic toolbox has been constructed to investigate the biology and pathogenic potential of these important human pathogens. In this review we summarize the tools and techniques that are currently available for the genetic manipulation of Borrelia, including the relapsing fever spirochetes, viewing them in the context of their utility and shortcomings. Our primary objective is to help researchers discern what is feasible and what is not practical when thinking about potential genetic experiments in Borrelia. We have summarized published methods and highlighted their critical elements, but we are not providing detailed protocols. Although many advances have been made since B. burgdorferi was first transformed over 25 years ago, some standard genetic tools remain elusive for Borrelia. We mention these limitations and why they persist, if known. We hope to encourage investigators to explore what might be possible, in addition to optimizing what currently can be achieved, through genetic manipulation of Borrelia.
... Further studies investigating variants of FhbA2, FHBP28, HcpA, and BpcA also provide evidence that multiple regions are involved in the interaction with FH (30,34,36,41). Of importance, infection studies utilizing a fhbA deletion mutant demonstrated that FhbA2 is the only FH-binding protein of B. hermsii and the absence of FhbA did not have an impact on serum resistance or infectivity of spirochetes, indicating functionally redundant roles played by other complementinteracting proteins as discussed below (46). ...
Relapsing fever (RF) is claimed a neglected arthropod-borne disease caused by a number of diverse human pathogenic Borrelia (B.) species. These RF borreliae are separated into the groups of tick-transmitted species including B. duttonii, B. hermsii, B. parkeri, B. turicatae, B. hispanica, B. persica, B. caucasica, and B. myiamotoi, and the louse-borne Borrelia species B. recurrentis. As typical blood-borne pathogens achieving high cell concentrations in human blood, RF borreliae (RFB) must outwit innate immunity, in particular complement as the first line of defense. One prominent strategy developed by RFB to evade innate immunity involves inactivation of complement by recruiting distinct complement regulatory proteins, e.g., C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), factor H (FH), FH-like protein-1 (FHL-1), and factor H-related proteins FHR-1 and FHR-2, or binding of individual complement components and plasminogen, respectively. A number of multi-functional, complement and plasminogen-binding molecules from distinct Borrelia species have previously been identified and characterized, exhibiting considerable heterogeneity in their sequences, structures, gene localization, and their capacity to bind host-derived proteins. In addition, RFB possess a unique system of antigenic variation, allowing them to change the composition of surface-exposed variable major proteins, thus evading the acquired immune response of the human host. This review focuses on the current knowledge of the immune evasion strategies by RFB and highlights the role of complement-interfering and infection-associated molecules for the pathogenesis of RFB.
... In fact, at the time of publication of the findings by Guyard et al., successful genetic manipulation of TBRF spirochetes had yet to be reported (Guyard et al., 2006;Battisti et al., 2008;Fine et al., 2011;Lopez et al., 2013). Moreover, genetic manipulation in TBRF spirochetes is still in its infancy, as the btpA mutants reported herein represent just the third publication of targeted mutagenesis in B. turicatae and the eighth publication utilizing targeted mutagenesis in all TBRF spirochetes (Battisti et al., 2008;Fine et al., 2011Fine et al., , 2014Lopez et al., 2013;Raffel et al., 2014;James et al., 2016;Krishnavajhala et al., 2017). Additionally, the btpA::aacC1 mutant is the first published use of a promoterless resistance marker for a mutagenesis approach in TBRF spirochetes. ...
Tick-borne relapsing fever (TBRF), characterized by recurring febrile episodes, is globally distributed and among the most common bacterial infections in some African countries. Despite the public health concern that this disease represents, little is known regarding the virulence determinants required by TBRF Borrelia during infection. Because the chromosomes of TBRF Borrelia show extensive colinearity with those of Lyme disease (LD) Borrelia, the exceptions represent unique genes encoding proteins that are potentially essential to the disparate enzootic cycles of these two groups of spirochetes. One such exception is a gene encoding an HtrA family protease, BtpA, that is present in TBRF Borrelia, but not in LD spirochetes. Previous work suggested that btpA orthologs may be important for resistance to stresses faced during mammalian infection. Herein, proteomic analyses of the TBRF spirochete, Borrelia turicatae, demonstrated that BtpA, as well as proteins encoded by adjacent genes in the B. turicatae genome, were produced in response to culture at mammalian body temperature, suggesting a role in mammalian infection. Further, transcriptional analyses revealed that btpA was expressed with the genes immediately upstream and downstream as part of an operon. To directly assess if btpA is involved in resistance to environmental stresses, btpA deletion mutants were generated. btpA mutants demonstrated no growth defect in response to heat shock, but were more sensitive to oxidative stress produced by t-butyl peroxide compared to wild-type B. turicatae. Finally, btpA mutants were fully infectious in a murine relapsing fever (RF) infection model. These results indicate that BtpA is either not required for mammalian infection, or that compensatory mechanisms exist in TBRF spirochetes to combat environmental stresses encountered during mammalian infection in the absence of BtpA.
... The used strains and models may influence this; recently, in a human FH transgenic mouse increased virulence of the S. pyogenes strain AP1 (which expresses protein H) was observed (39). Similarly, while several borrelial proteins with FH-binding capacity have been described (48), in some cases they may be dispensable for virulence (49). Furthermore, some microorganisms were shown to degrade FH (50)(51)(52). ...
Human-pathogenic microbes possess various means to avoid destruction by our immune system. These include interactions with the host complement system that may facilitate pathogen entry into cells and tissues, expression of molecules that defuse the effector complement components and complexes, and acquisition of host complement inhibitors to downregulate complement activity on the surface of the pathogen. A growing number of pathogenic microorganisms have acquired the ability to bind the complement inhibitor factor H (FH) from body fluids and thus hijack its host protecting function. In addition to FH, binding of FH-related (FHR) proteins was also demonstrated for several microbes. Initial studies assumed that these proteins are complement inhibitors similar to FH. However, recent evidence suggests that FHR proteins may rather enhance complement activation both directly and also by competing with the inhibitor FH for binding to certain ligands and surfaces. This mini review focuses on the role of the main alternative pathway regulator FH in host–pathogen interactions, as well as on the emerging role of the FHR proteins as enhancers of complement activation.
... However, loss-of-function strains currently can only be generated in B. burgdorferi. In addition, any redundant functions provided by other proteins involved in serum resistance in such a strain background may make the defect of a single gene undetectable (Coleman et al., 2008;Fine et al., 2014). Therefore, the alternative strategy is to ectopically produce these factors on the surface of the serum-susceptible spirochetes (gain-of-function strains). ...
Lyme disease and relapsing fever are caused by various Borrelia species. Lyme disease borreliae, the most common vector-borne pathogens in both the U.S. and Europe, are transmitted by Ixodes ticks and disseminate from the site of tick bites to tissues leading to erythema migrans skin rash, arthritis, carditis, and neuroborreliosis. Relapsing fever borreliae, carried by ticks and lice, trigger reoccurring fever episodes. Following transmission, spirochetes survive in the blood to induce bacteremia at the early stages of infection, which is thought to promote evasion of the host complement system. The complement system acts as an important innate immune defense mechanism in humans and vertebrates. Upon activation, the cleaved complement components form complexes on the pathogen surface to eventually promote bacteriolysis. The complement system is negatively modulated by a number of functionally diverse regulators to avoid tissue damage. To evade and inhibit the complement system, spirochetes are capable of binding complement components and regulators. Complement inhibition results in bacterial survival in serum (serum resistance) and is thought to promote bloodstream survival, which facilitates spirochete dissemination and disease manifestations. In this review, we discuss current methodologies to elucidate the mechanisms of Borrelia spp. that promote serum resistance and bloodstream survival, as well as novel methods to study factors responsible for bloodstream survival of Lyme disease borreliae that can be applied to relapsing fever borreliae. Understanding the mechanisms these pathogens utilize to evade the complement system will ultimately aid in the development of novel therapeutic strategies and disease prevention to improve human health.
... Two FHBPs, FhbA and BhCRASP-1, have been identified in B. hermsii strains YOR and HS1, respectively (152,153). However, binding FH is not as important for relapsing fever spirochetes to establish infection as it is for Lyme disease Borrelia (154,155). Further supporting the non-essential nature of binding FH, Woodman et al. (154) found that despite FhbA being surface exposed and strongly binding FH in vitro, only 16% of B. hermsii recovered from the blood of infected mice had detectable levels of bound FH. ...
The emerging pathogen, Borrelia miyamotoi, is a relapsing fever spirochete vectored by the same species of Ixodes ticks that carry the causative agents of Lyme disease in the US, Europe, and Asia. Symptoms caused by infection with B. miyamotoi are similar to a relapsing fever infection. However, B. miyamotoi has adapted to different vectors and reservoirs, which could result in unique physiology, including immune evasion mechanisms. Lyme Borrelia utilize a combination of Ixodes-produced inhibitors and native proteins [i.e., factor H-binding proteins (FHBPs)/complement regulator-acquiring surface proteins, p43, BBK32, BGA66, BGA71, CD59-like protein] to inhibit complement, while some relapsing fever spirochetes use C4b-binding protein and likely Ornithodoros-produced inhibitors. To evade the humoral response, Borrelia utilize antigenic variation of either outer surface proteins (Osps) and the Vmp-like sequences (Vls) system (Lyme borreliae) or variable membrane proteins (Vmps, relapsing fever borreliae). B. miyamotoi possesses putative FHBPs and antigenic variation of Vmps has been demonstrated. This review summarizes and compares the common mechanisms utilized by Lyme and relapsing fever spirochetes, as well as the current state of understanding immune evasion by B. miyamotoi.
... turicatae interactions, especially the effect of blood feeding (in vitro) versus spirochete growth in media on the expression of important B. turicatae genes. Based on their possible roles as virulence factors or related to the survival in the vector, the following B. turicatae genes were considered for our analysis: alp (Marcsisin et al. 2012), bipA (Lopez et al. 2013), fhbA (Fine et al. 2014), bta124 (Wilder et al. 2016), bta131 (Wilder et al. 2016), and bta116 (Wilder et al. 2016). The data from this study also provide new insights in to the dynamics of B. turicatae gene expression after acquisition and its colonization in various soft ticks developmental stages. ...
... The lower mRNA level of B. turicatae bipA and fhbA in fed ticks in comparison with the levels seen in the in vitro cultures suggests that these genes may not be essential for spirochete colonization in these ticks. Recent study has shown that B. hermsii FhbA is not required for infectivity in mice or for resistance to human complement in vitro (Lopez et al. 2008, Fine et al. 2014. Although the functional role for B. turicatae FhbA in the infection of a vertebrate host is yet to be explored, our study provides information that this molecule may not be essential for spirochete persistence in ticks. ...
In the Midwestern, Southwestern, and Southern part of the United States, the soft tick Ornithodoros turicata transmits the spirochete Borrelia turicatae, the causative agent of relapsing fever in humans. In this study, we report a simplified and an efficient method of in vitro feeding to evaluate O. turicata–B. turicatae interactions. Both nymphal and adult female ticks successfully acquired spirochetes upon in vitro feeding on the B. turicatae-infected blood. We also noted transstadial transmission of spirochetes to adult ticks that were molted from nymphs fed on B. turicatae-infected blood. A differential expression pattern for some of the B. turicatae genes was evident after acquisition and colonization of the vector. The levels of arthropod-associated lipoprotein Alp-mRNA were significantly upregulated and the mRNA levels of factor H binding protein FhbA and immunogenic protein BipA were significantly downregulated in the spirochetes after acquisition into ticks in comparison with spirochetes grown in culture medium. In addition, genes such as bta124 and bta116 were significantly upregulated in spirochetes in unfed ticks in comparison with the levels noted in spirochetes after acquisition. These findings represent an efficient in vitro blood-feeding method to study B. turicatae gene expression after acquisition and colonization in these ticks. In summary, we report that B. turicatae survive and develop in the tick host when acquired by in vitro feeding. We also report that B. turicatae genes are differentially expressed in ticks in comparison with the in vitro-grown cultures, indicating influence of tick environment on spirochete gene expression.
