The Cross-Talk between Spirochetal Lipoproteins and Immunity

Spirochetal diseases such as syphilis, Lyme disease, and leptospirosis are major threats to public health. However, the immunopathogenesis of these diseases has not been fully elucidated. Spirochetes interact with the host through various structural components such as lipopolysaccharides (LPS), surface lipoproteins, and glycolipids. Although spirochetal antigens such as LPS and glycolipids may contribute to the inflammatory response during spirochetal infections, spirochetes such as Treponema pallidum and Borrelia burgdorferi lack LPS. Lipoproteins are most abundant proteins that are expressed in all spirochetes and often determine how spirochetes interact with their environment. Lipoproteins are pro-inflammatory, may regulate responses from both innate and adaptive immunity and enable the spirochetes to adhere to the host or the tick midgut or to evade the immune system. However, most of the spirochetal lipoproteins have unknown function. Herein, the immunomodulatory effects of spirochetal lipoproteins are reviewed and are grouped into two main categories: effects related to immune evasion and effects related to immune activation. Understanding lipoprotein-induced immunomodulation will aid in elucidating innate immunopathogenesis processes and subsequent adaptive mechanisms potentially relevant to spirochetal disease vaccine development and to inflammatory events associated with spirochetal diseases.


INTRODUCTION
Spirochetes are the cause of important human diseases such as syphilis, Lyme disease, and leptospirosis that are major threats to public health (1). However, the immunopathogenesis of these diseases has not been fully elucidated. Tissue inflammation is characteristic of spirochetal diseases such as dermatitis in syphilis and Lyme disease, interstitial nephritis in leptospirosis, and periodontitis caused by oral treponemes (2,3). Spirochetes such as: Treponema pallidum (T. pallidum) and Borrelia burgdorferi (B. burgdorferi), the pathogens for syphilis and Lyme disease, respectively (1), may persist for prolonged periods despite the induced immune responses in the host (4)(5)(6). Several mechanisms may explain how spirochetes may evade host defenses such as intracellular sequestration of the spirochetes, the antigenic variation of the spirochetes, manipulation of host defenses to delay, and/or suppress the onset of effective immune responses and structural features of the outer membrane in spirochetes that contribute to immune evasion (7)(8)(9)(10)(11).
In bacteria, membrane lipoproteins are important virulence factors, pro-inflammatory agonists, enzymes, receptors, modular components of ATP binding cassette (ABC) transporters, and protective immune targets that regulate innate immunity (2,3). In contrast to other bacteria that do not express lipoproteins so abundantly (2,3,27), lipoproteins have an important role in the virulence of spirochetes since they are most abundant proteins that are expressed in all spirochetes (2,3,18,22,(27)(28)(29)(30)(31). The spirochetes express numerous lipoprotein genes [T. pallidum has >20 (24), B. burgdorferi has >100 lipoproteins (23), and approximately 8% of B. burgdorferi genes may encode lipoproteins (21, 23) and Leptospira spp. Have >140 lipoprotein genes (32)]. Examples of abundant lipoproteins in spirochetes include Tp47 of T. pallidum, OspA of B. burgdorferi, LipL32 of Leptospira species, and Vmp proteins of Borrelia species (Table 1). Finally, spirochetal lipoproteins have more prominent pro-inflammatory effects compared to other bacterial lipoproteins and synthetic lipopeptides (28). Surface-exposed lipoproteins often determine how spirochetes interact with their environment and immunity (Figure 1) (2,3,18,22,(27)(28)(29)(30)(31). Lipoproteins may be present in different cellular compartments (2,70,84) and their distribution varies among spirochetes (7,18,(85)(86)(87)(88) (Figure 1). The NH2-terminal lipopeptide region is the part of the lipoprotein that confers its immunologic activity since removal of this lipid component removed the immunoregulatory properties of these lipoproteins www.frontiersin.org FIGURE 1 | Structure of spirochetal membrane and lipoproteins. Similarly to gram-positive bacteria, the spirochetal cytoplasmic membrane is associated with the cell wall that consists of peptidoglycan. Similarly to Gram-negative bacteria, spirochetes also have an outer membrane, which is not attached to the peptidoglycan layer. Spirochetes differ phylogenetically from Gram-negative bacteria and interact with the host through various structural components such as lipopolysaccharides (LPS), surface lipoproteins and glycolipids that are present mostly in the outer membrane. LPS has not been identified in Borrelia and Treponema. The periplasmic space contains the flagellum. The distribution of lipoproteins varies among spirochetes and they may be present in different cellular compartments: the outer membrane, the extracellular and the periplasmic spaces. For example the pro-inflammatory lipoproteins of T. pallidum are located below its cell surface and thus do not interact directly with the immune system of the host. It has been suggested that uptake and degradation of T. pallidum releases lipoproteins and allows their interaction with receptors on immune cells leading to immune cell activation. Computational programs can predict spirochetal protein lipidation but do not determine the location of lipoproteins in the cells. Recently, developed fluorescence activated cell sorting (FACS) and surface proteolysis methods can be used to screen for lipoprotein localization. Right upper corner: structure of spirochetal lipoproteins. The finding of a cysteine residue after a signal peptide (+1) is suggestive evidence that a protein is lipidated. The spirochetal lipoproteins have a lipobox that is four amino acids in length and mediates NH2-terminal lipidation on a conserved cysteine residue. Lipoproteins interact with the phospholipids of membranes via three hydrophobic N-terminal acyl moieties (often palmitate; C16) attached to a N-terminal cysteine residue which may contribute to the localization of spirochetal lipoproteins. An analysis of the fatty acids of T. pallidum, B. burgdorferi, L. interrogans phospholipids and lipoproteins found that while fatty acids with different length side chains (C16 and C18) were found in phospholipids, palmitate (C16) predominated in the lipoproteins. The N-terminal tripalmitoyl-S-glyceryl-cysteine (Pam3Cys) lipid moiety is the part of the lipoprotein that confers its immunologic activity. C, cysteine; LPS, lipopolysaccharides.
Lipoproteins are environmentally regulated and may be expressed selectively during spirochetal infection (37, 50, 112-116) ( Table 1 in Supplementary Material). For example, the outersurface protein (Osp) A has a more important role in the pathogenesis of borrelial infection during the tick phase of B. burgdorferi and its expression is down regulated during the mammalian phase of B. burgdorferi infection (9). Thus, although OspA does not have a major role in regulation of host immunity in vivo, since it is not expressed during the later stages of borrelial infection, it has been used as a model to study in vitro the immunoregulatory effects of spirochetal lipoproteins (9). Herein, the immunomodulatory effects of spirochetal lipoproteins are reviewed and are grouped into two main categories: effects related to immune evasion and effects related to immune activation (Figure 2). Understanding these mechanisms will aid in elucidating the immunopathogenesis of chronic spirochetal diseases.

SPIROCHETES USE ANTIGENIC VARIATION OF SURFACE LIPOPROTEINS TO EVADE IMMUNITY
Antigenic variation in borrelias may result from recombination of variable large and small protein genes (122) and the diversity of Vmp lipoproteins allows these pathogens to evade the host immune response (2,3). Studies in immunocompromised hosts have suggested that the host immune responses have a major role in producing spirochetal antigenic variants (120).

SPIROCHETAL LIPOPROTEINS INHIBIT COMPLEMENT ACTIVATION
Spirochetes may evade immune responses by inhibiting complement, a major innate immune system of the host (132,133). Complement activation is caused by pathogen surface antigens such as LPS, antigen-antibody complexes, and binding of lectin to bacterial surfaces (134). Activation is regulated by host regulatory proteins, including factor H (FH) (134). Borrelia bind complement regulator FH and/or FH-like protein 1 (FHL-1) by directly interacting with Osp designated complement regulator-acquiring surface proteins (CRASPs) (135). Numerous surface spirochetal lipoproteins (outlined in Table 1; Table 1 in Supplementary Material; Figure 2) such as OspA, OspE, CspA may contribute to complement inhibition by binding to major complement regulatory proteins such as FH, FHL-1 C4b-binding protein (C4bp) and human C1 esterase inhibitor (C1-Inh) (48, 99, 136-147). Thus, spirochetal lipoproteins contribute to immune evasion through complement inhibition.

SPIROCHETAL LIPOPROTEINS MAY INHIBIT NEUTROPHIL FUNCTION
Except for complement resistance, inhibition of neutrophil function is another mechanism that B. burgdorferi uses to evade the immune system. OspB inhibits the phagocytosis and oxidative burst of human neutrophils enabling B. burgdorferi to resist phagocytosis and oxidative burst in areas such as joint, skin, and the nervous system (47-49  These lipoproteins may contribute to immune evasion through inhibition of complement, neutrophils, production of anti-inflammatory cytokines, evasion of antibody responses through antigenic variation and binding to other components of innate immunity (e.g., apolipoproteins). In addition, spirochetal lipoproteins may directly promote spirochetal tissue invasion and colonization and in combination with immune evasion may lead to increased spirochetal replication and tissue inflammation. Spirochetal lipoproteins may also directly and indirectly activate endothelial, epithelial cells, and immune cells that contribute to innate immune responses (neutrophils, monocytes, macrophages, and DCs) or adaptive immune responses (lymphocytes such as B cells and CD4 T helper cells). Collectively, these effects lead to increase inflammation in target tissues (e.g., skin) or adaptive autoimmune responses (e.g., arthritis) that contribute to the clinical manifestations of spirochetal diseases. IL, interleukin; TNF-a, tumor necrosis factor A.

(CR3)
, an adhesion molecule expressed on neutrophils that is involved in the interactions of Borrelia species with neutrophils (149), and OspA and OspB may bind to CR3 in a C3bi independent manner (150). Thus, different spirochetal lipoproteins may inhibit neutrophils to evade immune responses but others may activate neutrophils contributing to tissue inflammation.  (159). Thus, spirochetal lipoproteins induce immune responses in antigen presenting cells such as monocytes and macrophages through TLR-dependent and -independent mechanisms.

Spirochetal lipoproteins activate dendritic cells
Except for monocytes and macrophages, spirochetal lipoproteins may also activate other antigen presenting cells such as DCs. DCs are a major link between innate and adaptive immunity, since after activation, they up-regulate costimulatory molecules such as CD54 that interact with T cell receptors such as CD11a/CD18 within lymph nodes (162,163). Consistent with the hypothesis that lipoproteins are key pro-inflammatory mediators in spirochetal diseases, many studies have shown that treponemal lipoproteins and synthetic lipopeptides can up-regulate CD54 and contribute to DC activation (18, 33, 35-37, 41, 94). Phagocytosis of intact spirochetes, activation of TLRs at phagosomes and bacterial cell death may result in the release of treponemal lipoproteins and may also contribute to immune cell activation (163). Also B. burgdorferi lipoproteins increase chemotaxis of circulating plasmacytoid dendritic cells (pDCs) into skin (11) but do not activate pDCs in vitro and in vivo (58, 59).

SPIROCHETAL LIPOPROTEINS INDUCE INFLAMMATORY INFILTRATE INTO TARGET TISSUES AND ADAPTIVE IMMUNE RESPONSES IN VIVO THAT CONTRIBUTE TO CLINICAL MANIFESTATIONS OF SPIROCHETAL DISEASES
Although lipoproteins may activate neutrophils, macrophages, endothelial cells in vitro, they may also induce inflammatory infiltrate into target tissues during spirochetal infection in vivo (37, 50, 61, 94, 115,116,164). Lipoproteins may also contribute to the pathogenesis of the Jarisch-Herxheimer reaction, a transient immunological phenomenon that occurs during treatment of spirochetal infections (165,166). Injection of synthetic lipopeptides into the skin can also be used to study the immunomodulatory effects of spirochetal lipoproteins in vivo (94). Cutaneous injection of spirochetal lipopeptides and spirochetal skin infections elicit similar cellular infiltrate supporting the hypothesis that spirochetal lipoproteins recruit diverse leukocytes from peripheral blood into target tissues (10). Lipoprotein-responsive cells (167), such as endothelium (95,168), keratinocytes (169), and macrophages (170) induce chemotaxis of mixed cellular infiltrate that in combination with extravasating leukocytes further increase the tissue inflammatory response. Spirochetal lipoproteins activate in vivo antigen presenting cells (macrophages, DCs, CD4+ T cells) within the inflamed skin in spirochetal diseases (39, 40, 58, 59). Lipopeptides in combination with other antigens from spirochetes facilitate the transition from innate to prolonged adaptive immune responses that contribute to chronic manifestations of spirochetal diseases such as syphilis and Lyme disease (11). Consistent with these data from in vivo studies, in vitro studies have demonstrated that spirochetal lipoproteins may directly activate both B and T cells ( Table 1). These lipoprotein-induced adaptive immune responses may trigger autoimmune and vaccine immune responses ( Table 1). Thus, spirochetal lipoproteins induce initially innate immunity and then adaptive immunity through recruitment of spirochete-specific T cells and tissue inflammation that is associated with clinical manifestations of spirochetal disease such as Lyme arthritis (37, 50, 94, 115,116,164).

CONCLUSION
Lipoproteins are widely expressed by many pathogens and have pro-inflammatory effects . Thus, an understanding of how lipoproteins interact with the immune system will aid in the understanding of the pathogenesis of many infections including spirochetal infections. In addition, elucidating the molecular mechanisms of lipoprotein-induced immunomodulation (summarized in Figure 2) will lead to a greater understanding of the inflammatory processes, innate and adaptive immune responses associated with spirochetal diseases that may contribute to spirochetal disease vaccine development (163).

SUPPLEMENTAY MATERIAL
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