Subversion of the immune response of human pathogenic spirochetes

Abstract Spirochetes are a large group of prokaryotes that originated from Gram‐negative bacteria and are capable of causing a variety of human and animal infections. However, the pathogenesis of spirochetes remains unclear, as different types of spirochetes play pathogenic roles through different pathogenic substances and mechanisms. To survive and spread in the host, spirochetes have evolved complicated strategies to evade host immune responses. In this review, we aimed to provide a comprehensive overview of immune evasion strategies in spirochetes infection. These strategies can be explained from the following points: (i) Antigenic variation: random, unidirectional, and segmental conversion of the gene to evade immune surveillance; (ii) Overcoming the attack of the complement system: recruitment of host complement regulators, cleavage of complement components and inhibition of complement activation to evade immune defenses; (iii) Interfering with immune cells to regulating the immune system; (iv) Persistent infection: invading and colonizing the host cell to escape immune damage.

of syphilis, 6,7 Lyme disease, 8,9 and leptospirosis 10,11 have increased rapidly worldwide and posed major threats to public health.
Like with other pathogenic bacteria, a series of immune reactions occur when pathogenic spirochetes enter the body. The immune responses can be divided into innate and acquired immune responses.
When pathogen-associated molecular patterns (PAMPs) interact with pattern-recognition receptors (PRRs), these PRRs include Tolllike receptors (TLRs), nucleotide-binding oligomerization domain (Nod), RIG-like receptors (RLRs), C-type lectin receptors (CLRs), and so on. 12,13 The innate immune system is activated, with macrophages, dendritic cells (DCs), and neutrophils helping to identify the pathogen and subsequently activating the complement system to eliminate nonspecific pathogens. In addition, specific pathogens are then killed by the acquired immune system. Although there is an obvious immune response after spirochete infection, the pathogenic bacteria cannot be completely cleared, which is why it leads to persistent infection in vivo. In this review, we will focus on the immune evasion mechanism of spirochetes in terms of antigen variation, complement inhibition, and immune interference, and summarize how pathogenic spirochetes escape from the strictly controlled immune system and colonize the host to affect humans and animals alike. A deeper understanding of the immune escape mechanisms of spirochetes will help us to better understand why it is so challenging to eradicate spirochete infections and ultimately provide new insights for developing effective vaccines against them in the future.

| ANTI G ENI C VARIATI ON
It is generally accepted that antigenic variation is a common pathogenic mechanism adopted by bacterial, protozoan, and fungal pathogens to cope with host identification and defense. 14 It is commonly found in an evolutionary variety of obligate parasites, such as Neisseria gonorrhoeae (gonorrhea), Giardia lamblia (giardiasis or beaver fever), Treponema pallidum (syphilis), Mycoplasma pulmonis (mycoplasma pneumonia), Borrelia recurrentis (relapsing fever), or Borrelia burgdorferi (Lyme disease). In this part, we will consider B. burgdorferi as an example to illustrate the mechanism of antigen variation.
Lyme disease is a multisystem infectious disease caused by B. bugdorferi and is the most common vector-borne disease affecting humans in North America and temperate Eurasia. In the United States, Lyme disease is transmitted by hard-bodied ticks of the genus Ixodes. Infected ticks will spread B. burgdorferi to humans during feeding, which can lead to a local infection (erythema migrans) at the site of the tick bite. 15 The clinical manifestation is a chronic infection characterized by neuritis, arthritis, and myocarditis. B. burgdorferi has a highly evolved variable major protein (VMP)like sequence (vls) antigenic variation system, which possesses the ability to establish persistent infection by continuous variation of vlsE, a surface-bound lipoprotein. 16 The vls locus of B31 is located on the 28-kb linear plasmid (lp28-1). The vls locus comprises an expression site encoding the 35 kDa lipoprotein vlsE and a consecutive array of 15 silent vls cassettes (474-594 bp in length). The direction of these silent cassettes is opposite to the vlsE locus ( Figure 1A). 17 The vlsE locus and the silent vls cassettes are separated by a short intergenic region, and this intergenic space includes a 51-bp inverted repeat (IR) sequence. 18 Furthermore, a part of the promoter required for vlsE expression is located within this inverted repeat.
The expression region of vlsE is composed of a central variable cassette, and the constant region (CR) is located on both sides of the central variable cassette. There are 17-bp direct repeats (DR) at the junction of the variable and CRs and both ends of most silent cassettes ( Figure 1B). 19 The silent cassettes show high homology with the central variable cassette region of vlsE (90-96.1% nucleotide sequence identity). There are six variable regions and six invariant regions located in the central variable cassette region of vlsE, and most of the antigenic variation sequence differences are found in these six variable regions ( Figure 1C).
Antigenic variation is a random combination of the vlsE proteincoding site and the silent cassettes that lead to a difference in the expression of vlsE variants. In the vls system, B. burgdorferi can evade the host's acquired immunity via random, unidirectional, and segmental gene conversion. 16,20 Previous studies in infected mice 21 or rabbits 22 showed that while vlsE sequence variation occurs within 4 days and continues throughout infection, they could not be detected in vitro or the tick vector. 23 This suggests that vlsE variants only exist in mammals. Although vlsE through segmental gene conversion provides a large number of diversity variable sequences during antigenic variation to evade immune surveillance, more about its variation mechanism is still unclear. The proposed model for vlsE antigenic conversion indicated 24 that the vlsE central cassette regions are replaced by fragments of varied length and location in the silent cassettes. Bankhead 19 performed an in-depth analysis of the vlsE sequence changes and found that the vls antigenic variation system promotes varying length (short or long) recombination events in each cassette region. With the development of gene-conversion events, other template-independent changes also resulted in the amplification of vlsE variant sequences. The result of these cumulative changes is the generation of a new vlsE sequence with a mosaic structure ( Figure 1D). Thus, these mutated antigens prevent recognition by the host immune system. Moreover, it has been well documented that the lack of vlsE or vls genes residing on lp28-1 will cause the spirochete to lose its persistent infection. Other lp28-1 non-vls genes are not involved in the virulence, persistence, and recombination of vlsE. In other words, the vls locus and vlsE protein are key factors for immune escape and persistence of pathogens. [25][26][27] Similarly, T. pallidum has a corresponding protein gene (TprK) that causes persistent infection through antigenic variation, 28 but the mechanism is not yet known.
In recent years, some technical progress has been made in the study of the vls locus. These include the development of a newgeneration sequencing method 29 for the analysis of vlsE recombination switches and the mini-vls system 30 for the genetic manipulation of the vls locus. Through this information, we have deepened our understanding of the antigenic variation mechanism of spirochetes.
Although vlsE is flexible and variable in evolution, the locus has strictly conservative structural characteristics. These structures are indispensable in the process of antigenic variation. Therefore, whether we can inhibit the persistent infection by destroying the structure of the vls locus or using some genetic tools to achieve the effect of disease treatment remains to be further discussed.
Understanding the definite mechanism behind the antigenic variation of spirochetes may lay the foundation for intervention measures to inhibit infection. For now, however, we still have a long way to go to overcome the obstacles of gene manipulation.

| OVERCOMING THE AT TACK OF THE COMPLEMENT SYS TEM
The complement system is comprised of more than 50 glycoproteins, including plasma complement components, soluble proteins, membrane-bound proteins, and complement receptors. In innate immunity, the complement system forms an important line of defense against microbial invasion. Actually, the complement system  can be activated through three relatively independent and interrelated pathways to exert various biological effects such as regulating phagocytosis, cracking cells, mediating inflammation, regulating immune and clearing immune complexes. These three pathways are the classical pathway (CP), alternative pathway (AP), and lectin pathway (LP). All three pathways lead to the production of C3 invertase and C5 invertase that cleave C3 and C5, respectively, and pass through a common terminal pathway to form a membrane attack complex (MAC) ( Figure 2). After activation of complement molecules, the complement components and surface substance of the pathogens form a complex, which ultimately promotes bacterial dissolution. 31 In order to overcome the attack of the complement system, spirochetes take some evasion strategies that include acquiring host complement regulators, cracking complement components by binding of surface protein and Plg, and inhibiting complement activation by the interaction between the surface proteins of spirochetes and complement substances are introduced.

| Recruitment of host complement regulators
The activation of the complement system requires complement factors to perform normal physiological functions. If complement regulation is out of control, a large number of the complement factors will be consumed, which will lead to a decrease in the body's resistance to infection and also cause severe inflammation or damage to its tissues and cells. Therefore, the role of complement regulators is particularly important.

| Cleavage of complement components
When pathogenic microorganisms invade the body, the inactivated complement components in human serum will be activated in the complement system by antigen-antibody complexes.

| Inhibition of complement activation
The complement system can be activated by three pathways. Each of the complement components from these three pathways is critical, and the absence or downregulation of even one component will

| INTERFEREN CE WITH IMMUNE REG UL ATION
The immune response is that immune cells recognize, activate, proliferate, and produce immune substances under the stimulation of antigens, so as to mediate specific immune effects and finally eliminate invading pathogens. The body can maintain a relatively stable state through appropriate immune regulation. As is well known, immune responses can be divided into innate immunity and adaptive The results suggest that BBA57 can inhibit the activation of innate immune cells. 65 In addition, spirochetes seem to release significant nucleases to degrade the nuclear DNA of neutrophils and prevent them from being trapped and killed by NETs, which helps to spread in the host. 66 However, these specific mechanisms remain to be further studied. Results from these related studies have helped us to define the anti-phagocytosis and pro-apoptotic mechanism of spirochetes and provide a new perspective. effects such as arthritis. 77 In a mouse experiment, Zhang et al. 78 found that when Leptospira and TLR2 agonist Pam3csk4 were injected into hamsters, the IL-10 produced in the tissues of mice injected with Leptospira and Pam3csk4 was increased as compared with the group injected with Leptospira alone. Similarly, the IL-10 level of TLR2-deficient mice was lower than that of wild-type mice.

Dendritic cells and NKT cells are also essential cells in the innate
This suggests the use of TLR2 agonists to induce IL-10 production, IL-10 can downregulate the inflammatory system, thereby weakening the inflammatory response of the body. LipL32, as a major outer membrane protein of pathogenic Leptospira, is a TLR2 agonist and can induce a strong antibody response. 79 Like Leptospira, B. burgdorferi can also induce IL-10 to inhibit the production of inflammatory mediators by M/M and/or DCs in mice, and reduce the inflammatory response. 76,80 Regarding NKT cells, an important example is that spirochetes can directly interfere with NKT cells, which respond to CD1d glycolipids on the surface of spirochetes such as B. burgdorferi. 81 Although the exact mechanism of interference is still unclear, further studies are needed to understand the possible interactions between spirochetes and NKT cells.

| PER S IS TENT INFEC TI ON
The fibrinolytic (or Plg/Pla) system is an enzyme cascade consisting of many proteases and inhibitors that are involved in the production and regulation of Pla. Plg is transformed into Pla by tissue-type Plg activator (tPA) or urokinase-type Plg activator (uPA) in the fi- Plg, spirochetes can also stimulate human monocytes to secrete Pla activators that will be helpful to form the Pla on the bacterial surface. 83 On the other hand, pathogens can also indirectly promote The discovery of these new Plg-binding proteins will play an important role in the invasion and colonization of hosts. The introduction of more new recombinant proteins may deepen our understanding of the fibrinolytic system and the immunopathogenesis of spirochetes.

| CON CLUS ION
Spirochetes are prokaryotic microorganisms between bacteria and protozoa. It not only has the similar structure and biological characteristics of bacteria, including cell wall, binary fission, amorphous nucleus, and sensitivity to antibiotics (penicillin), but also are soft as protozoa. It can move flexibly by bending and contracting the elastic filaments between the cell wall and the cell membrane, but it differs from other pathogens such as fungi, viruses, and parasites in that it does not have a complete cell structure, nor is it a strictly intra-host parasitic organism. However, these pathogens all have a similar set of self-protection and independent immune evasion mechanisms that interact with the immune capacity of the host organism to form a certain balance, which is the result of their long-term co-evolution. Under the surveillance of the powerful and hostile immune system, spirochetes have developed many strategies to resist the detrimental effects of immune factors. In this work, we summarized several escape mechanisms of spirochetes, including antigenic variation, complement inhibition, and immune interference to subvert the immune response.
All the above-mentioned measures increase the possibility of spirochete survival in the host and lead to a persistent chronic infection. As we know, the vls locus has strictly conservative structural characteristics. With the successful construction of a mini-vls system, we speculate whether we can block the recombination switch or destroy the structure of the vls site or use other genetic tools to inhibit persistent infection, to achieve the effect of successful disease treatment that remains to be further investigated.
There are still several challenges concerning gene manipulation as a potential therapy. Moreover, we found that many escape mechanisms of spirochetes are closely associated with the spirilla surface proteins. In recent years, scientists have also developed some candidate vaccines for spirochetes using surface proteins. Our future studies are aimed at identifying more immune-related proteins to contribute to the development of vaccines against spirochetes.

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

AUTH O R CO NTR I B UTI O N S
Jielite Huang drafted the manuscript. Jinlin Chen and Yafeng Xie modified the manuscript. Zhuoran Liu conceived the idea.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.