Spirochete Research Labs
Spirochetes are an ancient and extremely successful eubacterial phylum characterized by distinctive helical or planar wave-form morphology and flagellar filaments confined to the periplasmic space. Spirochetes from the genera Leptospira, Treponema, and Borrelia are highly invasive pathogens that, as the agents of leptospirosis, syphilis (T. pallidum), Lyme disease (B. burgdorferi), and relapsing fever (B. hermsii, B. recurrentis, and others), pose public health problems of global dimensions. T. denticola and numerous other treponemal species, most of which remain uncultivated, are major components of the polymicrobial biofilms that cause periodontal disease. In recent years, the availability of genomic sequences for a number of spirochetes, improved methodologies for genetically manipulating these fastidious bacteria, and novel light and microscopic technologies have provided powerful new tools for studying the ultrastructures, genetics and parasitic strategies of these unusual pathogens.
Lyme Disease/Borrelia burgdorferi
The Lyme disease spirochete B. burgdorferi (Bb) is maintained in nature via an enzootic cycle in which it is transmitted to a mammalian host (typically the white-footed mouse Peromyscus leucopus) by the nymphal stage of its arthropod vector, the deer tick Ixodes scapularis, and then completes the cycle when it is acquired by a naïve larval tick feeding on an infected mouse. Infection of humans is accidental and is not required for persistence of the spirochete in nature. In order to transit between its arthropod and mammalian hosts during the nymphal and larval blood meals, spirochetes must decipher complex environmental cues delivered at the feeding site and, in response, undergo dramatic changes in their transcriptomes and proteomes. The principal objective of Lyme disease research conducted in the Radolf Laboratory is to understand these processes. The alternative sigma factor RpoS is a unifying genetic feature of this project. Signals delivered by the nymphal blood meal induce the expression of RpoS which, as the promoter-reading subunit of RNA polymerase, induces far-reaching changes in the bacterium’s transcriptome and proteome.
Critical to this work has been our development of green fluorescent protein (GFP) reporters that enable us to track live spirochetes in ticks and mice. Our live-imaging studies have fundamentally changed our understanding of the transmission process. In order to reach the mouse, spirochetes disseminate through the midgut into the salivary glands in order to access the salivary stream which they “ride” into the vertebrate host. We have found that dissemination of spirochetes in ticks is actually biphasic. In the first phase, which we have termed “adherence-mediated migration, spirochetes replicate in close association with differentiating midgut epithelial cells, “working” their way as aggregates or networks to the base of the epithelium. In the second phase, they transition into typically motile spirochetes, complete the penetration through the midgut, and then move on to the salivary glands en route to the mouse. Most recently, we have shown that spirochetes lacking RpoS cannot be transmitted by feeding nymphs. We are in the process of characterizing individual RpoS-dependent genes that are required for tick-to-mammal transmission.
Since Bb lacks orthologs of known exotoxins or the specialized secretory machinery required for the delivery of noxious molecules into host cells, it is widely accepted that the distinctive clinical signs and symptoms associated with Lyme disease result from the human host’s innate and adaptive immune responses to the bacterium. Monocytes and macrophages are considered to be two critical cellular elements of the innate immune response to Bb. Innate immune recognition of the spirochete by these cells was previously thought to result primarily from the interactions of the bacterium’s abundant outer membrane-associated lipoproteins with CD14 and Toll-like receptors (TLR) 1/2. However, experimental evidence generated in the Salazar laboratory has demonstrated that phagocytosed Bb induces inflammatory signals that differ both quantitatively and qualitatively from those generated by lipoproteins at the cell surface of monocytes/macrophages and dendritic cells. In our 2009 PLoS Pathogens publication (Salazar et al.), we demonstrated that internalized spirochetes induce transcription of interferon-β (IFN-β) and several type I interferon-stimulated genes (ISGs). In our 2011 PNAS publication (Cervantes et al.) we showed that recognition of borrelial ligands by monocytes involves a cooperative interaction between TLR2 and TLR8 signaling, and confirmed that endosomal TLR8 is solely responsible for IRF-7 mediated induction of IFN-β. More recently we proved that borrelial RNA is the principal TLR8 ligand and that in the case of Bb the TLR8-RNA interaction occurs solely in the phagosomal vacuole. Our combined observations have enabled us to formulate a new model for innate immune recognition of LD spirochetes. In this model, binding of unopsonized Bb to the monocyte/macrophage cell surface, through complement receptor 3 (CR3) (Hawley et al.) and other yet to be characterized phagocytic receptors, sets the stage for the broader immune signaling events that follow upon internalization of the bacterium and formation of the phagolysosome. As maturation of the phagosome proceeds and the spirochetes are degraded, TLR2 and TLR8 lining the vacuole sense spirochetal lipoproteins and borrelial nucleic acids, eliciting the production of pro- and anti-inflammatory cytokines, including TLR8-dependent induction of IFN-β.
The syphilis spirochete T. pallidum (Tp) harbors many resemblances to B. burgdorferi but actually employs a markedly different parasitic strategy. Whereas B. burgdorferi is an enzootic pathogen, Tp is an obligate pathogen of humans which cannot be cultivated in artificial medium. The modes of transmission of the two bacteria differ markedly as well: Tp is transmitted from person-to-person during sexual activity, whereas Bb is transmitted by an arthropod vector. Once within the host, Tp begins to replicate locally, eventually causing a genital ulcer called a chancre, the clinical hallmark of the primary stage of the disease. As the chancre develops, treponemes begin to make their way towards draining lymph nodes and blood vessels in order to spread systemically. Once within the blood, Tp is extremely adept at invading virtually every organ system in the body, including the central nervous system, and establishing persistent infection that can cause serious, even life threatening, complications. We have designated Tp “the stealth pathogen” because of its remarkable ability to evade host immune defenses.
Efforts in the Radolf Laboratory to explain Tp’s stealth pathogenicity have focused on the bacterium’s unusual molecular architecture. Over the years, we have generated abundant evidence that the Tp outer membrane differs markedly in structure and composition from its gram-negative counterparts (e.g., Escherichia coi). Not only does it lack lipopolysaccharide, the highly inflammatory glycolipid in the outer membranes of gram negative bacteria, it also contains a much lower density of integral membrane proteins that present few surface antigenic targets to the host immune system. Situated below the outer bilayer, where they are inaccessible to antibodies in intact organisms, are the bacterium’s major immunogens, many of which are periplasmic proteins tethered by N-terminal lipids to the cytoplasmic membrane. This work ushered in what we have termed “the quest” for Tp outer membrane proteins, a project that has been ongoing for more than 20 years. Why a quest? Because identifying rare outer membrane proteins is so difficult and requires extraordinary commitment. Fortunately, we now have much more powerful tools to complete the quest, one of which is the bacterium’s complete genomic sequence. Genome mining, however, isn’t as easy as it sounds because, with one exception, there are no proteins encoded by the Tp genome with sequence relatedness to well characterized outer membrane proteins of gram-negatives. This work is complicated further by the inability to cultivate the bacterium and the fragility of its outer membrane. In the past few years we have had considerable success using bioinformatics algorithms to identify outer membrane protein candidates. Why is this quest important? First, outer membrane proteins provide channels through which bacteria obtain nutrients. Second, we believe that these surface-exposed proteins, few as they are, are likely vaccine candidates. Given the explosive increase in syphilis cases in the United States and the world during the past decade, a vaccine would be a major weapon in our battle against this centuries old affliction of humans.
In order to fulfill its genetic destiny as a stealth pathogen, Tp must acquire nutrients in every milieu within its obligate human host in which it finds itself, while fending off the host’s attempts to undermine its homeostasis. Recognition of this metabolic war between pathogen and host led us to explore facets of Tp virulence that lie below its surface. One is transition metal acquisition. Transition metals, such as iron, manganese, and zinc, are essential for life but are present in mammalian body fluids at exceedingly low concentrations. Bacterial pathogens, Tp being no exception, employ highly developed strategems to obtain these nutrients. Our work along these lines has centered about characterizing two ABC transporters within the cytoplasmic membrane (Tro and Znu) that work cooperatively to meet the bacterium’s metal requirements. Lastly, our immune system uses toxic compounds, called reactive oxygen species, to kill bacteria. Tp has extremely robust enzymatic mechanisms for detoxifying reactive oxygen species. Understanding how these enzymes work and are regulated in response to host defenses is relevant to all bacterial diseases, not just syphilis.
The clinical manifestations of venereal syphilis reflect the propensity of Tp to disseminate systemically and to induce chronic inflammation in diverse tissues and organ systems. The appearance of the syphilitic chancre during primary syphilis typically emerges 2 to 4 weeks after the initial contact with the spirochete. By this time, spirochetes have disseminated to other organs and a tissue, including the skin, setting the stage for what is classically known as secondary syphilis (SS), the focus of our translational syphilis research. Paradoxically, despite the robust nature of the adaptive cellular and humoral immune responses typical of SS, which includes the production high titers opsonic antibodies, it takes weeks and in some cases months for host defenses to gain control of the invading pathogen, ultimately giving rise to the prolonged asymptomatic stage known as latent syphilis. Efforts to understand the duality of immune evasion and immune recognition in syphilis have been hindered by the inability to propagate the bacterium in vitro and the lack of a suitable inbred animal model for performing immunologic studies. To circumvent these problems and obtain information directly relevant to the disease process in humans, we have been studying SS, the stage in which the dichotomous features of syphilitic infection are clearly evident and specimens are readily obtainable. Our clinical studies are complemented by our Tp structural biology program and a powerful ex vivo PBMC/monocyte-Tp stimulation model.
Our analysis of Tp’s unique ultrastructure provides potential explanations for the paradoxical features of secondary syphilis. Unlike the outer membrane of gram-negative bacteria, that of Tp lacks the potent proinflammatory glycolipid lipopolysaccharide (LPS). In addition, freeze fracture microscopy studies have shown that the spirochete contains very few outer membrane integral membrane proteins (OMPs). Although Tp does contain an abundance of highly antigenic hydrophilic polypeptides, these molecules are tethered by covalently bound N-terminal lipids to the bacterium’s periplasmic inner membrane leaflet. This unusual topology enables intact spirochetes to avoid sensing by innate immune receptors (i.e., Toll-like receptors) present on tissue macrophages and dendritic cells (DCs), while protecting the surface of the organism from the high titers of anti-treponemal antibodies that are generated during SS. Inefficient antibody binding to the sparse spirochetal OMP opsonic targets is thought to allow a large proportion of spirochetes to shun antibody binding and opsonophagocytosis; a mechanism which we have previously demonstrated is an essential requirement for Tp-driven innate immune cell activation. Fortunately for the host, a progressively more robust adaptive immune response, including enhanced uptake and degradation of opsonized spirochetes by tissue based macrophages and activation of CD4+ and CD8+ T-cells, eventually leads to control of bacterial replication and lesion resolution. Our ongoing translational studies are designed to elucidate the duality of immune evasion and immune recognition of the syphilis spirochete as it occurs in SS, and which builds upon recent advances from the Radolf laboratory in understanding the spirochete’s unique ultrastructure and composition and the host antibody responses it elicits in its natural human host.
Based on our combined observations, we are now able to propose a revised early syphilis pathogenesis model that integrates innate and adaptive immune responses to the bacterium and also takes into account the spirochete’s immunoevasive countermeasures against host defenses. According to this model, spirochetes replicate at the site of initial inoculation unchecked by the innate immune surveillance system and rapidly disseminate to the skin and other tissues. At some point after initial entry of the bacterium increasing local spirochetal burdens allow a small number of organisms to be taken up by resident phagocytes, although this process is inefficient in the absence of opsonic antibodies. APCs containing phagocytosed spirochetes can then migrate onto draining lymph nodes where they present treponemal antigens to naïve CD4+ T cells and B-cells. Neo-sensitized T-helper cells traffic back into the primary lesion, where they recognize their cognate antigens and release IFN-γ. Clearance of organisms by IFN-γ activated tissue macrophages is markedly facilitated by the emergence of high titers of Tp-specific opsonic antibodies. In parallel events, while the chancre resolves, as soon as treponemal loads in the skin of early syphilis patients reach a sufficient density capable of triggering the local inflammatory response, SS skin lesions become clinically apparent. In contrast to the immunologic events that initially take place in the primary chancres, innate and adaptive immune responses in SS skin lesions appear to co-evolve in the presence of primed CD4+ and CD8+ T cells and high titers of opsonic antibodies. One would thus predict that these changes would be sufficient for the immune response to eradicate the bacterium. However, the paucity of OMP antigenic targets on the outer leaflet of the bacterium together with the emergence of Tp-subpopulations resistant to opsonophagocytosis, permits varying numbers of bacteria to avoid opsonization and uptake by skin macrophages. The low-level bacteremia which ensues allows the spirochete to avoid recognition by host innate and adaptive immune defenses in the blood compartment. Chronic spread of Tp into other tissues, including the bone marrow, could affect the development of myeloid progenitors of monocytes, DCs and NK-cells. We hypothesize that the predominance of CD8+ T cells in SS skin lesions reflects a deviated cellular immune response, which could be prompted by less efficient priming of CD4+ T cells in lymph nodes. Fortunately for the host, over time, the emergence of greater numbers of antigen-specific CD4+ and CD8+ T-cells, IFN-γ producing CD56+ NK-cells together with increasing titers of Tp-specific opsonic antibodies, allows the host to ultimately gain the upper hand against the bacterium. The complex balance shift between immune evasion and bacterial persistence to immune recognition and spirochetal clearance, will thus not only determine the intensity and duration of the clinical manifestations of venereal syphilis but also how long the spirochete can endure in blood and tissues.