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The preferential adaptation of pathogens to specific hosts, known as host tropism, evolves through host-pathogen interactions. Transmitted by ticks and maintained primarily in rodents and birds, the Lyme disease-causing bacterium Borrelia burgdorferi (Bb) is an ideal model to investigate the mechanisms of host tropism. In order to survive in hosts and escape complement-mediated clearance, a first-line host immune defense, Bb produces the outer surface protein CspZ that binds to the complement inhibitor factor H (FH) to facilitate bacterial dissemination in vertebrates. Despite high sequence conservation, CspZ variants vary in human FH-binding ability. Together with the FH polymorphisms found amongst vertebrate hosts, these findings raise a hypothesis that minor sequence variation in a bacterial outer surface protein confers dramatic differences in host- specific, FH-binding-mediated infectivity. We tested this hypothesis by determining the crystal structure of the CspZ-human FH complex, identifying a minor change localized in the FH-binding interface, and uncovered that the bird and rodent FH-specific binding activity of different CspZ variants directly impacts infectivity. Swapping the divergent loop region in the FH-binding interface between rodent- and bird-associated CspZ variants alters the ability to promote rodent- and bird-specific early-onset dissemination. By employing phylogenetic tree thinking, we correlated these loops and respective host-specific, complement-dependent phenotypes with distinct CspZ lineages and elucidated evolutionary mechanisms driving CspZ emergence. Our multidisciplinary work provides mechanistic insights into how a single, short pathogen protein motif could greatly impact host tropism.AUTHOR SUMMARYLyme disease presents a suitable model for the investigation of host tropism – a pathogen’s ability to colonize and survive in different host species – since its causative agent, the spirochete Borrelia burgdorferi (Bb) is transmitted by ticks and maintained in rodent and bird reservoir hosts. In order to survive in vertebrates and escape from killing by complement, a first-line host immune defense, Bb produces the outer surface protein CspZ that binds the complement inhibitor factor H (FH) to promote infection. Protein sequence conservation seems to be linked to FH-binding activity divergence, raising the hypothesis that even minor variation can confer host-specific, FH- binding-mediated infectivity. Our work shows that that this minor variation is located in a loop in the CspZ protein localized in the CspZ-FH binding interface. Our functional experiments prove that this loop promotes bird- or rodent-specific FH-binding activity and infectivity. Swapping loops between rodent- and bird-associated CspZ variants alters their capability to confer host- specific dissemination. We further investigated the evolutionary mechanisms driving the emergence of the CspZ loop-mediated, host-dependent complement evasion. This multifaceted work demonstrates how a single, short protein motif can significantly impact host tropism.

Lyme disease (LD) is caused by the spirochete Borrelia burgdorferi sensu lato (Bbsl). After transmission to humans by ticks, Bbsl spreads to multiple organs, leading to arthritis, carditis, and neuroborreliosis. No effective prophylaxis against human LD prior to tick exposure is currently available. Thus, a pre-exposure prophylaxis (PrEP) against LD is needed. The establishment of LD bacteria at diverse sites is dictated partly by the binding of Bbsl to proteoglycans (PGs) and glycosaminoglycans (GAGs) in tissues. The drug heparin is structurally similar to these GAGs and inhibits Bbsl attachment to PGs, GAGs, cells, and tissues, suggesting its potential to prevent LD. However, the anticoagulant activity of heparin often results in hemorrhage, hampering the development of this compound as LD PrEP. We have previously synthesized a non-anticoagulant version of heparin (NACH), which was verified for safety in mice and humans. Here, we showed that NACH blocks Bbsl attachment to PGs, GAGs, and mammalian cells. We also found that treating mice with NACH prior to the exposure of ticks carrying Bbsl followed by continuous administration of this compound prevents tissue colonization by Bbsl. Furthermore, NACH-treated mice develop greater levels of IgG and IgM against Bbsl at early stages of infection, suggesting that the upregulation of antibody immune responses may be one of the mechanisms for NACH-mediated LD prevention. This is one of the first studies examining the ability of a heparin-based compound to prevent LD prior to tick exposure. The information presented might also be extended to prevent other infectious diseases agents.

Lyme borreliosis is caused by multiple species of the spirochete bacteria Borrelia burgdorferi sensu lato. The spirochetes are transmitted by ticks to vertebrate hosts, including small- and medium-sized mammals, birds, reptiles, and humans. Strain-to-strain variation in host-specific infectivity has been documented, but the molecular basis that drives this differentiation is still unclear. Spirochetes possess the ability to evade host immune responses and colonize host tissues to establish infection in vertebrate hosts. In turn, hosts have developed distinct levels of immune responses when invaded by different species/strains of Lyme borreliae. Similarly, the ability of Lyme borreliae to colonize host tissues varies among different spirochete species/strains. One potential mechanism that drives this strain-to-strain variation of immune evasion and colonization is the polymorphic outer surface proteins produced by Lyme borreliae. In this review, we summarize research on strain-to-strain variation in host competence and discuss the evidence that supports the role of spirochete-produced protein polymorphisms in driving this variation in host specialization. Such information will provide greater insights into the adaptive mechanisms driving host and Lyme borreliae association, which will lead to the development of interventions to block pathogen spread and eventually reduce Lyme borreliosis health burden.

The spirochete Borrelia burgdorferisensu lato is the causative agent of Lyme disease (LD). The spirochetes produce the CspZ protein that binds to a complement regulator, factor H (FH). Such binding downregulates activation of host complement to facilitate spirochete evasion of complement killing. However, vaccination with CspZ does not protect against LD infection. In this study, we demonstrated that immunization with CspZ-YA, a CspZ mutant protein with no FH-binding activity, protected mice from infection by several spirochete genotypes introduced via tick feeding. We found that the sera from CspZ-YA-vaccinated mice more efficiently eliminated spirochetes and blocked CspZ FH-binding activity than sera from CspZ-immunized mice. We also found that vaccination with CspZ, but not CspZ-YA, triggered the production of anti-FH antibodies, justifying CspZ-YA as an LD vaccine candidate. The mechanistic and efficacy information derived from this study provides insights into the development of a CspZ-based LD vaccine.

Lyme borreliosis, the most common vector-borne illness in Europe and the United States, is caused by spirochetes of the Borrelia burgdorferi sensu lato complex and transmitted by Ixodes ticks. In humans, the spirochetes disseminate from the tick bite site to multiple tissues, leading to serious clinical manifestations. The ability of spirochetes to survive in ticks during blood feeding is thought to be essential for Lyme borreliae to be transmitted to different vertebrate hosts. This ability is partly attributed to several B. burgdorferi proteins, including BBA52 and Lp6.6, which promote spirochete survival in nymphal ticks feeding on mice. One of the strategies to identify such proteins without using live animals is to feed B. burgdorferi-infected ticks on blood via artificial feeding chambers. In previous studies, ticks were only fed on bovine blood in the feeding chambers. In this study, we used this chamber model and showed that I. scapularis ticks will not only acquire bovine blood but human and quail blood as well. The latter two are the incidental host and an avian host of Lyme borreliae, respectively. We also investigated the roles that BBA52 and Lp6.6 play in promoting spirochete survival in nymphal ticks fed on human or quail blood. After feeding on human blood, spirochete burdens in ticks infected with an lp6.6-deficient B. burgdorferi were significantly reduced, while bba52-deficient spirochete burdens in ticks remained unchanged, similar to the wild-type strain. No strain showed a change in spirochete burdens in ticks fed on quail blood. These results indicate that Lp6.6 plays a role for B. burgdorferi in nymphs fed on human but not quail blood. Such information also demonstrates that the artificial feeding chamber is a powerful tool to identify B. burgdorferi proteins that promote vertebrate host blood-specific spirochete survival in I. scapularis ticks.

Pathogens possess the ability to adapt and survive in some host species but not in others–an ecological trait known as host tropism. Transmitted through ticks and carried mainly by mammals and birds, the Lyme disease (LD) bacterium is a well-suited model to study such tropism. Three main causative agents of LD, Borrelia burgdorferi, B. afzelii, and B. garinii, vary in host ranges through mechanisms eluding characterization. By feeding ticks infected with different Borrelia species, utilizing feeding chambers and live mice and quail, we found species-level differences in bacterial transmission. These differences localize on the tick blood meal, and specifically complement, a defense in vertebrate blood, and a polymorphic bacterial protein, CspA, which inactivates complement by binding to a host complement inhibitor, Factor H (FH). CspA selectively confers bacterial transmission to vertebrates that produce FH capable of allele-specific recognition. CspA is the only member of the Pfam54 gene family to exhibit host-specific FH-binding. Phylogenetic analyses revealed convergent evolution as the driver of such uniqueness, and that FH-binding likely emerged during the last glacial maximum. Our results identify a determinant of host tropism in Lyme disease infection, thus defining an evolutionary mechanism that shapes host-pathogen associations., Author summary The evolution of pathogens in response to a host environment often leads to a specificity of these pathogens to survive in particular hosts, known as host tropism. However, the mechanisms of host tropism remain unclear. The Lyme disease (LD) bacterium is an ideal model to study such mechanisms because the causative agent of this disease (bacteria species) are transmitted by ticks and carried by multiple vertebrates, including mammals and birds. We found that different LD bacteria species vary in their ability to survive in mice and quail, and in ticks fed on human or quail blood after transmission. We also showed that such a variation is dependent on whether tick blood meals have functional complement, an innate defense in vertebrate blood. We attributed such species-to-species differences of transmission to the polymorphisms of a bacterial complement inactivating protein, CspA: CspA, by inactivating vertebrate complement in tick blood meals in a species-specific manner, allows bacterial transmission to selective vertebrates. We further demonstrated that this CspA-mediated bacterium species-specific complement evasion arose through convergent evolution, as an evolutionary mechanism to permit such vertebrate-bacteria interaction. The mechanisms defined in this multi-disciplinary work provide new insights into the host-pathogen interactions.

Borrelia burgdorferi conserved gene products BB0406 and BB0405, members of a common B. burgdorferi paralogous gene family, share 59% similarity. Although both gene products can function as potential porins, only BB0405 is essential for infection. Here we show that, despite sequence homology and coexpression from the same operon, both proteins differ in their membrane localization attributes, antibody accessibility, and immunogenicity in mice. BB0406 is required for spirochete survival in mammalian hosts, particularly for the disseminated infection in distant organs. We identified that BB0406 interacts with laminin, one of the major constituents of the vascular basement membrane, and facilitates spirochete transmigration across host endothelial cell barriers. A better understanding of how B. burgdorferi transmigrates through dermal and tissue vascular barriers and establishes disseminated infections will contribute to the development of novel therapeutics to combat early infection.

Outer surface protein A (OspA) is a Borrelia lipoprotein and an established Lyme disease vaccine target. Admixing non-lipidated, recombinant B. burgdorferi OspA with liposomes containing cobalt porphyrin-phospholipid (CoPoP) resulted in rapid, particulate surface display of the conformationally intact antigen. Particleization was serum-stable and led to enhanced antigen uptake in murine macrophages in vitro. Mouse immunization using CoPoP liposomes that also contained a synthetic monophosphoryl lipid A (PHAD) elicited a Th1-biased OspA antibody response with higher IgG production compared to other vaccine adjuvants. Antibodies were reactive with intact B. burgdorferi spirochetes and Borrelia lysates, and induced complement-mediated borreliacidal activity in vitro. One year after initial immunization, mice maintained high levels of circulating borreliacidal antibodies capable of blocking B. burgdorferi transmission from infected ticks to human blood in a feeding chamber.

Complement is a key first line innate host defense system in the blood of vertebrates. Upon activation, this powerful defense mechanism can elicit inflammatory responses, lyse non-self-cells, or mark them for opsonophagocytic removal. Blood-feeding arthropods thus require the ability to block host complement activation in the bloodmeal to prevent undesired cell or tissue damage during feeding. The soft tick Ornithodoros moubata produces a complement inhibitory protein, OmCI. This protein binds to a mammalian complement protein C5 and blocks further activation of complement cascades, which results in the prevention of complement-mediated bacterial killing through membrane attack complex. Interestingly, the amino acids involved in OmCI binding are highly conserved among mammalian and avian C5, but the ability of this protein to inhibit the complement from birds remains unclear. Here we demonstrated that OmCI is capable of preventing quail complement-mediated erythrocyte lysis, inhibiting the capability of this animal's complement to eliminate a serum-sensitive Lyme disease bacterial strain. We also found that the ability of OmCI to inhibit quail complement-mediated killing of Lyme disease bacteria can be extended to different domestic and wild birds. Our results illustrate the utility of OmCI to block bird complement. These results provide the foundation for further use of this protein as a tool to study the molecular basis of avian complement and pathogen evasion to such a defense mechanism.

Borrelia burgdorferi sensu lato (Bbsl), the causative agent of Lyme disease, establishes an initial infection in the host’s skin following a tick bite, and then disseminates to distant organs, leading to multisystem manifestations. Tick-to-vertebrate host transmission requires that Bbsl survives during blood feeding. Complement is an important innate host defense in blood and interstitial fluid. Bbsl produces a polymorphic surface protein, CspA, that binds to a complement regulator, Factor H (FH) to block complement activation in vitro. However, the role that CspA plays in the Bbsl enzootic cycle remains unclear. In this study, we demonstrated that different CspA variants promote spirochete binding to FH to inactivate complement and promote serum resistance in a host-specific manner. Utilizing a tick-to-mouse transmission model, we observed that a cspA-knockout B. burgdorferi is eliminated from nymphal ticks in the first 24 hours of feeding and is unable to be transmitted to naïve mice. Conversely, ectopically producing CspA derived from B. burgdorferi or B. afzelii, but not B. garinii in a cspA-knockout strain restored spirochete survival in fed nymphs and tick-to-mouse transmission. Furthermore, a CspA point mutant, CspA-L246D that was defective in FH-binding, failed to survive in fed nymphs and at the inoculation site or bloodstream in mice. We also allowed those spirochete-infected nymphs to feed on C3-/- mice that lacked functional complement. The cspA-knockout B. burgdorferi or this mutant strain complemented with cspA variants or cspA-L246D was found at similar levels as wild type B. burgdorferi in the fed nymphs and mouse tissues. These novel findings suggest that the FH-binding activity of CspA protects spirochetes from complement-mediated killing in fed nymphal ticks, which ultimately allows Bbsl transmission to mammalian hosts., Author summary Lyme disease, the most common vector-borne disease in the United States, is caused by the bacterium, Borrelia burgdorferi. This bacterium is transmitted to humans via the bite of a tick, and then spreads from the bite site to multiple tissues. Tick-to-human transmission of this bacterium requires bacterial survival in fed ticks because the blood and body fluid that ticks consume contain complement, an important mechanism that kills bacteria. To prevent host cell damage by this powerful mechanism, vertebrate animals produce factor H to inhibit complement. Lyme disease bacteria produce a surface protein CspA that binds to factor H to inhibit complement, but the role that CspA-mediated factor H-binding activity plays in tick-to-human transmission remains unexplained. To investigate this, we infected mice with Lyme disease strains that were identical except for the CspA variant or mutant with no factor H-binding ability they produced. We found that the factor H-binding activity of CspA appears to prevent the bacteria from being killed by complement in fed nymphal ticks. Such ability further facilitates bacterial transmission to mice. These results will promote the development of strategies against CspA to intervene in Lyme disease bacterial transmission from ticks to humans.

Some adult odonates resist parasitism by larval water mites ( Arrenurus spp.) with melanotic encapsulation, in which the mite’s stylestome is clogged and the mite starves. In summer 2014, we counted the engorged and resisted mites on 2,729 adult odonates sampled by aerial net at 11 water bodies in Greenville Co. and Pickens Co., SC, and tested the hypothesis that the frequency and intensity of resistance correlates with parasite prevalence (the percentage of parasitized hosts). Resistance prevalence (the percentage of parasitized hosts that resisted at least one mite) varied significantly among host species, exceeding 60% for Argia fumipennis (Burmeister) and Celithemis fasciata Kirby but less than 20% for other species. However, neither resistance prevalence nor mean resistance intensity (mean percentage of resisted mites on resisting hosts) correlated with parasite prevalence. We described potential effects of parasitism on host development of A. fumipennis and Pachydiplax longipennis (Burmeister) by comparing the percent asymmetry of forewing lengths between parasitized and unparasitized individuals. There was no significant difference in asymmetry for either males or females of A. fumipennis , or males of Pa. longipennis (females were not sampled). We also evaluated differences in melanotic encapsulation between A. fumipennis , which readily encapsulates mites in nature, and Pa. longipennis . We inserted a 2.0-mm piece of sterile monofilament line into the thorax of captured individuals for 24 h and compared mean gray value scores of inserted and emergent ends using Image-J software. There was no difference in melanotic encapsulation between species.