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1. Phylogenomic Diversity Elucidates Mechanistic Insights into Lyme Borreliae-Host Association

   https://doi.org/10.1128/msystems.00488-22  

   Combs, Matthew; Marcinkiewicz, Ashley L.; Dupuis, Alan P.; Davis, April D.; Lederman, Patricia; Nowak, Tristan A.; Stout, Jessica L.; Strle, Klemen; Fingerle, Volker; Margos, Gabriele; et al (August 2022, mSystems)

   Gilbert, Jack A. (Ed.)

   ABSTRACT Host association—the selective adaptation of pathogens to specific host species—evolves through constant interactions between host and pathogens, leaving a lot yet to be discovered on immunological mechanisms and genomic determinants. The causative agents of Lyme disease (LD) are spirochete bacteria composed of multiple species of the Borrelia burgdorferi sensu lato complex, including B. burgdorferi ( Bb ), the main LD pathogen in North America—a useful model for the study of mechanisms underlying host-pathogen association. Host adaptation requires pathogens’ ability to evade host immune responses, such as complement, the first-line innate immune defense mechanism. We tested the hypothesis that different host-adapted phenotypes among Bb strains are linked to polymorphic loci that confer complement evasion traits in a host-specific manner. We first examined the survivability of 20 Bb strains in sera in vitro and/or bloodstream and tissues in vivo from rodent and avian LD models. Three groups of complement-dependent host-association phenotypes emerged. We analyzed complement-evasion genes, identified a priori among all strains and sequenced and compared genomes for individual strains representing each phenotype. The evolutionary history of ospC loci is correlated with host-specific complement-evasion phenotypes, while comparative genomics suggests that several gene families and loci are potentially involved in host association. This multidisciplinary work provides novel insights into the functional evolution of host-adapted phenotypes, building a foundation for further investigation of the immunological and genomic determinants of host association. IMPORTANCE Host association is the phenotype that is commonly found in many pathogens that preferential survive in particular hosts. The Lyme disease (LD)-causing agent, B. burgdorferi ( Bb ), is an ideal model to study host association, as Bb is mainly maintained in nature through rodent and avian hosts. A widespread yet untested concept posits that host association in Bb strains is linked to Bb functional genetic variation conferring evasion to complement, an innate defense mechanism in vertebrate sera. Here, we tested this concept by grouping 20 Bb strains into three complement-dependent host-association phenotypes based on their survivability in sera and/or bloodstream and distal tissues in rodent and avian LD models. Phylogenomic analysis of these strains further correlated several gene families and loci, including ospC , with host-specific complement-evasion phenotypes. Such multifaceted studies thus pave the road to further identify the determinants of host association, providing mechanistic insights into host-pathogen interaction. 

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2. Utilizing Two Borrelia bavariensis Isolates Naturally Lacking the PFam54 Gene Array To Elucidate the Roles of PFam54-Encoded Proteins

   https://doi.org/10.1128/AEM.01555-21  

   Rollins, Robert E.; Wülbern, Janna; Röttgerding, Florian; Nowak, Tristan A.; Hepner, Sabrina; Fingerle, Volker; Margos, Gabriele; Lin, Yi-Pin; Kraiczy, Peter; Becker, Noémie S. (March 2022, Applied and Environmental Microbiology)

   Rudi, Knut (Ed.)

   ABSTRACT Lyme borreliosis is the most common vector-borne disease in the Northern Hemisphere, caused by spirochetes belonging to the Borrelia burgdorferi sensu lato species complex, which are transmitted by ixodid ticks. B. burgdorferi sensu lato species produce a family of proteins on the linear plasmid 54 (PFam54), some of which confer the functions of cell adhesion and inactivation of complement, the first line of host defense. However, the impact of PFam54 in promoting B. burgdorferi sensu lato pathogenesis remains unclear because of the hurdles to simultaneously knock out all PFam54 proteins in a spirochete. Here, we describe two Borrelia bavariensis strains, PBN and PNi, isolated from patients naturally lacking PFam54 but maintaining the rest of the genome with greater than 95% identity to the reference B. bavariensis strain, PBi. We found that PBN and PNi less efficiently survive in human serum than PBi. Such defects were restored by introducing two B. bavariensis PFam54 recombinant proteins, BGA66 and BGA71, confirming the role of these proteins in providing complement evasion of B. bavariensis . Further, we found that all three strains remain detectable in various murine tissues 21 days post-subcutaneous infection, supporting the nonessential role of B. bavariensis PFam54 in promoting spirochete persistence. This study identified and utilized isolates deficient in PFam54 to associate the defects with the absence of these proteins, building the foundation to further study the role of each PFam54 protein in contributing to B. burgdorferi sensu lato pathogenesis. IMPORTANCE To establish infections, Lyme borreliae utilize various means to overcome the host’s immune system. Proteins encoded by the PFam54 gene array play a role in spirochete survival in vitro and in vivo . Moreover, this gene array has been described in all currently available Lyme borreliae genomes. By investigating the first two Borrelia bavariensis isolates naturally lacking the entire PFam54 gene array, we showed that both patient isolates display an increased susceptibility to human serum, which can be rescued in the presence of two PFam54 recombinant proteins. However, both isolates remain infectious to mice after intradermal inoculation, suggesting the nonessential role of PFam54 during the long-term, but may differ slightly in the colonization of specific tissues. Furthermore, these isolates show high genomic similarity to type strain PBi (>95%) and could be used in future studies investigating the role of each PFam54 protein in Lyme borreliosis pathogenesis. 

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3. The Factor H-Binding Site of CspZ as a Protective Target against Multistrain, Tick-Transmitted Lyme Disease

   https://doi.org/10.1128/IAI.00956-19  

   Marcinkiewicz, Ashley L.; Lieknina, Ilva; Yang, Xiuli; Lederman, Patricia L.; Hart, Thomas M.; Yates, Jennifer; Chen, Wen-Hsiang; Bottazzi, Maria Elena; Mantis, Nicholas J.; Kraiczy, Peter; et al (April 2020, Infection and Immunity)

   ABSTRACT The spirochete Borrelia burgdorferi sensu 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. 

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4. Structural evolution of an immune evasion determinant shapes Lyme borreliae host tropism

   https://doi.org/10.1101/2022.09.13.507797  

   Marcinkiewicz AL; Brangulis K; Dupuis AP II; Hart TH; Zamba-Campero M; Nowak TA; Stout JL; Akopjana I; Kazaks A; Bogans J; et al (September 2022, bioRxiv)

   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. 

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5. Cellular and immunological mechanisms influence host-adapted phenotypes in a vector-borne microparasite

   https://doi.org/10.1098/rspb.2021.2087  

   Lin, Yi-Pin; Tufts, Danielle M.; Combs, Matthew; Dupuis, Alan P.; Marcinkiewicz, Ashley L.; Hirsbrunner, Andrew D.; Diaz, Alexander J.; Stout, Jessica L.; Blom, Anna M.; Strle, Klemen; et al (February 2022, Proceedings of the Royal Society B: Biological Sciences)

   Predicting pathogen emergence and spillover risk requires understanding the determinants of a pathogens' host range and the traits involved in host competence. While host competence is often considered a fixed species-specific trait, it may be variable if pathogens diversify across hosts. Balancing selection can lead to maintenance of pathogen polymorphisms (multiple-niche-polymorphism; MNP). The causative agent of Lyme disease, Borrelia burgdorferi ( Bb ), provides a model to study the evolution of host adaptation, as some Bb strains defined by their outer surface protein C ( ospC ) genotype, are widespread in white-footed mice and others are associated with non-rodent vertebrates (e.g. birds). To identify the mechanisms underlying potential strain × host adaptation, we infected American robins and white-footed mice, with three Bb strains of different ospC genotypes. Bb burdens varied by strain in a host-dependent fashion, and strain persistence in hosts largely corresponded to Bb survival at early infection stages and with transmission to larvae (i.e. fitness). Early survival phenotypes are associated with cell adhesion, complement evasion and/or inflammatory and antibody-mediated removal of Bb, suggesting directional selective pressure for host adaptation and the potential role of MNP in maintaining OspC diversity. Our findings will guide future investigations to inform eco-evolutionary models of host adaptation for microparasites. 

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