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

   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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4. VlsE, the nexus for antigenic variation of the Lyme disease spirochete, also mediates early bacterial attachment to the host microvasculature under shear force

   https://doi.org/10.1371/journal.ppat.1010511  

   Tan, Xi ; Lin, Yi-Pin ; Pereira, Michael J. ; Castellanos, Mildred ; Hahn, Beth L. ; Anderson, Phillip ; Coburn, Jenifer ; Leong, John M. ; Chaconas, George ( May 2022 , PLOS Pathogens)

   Skare, Jon T. (Ed.)

   Hematogenous dissemination is a critical step in the evolution of local infection to systemic disease. The Lyme disease (LD) spirochete, which efficiently disseminates to multiple tissues, has provided a model for this process, in particular for the key early event of pathogen adhesion to the host vasculature. This occurs under shear force mediated by interactions between bacterial adhesins and mammalian cell-surface proteins or extracellular matrix (ECM). Using real-time intravital imaging of the Lyme spirochete in living mice, we previously identified BBK32 as the first LD spirochetal adhesin demonstrated to mediate early vascular adhesion in a living mouse; however, deletion of bbk32 resulted in loss of only about half of the early interactions, suggesting the existence of at least one other adhesin (adhesin-X) that promotes early vascular interactions. VlsE, a surface lipoprotein, was identified long ago by its capacity to undergo rapid antigenic variation, is upregulated in the mammalian host and required for persistent infection in immunocompetent mice. In immunodeficient mice, VlsE shares functional overlap with OspC, a multi-functional protein that displays dermatan sulfate-binding activity and is required for joint invasion and colonization. In this research, using biochemical and genetic approaches as well as intravital imaging, we have identified VlsE as adhesin-X; it is a dermatan sulfate (DS) adhesin that efficiently promotes transient adhesion to the microvasculature under shear force via its DS binding pocket. Intravenous inoculation of mice with a low-passage infectious B .  burgdorferi strain lacking both bbk32 and vlsE almost completely eliminated transient microvascular interactions. Comparative analysis of binding parameters of VlsE, BBK32 and OspC provides a possible explanation why these three DS adhesins display different functionality in terms of their ability to promote early microvascular interactions. 

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5. Comparative transcriptome analysis of Peromyscus leucopus and C3H mice infected with the Lyme disease pathogen

   https://doi.org/10.3389/fcimb.2023.1115350  

   Gaber, Alhussien M. ; Mandric, Igor ; Nitirahardjo, Caroline ; Piontkivska, Helen ; Hillhouse, Andrew E. ; Threadgill, David W. ; Zelikovsky, Alex ; Rogovskyy, Artem S. ( April 2023 , Frontiers in Cellular and Infection Microbiology)

   Lyme disease (LD), the most prevalent tick-borne disease of humans in the Northern Hemisphere, is caused by the spirochetal bacterium of Borreliella burgdorferi ( Bb ) sensu lato complex. In nature, Bb spirochetes are continuously transmitted between Ixodes ticks and mammalian or avian reservoir hosts. Peromyscus leucopus mice are considered the primary mammalian reservoir of Bb in the United States. Earlier studies demonstrated that experimentally infected P. leucopus mice do not develop disease. In contrast, C3H mice, a widely used laboratory strain of Mus musculus in the LD field, develop severe Lyme arthritis. To date, the exact tolerance mechanism of P. leucopus mice to Bb -induced infection remains unknown. To address this knowledge gap, the present study has compared spleen transcriptomes of P. leucopus and C3H/HeJ mice infected with Bb strain 297 with those of their respective uninfected controls. Overall, the data showed that the spleen transcriptome of Bb -infected P. leucopus mice was much more quiescent compared to that of the infected C3H mice. To date, the current investigation is one of the few that have examined the transcriptome response of natural reservoir hosts to Borreliella infection. Although the experimental design of this study significantly differed from those of two previous investigations, the collective results of the current and published studies have consistently demonstrated very limited transcriptomic responses of different reservoir hosts to the persistent infection of LD pathogens. Importance The bacterium Borreliella burgdorferi ( Bb ) causes Lyme disease, which is one of the emerging and highly debilitating human diseases in countries of the Northern Hemisphere. In nature, Bb spirochetes are maintained between hard ticks of Ixodes spp. and mammals or birds. In the United States, the white-footed mouse, Peromyscus leucopus , is one of the main Bb reservoirs. In contrast to humans and laboratory mice (e.g., C3H mice), white-footed mice rarely develop clinical signs (disease) despite being (persistently) infected with Bb . How the white-footed mouse tolerates Bb infection is the question that the present study has attempted to address. Comparisons of genetic responses between Bb -infected and uninfected mice demonstrated that, during a long-term Bb infection, C3H mice reacted much stronger, whereas P. leucopus mice were relatively unresponsive. 

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