A selective antibiotic for Lyme disease.

Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment.

A Novel Treponema pallidum Subspecies pallidum Strain Associated With a Painful Oral Lesion Is a Member of a Potentially Emerging Nichols-Related Subgroup.

BACKGROUND: Early syphilitic lesions are typically painless; however, several recent case studies have included patients with tender lesions and no evidence of concurrent infections. Here we present the manifestations and serological and molecular findings of a patient from New York State with a painful tongue lesion. METHODS: The diagnosis of syphilis was based on a combination of physical examination, serologic, pathologic, and immunohistochemical findings. DNA obtained from a formalin-fixed, paraffin-embedded biopsy was used to characterize the infecting pathogen using polymerase chain reaction, multilocus sequence typing, and whole-genome sequencing methods. RESULTS: Polymerase chain reaction and multilocus sequence typing of the biopsy specimen confirmed infection with T. pallidum subspecies pallidum ( T. pallidum ) of the Nichols cluster. Whole-genome sequencing analysis of this strain (herein called NYMC01) showed that it contained 17 unique single nucleotide variations and 4 more complex genetic differences; this novel genotype matched only 2 specimens, both from a patient in Seattle, Washington. The presence of this rare genotype in 2 geographically distinct locations suggests the potential emergence and spread of a new subgroup of the Nichols cluster. CONCLUSIONS: To our knowledge, this is the first genomic sequence obtained from a T. pallidum strain linked to a painful lesion, and the third description of whole-genome sequencing of T. pallidum from formalin-fixed, paraffin-embedded tissue. Analysis of additional specimens may reveal that the NYMC01-related genotype represents an emerging T. pallidum subgroup and may also aid in determining whether the painful clinical presentation of primary syphilis is related to specific T. pallidum genotypes.

Comparison of transcriptional profiles of Treponema pallidum during experimental infection of rabbits and in vitro culture: Highly similar, yet different.

Treponema pallidum ssp. pallidum, the causative agent of syphilis, can now be cultured continuously in vitro utilizing a tissue culture system, and the multiplication rates are similar to those obtained in experimental infection of rabbits. In this study, the RNA transcript profiles of the T. pallidum Nichols during in vitro culture and rabbit infection were compared to examine whether gene expression patterns differed in these two environments. To this end, RNA preparations were converted to cDNA and subjected to RNA-seq using high throughput Illumina sequencing; reverse transcriptase quantitative PCR was also performed on selected genes for validation of results. The transcript profiles in the in vivo and in vitro environments were remarkably similar, exhibiting a high degree of concordance overall. However, transcript levels of 94 genes (9%) out of the 1,063 predicted genes in the T. pallidum genome were significantly different during rabbit infection versus in vitro culture, varying by up to 8-fold in the two environments. Genes that exhibited significantly higher transcript levels during rabbit infection included those encoding multiple ribosomal proteins, several prominent membrane proteins, glycolysis-associated enzymes, replication initiator DnaA, rubredoxin, thioredoxin, two putative regulatory proteins, and proteins associated with solute transport. In vitro cultured T. pallidum had higher transcript levels of DNA repair proteins, cofactor synthesis enzymes, and several hypothetical proteins. The overall concordance of the transcript profiles may indicate that these environments are highly similar in terms of their effects on T. pallidum physiology and growth, and may also reflect a relatively low level of transcriptional regulation in this reduced genome organism.

YebC regulates variable surface antigen VlsE expression and is required for host immune evasion in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease pathogen causes persistent infection by evading the host immune response. Differential expression of the surface-exposed lipoprotein VlsE that undergoes antigenic variation is a key immune evasion strategy employed by B. burgdorferi. Most studies focused on the mechanism of VlsE antigen variation, but little is known about VlsE regulation and factor(s) that regulates differential vlsE expression. In this study, we investigated BB0025, a putative YebC family transcriptional regulator (and hence designated BB0025 as YebC of B. burgdorferi herein). We constructed yebC mutant and complemented strain in an infectious strain of B. burgdorferi. The yebC mutant could infect immunocompromised SCID mice but not immunocompetent mice, suggesting that YebC plays an important role in evading host adaptive immunity. RNA-seq analyses identified vlsE as one of the genes whose expression was most affected by YebC. Quantitative RT-PCR and Western blot analyses confirmed that vlsE expression was dependent on YebC. In vitro, YebC and VlsE were co-regulated in response to growth temperature. In mice, both yebC and vlsE were inversely expressed with ospC in response to the host adaptive immune response. Furthermore, EMSA proved that YebC directly binds to the vlsE promoter, suggesting a direct transcriptional control. These data demonstrate that YebC is a new regulator that modulates expression of vlsE and other genes important for spirochetal infection and immune evasion in the mammalian host.

The intergenic small non-coding RNA ittA is required for optimal infectivity and tissue tropism in Borrelia burgdorferi.

Post-transcriptional regulation via small regulatory RNAs (sRNAs) has been implicated in diverse regulatory processes in bacteria, including virulence. One class of sRNAs, termed trans-acting sRNAs, can affect the stability and/or the translational efficiency of regulated transcripts. In this study, we utilized a collaborative approach that employed data from infection with the Borrelia burgdorferi Tn library, coupled with Tn-seq, together with borrelial sRNA and total RNA transcriptomes, to identify an intergenic trans-acting sRNA, which we designate here as ittA for infectivity-associated and tissue-tropic sRNA locus A. The genetic inactivation of ittA resulted in a significant attenuation in infectivity, with decreased spirochetal load in ear, heart, skin and joint tissues. In addition, the ittA mutant did not disseminate to peripheral skin sites or heart tissue, suggesting a role for ittA in regulating a tissue-tropic response. RNA-Seq analysis determined that 19 transcripts were differentially expressed in the ittA mutant relative to its genetic parent, including vraA, bba66, ospD and oms28 (bba74). Subsequent proteomic analyses also showed a significant decrease of OspD and Oms28 (BBA74) proteins. To our knowledge this is the first documented intergenic sRNA that alters the infectivity potential of B. burgdorferi.

Lyme Disease Pathogenesis.

Lyme disease Borrelia are obligately parasitic, tick- transmitted, invasive, persistent bacterial pathogens that cause disease in humans and non-reservoir vertebrates primarily through the induction of inflammation. During transmission from the infected tick, the bacteria undergo significant changes in gene expression, resulting in adaptation to the mammalian environment. The organisms multiply and spread locally and induce inflammatory responses that, in humans, result in clinical signs and symptoms. Borrelia virulence involves a multiplicity of mechanisms for dissemination and colonization of multiple tissues and evasion of host immune responses. Most of the tissue damage, which is seen in non-reservoir hosts, appears to result from host inflammatory reactions, despite the low numbers of bacteria in affected sites. This host response to the Lyme disease Borrelia can cause neurologic, cardiovascular, arthritic, and dermatologic manifestations during the disseminated and persistent stages of infection. The mechanisms by which a paucity of organisms (in comparison to many other infectious diseases) can cause varied and in some cases profound inflammation and symptoms remains mysterious but are the subjects of diverse ongoing investigations. In this review, we provide an overview of virulence mechanisms and determinants for which roles have been demonstrated in vivo, primarily in mouse models of infection.

Genome-wide screen identifies novel genes required for Borrelia burgdorferi survival in its Ixodes tick vector.

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is maintained in a complex biphasic life cycle, which alternates between tick and vertebrate hosts. To successfully survive and complete its enzootic cycle, B. burgdorferi adapts to diverse hosts by regulating genes required for survival in specific environments. Here we describe the first ever use of transposon insertion sequencing (Tn-seq) to identify genes required for B. burgdorferi survival in its tick host. We found that insertions into 46 genes resulted in a complete loss of recovery of mutants from larval Ixodes ticks. Insertions in an additional 56 genes resulted in a >90% decrease in fitness. The screen identified both previously known and new genes important for larval tick survival. Almost half of the genes required for survival in the tick encode proteins of unknown function, while a significant portion (over 20%) encode membrane-associated proteins or lipoproteins. We validated the results of the screen for five Tn mutants by performing individual competition assays using mutant and complemented strains. To better understand the role of one of these genes in tick survival, we conducted mechanistic studies of bb0017, a gene previously shown to be required for resistance against oxidative stress. In this study we show that BB0017 affects the regulation of key borrelial virulence determinants. The application of Tn-seq to in vivo screening of B. burgdorferi in its natural vector is a powerful tool that can be used to address many different aspects of the host pathogen interaction.

Cryo-electron tomography of periplasmic flagella in Borrelia burgdorferi reveals a distinct cytoplasmic ATPase complex.

Periplasmic flagella are essential for the distinct morphology and motility of spirochetes. A flagella-specific type III secretion system (fT3SS) composed of a membrane-bound export apparatus and a cytosolic ATPase complex is responsible for the assembly of the periplasmic flagella. Here, we deployed cryo-electron tomography (cryo-ET) to visualize the fT3SS machine in the Lyme disease spirochete Borrelia burgdorferi. We show, for the first time, that the cytosolic ATPase complex is attached to the flagellar C-ring through multiple spokes to form the "spoke and hub" structure in B. burgdorferi. This structure not only strengthens structural rigidity of the round-shaped C-ring but also appears to rotate with the C-ring. Our studies provide structural insights into the unique mechanisms underlying assembly and rotation of the periplasmic flagella and may provide the basis for the development of novel therapeutic strategies against several pathogenic spirochetes.

In Vitro Susceptibility of Treponema pallidum subsp. pallidum to Doxycycline.

Doxycycline is regarded as an effective therapy for early syphilis, and there is increasing interest in using doxycycline for prophylaxis of this infection. However, the MIC of doxycycline for Treponema pallidum subsp. pallidum has not been reported previously. In this study, an in vitro culture system was utilized to determine that the MIC of doxycycline is 0.06 to 0.10 µg/ml for four strains of T. pallidum subsp. pallidum (Nichols, SS14, UW231B, and UW249B). The Nichols strain cultured in vitro with doxycycline was also tested for infectivity in rabbits, and the minimum bactericidal concentration (MBC) was found to be ≤0.1 µg/ml using this method. The low MIC and MBC values are consistent with the previously demonstrated clinical efficacy of doxycycline for the treatment of early syphilis. This study represents the first report of the in vitro susceptibility of T. pallidum to doxycycline, and the resulting information may be useful in the consideration of doxycycline for use in prevention of syphilis.

BBB07 contributes to, but is not essential for, Borrelia burgdorferi infection in mice.

Borrelia burgdorferi, a causative agent of Lyme disease, encodes a protein BBB07 on the genomic plasmid cp26. BBB07 was identified as a candidate integrin ligand based on the presence of an RGD tripeptide motif, which is present in a number of mammalian ligands for ß1 and ß3 integrins . Previous work demonstrated that BBB07 in recombinant form binds to ß1 integrins and induces inflammatory responses in synovial cells in culture. Several transposon mutants in bbb07 were attenuated in an in vivo screen of the transposon library in mice. We therefore tested individual transposon mutant clones in single-strain infections in mice and found that they were attenuated in terms of ID50 but did not have significantly reduced tissue burdens in mice. Based on data presented here we conclude that BBB07 is not essential for, but does contribute to, B. burgdorferi infectivity in mice.

Hiding in Plain Sight: Colonic Spirochetosis in Humans.

In 1967, Harland and Lee made a startling discovery: in some humans, the colonic epithelium is covered with a "forest" of spirochetes (W. A. Harlan, and F. D. Lee, Br Med J 3:718-719, 1967, https://doi.org/10.1136/bmj.3.5567.718). In this issue of Journal of Bacteriology, Thorell et al. present a systematic analysis of the prevalence and diversity of the spirochetes Brachyspira aalborgi and Brachyspira pilosicoli in the human colon. These and prior studies provide avenues toward resolving important questions: what bacterial and host parameters contribute to this extensive colonization, and what impact does it have on human health?

A high-throughput genetic screen identifies previously uncharacterized Borrelia burgdorferi genes important for resistance against reactive oxygen and nitrogen species.

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is exposed to reactive oxygen and nitrogen species (ROS and RNS) in both the tick vector and vertebrate reservoir hosts. B. burgdorferi contains a limited repertoire of canonical oxidative stress response genes, suggesting that novel gene functions may be important for protection of B. burgdorferi against ROS or RNS exposure. Here, we use transposon insertion sequencing (Tn-seq) to conduct an unbiased search for genes involved in resistance to nitric oxide, hydrogen peroxide, and tertiary-butyl hydroperoxide in vitro. The screens identified 66 genes whose disruption resulted in increased susceptibility to at least one of the stressors. These genes include previously characterized mediators of ROS and RNS resistance (including components of the nucleotide excision repair pathway and a subunit of a riboflavin transporter), as well as novel putative resistance candidates. DNA repair mutants were among the most sensitive to RNS in the Tn-seq screen, and survival assays with individual Tn mutants confirmed that the putative ribonuclease BB0839 is involved in resistance to nitric oxide. In contrast, mutants lacking predicted inner membrane proteins or transporters were among the most sensitive to ROS, and the contribution of three such membrane proteins (BB0017, BB0164, and BB0202) to ROS sensitivity was confirmed using individual Tn mutants and complemented strains. Further analysis showed that levels of intracellular manganese are significantly reduced in the Tn::bb0164 mutant, identifying a novel role for BB0164 in B. burgdorferi manganese homeostasis. Infection of C57BL/6 and gp91phox-/- mice with a mini-library of 39 Tn mutants showed that many of the genes identified in the in vitro screens are required for infectivity in mice. Collectively, our data provide insight into how B. burgdorferi responds to ROS and RNS and suggests that this response is relevant to the in vivo success of the organism.

Global Tn-seq analysis of carbohydrate utilization and vertebrate infectivity of Borrelia burgdorferi.

Borrelia burgdorferi maintains a complex life cycle between tick and vertebrate hosts. Although some genes have been identified as contributing to bacterial adaptation in the different hosts, the list is incomplete. In this manuscript, we report the first use of transposon mutagenesis combined with high-throughput sequencing (Tn-seq) in B. burgdorferi. We utilize the technique to investigate mechanisms of carbohydrate utilization in B. burgdorferi and the role of carbohydrate metabolism during mouse infection. We performed genetic fitness analyses to identify genes encoding factors contributing to growth on glucose, maltose, mannose, trehalose and N-acetyl-glucosamine. We obtained insight into the potential functions of proteins predicted to be involved in carbohydrate utilization and identified additional factors previously unrecognized as contributing to the metabolism of the tested carbohydrates. Strong phenotypes were observed for the putative carbohydrate phosphotransferase transporters BB0408 and BBB29 as well as the response regulator Rrp1. We further validated Tn-seq for use in mouse studies and were able to correctly identify known infectivity factors as well as additional transporters and genes on lp54 that may contribute to optimal mouse infection. As such, this study establishes Tn-seq as a powerful method for both in vitro and in vivo studies of B. burgdorferi.

Cryoelectron tomography reveals the sequential assembly of bacterial flagella in Borrelia burgdorferi.

Periplasmic flagella are essential for the distinctive morphology, motility, and infectious life cycle of the Lyme disease spirochete Borrelia burgdorferi. In this study, we genetically trapped intermediates in flagellar assembly and determined the 3D structures of the intermediates to 4-nm resolution by cryoelectron tomography. We provide structural evidence that secretion of rod substrates triggers remodeling of the central channel in the flagellar secretion apparatus from a closed to an open conformation. This open channel then serves as both a gateway and a template for flagellar rod assembly. The individual proteins assemble sequentially to form a modular rod. The hook cap initiates hook assembly on completion of the rod, and the filament cap facilitates filament assembly after formation of the mature hook. Cryoelectron tomography and mutational analysis thus combine synergistically to provide a unique structural blueprint of the assembly process of this intricate molecular machine in intact cells.

Phosphoenolpyruvate Phosphotransferase System Components Modulate Gene Transcription and Virulence of Borrelia burgdorferi.

The phosphoenolpyruvate phosphotransferase system (PEP-PTS) and adenylate cyclase (AC) IV (encoded by BB0723 [cyaB]) are well conserved in different species of Borrelia. However, the functional roles of PEP-PTS and AC in the infectious cycle of Borrelia have not been characterized previously. We examined 12 PEP-PTS transporter component mutants by needle inoculation of mice to assess their ability to cause mouse infection. Transposon mutants with mutations in the EIIBC components (ptsG) (BB0645, thought to be involved in glucose-specific transport) were unable to cause infection in mice, while all other tested PEP-PTS mutants retained infectivity. Infectivity was partially restored in an in trans-complemented strain of the ptsG mutant. While the ptsG mutant survived normally in unfed as well as fed ticks, it was unable to cause infection in mice by tick transmission, suggesting that the function of ptsG is essential to establish infection by either needle inoculation or tick transmission. In Gram-negative organisms, the regulatory effects of the PEP-PTS are mediated by adenylate cyclase and cyclic AMP (cAMP) levels. A recombinant protein encoded by B. burgdorferi BB0723 (a putative cyaB homolog) was shown to have adenylate cyclase activity in vitro; however, mutants with mutations in this gene were fully infectious in the tick-mouse infection cycle, indicating that its function is not required in this process. By transcriptome analysis, we demonstrated that the ptsG gene may directly or indirectly modulate gene expression of Borrelia burgdorferi. Overall, the PEP-PTS glucose transporter PtsG appears to play important roles in the pathogenesis of B. burgdorferi that extend beyond its transport functions.

Molecular architecture of the bacterial flagellar motor in cells.

The flagellum is one of the most sophisticated self-assembling molecular machines in bacteria. Powered by the proton-motive force, the flagellum rapidly rotates in either a clockwise or counterclockwise direction, which ultimately controls bacterial motility and behavior. Escherichia coli and Salmonella enterica have served as important model systems for extensive genetic, biochemical, and structural analysis of the flagellum, providing unparalleled insights into its structure, function, and gene regulation. Despite these advances, our understanding of flagellar assembly and rotational mechanisms remains incomplete, in part because of the limited structural information available regarding the intact rotor-stator complex and secretion apparatus. Cryo-electron tomography (cryo-ET) has become a valuable imaging technique capable of visualizing the intact flagellar motor in cells at molecular resolution. Because the resolution that can be achieved by cryo-ET with large bacteria (such as E. coli and S. enterica) is limited, analysis of small-diameter bacteria (including Borrelia burgdorferi and Campylobacter jejuni) can provide additional insights into the in situ structure of the flagellar motor and other cellular components. This review is focused on the application of cryo-ET, in combination with genetic and biophysical approaches, to the study of flagellar structures and its potential for improving the understanding of rotor-stator interactions, the rotational switching mechanism, and the secretion and assembly of flagellar components.

Understanding barriers to Borrelia burgdorferi dissemination during infection using massively parallel sequencing.

Borrelia burgdorferi is an invasive spirochete that can cause acute and chronic infections in the skin, heart, joints, and central nervous system of infected mammalian hosts. Little is understood about where the bacteria encounter the strongest barriers to infection and how different components of the host immune system influence the population as the infection progresses. To identify population bottlenecks in a murine host, we utilized Tn-seq to monitor the composition of mixed populations of B. burgdorferi during infection. Both wild-type mice and mice lacking the Toll-like receptor adapter molecule MyD88 were infected with a pool of infectious B. burgdorferi transposon mutants with insertions in the same gene. At multiple time points postinfection, bacteria were isolated from the mice and the compositions of the B. burgdorferi populations at the injection site and in distal tissues determined. We identified a population bottleneck at the site of infection that significantly altered the composition of the population. The magnitude of this bottleneck was reduced in MyD88(-/-) mice, indicating a role for innate immunity in limiting early establishment of B. burgdorferi infection. There is not a significant bottleneck during the colonization of distal tissues, suggesting that founder effects are limited and there is not a strict limitation on the number of organisms able to initiate populations at distal sites. These findings further our understanding of the interactions between B. burgdorferi and its murine host in the establishment of infection and dissemination of the organism.

Peaceful coexistence amongst Borrelia plasmids: getting by with a little help from their friends?

Borrelia species comprise a unique genus of bacterial pathogens. These organisms contain a segmented genome with up to two dozen plasmids ranging in size from 5 kb up to about 200 kb. The plasmids have also been referred to as mini-chromosomes or essential genetic elements, as some of them carry information important for infection of vertebrates or for survival in the tick vector. Most of the plasmids are linear with covalently closed hairpin telomeres and these linear plasmids are in a constant state of genetic rearrangement. The mechanisms of plasmid replication, maintenance and partitioning remain largely obscure and are complicated by a long doubling time, the requirement for expensive media and inefficient genetic manipulation. A set of five parologous protein families (PFs) are believed to confer the ability for autonomous replication and plasmid maintenance. The number of plasmids also complicates analyses because of the possibility that PFs from one plasmid may sometimes function in trans on other plasmids. Two papers in the last year have moved the field forward and their combined data suggest that trans complementation amongst Borrelia plasmids may sometimes occur.

Strict Conservation yet Non-Essential Nature of Plasmid Gene bba40 in the Lyme Disease Spirochete Borrelia burgdorferi.

The highly segmented genome of Borrelia burgdorferi, the tick-borne bacterium that causes Lyme disease, is composed of a linear chromosome and more than 20 co-existing endogenous plasmids. Many plasmid-borne genes are unique to B. burgdorferi and some have been shown to provide essential functions at discrete points of the infectious cycle between a tick vector and rodent host. In this study, we investigated the role of bba40, a highly conserved and differentially expressed gene on a ubiquitous linear plasmid of B. burgdorferi. In a prior genome-wide analysis, inactivation of bba40 by transposon insertion was linked with a noninfectious phenotype in mice, suggesting that conservation of the gene in the Lyme disease spirochete reflected a critical function of the encoded protein. To address this hypothesis, we moved the bba40::Tn allele into a similar wild-type background and compared the phenotypes of isogenic wild-type, mutant and complemented strains in vitro and throughout the in vivo mouse/tick infectious cycle. In contrast to the previous study, we identified no defect in the ability of the bba40 mutant to colonize the tick vector or murine host, or to be efficiently transmitted between them. We conclude that bba40 joins a growing list of unique, highly conserved, yet fully dispensable plasmid-borne genes of the Lyme disease spirochete. We infer that the experimental infectious cycle, while including the tick vector and murine host, lacks key selective forces imposed during the natural enzootic cycle. IMPORTANCE The key finding of this study contradicts our premise that the ubiquitous presence and strict sequence conservation of a unique gene in the Lyme disease spirochete, Borrelia burgdorferi, reflect a critical role in either the murine host or tick vector in which these bacteria are maintained in nature. Instead, the outcome of this investigation illustrates the inadequate nature of the experimental infectious cycle currently employed in the laboratory to fully model the enzootic cycle of the Lyme disease spirochete. This study also highlights the importance of complementation for accurate interpretation of mutant phenotypes in genetic studies of Borrelia burgdorferi.

Clonal isolates of Treponema pallidum subsp. pallidum Nichols provide evidence for the occurrence of microevolution during experimental rabbit infection and in vitro culture.

The recent development of a system for long-term in vitro culture of the syphilis spirochete, Treponema pallidum subsp. pallidum, has introduced the possibility of detailed genetic analysis of this bacterium. In this study, the in vitro culture system was used to isolate and characterize clonal populations of T. pallidum subsp. pallidum Nichols, the most widely studied strain. In limiting dilutions experiments, it was possible to establish cultures with inocula as low as 0.5 T. pallidum per well despite the long generation time (~35 to 40 hours) of this organism. Six Nichols strain clones isolated by limiting dilution were characterized in detail. All clones exhibited indistinguishable morphology and motility, highly similar in vitro multiplication rates, and comparable infectivity in the rabbit model (ID50 ≤ 100 bacteria). Genomic sequencing revealed sequence heterogeneity in the form of insertions or deletions at 5 sites, single nucleotide variations at 20 sites, and polynucleotide (polyG/C) tract length differences at 22 locations. Genomic sequences of the uncloned Nichols strain preparations propagated in rabbits or in vitro cultures exhibited substantial heterogeneity at these locations, indicating coexistence of many varied 'clonotypes' within these populations. Nearly all genetic variations were specific for the Nichols strain and were not detected in the >280 T. pallidum genomic sequences that are currently available. We hypothesize that these Nichols strain-specific sequence variations arose independently either during human infection or within the 110 years since the strain's initial isolation, and thus represent examples of microevolution and divergence.