FliL ring enhances the function of periplasmic flagella.

SignificanceHow flagella sense complex environments and control bacterial motility remain fascinating questions. Here, we deploy cryo-electron tomography to determine in situ structures of the flagellar motor in wild-type and mutant cells of Borrelia burgdorferi, revealing that three flagellar proteins (FliL, MotA, and MotB) form a unique supramolecular complex in situ. Importantly, FliL not only enhances motor function by forming a ring around the stator complex MotA/MotB in its extended, active conformation but also facilitates assembly of the stator complex around the motor. Our in situ data provide insights into how cooperative remodeling of the FliL-stator supramolecular complex helps regulate the collective ion flux and establishes the optimal function of the flagellar motor to guide bacterial motility in various environments.

A Borrelia burgdorferi LptD homolog is required for flipping of surface lipoproteins through the spirochetal outer membrane.

Borrelia spirochetes are unique among diderm bacteria in their lack of lipopolysaccharide (LPS) in the outer membrane (OM) and their abundance of surface-exposed lipoproteins with major roles in transmission, virulence, and pathogenesis. Despite their importance, little is known about how surface lipoproteins are translocated through the periplasm and the OM. Here, we characterized Borrelia burgdorferi BB0838, a distant homolog of the OM LPS assembly protein LptD. Using a CRISPR interference approach, we showed that BB0838 is required for cell growth and envelope stability. Upon BB0838 knockdown, surface lipoprotein OspA was retained in the inner leaflet of the OM, as determined by its inaccessibility to in situ proteolysis but its presence in OM vesicles. The topology of the OM porin/adhesin P66 remained unaffected. Quantitative mass spectrometry of the B. burgdorferi membrane-associated proteome confirmed the selective periplasmic retention of surface lipoproteins under BB0838 knockdown conditions. Additional analysis identified a single in situ protease-accessible BB0838 peptide that mapped to a predicted ß-barrel surface loop. Alphafold Multimer modeled a B. burgdorferi LptB2 FGCAD complex spanning the periplasm. Together, this suggests that BB0838/LptDBb facilitates the essential terminal step in spirochetal surface lipoprotein secretion, using an orthologous OM component of a pathway that secretes LPS in proteobacteria.

Development of an mRNA-lipid nanoparticle vaccine against Lyme disease.

Lyme disease is the most common vector-borne infectious disease in the United States, in part because a vaccine against it is not currently available for humans. We propose utilizing the lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) platform to generate a Lyme disease vaccine like the successful clinical vaccines against SARS-CoV-2. Of the antigens expressed by Borrelia burgdorferi, the causative agent of Lyme disease, outer surface protein A (OspA) is the most promising candidate for vaccine development. We have designed and synthesized an OspA-encoding mRNA-LNP vaccine and compared its immunogenicity and protective efficacy to an alum-adjuvanted OspA protein subunit vaccine. OspA mRNA-LNP induced superior humoral and cell-mediated immune responses in mice after a single immunization. These potent immune responses resulted in protection against bacterial infection. Our study demonstrates that highly efficient mRNA vaccines can be developed against bacterial targets.

Grouped follicular secondary syphilis: Case report and review of literature.

Secondary syphilis typically presents with macular, maculopapular or papular lesions, sometimes with systemic symptoms; however, there are some less common cutaneous presentations which can result in several differential diagnoses. We report the case of a 25-year-old man with the recent onset of a symmetric eruption of grouped follicular papules, for which syphilis was not originally considered. Histopathology revealed non-caseating granulomas with a lichenoid infiltrate. Subsequent spirochete immunostaining was positive, and further physical examination revealed moth-eaten alopecia, confirming the diagnosis of secondary syphilis.

Impact of Extracellular DNA on Architectural Parameters of Leptospira biflexa Biofilm.

Extracellular DNA (eDNA) is a major component of bacterial biofilms. In this study, we performed a three-dimensional analysis of Leptospira biofilm using advanced imaging by confocal laser scanning microscopy (CLSM) and multi-parameter analysis by COMSTAT 2 software, with quantification of Leptospira and eDNA fluorescence. To investigate the role of eDNA in Leptospira biofilm, we treated Leptospira biflexa biofilms with DNase I enzyme (DNase), which digested eDNA, and compared DNase treated biofilms and controls. There was a significant reduction of the biomass of biofilms treated with DNase, by spectrophotometry and COMSTAT analysis. The multiparameter analysis evidenced for DNase-treated biofilms a significant decrease in the surface area and the average thickness; opposing to a significant augmentation of the surface/biovolume ratio and the roughness coefficient (Ra*), when compared to controls. We analyzed the parameters of DNase-treated biofilms by Pearson's correlation coefficient and found significant positive correlations between biomass and average thickness; biomass and surface area; surface area and average thickness. On the other hand, there were significant negative correlations between Ra* and biomass; Ra* and average thickness; Ra* and surface area. These findings suggest that eDNA digestion results in biofilm instability and alteration of the three-dimensional architecture, justifying the negative correlation between Ra* and the above-mentioned parameters. In conclusion, our study showed that eDNA digestion produced a massive structural loss, instability, and dramatic changes in the three-dimensional architecture of Leptospira biflexa biofilm. These findings contribute to a better understanding of the role of eDNA and highlight the importance of eDNA as a key component in Leptospira biofilms.

Characterization of Treponema denticola Major Surface Protein (Msp) by Deletion Analysis and Advanced Molecular Modeling.

Treponema denticola, a keystone pathogen in periodontitis, is a model organism for studying Treponema physiology and host-microbe interactions. Its major surface protein Msp forms an oligomeric outer membrane complex that binds fibronectin, has cytotoxic pore-forming activity, and disrupts several intracellular processes in host cells. T. denticola msp is an ortholog of the Treponema pallidum tprA to -K gene family that includes tprK, whose remarkable in vivo hypervariability is proposed to contribute to T. pallidum immune evasion. We recently identified the primary Msp surface-exposed epitope and proposed a model of the Msp protein as a ß-barrel protein similar to Gram-negative bacterial porins. Here, we report fine-scale Msp mutagenesis demonstrating that both the N and C termini as well as the centrally located Msp surface epitope are required for native Msp oligomer expression. Removal of as few as three C-terminal amino acids abrogated Msp detection on the T. denticola cell surface, and deletion of four residues resulted in complete loss of detectable Msp. Substitution of a FLAG tag for either residues 6 to 13 of mature Msp or an 8-residue portion of the central Msp surface epitope resulted in expression of full-length Msp but absence of the oligomer, suggesting roles for both domains in oligomer formation. Consistent with previously reported Msp N-glycosylation, proteinase K treatment of intact cells released a 25 kDa polypeptide containing the Msp surface epitope into culture supernatants. Molecular modeling of Msp using novel metagenome-derived multiple sequence alignment (MSA) algorithms supports the hypothesis that Msp is a large-diameter, trimeric outer membrane porin-like protein whose potential transport substrate remains to be identified. IMPORTANCE The Treponema denticola gene encoding its major surface protein (Msp) is an ortholog of the T. pallidum tprA to -K gene family that includes tprK, whose remarkable in vivo hypervariability is proposed to contribute to T. pallidum immune evasion. Using a combined strategy of fine-scale mutagenesis and advanced predictive molecular modeling, we characterized the Msp protein and present a high-confidence model of its structure as an oligomer embedded in the outer membrane. This work adds to knowledge of Msp-like proteins in oral treponemes and may contribute to understanding the evolutionary and potential functional relationships between T. denticola Msp and the orthologous T. pallidum Tpr proteins.

Agglutination of Borreliella burgdorferi by Transmission-Blocking OspA Monoclonal Antibodies and Monovalent Fab Fragments.

Lyme disease vaccines based on recombinant Outer surface protein A (OspA) elicit protective antibodies that interfere with tick-to-host transmission of the disease-causing spirochete Borreliella burgdorferi. Another hallmark of OspA antisera and certain OspA monoclonal antibodies (MAbs) is their capacity to induce B. burgdorferi agglutination in vitro, a phenomenon first reported more than 30 years ago but never studied in molecular detail. In this report, we demonstrate that transmission-blocking OspA MAbs, individually and in combination, promote dose-dependent and epitope-specific agglutination of B. burgdorferi. Agglutination occurred within minutes and persisted for hours. Spirochetes in the core of the aggregates exhibited evidence of outer membrane (OM) stress, revealed by propidium iodide uptake. The most potent agglutinator was the mouse MAb LA-2, which targets the OspA C terminus (ß-strands 18 to 20). Human MAb 319-44, which also targets the OspA C terminus (ß-strand 20), and 857-2, which targets the OspA central ß-sheet (strands 8 to 10), were less potent agglutinators, while MAb 221-7, which targets ß-strands 10 to 11, had little to no measurable agglutinating activity, even though its affinity for OspA exceeded that of LA-2. Remarkably, monovalent Fab fragments derived from LA-2, and to a lesser degree 319-44, retained the capacity to induce B. burgdorferi aggregation and OM stress, a particularly intriguing observation considering that "LA-2-like" Fabs have been shown to experimentally entrap B. burgdorferi within infected ticks and prevent transmission during feeding to a mammalian host. It is therefore tempting to speculate that B. burgdorferi aggregation triggered by OspA-specific antibodies in vitro may in fact reflect an important biological activity in vivo.

Motility of the Zoonotic Spirochete Leptospira: Insight into Association with Pathogenicity.

If a bacterium has motility, it will use the ability to survive and thrive. For many pathogenic species, their motilities are a crucial virulence factor. The form of motility varies among the species. Some use flagella for swimming in liquid, and others use the cell-surface machinery to move over solid surfaces. Spirochetes are distinguished from other bacterial species by their helical or flat wave morphology and periplasmic flagella (PFs). It is believed that the rotation of PFs beneath the outer membrane causes transformation or rolling of the cell body, propelling the spirochetes. Interestingly, some spirochetal species exhibit motility both in liquid and over surfaces, but it is not fully unveiled how the spirochete pathogenicity involves such amphibious motility. This review focuses on the causative agent of zoonosis leptospirosis and discusses the significance of their motility in liquid and on surfaces, called crawling, as a virulence factor.

Comparison of Lyme Disease in the United States and Europe.

Lyme disease, or Lyme borreliosis, is the most common tickborne disease in the United States and Europe. In both locations, Ixodes species ticks transmit the Borrelia burgdorferi sensu lato bacteria species responsible for causing the infection. The diversity of Borrelia species that cause human infection is greater in Europe; the 2 B. burgdorferi s.l. species collectively responsible for most infections in Europe, B. afzelii and B. garinii, are not found in the United States, where most infections are caused by B. burgdorferi sensu stricto. Strain differences seem to explain some of the variation in the clinical manifestations of Lyme disease, which are both minor and substantive, between the United States and Europe. Future studies should attempt to delineate the specific virulence factors of the different species of B. burgdorferi s.l. responsible for these variations in clinical features.

Characterization of 6S RNA in the Lyme disease spirochete.

6S RNA binds to RNA polymerase and regulates gene expression, contributing to bacterial adaptation to environmental stresses. In this study, we examined the role of 6S RNA in murine infectivity and tick persistence of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi. B. burgdorferi 6S RNA (Bb6S RNA) binds to RNA polymerase, is expressed independent of growth phase or nutrient stress in culture, and is processed by RNase Y. We found that rny (bb0504), the gene encoding RNase Y, is essential for B. burgdorferi growth, while ssrS, the gene encoding 6S RNA, is not essential, indicating a broader role for RNase Y activity in the spirochete. Bb6S RNA regulates expression of the ospC and dbpA genes encoding outer surface protein C and decorin binding protein A, respectively, which are lipoproteins important for host infection. The highest levels of Bb6S RNA are found when the spirochete resides in unfed nymphs. ssrS mutants lacking Bb6S RNA were compromised for infectivity by needle inoculation, but injected mice seroconverted, indicating an ability to activate the adaptive immune response. ssrS mutants were successfully acquired by larval ticks and persisted through fed nymphs. Bb6S RNA is one of the first regulatory RNAs identified in B. burgdorferi that controls the expression of lipoproteins involved in host infectivity.

Mucosal eosinophilic infiltration may be a characteristic of human intestinal spirochetosis.

BACKGROUND: Human intestinal spirochetosis (HIS) is an infectious disease of large intestines caused by Brachyspira species, and most HIS cases are asymptomatic or exhibit mild intestinal symptoms. The host reaction to HIS remains unclear, and we examined HIS-related mucosal inflammatory features histologically. METHODS: From the archival HIS cases in a single medical center, 24 endoscopically taken specimens from 14 HIS cases (male:female = 10:4; 28-73 yrs) were selected as not containing polypoid or neoplastic lesions. Stromal neutrophils, eosinophils, and mast cells, and intraepithelial neutrophils and eosinophils, (sNeu, sEo, sMast, iNeu, and iEo, respectively) were counted, and the presence or absence of lymphoid follicles/aggregates (LFs) was also examined. Association of the above inflammation parameters and spirochetal infection parameters (such as degrees of characteristic fringe distribution, of spirochetal cryptal invasion, and of spirochetal intraepithelial invasion) were also analysed. RESULTS: iNeu was observed in 29.2%, iEo in 58.3%, and LFs in 50.0% of the specimens. Maximal counts of sNeu, sEo, sMast, iNeu, and iEo averaged 8.4, 21.5, 6.0, 0.5 and 1.5, respectively. Strong correlation between the maximum counts of iNeu and iEo (p < 0.001, r = 0.81), and correlations between those of iEo and sNeu (p = 0.0012, r = 0.62) and between those of iEo and sEo (p = 0.026, r = 0.45) were observed. iNeu was influenced by fringe formation (p < 0.05) and spirochetal crypt involvement (p < 0.05). CONCLUSIONS: HIS was accompanied by inflammatory reactions, and among these, mucosal eosinophilic infiltration may be a central indicator and host reaction of HIS.

Underrecognized Tickborne Illnesses: Borrelia Miyamotoi and Powassan Virus.

Over the past 2 decades, tickborne disease has been increasingly recognized as a threat to humans as a result of the growing geographic range of ticks. This review describes 2 tickborne diseases, Borrelia miyamotoi and Powassan virus, that likely have a significant impact on humans, yet are underdiagnosed compared to most other tickborne diseases. We performed a literature search from 2015 to 2020. Borrelia miyamotoi is a tickborne pathogen that infects and co-infects ticks along with other pathogens, including Borrelia burgdorferi. Because B miyamotoi infects the same Ixodes ticks as B burgdorferi, B miyamotoi may cover a similar geographic range. B miyamotoi infection may be underdiagnosed for 2 reasons. First, a presumptive treatment approach to Lyme disease may result in B miyamotoi infection treatment without identification of the actual cause. Second, the absence of readily available testing and diagnostic criteria makes it difficult to diagnose B miyamotoi infection. Powassan virus is a tickborne flavivirus similar to the dengue virus. Powassan virus disease appears to have an asymptomatic or minimally symptomatic presentation in most people but can cause devastating and fatal encephalitis. The Powassan virus may be transmitted in less than 15 min of tick feeding. Powassan virus disease is a difficult diagnosis because testing capabilities are limited and because there may be co-infection with other tickborne pathogens.

A pleiotropic role of FlaG in regulating the cell morphogenesis and flagellar homeostasis at the cell poles of Treponema denticola.

FlaG homologue has been found in several bacteria including spirochetes; however, its function is poorly characterised. In this report, we investigated the role of TDE1473, a putative FlaG, in the spirochete Treponema denticola, a keystone pathogen of periodontitis. TDE1473 resides in a large gene operon that is controlled by a σ70 -like promoter and encodes a putative FlaG protein of 123 amino acids. TDE1473 can be detected in the periplasmic flagella (PFs) of T. denticola, suggesting that it is a flagella-associated protein. Consistently, in vitro studies demonstrate that the recombinant TDE1473 interacts with the PFs in a dose-dependent manner and that such an interaction requires FlaA, a flagellar filament sheath protein. Deletion of TDE1473 leads to long and less motile mutant cells. Cryo-electron tomography analysis reveal that the wild-type cells have 2-3 PFs with nearly homogenous lengths (ranging from 3 to 6 µm), whereas the mutant cells have less intact PFs with disparate lengths (ranging from 0.1 to 9 µm). The phenotype of T. denticola TDE1473 mutant reported here is different from its counterparts in other bacteria, which provides insight into further understanding the role of FlaG in the regulation of bacterial cell morphogenesis and flagellation.

A small intergenic region of lp17 is required for evasion of adaptive immunity and induction of pathology by the Lyme disease spirochete.

The causative agent of Lyme disease, Borrelia burgdorferi, harbours a single linear chromosome and upwards of 23 linear and circular plasmids. Only a minority of these plasmids, including linear plasmid 17, are maintained with near-absolute fidelity during extended in vitro passage, and characterisation of any putative virulence determinants they encode has only recently begun. In this work, a mutant lacking a ~4.7 kb fragment of lp17 was studied. Colonisation of murine tissues by this lp17 mutant was significantly impaired, as was the ability to induce carditis and arthritis. The deficiency in tissue colonisation was alleviated in severe combined immunodeficient (SCID) mice, implicating a role for this plasmid region in adaptive immune evasion. Through genetic complementation, the mutant phenotype could be fully attributed to a 317 bp intergenic region that corresponds to the discontinued bbd07 ORF and upstream sequence. The intergenic region was found to be transcriptionally active, and mutant spirochetes lacking this region exhibited an overall difference in the antigenic profile during infection of an immunocompetent murine host. Overall, this study is the first to provide evidence for the involvement of lp17 in colonisation of joint and heart tissues, along with the associated pathologies caused by the Lyme disease spirochete.

The Spirochete Brachyspira pilosicoli, Enteric Pathogen of Animals and Humans.

Brachyspira pilosicoli is a slow-growing anaerobic spirochete that colonizes the large intestine. Colonization occurs commonly in pigs and adult chickens, causing colitis/typhlitis, diarrhea, poor growth rates, and reduced production. Colonization of humans also is common in some populations (individuals living in village and peri-urban settings in developing countries, recent immigrants from developing countries, homosexual males, and HIV-positive patients), but the spirochete rarely is investigated as a potential human enteric pathogen. In part this is due to its slow growth and specialized growth requirements, meaning that it is not detectable in human fecal samples using routine diagnostic methods. Nevertheless, it has been identified histologically attached to the colon and rectum in patients with conditions such as chronic diarrhea, rectal bleeding, and/or nonspecific abdominal discomfort, and one survey of Australian Aboriginal children showed that colonization was significantly associated with failure to thrive. B. pilosicoli has been detected in the bloodstream of elderly patients or individuals with chronic conditions such as alcoholism and malignancies. This review describes the spirochete and associated diseases. It aims to encourage clinicians and clinical microbiologists to consider B. pilosicoli in their differential diagnoses and to develop and use appropriate diagnostic protocols to identify the spirochete in clinical specimens.

Infection resistance and tolerance in Peromyscus spp., natural reservoirs of microbes that are virulent for humans.

The widely-distributed North American species Peromyscus leucopus and P. maniculatus of cricetine rodents are, between them, important natural reservoirs for several zoonotic diseases of humans: Lyme disease, human granulocytic anaplasmosis, babesiosis, erhlichiosis, hard tickborne relapsing fever, Powassan virus encephalitis, hantavirus pulmonary syndrome, and plague. While these infections are frequently disabling and sometimes fatal for humans, the peromyscines display little pathology and apparently suffer few consequences, even when prevalence of persistent infection in a population is high. While these Peromyscus spp. are unable to clear some of the infections, they appear to have partial resistance, which limits the burden of the pathogen. In addition, they display traits of infection tolerance, which reduces the damage of the infection. Research on these complementary resistance and tolerance phenomena in Peromyscus has relevance both for disease control measures targeting natural reservoirs and for understanding the mechanisms of the comparatively greater sickness of many humans with these and other infections.

Localization of Outer Membrane Proteins in Treponema denticola by Quantitative Proteome Analyses of Outer Membrane Vesicles and Cellular Fractions.

The identification and localization of outer membrane proteins (Omps) and lipoproteins in pathogenic treponemes such as T. denticola (periodontitis) and T. pallidum (syphilis) has been challenging. In this study, label-free quantitative proteomics using MaxQuant was applied to naturally produced outer membrane vesicles (OMVs) and cellular fractions to identify 1448 T. denticola proteins. Of these, 90 proteins were localized to the outer membrane (OM) comprising 59 lipoproteins, 25 ß-barrel proteins, and six other putative OM-associated proteins. Twenty-eight lipoproteins were localized to the inner membrane (IM), and 43 proteins were assigned to the periplasm. The signal cleavage regions of the OM and IM lipoprotein sequences were different and may reveal the signals for their differential localization. Proteins significantly enriched in OMVs included dentilisin, proteins containing leucine-rich repeats, and several lipoproteins containing FGE-sulfatase domains. Blue native PAGE analysis enabled the native size of the dentilisin complex and Msp to be determined and revealed that the abundant ß-barrel Omps TDE2508 and TDE1717 formed large complexes. In addition to the large number of integral Omps and potentially surface-located lipoproteins identified in T. denticola, many such proteins were also newly identified in T. pallidum through homology, generating new targets for vaccine development in both species.

Treponeme-Associated Hoof Disease of Free-Ranging Elk ( Cervus elaphus) in Southwestern Washington State, USA.

A novel foot disease in free-ranging elk ( Cervus elaphus) in southwestern Washington State emerged in 2008 and spread throughout the region. Initial studies showed adult elk had chronic hoof overgrowth, sole ulcers, and sloughed hoof capsules, but no cause was determined. To identify possible causes and characterize the earliest lesions, 9-, 7-, and 3-month-old elk were collected. Nine-month-old elk had sole ulcers (3/9 elk) and sloughed/overgrown hoof capsules (4/9 elk) similar to adults. Histologically, lesions consisted of coronary, heel bulb, and interdigital ulcers with suppurative inflammation, epithelial hyperplasia, deeply invasive spirochetes, and underrunning of the hoof capsule and heel-sole junction. Spirochetes were identified as Treponema via immunohistochemistry and polymerase chain reaction (PCR). Seven-month-old elk had similar underrunning foot ulcers (6/8 elk) with Treponema identified in all lesions but no chronic overgrowth or sloughed hoof capsules. Three-month-old calves had superficial coronary erosions with no inflammation or identifiable spirochetes (3/5 elk) but were culture/PCR positive for Treponema, suggesting possible early lesions. Lesions from 9- and 7-month-old elk included aerobic and anaerobic bacteria, many of which are associated with infectious foot disease in livestock. Antibody enzyme-linked immunosorbent assay of 7- and 3-month-old elk from the enzootic region showed a trend toward increased Treponema antibody titers compared to normal control elk from outside the region, further supporting the significance of Treponema in the pathogenesis of foot disease. Treponeme-associated hoof disease (TAHD) in elk, a debilitating and progressive condition, shares similarities to bovine digital dermatitis and contagious ovine digital dermatitis.

Syphilis of the oropharynx: Case series of "The Great Masquerader".

OBJECTIVE: Syphilis is a sexually transmitted infection with various presentations. Although, oropharyngeal manifestations are known to occur, the purpose of this study is to present the first case series in which the lesions were initially mistaken for human-papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC). METHODS: A multi-institutional retrospective review. RESULTS: Six cases of oropharyngeal syphilis were initially thought to be secondary to OPSCC due to presentation. Symptoms were vague and exam findings consisted of either a tonsillar or base of tongue mass, or lymphadenopathy. Biopsies were negative for OPSCC. Further workup diagnosed syphilis, with resolution of symptoms and lesions after antibiotic treatment. CONCLUSIONS: Head and neck manifestations of syphilis have been reported in the literature. However, this is the first series reporting on oropharyngeal syphilis masquerading as HPV-related OPSCC. Ultimately, otolaryngologists must maintain a high suspicion for syphilis in order to ensure prompt diagnosis and treatment.