BackgroundTreponema pallidum subspecies pallidum causes systemic syphilis, exclusively infects humans in nature and can persist for decades in the absence of treatment despite generating robust adaptive immune responses. The T. pallidum repeat (Tpr) family of outer membrane proteins are immunogenic and are implicated in immune evasion, indicating them to be virulence factors displayed on the spirochete surface. Long-term survival of T. pallidum is largely attributed to sparse surface-exposed outer membrane proteins and antigenic variation in the major surface protein TprK through phase variation. This mechanism has been studied for decades; however, the functions of Tprs of this extracellular pathogen are not yet experimentally determined.MethodsIn this study, the localization and functional roles of TprD and TprK were investigated using a heterologous spirochete expression system and a gain-of-function approach by employing a non-infectious, non-adherent Borrelia burgdorferi B314 strain. Opsonophagocytosis of engineered B. burgdorferi as well as of T. pallidum Nichols and SS14 strains was evaluated using J774A.1 macrophages and mouse antibodies raised against predicted surface-exposed loops of TprD and TprK using IncuCyte system.ResultsBoth TprD and TprK were found to be surface exposed in engineered B. burgdorferi and infectious T. pallidum strains. Expression of these proteins conferred adherence to several mammalian cell lines in vitro. In addition, antibodies we generated recognized TprD and TprK on the surface of spirochetes and significantly enhanced macrophage-mediated opsonophagocytosis.ConclusionOur findings here demonstrate that TprD and TprK function as T. pallidum adhesins that are also targets of opsonic antibodies. These Tprs likely facilitate tissue colonization during infection, while also rendering the pathogen susceptible to immune clearance. Our findings support inclusion of TprD and TprK as components of a multivalent vaccine against syphilis.

BackgroundPseudomonas aeruginosa is a ubiquitous organism that adapts well in different environments. It is an opportunistic bacterial pathogen that produces a wide range of virulence factors, colonizes lungs to cause pneumonia, causes non-healing wounds especially in burn victims, and is a major culprit in destructive keratitis. It can reach the cornea through reusable, extended-use contact lenses and by contaminated eyedrops and artificial tears. Secreted proteases of P. aeruginosa together with pyocyanin metabolite, which inhibits Serine Protease Inhibitors (Serpins) activity contribute to severe tissue damage during infection.MethodsPseudomonas aeruginosa strains isolated from different clinical sites were obtained from different researchers and clinicians for this study. We examined P. aeruginosa strains and secretion defective and prpL knockout mutants in PA64481 strain for lysyl endopeptidase (PrpL, a serine protease that cleaves after a lysine residue) activity using serine protease specific D-Val-Leu-Lys -p-nitroanalide substrate. We also determined pyocyanin production in these strains.ResultsExamination of secreted milieu from P. aeruginosa showed that 41 corneal isolates had detectable lysyl endopeptidase activity associated with PrpL at levels significantly higher than by 27 non-corneal isolates. We found that PrpL is secreted by the xcp-based type II secretion system. Bacterial culture supernatants displaying higher PrpL activity and not low secreted PrpL levels disrupted corneal epithelial cell monolayers in vitro, which is consistent with a role of this protease in destructive keratitis. Many examined strains also produced high levels of pyocyanin.ConclusionThis retrospective examination of clinical P. aeruginosa suggests that high levels of PrpL and pyocyanin-producing isolates are more prevalent among corneal isolates and could enhance tissue damage during infection. Supporting this premise, corneal epithelial cell monolayers disrupted by high PrpL-producing strains but remained intact after treatment with P. aeruginosa mutants culture supernatants that lack or have reduced secreted PrpL.

Treponema pallidum subspecies pallidum ( T. pallidum ) causes systemic syphilis, exclusively infects humans in nature and can persist for decades in the absence of treatment despite generating robust adaptive immune responses. The T. pallidum repeat (Tpr) family of outer membrane proteins are immunogenic but have been implicated in immune evasion due to antigenic variation, indicating that Tprs are virulence factors displayed on the spirochetes surface. Long-term survival of T. pallidum is largely attributed to the sparse surface-exposed outer membrane proteins and antigenic variation exhibited by the major surface protein TprK, which undergoes phase variation. Mechanism of antigenic variation has been studied for decades; however, functions of Tprs in this extracellular pathogen have not been experimentally determined. In this study, we determined TprD and TprK location, their role in adherence and in clearance of T. pallidum by macrophages. Using our now established heterologous surrogate system and gain-in-function approach using non-adherent related spirochete, the B314 strain of Borrelia burgdorferi , we show that both TprD and TprK are surface exposed to some extent on engineered B. burgdorferi as well as on T. pallidum Nichols and SS14 strains. We further demonstrate that both proteins mediate adherence to different mammalian cells in vitro and mouse antibodies generated against TprD and TprK putative surface loops bind spirochetes and promote J774A.1 macrophages-mediated opsonophagocytosis. Thus, surface-exposed adhesins TprD and TprK of T. pallidum contribute to binding to different types of cells which likely reflect the pathogen’s ability to colonize different tissues, and they are also targets of opsonic antibodies. IMPORTANCE Syphilis remains a major global public health challenge and is exacerbated by the rising number of cases of congenital infection and increased risk of HIV acquisition and transmission in syphilitic patients. A critical barrier to improving understanding of the molecular basis of syphilis pathogenesis owe to fragility of T. pallidum , inability to grow it in pure culture, and difficulty in generating knockout mutants in predicted virulence factors due to their possible essential role in spirochetes viability. Our findings provide experimental evidence linking Tpr proteins to host cell adherence, their ability to generate humoral immune response in the rabbit model of infection as well as in humans which could facilitate clearance by macrophages. Demonstration of TprD and TprK as the targets of opsonic antibodies here highlight their potential as protective immunogens and emphasizes importance of their inclusion in the cocktail to produce effective vaccine against syphilis.

The Lyme disease spirochete, Borrelia burgdorferi, requires cholesterol to grow. The spirochete acquires cholesterol from the host to form cholesterol glycolipids, which are then incorporated into the spirochete's membrane. This study aimed to determine whether higher levels of serum cholesterol could facilitate the infection and contribute to the pathogenesis of Lyme disease. We investigated the effect of acute and chronic hypercholesterolemia on spirochetal infection in C3H/HeJ mice fed a high-fat diet (HF) compared to mice fed a control diet. The course of infection in mice was followed for 3 weeks (short-term effects) and 16 weeks post-infection (longterm effects) by measuring spirochete bioluminescence in vivo. At the endpoint, bacterial burden was measured in tissues by real-time PCR, and histology was performed to assess differences in the inflammatory response. Between days 10 and 14 post-infection, live imaging showed that mice on a HF diet presented a significantly higher spirochetal burden and greater dissemination than the controls. These differences were transient and restricted to the first two weeks of infection, without long-term effect observed. Histology showed no significant differences in inflammation between HF and control mice. However, qPCR showed that mice fed with HF diet had a higher B. burgdorferi burden in tissues, including heart, visceral, and subcutaneous fat. These findings revealed that high cholesterol levels resulting from a HF diet led to increases in spirochetal burden and dissemination early in the infection, suggesting that cholesterol may contribute to spirochete persistence and associated Lyme disease symptoms.

Babesia microti is increasingly co-transmitted with Lyme disease-causing Borrelia burgdorferi by ticks in the endemic regions of the United States. Infection in mice by this parasite mirrors human babesiosis, such as anemia, splenomegaly, alterations in splenic leukocyte balance, and diminished humoral immunity associated with enhanced Lyme disease manifestations at the acute phase. To evaluate the long-term survival of B. burgdorferi and B. microti in mice and their effects on pathogenesis, we conducted a 16-week infection experiment in C3H/HeJ mice. All mice infected with B. microti, irrespective of the co-infection, displayed a low-level, albeit microscopically detectable, parasitemia in both male and female mice even after 16 weeks post-infection. Splenomegaly was detected at this stage in both male and female mice and was significantly higher in females infected solely with B. microti compared to co-infected mice, likely due to a greater peak parasitemia at the acute phase of infection and persistent splenic manifestations in these mice. Interestingly, B. microti disrupted the blood-brain barrier (BBB) in mice, as reported during cerebral malaria caused by Plasmodium falciparum. Furthermore, B. burgdorferi colonization could be detected until 16 weeks of infection, while more pronounced Lyme inflammatory arthritis was observed at 4 weeks post-infection. This study underscores the complex interactions between B. microti and B. burgdorferi to affect each disease, highlighting the potential implications for vaccine development against Lyme spirochetes and therapeutic management of co-infected individuals.IMPORTANCETick-borne co-infections are becoming increasingly prevalent worldwide due to simultaneous or sequential acquisition and transmission by Ixodes species ticks during their blood meal. We reported that B. burgdorferi and B. microti co-infection reciprocally affects these pathogens during the acute phase of infection; however, the effect of co-infections on microbial long-term persistence in the murine model was not previously investigated. In this study, we have filled a critical lacuna in understanding the interactions between these two pathogens at different stages of infection and their effects on the host and disease manifestations in mice. Our investigation provides insights into their pathogenicity to allow the development of effective vaccines and successful antimicrobials against these tick-borne co-infections.

Apicomplexan protozoan parasite Babesia microti (Bm) and spirochete Borrelia burgdorferi are tick-transmitted pathogens that cause babesiosis and Lyme disease and increasingly cause coinfections. More pronounced Lyme arthritis occurs during the acute phase of coinfection compared to B. burgdorferi alone. In susceptible C3H mice, Bm parasitemia was quantified by microscopic examination of blood smears while live imaging of bioluminescent B. burgdorferi N40 strain-infected mice allowed monitoring of disseminated infection. Furthermore, proteomic analyses of blood samples unveiled nuanced temporal host gene expression. At 2 weeks postinfection, 31, 96, 76, and 22 unique proteins were detected in naïve and N40+Bm-, N40-, and Bm-infected mice, respectively, while 3359 common proteins were identified across all groups. The proteomic landscape showed a significant overlap between naive and Bm-infected mice with the most pronounced differences from the coinfected group. Using fold change scatter plots, upregulation of proteins associated with cellular and metabolic processes was noticed particularly in the coinfected mice. At 4 weeks, proteomic profiles among naive, Bm, and coinfection mice demonstrate distinct host responses in blood. The overlap diminished further at 16 weeks with stage-specific clustering of proteins observed. Our findings illustrate intricate interactions between these two pathogens and valuable host proteome dynamics during infection.

ABSTRACT Background: Pseudomonas aeruginosa is a ubiquitous organism that adapts well in different environments. It is an opportunistic bacterial pathogen that produces a wide range of virulence factors, colonizes lungs to cause pneumonia, causes non-healing wounds especially in burn victims, and is a major culprit in destructive keratitis. It can reach the cornea through reusable, extended-use contact lenses and by contaminated eyedrops and artificial tears. Secreted proteases of P. aeruginosa together with pyocyanin metabolite, which inhibits Serine Protease Inhibitors (Serpins) activity contribute to severe tissue damage during infection. Methods: P. aeruginosa strains isolated from different clinical sites were obtained from different researchers and clinicians for this study. We examined P. aeruginosa strains and secretion defective and prpL knockout mutants in PA64481 strain for lysyl endopeptidase (PrpL, a serine protease that cleaves after a lysine residue) activity using serine protease specific D-Val-Leu-Lys -p-nitroanalide substrate. We also determined pyocyanin production in these strains. Results: Examination of secreted milieu from P. aeruginosa showed that 41 corneal isolates had detectable lysyl endopeptidase activity associated with PrpL at levels significantly higher than by 27 non-corneal isolates. We found that PrpL is secreted by the xcp-based type II secretion system. Bacterial culture supernatants displaying higher PrpL activity and not low secreted PrpL levels disrupted corneal epithelial cell monolayers in vitro, which is consistent with a role of this protease in destructive keratitis. Many examined strains also produced high levels of pyocyanin. Conclusions: This retrospective examination of clinical P. aeruginosa suggests that high levels of PrpL and pyocyanin-producing isolates are more prevalent among corneal isolates and could enhance tissue damage during infection. Supporting this premise, corneal epithelial cell monolayers disrupted by high PrpL-producing strains but remained intact after treatment with P. aeruginosa mutants culture supernatants that lack or have reduced secreted PrpL. ### Competing Interest Statement The authors have declared no competing interest.

The major human spirochetal pathogens (Leptospira, Borrelia, and Treponema) are difficult to diagnose and lack vaccines to prevent infections. Infection by these spirochetes does not generate general protective immunity, allowing reinfection by different strains to occur. These stealth pathogens have uncommon physiology, pathogenesis, and clinical presentations and possess unique immune evasion mechanisms to facilitate their host adaptation and persistence. Collectively, host-spirochete interactions orchestrate systemic infections in a manner distinct from organ- and tissue-specific diseases caused by many bacterial pathogens. Difficulties in growing and genetic manipulation of infectious spirochetes have hindered the full understanding of their virulence factors despite decades to centuries of research. This article highlights the current understanding of the intricacies of spirochetal pathogenesis and diseases. Our comprehensive review of the progress versus gaps in knowledge lays a foundation for researchers to direct their studies toward the development of effective diagnostics and vaccines to protect patients from serious, chronic spirochetal diseases.

Objective: Objective of this study was to explore factors and outcomes associated with gestational syphilis in Peru.Methods: Women from the miscarriage, vaginal delivery, and C-section wards from a large maternity hospital in Lima with, or without syphilis diagnosis were enrolled and their pregnancy outcomes compared. Maternal syphilis status using maternal blood and child serostatus using cord blood were determined by rapid plasma reagin (RPR) and rapid syphilis tests (RST). Newborns’ clinical records were used to determine congenital syphilis.Results: Among 340 women samples, 197 were positive and 143 were negative for RPR/RST. Antibody titers in sera from cord and maternal blood were comparable with RPR titers highly correlated (rho: 0·82, p<0·001). Young age (p=0·009) and lower birth weight (p=0·029) were associated with gestational syphilis. Among women with gestational syphilis, 76% had received proper treatment. Mothers of all newborns with congenital syphilis also received appropriate treatment. Treatment of their sexual partners was not documented.Conclusions: Syphilis during pregnancy remains a major cause of the fetal loss and devastating effects of congenital syphilis in newborns.

Lyme disease is the most prevalent tick-borne infection caused by Borrelia burgdorferi bacteria in North America. Other Borrelia species are predominately the cause of this disease in Eurasia with some distinct and various overlapping manifestations. Consequently, caution must be exercised when comparing the disease and its manifestations and treatment regimens in North America and Europe. Diagnosis of the early Lyme disease remains difficult using the currently FDA approved serological tests in the absence of a reported tick bite or of erythema migrans in many individuals, non-specific initial symptoms, and the absence of detectable anti-Borrelia antibodies in the prepatent period of infection. Furthermore, it is difficult to distinguish persistence of infection and disease versus reinfection in the endemic regions of Lyme disease by serological assays. If early infection remains untreated, spirochetes can disseminate and could affect various organs in the body with a variety of disease manifestations including arthralgias and musculoskeletal pain, neurologic symptoms and anomalies, and acrodermatitis chronicum atrophicans (ACA) in Europe. Although most patients recover after antibiotic treatment, an estimated ∼10–20% patients in the United States show persistence of symptoms known as post-treatment Lyme disease syndrome (PTLDS). The causes and biomarkers of PTLDS are not well-defined; however, several contributing factors with inconsistent degree of supporting evidence have been suggested. These include antigenic debris, dysregulation of immunological response, bacterial persisters, or combination of these features. This review highlights currently employed treatment approaches describing different antimicrobials used, and vaccine candidates tried to prevent B. burgdorferi infection.

Transplacental transmission of syphilis causing spirochete, Treponema pallidum subspecies pallidum, from mother to child results in congenital syphilis, an ever-expanding devastating disease worldwide. Although adverse effects of untreated gestational Lyme disease, caused by a related spirochete, Borrelia burgdorferi on fetus viability and development have been observed, cases of congenital Lyme disease are not reported. In this study, we show that B. burgdorferi colonizes mammary glands of C3H mice only postpartum; however, neither transmission of these spirochetes from dams-to-pups occurs nor congenital Lyme disease is observed in pups.

Lyme disease is caused by Borrelia burgdorferi, and the pathogenesis of the disease is complex with both bacterial and host factors contributing to inflammatory responses. Lyme disease affects different organs including joints and results in arthritis. Immune responses stimulated by B. burgdorferi through toll-like receptors cause infiltration of leukocytes, which produce inflammatory cytokines and facilitate spirochete clearance. However, arthritic manifestations and chronic fatigue syndrome-like symptoms persist long after completion of antibiotic treatment regimens in a significant number of patients. To counter the effects of inflammation, treatment by non-steroidal anti-inflammatory drugs, hydroxychloroquine, or synovectomy to eradicate inflammatory arthritis in the involved joint could be employed; however, they often have long-term consequences. Acupuncture has been used for a long time in Asian medicine to diminish pain during various ailments, but the effects and its mechanism are just beginning to be explored. Control of inflammation by neuronal stimulation has been exploited as a systemic therapeutic intervention to arrest inflammatory processes. Our objective was to determine whether activation of the sciatic–vagal network by electroacupuncture on ST36 acupoint, which is used to control systemic inflammation in experimental models of infectious disorders such as endotoxemia, can also alleviate Lyme arthritis symptoms in mice. This aim was further strengthened by the reports that sciatic–vagal neuronal network stimulation can lead to dopamine production in the adrenal medulla and moderate the production of inflammatory factors. We first assessed whether electroacupuncture affects spirochete colonization to attenuate Lyme arthritis. Interestingly, bioluminescent B. burgdorferi burden detected by live imaging and qPCR were similar in electroacupuncture- and mock-treated mice, while electroacupuncture induced a lasting anti-inflammatory effect on mice. Despite the discontinuation of treatment at 2 weeks, the simultaneous decrease in neutrophils in the joints and inflammatory cytokine levels throughout the body at 4 weeks suggests a systemic and persistent effect of electroacupuncture that attenuates Lyme arthritis. Our results suggest that electroacupuncture-mediated anti-inflammatory responses could offer promising healthcare benefits in patients suffering from long-term Lyme disease manifestations.

A wide range of protozoan pathogens either transmitted by vectors (Plasmodium, Babesia, Leishmania and Trypanosoma), by contaminated food or water (Entamoeba and Giardia), or by sexual contact (Trichomonas) invade various organs in the body and cause prominent human diseases, such as malaria, babesiosis, leishmaniasis, trypanosomiasis, diarrhea, and trichomoniasis. Humans are frequently exposed to multiple pathogens simultaneously, or sequentially in the high-incidence regions to result in co-infections. Consequently, synergistic or antagonistic pathogenic effects could occur between microbes that also influences overall host responses and severity of diseases. The co-infecting organisms can also follow independent trajectory. In either case, co-infections change host and pathogen metabolic microenvironments, compromise the host immune status, and affect microbial pathogenicity to influence tissue colonization. Immunomodulation by protozoa often adversely affects cellular and humoral immune responses against co-infecting bacterial pathogens and promotes bacterial persistence, and result in more severe disease symptoms. Although co-infections by protozoa and viruses also occur in humans, extensive studies are not yet conducted probably because of limited animal model systems available that can be used for both groups of pathogens. Immunosuppressive effects of protozoan infections can also attenuate vaccines efficacy, weaken immunological memory development, and thus attenuate protection against co-infecting pathogens. Due to increasing occurrence of parasitic infections, roles of acute to chronic protozoan infection on immunological changes need extensive investigations to improve understanding of the mechanistic details of specific immune responses alteration. In fact, this phenomenon should be seriously considered as one cause of breakthrough infections after vaccination against both bacterial and viral pathogens, and for the emergence of drug-resistant bacterial strains. Such studies would facilitate development and implementation of effective vaccination and treatment regimens to prevent or significantly reduce breakthrough infections.

Tick-borne pathogens such as species of Borrelia, Babesia, Anaplasma, Rickettsia, and Ehrlichia are widespread in the United States and Europe among wildlife, in passerines as well as in domestic and farm animals. Transmission of these pathogens occurs by infected ticks during their blood meal, carnivorism, and through animal bites in wildlife, whereas humans can become infected either by an infected tick bite, through blood transfusion and in some cases, congenitally. The reservoir hosts play an important role in maintaining pathogens in nature and facilitate transmission of individual pathogens or of multiple pathogens simultaneously to humans through ticks. Tick-borne co-infections were first reported in the 1980s in white-footed mice, the most prominent reservoir host for causative organisms in the United States, and they are becoming a major concern for public health now. Various animal infection models have been used extensively to better understand pathogenesis of tick-borne pathogens and to reveal the interaction among pathogens co-existing in the same host. In this review, we focus on the prevalence of these pathogens in different reservoir hosts, animal models used to investigate their pathogenesis and host responses they trigger to understand diseases in humans. We also documented the prevalence of these pathogens as correlating with the infected ticks' surveillance studies. The association of tick-borne co-infections with other topics such as pathogens virulence factors, host immune responses as they relate to diseases severity, identification of vaccine candidates, and disease economic impact are also briefly addressed here.

Malaria caused by Plasmodium species and transmitted by Anopheles mosquitoes affects large human populations, while Ixodes ticks transmit Babesia species and cause babesiosis. Babesiosis in animals has been known as an economic drain, and human disease has also emerged as a serious healthcare problem in the last 20-30 years. There is limited literature available regarding pathogenesis, immunity, and disease caused by Babesia spp. with their genomes sequenced only in the last decade. Therefore, using previous studies on Plasmodium as the foundation, we have compared similarities and differences in the pathogenesis of Babesia and host immune responses. Sexual life cycles of these two hemoparasites in their respective vectors are quite similar. An adult Anopheles female can take blood meal several times in its life such that it can both acquire and transmit Plasmodia to hosts. Since each tick stage takes blood meal only once, transstadial horizontal transmission from larva to nymph or nymph to adult is essential for the release of Babesia into the host. The initiation of the asexual cycle of these parasites is different because Plasmodium sporozoites need to infect hepatocytes before egressed merozoites can infect erythrocytes, while Babesia sporozoites are known to enter the erythrocytic cycle directly. Plasmodium metabolism, as determined by its two- to threefold larger genome than different Babesia, is more complex. Plasmodium replication occurs in parasitophorous vacuole (PV) within the host cells, and a relatively large number of merozoites are released from each infected RBC after schizogony. The Babesia erythrocytic cycle lacks both PV and schizogony. Cytoadherence that allows the sequestration of Plasmodia, primarily P. falciparum in different organs facilitated by prominent adhesins, has not been documented for Babesia yet. Inflammatory immune responses contribute to the severity of malaria and babesiosis. Antibodies appear to play only a minor role in the resolution of these diseases; however, cellular and innate immunity are critical for the clearance of both pathogens. Inflammatory immune responses affect the severity of both diseases. Macrophages facilitate the resolution of both infections and also offer cross-protection against related protozoa. Although the immunosuppression of adaptive immune responses by these parasites does not seem to affect their own clearance, it significantly exacerbates diseases caused by coinfecting bacteria during coinfections.

Toll-like receptors (TLRs) are a class of membrane-spanning proteins of host cells. TLR2 and TLR4 are displayed on the surface of macrophages, neutrophils and dendritic cells and recognise structurally conserved microbial signatures defined as Pathogen associated molecular patterns (PAMPs). C3H mice are susceptible to tick-borne pathogens; Lyme disease causing Borrelia burgdorferi that manifests arthritis and carditis and Apicomplexan protozoan, Babesia microti (Bm) that causes significant parasitemia associated with erythrocytopenia and haemoglobinuria. B. burgdorferi lacks typical TLR4 ligand lipopolysaccharides (LPS) and Bm TLR ligand(s) remain unknown. Only Borrelia lipoproteins that signal through TLR2 are established as PAMPs of these pathogens for TLR2/TLR4. Infection of C3H mice with each pathogen individually resulted in increase in the percentage of splenic B, T and FcR+ cells while their co-infection significantly diminished levels of these cells and caused increased B. burgdorferi burden in the specific organs. The most pronounced inflammatory arthritis was observed in co-infected C3H/HeJ mice. Parasitemia levels and kinetics of resolution of Bm in both mice strains were not significantly different. Transfected HEK293 cells showed pronounced signalling by B. burgdorferi through TLR2 and to some extent by TLR4 while Bm and infected erythrocytes did not show any response confirming our results in mice.

Lyme borreliosis is the most prevalent vector-borne disease in northern hemisphere. Borrelia burgdorferi sensu lato spirochetes are transmitted by Ixodes species ticks. During a blood meal, these spirochetes are inoculated into the skin where they multiply and often spread to various target organs: disseminated skin sites, the central nervous system, the heart and large joints. The usual diagnosis of this disease relies on serological tests. However, in patients presenting persistent clinical manifestations, this indirect diagnosis is not capable of detecting an active infection. If the serological tests are positive, it only proves that exposure of an individual to Lyme spirochetes had occurred. Although culture and quantitative PCR detect active infection, currently used tests are not sensitive enough for wide-ranging applications. Animal models have shown that B. burgdorferi persists in the skin. We present here our targeted proteomics results using infected mouse skin biopsies that facilitate detection of this pathogen. We have employed several novel approaches in this study. First, the effect of lidocaine, a local anesthetic used for human skin biopsy, on B. burgdorferi presence was measured. We further determined the impact of topical corticosteroids to reactivate Borrelia locally in the skin. This local immunosuppressive compound helps follow-up detection of spirochetes by proteomic analysis of Borrelia present in the skin. This approach could be developed as a novel diagnostic test for active Lyme borreliosis in patients presenting disseminated persistent infection. Although our results using topical corticosteroids in mice are highly promising for recovery of spirochetes, further optimization will be needed to translate this strategy for diagnosis of Lyme disease in patients.

Syphilis is a global, re-emerging sexually transmitted infection and congenital syphilis remains a major cause of adverse pregnancy outcomes due to bacterial infection in developing nations with a high rate of fetus loss. The molecular mechanisms involved in pathogenesis of the causative agent, Treponema pallidum subsp. pallidum remain poorly understood due to the difficulties of working with this pathogen, including the inability to grow it in pure culture. To reduce the spread of syphilis, we must first increase our knowledge of the virulence factors of T. pallidum and their contribution to syphilis manifestations. Tp0954 was predicted to be a surface lipoprotein of T. pallidum. Therefore, we experimentally demonstrated that Tp0954 is indeed a surface protein and further investigated its role in mediating bacterial attachment to various mammalian host cells. We found that expression of Tp0954 in a poorly adherent, but physiologically related derivative strain of the Lyme disease causing spirochete Borrelia burgdorferi B314 strain promotes its binding to epithelial as well as non-epithelial cells including glioma and placental cell lines. We also found that Tp0954 expression facilitates binding of this strain to purified dermatan sulfate and heparin, and also that bacterial binding to mammalian cell lines is mediated by the presence of heparan sulfate and dermatan sulfate in the extracellular matrix of the specific cell lines. These results suggest that Tp0954 may be involved not only in initiating T. pallidum infection by colonizing skin epithelium, but it may also contribute to disseminated infection and colonization of distal tissues. Significantly, we found that Tp0954 promotes binding to the human placental choriocarcinoma BeWo cell line, which is of trophoblastic endocrine cell type, as well as human placental tissue sections, suggesting its role in placental colonization and possible contribution to transplacental transmission of T. pallidum. Altogether, these novel findings offer an important step toward unraveling syphilis pathogenesis, including placental colonization and T. pallidum vertical transmission from mother to fetus during pregnancy.

The first line therapy for Lyme disease is treatment with doxycycline, amoxicillin, or cefuroxime. In endemic regions, the persistence of symptoms in many patients after completion of antibiotic treatment remains a major healthcare concern. The causative agent of Lyme disease is a spirochete, Borrelia burgdorferi, an extreme auxotroph that cannot exist under free-living conditions and depends upon the tick vector and mammalian hosts to fulfill its nutritional needs. Despite lacking all major biosynthetic pathways, B. burgdorferi uniquely possesses three homologous and functional methylthioadenosine/S-adenosylhomocysteine nucleosidases (MTANs: Bgp, MtnN, and Pfs) involved in methionine and purine salvage, underscoring the critical role these enzymes play in the life cycle of the spirochete. At least one MTAN, Bgp, is exceptional in its presence on the surface of Lyme spirochetes and its dual functionality in nutrient salvage and glycosaminoglycan binding involved in host-cell adherence. Thus, MTANs offer highly promising targets for discovery of new antimicrobials. Here we report on our studies to evaluate five nucleoside analogs for MTAN inhibitory activity, and cytotoxic or cytostatic effects on a bioluminescently engineered strain of B. burgdorferi. All five compounds were either alternate substrates and/or inhibitors of MTAN activity, and reduced B. burgdorferi growth. Two inhibitors: 5′-deoxy-5′-iodoadenosine (IADO) and 5′-deoxy-5′-ethyl-immucillin A (dEt-ImmA) showed bactericidal activity. Thus, these inhibitors exhibit high promise and form the foundation for development of novel and effective antimicrobials to treat Lyme disease.