A family of conserved bacterial virulence factors dampens interferon responses by blocking calcium signaling.

Interferons (IFNs) induce an antimicrobial state, protecting tissues from infection. Many viruses inhibit IFN signaling, but whether bacterial pathogens evade IFN responses remains unclear. Here, we demonstrate that the Shigella OspC family of type-III-secreted effectors blocks IFN signaling independently of its cell death inhibitory activity. Rather, IFN inhibition was mediated by the binding of OspC1 and OspC3 to the Ca2+ sensor calmodulin (CaM), blocking CaM kinase II and downstream JAK/STAT signaling. The growth of Shigella lacking OspC1 and OspC3 was attenuated in epithelial cells and in a murine model of infection. This phenotype was rescued in both models by the depletion of IFN receptors. OspC homologs conserved in additional pathogens not only bound CaM but also inhibited IFN, suggesting a widespread virulence strategy. These findings reveal a conserved but previously undescribed molecular mechanism of IFN inhibition and demonstrate the critical role of Ca2+ and IFN targeting in bacterial pathogenesis.

Gac Is a Transcriptional Repressor of the Lyme Disease Spirochete's OspC Virulence-Associated Surface Protein.

The OspC outer-surface lipoprotein is essential for the Lyme disease spirochete's initial phase of vertebrate infection. Bacteria within the midguts of unfed ticks do not express OspC but produce high levels when ticks begin to ingest blood. Lyme disease spirochetes cease production of OspC within 1 to 2 weeks of vertebrate infection, and bacteria that fail to downregulate OspC are cleared by host antibodies. Thus, tight regulation of OspC levels is critical for survival of Lyme borreliae and, therefore, an attractive target for development of novel treatment strategies. Previous studies determined that a DNA region 5' of the ospC promoter, the ospC operator, is required for control of OspC production. Hypothesizing that the ospC operator may bind a regulatory factor, DNA affinity pulldown was performed and identified binding by the Gac protein. Gac is encoded by the C-terminal domain of the gyrA open reading frame from an internal promoter, ribosome-binding site, and initiation codon. Our analyses determined that Gac exhibits a greater affinity for ospC operator and promoter DNAs than for other tested borrelial sequences. In vitro and in vivo analyses demonstrated that Gac is a transcriptional repressor of ospC. These results constitute a substantial advance to our understanding of the mechanisms by which the Lyme disease spirochete controls production of OspC. IMPORTANCE Borrelia burgdorferi sensu lato requires its surface-exposed OspC protein in order to establish infection in humans and other vertebrate hosts. Bacteria that either do not produce OspC during transmission or fail to repress OspC after infection is established are rapidly cleared by the host. Herein, we identified a borrelial protein, Gac, that exhibits preferential affinity to the ospC promoter and 5' adjacent DNA. A combination of biochemical analyses and investigations of genetically manipulated bacteria demonstrated that Gac is a transcriptional repressor of ospC. This is a substantial advance toward understanding how the Lyme disease spirochete controls production of the essential OspC virulence factor and identifies a novel target for preventative and curative therapies.

Borrelia burgdorferi Outer Surface Protein C Is Not the Sole Determinant of Dissemination in Mammals.

Lyme disease in the United States is most often caused by Borrelia burgdorferi sensu stricto. After a tick bite, the patient may develop erythema migrans at that site. If hematogenous dissemination occurs, the patient may then develop neurologic manifestations, carditis, or arthritis. Host-pathogen interactions include factors that contribute to hematogenous dissemination to other body sites. Outer surface protein C (OspC), a surface-exposed lipoprotein of B. burgdorferi, is essential during the early stages of mammalian infection. There is a high degree of genetic variation at the ospC locus, and certain ospC types are more frequently associated with hematogenous dissemination in patients, suggesting that OspC may be a major contributing factor to the clinical outcome of B. burgdorferi infection. In order to evaluate the role of OspC in B. burgdorferi dissemination, ospC was exchanged between B. burgdorferi isolates with different capacities to disseminate in laboratory mice, and these strains were then tested for their ability to disseminate in mice. The results indicated that the ability of B. burgdorferi to disseminate in mammalian hosts does not depend on OspC alone. The complete genome sequences of two closely related strains of B. burgdorferi with differing dissemination phenotypes were determined, but a specific genetic locus that could explain the differences in the phenotypes could not be definitively identified. The animal studies performed clearly demonstrated that OspC is not the sole determinant of dissemination. Future studies of the type described here with additional borrelial strains will hopefully clarify the genetic elements associated with hematogenous dissemination.

The Rrp2-RpoN-RpoS pathway plays an important role in the blood-brain barrier transmigration of the Lyme disease pathogen.

Lyme disease, caused by Borrelia (or Borreliella) burgdorferi, is a complex multisystemic disorder that includes Lyme neuroborreliosis resulting from the invasion of both the central and peripheral nervous systems. However, factors that enable the pathogen to cross the blood-brain barrier (BBB) and invade the central nervous system (CNS) are still not well understood. The objective of this study was to identify the B. burgdorferi factors required for BBB transmigration. We utilized a transwell BBB model based on human brain-microvascular endothelial cells and focused on investigating the Rrp2-RpoN-RpoS pathway, a central regulatory pathway that is essential for mammalian infection by B. burgdorferi. Our results demonstrated that the Rrp2-RpoN-RpoS pathway is crucial for BBB transmigration. Furthermore, we identified OspC, a major surface lipoprotein controlled by the Rrp2-RpoN-RpoS pathway, as a significant contributor to BBB transmigration. Constitutive production of OspC in a mutant defective in the Rrp2-RpoN-RpoS pathway did not rescue the impairment in BBB transmigration, indicating that this pathway controls additional factors for this process. Two other major surface lipoproteins controlled by this pathway, DbpA/B and BBK32, appeared to be dispensable for BBB transmigration. In addition, both the surface lipoprotein OspA and the Rrp1 pathway, which are required B. burgdorferi colonization in the tick vector, were found not required for BBB transmigration. Collectively, our findings using in vitro transwell assays uncover another potential role of the Rrp2-RpoN-RpoS pathway in BBB transmigration of B. burgdorferi and invasion into the CNS.

Assessing and validating housekeeping genes in normal, cancerous, and polycystic human ovaries.

PURPOSE: Housekeeping genes (HKGs), reference or endogenous control genes, are vital to normalize mRNA levels between different samples. Since using inappropriate HKGs can lead to unreliable results, selecting the proper ones is critical for gene expression studies. To this end, normal human ovaries, as well as those from patients diagnosed with ovarian endometrioid adenocarcinoma (OEA), ovarian mucinous adenocarcinoma (OMA), ovarian serous papillary carcinoma (OSPC), and polycystic ovary syndrome (PCOS), were used to identify the most suitable housekeeping genes. METHODS: RNA was isolated from 5 normal human ovaries (52-79 years of age), 9 cancerous ovaries (3 OEA, 3 OMA, 3 OSPC; 49-75 years of age), and 4 PCOS ovaries (18-35 years of age) in women undergoing hysterectomy. cDNA was synthesized using a whole transcriptome kit, and quantitative real-time PCR was performed using TaqMan array 96-well plates containing 32 human endogenous controls in triplicate. RESULTS: Among 32 HKGs studied, RPS17, RPL37A, PPIA, 18srRNA, B2M, RPLP0, RPLP30, HPRT1, POP4, CDKN1B, and ELF1 were selected as the best reference genes. CONCLUSIONS: This study confirms recent investigations demonstrating that conventional HKGs, such as GAPDH and beta-actin, are not suitable reference genes for specific pathological conditions, emphasizing the importance of determining the best HKGs on a case-by-case basis and according to tissue type. Our results have identified reliable HKGs for studies of normal human ovaries and those affected by OEA, OMA, OSPC, or PCOS, as well as combined studies of control subjects vs. each cancer or PCOS group.

Identification of interactions among host and bacterial proteins and evaluation of their role early during Shigella flexneri infection.

Shigella species cause diarrhoea by invading and spreading through the epithelial layer of the human colon. The infection triggers innate immune responses in the host that the bacterium combats by translocating into the host cell cytosol via a type 3 secretion system bacterial effector proteins that interfere with host processes. We previously demonstrated that interaction of the Shigella type 3 secreted effector protein IcsB with the host protein Toca-1 inhibits the innate immune response microtubule-associated protein light-chain 3 (LC3)-associated phagocytosis, and that IcsB interaction with Toca-1 is required for inhibition of this host response. Here, we show that Toca-1 in vitro precipitated not only IcsB, but also the type 3 secreted proteins OspC3, IpgD and IpaB. OspC3 and IpgD precipitation with Toca-1 was dependent on IcsB. Early during infection, most of these proteins localized near intracellular Shigella. We examined whether interactions among these proteins restrict innate host cell responses other than LC3-associated phagocytosis. In infected cells, OspC3 blocks production and secretion of the mature pro-inflammatory cytokine IL-18; however, we found that interaction of OspC3 with IcsB, either directly or indirectly via Toca-1, was not required for OspC3-mediated restriction of IL-18 production. These results indicate that interactions of the host protein Toca-1 with a subset of type 3 effector proteins contribute to the established function of some, but not all involved, effector proteins.

Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival.

Borrelia burgdorferi (Bb) is the causative agent of Lyme disease in the United States, a disease that can result in carditis, and chronic and debilitating arthritis and/or neurologic symptoms if left untreated. Bb survives in the midgut of the Ixodes scapularis tick, or within tissues of immunocompetent hosts. In the early stages of infection, the bacteria are present in the bloodstream where they must resist clearance by the innate immune system of the host. We have found a novel role for outer surface protein C (OspC) from B. burgdorferi and B. garinii in interactions with the complement component C4b and bloodstream survival in vivo. Our data show that OspC inhibits the classical and lectin complement pathways and competes with complement protein C2 for C4b binding. Resistance to complement is important for maintenance of the lifecycle of Bb, enabling survival of the pathogen within the host as well as in the midgut of a feeding tick when ospC expression is induced.

Multistrain Infections with Lyme Borreliosis Pathogens in the Tick Vector.

Mixed or multiple-strain infections are common in vector-borne diseases and have important implications for the epidemiology of these pathogens. Previous studies have mainly focused on interactions between pathogen strains in the vertebrate host, but little is known about what happens in the arthropod vector. Borrelia afzelii and Borrelia garinii are two species of spirochete bacteria that cause Lyme borreliosis in Europe and that share a tick vector, Ixodes ricinus Each of these two tick-borne pathogens consists of multiple strains that are often differentiated using the highly polymorphic ospC gene. For each Borrelia species, we studied the frequencies and abundances of the ospC strains in a wild population of I. ricinus ticks that had been sampled from the same field site over a period of 3 years. We used quantitative PCR (qPCR) and 454 sequencing to estimate the spirochete load and the strain diversity within each tick. For B. afzelii, there was a negative relationship between the two most common ospC strains, suggesting the presence of competitive interactions in the vertebrate host and possibly the tick vector. The flat relationship between total spirochete abundance and strain richness in the nymphal tick indicates that the mean abundance per strain decreases as the number of strains in the tick increases. Strains with the highest spirochete load in the nymphal tick were the most common strains in the tick population. The spirochete abundance in the nymphal tick appears to be an important life history trait that explains why some strains are more common than others in nature. IMPORTANCE: Lyme borreliosis is the most common vector-borne disease in the Northern Hemisphere and is caused by spirochete bacteria that belong to the Borrelia burgdorferi sensu lato species complex. These tick-borne pathogens are transmitted among vertebrate hosts by hard ticks of the genus Ixodes Each Borrelia species can be further subdivided into genetically distinct strains. Multiple-strain infections are common in both the vertebrate host and the tick vector and can result in competitive interactions. To date, few studies on multiple-strain vector-borne pathogens have investigated patterns of cooccurrence and abundance in the arthropod vector. We demonstrate that the abundance of a given strain in the tick vector is negatively affected by the presence of coinfecting strains. In addition, our study suggests that the spirochete abundance in the tick is an important life history trait that can explain why some strains are more common in nature than others.

Histopathology and immunophenotype of acrodermatitis chronica atrophicans correlated with ospA and ospC genotypes of Borrelia species.

BACKGROUND: Chronic cutaneous borreliosis (acrodermatitis chronica atrophicans, ACA) is a relatively rare manifestation of borreliosis attributed mainly to Borrelia afzelii. Chronic borreliosis has been associated with ospA and ospC genotypes. Literature on molecular investigations of Borrelia in lesions of ACA is scant. METHODS: Histopathological and immmunohistochemical features in 22 biopsies of ACA (16 patients) were examined. Paraffin-embedded biopsies were analyzed with polymerase chain reaction (PCR) assays targeting ospA and ospC genes, sequencing and phylogenetic analysis. RESULTS: Genotyping of ospA identified B. afzelii, serotype 2, in 12 of 16 patients. ospC-PCR was positive in seven patients revealing genotypes Af5 (n = 4), Af2 (n = 2) and Af6 (n = 1). Histopathologically, interstitial granulomatous infiltrates (CD68 positive) were common, combined with thickened collagen bundles and band-like infiltrates of CD4 positive T lymphocytes. Plasma cells were sparse/absent in 9 of 22 specimens even on staining with CD138. On CD34-staining, interstitial fibroblasts were often reduced akin to the situation in morphea. CONCLUSIONS: With assays targeting ospA and ospC genes we confirmed from paraffin-embedded biopsies that B. afzelii, serotype 2, osp C groups Af5, Af2 and Af6 is the main cause of ACA. Specimens commonly showed a combination of band-like T-cell-rich infiltrates with interstitial granulomatous features, a pattern previously under-recognized in ACA. This finding was particularly typical for lesions infected with ospC genotype Af5.

Shigella induces stress granule formation by ADP-riboxanation of the eIF3 complex.

Under stress conditions, translationally stalled mRNA and associated proteins undergo liquid-liquid phase separation and condense into cytoplasmic foci called stress granules (SGs). Many viruses hijack SGs for their pathogenesis; however, whether pathogenic bacteria also exploit this pathway remains unknown. Here, we report that members of the OspC family of Shigella flexneri induce SG formation in infected cells. Mechanistically, the OspC effectors target multiple subunits of the host translation initiation factor 3 complex by ADP-riboxanation. The modification of eIF3 leads to translational arrest and thus the formation of SGs. Furthermore, OspC-mediated SGs are beneficial for S. flexneri replication within infected host cells, and bacterial strains unable to induce SGs are attenuated for virulence in a murine model of infection. Our findings reveal a mechanism by which bacterial pathogens induce SG assembly by inactivating host translational machinery and promote bacterial proliferation in host cells.

Classical Borrelia Serology Does Not Aid in the Diagnosis of Persistent Symptoms Attributed to Lyme Borreliosis: A Retrospective Cohort Study.

OBJECTIVE: The diagnosis of Lyme borreliosis is based on two-tier testing using an ELISA and Western blot. About 5-10% of patients report persistent symptoms of unknown etiology after treatment, resulting in substantial difficulties in further diagnostic workup. This paper presents a study aimed at determining whether serology can differentiate between patients with persistent symptoms attributed to Lyme and other patients with Lyme borreliosis. METHODS: A retrospective cohort study included 162 samples from four subgroups: patients with persistent symptoms of Lyme (PSL), early Lyme borreliosis with erythema migrans (EM), patients tested in a general practitioner setting (GP), and healthy controls (HC). ELISA, Western blots, and multiplex assays from different manufacturers were used to determine inter-test variations in PSL and to compare reactivity against Borrelia-specific antigens among the groups. RESULTS: In comparing the IgG and IgM reactivity by Western blot, IgG was more often positive in the PSL group than in the GP group. The individual antigen reactivity was similar between the PSL and EM or GP groups. Inter-test agreement among the manufacturers was variable, and agreement was higher for IgG testing compared to IgM. CONCLUSIONS: Serological testing is unable to define the subgroup of patients with persistent symptoms attributed to Lyme borreliosis. Additionally, the current two-tier testing protocol shows a large variance among different manufacturers in these patients.

MHC class II genotype-by-pathogen genotype interaction for infection prevalence in a natural rodent-Borrelia system.

MHC genes are extraordinarily polymorphic in most taxa. Host-pathogen coevolution driven by negative frequency-dependent selection (NFDS) is one of the main hypotheses for the maintenance of such immunogenetic variation. Here, we test a critical but rarely tested assumption of this hypothesis-that MHC alleles affect resistance/susceptibility to a pathogen in a strain-specific way, that is, there is a host genotype-by-pathogen genotype interaction. In a field study of bank voles naturally infected with the tick-transmitted bacterium Borrelia afzelii, we tested for MHC class II (DQB) genotype-by-B. afzelii strain interactions for infection prevalence between 10 DQB alleles and seven strains. One allele (DQB*37) showed an interaction, such that voles carrying DQB*37 had higher prevalence of two strains and lower prevalence of one strain than individuals without the allele. These findings were corroborated by analyses of strain composition of infections, which revealed an effect of DQB*37 in the form of lower ß diversity among infections in voles carrying the allele. Taken together, these results provide rare support at the molecular genetic level for a key assumption of models of antagonistic coevolution through NFDS.

Genetic variation of Borreliella burgdorferi in Fairfax County, Virginia, targeting the OspC gene in white-footed mice.

Outer surface protein C (OspC) is a commonly used marker in population studies of Borreliella to differentiate types and establish evolution over time. Investigating the ospC genetic types of Borreliella burgdorferi across multiple organ tissues of white-footed mice has the potential to contribute to our understanding of Lyme disease and the wide spectrum of clinical presentation associated with infection. In this study, five unique tissue types were sampled from 90 mice and screened for B. burgdorferi infections. This initial screening revealed a 63% overall B. burgdorferi infection rate in the mice collected (57/90). A total of 163 tissues (30.4%) tested positive for B. burgdorferi infections and when mapped to Borreliella types, 143,894 of the initial 322,480 reads mapped to 10 of the reference sequences in the ospC strain library constructed for this study at a 97% MOI. Two tissue types, the ear and the tongue, each accounted for 90% of the observed Borreliella sequence diversity in the tissue samples surveyed. The largest amount of variation was observed in an individual ear tissue sample with six ospC sequence types, which is equivalent to 60% of the observed variation seen across all tested specimens, with statistically significant associations observed between tissue type and detected Borreliella. There is strong evidence for genetic variability in B. burgdorferi within local white-footed mouse populations and even within individual hosts by tissue type. These findings may shed light on drivers of infection sequalae in specific tissues in humans and highlights the need for expanded surveillance on the epigenetics of B. burgdorferi across reservoirs, ticks, and infected patients.

Calmodulin Binding Activates Chromobacterium CopC Effector to ADP-Riboxanate Host Apoptotic Caspases.

Blocking host cell death is an important virulence strategy employed by many bacterial pathogens. We recently reported that Shigella flexneri inhibits host pyroptosis by delivering a type III secretion system (T3SS) effector OspC3 that catalyzes a novel arginine ADP-riboxanation modification on caspase-4/11. Here, we investigated the OspC3 homologue CopC from Chromobacterium violaceum, an opportunistic but sometimes deadly bacterial pathogen. CopC bears the same arginine ADP-riboxanase activity as OspC3, but with a different substrate specificity. Through proteomic analysis, we first identified host calmodulin (CaM) as a binding partner of CopC. The analyses additionally revealed that CopC preferably modifies apoptotic caspases including caspase-7, -8 and -9. This results in suppression of both extrinsic and intrinsic apoptosis programs in C. violaceum-infected cells. Biochemical reconstitution showed that CopC requires binding to CaM, specifically in the calcium-free state, to achieve efficient ADP-riboxanation of the caspases. We determined crystal structure of the CaM-CopC-CASP7 ternary complex, which illustrates the caspase recognition mechanism and a unique CaM-binding mode in CopC. Structure-directed mutagenesis validated the functional significance of CaM binding for stimulating CopC modification of its caspase substrates. CopC adopts an ADP-ribosyltransferase-like fold with a unique His-Phe-Glu catalytic triad, featuring two acidic residues critical for site-specific arginine ADP-riboxanation. Our study expands and deepens our understanding of the OspC family of ADP-riboxanase effectors. IMPORTANCE Programmed cell death is a suicidal defense mechanism for eukaryotes to combat pathogen infection. In the evolutionary arms race with the host, bacteria are endowed with ingenious tactics to block host cell death to facilitate their replication. Here, we report that the C. violaceum effector CopC ADP-riboxanates caspase-7/8/9, enabled by interacting with the host factor calmodulin, to block host cell apoptosis, illustrating a unique and sophisticated strategy adopted by the pathogen to counteract host defense.

Strong interactions between Salp15 homologues from the tick I. ricinus and distinct types of the outer surface OspC protein from Borrelia.

Ticks belonging to the genus Ixodes are parasites feeding on vertebrate blood and vectors for many pathogenic microbes, including Borrelia burgdorferi sensu lato spirochetes, the causative agent of Lyme borreliosis. The tick saliva contains a mixture of bioactive molecules showing a wide range of properties for efficient engorgement. One of the most extensively studied components of tick saliva is a 15-kDa salivary gland protein (Salp15) from Ixodes scapularis. This multifunctional protein suppresses the immune response of hosts through pleiotropic action on a few crucial defense pathways. Salp15 and its homologue from I. ricinus Iric1 have been also shown to bind to Borrelia burgdorferi sensu stricto outer surface protein C (OspC) permitting the spirochetes to evade antibody-mediated killing in the human host. Further studies revealed that Salp15 and Iric1 protected B. burgdorferi s. s. and B. garinii expressing OspC against the complement system. OspC is the most variable protein on the outer surface of Borrelia, which in addition to Salp15 can also bind other ligands, such as plasminogen, fibrinogen, fibronectin or complement factor 4. So far several OspC variants produced by B. burgdorferi s. l. spirochetes were shown to be capable of binding Salp15 or its homologue, but the protection against borreliacidal antibodies has only been proven in the case of B. burgdorferi s. s. The question of Salp15 contribution to Borrelia survival during the infection has been comprehensively studied during the last decades. In contrast, the organization of the OspC-Salp15 complex has been poorly explored. This report describes the binding between three Salp15 homologues from the tick Ixodes ricinus (Iric1, Iric2 and Iric3) and OspC from four B. burgdorferi sensu lato strains in terms of the binding parameters, analyzed with two independent biophysical methods - Microscale thermophoresis (MST) and Biolayer interferometry (BLI). The results of both experiments show a binding constant at the nanomolar level, which indicates very strong interactions. While the Iric1-OspC binding has been reported before, we show in this study that also Iric2 and Iric3 are capable of OspC binding with high affinity. This observation suggests that these two Salp15 homologues might be used by B. burgdorferi s. l. in a way analogous to Iric1. A comparison of the results from the two methods let us propose that N-terminal immobilization of OspC significantly increases the affinity between the two proteins. Finally, our results indicate that the Iric binding site is located in close proximity of the OspC epitopes recognized by human antibodies, which may have important biological and medical implications.

Persistent Anti-Borrelia IgM Antibodies without Lyme Borreliosis in the Clinical and Immunological Context.

The aim of the study was to investigate the etiology of persistent IgM antibodies against Borrelia burgdorferi sensu lato (sl) and to analyze their association with nonspecific symptoms. The study group comprised individuals with persistent IgM antibodies in the absence of IgG. The relation between ELISA values and time elapsed since past erythema migrans (EM) was analyzed. Previous antibiotic treatments were assessed. The association between persistent IgM and nonspecific symptoms was evaluated statistically. Specificity of IgM antibodies for outer surface protein C (OspC) of B. burgdorferi sl was examined by immunoblotting. Further, we investigated the cross-reactivity with Borrelia-unrelated proteins. Fifty-nine patients (46 women; 78%) were included in the study group. The mean IgM-ELISA values did not change significantly during follow-up (median 6.2 months). The mean ELISA value in the study group was dependent on time elapsed since past EM. Nonspecific symptoms improved significantly more often in patients with lower IgM ELISA results. Persistent IgM antibodies were specific for the C-terminal PKKP motif of OspC. Cross-reacting C-terminal PKKP antigens from both human and prokaryotic origins were identified. We demonstrate that the C-terminal PKKP motif plays a main role for the reactivity of persistent Borrelia IgM toward OspC. However, cross-reactivity to other eukaryotic and/or prokaryotic antigens may hamper the specificity of OspC in the serological diagnosis of Lyme borreliosis. Lack of improvement of nonspecific symptoms was associated with higher IgM ELISA values. IMPORTANCE The reactivity of human IgM with the outer surface protein C (OspC) of Borrelia burgdorferi sensu lato is frequently used to detect Borrelia specific IgM in commercial immunoassays, and such antibodies usually occur in the early phase of the infection. We identified a group of individuals with persistent Borrelia IgM without symptoms of Lyme borreliosis. We used their sera to demonstrate that the C-terminal epitope of OspC binds the IgM. Strikingly, we found that the same epitope occurs also in certain proteins of human and environmental origin; the latter include other bacteria and food plants. Our experimental data show that these Borrelia-unrelated proteins cross-react with the OpsC-specific IgM. This knowledge is important for the development of serologic assays for Lyme borreliosis and provides a cross-reactive explanation for the persistence of Borrelia-IgM.

Cross-reactivity of antibody responses to Borrelia afzelii OspC: Asymmetry and host heterogeneity.

The tick-transmitted bacterium Borrelia afzelii consists of a number of antigenically different strains - often defined by outer surface protein C (OspC) genotype - that coexist at stable frequencies in host populations. To investigate how host antibody responses affect strain coexistence, we measured antibody cross-reactivity to three different OspC types (OspC 2, 3 and 9) in three different strains of laboratory mice (BALB/c, C3H and C57BL/6). The extent of cross-reactivity differed between mouse strains, being higher in C3H than BALB/c and C57BL/6. In one of three pairwise comparisons of OspC types (OspC2 vs OspC9), there was evidence for asymmetry of cross-reactivity, with antibodies to OspC2 cross-reacting more strongly with OspC9 than vice versa. These results indicate that the extent of antibody-mediated competition between OspC types may depend on the composition of the host population, and that such competition may be asymmetric. We discuss the implications of these results for understanding the coexistence of OspC types.

Tick-Tattoo: DNA Vaccination Against B. burgdorferi or Ixodes scapularis Tick Proteins.

Introduction: Borrelia burgdorferi sensu lato (sl) is the causative agent of Lyme borreliosis. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines. DNA tattoo vaccination with B. afzelii strain PKo OspC in mice has proven to be fully protective against B. afzelii syringe challenge and induces a favorable humoral immunity compared to recombinant protein vaccination. Alternatively, several recombinant protein vaccines based on tick proteins have shown promising effect in tick-bite infection models. In this study, we evaluated the efficacy of DNA vaccines against Borrelia OspC or tick antigens in a tick-bite infection model. Method: We vaccinated C3H/HeN mice with OspC using a codon-optimized DNA vaccine or with recombinant protein. We challenged these mice with B. burgdorferi sensu stricto (ss)-infected Ixodes scapularis nymphs. Subsequently, we vaccinated C3H/HeN mice with DNA vaccines coding for tick proteins for which recombinant protein vaccines have previously resulted in interference with tick feeding and/or Borrelia transmission: Salp15, tHRF, TSLPI, and Tix-5. These mice were also challenged with B. burgdorferi ss infected Ixodes scapularis nymphs. Results: DNA tattoo and recombinant OspC vaccination both induced total IgG responses. Borrelia cultures and DNA loads of skin and bladder remained negative in the mice vaccinated with OspC DNA vaccination, except for one culture. DNA vaccines against tick antigens Salp15 and Tix-5 induced IgG responses, while those against tHRF and TSLPI barely induced any IgG response. In addition, Borrelia cultures, and DNA loads from mice tattooed with DNA vaccines against tick proteins TSLPI, Salp15, tHRF, and Tix-5 were all positive. Conclusion: A DNA tattoo vaccine against OspC induced high specific IgG titers and provided near total protection against B. burgdorferi ss infection by tick challenge. In contrast, DNA tattoo vaccines against tick proteins TSLPI, Salp15, tHRF, and Tix-5 induced low to moderate IgG titers and did not provide protection. Therefore, DNA tattoo vaccination does not seem a suitable vaccine strategy to identify, or screen for, tick antigens for anti-tick vaccines. However, DNA tattoo vaccination is a straightforward and effective vaccination platform to assess novel B. burgdorferi sl antigen candidates in a relevant tick challenge model.

Corrigendum: Role of HK2 in the Enzootic Cycle of Borrelia burgdorferi.

[This corrects the article DOI: 10.3389/fmed.2020.573648.].

Antibody profiling of a Borreliella burgdorferi (Lyme disease) C6 antibody positive, symptomatic Rottweiler and her pups.

Lyme disease (LD) is a tick-transmitted disease caused by Borreliella burgdorferi (Bb). Temporal studies of maternal antibody (Ab) profiles in Bb infected pregnant dogs and their pups have not been conducted. In this study, Ab profiles of a client-owned Bb C6 Ab positive Rottweiler and her nine pups were assessed. The dam presented with lameness 12 days prior to parturition and was C6 Ab positive with a Quant C6 Ab concentration of 237U/mL. Treatment with amoxicillin was initiated and 11 days later nine pups were delivered. Screening of the sera from the dam and pups against Bb cell lysates and a panel of antigens revealed similar immunoreactivity profiles. While antigen-specific IgG and IgM reactivity persisted in the dam for at least 7 months, a rapid decline in IgG specific for BBA36, BBK53, BB0238, BBA73 and outer surface protein (Osp) E in the pups occurred between days 29 and 52 post-parturition. In contrast, Ab specific for DbpA and the diagnostic antigens VlsE (C6) and OspF, remained elevated in the pups. Sera from the dam displayed potent complement-dependent bactericidal activity against Bb. Sera from the pups was also bactericidal but primarily through a complement-independent mechanism. Lastly, single dose vaccination of the dam at day 51 post-parturition with a LD subunit vaccine consisting of OspA and an OspC chimeritope triggered a broad anti-OspC Ab response indicative of an anamnestic response. Although this study focused on a single case, these findings add to our knowledge of maternal Ab profiles and will aid the interpretation of serological assays in pups delivered by a Bb C6 Ab positive dog.