Harnessing RNA Technology to Advance Therapeutic Vaccine Antigens against Chagas Disease.

Chagas disease (CD) (American trypanosomiasis caused by Trypanosoma cruzi) is a parasitic disease endemic in 21 countries in South America, with increasing global spread. When administered late in the infection, the current antiparasitic drugs do not prevent the onset of cardiac illness leading to chronic Chagasic cardiomyopathy. Therefore, new therapeutic vaccines or immunotherapies are under development using multiple platforms. In this study, we assessed the feasibility of developing an mRNA-based therapeutic CD vaccine targeting two known T. cruzi vaccine antigens (Tc24─a flagellar antigen and ASP-2─an amastigote antigen). We present the mRNA engineering steps, preparation, and stability of the lipid nanoparticles and evaluation of their uptake by dendritic cells, as well as their biodistribution in c57BL/J mice. Furthermore, we assessed the immunogenicity and efficacy of two mRNA-based candidates as monovalent and bivalent vaccine strategies using an in vivo chronic mouse model of CD. Our results show several therapeutic benefits, including reductions in parasite burdens and cardiac inflammation, with each mRNA antigen, especially with the mRNA encoding Tc24, and Tc24 in combination with ASP-2. Therefore, our findings demonstrate the potential of mRNA-based vaccines as a therapeutic option for CD and highlight the opportunities for developing multivalent vaccines using this approach.

Adaptation of Chagas Disease Screening Recommendations for a Community of At-risk HIV in the United States.

Chagas disease (CD), caused by Trypanosoma cruzi, is underdiagnosed in the United States. Improved screening strategies are needed, particularly for people at risk for life-threatening sequelae of CD, including people with human immunodeficiency virus (HIV, PWH). Here we report results of a CD screening strategy applied at a large HIV clinic serving an at-risk population.

Localized cardiac small molecule trajectories and persistent chemical sequelae in experimental Chagas disease.

Post-infectious conditions present major health burdens but remain poorly understood. In Chagas disease (CD), caused by Trypanosoma cruzi parasites, antiparasitic agents that successfully clear T. cruzi do not always improve clinical outcomes. In this study, we reveal differential small molecule trajectories between cardiac regions during chronic T. cruzi infection, matching with characteristic CD apical aneurysm sites. Incomplete, region-specific, cardiac small molecule restoration is observed in animals treated with the antiparasitic benznidazole. In contrast, superior restoration of the cardiac small molecule profile is observed for a combination treatment of reduced-dose benznidazole plus an immunotherapy, even with less parasite burden reduction. Overall, these results reveal molecular mechanisms of CD treatment based on simultaneous effects on the pathogen and on host small molecule responses, and expand our understanding of clinical treatment failure in CD. This link between infection and subsequent persistent small molecule perturbation broadens our understanding of infectious disease sequelae.

Atypical initial cleavage patterns minimally impact rhesus macaque in vitro embryo morphokinetics and embryo outgrowth development†.

Embryo morphokinetic analysis through time-lapse embryo imaging is envisioned as a method to improve selection of developmentally competent embryos. Morphokinetic analysis could be utilized to evaluate the effects of experimental manipulation on pre-implantation embryo development. The objectives of this study were to establish a normative morphokinetic database for in vitro fertilized rhesus macaque embryos and to assess the impact of atypical initial cleavage patterns on subsequent embryo development and formation of embryo outgrowths. The cleavage pattern and the timing of embryo developmental events were annotated retrospectively for unmanipulated in vitro fertilized rhesus macaque blastocysts produced over four breeding seasons. Approximately 50% of the blastocysts analyzed had an abnormal early cleavage event. The time to the initiation of embryo compaction and the time to completion of hatching was significantly delayed in blastocysts with an abnormal early cleavage event compared to blastocysts that had cleaved normally. Embryo hatching, attachment to an extracellular matrix, and growth during the implantation stage in vitro was not impacted by the initial cleavage pattern. These data establish normative morphokinetic parameters for in vitro fertilized rhesus macaque embryos and suggest that cleavage anomalies may not impact embryo implantation rates following embryo transfer.

Heterologous mRNA-protein vaccination with Tc24 induces a robust cellular immune response against Trypanosoma cruzi, characterized by an increased level of polyfunctional CD8+ T-cells.

Tc24 is a Trypanosoma cruzi-derived flagellar protein that, when formulated with a TLR-4 agonist adjuvant, induces a balanced immune response in mice, elevating IgG2a antibody titers and IFN-γ levels. Furthermore, vaccination with the recombinant Tc24 protein can reduce parasite levels and improve survival during acute infection. Although some mRNA vaccines have been proven to elicit a stronger immune response than some protein vaccines, they have not been used against T. cruzi. This work evaluates the immunogenicity of a heterologous prime/boost vaccination regimen using protein and mRNA-based Tc24 vaccines. Mice (C57BL/6) were vaccinated twice subcutaneously, three weeks apart, with either the Tc24-C4 protein + glucopyranosyl A (GLA)-squalene emulsion, Tc24 mRNA Lipid Nanoparticles, or with heterologous protein/mRNA or mRNA/protein combinations, respectively. Two weeks after the last vaccination, mice were euthanized, spleens were collected to measure antigen-specific T-cell responses, and sera were collected to evaluate IgG titers and isotypes. Heterologous presentation of the Tc24 antigen generated antigen-specific polyfunctional CD8+ T cells, a balanced Th1/Th2/Th17 cytokine profile, and a balanced humoral response with increased serum IgG, IgG1 and IgG2c antibody responses. We conclude that heterologous vaccination using Tc24 mRNA to prime and Tc24-C4 protein to boost induces a broad and robust antigen-specific immune response that was equivalent or superior to two doses of a homologous protein vaccine, the homologous mRNA vaccine and the heterologous Tc24-C4 Protein/mRNA vaccine.

The impact of vaccine-linked chemotherapy on liver health in a mouse model of chronic Trypanosoma cruzi infection.

Background: Chagas disease, chronic infection with Trypanosoma cruzi, mainly manifests as cardiac disease. However, the liver is important for both controlling parasite burdens and metabolizing drugs. Notably, high doses of anti-parasitic drug benznidazole (BNZ) causes liver damage. We previously showed that combining low dose BNZ with a prototype therapeutic vaccine is a dose sparing strategy that effectively reduced T. cruzi induced cardiac damage. However, the impact of this treatment on liver health is unknown. Therefore, we evaluated several markers of liver health after treatment with low dose BNZ plus the vaccine therapy in comparison to a curative dose of BNZ. Methodology: Female BALB/c mice were infected with a bioluminescent T. cruzi H1 clone for approximately 70 days, then randomly divided into groups of 15 mice each. Mice were treated with a 25mg/kg BNZ, 25µg Tc24-C4 protein/5µg E6020-SE (Vaccine), 25mg/kg BNZ followed by vaccine, or 100mg/kg BNZ (curative dose). At study endpoints we evaluated hepatomegaly, parasite burden by quantitative PCR, cellular infiltration by histology, and expression of B-cell translocation gene 2(BTG2) and Peroxisome proliferator-activated receptor alpha (PPARα) by RT-PCR. Levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were quantified from serum. Results: Curative BNZ treatment significantly reduced hepatomegaly, liver parasite burdens, and the quantity of cellular infiltrate, but significantly elevated serum levels of ALT, AST, and LDH. Low BNZ plus vaccine did not significantly affect hepatomegaly, parasite burdens or the quantity of cellular infiltrate, but only elevated ALT and AST. Low dose BNZ significantly decreased expression of both BTG2 and PPARα, and curative BNZ reduced expression of BTG2 while low BNZ plus vaccine had no impact. Conclusions: These data confirm toxicity associated with curative doses of BNZ and suggest that the dose sparing low BNZ plus vaccine treatment better preserves liver health.

Microbiome Alterations Driven by Trypanosoma cruzi Infection in Two Disjunctive Murine Models.

Alterations caused by Trypanosoma cruzi in the composition of gut microbiome may play a vital role in the host-parasite interactions that shapes physiology and immune responses against infection. Thus, a better understanding of this parasite-host-microbiome interaction may yield relevant information in the comprehension of the pathophysiology of the disease and the development of new prophylactic and therapeutic alternatives. Therefore, we implemented a murine model with two mice strains (BALB/c and C57BL/6) to evaluate the impact of Trypanosoma cruzi (Tulahuen strain) infection on the gut microbiome utilizing cytokine profiling and shotgun metagenomics. Higher parasite burdens were observed in cardiac and intestinal tissues, including changes in anti-inflammatory (interleukin-4 [IL-4] and IL-10) and proinflammatory (gamma interferon, tumor necrosis factor alpha, and IL-6) cytokines. Bacterial species such as Bacteroides thetaiotaomicron, Faecalibaculum rodentium, and Lactobacillus johnsonii showed a decrease in relative abundance, while Akkermansia muciniphila and Staphylococcus xylosus increased. Likewise, as infection progressed, there was a decrease in gene abundances related to metabolic processes such as lipid synthesis (including short-chain fatty acids) and amino acid synthesis (including branched-chain amino acids). High-quality metagenomic assembled genomes of L. johnsonii and A. muciniphila among other species were reconstructed, confirming, functional changes associated with metabolic pathways that are directly affected by the loss of abundance of specific bacterial taxa. IMPORTANCE Chagas disease (CD) is caused by the protozoan Trypanosoma cruzi, presenting acute and chronic phases where cardiomyopathy, megaesophagus, and/or megacolon stand out. During the course of its life cycle, the parasite has an important gastrointestinal tract transit that leads to severe forms of CD. The intestinal microbiome plays an essential role in the immunological, physiological, and metabolic homeostasis of the host. Therefore, parasite-host-intestinal microbiome interactions may provide information on certain biological and pathophysiological aspects related to CD. The present study proposes a comprehensive evaluation of the potential effects of this interaction based on metagenomic and immunological data from two mice models with different genetic, immunological, and microbiome backgrounds. Our findings suggest that there are alterations in the immune and microbiome profiles that affect several metabolic pathways that can potentially promote the infection's establishment, progression, and persistence. In addition, this information may prove essential in the research of new prophylactic and therapeutic alternatives for CD.

A short-term method to evaluate anti-leishmania drugs by inhibition of stage differentiation in Leishmania mexicana using flow cytometry.

Leishmaniasis is a vector-borne neglected tropical disease caused by the Leishmania spp. Parasite. The disease is transmitted to humans and animals by the bite of infected female sandflies during the ingestion of bloodmeal. Because current drug treatments induce toxicity and parasite resistance, there is an urgent need to evaluate new drugs. Most therapeutics target the differentiation of promastigotes to amastigotes, which is necessary to maintain Leishmania infection. However, in vitro assays are laborious, time-consuming, and depend on the experience of the technician. In this study, we aimed to establish a short-term method to assess the differentiation status of Leishmania mexicana (L. mexicana) using flow cytometry. Here, we showed that flow cytometry provides a rapid means to quantify parasite differentiation in cell culture as reliably as light microscopy. Interestingly, we found using flow cytometry that miltefosine reduced promastigote-to-amastigote differentiation of L. mexicana. We conclude that flow cytometry provides a means to rapidly assay the efficacy of small molecules or natural compounds as potential anti-leishmanials.

A broadly distributed predicted helicase/nuclease confers phage resistance via abortive infection.

There is strong selection for the evolution of systems that protect bacterial populations from viral attack. We report a single phage defense protein, Hna, that provides protection against diverse phages in Sinorhizobium meliloti, a nitrogen-fixing alpha-proteobacterium. Homologs of Hna are distributed widely across bacterial lineages, and a homologous protein from Escherichia coli also confers phage defense. Hna contains superfamily II helicase motifs at its N terminus and a nuclease motif at its C terminus, with mutagenesis of these motifs inactivating viral defense. Hna variably impacts phage DNA replication but consistently triggers an abortive infection response in which infected cells carrying the system die but do not release phage progeny. A similar host cell response is triggered in cells containing Hna upon expression of a phage-encoded single-stranded DNA binding protein (SSB), independent of phage infection. Thus, we conclude that Hna limits phage spread by initiating abortive infection in response to a phage protein.

Vaccine-linked chemotherapy improves cardiac structure and function in a mouse model of chronic Chagas disease.

Introduction: Chagas disease, caused by chronic infection with the protozoan parasite Trypanosoma cruzi, affects 6-7 million people worldwide. The major clinical manifestation of Chagas disease is chronic Chagasic cardiomyopathy (CCC), which encompasses a spectrum of symptoms including arrhythmias, hypertrophy, dilated cardiomyopathy, heart failure, and sudden death. Current treatment is limited to two antiparasitic drugs, benznidazole (BNZ) and nifurtimox, but both have limited efficacy to halt the progression of CCC. We developed a vaccine-linked chemotherapy strategy using our vaccine consisting of recombinant Tc24-C4 protein and a TLR-4 agonist adjuvant in a stable squalene emulsion, in combination with low dose benznidazole treatment. We previously demonstrated in acute infection models that this strategy parasite specific immune responses, and reduced parasite burdens and cardiac pathology. Here, we tested our vaccine-linked chemotherapy strategy in a mouse model of chronic T. cruzi infection to evaluate the effect on cardiac function. Methods: Female BALB/c mice infected with 500 blood form T. cruzi H1 strain trypomastigotes were treated beginning 70 days after infection with a low dose of BNZ and either low or high dose of vaccine, in both sequential and concurrent treatments streams. Control mice were untreated, or administered only one treatment. Cardiac health was monitored throughout the course of treatment by echocardiography and electrocardiograms. Approximately 8 months after infection, endpoint histopathology was performed to measure cardiac fibrosis and cellular infiltration. Results: Vaccine-linked chemotherapy improved cardiac function as evidenced by amelioration of altered left ventricular wall thickness, left ventricular diameter, as well as ejection fraction and fractional shortening by approximately 4 months of infection, corresponding to two months after treatment was initiated. At study endpoint, vaccine-linked chemotherapy reduced cardiac cellular infiltration, and induced significantly increased antigen specific IFN-γ and IL-10 release from splenocytes, as well as a trend toward increased IL-17A. Discussion: These data suggest that vaccine-linked chemotherapy ameliorates changes in cardiac structure and function induced by infection with T. cruzi. Importantly, similar to our acute model, the vaccine-linked chemotherapy strategy induced durable antigen specific immune responses, suggesting the potential for a long lasting protective effect. Future studies will evaluate additional treatments that can further improve cardiac function during chronic infection.

Limosilactobacillus reuteri administration alters the gut-brain-behavior axis in a sex-dependent manner in socially monogamous prairie voles.

Research on the role of gut microbiota in behavior has grown dramatically. The probiotic L. reuteri can alter social and stress-related behaviors - yet, the underlying mechanisms remain largely unknown. Although traditional laboratory rodents provide a foundation for examining the role of L. reuteri on the gut-brain axis, they do not naturally display a wide variety of social behaviors. Using the highly-social, monogamous prairie vole (Microtus ochrogaster), we examined the effects of L. reuteri administration on behaviors, neurochemical marker expression, and gut-microbiome composition. Females, but not males, treated with live L. reuteri displayed lower levels of social affiliation compared to those treated with heat-killed L. reuteri. Overall, females displayed a lower level of anxiety-like behaviors than males. Live L. reuteri-treated females had lower expression of corticotrophin releasing factor (CRF) and CRF type-2-receptor in the nucleus accumbens, and lower vasopressin 1a-receptor in the paraventricular nucleus of the hypothalamus (PVN), but increased CRF in the PVN. There were both baseline sex differences and sex-by-treatment differences in gut microbiome composition. Live L. reuteri increased the abundance of several taxa, including Enterobacteriaceae, Lachnospiraceae NK4A136, and Treponema. Interestingly, heat-killed L. reuteri increased abundance of the beneficial taxa Bifidobacteriaceae and Blautia. There were significant correlations between changes in microbiota, brain neurochemical markers, and behaviors. Our data indicate that L. reuteri impacts gut microbiota, gut-brain axis and behaviors in a sex-specific manner in socially-monogamous prairie voles. This demonstrates the utility of the prairie vole model for further examining causal impacts of microbiome on brain and behavior.

Localized cardiac metabolic trajectories and post-infectious metabolic sequelae in experimental Chagas disease.

Post-infectious conditions, where clinical symptoms fail to resolve even after pathogen clearance, present major health burdens. However, the mechanisms involved remain poorly understood. In Chagas disease (CD), caused by the parasite Trypanosoma cruzi, antiparasitic agents can clear T. cruzi but late-stage treatment does not improve clinical cardiac outcomes. In this study, we revealed differential metabolic trajectories of cardiac regions during T. cruzi infection, matching sites of clinical symptoms. Incomplete, region-specific, cardiac metabolic restoration was observed in animals treated with the antiparasitic benznidazole, even though parasites were successfully cleared. In contrast, superior metabolic restoration was observed for a combination treatment of reduced-dose benznidazole plus an immunotherapy (Tc24-C4 T. cruzi flagellar protein and TLR4 agonist adjuvant), even though parasite burden reduction was lower. Overall, these results provide a mechanism to explain prior clinical treatment failures in CD and to test novel candidate treatment regimens. More broadly, our results demonstrate a link between persistent metabolic perturbation and post-infectious conditions, with broad implications for our understanding of post-infectious disease sequelae.

Immunomodulatory proteins from hookworms reduce cardiac inflammation and modulate regulatory responses in a mouse model of chronic Trypanosoma cruzi infection.

Introduction: Hookworms are parasitic helminths that secrete a variety of proteins that induce anti-inflammatory immune responses, stimulating increased CD4 + Foxp3+ regulatory T cells and IL-10 production. Hookworm-derived recombinant proteins AIP-1 and AIP-2 have been shown to reduce inflammation in mouse models of inflammatory bowel disease and inflammatory airway disease by inducing CD4+Foxp3+ cells and IL-10 production. In contrast, chronic infection with the protozoal parasite Trypanosoma cruzi, the causative agent of Chagas disease, leads to chronic inflammation in tissues. Persistence of the parasites in tissues drives chronic low-grade inflammation, with increased infiltration of inflammatory cells into the heart, accompanied by increased production of inflammatory cytokines. There are no current antiparasitic drugs that effectively reduce or prevent chronic myocarditis caused by the onset of Chagas disease, thus new therapies are urgently needed. Therefore, the impact of AIP-1 and AIP-2 on myocarditis was investigated in a mouse model of chronic T. cruzi infection. Methods: Female BALB/c mice infected with bioluminescent T. cruzi H1 strain trypomastigotes for 70 days were treated once daily for 7 days with 1mg/kg AIP-1 or AIP-2 protein by intraperitoneal injection. Control mice were left untreated or treated once daily for 14 days with 25mg/kg aspirin in drinking water. At 84 days of infection, splenocytes, cardiac tissue and serum were collected for evaluation. Results: Treatment with both AIP-1 and AIP-2 proteins significantly reduced cardiac cellular infiltration, and reduced cardiac levels of IFNγ, IL-6 and IL-2. AIP-2 treatment reduced cardiac expression of COX-2. Further, while incubation with AIP-1 and AIP-2 proteins did not induce a significant upregulation of an immunoregulatory phenotype in dendritic cells (DC), there was a modest upregulation of CD11c +CD11b+MHCII+SIRPα+ expression, suggesting a regulatory phenotype. Ex-vivo stimulation of splenocytes from the treatment groups with AIP-1 loaded DC induced reduced levels of cytotoxic and pro-inflammatory T cells, stimulation with AIP-2 loaded DC specifically induced enhanced levels of CD4+CD25+Foxp3+ regulatory T cells among treatment groups. Discussion: All in vivo and in vitro results demonstrate that hookworm-derived AIP-1 and AIP-2 proteins reduce T. cruzi induced cardiac inflammation, possibly through multiple anti-inflammatory mechanisms.

A novel multi-epitope recombinant protein elicits an antigen-specific CD8+ T cells response in Trypanosoma cruzi-infected mice.

About 6.5 million people worldwide are afflicted by Chagas disease, which is caused by the protozoan parasite Trypanosoma cruzi. The development of a therapeutic vaccine to prevent the progression of Chagasic cardiomyopathy has been proposed as an alternative for antiparasitic chemotherapy. Bioinformatics tools can predict MHC class I CD8 + epitopes for inclusion in a single recombinant protein with the goal to develop a multivalent vaccine. We expressed a novel recombinant protein Tc24-C4.10E harboring ten nonameric CD8 + epitopes and using Tc24-C4 protein as scaffold to evaluate the therapeutic effect in acute T. cruzi infection. T. cruzi-infected mice were immunized with Tc24-C4.10E or Tc24-C4 in a 50-day model of acute infection. Tc24-C4.10E-treated mice showed a decreased parasitemia compared to the Tc24-C4 (non-adjuvant) immunized mice or control group. Moreover, Tc24-C4.10E induced a higher stimulation index of CD8 + T cells producing IFNγ and IL-4 cytokines. These results suggest that the addition of the MHC Class I epitopes to Tc24-C4 can synergize the antigen-specific cellular immune responses, providing proof-of-concept that this approach could lead to the development of a promising vaccine candidate for Chagas disease.

Preclinical advances and the immunophysiology of a new therapeutic Chagas disease vaccine.

INTRODUCTION: Chronic infection with the protozoal parasite Trypanosoma cruzi leads to a progressive cardiac disease, known as chronic Chagasic cardiomyopathy (CCC). A new therapeutic Chagas disease vaccine is in development to augment the existing antiparasitic chemotherapy drugs. AREAS COVERED: We report on our current understanding of the underlying immunologic and physiologic mechanisms that lead to CCC, including parasite immune escape mechanisms that allow persistence and the subsequent inflammatory and fibrotic processes that lead to clinical disease. We report on vaccine design and the observed immunotherapeutic effects including induction of a balanced TH1/TH2/TH17 immune response that leads to reduced parasite burdens and tissue pathology. Furthermore, we report vaccine-linked chemotherapy, a dose-sparing strategy to further reduce parasite burdens and tissue pathology. EXPERT OPINION: Our vaccine-linked chemotherapeutic approach is a multimodal treatment strategy, addressing both the parasite persistence and the underlying deleterious host inflammatory and fibrotic responses that lead to cardiac dysfunction. In targeting treatment towards patients with chronic indeterminate or early determinate Chagas disease, this vaccine-linked chemotherapeutic approach will be highly economical and will reduce the global disease burden and deaths due to CCC.

Modeling genetic diseases in nonhuman primates through embryonic and germline modification: Considerations and challenges.

Genetic modification of the embryo or germ line of nonhuman primates is envisioned as a method to develop improved models of human disease, yet the promise of such animal models remains unfulfilled. Here, we discuss current methods and their limitations for producing nonhuman primate genetic models that faithfully genocopy and phenocopy human disease. We reflect on how to ethically maximize the translational relevance of such models in the search for new therapeutic strategies to treat human disease.

Mucosal Vaccination With Recombinant Tm-WAP49 Protein Induces Protective Humoral and Cellular Immunity Against Experimental Trichuriasis in AKR Mice.

Trichuriasis is one of the most common neglected tropical diseases of the world's poorest people. A recombinant vaccine composed of Tm-WAP49, an immunodominant antigen secreted by adult Trichuris stichocytes into the mucosa of the cecum to which the parasite attaches, is under development. The prototype is being evaluated in a mouse model of Trichuris muris infection, with the ultimate goal of producing a mucosal vaccine through intranasal delivery. Intranasal immunization of mice with Tm-WAP49 formulated with the adjuvant OCH, a truncated analog of alpha-GalCer with adjuvanticity to stimulate natural killer T cells (NKT) and mucosal immunity, induced significantly high levels of IgG and its subclasses (IgG1 and IgG2a) in immunized mice. This also resulted in a significant reduction of worm burden after challenge with T. muris-infective eggs. The addition of QS-21 adjuvant to this vaccine formulation further reduced worm counts. The improved protection from the dual-adjuvanted vaccine correlated with higher serum antibody responses (IgG, IgG1, IgG2a, IgA) as well as with the induction of antigen-specific IgA in the nasal mucosa. It was also associated with the robust cellular responses including functional subsets of CD4 T cells producing IL-4, and cytotoxic CD8 T cells expressing granzyme B. The worm reduction achieved by mucosal immunization was higher than that induced by subcutaneous immunization. Intranasal immunization also induced a significantly higher nasal mucosa-secreted antigen-specific IgA response, as well as higher functional cellular responses including CD4+IL4+ (Th1) and CD8+GnzB+ (Th2) T cells, and antigen-specific INFγ-producing T cells in both spleen and MLNs and antibody-producing B cells (CD19+B220+/B220+GL7+). Mucosal immunization further induced long-term T lymphocyte memory with increased central (CD62L+CD44+) and effector (CD62L-CD44+) memory subsets of both CD4 and CD8 T cells at 60 days after the last immunization. In summary, intranasal immunization with recombinant Tm-WAP49 protein induced strong protection versus murine trichuriasis. It represents a promising vaccination approach against intestinal nematodes.

'Candidatus Liberibacter asiaticus'-Encoded BCP Peroxiredoxin Suppresses Lipopolysaccharide-Mediated Defense Signaling and Nitrosative Stress In Planta.

The lipopolysaccharides (LPS) of gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic 'Candidatus Liberibacter' spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on nonhost tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host orange (Citrus sinensis), confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione pool, callose deposition, and activation of the salicylic acid and azelaic acid (AzA) signaling networks. Transient expression of 'Ca. L. asiaticus' bacterioferritin comigratory protein (BCP) peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that 'Ca. L. asiaticus' BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. 'Ca. L. asiaticus' BCP peroxiredoxin (i) attenuates NO-mediated SAR signaling and (ii) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of 'Ca. L. asiaticus' acquisition and transmission by fecund psyllids throughout the limited flush period in citrus.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Tad pilus-mediated twitching motility is essential for DNA uptake and survival of Liberibacters.

Axenically cultured Liberibacter crescens (Lcr) is a closely related surrogate for uncultured plant pathogenic species of the genus Liberibacter, including 'Candidatus L. asiaticus' (CLas) and 'Ca. L. solanacearum' (CLso). All Liberibacters encode a completely conserved gene repertoire for both flagella and Tad (Tight Adherence) pili and all are missing genes critical for nucleotide biosynthesis. Both flagellar swimming and Tad pilus-mediated twitching motility in Lcr were demonstrated for the first time. A role for Tad pili in the uptake of extracellular dsDNA for food in Liberibacters was suspected because both twitching and DNA uptake are impossible without repetitive pilus extension and retraction, and no genes encoding other pilus assemblages or mechanisms for DNA uptake were predicted to be even partially present in any of the 35 fully sequenced Liberibacter genomes. Insertional mutations of the Lcr Tad pilus genes cpaA, cpaB, cpaE, cpaF and tadC all displayed such severely reduced growth and viability that none could be complemented. A mutation affecting cpaF (motor ATPase) was further characterized and the strain displayed concomitant loss of twitching, viability and reduced periplasmic uptake of extracellular dsDNA. Mutations of comEC, encoding the inner membrane competence channel, had no effect on either motility or growth but completely abolished natural transformation in Lcr. The comEC mutation was restored by complementation using comEC from Lcr but not from CLas strain psy62 or CLso strain RS100, indicating that unlike Lcr, these pathogens were not naturally competent for transformation. This report provides the first evidence that the Liberibacter Tad pili are dynamic and essential for both motility and DNA uptake, thus extending their role beyond surface adherence.

Myelin Basic Protein and Cardiac Sympathetic Neurodegeneration in Nonhuman Primates.

Minimal myelination is proposed to be a contributing factor to the preferential nigral neuronal loss in Parkinson's disease (PD). Similar to nigral dopaminergic neurons, sympathetic neurons innervating the heart have long, thin axons which are unmyelinated or minimally myelinated. Interestingly, cardiac sympathetic loss in PD is heterogeneous across the heart, yet the spatial relationship between myelination and neurodegeneration is unknown. Here, we report the mapping of myelin basic protein (MBP) expression across the left ventricle of normal rhesus macaques (n = 5) and animals intoxicated with systemic 6-OHDA (50 mg/kg iv) to model parkinsonian cardiac neurodegeneration (n = 10). A subset of 6-OHDA-treated rhesus received daily dosing of pioglitazone (5 mg/kg po; n = 5), a PPARγ agonist with neuroprotective properties. In normal animals, MBP-immunoreactivity (-ir) was identified surrounding approximately 14% of axonal fibers within nerve bundles of the left ventricle, with more myelinated nerve fibers at the base level of the left ventricle than the apex (p < 0.014). Greater MBP-ir at the base was related to a greater number of nerve bundles at that level relative to the apex (p < 0.05), as the percent of myelinated nerve fibers in bundles was not significantly different between levels of the heart. Cardiac sympathetic loss following 6-OHDA was associated with decreased MBP-ir in cardiac nerve bundles, with the percent decrease of MBP-ir greater in the apex (84.5%) than the base (52.0%). Interestingly, cardiac regions and levels with more MBP-ir in normal animals showed attenuated sympathetic loss relative to areas with less MBP-ir in 6-OHDA + placebo (r = -0.7, p < 0.014), but not in 6-OHDA + pioglitazone (r = -0.1) subjects. Our results demonstrate that myelination is present around a minority of left ventricle nerve bundle fibers, is heterogeneously distributed in the heart of rhesus macaques, and has a complex relationship with cardiac sympathetic neurodegeneration and neuroprotection.