RESUMEN
How intracellular bacteria subvert the major histocompatibility complex (MHC) class I pathway is poorly understood. Here, we show that the obligate intracellular bacterium Orientia tsutsugamushi uses its effector protein, Ank5, to inhibit nuclear translocation of the MHC class I gene transactivator, NLRC5, and orchestrate its proteasomal degradation. Ank5 uses a tyrosine in its fourth ankyrin repeat to bind the NLRC5 N-terminus while its F-box directs host SCF complex ubiquitination of NLRC5 in the leucine-rich repeat region that dictates susceptibility to Orientia- and Ank5-mediated degradation. The ability of O. tsutsugamushi strains to degrade NLRC5 correlates with ank5 genomic carriage. Ectopically expressed Ank5 that can bind but not degrade NLRC5 protects the transactivator during Orientia infection. Thus, Ank5 is an immunoevasin that uses its bipartite architecture to rid host cells of NLRC5 and reduce surface MHC class I molecules. This study offers insight into how intracellular pathogens can impair MHC class I expression.
Asunto(s)
Antígenos de Histocompatibilidad Clase I , Péptidos y Proteínas de Señalización Intracelular , Orientia tsutsugamushi , Orientia tsutsugamushi/metabolismo , Orientia tsutsugamushi/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Citoplasma/metabolismo , Células HEK293 , Proteolisis , Tifus por Ácaros/inmunología , Tifus por Ácaros/microbiología , Tifus por Ácaros/metabolismo , Ratones , Ubiquitinación , Interacciones Huésped-Patógeno/inmunologíaRESUMEN
Scrub typhus, a vector-borne bacterial infection, is an important but neglected disease globally. Accurately characterizing the burden is challenging because of nonspecific symptoms and limited diagnostics. Prior seroepidemiology studies have struggled to find consensus cutoffs that permit comparisons of estimates across contexts and time. In this study, we present a novel approach that does not require a cutoff and instead uses information about antibody kinetics after infection to estimate seroincidence. We use data from three cohorts of scrub typhus patients in Chiang Rai, Thailand, and Vellore, India, to characterize antibody kinetics after infection and two population serosurveys in the Kathmandu Valley, Nepal, and Tamil Nadu, India, to estimate seroincidence. The samples were tested for IgM and IgG responses to Orientia tsutsugamushi-derived recombinant 56-kDa antigen using commercial enzyme-linked immunosorbent assay kits. We used Bayesian hierarchical models to characterize antibody responses after scrub typhus infection and used the joint distributions of the peak antibody titers and decay rates to estimate population-level incidence rates in the cross-sectional serosurveys. Median responses persisted above an optical density (OD) of 1.8 for 23.6 months for IgG and an OD of 1 for 4.5 months for IgM. Among 18- to 29-year-olds, the seroincidence was 10 per 1,000 person-years (95% CI, 5-19) in Tamil Nadu, India, and 14 per 1,000 person-years (95% CI: 10-20) in the Kathmandu Valley, Nepal. When seroincidence was calculated with antibody decay ignored, the disease burden was underestimated by more than 50%. The approach can be deployed prospectively, coupled with existing serosurveys, or leverage banked samples to efficiently generate scrub typhus seroincidence estimates.
Asunto(s)
Anticuerpos Antibacterianos , Inmunoglobulina G , Inmunoglobulina M , Orientia tsutsugamushi , Tifus por Ácaros , Tifus por Ácaros/epidemiología , Tifus por Ácaros/inmunología , Humanos , India/epidemiología , Estudios Seroepidemiológicos , Nepal/epidemiología , Inmunoglobulina G/sangre , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Orientia tsutsugamushi/inmunología , Inmunoglobulina M/sangre , Incidencia , Adulto , Masculino , Femenino , Tailandia/epidemiología , Adolescente , Adulto Joven , Teorema de Bayes , Persona de Mediana EdadRESUMEN
Scrub typhus is an acute febrile disease due to Orientia tsutsugamushi (Ot) infection and can be life-threatening with organ failure, hemorrhage, and fatality. Yet, little is known as to how the host reacts to Ot bacteria at early stages of infection; no reports have addressed the functional roles of type I versus type II interferon (IFN) responses in scrub typhus. In this study, we used comprehensive intradermal (i.d.) inoculation models and two clinically predominant Ot strains (Karp and Gilliam) to uncover early immune events. Karp infection induced sequential expression of Ifnb and Ifng in inflamed skin and draining lymph nodes at days 1 and 3 post-infection. Using double Ifnar1-/-Ifngr1-/- and Stat1-/- mice, we found that deficiency in IFN/STAT1 signaling resulted in lethal infection with profound pathology and skin eschar lesions, which resembled to human scrub typhus. Further analyses demonstrated that deficiency in IFN-γ, but not IFN-I, resulted in impaired NK cell and macrophage activation and uncontrolled bacterial growth and dissemination, leading to metabolic dysregulation, excessive inflammatory cell infiltration, and exacerbated tissue damage. NK cells were found to be the major cellular source of innate IFN-γ, contributing to the initial Ot control in the draining lymph nodes. In vitro studies with dendritic cell cultures revealed a superior antibacterial effect offered by IFN-γ than IFN-ß. Comparative in vivo studies with Karp- and Gilliam-infection revealed a crucial role of IFN-γ signaling in protection against progression of eschar lesions and Ot infection lethality. Additionally, our i.d. mouse models of lethal infection with eschar lesions are promising tools for immunological study and vaccine development for scrub typhus.
Asunto(s)
Interferón gamma , Orientia tsutsugamushi , Tifus por Ácaros , Transducción de Señal , Animales , Ratones , Modelos Animales de Enfermedad , Interferón gamma/metabolismo , Interferón gamma/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Orientia tsutsugamushi/inmunología , Tifus por Ácaros/inmunología , Tifus por Ácaros/microbiología , Piel/microbiología , Piel/patología , Piel/inmunología , Factor de Transcripción STAT1/metabolismo , Receptor de Interferón gamma/genética , Receptor de Interferón gamma/metabolismoRESUMEN
Scrub typhus, a potentially life-threatening infectious disease, is attributed to bacteria Orientia tsutsugamushi (O. tsutsugamushi). The transmission of this illness to humans occurs through the bite of infected chiggers, which are the larval forms of mites belonging to the genus Leptotrombidium. In this research, we developed a subunit vaccine specifically designed to target outer membrane proteins. Immunodominant cytotoxic T-lymphocytes (CTLs), B- lymphocytes (BCLs), and major histocompatibility complex (MHC)- II epitopes were identified using machine learning and bioinformatics approaches. These epitopes were arranged in different combinations with the help of suitable linkers like AAY, KK, GPGPG and adjuvant (cholera toxin B) that resulted in a vaccine construct. Physiochemical properties were assessed, where the predicted solubility (0.571) was higher than threshold value. Tertiary structure was predicted using I-TASSER web server and evaluated using Ramachandran plot (94 % residues in most favourable region) and z-score (-6.04), which had shown the structure to have good stability and residue arrangement. Molecular docking with immune receptors, Toll-like receptor (TLR)-2 and -4 showed good residue interaction with 13 and 5 hydrogen bonds respectively. Molecular dynamics simulations of receptor-ligand complex provided the idea about the strong interaction having 1.524751 × 10-5 eigenvalue. Amino acid sequence of vaccine was converted to nucleotide sequence and underwent codon optimization. The optimized codon sequence was used for in-silico cloning, which provided idea about the possibility of synthesis of vaccine using E. coli as host. Overall, this study provided a promising blueprint for a scrub typhus vaccine, although experimental validation is needed for confirmation. Furthermore, it is crucial to acknowledge that while bioinformatics provides valuable insights, in-vitro and in-vivo studies are imperative for a comprehensive evaluation of vaccine candidate. Thus, the integration of computational predictions with empirical research is essential to validate the efficacy, safety, and real-world applicability of the designed vaccine against Scrub Typhus. Nevertheless, the findings are good to carry forward for in-vitro and in-vivo investigations.
Asunto(s)
Epítopos de Linfocito B , Epítopos de Linfocito T , Orientia tsutsugamushi , Tifus por Ácaros , Vacunas de Subunidad , Tifus por Ácaros/inmunología , Tifus por Ácaros/prevención & control , Orientia tsutsugamushi/inmunología , Humanos , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Vacunas de Subunidad/inmunología , Simulación del Acoplamiento Molecular , Vacunas Bacterianas/inmunología , Simulación por Computador , Biología Computacional/métodos , Linfocitos T Citotóxicos/inmunología , Aprendizaje Automático , Linfocitos B/inmunología , Receptor Toll-Like 2/inmunologíaRESUMEN
Infection with Orientia tsutsugamushi, an obligate intracellular bacterium, can cause mild or severe scrub typhus. Some patients develop acute lung injury, multi-organ failure, and fatal infection; however, little is known regarding key immune mediators that mediate infection control or disease pathogenesis. Using murine models of scrub typhus, we demonstrated in this study the requirement of TNF-TNFR signaling in protective immunity against this infection. Mice lacking both TNF receptors (TNFR1 and TNFR2) were highly susceptible to O. tsutsugamushi infection, displaying significantly increased tissue bacterial burdens and succumbing to infection by day 9, while most wild-type mice survived through day 20. This increased susceptibility correlated with poor activation of cellular immunity in inflamed tissues. Flow cytometry of lung- and spleen-derived cells revealed profound deficiencies in total numbers and activation status of NK cells, neutrophils, and macrophages, as well as CD4 and CD8 T cells. To define the role of individual receptors in O. tsutsugamushi infection, we used mice lacking either TNFR1 or TNFR2. While deficiency in either receptor alone was sufficient to increase host susceptibility to the infection, TNFR1 and TNFR2 played a distinct role in cellular responses. TNF signaling through TNFR1 promoted inflammatory responses and effector T cell expansion, while TNFR2 signaling was associated with anti-inflammatory action and tissue homeostasis. Moreover, TNFRs played an intrinsic role in CD8+ T cell activation, revealing an indispensable role of TNF in protective immunity against O. tsutsugamushi infection.
Asunto(s)
Orientia tsutsugamushi , Receptores Tipo II del Factor de Necrosis Tumoral , Receptores Tipo I de Factores de Necrosis Tumoral , Tifus por Ácaros , Animales , Ratones , Ratones Endogámicos C57BL , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Tifus por Ácaros/inmunologíaRESUMEN
Scrub typhus (ST), also known as tsutsugamushi disease and caused by rickettsia Orientia tsutsugamushi, is an underestimated fatal epidemic in the Asia-Pacific region, resulting in a million human infections each year. ST is easily misdiagnosed as clinical diagnosis is based on non-specific skin eschar and flu-like symptoms. Thus, the lack of accurate, convenient, and low-cost detection methods for ST poses a global health threat. To address this problem, we adopted baculovirus surface-display technology to express three variants of TSA56, the major membrane antigen of O. tsutsugamushi, as well as the passenger domain of ScaC (ScaC-PD), on insect Sf21 cell surfaces rather than biosafety level 3 bacteria in an enzyme-linked immunosorbent assay (ELISA). Recombinant TSA56 and ScaC-PD were all properly expressed and displayed on Sf21 cells. Our cell-based ELISA comprising the four antigen-displaying cell types interacted with monoclonal antibodies as well as serum samples from ST-positive field-caught rats. This cell-based ELISA presented high accuracy (96.3%), sensitivity (98.6%), and specificity (84.6%) when tested against the ST-positive rat sera. Results of a pilot study using human sera were also highly consistent with the results of immunofluorescence analyses. By adopting this approach, we circumvented complex purification and refolding processes required to generate recombinant O. tsutsugamushi antigens and reduced the need for expensive equipment and extensively trained operators. Thus, our system has the potential to become a widely used serological platform for diagnosing ST.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Orientia tsutsugamushi/inmunología , Tifus por Ácaros/diagnóstico , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Baculoviridae/genética , Línea Celular , Técnicas de Visualización de Superficie Celular , Ensayo de Inmunoadsorción Enzimática , Humanos , Ratones , Ratas , Proteínas Recombinantes/inmunología , Tifus por Ácaros/sangre , Tifus por Ácaros/inmunología , SpodopteraRESUMEN
Scrub typhus is an acute infectious disease caused by the bacterium Orientia tsutsugamushi, which is widely distributed in northern, southern, and eastern Asia. Early diagnosis is essential because the average case fatality rate is usually >10% but can be as high as 45% if antimicrobial treatment is delayed. Although an O. tsutsugamushi 56-kDa type-specific antigen (TSA) is commonly used for serological diagnosis of scrub typhus, the 56-kDa TSA shows variations among O. tsutsugamushi strains, which may lead to poor diagnostic results. Therefore, the discovery of new antigenic proteins may improve diagnostic accuracy. In this study, we identified an O. tsutsugamushi 27 kDa antigen through an immunoinformatic approach and verified its diagnostic potential using patient samples. Compared with the O. tsutsugamushi 56-kDa antigen, the new 27-kDa antigen showed better diagnostic specificity with similar diagnostic sensitivity. Therefore, the O. tsutsugamushi 27-kDa antigen shows potential as a novel serological diagnostic antigen for scrub typhus, providing higher diagnostic accuracy for O. tsutsugamushi than the 56-kDa antigen.
Asunto(s)
Antígenos Bacterianos/sangre , Antígenos Bacterianos/inmunología , Orientia tsutsugamushi/genética , Orientia tsutsugamushi/aislamiento & purificación , Tifus por Ácaros/diagnóstico , Tifus por Ácaros/inmunología , Pruebas Serológicas/métodos , Voluntarios Sanos , Humanos , República de CoreaRESUMEN
Orientia (O.) tsutsugamushi, the causative agent of scrub typhus, is a neglected, obligate intracellular bacterium that has a prominent tropism for monocytes and macrophages. Complications often involve the lung, where interstitial pneumonia is a typical finding. The severity of scrub typhus in humans has been linked to altered plasma concentrations of chemokines which are known to act as chemoattractants for myeloid cells. The trafficking and function of monocyte responses is critically regulated by interaction of the CC chemokine ligand 2 (CCL2) and its CC chemokine receptor CCR2. In a self-healing mouse model of intradermal infection with the human-pathogenic Karp strain of O. tsutsugamushi, we investigated the role of CCR2 on bacterial dissemination, development of symptoms, lung histology and monocyte subsets in blood and lungs. CCR2-deficient mice showed a delayed onset of disease and resolution of symptoms, higher concentrations and impaired clearance of bacteria in the lung and the liver, accompanied by a slow infiltration of interstitial macrophages into the lungs. In the blood, we found an induction of circulating monocytes that depended on CCR2, while only a small increase in Ly6Chi monocytes was observed in CCR2-/- mice. In the lung, significantly higher numbers of Ly6Chi and Ly6Clo monocytes were found in the C57BL/6 mice compared to CCR2-/- mice. Both wildtype and CCR2-deficient mice developed an inflammatory milieu as shown by cytokine and inos/arg1 mRNA induction in the lung, but with delayed kinetics in CCR2-deficient mice. Histopathology revealed that infiltration of macrophages to the parenchyma, but not into the peribronchial tissue, depended on CCR2. In sum, our data suggest that in Orientia infection, CCR2 drives blood monocytosis and the influx and activation of Ly6Chi and Ly6Clo monocytes into the lung, thereby accelerating bacterial replication and development of interstitial pulmonary inflammation.
Asunto(s)
Antígenos Ly/metabolismo , Pulmón/microbiología , Macrófagos/microbiología , Monocitos/microbiología , Orientia tsutsugamushi/patogenicidad , Receptores CCR2/deficiencia , Tifus por Ácaros/microbiología , Animales , Carga Bacteriana , Quimiotaxis de Leucocito , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Patógeno , Hígado/inmunología , Hígado/metabolismo , Hígado/microbiología , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/inmunología , Monocitos/metabolismo , Orientia tsutsugamushi/crecimiento & desarrollo , Orientia tsutsugamushi/inmunología , Receptores CCR2/genética , Tifus por Ácaros/genética , Tifus por Ácaros/inmunología , Tifus por Ácaros/metabolismoRESUMEN
Background: Dendritic cells (DCs) are specialized antigen-presenting cells known to bridge innate and adaptive immune reactions. However, the relationship between circulating DCs and Orientia tsutsugamushi infection is unclear. Therefore, this study aimed to examine the level and function of plasmacytoid DCs (pDCs) and conventional DCs (cDCs), two subsets of circulating DCs, in scrub typhus patients. Methods: The study included 35 scrub typhus patients and 35 healthy controls (HCs). pDC and cDC levels, CD86 and CD274 expression, and cytokine levels were measured using flow cytometry. Results: Circulating pDC and cDC levels were found to be significantly reduced in scrub typhus patients, which were correlated with disease severity. The patients displayed increased percentages of CD86+ pDCs, CD274+ pDCs, and CD274+ cDCs in the peripheral blood. The alterations in the levels and surface phenotypes of pDCs and cDCs were recovered in the remission state. In addition, the production of interferon (IFN)-α and tumor necrosis factor (TNF)-α by circulating pDCs, and interleukin (IL)-12 and TNF-α by circulating cDCs was reduced in scrub typhus patients. Interestingly, our in vitro experiments showed that the percentages of CD86+ pDCs, CD274+ pDCs, and CD274+ cDCs were increased in cultures treated with cytokines including IFN-γ, IL-12, and TNF-α. Conclusions: This study demonstrates that circulating pDCs and cDCs are numerically deficient and functionally impaired in scrub typhus patients. In addition, alterations in the expression levels of surface phenotypes of pDCs and cDCs could be affected by pro-inflammatory cytokines.
Asunto(s)
Células Dendríticas/inmunología , Tifus por Ácaros/inmunología , Adulto , Anciano , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Orientia tsutsugamushi is an obligately intracellular bacterium and the etiological agent of scrub typhus. The lung is a major target organ of infection, displaying type 1-skewed proinflammatory responses. Lung injury and acute respiratory distress syndrome are common complications of severe scrub typhus; yet, their underlying mechanisms remain unclear. In this study, we investigated whether the C-type lectin receptor (CLR) Mincle contributes to immune recognition and dysregulation. Following lethal infection in mice, we performed pulmonary differential expression analysis with NanoString. Of 671 genes examined, we found 312 significantly expressed genes at the terminal phase of disease. Mincle (Clec4e) was among the top 5 greatest up-regulated genes, accompanied with its signaling partners, type 1-skewing chemokines (Cxcr3, Ccr5, and their ligands), as well as Il27. To validate the role of Mincle in scrub typhus, we exposed murine bone marrow-derived macrophages (MΦ) to live or inactivated O. tsutsugamushi and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. We found that while heat-killed bacteria stimulated transitory Mincle expression, live bacteria generated a robust response in MΦ, which was validated by indirect immunofluorescence and western blot. Notably, infection had limited impact on other tested CLRs or TLRs. Sustained proinflammatory gene expression in MΦ (Cxcl9, Ccl2, Ccl5, Nos2, Il27) was induced by live, but not inactivated, bacteria; infected Mincle-/- MΦ significantly reduced proinflammatory responses compared with WT cells. Together, this study provides the first evidence for a selective expression of Mincle in sensing O. tsutsugamushi and suggests a potential role of Mincle- and IL-27-related pathways in host responses to severe infection. Additionally, it provides novel insight into innate immune recognition of this poorly studied bacterium.
Asunto(s)
Inmunidad Innata/inmunología , Lectinas Tipo C/inmunología , Proteínas de la Membrana/inmunología , Tifus por Ácaros/inmunología , Animales , Regulación de la Expresión Génica/inmunología , Inflamación/inmunología , Ratones , Ratones Endogámicos C57BL , Orientia tsutsugamushi/inmunologíaRESUMEN
BACKGROUND: Scrub typhus is a neglected tropical disease that threatens more than one billion people. If antibiotic therapy is delayed, often due to mis- or late diagnosis, the case fatality rate can increase considerably. Scrub typhus is caused by the obligate intracellular bacterium, Orientia tsutsugamushi, which invades phagocytes and endothelial cells in vivo and diverse tissue culture cell types in vitro. The ability of O. tsutsugamushi to replicate in the cytoplasm indicates that it has evolved to counter eukaryotic host cell immune defense mechanisms. The transcription factor, NF-κB, is a tightly regulated initiator of proinflammatory and antimicrobial responses. Typically, the inhibitory proteins p105 and IκBα sequester the NF-κB p50:p65 heterodimer in the cytoplasm. Canonical activation of NF-κB via TNFα involves IKKß-mediated serine phosphorylation of IκBα and p105, which leads to their degradation and enables NF-κB nuclear translocation. A portion of p105 is also processed into p50. O. tsutsugamushi impairs NF-κB translocation into the nucleus, but how it does so is incompletely defined. PRINCIPAL FINDINGS: Western blot, densitometry, and quantitative RT-PCR analyses of O. tsutsugamushi infected host cells were used to determine if the pathogen's ability to inhibit NF-κB is linked to modulation of p105. Results demonstrate that p105 levels are elevated several-fold in O. tsutsugamushi infected HeLa and RF/6A cells with only a nominal increase in p50. The O. tsutsugamushi-stimulated increase in p105 is bacterial dose- and protein synthesis-dependent, but does not occur at the level of host cell transcription. While TNFα-induced phosphorylation of p105 serine 932 proceeds unhindered in infected cells, p105 levels remain elevated and NF-κB p65 is retained in the cytoplasm. CONCLUSIONS: O. tsutsugamushi specifically stabilizes p105 to inhibit the canonical NF-κB pathway, which advances understanding of how it counters host immunity to establish infection.
Asunto(s)
Proteínas Bacterianas/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Orientia tsutsugamushi/metabolismo , Orientia tsutsugamushi/patogenicidad , Factor de Transcripción ReIA/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Células HeLa , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/fisiología , Humanos , Orientia tsutsugamushi/inmunología , Tifus por Ácaros/inmunología , Tifus por Ácaros/microbiología , Activación Transcripcional , Factor de Necrosis Tumoral alfa/metabolismo , Virulencia/genética , Virulencia/inmunología , Virulencia/fisiologíaRESUMEN
Scrub typhus is a fatal zoonotic disease caused by Orientia tsutsugamushi. This disease is accompanied by systemic vasculitis, lymphadenopathy, headache, myalgia, and eschar. In recent studies, a novel strain that is resistant to current medical treatment was identified in Thailand. Thus, the development of new specific drugs for scrub typhus is needed. However, the exact molecular mechanism governing the progression of scrub typhus has not been fully elucidated. To understand disease-related genetic factors and mechanisms associated with the progression of scrub typhus, we performed a genome-wide association study (GWAS) in scrub typhus-infected patients and found a scrub typhus-related signaling pathway by molecular interaction search tool (MIST) and PANTHER. We identified eight potent scrub typhus-related single nucleotide polymorphisms (SNPs) located on the PRMT6, PLGLB2, DTWD2, BATF, JDP2, ONECUT1, WDR72, KLK, MAP3K7, and TGFBR2 genes using a GWAS. We also identified 224 genes by analyzing protein-protein interactions among candidate genes of scrub typhus and identified 15 signaling pathways associated with over 10 genes by classifying these genes according to signaling pathways. The signaling pathway with the largest number of associated genes was the gonadotropin-releasing hormone receptor pathway, followed by the TGF-beta signaling pathway and the apoptosis signaling pathway. To the best of our knowledge, this report describes the first GWAS in scrub typhus.
Asunto(s)
Sitios Genéticos/inmunología , Estudio de Asociación del Genoma Completo/métodos , Tifus por Ácaros/inmunología , Humanos , Persona de Mediana Edad , Transducción de SeñalRESUMEN
BACKGROUND: The mechanisms that control local and systemic inflammation in scrub typhus have only been partially elucidated. The wingless (Wnt) signaling pathways are emerging as important regulators of inflammation and infection, but have not been investigated in scrub typhus. METHODOLOGY/PRINCIPAL FINDINGS: Plasma levels of secreted Wnt antagonists (i.e. DKK-1, sFRP-3, WIF-1 and SOST) were analyzed in patients with scrub typhus (n = 129), patients with similar febrile illness without O. tsutsugamushi infection (n = 31), febrile infectious disease controls, and in healthy controls (n = 31) from the same area of South India, and were correlated to markers of inflammation, immune and endothelial cell activation as well as for their association with organ specific dysfunction and mortality in these patients. We found i) Levels of SOST and in particular sFRP-3 and WIF-1 were markedly increased and DKK-1 decreased in scrub typhus patients at admission to the hospital compared to healthy controls. ii) In recovering scrub typhus patients, SOST, sFRP-3 and WIF-1 decreased and DKK-1 increased. iii) SOST was positively correlated with markers of monocyte/macrophage and endothelial/vascular activation as well as with renal dysfunction and poor outcome iv) Finally, regulation of Wnt pathways by O. tsutsugamushi in vitro in monocytes and ex vivo in mononuclear cells isolated from patients with scrub typhus, as evaluated by gene expression studies available in public repositories, revealed markedly attenuated canonical Wnt signaling. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that scrub typhus is characterized by attenuated Wnt signaling possibly involving dysregulated levels of several secreted pathway antagonists. The secreted Wnt antagonist SOST was strongly associated with renal dysfunction and poor prognosis in these patients.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/sangre , Orientia tsutsugamushi/fisiología , Tifus por Ácaros/sangre , Proteínas Wnt/sangre , Adolescente , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Humanos , India , Inflamación/inmunología , Modelos Lineales , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Tifus por Ácaros/inmunología , Transducción de Señal , Proteínas Wnt/antagonistas & inhibidores , Adulto JovenRESUMEN
BACKGROUND: Scrub typhus is a dominant cause of febrile illness in many parts of Asia. Immunity is limited by the great strain diversity of Orientia tsutsugamushi. It is unclear whether previous infection protects from severe infection or enhances the risk. METHODS/PRINCIPAL FINDINGS: We studied IgG antibody levels against O. tsutsugamushi at presentation in 636 scrub typhus patients using enzyme-linked immunosorbent assays (ELISA). The association between ELISA optical density (OD) and risk of severe infection was modelled using Poisson regression. OD was categorised as low (<1.0), intermediate (1.0 to 2.9), and high (≥3.0). OD was also modelled as a continuous variable (cubic spline). Median age of cases was 41 years (range 0-85), with 37% having severe infection. Compared to the low category, the age-adjusted risk of severe infection was 1.5 times higher in the intermediate category (95%CI 1.2, 1.9), and 1.3 times higher in the high category (95%CI 1.0, 1.7). The effect was stronger in cases <40 years, doubling the risk in the intermediate and high categories compared to the low category. The effect was more pronounced in cases tested within 7 days of fever onset when IgG ODs are more likely to reflect pre-infection levels. CONCLUSIONS/SIGNIFICANCE: Intermediate and high IgG antibody levels at the time of diagnosis are associated with a higher risk of severe scrub typhus infection. The findings may be explained by severe infection eliciting an accelerated IgG response or by previous scrub typhus infection enhancing the severity of subsequent episodes.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Protección Cruzada/inmunología , Inmunoglobulina G/sangre , Orientia tsutsugamushi/inmunología , Tifus por Ácaros/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Estudios Transversales , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , India/epidemiología , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Estudios Retrospectivos , Riesgo , Tifus por Ácaros/inmunología , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Scrub typhus, a vector-borne rickettsiosis, is the leading treatable cause of non-malarial febrile illness in Asia. The myriad of typical and atypical features poses a clinical conundrum. We aimed to study the clinical and laboratory profile of children with scrub typhus infection diagnosed by IgM ELISA. Data of children < 12 years presenting with undifferentiated fever to the pediatric services of a tertiary teaching institute between January 2012 and December 2018 were retrieved. Children with seropositive IgM ELISA (InBios International Kit, Seattle, WA) for scrub typhus were enrolled in the study. Clinical features, laboratory investigations, treatment received, and the outcome recorded were obtained. Objective evidence of organ dysfunction was taken as severe scrub typhus. In total, 262 children were diagnosed with scrub typhus. The mean age was 5 years, with male preponderance (65%). And, 13 children presented during infancy. Fever was universal, and generalized lymphadenopathy (93.5%) and hepatomegaly (70%) were the common clinical signs. Eschar was identified in 31%, with greater predilection for groin and axilla. Thrombocytopenia was striking in one-third of children. Also, 25 children (9.5%) had severe scrub typhus and 18 required intensive care stay. Elevated aspartate aminotransferase enzyme levels was a predictor of severity ([OR 3.9], P value 0.005) by multivariate analysis. Lymphadenopathy was found significantly associated with eschar (P < 0.005). No mortality was recorded. This 6-year study underscores the varied spectrum of pediatric scrub typhus infection. Zero mortality in our cohort signifies the excellent outcome with judicious first-line antibiotics.
Asunto(s)
Orientia tsutsugamushi/patogenicidad , Tifus por Ácaros/epidemiología , Tifus por Ácaros/fisiopatología , Adolescente , Antibacterianos , Niño , Preescolar , Femenino , Interacciones Huésped-Patógeno , Humanos , India/epidemiología , Lactante , Laboratorios , Masculino , Orientia tsutsugamushi/inmunología , Estudios Prospectivos , Estudios Retrospectivos , Tifus por Ácaros/tratamiento farmacológico , Tifus por Ácaros/inmunología , Centros de Atención Terciaria/estadística & datos numéricosRESUMEN
Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning to study the transcriptional architecture of Ot, we find evidence for wide-spread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/inmunología , Interacciones Huésped-Patógeno/inmunología , Enfermedades Desatendidas/inmunología , Orientia tsutsugamushi/genética , Tifus por Ácaros/inmunología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Estudios de Factibilidad , Femenino , Genoma Bacteriano , Células Endoteliales de la Vena Umbilical Humana , Humanos , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Secuencias Repetitivas Esparcidas/genética , Ratones , Enfermedades Desatendidas/microbiología , Orientia tsutsugamushi/inmunología , Orientia tsutsugamushi/patogenicidad , Proteómica , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , ARN Bacteriano/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , RNA-Seq , Tifus por Ácaros/microbiología , Transcripción Genética , Secuenciación del ExomaRESUMEN
Nine criteria regarding the infectious agent, mode of transmission, portal of entry, route of spread, target organs, target cells, pathologic lesions, incubation period, and modifiable spectrum of disease and outcomes appropriate to the intended experimental purpose are described. To provide context for each criterion, mouse models of two vector-borne zoonotic infectious diseases, scrub typhus and dengue, are summarized. Application of the criteria indicates that intravenous inoculation of Orientia tsutsugamushi into inbred mice is the best current model for life-threatening scrub typhus, and intradermal inoculation accurately models sublethal human scrub typhus, whereas the immunocompromised mouse models of dengue provide disease outcomes most closely associated with human dengue. In addition to addressing basic questions of immune and pathogenic mechanisms, mouse models are useful for preclinical testing of experimental vaccines and therapeutics. The nine criteria serve as guidelines to evaluate and compare models of vector-borne infectious diseases.
Asunto(s)
Virus del Dengue/inmunología , Dengue/inmunología , Modelos Animales de Enfermedad , Huésped Inmunocomprometido , Orientia tsutsugamushi/inmunología , Tifus por Ácaros/inmunología , Animales , Dengue/patología , Dengue/virología , Virus del Dengue/patogenicidad , Humanos , Periodo de Incubación de Enfermedades Infecciosas , Inyecciones Intradérmicas , Inyecciones Intravenosas , Hígado/microbiología , Hígado/virología , Tejido Linfoide/microbiología , Tejido Linfoide/virología , Ratones , Ratones Noqueados , Orientia tsutsugamushi/patogenicidad , Tifus por Ácaros/microbiología , Tifus por Ácaros/patología , Bazo/microbiología , Bazo/virologíaRESUMEN
Orientia tsutsugamushi infection can cause acute lung injury and high mortality in humans; however, the underlying mechanisms are unclear. Here, we tested a hypothesis that dysregulated pulmonary inflammation and Tie2-mediated endothelial malfunction contribute to lung damage. Using a murine model of lethal O. tsutsugamushi infection, we demonstrated pathological characteristics of vascular activation and tissue damage: 1) a significant increase of ICAM-1 and angiopoietin-2 (Ang2) proteins in inflamed tissues and lung-derived endothelial cells (EC), 2) a progressive loss of endothelial quiescent and junction proteins (Ang1, VE-cadherin/CD144, occuludin), and 3) a profound impairment of Tie2 receptor at the transcriptional and functional levels. In vitro infection of primary human EC cultures and serum Ang2 proteins in scrub typhus patients support our animal studies, implying endothelial dysfunction in severe scrub typhus. Flow cytometric analyses of lung-recovered cells further revealed that pulmonary macrophages (MΦ) were polarized toward an M1-like phenotype (CD80+CD64+CD11b+Ly6G-) during the onset of disease and prior to host death, which correlated with the significant loss of CD31+CD45- ECs and M2-like (CD206+CD64+CD11b+Ly6G-) cells. In vitro studies indicated extensive bacterial replication in M2-type, but not M1-type, MΦs, implying the protective and pathogenic roles of M1-skewed responses. This is the first detailed investigation of lung cellular immune responses during acute O. tsutsugamushi infection. It uncovers specific biomarkers for vascular dysfunction and M1-skewed inflammatory responses, highlighting future therapeutic research for the control of this neglected tropical disease.
Asunto(s)
Angiopoyetina 2/metabolismo , Células Endoteliales/patología , Orientia tsutsugamushi/crecimiento & desarrollo , Neumonía/patología , Receptor TIE-2/metabolismo , Tifus por Ácaros/patología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Macrófagos/inmunología , Macrófagos/microbiología , Ratones Endogámicos C57BL , Neumonía/inmunología , Tifus por Ácaros/inmunologíaRESUMEN
Scrub typhus is an acute vector-borne disease caused by infection with the intracellular gram-negative bacterium Orientia tsutsugamushi (Ot). The rapid production of an efficient vaccine against Ot using novel strategies is required because of the global increase in mortality caused by these infections; however, no commercial vaccine is currently available. Ot induces T-cell-mediated immunogenic responses upon infection; therefore, a new rapidly producible vaccine that maximizes T-cell responses against Ot is required. In this study, we sought to develop a model vaccine platform for T-cell-mediated Ot infection using T-cell-immunity associated Salmonella-derived extracellular vesicles (EVs). For this purpose, we optimized DNA sequences encoding the full-length Ot proteins, TSA56, ScaA, ScaC, ScaD, and ScaE, and their expression in Salmonella. The sequences were incorporated into a new platform vector, pKST, which ectopically and concurrently produces Ot proteins and EVs. Expression analysis using pKST-antigen plasmids showed that TSA56 and ScaC produced antigen-associated EVs and showed strong T-cell immunogenic responses. We found that mice vaccinated with EVs derived from TSA56-expressing cells were protected from Salmonella-induced mortality. Therefore, our findings showed that Salmonella EV-associated antigen is a model platform for T-cell immune response infections. Our system could help prepare EV-antigen vaccines against scrub typhus in an easy and rapid manner.