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1.
Gut Microbes ; 16(1): 2297897, 2024.
Article in English | MEDLINE | ID: mdl-38189373

ABSTRACT

Cryptosporidiosis is a major cause of severe diarrheal disease in infants from resource poor settings. The majority of infections are caused by the human-specific pathogen C. hominis and absence of in vitro growth platforms has limited our understanding of host-pathogen interactions and development of effective treatments. To address this problem, we developed a stem cell-derived culture system for C. hominis using human enterocytes differentiated under air-liquid interface (ALI) conditions. Human ALI cultures supported robust growth and complete development of C. hominis in vitro including all life cycle stages. Cryptosporidium infection induced a strong interferon response from enterocytes, possibly driven, in part, by an endogenous dsRNA virus in the parasite. Prior infection with Cryptosporidium induced type III IFN secretion and consequently blunted infection with Rotavirus, including live attenuated vaccine strains. The development of hALI provides a platform for further studies on human-specific pathogens, including clinically important coinfections that may alter vaccine efficacy.


Subject(s)
Cryptosporidiosis , Cryptosporidium , Gastrointestinal Microbiome , Rotavirus , Infant , Humans , Interferon Lambda , Epithelial Cells , Zea mays
2.
bioRxiv ; 2023 Sep 06.
Article in English | MEDLINE | ID: mdl-37693422

ABSTRACT

Cryptosporidiosis is a major cause of severe diarrheal disease in infants from resource poor settings. The majority of infections are caused by the human-specific pathogen C. hominis and absence of in vitro growth platforms has limited our understanding of host-pathogen interactions and development of effective treatments. To address this problem, we developed a stem cell-derived culture system for C. hominis using human enterocytes differentiated under air-liquid interface (ALI) conditions. Human ALI cultures supported robust growth and complete development of C. hominis in vitro including all life cycle stages. C. hominis infection induced a strong interferon response from enterocytes, likely driven by an endogenous dsRNA virus in the parasite. Prior infection with Cryptosporidium induced type III IFN secretion and consequently blunted infection with Rotavirus, including live attenuated vaccine strains. The development of hALI provides a platform for further studies on human-specific pathogens, including clinically important coinfections that may alter vaccine efficacy.

3.
Microorganisms ; 11(4)2023 Apr 18.
Article in English | MEDLINE | ID: mdl-37110479

ABSTRACT

Host immune responses are required for the efficient control of cryptosporidiosis. Immunity against Cryptosporidium infection has been best studied in mice, where it is mediated by both innate and adaptive immune responses. Dendritic cells are the key link between innate and adaptive immunity and participate in the defense against Cryptosporidium infection. While the effector mechanism varies, both humans and mice rely on dendritic cells for sensing parasites and restricting infection. Recently, the use of mouse-adapted strains C. parvum and mouse-specific strain C. tyzzeri have provided tractable systems to study the role of dendritic cells in mice against this parasite. In this review, we provide an overview of recent advances in innate immunity acting during infection with Cryptosporidium with a major focus on the role of dendritic cells in the intestinal mucosa. Further work is required to understand the role of dendritic cells in the activation of T cells and to explore associated molecular mechanisms. The identification of Cryptosporidium antigen involved in the activation of Toll-like receptor signaling in dendritic cells during infection is also a matter of future study. The in-depth knowledge of immune responses in cryptosporidiosis will help develop targeted prophylactic and therapeutic interventions.

4.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Article in English | MEDLINE | ID: mdl-34462359

ABSTRACT

Oral infection with Toxoplasma gondii results in dysbiosis and enteritis, both of which revert to normal during chronic infection. However, whether infection leaves a lasting impact on mucosal responses remains uncertain. Here we examined the effect of the chemical irritant dextran sodium sulfate (DSS) on intestinal damage and wound healing in chronically infected mice. Our findings indicate that prior infection with T. gondii exacerbates damage to the colon caused by DSS and impairs wound healing by suppressing stem cell regeneration of the epithelium. Enhanced tissue damage was attributable to inflammatory monocytes that emerge preactivated from bone marrow, migrate to the intestine, and release inflammatory mediators, including nitric oxide. Tissue damage was reversed by neutralization of inflammatory monocytes or nitric oxide, revealing a causal mechanism for tissue damage. Our findings suggest that chronic infection with T. gondii enhances monocyte activation to increase inflammation associated with a secondary environmental insult.


Subject(s)
Colitis/complications , Toxoplasmosis/complications , Animals , Chronic Disease , Disease Susceptibility , Gastrointestinal Microbiome , Intestinal Mucosa/pathology , Mice , Mice, Inbred C57BL , Monocytes/pathology , Regeneration , Stem Cells/pathology
5.
mSphere ; 4(1)2019 02 27.
Article in English | MEDLINE | ID: mdl-30814319

ABSTRACT

Microneme (MIC) proteins play important roles in the recognition, adhesion, and invasion of host cells by Toxoplasma gondii Previous studies have shown that MIC proteins are highly immunogenic in the mouse and recognized by human serum antibodies. Here we report that T. gondii antigens MIC1, MIC3, MIC4, and MIC6 were capable of inducing memory responses leading to production of gamma interferon (IFN-γ) by T cells from T. gondii-infected mice. Production of IFN-γ was demonstrated using enzyme-linked immunosorbent spot (ELISPOT) assay and also intracellular cytokine staining. All four MIC antigens displayed very high sensitivity (100%) and specificity (86 to 100%) for detecting chronic infection. Interestingly, IFN-γ was produced by both CD4+ and CD8+ T cells in BALB/c mice but primarily by CD4+ T cells in C57BL/6 mice. Phenotypic characterization of IFN-γ-producing CD4+ and CD8+ T cells in BALB/c mice and CD4+ T cells in C57BL/6 mice revealed effector memory T cells (CD44hi CD62Llo) as the predominant cells that contributed to IFN-γ production in response to MIC antigens. Effector memory responses were seen in mice of different major histocompatibility complex class II (MHC-II) haplotypes, suggesting that MIC antigens contain epitopes that are broadly recognized.IMPORTANCE Current diagnosis of toxoplasmosis relies almost exclusively on antibody detection, and while detection of IgG provides a useful estimate of prior infection, it does not alone indicate immune status. In contrast, detection of IFN-γ responses to T. gondii antigens has been used to monitor immune responsiveness in HIV-infected patients, thus providing valuable predictions about the potential for disease reactivation. However, specific T. gondii antigens that can be used in assays to detect cellular immunity remain largely undefined. In this study, we examined the diagnostic potential of microneme antigens of T. gondii using IFN-γ detection assays. Our findings demonstrate that MIC antigens (MIC1, MIC3, MIC4, and MIC6) elicit IFN-γ responses from memory T cells in chronically infected mice. Monitoring IFN-γ production by T cells stimulated with MIC antigens provided high sensitivity and specificity for detection of T. gondii infection in mice. Taken together, these studies suggest that microneme antigens might be useful as an adjunct to serological testing to monitor immune status during infection.


Subject(s)
Immunologic Memory , Protozoan Proteins/immunology , T-Lymphocytes/immunology , Toxoplasmosis/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Adhesion Molecules/immunology , Chronic Disease , Female , Immunity, Cellular , Interferon-gamma/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Specific Pathogen-Free Organisms , Toxoplasma
6.
Tuberculosis (Edinb) ; 106: 16-24, 2017 09.
Article in English | MEDLINE | ID: mdl-28802400

ABSTRACT

Cell wall of Mycobacterium tuberculosis (M.tb) is a major source of immunogenic proteins that can be tested as vaccine candidates. MymA (Rv3083), a 55 kDa M.tb flavin containing monooxygenase, is involved in modification of mycolic acids during acidic shock following M.tb internalization in macrophage. In this study, we have investigated the role of this cell wall associated protein in activation of macrophages by toll like receptor (TLRs) engagement and subsequent signaling. Our results showed that MymA stimulation of THP1 cells and human monocyte derived macrophages (MDM) lead to upregulation of TLR2 and co-stimulatory molecules CD40, CD80, CD86 and HLA-DR. This upregulation is partially reduced by TLR2 blocking antibodies. The activation of macrophage following MymA stimulation also resulted in release of proinflammatory cytokines, TNF-α and IL-12. Moreover, MymA also polarized the immune response towards TH1 as shown by an increased IFN-γ level in the supernatant of stimulated peripheral blood mononuclear cells (PBMC). In consensus with the TLR2 signaling involving MyD88 and NF-κB, we also observed several fold increase in mRNA for TLR2, MyD88 and NF-κB on MymA induction of THP-1 and MDM by qRT-PCR. The increased production of NF-κB following recognition of MymA by TLR2 was further confirmed by HEK-TLR2 reporter cell line colorimetric assay. In conclusion, immunological evaluation revealed that MymA is a TLR2 agonist that upregulates signaling via MyD88 and NF-κB in macrophages to stimulate the release of proinflammatory cytokines. The MymA protein should be investigated further for expression in recombinant BCG as a pre-exposure vaccine or as a post-exposure subunit vaccine candidate.


Subject(s)
Bacterial Proteins/immunology , Lymphocyte Activation , Macrophage Activation , Macrophages/immunology , Oxygenases/immunology , Th1 Cells/drug effects , Toll-Like Receptor 2/agonists , Tuberculosis Vaccines/immunology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cytokines/metabolism , HEK293 Cells , Humans , Inflammation Mediators/metabolism , Macrophages/metabolism , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Oxygenases/genetics , Oxygenases/metabolism , Protein Binding , Signal Transduction/drug effects , THP-1 Cells , Th1 Cells/immunology , Th1 Cells/metabolism , Time Factors , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/metabolism , Tuberculosis Vaccines/genetics , Tuberculosis Vaccines/metabolism , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology , Vaccines, Subunit/metabolism
7.
Chem Biol Drug Des ; 89(1): 152-160, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27539124

ABSTRACT

Tuberculosis is a global health problem especially with the emergence of drug-resistant Mycobacterium tuberculosis strains, creating an urgent need to identify new drug targets. The mycobacterial cell wall is an attractive target for chemotherapeutic agents. Gene products of mymA operon are known to be required for the maintenance of cell wall and play an important role in persistence, thus making them important drug targets. This study was undertaken to biochemically characterize the MymA as a flavin-containing monooxygenase (FMO). Our results established its enzymatic activity in vitro and found that the mycobacterial FMO requires NADPH and FAD as cofactors, similar to other characterized bacterial FMOs. The enzyme follows Michaelis-Menten kinetics to catalyze substrates such as trimethylamine and thiourea. We also propose that MymA could be one of the targets of the antituberculosis drug, isoniazid (INH), which is a cell wall inhibitor. Molecular docking studies revealed that INH targeted NADPH-binding site of the MymA. Further, experimental validation revealed that INH inhibits MymA with the IC50 value of 4.9 µm. Thus, this study characterizes for the first time that MymA is a mycobacterial FMO, which may be a target of INH.


Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/drug effects , Flavins/analysis , Isoniazid/pharmacology , Mixed Function Oxygenases/drug effects , Amino Acid Sequence , Antitubercular Agents/chemistry , Bacterial Proteins/chemistry , Isoniazid/chemistry , Mixed Function Oxygenases/chemistry , Molecular Docking Simulation , Sequence Homology, Amino Acid
8.
Infect Genet Evol ; 44: 182-189, 2016 10.
Article in English | MEDLINE | ID: mdl-27389362

ABSTRACT

Limited efficacy of Bacillus Calmette-Guérin vaccine has raised the need to explore other immunogenic candidates to develop an effective vaccine against Mycobacterium tuberculosis (Mtb). Both CD4+ and CD8+ T cells play a critical role in host immunity to Mtb. Infection of macrophages with Mtb results in upregulation of mymA operon genes thereby suggesting their importance as immune targets. In the present study, after exclusion of self-peptides mymA operon proteins of Mtb were analyzed in silico for the presence of Human Leukocyte Antigen (HLA) Class I and Class II binding peptides using Bioinformatics and molecular analysis section, NetMHC 3.4, ProPred and Immune epitope database software. Out of 56 promiscuous epitopes obtained, 41 epitopes were predicted to be antigenic for MHC Class I. In MHC Class II, out of 336 promiscuous epitopes obtained, 142 epitopes were predicted to be antigenic. The comparative bioinformatics analysis of mymA operon proteins found Rv3083 to be the best vaccine candidate. Molecular docking was performed with the most antigenic peptides of Rv3083 (LASGAASVV with alleles HLA-B51:01, HAATSGTLI with HLA-A02, IVTATGLNI and EKIHYGLKVNTA with HLA-DRB1_01:01) to study the structural basis for recognition of peptides by various HLA molecules. The software binding prediction was validated by the obtained molecular docking score of peptide-HLA complex. These peptides can be further investigated for their immunological relevance in patients of tuberculosis using major histocompatibility complex tetramer approach.


Subject(s)
Bacterial Proteins/metabolism , Epitopes, T-Lymphocyte/metabolism , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class I/immunology , Mycobacterium tuberculosis/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Carboxylic Ester Hydrolases/immunology , Carboxylic Ester Hydrolases/metabolism , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class II/metabolism , Humans , Immunogenicity, Vaccine , Molecular Docking Simulation , Virulence Factors/immunology , Virulence Factors/metabolism
9.
Immunol Cell Biol ; 92(9): 741-6, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24983458

ABSTRACT

Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, is an intracellular bacterium capable of surviving and persisting within host mononuclear cells. The host response against tubercle bacilli is dominated by fine-tuned interaction of innate and adaptive immune responses. Toll-like receptors (TLRs) play a critical role in the formation of this immune response by facilitating in elaboration of protective T helper type 1 (Th1) cytokines and microbicidal molecules, but the intracellular persistence of M. tuberculosis in the phagosome and processing and presentation of TLR ligands by host antigen-presenting cell leads to continuous and chronic TLR2 signaling. The prolonged stimulation of TLR ultimately results in elaboration of immunosuppressive cytokines and downregulation of antigen presentation by major histocompatibility complex (MHC) class II and therefore becomes beneficial for M. tuberculosis, resulting in its continued survival inside macrophages. An understanding of the host-pathogen interaction in tuberculosis is important to delineate the mechanisms that can modulate the immune response toward protection. This review focuses on the role of TLRs in immune response and immune evasion and how M. tuberculosis maintains its dominance over the host during infection. A precise understanding of the TLRs and M. tuberculosis interaction will undoubtedly lead to the development of novel therapies to combat tuberculosis.


Subject(s)
Host-Pathogen Interactions/immunology , Immune Evasion/immunology , Mycobacterium tuberculosis/immunology , Toll-Like Receptors/immunology , Tuberculosis/immunology , Animals , Humans , Macrophages/immunology
10.
Vaccine ; 32(6): 712-6, 2014 Feb 03.
Article in English | MEDLINE | ID: mdl-24300592

ABSTRACT

Tuberculosis remains a great health threat to the world among infectious diseases particularly with the advent of human immunodeficiency virus and emergence of drug resistant strains. In the light of the inconsistent efficacy imparted by the only currently available pre-exposure vaccine bacillus Calmette-Guerin BCG, the development of an improved TB vaccine is a very high international research priority. Vaccine candidates currently in clinical trials are also pre-exposure vaccines that aim to prevent active tuberculosis during an individual's lifetime. According to World Health Organization approximately a third of the world's population is latently infected with Mycobacterium tuberculosis. Dormancy or latency of Mycobacteria is associated with the formation of granuloma with poorly perfused interior leading to expression of genes which help them survive in this hostile environment. A group of about 50 genes belonging to the DosR regulon also known as latency antigens are expressed by Mycobacteria when they are persisting in the immuno-competent host. An understanding of the immunological effects produced by products of these latency induced genes may help in making a more potent vaccine. Incorporation of latency antigens into improved (live or subunit) vaccines may enhance the impact of these vaccines in which BCG priming can be followed by multisubunit protein boosting. These vaccines could act as post exposure vaccines for containment and prevention of latent TB activation. This heterologous boosting of BCG-primed immunity will be able to stimulate the known immune correlates of protective immunity against M. tuberculosis i.e. TH1 cells (CD4(+) and CD8(+) T cells) mediated immune responses with cytokines such as IFN-γ and TNF-α⋅ In our review we have analysed and compared the immunogenic potential of various latency-associated antigens of the DosR regulon in line with the current strategy of developing a recombinant post exposure booster vaccine.


Subject(s)
Antigens, Bacterial/immunology , Latent Tuberculosis/immunology , Tuberculosis Vaccines/immunology , Tuberculosis/prevention & control , Animals , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Bacterial Proteins/immunology , DNA-Binding Proteins , Humans , Mycobacterium tuberculosis , Protein Kinases/genetics , Protein Kinases/immunology , Regulon , Vaccines, Synthetic/immunology
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