Papers by Bharathi Krishnan
Background: We recently reported the identification of Bacillus sp. NRRL B-14911 that induces hea... more Background: We recently reported the identification of Bacillus sp. NRRL B-14911 that induces heart autoimmunity by generating cardiac-reactive T cells through molecular mimicry. This marine bacterium was originally isolated from the Gulf of Mexico, but no associations with human diseases were reported. Therefore, to characterize its biological and medical significance, we sought to determine and analyze the complete genome sequence of Bacillus sp. NRRL B-14911. Results: Based on the phylogenetic analysis of 16S ribosomal RNA (rRNA) genes, sequence analysis of the 16S-23S rDNA intergenic transcribed spacers, phenotypic microarray, and matrix-assisted laser desorption ionization time-offlight mass spectrometry, we propose that this organism belongs to the species Bacillus infantis, previously shown to be associated with sepsis in a newborn child. Analysis of the complete genome of Bacillus sp. NRRL B-14911 revealed several virulence factors including adhesins, invasins, colonization ...
Identification of Bacillus sp. NRRL B-14911 spores. Bacterial smear was stained with malachite gr... more Identification of Bacillus sp. NRRL B-14911 spores. Bacterial smear was stained with malachite green and safranin as described in the Methods section and examined under oil immersion microscope. Arrows indicate round and symmetrical endospores present both within and outside the bacteria. Original magnification: 100x. Figure S2. The genes encoded by the plasmid of Bacillus sp. NRRL B-14911. The plasmid sequence was generated from the sequence data obtained by sequencing of the genomic DNA from Bacillus sp. NRRL B-14911 in long-reads. The genes encoded by the plasmid were annotated as described for the bacterial chromosome (see methods).Figure S3. Alignment of contigs previously reported for Bacillus sp. NRRL B-14911 to the new long-read-based finished assembly. The inner ring (blue) represents contigs assembled from short-reads without any scaffolding (middle panel in Figure 1) as aligned to our de novo assembly based on long-reads. The outer ring (pink and black) represents alignme...
Journal of immunology (Baltimore, Md. : 1950), Jan 11, 2017
Sarcoplasmic/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA)2a, a critical regulator ... more Sarcoplasmic/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA)2a, a critical regulator of calcium homeostasis, is known to be decreased in heart failure. Patients with myocarditis or dilated cardiomyopathy develop autoantibodies to SERCA2a suggesting that they may have pathogenetic significance. In this report, we describe epitope mapping analysis of SERCA2a in A/J mice that leads us to make five observations: 1) SERCA2a contains multiple T cell epitopes that induce varying degrees of myocarditis. One epitope, SERCA2a 971-990, induces widespread atrial inflammation without affecting noncardiac tissues; the cardiac abnormalities could be noninvasively captured by echocardiography, electrocardiography, and magnetic resonance microscopy imaging. 2) SERCA2a 971-990-induced disease was associated with the induction of CD4 T cell responses and the epitope preferentially binds MHC class II/IAk rather than IEk By creating IAk/and IEk/SERCA2a 971-990 dextramers, the T cell response...
The American Journal of Pathology, 2016
Heart failure, a leading cause of death in humans, can emanate from myocarditis. Although most in... more Heart failure, a leading cause of death in humans, can emanate from myocarditis. Although most individuals with myocarditis recover spontaneously, some develop chronic dilated cardiomyopathy. Myocarditis may result from both infectious and noninfectious causes, including autoimmune responses to cardiac antigens. In support of this notion, intracellular cardiac antigens, like cardiac myosin heavy chain-a, cardiac troponin-I, and adenine nucleotide translocator 1 (ANT 1), have been identified as autoantigens in cardiac autoimmunity. Herein, we demonstrate that ANT 1 can induce autoimmune myocarditis in A/J mice by generating autoreactive T cells. We show that ANT 1 encompasses multiple immunodominant epitopes (namely, ANT 1 21-40, ANT 1 31-50, ANT 1 171-190, and ANT 1 181-200). Although all four peptides induce comparable T-cell responses, only ANT 1 21-40 was found to be a major myocarditogenic epitope in immunized animals. The myocarditis-inducing ability of ANT 1 21-40 was associated with the generation of T cells producing predominantly IL-17A, and the antigen-sensitized T cells could transfer the disease to naïve recipients. These data indicate that cardiac mitochondrial proteins can be target autoantigens in myocarditis, supporting the notion that the antigens released as a result of primary damage may contribute to the persistence of chronic inflammation through autoimmunity.
BMC Genomics, 2016
Background: We recently reported the identification of Bacillus sp. NRRL B-14911 that induces hea... more Background: We recently reported the identification of Bacillus sp. NRRL B-14911 that induces heart autoimmunity by generating cardiac-reactive T cells through molecular mimicry. This marine bacterium was originally isolated from the Gulf of Mexico, but no associations with human diseases were reported. Therefore, to characterize its biological and medical significance, we sought to determine and analyze the complete genome sequence of Bacillus sp. NRRL B-14911. Results: Based on the phylogenetic analysis of 16S ribosomal RNA (rRNA) genes, sequence analysis of the 16S-23S rDNA intergenic transcribed spacers, phenotypic microarray, and matrix-assisted laser desorption ionization time-offlight mass spectrometry, we propose that this organism belongs to the species Bacillus infantis, previously shown to be associated with sepsis in a newborn child. Analysis of the complete genome of Bacillus sp. NRRL B-14911 revealed several virulence factors including adhesins, invasins, colonization factors, siderophores and transporters. Likewise, the bacterial genome encodes a wide range of methyl transferases, transporters, enzymatic and biochemical pathways, and insertion sequence elements that are distinct from other closely related bacilli. Conclusions: The complete genome sequence of Bacillus sp. NRRL B-14911 provided in this study may facilitate genetic manipulations to assess gene functions associated with bacterial survival and virulence. Additionally, this bacterium may serve as a useful tool to establish a disease model that permits systematic analysis of autoimmune events in various susceptible rodent strains.
The Journal of Immunology, May 1, 2014
We recently discovered that Bacillus spp., NRRL B-14911, a marine microbe, has the ability to ind... more We recently discovered that Bacillus spp., NRRL B-14911, a marine microbe, has the ability to induce myocarditis through molecular mimicry. The bacterium contains a mimicry epitope designated, BAC 25-40, and it induces autoimmune myocarditis by generating cross-reactive T cells for cardiac myosin heavy chain-alpha 334-352 (Myhc). We have analysed the complete genome sequence leading us to identify this microorganism as Bacillus infantis NRRL B-14911, a close relative of the strain that has been isolated in sepsis of newborn infants. Conditions have been optimized for the bacterium to grow in a Zobell marine medium at 37oC in an aerobic environment. Our preliminary studies indicate that A/J mice infected with Bacillus infantis NRRL B-14911 show the generation of CD4 T cells that can react with both BAC 25-40 and Myhc, as determined by major histocompatibility complex class II/IAk tetramer staining. We propose that Bacillus infantis NRRL B-14911-induced myocarditis can serve as a useful disease model to study the immune-mediated mechanisms of bacterial myocarditis as they relate to molecular mimicry and/or epitope spreading.
Scandinavian Journal of Immunology, 2015
The advent of major histocompatibility complex (MHC) tetramer technology has been a major contrib... more The advent of major histocompatibility complex (MHC) tetramer technology has been a major contribution to T cell immunology, because tetramer reagents permit detection of antigen-specific T cells at the single-cell level in heterogeneous populations by flow cytometry. However, unlike MHC class I tetramers, the utility of MHC class II tetramers has been less frequently reported. MHC class II tetramers can be used successfully to enumerate the frequencies of antigen-specific CD4 T cells in cells activated in vitro, but their use for ex vivo analyses continues to be a problem, due in part to their activation dependency for binding with T cells. To circumvent this problem, we recently reported the creation of a new generation of reagents called MHC class II dextramers, which were found to be superior to their counterparts. In this review, we discuss the utility of class II dextramers visa -vis tetramers, with respect to their specificity and sensitivity, including potential applications and limitations.
Cytokine
Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells. However, unl... more Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells. However, unlike CD8 T cells, CD4 T cells – especially self-reactive cells – are challenging to detect with major histocompatibility complex (MHC) class II tetramers because of their low frequencies, and low affinities of their T cell receptors to MHC-peptide complexes. In our efforts to improve the sensitivity of MHC class II tetramers, we recently created next-generation tetramers, designated ‘dextramers’. The dextramer reagents were helpful tools to detect and study the functionalities of autoreactive CD4 T cells in several murine autoimmune disease models. These include experimental autoimmune encephalomyelitis induced with proteolipid protein 139–151, and myelin oligodendrocyte glycoprotein 35–55; and myocarditis induced with cardiac myosin heavy chain (Myhc)-α 334–352 and coxsackievirus B3 (CVB). Here, we report five key findings. (i) dextramers and not tetramers, permitted us to enumerate the precursor frequencies of antigen-specific CD4 T cells ex vivo in the brain and heart by flow cytometry; (ii) direct staining with dextramers was sufficient to detect antigen-reactive T cells in situ by confocal microscopy; (iii) using Myhc-α 334–352 dextramers, for the first time, we showed that CVB infection leads to the generation of pathogenic cardiac myosin-specific CD4 T cells; (iv) dextramers were powerful tools to derive antigen-specific T cell hybridomas including evaluation of T cell markers, T cell receptors and their vβ usage in a single step by flow cytometry; and (v) characterization of pathogenic potential of dextramer+ cells based on cytokine-secretion and chemokine receptor expression led us to conclude that autoreactive T cells sensitized with antigens derived from different organ systems do not have common cytokine signatures. The implications of these observations will be discussed.
Journal of immunological methods, Jan 9, 2015
Antigen-specific, T cell hybridomas are useful to study the cellular, molecular and functional ev... more Antigen-specific, T cell hybridomas are useful to study the cellular, molecular and functional events, but their generation is a lengthy process. Thus, there is a need to develop robust methods to generate the hybridoma clones rapidly in a short period of time. To this end, we have demonstrated a novel approach using major histocompatibility complex (MHC) class II dextramers to generate T cell hybridomas for an autoantigen, proteolipid protein (PLP) 139-151. Using MHC class II dextramers assembled with PLP 139-151 as screening and sorting tools, we successfully obtained mono antigen-specific clones within seven to eight weeks. In conjunction with other T cell markers, dextramers permitted phenotypic characterization of hybridoma clones for their antigen specificity in a single step by flow cytometry. Importantly, we achieved successful fusions using dextramer(+) cells sorted by flow cytometry as a starting population, resulting in direct identification of multiple antigen-specific c...
Frontiers in immunology, 2017
Myocarditis/dilated cardiomyopathy (DCM) patients can develop autoantibodies to various cardiac a... more Myocarditis/dilated cardiomyopathy (DCM) patients can develop autoantibodies to various cardiac antigens and one major antigen is β1-adrenergic receptor (β1AR). Previous reports indicate that animals immunized with a β1AR fragment encompassing, 197-222 amino acids for a prolonged period can develop DCM by producing autoantibodies, but existence of T cell epitopes, if any, were unknown. Using A/J mice that are highly susceptible to lymphocytic myocarditis, we have identified β1AR 171-190, β1AR 181-200, and β1AR 211-230 as the major T cell epitopes that bind major histocompatibility complex class II/IAk or IEk alleles, and by creating IAk and IEk dextramers, we demonstrate that the CD4 T cell responses to be antigen-specific. Of note, all the three epitopes were found also to stimulate CD8 T cells suggesting that they can act as common epitopes for both CD4 and CD8 T cells. While, all epitopes induced only mild myocarditis, the disease-incidence was enhanced in animals immunized with ...
Journal of immunological methods, Jan 9, 2015
Antigen-specific, T cell hybridomas are useful to study the cellular, molecular and functional ev... more Antigen-specific, T cell hybridomas are useful to study the cellular, molecular and functional events, but their generation is a lengthy process. Thus, there is a need to develop robust methods to generate the hybridoma clones rapidly in a short period of time. To this end, we have demonstrated a novel approach using major histocompatibility complex (MHC) class II dextramers to generate T cell hybridomas for an autoantigen, proteolipid protein (PLP) 139-151. Using MHC class II dextramers assembled with PLP 139-151 as screening and sorting tools, we successfully obtained mono antigen-specific clones within seven to eight weeks. In conjunction with other T cell markers, dextramers permitted phenotypic characterization of hybridoma clones for their antigen specificity in a single step by flow cytometry. Importantly, we achieved successful fusions using dextramer(+) cells sorted by flow cytometry as a starting population, resulting in direct identification of multiple antigen-specific c...
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Papers by Bharathi Krishnan