Polyaminated, acetylated and stop codon readthrough of recombinant Francisella tularensis universal stress protein in Escherichia coli.

Recombinant Francisella tularensis universal stress protein with a C-terminal histidine-tag (rUsp/His6) was expressed in Escherichia coli. Endogenous F. tularensis Usp has a predicted molecular mass of 30 kDa, but rUsp/His6 had an apparent molecular weight of 33 kDa based on Western blot analyses. To determine the source of the higher molecular weight for rUsp/His6, post translational modifications were examined. Tryptic peptides of purified rUsp/His6 were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) and fragmentation spectra were searched for acetylated lysines and polyaminated glutamines. Of the 24 lysines in rUsp/His6, 10 were acetylated (K63, K68, K72, K129, K175, K201, K208, K212, K233, and K238) and three of the four glutamines had putrescine, spermidine and spermine adducts (Q55, Q60 and Q267). The level of post-translational modification was substoichiometric, eliminating the possibility that these modifications were the sole contributor to the 3 kDa extra mass of rUsp/His6. LC-MS/MS revealed that stop codon readthrough had occurred resulting in the unexpected addition of 20 extra amino acids at the C-terminus of rUsp/His6, after the histidine tag. Further, the finding of polyaminated glutamines in rUsp/His6 indicated that E. coli is capable of transglutaminase activity.

Arginine Catabolism and Polyamine Biosynthesis Pathway Disparities Within Francisella tularensis Subpopulations.

Francisella tularensis is a highly infectious zoonotic pathogen with as few as 10 organisms causing tularemia, a disease that is fatal if untreated. Although F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B) share over 99.5% average nucleotide identity, notable differences exist in genomic organization and pathogenicity. The type A clade has been further divided into subtypes A.I and A.II, with A.I strains being recognized as some of the most virulent bacterial pathogens known. In this study, we report on major disparities that exist between the F. tularensis subpopulations in arginine catabolism and subsequent polyamine biosynthesis. The genes involved in these pathways include the speHEA and aguAB operons, along with metK. In the hypervirulent F. tularensis A.I clade, such as the A.I prototype strain SCHU S4, these genes were found to be intact and highly transcribed. In contrast, both subtype A.II and type B strains have a truncated speA gene, while the type B clade also has a disrupted aguA and truncated aguB. Ablation of the chromosomal speE gene that encodes a spermidine synthase reduced subtype A.I SCHU S4 growth rate, whereas the growth rate of type B LVS was enhanced. These results demonstrate that spermine synthase SpeE promotes faster replication in the F. tularensis A.I clade, whereas type B strains do not rely on this enzyme for in vitro fitness. Our ongoing studies on amino acid and polyamine flux within hypervirulent A.I strains should provide a better understanding of the factors that contribute to F. tularensis pathogenicity.

Production of Autoreactive Heavy Chain-Only Antibodies in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the overproduction of high-affinity autoreactive antibodies. Here, we show that more than 65.8% of 222 recombinant antibodies derived from 8 SLE patients can be secreted as heavy chain-only antibodies (HCAbs) when expressed in HEK-293T cells. The secretion of HCAbs follows the conventional endoplasmic reticulum-Golgi apparatus pathway, despite triggering a weaker unfolded protein response (UPR). Many of the purified SLE HCAbs remain autoreactive and have an even higher affinity for dsDNA, Sm, nucleosome, and cardiolipin than HCAbs from healthy individuals. Extended analyses of the CDR3 region and the heavy chain variable (VH) region of HCAb F3 show that the VH region is responsible for IgH secretion, while the CDR3 region determines its reactivity. Such a high frequency of HCAb secretion cannot fully concur with our current understanding of antibody assembly and secretion. The presence of a large proportion of autoreactive HCAbs in SLE reveals a novel mechanism for the generation of autoreactive antibodies in lupus.

Genetic Alleles Associated with SLE Susceptibility and Clinical Manifestations in Hispanic Patients from the Dominican Republic.

PURPOSE: Systemic lupus erythematosus (SLE) is a complex autoimmune disease with marked disparities in prevalence and disease severity among different ethnic groups. The purpose of this study is to characterize a Latin American cohort and identify genetic risk factors for developing SLE and its end-organ manifestations in this Latin Hispanic cohort. METHODS: A total of 201 SLE cases and 205 non-diseased controls were recruited in the Dominican Republic (DR). Cases were defined according to the 1997 revised American College of Rheumatology criteria for the classification of SLE. Genomic DNA was prepared from whole blood and applied to genotyping analyses for 42 single nucleotide polymorphisms (SNPs) that have been implicated in autoimmune diseases, including SLE, in other ethnic populations. Data were analyzed by Fisher's Exact Probability Test. RESULTS: In this cohort, SNP rs9271366 (tag SNP for HLA-DRB1*15:01) confers the highest risk for SLE among the 13 MHC gene alleles that display association with SLE (p = 8.748E-10; OR = 3.5). Among the 26 non-MHC gene alleles analyzed, SNP rs2476601 in PTPN22 gene confers the highest risk for SLE (p = 0.0001; OR = 5.6). ITGAM, TNFSF4, TNIP1, STAT4, CARD11, BLK, and TNXB gene alleles were confirmed as SLE-susceptible alleles in the DR cohort. However, IRF5 and TNFAIP3 gene alleles, established risk factors for SLE in populations of European and Asian ancestry, are not significantly associated with SLE in this cohort. We also defined a novel HLA-DRA haplotype that confers an increased risk for lupus nephritis (LN) and alleles in HLA-DRA2 and TNFSF4 genes as genetic risk factors for developing neuropsychiatric (NP) SLE. CONCLUSION: Our data suggest that the Latin American population shares some common genetic risk factors for SLE as other populations, but also has distinct risk gene alleles that contribute to SLE susceptibility and development of LN and NPSLE. This is the first study focusing on genetic risk factors for SLE in the DR, a Latin American population that has never been characterized before.

Characteristics of Plasmablast Repertoire in Chronically HIV-Infected Individuals for Immunoglobulin H and L Chain Profiled by Single-Cell Analysis.

Characterization of the diversified immunoglobulin (Ig) repertoire may provide insight into pathways that shape an efficient antibody (Ab) repertoire for immune response against human immunodeficiency virus (HIV) infection. This study aimed to profile characteristics of the plasmablast repertoire during chronic HIV infection. Ig variable regions of plasmablasts from both chronically HIV-infected donors (HIVDs) previously treated with antiretroviral therapy (ART) and healthy donors (HDs) were amplified by single-cell PCR to establish the basis for further repertoire analysis. We compared the plasmablast repertoires expressed in multiple chronically HIVDs after ART treatment cessation and HDs. We also examined the non-productive repertoire to identify the indication of the immediate products of the rearrangement machinery without an impact of selection during HIV infection. We found multiple differences between the productive repertoires of HIVD and HD subjects, including biased usages of VH3-49, VH1-2, VH3-33, VH3-74, and VH5-51 in VH and D1-7, D1-14, D1-20, and D5-5/18 in D segments in the HIVD group, as well as shorter and preferential glycine usages in CDRH3 regions. Gene selections were also detected in light chains. Notably, differences between productive rearrangements of HIVDs and HDs outnumbered those between productive and non-productive rearrangements within HIVDs. HIV infection may exert a dominant impact on the development of the plasmablast repertoire. The impact of selection is of limited significance in shaping the plasmablast repertoire. Overall, the data indicate that the environment in which the plasmablasts live can affect the distribution of the VH and VL genes in the repertoire and the amino acid compositions of the expressed Abs.

Circulating Plasmablasts from Chronically Human Immunodeficiency Virus-Infected Individuals Predominantly Produce Polyreactive/Autoreactive Antibodies.

Understanding the B-cell response during chronic human immunodeficiency virus (HIV) infection is essential for eliciting broad and potent neutralizing antibodies (Abs). In this study, we analyzed the plasmablast repertoire of chronically HIV-infected individuals in combination with antiretroviral therapy (ART). Among the obtained 72 recombinant monoclonal antibodies (mAbs), 27.8% weakly bound to HIV gp140 and were non-neutralizing. Remarkably, 56.9% were polyreactive and 55.6% were autoreactive. The prominent feature of being polyreactive/autoreactive is not limited to anti-gp140 Abs. Furthermore, these polyreactive/autoreactive Abs displayed striking cross-reactivity with DWEYS in the N-methyl-d-aspartate receptor (NMDAR), and this binding induced SH-SY5Y cell apoptosis. We also found higher frequencies of VH4-34 utilization and VH replacement in the plasmablast repertoire of chronically HIV-infected individuals, which may contribute to the generation of poly/autoreactive Abs. Taken together, these data demonstrate that circulating plasmablasts in chronically HIV-infected individuals experienced with ART predominantly produce poly/autoreactive Abs with minimal anti-HIV neutralizing capacity and potential cross-reactivity with autoantigens. This may represent another dysfunction of B cells during chronic HIV infection.

Autoantibodies From Single Circulating Plasmablasts React With Citrullinated Antigens and Porphyromonas gingivalis in Rheumatoid Arthritis.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA). However, the molecular basis for ACPA production is still unclear. The purpose of this study was to determine if circulating plasmablasts from RA patients produce ACPAs and whether Porphyromonas gingivalis facilitates the generation of ACPAs. METHODS: Using a single-cell antibody cloning approach, we generated 217 and 110 monoclonal recombinant antibodies from circulating plasmablasts from 7 RA patients and 4 healthy controls, respectively. Antibody reactivity with citrullinated antigens was tested by a second-generation anti-cyclic citrullinated peptide (anti-CCP) kit and by enzyme-linked immunosorbent assays (ELISAs) against citrullinated human antigens. Antibody reactivity with P gingivalis was tested by ELISAs against outer membrane antigens (OMAs) and citrullinated enolase from P gingivalis. RESULTS: Approximately 19.5% of plasmablast-derived antibodies from anti-CCP-positive RA patients, but none from 1 anti-CCP-negative RA patient or the healthy controls, specifically recognized citrullinated antigens. The immunoglobulin genes encoding these ACPAs were highly mutated, with increased ratios of replacement mutations to silent mutations, suggesting the involvement of active antigen selection in ACPA generation. Interestingly, 63% of the ACPAs cross-reacted with OMAs and/or citrullinated enolase from P gingivalis. The reactivity of ACPAs against citrullinated proteins from P gingivalis was confirmed by immunoblotting and mass spectrometry. Furthermore, some germline-reverted ACPAs retained their reactivity with P gingivalis antigens but completely lost their reactivity with citrullinated human antigens. CONCLUSION: These results suggest that circulating plasmablasts in RA patients produce ACPAs and that this process may be facilitated by anti-P gingivalis immune responses.

Microbial Infection and Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a complex autoimmune disease affecting 1-2% of general worldwide population. The etiopathogenesis of RA involves the interplay of multiple genetic risk factors and environmental triggers. Microbial infections are believed to play an important role in the initiation and perpetuation of RA. Recent clinical studies have shown the association of microbial infections with RA. Accumulated studies using animal models have also found that microbial infections can induce and/or exaggerate the symptoms of experimental arthritis. In this review, we have identified the most common microbial infections associated with RA in the literature and summarized the current evidence supporting their pathogenic role in RA. We also discussed the potential mechanisms whereby infection may promote the development of RA, such as generation of neo-autoantigens, induction of loss of tolerance by molecular mimicry, and bystander activation of the immune system.