Dysregulation of Stress-Induced Translational Control by Porphyromonas gingivalis in Host Cells.

Porphyromonas gingivalis contributes to the chronic oral disease periodontitis, triggering the activation of host inflammatory responses, inducing cellular stresses such as oxidation. During stress, host cells can activate the Integrated Stress Response (ISR), a pathway which determines cellular fate, by either downregulating protein synthesis and initiating a stress-response gene expression program, or by initiating programmed cell death. Recent studies have implicated the ISR within both host antimicrobial defenses and the pathomechanism of certain microbes. In this study, using a combination of immunofluorescence confocal microscopy and immunoblotting, the molecular mechanisms by which P. gingivalis infection alters translation attenuation during oxidative stress-induced activation of the ISR in oral epithelial cells were investigated. P. gingivalis infection alone did not result in ISR activation. In contrast, infection coupled with stress caused differential stress granule formation and composition. Infection heightened stress-induced translational repression independently of core ISR mediators. Heightened translational repression during stress was observed with both P. gingivalis-conditioned media and outer membrane vesicles, implicating a secretory factor in this exacerbated translational repression. The effects of gingipain inhibitors and gingipain-deficient P. gingivalis mutants confirmed these pathogen-specific proteases as the effector of exacerbated translational repression. Gingipains are known to degrade the mammalian target of rapamycin (mTOR) and the findings of this study implicate the gingipain-mTOR axis as the effector of host translational dysregulation during stress.

Bacterial Manipulation of the Integrated Stress Response: A New Perspective on Infection.

Host immune activation forms a vital line of defence against bacterial pathogenicity. However, just as hosts have evolved immune responses, bacteria have developed means to escape, hijack and subvert these responses to promote survival. In recent years, a highly conserved group of signalling cascades within the host, collectively termed the integrated stress response (ISR), have become increasingly implicated in immune activation during bacterial infection. Activation of the ISR leads to a complex web of cellular reprogramming, which ultimately results in the paradoxical outcomes of either cellular homeostasis or cell death. Therefore, any pathogen with means to manipulate this pathway could induce a range of cellular outcomes and benefit from favourable conditions for long-term survival and replication. This review aims to outline what is currently known about bacterial manipulation of the ISR and present key hypotheses highlighting areas for future research.

Role of OmpA2 surface regions of Porphyromonas gingivalis in host-pathogen interactions with oral epithelial cells.

Outer membrane protein A (OmpA) is a key outer membrane protein found in Gram-negative bacteria that contributes to several crucial processes in bacterial virulence. In Porphyromonas gingivalis, OmpA is predicted as a heterotrimer of OmpA1 and OmpA2 subunits encoded by adjacent genes. Here we describe the role of OmpA and its individual subunits in the interaction of P. gingivalis with oral cells. Using knockout mutagenesis, we show that OmpA2 plays a significant role in biofilm formation and interaction with human epithelial cells. We used protein structure prediction software to identify extracellular loops of OmpA2, and determined these are involved in interactions with epithelial cells as evidenced by inhibition of adherence and invasion of P. gingivalis by synthetic extracellular loop peptides and the ability of the peptides to mediate interaction of latex beads with human cells. In particular, we observe that OmpA2-loop 4 plays an important role in the interaction with host cells. These data demonstrate for the first time the important role of P. gingivalis OmpA2 extracellular loops in interaction with epithelial cells, which may help design novel peptide-based antimicrobial therapies for periodontal disease.

Beta-hexosaminidase activity of the oral pathogen Tannerella forsythia influences biofilm formation on glycoprotein substrates.

Tannerella forsythia is an important pathogen in periodontal disease. Previously, we showed that its sialidase activity is key to utilization of sialic acid from a range of human glycoproteins for biofilm growth and initial adhesion. Removal of terminal sialic acid residues often exposes ß-linked glucosamine or galactosamine, which may also be important adhesive molecules. In turn, these residues are often removed by a group of enzymes known as ß-hexosaminidases. We show here that T. forsythia has the ability to cleave glucosamine and galactosamine from model substrates and that this activity can be inhibited by the hexosaminidase inhibitor PugNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate). We now demonstrate for the first time that ß-hexosaminidase activity plays a role in biofilm growth on glycoprotein-coated surfaces because biofilm growth and initial cell adhesion are inhibited by PugNAc. In contrast, adhesion to siallo-glycoprotein-coated surfaces is unaltered by PugNAc in the absence of sialidase activity (using a sialidase-deficient mutant) or surprisingly on the clinically relevant substrates saliva or serum. These data indicate that ß-hexosaminidase activity has a significant role in biofilm formation in combination with sialidase activity in the biofilm lifestyle of T. forsythia.

A single-nucleotide polymorphism in the human ITGB3 gene is associated with the platelet-specific alloantigen Va (HPA-17bw) involved in fetal maternal alloimmune thrombocytopenia.

BACKGROUND: The previously reported platelet (PLT)-specific antigen, Va(a), was defined by an alloantibody detected in the serum sample of a mother who delivered an infant displaying symptoms of severe fetal maternal alloimmune thrombocytopenia (FMAIT). This PLT antigen was localized to the integrin alphaIIbbeta3 (GPIIbIIIa) but its genetic basis was not defined. STUDY DESIGN AND METHODS: Genomic ITGA2B (alphaIIb) and ITGB3 (beta3) DNA from a Va(a)-positive individual were sequenced to identify potential polymorphisms underlying the Va(a) antigen. Recombinant beta3 integrin carrying the putative mutation was then used to assess serologic reactivity with the original maternal Va(a) antiserum. RESULTS: A single-nucleotide polymorphism (SNP; C622>T) in exon 5 of ITGB3 resulting in the replacement of threonine with methionine at residue 195 of the mature beta3 was identified. Calmodulin-tagged soluble recombinant beta3 encoding Met195 was produced in S2 cells and found to react specifically with the Va(a) antiserum. CONCLUSION: With the use of a combination of DNA sequencing and recombinant antigen expression, the molecular basis of the PLT-specific Va(a) antigen (HPA-17bw), a low-frequency antigen implicated in FMAIT, has been resolved. These data further demonstrate the value of using recombinant beta3 peptides for the detection of rare but clinically relevant antibodies.

HPA-1a antibody potency and bioactivity do not predict severity of fetomaternal alloimmune thrombocytopenia.

BACKGROUND: The antenatal management of fetomaternal alloimmune thrombocytopenia (FMAIT) due to HPA-1a antibodies remains controversial, and a test identifying pregnancies that do not require therapy would be of clinical value. STUDY DESIGN AND METHODS: The statistical correlation was analyzed between clinical outcome and 1) anti-HPA-1a potency in maternal serum samples determined by a monoclonal antibody immobilization of platelet (PLT) antigen assay with an international anti-HPA-1a potency standard and 2) anti-HPA-1a biological activity measured by a monocyte chemiluminescence (CL) assay. RESULTS: A total of 133 pregnancies with FMAIT due to anti-HPA-1a were analyzed. In 97 newly diagnosed cases, there was no difference in antibody potency or CL signal between cases with intracranial hemorrhage (ICH; n = 15), those with no ICH but a PLT count of less than 20 x 10(9) per L (n = 52), and those with a PLT count of at least 20 x 10(9) per L (n = 30). In 22 previously known pregnancies, the positive predictive value of maternal anti-HPA-1a of greater than 30 IU per mL for a PLT count of less than 20 x 10(9) per L was 90 percent, but the negative predictive value was only 66 percent. Antibody potency tended to stay stable throughout pregnancy (n = 16) and from one pregnancy to the next (n = 16). CONCLUSION: Neither severe thrombocytopenia nor ICH in HPA-1a-alloimmunized pregnancies can be predicted with sufficient sensitivity and specificity for clinical application from maternal anti-HPA-1a potency or bioactivity.

Molecular characterization of the variable domains of an alphaIIbbeta3-specific immunoglobulin M kappa platelet cold agglutinin in a follicular lymphoma patient with treatment refractory autoimmune thrombocytopenia: idiotypic overlap between alphaIIbbeta3 integrin antibodies.

BACKGROUND: Cold hemagglutinins are generally immunoglobulin M (IgM) kappa antibodies reactive at temperatures below 37 degrees C and if of high titer may cause hemolysis. Platelet (PLT) cold agglutinins (CAs) are rare and poorly characterized. A detailed molecular characterization of the variable domains of a pathologic, PLT-reactive, CA is presented. CASE REPORT: A 70-year-old woman was admitted with rectal bleeding accompanied by widespread petechiae, bruising, tongue and buccal mucosa bleeding, and epistaxes and proved refractory to HLA- and HPA-matched PLTs. Detailed investigation showed monoclonal heavy-chain gene rearrangement with an IgM paraprotein of 3.3 g per L and a trace of kappa Bence Jones protein in the urine, compatible with a diagnosis of secretory B-cell non-Hodgkin's lymphoma (B-NHL). PLT antibody (PAIg) investigations revealed a potent IgM kappa PLT CA. Sequencing of the rearranged variable domain genes of the malignant clone together with idiotype-specific antibodies obtained by DNA-based immunization of rabbits and matrix-assisted laser desorption/ionization-time-of-flight analysis of the PAIgM provided a irrefutable link between the thrombocytopenia, the IgM paraprotein, and the PAIgM against alphaIIbbeta3. The thrombocytopenia and bleeding were refractory to standard treatment and PLT transfusion, but treatment with rituximab resulted in a recovery of the PLT count and a complete remission of B-NHL. CONCLUSION: The IgM kappa paraprotein derived from the malignant B-cell clone was a potent and clinically significant CA against alphaIIbbeta3. The testing for PLT CAs in patients with a paraprotein and refractory to matched PLTs may aid the selection of appropriate treatment.

Production of calmodulin-tagged proteins in Drosophila Schneider S2 cells: a novel system for antigen production and phage antibody isolation.

We report the development of an expression system for the production of soluble, calmodulin (CaM)-tagged proteins in Drosophila Schneider S2 cells and the subsequent use of these proteins for the selection of phage displayed antibodies. The CaM-tag permitted the purification of recombinant protein to >90% purity in a single step at yields of >20 mg/l. Using platelet glycoprotein VI (GP6) as a model, we demonstrated that the recombinant CaM-tagged protein was post-translationally N-glycosylated and had identical ligand specificity to native protein. A novel selection strategy, exploiting the CaM tag, was then used to isolate four single chain Fv fragments (scFvs) specific for GP6 from a non-immune phage display library. In contrast to other selection methods, which can result in antibodies that do not recognise native protein, all of the scFvs we selected bound cell surface expressed GP6. In conclusion, the production of CaM-tagged proteins in Drosophila Schneider S2 cells and the selection strategy reported here offer advantages over previously published methods, including simple culture conditions, rapid protein purification, specific elution of phage antibodies and preferential selection of phage antibodies that recognise native, cell surface expressed protein.

Platelet integrin alpha2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display.

Antibodies are a powerful tool for structure/function studies of platelet proteins. However, classic immunisation frequently elicits antibody responses against domains of minor functional interest. Robust strategies to generate antibodies against defined domains would be of significant interest in post-genome research. In this study, we report a new strategy using a combination of DNA vaccination and V gene phage display that allows the rapid generation of domain specific single-chain Fv antibodies (scFvs). This system was validated using the I-domain of alpha2 integrin as a model. The alpha2beta1 integrin, which is expressed on many cell types, is the dominant collagen attachment receptor on platelets, functioning in close interplay with the collagen signalling receptor glycoprotein VI. A novel set of I-domain specific antibodies was obtained by a DNA vaccination/V gene repertoire cloning approach. Mice were first immunised with a DNA vaccine in which the alpha2 I-domain is expressed as a fusion protein with fragment C of tetanus toxoid (FrC-TT). Then the heavy and kappa light chain variable gene repertoires were rescued from immune splenocytes using antibody phage display. A total of four alpha2 I-domain specific scFvs were isolated by selection on recombinant I-domain or native platelet alpha2beta1 integrin. Characterisation of the scFvs indicated that they recognised distinct epitopes that had profound differences in accessibility between native and recombinant I-domain. Our data suggest DNA immunisation and phage display represent versatile alternatives to protein immunisation and hybridoma-fusion techniques for the isolation of recombinant antibody reagents. This approach will be particularly useful for the generation of domain or splice-variant specific antibodies that recognise native protein.