Elena B. Lugli

Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis

Background Rheumatoid arthritis (RA) is characterised by autoimmunity to citrullinated proteins, and there is increasing epidemiologic evidence linking Porphyromonas gingivalis to RA. P gingivalis is apparently unique among periodontal pathogens in possessing a citrullinating enzyme, peptidylarginine deiminase (PPAD) with the potential to generate antigens driving the autoimmune response. Objectives To examine the immune response to PPAD in patients with RA, individuals with periodontitis (PD) and controls (without arthritis), confirm PPAD autocitrullination and identify the modified arginine residues. Methods PPAD and an inactivated mutant (C351A) were cloned and expressed and autocitrullination of both examined by immunoblotting and mass spectrometry. ELISAs using PPAD, C351A and another P gingivalis protein arginine gingipain (RgpB) were developed and antibody reactivities examined in patients with RA (n=80), individuals with PD (n=44) and controls (n=82). Results Recombinant PPAD was a potent citrullinating enzyme. Antibodies to PPAD, but not to Rgp, were elevated in the RA sera (median 122 U/ml) compared with controls (median 70 U/ml; p<0.05) and PD (median 60 U/ml; p<0.01). Specificity of the anti-peptidyl citrullinated PPAD response was confirmed by the reaction of RA sera with multiple epitopes tested with synthetic citrullinated peptides spanning the PPAD molecule. The elevated antibody response to PPAD was abolished in RA sera if the C351A mutant was used on ELISA. Conclusions The peptidyl citrulline-specific immune response to PPAD supports the hypothesis that, as a bacterial protein, it might break tolerance in RA, and could be a target for therapy.

Analysis of Genetic Diversity at the arom Locus in Isolates of Pneumocystis carinii

ABSTRACT. The DNA sequences of a portion of the 5‐enolpyruvyl shikimate phosphate synthase domain of the arom gene, encoding the pentafunctional AROM protein, were determined from isolates of Pneumocystis carinii from five mammalian host species (rat, human, ferret, rabbit and mouse). High levels of genetic divergence were found among P. carinii derived from different hosts species, 7–22% at the DNA sequence level, and 7–26% at the derived amino acid sequence level. Two separate and distinct sequences were isolated from infected ferret lungs. Low levels of divergence were seen in human‐derived organisms.

Trypanosome lytic factors: novel mediators of human innate immunity

A novel trypanosome lytic factor (TLF) has been characterized that protects humans from infection by Trypanosoma brucei brucei. The mechanism of trypanolysis is unknown; contrary to one hypothesis, TLF does not kill trypanosomes by generating oxygen radicals. However, these trypanosomes become human-infective when they express a serum-resistance-associated gene.

Autoantibodies to human endogenous retrovirus-K are frequently detected in health and disease and react with multiple epitopes

A number of studies have found increased levels of antibodies to human endogenous retroviruses (HERVs) in autoimmune rheumatic diseases. It is not clear whether this immune response is driven by the HERV itself or by cross‐reactions with an exogenous virus or an autoantigen. To address this question, we examined the antibody response to the Env protein of two closely related members of the HERV‐K family, HERV‐K10 and IDDMK1,222. By immunoblotting of recombinant proteins, antibodies were found in 32–47% of 84 sera from patients with autoimmune rheumatic disease, and 29% of 35 normal controls. Epitope mapping with overlapping 15mers identified multiple reactive peptides on both antigens, with one (GKTCPKEIPKGSKNT) containing immunodominant epitope(s). By ELISA, the median titre of antibody to this peptide was significantly increased in 39 patients with SLE compared to 39 healthy controls and 86 patients with other rheumatic diseases (P < 0·003).

A Pneumocystis carinii multi-gene family with homology to subtilisin-like serine proteases

Copies of multi-gene family, named PRT1 (protease 1), encoding a subtilisin-like serine protease were cloned from the opportunistic fungal pathogen Pneumocystis carinii. Comparison of the nucleotide sequence of a genomic clone and a cDNA clone of PRT1 from P. carinii f. sp. carinii revealed the presence of seven short introns. Several different domains were predicted from the deduced amino acid sequence: an N-terminal hydrophobic signal sequence, a pro-domain, a subtilisin-like catalytic domain, a P-domain (essential for proteolytic activity), a proline-rich domain, a serine/threonine-rich domain and a C-terminal hydrophobic domain. The catalytic domain showed high homology to other eukaryotic subtilisin-like serine proteases and possessed the three essential residues of the catalytic active site. Karyotypic analysis showed that PRT1 was a multi-gene family, copies of which were present on all but one of the P. carinii f. sp. carinii chromosomes. The different copies of the PRT1 genes showed nucleotide sequence heterogeneity, the highest level of divergence being in the proline-rich domain, which varied in both length and composition. Some copies of PRT1 were contiguous with genes encoding the P. carinii major surface glycoprotein.

Expression of citrulline and homocitrulline residues in the lungs of non-smokers and smokers: implications for autoimmunity in rheumatoid arthritis.

IntroductionSmoking is a well-established risk factor for rheumatoid arthritis (RA), and it has been proposed that smoking-induced citrullination renders autoantigens immunogenic. To investigate this mechanism, we examined human lung tissue from 40 subjects with defined smoking status, with or without chronic obstructive pulmonary disease (COPD), and control tissues from other organs for citrullinated proteins and the deiminating enzymes peptidylarginine deiminase type-2 (PAD2) and -4 (PAD4).MethodsLung tissue samples, dissected from lobectomy specimens from 10 never smokers, 10 smokers without airflow limitation, 13 COPD smokers and eight COPD ex-smokers, and control tissue samples (spleen, skeletal muscle, liver, ovary, lymph node, kidney and heart), were analysed for citrullinated proteins, PAD2 and PAD4 by immunoblotting. Citrulline and homocitrulline residues in enolase and vimentin were analysed by partial purification by gel electrophoresis followed by mass spectrometry in 12 of the lung samples and one from each control tissues. Band intensities were scored semi-quantitatively and analysed by two-tailed Mann-Whitney T-test.ResultsWithin the lung tissue samples, citrullinated proteins, PAD2 and PAD4 were found in all samples, with an increase in citrullination in COPD (P = 0.039), but minimal difference between smokers and non-smokers (P = 0.77). Citrullination was also detected at lower levels in the tissues from other organs, principally in lymph node, kidney and skeletal muscle. Mass spectrometry of the lung samples showed that vimentin was citrullinated at positions 71, 304, 346, 410 and 450 in non-smokers and smokers both with and without COPD. A homocitrulline at position 104 was found in four out of six COPD samples and one out of six non-COPD. Citrulline-450 was also found in three of the control tissues. There were no citrulline or homocitrulline residues demonstrated in α-enolase.ConclusionsWe have shown evidence of citrullination of vimentin, a major autoantigen in RA, in both non-smokers and smokers. The increase in citrullinated proteins in COPD suggests that citrullination in the lungs of smokers is mainly due to inflammation. The ubiquity of citrullination of vimentin in the lungs and other tissues suggests that the relationship between smoking and autoimmunity in RA may be more complex than previously thought.

Cell surface protease PRT1 identified in the fungal pathogen Pneumocystis carinii

The subtelomeric regions of the chromosomes of many organisms contain gene families that allow adaptation to a changing environment. In a number of parasites, these subtelomeric gene families encode cell surface proteins that undergo antigenic variation. Proteases are another important virulence determinant in pathogenic microorganisms. We report the localization of the PRT1 protease of the pathogenic fungus Pneumocystis carinii sp. f. carinii, encoded by a subtelomeric gene family, to the cell surface of both the trophozoite and the cyst phase of the organism. Using anti‐PRT1 antiserum, we demonstrated specificity to P. carinii sp. f. carinii in sections of infected rat lungs and, using immunofluorescence, we showed that the PRT1 protease has the characteristic distribution of a surface protein. The anti‐PRT1 antiserum showed cross‐reactivity with a number of P. carinii sp. f. carinii proteins migrating between 185 kDa and 28 kDa, the majority migrating between 42 kDa and 52 kDa, a region that has been shown by serological studies to contain important immunodominant P. carinii proteins. Cross‐reactivity was also observed with P. carinii sp. f. hominis proteins. We have also cloned a portion of the catalytic domain of PRT1 from P. carinii sp. f. hominis, P. carinii sp. f. muris and P. carinii sp. f. rattus. Our data suggest that the PRT1 protease plays an important role in the pathogenicity of P. carinii.

Natural immunity to human African trypanosomiasis: trypanosome lytic factors and the blood incubation infectivity test

This review focuses on the epidemiology of human African trypanosomiasis: why it occurs in humans, the current methods of surveillance, and the drugs available to treat it. Emphasis is placed on the identification of human-infective trypanosomes by the blood incubation infectivity test. This test distinguishes between trypanosomes that are non-infective for humans and those that are potentially infective. Currently the test requires incubation of parasites with human serum before injection into mice; any surviving parasites are considered human-infective. The factors in serum that kill all non-human-infective parasites are known as trypanosome lytic factors. The paper details the biochemistry of these factors and recommends standardization of the test based on current knowledge. This test can be used to screen animals with trypanosomiasis, in order to evaluate their role during endemic and epidemic human African trypanosomiasis.

Is Citrullination the Missing Link between Periodontal Disease and Rheumatoid Arthritis

Connections between periodontitis (PD) and rheumatoid arthritis (RA) are suggested by an increased prevalence of PD in RA, shared environmental and genetic risk factors, and correlating levels of severity when the two diseases occur together. Here, we compare and contrast the results from numerous studies documenting this association, and highlight the involvement of citrullination in the development of autoimmunity leading to the destructive pathology that characterizes RA. The contribution of citrullination to RA may occur in at least three distinct phases: (i) the initial breakdown of tolerance leading to autoimmunity; (ii) the maturation of the citrulline specificity of the autoantibody response; and (iii) the pro-inflammatory effect of citrullinated proteins themselves in established disease. We conclude that citrullination is more than a ‘missing link’; rather, it is an active process in the evolution of low levels of autoimmunity found in PD into the pathogenic anti-citrullinated protein response specific to RA.

Autodeimination of Porphyromonas gingivalis peptidylarginine deiminase: a novel antigen in rheumatoid arthritis

Backgroundand objectives Porphyromonas gingivalis, a major periodontal pathogen, has been implicated in the pathogenesis of rheumatoid arthritis (RA). It is the only pathogenic prokaryote known to possess a bacterial peptidylarginine deiminase (PPAD), an enzyme that catalyses the post-translational modification of arginine to citrulline. The enzyme is surface expressed and citrullinates c-terminal arginines of human fibrinogen and enolase peptides. Here, the authors show that PPAD is capable of autocitrullination and able to raise an immune response in RA. Materials and methods PPAD was expressed as a GST/His-tagged fusion protein using a bacterial expression system. Site-directed mutagenesis using a PCR based method was used to create a PPAD mutant (mPPAD) with a single residue mutation (cysteine 351 to alanine) and with no enzymatic activity. Citrullination was determined by immunoblotting with an antimodified citrullinated antibody kit (AMC, New York, NY, USA). Mass spectrophotometry was carried out to verify PPAD-GST citrullination. ELISAs using recombinant PPAD and recombinant mPPAD were developed to test sera from patients with periodontitis (PD) (n=44), RA (n=82) and healthy controls (C) (n=80). Reactivity was expressed as arbitrary units per ml (AU/ml) using a standard curve. Results Sera from patients with RA, PD and healthy controls were tested for IgG antibodies to PPAD and showed that RA sera have a significantly elevated antiPPAD IgG response (mean 182.4 AU/ml) compared to PD (mean 96.4AU/ml; p<0.01) and healthy control sera (mean 99.2AU/ml; p<0.05). An ELISA with the enzymatically non-active, C351A mutated PPAD (mPPAD), showed that the antibody response in the RA group was no longer significantly elevated compared to the antibody responses in the PD and healthy control sera. Antimodified citrulline immunoblotting demonstrated that recombinant PPAD was citrullinated whereas the mPPAD was not. Citrullination of PPAD was confirmed by mass spectrophotometry which showed that 7 of the 18 arginines in PPAD were citrullinated. All citrullines detected were internal and no c-terminal citrullination was observed. Conclusions The authors present evidence that PPAD is capable of autocitrullination and its target site is not restricted to c-terminal peptide arginines. The antibody response to autocitrullinated PPAD was significantly elevated in RA patients compared to PD patients and controls. This indicates that PPAD is a potential novel antigen in RA and may form part of the anticitrullinated protein antigen response seen in this disease, thus substantiating a possible link between PD and RA.