Induction of Susceptibility to Disseminated Infection with IgA1 Protease-Producing Encapsulated Pathogens Streptococcus pneumoniae, Haemophilus influenzae Type b, and Neisseria meningitidis.

Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae are the principal causes of bacterial meningitis. It is unexplained why only occasional individuals develop invasive infection, while the vast majority remain healthy and develop immunity when encountering these pathogens. A capsular polysaccharide and an IgA1 protease are common to these pathogens. We tested the hypothesis that patients are primed to susceptibility to invasive infection by other bacteria that express the same capsular polysaccharide but no IgA1 protease. Thereby, the subsequently colonizing pathogen may protect its surface with IgA1 protease-generated Fab fragments of IgA1 devoid of Fc-mediated effector functions. Military recruits who remained healthy when acquiring meningococci showed a significant response of inhibitory antibodies against the IgA1 protease of the colonizing clone concurrent with serum antibodies against its capsular polysaccharide. At hospitalization, 70.8% of meningitis patients carried fecal bacteria cross-reactive with the capsule of the actual pathogen, in contrast to 6% of controls (P < 0.0001). These were Escherichia coli K100, K1, and K92 in patients with infection caused by H. influenzae type b and N. meningitidis groups B and C, respectively. This concurred with a significant IgA1 response to the capsule but not to the IgA1 protease of the pathogen. The demonstrated multitude of relationships between capsular types and distinct IgA1 proteases in pneumococci suggests an alternative route of immunological priming associated with recombining bacteria. The findings support the model and offer an explanation for the rare occurrence of invasive diseases in spite of the comprehensive occurrence of the pathogens. IMPORTANCE Why some individuals develop invasive infection, including meningitis, with Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae type b is unexplained. The vast majority of humans are colonized with the three pathogens but remain healthy and develop immunity. The findings of this study support the hypothesis that patients are primed for disease by time-shifted acquisition of two different bacteria, an immunogenic commensal followed by the pathogen, but both expressing the same capsular polysaccharide. The IgA1 protease common to the three pathogens cleaves the preexisting IgA1 antibodies induced by the commensal. This eliminates Fc-mediated protective mechanisms and releases capsule-binding monomeric Fab fragments that enhance bacterial adherence and block access of other isotypes of antibody molecules. This concept provides new insight into the pathogenesis of bacterial meningitis and potential new strategies for prevention.

Circulating antibodies against leukotoxin A as marker of periodontitis Grades B and C and oral infection with Aggregatibacter actinomycetemcomitans.

BACKGROUND: The facultative bacterium Aggregatibacter actinomycetemcomitans (Aa) is strongly associated with periodontitis and is occasionally found in periodontally healthy subjects. We aimed to determine the prevalence of salivary Aa among patients with either periodontitis Grade B (periodontitis-B) or Grade C (periodontitis-C), periodontally healthy controls (HCs), and to determine if systemic antibodies against Aa or its virulence factor leukotoxin A (LtxA) may serve as biomarkers that reveal the oral presence of the bacterium and discriminate subjects with periodontitis-C, periodontitis-B, or no periodontitis from each other. METHODS: Serum and unstimulated saliva samples were collected from patients with periodontitis-C (n = 27), patients with periodontitis-B (n = 34), and HCs (n = 28). Serum level of immunoglobulin G antibodies to fragmented whole Aa and to LtxA were quantified using a bead-based assay. Aa was identified in saliva using quantitative polymerase chain reaction (qPCR). All analyses were adjusted for age, sex, and current smoking status. RESULTS: Aa was present in saliva from 11% of HCs, in 32% of patients with periodontitis-B (P = 0.04 versus HCs), and in 37% of patients with periodontitis-C (P = 0.02 versus HCs). Serum antibodies to fragments of Aa associated significantly with periodontitis-C (P = 0.03), while serum anti-LtxA antibodies associated with both periodontitis-B and periodontitis-C (P = 0.002 and P = 9×10-4 , respectively). Moreover, a significant association between serum anti-LtxA antibodies and Aa count in saliva was observed (P = 0.001). On the basis of serum anti-LtxA antibody levels, patients with periodontitis could be discriminated from HCs (AUC = 0.74 in ROC curve-analysis, P = 0.0003), and carriers of Aa could be discriminated from non-carriers (AUC = 0.78, P <0.0001). CONCLUSIONS: Aa is highly prevalent in saliva of patients with periodontitis-B or periodontitis-C. Systemic immunoglobulin G antibodies against LtxA distinguish patients with periodontitis, regardless of grade, from HCs, while their quantity reflects the concurrent bacterial burden in the oral cavity.

Associations of Antibodies Targeting Periodontal Pathogens With Subclinical Coronary, Carotid, and Peripheral Arterial Atherosclerosis in Rheumatoid Arthritis.

OBJECTIVE: Both periodontal disease and cardiovascular disease (CVD) are overrepresented in rheumatoid arthritis (RA). This study was undertaken to investigate the contribution of periodontal pathogens to CVD in RA. METHODS: RA patients underwent assessments of coronary artery calcification (CAC), carotid intima-media thickness and plaque, and ankle-brachial index via computed tomography, ultrasound, and Doppler ultrasound, respectively. Sera were assayed for antibodies targeting Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans serotype B (Aa), and Aa-derived leukotoxin A (LtxA). Associations of antibodies against these periodontal pathogens with measures of atherosclerosis were explored using generalized linear models. RESULTS: Among 197 RA patients, anti-Pg was detected in 72 patients (37%), anti-Aa in 41 patients (21%), and anti-LtxA in 84 patients (43%). Adjusting for relevant confounders and reported tooth loss, the mean CAC score was 90% higher in those with anti-Aa and/or anti-LtxA compared with those without either antibody (19 units versus 10 units; P = 0.033). The adjusted odds of CAC ≥100 units were 2.23-fold higher in those with anti-Aa and/or anti-LtxA compared with those without either antibody (P = 0.040). Anti-Aa and/or anti-LtxA seropositivity was significantly associated with all other assessed measures of atherosclerosis except carotid plaque. Anti-Pg was not associated with any measure of atherosclerosis. Higher swollen joint count was associated with CAC exclusively in the group with anti-Aa and/or anti-LtxA. CONCLUSION: Immunoreactivity against Aa and/or its major virulence factor LtxA was associated with atherosclerosis in multiple vascular beds of RA patients and amplified the effect of swollen joints on coronary atherosclerosis, suggesting a role for treatment/prevention of periodontal disease in the prevention of CVD in RA.

Differential Cell Lysis Among Periodontal Strains of JP2 and Non-JP2 Genotype of Aggregatibacter actinomycetemcomitans Serotype B Is Not Reflected in Dissimilar Expression and Production of Leukotoxin.

Leukotoxic potential of Aggregatibacter actinomycetemcomitans strains has been studied by the use of several methods, and results differ depending on the methods used. The aim of the present study was to perform a comprehensive examination of the leukotoxic potential of a collection of A. actinomycetemcomitans strains by use of three quantitative methods, Western blotting, ELISA, and mRNA expression assay and compare these results with previous data obtained by a cell lysis assay. A higher leukotoxic potential among JP2 genotype strains compared to non-JP2 genotype strains of A. actinomycetemcomitans was found by Western blotting, ELISA and mRNA expression assay. Leukotoxicity as determined by cell lysis assay showed a variation among strains examined, not only depending on being part of JP2 genotype vs. non-JP2 genotype group of A. actinomycetemcomitans. The leukotoxicity of A. actinomycetemcomitans strains as determined by cell lysis assay did not correspond to the leukotoxic potential of A. actinomycetemcomitans strains as determined by three quantitative methods. A comparison of the results obtained by ELISA and mRNA expression assay showed a reasonable correlation between these two methods. It seems important to use more than one method to assess the LtxA-related virulence capacity of A. actinomycetemcomitans in order to obtain comprehensive understanding of the leukotoxic potential of A. actinomycetemcomitans strains.

Immunoglobulin G antibodies against Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans in cardiovascular disease and periodontitis.

Objectives: The aim was to elucidate whether levels of circulating antibodies to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis correlate to loss of attachment, as a marker for periodontitis and cardiovascular disease (CVD). Design: Sera were collected from 576 participants of the Danish Health Examination Survey (DANHES). Immunoglobulin G antibodies against lipopolysaccharide (LPS) and protein antigens from the a, b and c serotypes of A. actinomycetemcomitans and P. gingivalis were quantified by titration in ELISA plates coated with a mixture of antigens prepared by disintegration of bacteria. Results: Levels of antibodies against P. gingivalis (OR = 1.48) and A. actinomycetemcomitans (1.31) associated with periodontitis, as determined by univariable logistic regression analysis. These antibody levels also associated with CVD (1.17 and 1.37), respectively, However, after adjusting for other risk factors, including age, smoking, gender, alcohol consumption, overweight, and level of education using multivariable logistic regression analysis, only increasing body mass index (BMI; 1.09), previous smoking (1.99), and increasing age (decades) (2.27) remained associated with CVD. Increased levels of antibodies against P. gingivalis (1.34) remained associated with periodontitis after adjusting for other risk factors. Conclusions: CVD and periodontitis were associated with levels of IgG antibodies to P. gingivalis or A. actinomycetemcomitans in univariable analyses, but only the association of P. gingivalis antibody levels with periodontitis reached statistical significance after adjustment for common confounders. Age, in particular, influenced this relationship.

Aggregatibacter actinomycetemcomitans-induced hypercitrullination links periodontal infection to autoimmunity in rheumatoid arthritis.

A bacterial etiology of rheumatoid arthritis (RA) has been suspected since the beginnings of modern germ theory. Recent studies implicate mucosal surfaces as sites of disease initiation. The common occurrence of periodontal dysbiosis in RA suggests that oral pathogens may trigger the production of disease-specific autoantibodies and arthritis in susceptible individuals. We used mass spectrometry to define the microbial composition and antigenic repertoire of gingival crevicular fluid in patients with periodontal disease and healthy controls. Periodontitis was characterized by the presence of citrullinated autoantigens that are primary immune targets in RA. The citrullinome in periodontitis mirrored patterns of hypercitrullination observed in the rheumatoid joint, implicating this mucosal site in RA pathogenesis. Proteomic signatures of several microbial species were detected in hypercitrullinated periodontitis samples. Among these, Aggregatibacter actinomycetemcomitans (Aa), but not other candidate pathogens, induced hypercitrullination in host neutrophils. We identified the pore-forming toxin leukotoxin A (LtxA) as the molecular mechanism by which Aa triggers dysregulated activation of citrullinating enzymes in neutrophils, mimicking membranolytic pathways that sustain autoantigen citrullination in the RA joint. Moreover, LtxA induced changes in neutrophil morphology mimicking extracellular trap formation, thereby releasing the hypercitrullinated cargo. Exposure to leukotoxic Aa strains was confirmed in patients with RA and was associated with both anticitrullinated protein antibodies and rheumatoid factor. The effect of human lymphocyte antigen-DRB1 shared epitope alleles on autoantibody positivity was limited to RA patients who were exposed to Aa These studies identify the periodontal pathogen Aa as a candidate bacterial trigger of autoimmunity in RA.

Bacterial RTX toxins allow acute ATP release from human erythrocytes directly through the toxin pore.

ATP is as an extracellular signaling molecule able to amplify the cell lysis inflicted by certain bacterial toxins including the two RTX toxins α-hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans. Inhibition of P2X receptors completely blocks the RTX toxin-induced hemolysis over a larger concentration range. It is, however, at present not known how the ATP that provides the amplification is released from the attacked cells. Here we show that both HlyA and LtxA trigger acute release of ATP from human erythrocytes that preceded and were not caused by cell lysis. This early ATP release did not occur via previously described ATP-release pathways in the erythrocyte. Both HlyA and LtxA were capable of triggering ATP release in the presence of the pannexin 1 blockers carbenoxolone and probenecid, and the HlyA-induced ATP release was found to be similar in erythrocytes from pannexin 1 wild type and knock-out mice. Moreover, the voltage-dependent anion channel antagonist TRO19622 had no effect on ATP release by either of the toxins. Finally, we showed that both HlyA and LtxA were able to release ATP from ATP-loaded lipid (1-palmitoyl-2-oleoyl-phosphatidylcholine) vesicles devoid of any erythrocyte channels or transporters. Again we were able to show that this happened in a non-lytic fashion, using calcein-containing vesicles as controls. These data show that both toxins incorporate into lipid vesicles and allow ATP to be released. We suggest that both toxins cause acute ATP release by letting ATP pass the toxin pores in both human erythrocytes and artificial membranes.

Sialic acid residues are essential for cell lysis mediated by leukotoxin from Aggregatibacter actinomycetemcomitans.

Leukotoxin (LtxA) from Aggregatibacter actinomycetemcomitans is known to target and lyse ß2-integrin-expressing cells such as polymorphonuclear leukocytes and macrophages. LtxA is an important virulence factor that facilitates chronic inflammation and is strongly associated with a fast-progressing form of periodontitis caused by the JP2 clone of the bacterium. Here, we show that sialic acid residues are important for LtxA-induced cell lysis, regardless of whether the cell express ß2-integrin or not. Clearly, removal of sialic acid groups significantly reduces a ß2-integrin-specific LtxA-induced lysis. Moreover, sialic acid presented on alternative proteins, such as, for instance, on erythrocytes that do not express ß2-integrin, also makes the cells more sensitive to LtxA. The data also illustrate the importance of the negative charge in order for the sialic acid to associate LtxA with the membrane. Removal of sialic acid is in itself sufficient to significantly reduce the negative charge on the erythrocytes. Moreover, we found that on human erythrocytes there is a positive association between the sensitivity to LtxA and the amount of negative charge caused by sialic acid. Interestingly, these features are not shared by all RTX toxins, since α-hemolysin from Escherichia coli induced cell lysis of both ß2-integrin-expressing and nonexpressing cells and this lysis is independent of the presence of sialic acid residues. In conclusion, LtxA not only is cytotoxic to ß2-integrin-expressing cells but can potentially initiate cell lysis in all cells that present a sufficient density of sialic acid groups on their plasma membrane.

Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: interactions with human ß2 integrins and erythrocytes.

Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. The known receptor for LtxA on leukocytes is integrin α(L)ß(2) (LFA-1 or CD11a/CD18). However, the molecular mechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has proven difficult to prepare for experiments as free of contaminants and with its native structure. Here, we describe a protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturing procedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotron radiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content, and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonas with a known three dimensional structure supports the presence of a so-called beta-ladder domain in the C-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express ß(2) integrin. Cells expressing α(M)ß(2) (CD11b/CD18) or α(X)ß(2) (CD11c/CD18) were killed as efficiently as cells expressing α(L)ß(2). Erythrocytes, which do not express ß(2) integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the ß(2) chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins α(M)ß(2) and α(X)ß(2) as novel receptors for LtxA.

Leukotoxin from Aggregatibacter actinomycetemcomitans causes shrinkage and P2X receptor-dependent lysis of human erythrocytes.

Leukotoxin (LtxA) is a virulence factor secreted by the bacterium Aggregatibacter actinomycetemcomitans, which can cause localized aggressive periodontitis and endocarditis. LtxA belongs to the repeat-in-toxin (RTX) family of exotoxins of which other members inflict lysis by formation of membrane pores. Recently, we documented that the haemolytic process induced by another RTX toxin [α-haemolysin (HlyA) from Escherichia coli] requires P2X receptor activation and consists of sequential cell shrinkage and swelling. In contrast, the cellular and molecular mechanisms of LtxA-mediated haemolysis are not fully understood. Here, we investigate the effect of LtxA on erythrocyte volume and whether P2 receptors also play a part in LtxA-mediated haemolysis. We observed that LtxA initially decreases the cell size, followed by a gradual rise in volume until the cell finally lyses. Moreover, LtxA triggers phosphatidylserine (PS) exposure in the erythrocyte membrane and both the shrinkage and the PS-exposure is preceded by increments in the intracellular Ca(2+) concentration ([Ca(2+)](i)). Interestingly, LtxA-mediated haemolysis is significantly potentiated by ATP release and P2X receptor activation in human erythrocytes. Furthermore, the LtxA-induced [Ca(2+)](i) increase and following volume changes partially depend on P2 receptor activation. Theseobservations imply that intervention against local P2-mediated auto- and paracrine signalling may prevent LtxA-mediated cell damage.

Proteomic and immunoproteomic analysis of Aggregatibacter actinomycetemcomitans JP2 clone strain HK1651.

The proteome of Aggregatibacter actinomycetemcomitans HK1651 (JP2 clone) and immunoreactive antigens were studied by two-dimensional (2D) gel electrophoresis, matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS), and 2D immunoblotting. The highly leukotoxic JP2 clone of A. actinomycetemcomitans is strongly associated with aggressive periodontitis (AgP) in adolescents of North-West African descent and the pathogenicity of this bacterium is of major interest. Hence, we developed a comprehensive 2D proteome reference map of A. actinomycetemcomitans proteins with 167 identified spots representing 114 different proteins of which 15 were outer membrane proteins. To unravel immunoreactive antigens, we applied 2D-gel and subsequent immunoblotting analyses using sera from five individuals with A. actinomycetemcomitans infections and one healthy control. The analysis revealed 32 immunoreactive proteins. Antibodies to two outer membrane proteins, YaeT (85 kDa) and Omp39 (39 kDa), not previously described as immunoreactive, were found only in subjects with current or previous A. actinomycetemcomitans JP2 infection. Further proteome-based studies of A. actinomycetemcomitans combined with analyses of the humoral immune response and targeted against outer membrane proteins may provide important insight into the host relationship of this important pathogen.

[Periodontitis is one of the most commonly occurring inflammatory diseases]. / Marginal parodontitis er en af de almindeligste inflammatoriske sygdomme.

Periodontitis is a common inflammatory disease characterized by breakdown of the structure supporting the tooth. The disease, which is caused by bacteria colonizing the root surface, occurs with a marked difference in individual susceptibility and presents as chronic and aggressive forms of the disease. A number of medical disorders act as predisposing factors, and periodontitis may influence the course of some diseases, including coronary heart disease and diabetes.

Immunoglobulin levels and phagocytes in the cervical mucus plug at term of pregnancy.

BACKGROUND: To characterize the potential for adaptive immune protection in cervical mucus plugs with respect to immunoglobulin isotypes and effector cells (phagocytes). METHODS: Thirty-one cervical mucus plugs were collected from healthy women in labor at term. The cervical mucus plugs were allocated either to analysis of immunoglobulins by enzyme-linked immunosorbent assay (ELISA), gel chromatography and Western blotting (n = 20) or to microscopical, including immunocytochemical, analyses. The levels of immunoglobulin in the plugs were compared to the levels in 10 samples of ovulatory cervical mucus from nonpregnant women. RESULTS: In the cervical mucus plugs, levels of immunoglobulin G (IgG) [median 3270 microg/mL (100-14 500)] and IgA [540 (22-2820)], but not IgM [30.5 (1.0-160)], were significantly elevated compared to cervical mucus from nonpregnant women (p < 0.02 for IgG and IgA). The IgG : IgA ratio in the plugs was also elevated (p < 0.02). The proportion of secretory immunoglobulin A (SIgA) relative to total IgA in the plugs ranged from 16 to 65% (n = 5). IgA and IgG were largely intact. Microscopically, the vagina-proximal part of the cervical mucus plugs contained bacteria and was rich in cells, mainly phagocytes. Conversely, the uterine part contained few cells. CONCLUSION: The high immunoglobulin levels in combination with the presence of phagocytes suggest a potential for adaptive immune defense in the cervical mucus plug, which, together with innate immune factors, may act as an immunological gatekeeper protecting the fetomaternal unit against infection from the vagina.

Bet v 1-specific IgA increases during the pollen season but not after a single allergen challenge in children with birch pollen-induced intermittent allergic rhinitis.

Allergen-specific immunoglobulins of the Immunoglobulin A (IgA) type have been found in the nasal fluid of patients with allergic rhinitis. IgA may play a protective role, but there are also data which show that allergen-specific IgA can induce eosinophil degranulation. The aim of this study was to quantitate Bet v 1-specific IgA in relation to total IgA in the nasal fluid of children with birch pollen-induced intermittent allergic rhinitis and healthy controls, after allergen challenge and during the natural pollen season. Eosinophil cationic protein (ECP), Bet v 1-specific IgA and total IgA were analyzed in nasal fluids from 30 children with birch pollen-induced intermittent allergic rhinitis and 30 healthy controls. Samples were taken before the pollen season, after challenge with birch pollen and during the pollen season, before and after treatment with nasal steroids. During the pollen season, but not after nasal allergen challenge, Bet v 1-specific IgA increased in relation to total IgA in children with allergic rhinitis. No change was found in the healthy controls. The ratio of Bet v 1-specific IgA to total IgA increased from 0.1 x 10(-3) (median) to 0.5 x 10(-3) in the allergic children, p < 0.001. No change was seen after treatment with nasal steroids, although symptoms, ECP and eosinophils were reduced. In conclusion, allergen-specific IgA in relation to total IgA increases in nasal fluids during the pollen season in allergic children but not in healthy controls. These findings are compatible with the hypothesis that allergen-specific IgA plays a role in the allergic inflammation and further studies are needed to clarify the functional role of these allergen-specific antibodies.

Isolation and detection of human IgA using a streptococcal IgA-binding peptide.

Bacterial proteins that bind to the Fc part of IgG have found widespread use in immunology. A similar protein suitable for the isolation and detection of human IgA has not been described. Here, we show that a 50-residue synthetic peptide, designated streptococcal IgA-binding peptide (Sap) and derived from a streptococcal M protein, can be used for single-step affinity purification of human IgA. High affinity binding of IgA required the presence in Sap of a C-terminal cysteine residue, not present in the intact M protein. Passage of human serum through a Sap column caused depletion of >99% of the IgA, and elution of the column allowed quantitative recovery of highly purified IgA, for which the proportions of the IgA1 and IgA2 subclasses were the same as in whole serum. Moreover, immobilized Sap could be used for single-step purification of secretory IgA of both subclasses from human saliva, with a recovery of approximately 45%. The Sap peptide could also be used to specifically detect IgA bound to Ag. Together, these data indicate that Sap is a versatile Fc-binding reagent that may open new possibilities for the characterization of human IgA.

The Clostridium ramosum IgA proteinase represents a novel type of metalloendopeptidase.

Clostridium ramosum is part of the normal flora in the human intestine. Some strains produce an IgA proteinase that specifically cleaves human IgA1 and the IgA2m(1) allotype. This prolylendopeptidase was purified from a broth culture supernatant, and N-terminal sequences of the native protein and tryptic fragments thereof were determined. A fragment of the iga gene encoding the IgA proteinase was isolated using degenerate primers in PCR, and the complete gene was obtained by inverse PCR. The identity of the iga gene was confirmed by heterologous expression in Escherichia coli. The deduced amino acid sequence indicated a signal peptide of 30 residues and a secreted proteinase of 133,828 Da. A typical Gram-positive cell wall anchor motif was identified in the C terminus. The presence of a putative zinc-binding motif His-Glu-Phe-Gly-His together with inhibition studies indicate that the proteinase belongs to the zinc-dependent metalloproteinases. However, the sequence of the C. ramosum IgA proteinase shows no overall similarity to other proteins except for significant identity around the zinc-binding motif with family M6 of metalloendopeptidases, and the unique sequence of the IgA proteinase in this area presumably establishes a new subfamily. The GC percentage of the iga gene is significantly higher than that for the entire genome of C. ramosum, suggesting that the gene was acquired recently in evolution.