Knud Poulsen

Biological significance of IgA1 proteases in bacterial colonization and pathogenesis: critical evaluation of experimental evidence*

IgA1 protease activity, which allows bacteria to cleave human IgA1 in the hinge region, represents a striking example of convergent evolution of a specific property in bacteria. Although it has been known since 1979 that IgA1 protease is produced by the three leading causes of bacterial meningitis in addition to important urogenital pathogens and some members of the oropharyngeal flora, the exact role of this enzyme in bacterial pathogenesis is still incompletely understood owing to lack of a satisfactory animal model. Cleavage of IgA1 by these post‐proline endopeptidases efficiently separates the monomeric antigen‐binding fragments from the secondary effector functions of the IgA1 antibody molecule. Several in vivo and in vitro observations indicate that the enzymes are important for the ability of bacteria to colonize mucosal membranes in the presence of S‐IgA antibodies. Furthermore, the extensive cleavage of IgA sometimes observed in vivo, suggests that IgA1 protease activity results in a local functional IgA deficiency that may facilitate colonization of other microorganisms and the penetration of potential allergens. It has been hypothesized that IgA1 protease activity of Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae, under special immunological circumstances, allows these bacteria to take advantage of specific IgA1 antibodies in a strategy to evade other immune factors of the human body. The decisive factor is the balance between IgA antibodies against surface antigens of the respective bacteria and their IgA1 protease. Recent studies have shown that serine‐type IgA1 proteases of H. influenzae, meningococci, and gonococci belong to a family of proteins used by a diverse group of Gramnegative bacteria for colonization and invasion.

Risk of aggressive periodontitis in adolescent carriers of the JP2 clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans in Morocco: a prospective longitudinal cohort study.

BACKGROUND Periodontitis is a loss of supporting connective tissue and alveolar bone around teeth, and if it occurs in an aggressive form it can lead to tooth loss before the age of 20 years. Although the cause of periodontitis in general remains elusive, a particular clone (JP2) of the gram-negative rod Aggregatibacter (Actinobacillus) actinomycetemcomitans is considered a possible aetiological agent of the aggressive form in adolescents living in or originating from north and west Africa, where the disease is highly prevalent. We did a population-based longitudinal study of adolescents to assess the role of the JP2 clone in the initiation of aggressive periodontitis. METHODS A total of 700 adolescents from public schools in Rabat, Morocco, were enrolled in the study. We used PCR to detect A actinomycetemcomitans in plaque samples (taken from molar and incisor sites) and to differentiate between the JP2 clone and other non-JP2 genotypes of the bacterium. 18 individuals were found to already have periodontitis and were excluded. The 682 periodontally healthy adolescents (mean age 12.5 years; SD 1.0) were classified according to their A actinomycetemcomitans carrier status at baseline. After 2 years, 428 (62.8%) individuals returned for re-examination, which included recording of periodontal attachment loss measured from the cemento-enamel junction to the bottom of the periodontal pockets of all teeth present. FINDINGS Individuals who carried the JP2 clone of A actinomycetemcomitans alone (relative risk 18.0; 95% CI 7.8-41.2, p<0.0001) or together with non-JP2 clones of A actinomycetemcomitans (12.4; 5.2-29.9, p<0.0001) had a significantly increased risk of periodontal attachment loss. A much less pronounced disease risk was found in those carrying non-JP2 clones only (3.0; 1.3-7.1, p=0.012). INTERPRETATION The JP2 clone of A actinomycetemcomitans is likely to be an important aetiological agent in initiation of periodontal attachment loss in children and adolescents. Co-occurrence of non-JP2 clones of A actinomycetemcomitans reduces the risk of development of periodontitis, suggesting competition for the ecological niche between the JP2 and non-JP2 clones of this species.

Distinct Pathways of Mannan-Binding Lectin (MBL)- and C1-Complex Autoactivation Revealed by Reconstitution of MBL with Recombinant MBL-Associated Serine Protease-2

Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.

Evolution of Streptococcus pneumoniae and its close commensal relatives.

Streptococcus pneumoniae is a member of the Mitis group of streptococci which, according to 16S rRNA-sequence based phylogenetic reconstruction, includes 12 species. While other species of this group are considered prototypes of commensal bacteria, S. pneumoniae is among the most frequent microbial killers worldwide. Population genetic analysis of 118 strains, supported by demonstration of a distinct cell wall carbohydrate structure and competence pheromone sequence signature, shows that S. pneumoniae is one of several hundred evolutionary lineages forming a cluster separate from Streptococcus oralis and Streptococcus infantis. The remaining lineages of this distinct cluster are commensals previously collectively referred to as Streptococcus mitis and each represent separate species by traditional taxonomic standard. Virulence genes including the operon for capsule polysaccharide synthesis and genes encoding IgA1 protease, pneumolysin, and autolysin were randomly distributed among S. mitis lineages. Estimates of the evolutionary age of the lineages, the identical location of remnants of virulence genes in the genomes of commensal strains, the pattern of genome reductions, and the proportion of unique genes and their origin support the model that the entire cluster of S. pneumoniae, S. pseudopneumoniae, and S. mitis lineages evolved from pneumococcus-like bacteria presumably pathogenic to the common immediate ancestor of hominoids. During their adaptation to a commensal life style, most of the lineages gradually lost the majority of genes determining virulence and became genetically distinct due to sexual isolation in their respective hosts.

Epidemic Population Structure of Pseudomonas aeruginosa: Evidence for a Clone That Is Pathogenic to the Eye and That Has a Distinct Combination of Virulence Factors

ABSTRACT The genetic structure of a population of Pseudomonas aeruginosa, isolated from patients with keratitis, endophthalmitis, and contact lens-associated red eye, contact lens storage cases, urine, ear, blood, lungs, wounds, feces, and the environment was determined by multilocus enzyme electrophoresis. The presence and characteristics of virulence factors were determined by restriction fragment length polymorphism analysis with DNA probes for lasA, lasB,aprA, exoS, exoT,exoU, and ctx and by zymography of staphylolysin, elastase, and alkaline protease. These analyses revealed an epidemic population structure of P. aeruginosa, characterized by frequent recombination in which a particular successful clone may increase, predominate for a time, and then disasappear as a result of recombination. Epidemic clones were found among isolates from patients with keratitis. They were characterized by high activity of a hitherto-unrecognized size variant of elastase, high alkaline protease activity, and possession of the exoUgene encoding the cytotoxic exoenzyme U. These virulence determinants are not exclusive traits in strains causing keratitis, as strains with other properties may cause keratitis in the presence of predisposing conditions. There were no uniform patterns of characteristics of isolates from other types of infection; however, all strains from urinary tract infections possessed the exoS gene, all strains from environment and feces and the major part of keratitis and wound isolates exhibited high elastase and alkaline protease activity, and all strains from feces showed high staphylolysin activity, indicating that these virulence factors may be important in the pathogenesis of these infectious diseases.

Early-onset Periodontitis in Morocco is Associated with the Highly Leukotoxic Clone of Actinobacillus actinomycetemcomitans

A particular clone (JP2) of Actinobacillus actinomycetemcomitans with increased leukotoxin production has been isolated from individuals with early-onset periodontitis (EOP). The aim of this study was to determine the frequency of carriers of this clone and its association with EOP in Moroccan schoolchildren. Of 217 plaque samples, 131 (60.4%) were culture-positive for A. actinomycetemcomitans. A total of 19 of these isolates had a 530-bp deletion in the leukotoxin promoter region characteristic of the JP2 clone. A strong association between the presence of A. actinomycetemcomitans with the 530-bp deletion and EOP was found (adjusted OR = 29.4; 95% CI = 8.3 - 104.4; p < 0.0005), while no association could be demonstrated between the presence of A. actinomycetemcomitans without the deletion and EOP (adjusted OR = 1.3; 95% CI = 0.5 - 2.9; p = 0.750). The study demonstrates that the endemic presence, in a human population, of the highly leukotoxic JP2 clone may result in an unusually high prevalence of EOP.

Population structure of Actinobacillus actinomycetemcomitans: a framework for studies of disease-associated properties.

The Actinobacillus actinomycetemcomitans population consists of a large number of clones among which the ubiquitous leukotoxin gene operon appears very homogeneous. Population genetic analyses performed by multilocus enzyme electrophoresis together with DNA fingerprinting and analyses of genomic DNA restriction fragment length polymorphisms (RFLP) on 97 strains isolated over a period of 45 years revealed that each of the serotypes a, b, c, d and e comprise genetically isolated subpopulations and that successful horizontal transfer of genomic DNA between strains of different serotypes appears to be extremely rare in vivo. In contrast, recombination between strains of the same serotype in general appears to take place in nature. The results provide evidence that non-serotypeable strains are serotype antigen-deficient variants originating from strains of the known serotypes. Serotype b and c strains may contain transmittable DNA sequences not found in strains of the other serotypes.

Emergence and Global Dissemination of Host-Specific Streptococcus agalactiae Clones

ABSTRACT To examine the global diversity of Streptococcus agalactiae (group B streptococci [GBS]) and to elucidate the evolutionary processes that determine its population genetics structure and the reported changes in host tropism and infection epidemiology, we examined a collection of 238 bovine and human isolates from nine countries on five continents. Phylogenetic analysis based on the sequences of 15 housekeeping genes combined with patterns of virulence-associated traits identified a genetically heterogeneous core population from which virulent lineages occasionally emerge as a result of recombination affecting major segments of the genome. Such lineages, like clonal complex 17 (CC17) and two distinct clusters of CC23, are exclusively adapted to either humans or cattle and successfully spread globally. The recent emergence and expansion of the human-associated and highly virulent sequence type 17 (ST17) could conceivably account, in part, for the increased prevalence of neonatal GBS infections after 1960. The composite structure of the S. agalactiae genome invalidates phylogenetic inferences exclusively based on multilocus sequence typing (MLST) data and thereby the previously reported conclusion that the human-associated CC17 emerged from the bovine-associated CC67. IMPORTANCE Group B streptococci (GBS) (Streptococcus agalactiae) have long been recognized as important causes of mastitis in cattle. After 1960, GBS also became the most prevalent cause of invasive and often fatal infections in newborns. At the same time, GBS are carried by a substantial proportion of healthy individuals. The aims of this study were to elucidate the genetic mechanisms that lead to diversification of the GBS population and to examine the relationship between virulence and host preference of evolutionary lineages of GBS. Genetic analysis of GBS isolates from worldwide sources demonstrated epidemic clones adapted specifically to either the human or bovine host. Such clones seem to emerge from a genetically heterogeneous core population as a result of recombination affecting major segments of the genome. Emergence and global spread of certain clones explain, in part, the change in epidemiology of GBS disease and may have implications for prevention. Group B streptococci (GBS) (Streptococcus agalactiae) have long been recognized as important causes of mastitis in cattle. After 1960, GBS also became the most prevalent cause of invasive and often fatal infections in newborns. At the same time, GBS are carried by a substantial proportion of healthy individuals. The aims of this study were to elucidate the genetic mechanisms that lead to diversification of the GBS population and to examine the relationship between virulence and host preference of evolutionary lineages of GBS. Genetic analysis of GBS isolates from worldwide sources demonstrated epidemic clones adapted specifically to either the human or bovine host. Such clones seem to emerge from a genetically heterogeneous core population as a result of recombination affecting major segments of the genome. Emergence and global spread of certain clones explain, in part, the change in epidemiology of GBS disease and may have implications for prevention.

IgA nephropathy and Henoch-Schoenlein purpura nephritis: aberrant glycosylation of IgA1, formation of IgA1-containing immune complexes, and activation of mesangial cells.

IgA1 in the circulation and glomerular deposits of patients with IgA nephropathy (IgAN) is aberrantly glycosylated; the hinge-region O-linked glycans are galactose-deficient. The circulating IgA1 of patients with Henoch-Schoenlein purpura nephritis (HSPN) has a similar defect. This aberrancy exposes N-acetylgalactosamine-containing neoepitopes recognized by naturally occurring IgG or IgA1 antibodies resulting in formation of immune complexes. IgA1 contains up to six O-glycosylation sites per heavy chain; it is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We sought to define the aberrant glycosylation of a galactose-deficient IgA1 myeloma protein and analyze the formation of the immune complexes and their biological activities. Supplementation of serum or cord-blood serum with this IgA1 protein resulted in formation of new IgA1 complexes. These complexes stimulated proliferation of cultured human mesangial cells, as did the naturally-occurring IgA1-containing complexes from sera of patients with IgAN and HSPN. Uncomplexed IgA1 did not affect cellular proliferation. Using specific proteases, lectin Western blots, and mass spectrometry, we determined the O-glycosylation sites in the hinge region of the IgA1 myeloma protein and IgA1 proteins from sera of IgAN patients. The IgA1 myeloma protein had galactose-deficient sites at residues 228 and/or 230 and 232. These sites reacted with IgG specific to galactose-deficient IgA1. IgA1 from the IgAN patients had galactose-deficient O-glycans at the same residues. In summary, we identified the neoepitopes on IgA1 responsible for formation of the pathogenic immune complexes. These studies may lead to development of noninvasive diagnostic assays and future disease-specific therapy.

Resident aerobic microbiota of the adult human nasal cavity.

Recent evidence strongly suggests that the microbiota of the nasal cavity plays a crucial role in determining the reaction patterns of the mucosal and systemic immune system. However, little is known about the normal microbiota of the nasal cavity. The purpose of this study was to determine the microbiota in different parts of the nasal cavity and to develop and evaluate methods for this purpose. Samples were collected from 10 healthy adults by nasal washes and by swabbing of the mucosa through a sterile introduction device. Both methods gave results that were quantitatively and qualitatively reproducible, and revealed significant differences in the density of the nasal microbiota between individuals. The study revealed absence of Gram‐negative bacteria that are regular members of the commensal microbiota of the pharynx. Likewise, viridans type streptococci were sparsely represented. The nasal microbiota was dominated by species of the genera Corynebacterium, Aureobacterium, Rho‐dococcus, and Staphylococcus, including S. epidermis, S. capitis, S. hominis, S. haemolyticus, S. lugdu‐nensis and S. warneri. These studies show that the microbiota of the nasal cavity of adults is strikingly different from that of the pharynx, and that the nasal cavity is a primary habitat for several species of diphtheroids recognized as opportunistic pathogens. Under special circumstances, single species, including IgAl protease‐producing bacteria, may become predominant in a restricted area of the nasal mucosa.