Antibiotics inhibit tumor and disease activity in cutaneous T-cell lymphoma.

It has been proposed that CD4 T-cell responses to Staphylococcus aureus (SA) can inadvertently enhance neoplastic progression in models of skin cancer and cutaneous T-cell lymphoma (CTCL). In this prospective study, we explored the effect of transient antibiotic treatment on tumor cells and disease activity in 8 patients with advanced-stage CTCL. All patients experienced significant decrease in clinical symptoms in response to aggressive, transient antibiotic treatment. In some patients, clinical improvements lasted for more than 8 months. In 6 of 8 patients, a malignant T-cell clone could be identified in lesional skin, and a significant decrease in the fraction of malignant T cells was observed following antibiotics but an otherwise unchanged treatment regimen. Immunohistochemistry, global messenger RNA expression, and cell-signaling pathway analysis indicated that transient aggressive antibiotic therapy was associated with decreased expression of interleukin-2 high-affinity receptors (CD25), STAT3 signaling, and cell proliferation in lesional skin. In conclusion, this study provides novel evidence suggesting that aggressive antibiotic treatment inhibits malignant T cells in lesional skin. Thus, we provide a novel rationale for treatment of SA in advanced CTCL.

The oral microbiome - friend or foe?

The microbiome and the human body constitute an integrated superorganism, which is the result of millions of years of coevolution with mutual adaptation and functional integration, and confers significant benefits for both parties. This evolutionary process has resulted in a highly diverse oral microbiome, which covers the full spectrum of acidogenic, aciduric, inflammatory, and anti-inflammatory properties. The relative proportions of members of the microbiome are affected by factors associated with modern life, such as general diet patterns, sugar consumption, tobacco smoking, oral hygiene, use of antibiotics and other antimicrobials, and vaccines. A perturbed balance in the oral microbiome may result in caries, periodontal disease, or candidiasis, and oral bacteria passively transferred to normally sterile parts of the body may cause extra-oral infections. Nevertheless, it should never be our goal to eliminate the oral microbiome, but rather we have to develop ways to re-establish a harmonious coexistence that is lost because of the modern lifestyle. With regard to oral diseases, this goal can normally be achieved by optimal oral hygiene, exposure to fluoride, reduction of sucrose consumption, stimulation of our innate immune defense, smoking cessation, and control of diabetes.

Staphylococcal enterotoxin A (SEA) stimulates STAT3 activation and IL-17 expression in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient-derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region ß chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk-dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis.

Staphylococcal enterotoxins stimulate lymphoma-associated immune dysregulation.

Patients with cutaneous T-cell lymphoma (CTCL) are frequently colonized with Staphylococcus aureus (SA). Eradication of SA is, importantly, associated with significant clinical improvement, suggesting that SA promotes the disease activity, but the underlying mechanisms remain poorly characterized. Here, we show that SA isolates from involved skin express staphylococcal enterotoxins (SEs) that induce crosstalk between malignant and benign T cells leading to Stat3-mediated interleukin-10 (IL-10) production by the malignant T cells. The SEs did not stimulate the malignant T cells directly. Instead, SEs triggered a cascade of events involving cell-cell and asymmetric cytokine interactions between malignant and benign T cells, which stimulated the malignant T cells to express high levels of IL-10. Much evidence supports that malignant activation of the Stat3/IL-10 axis plays a key role in driving the immune dysregulation and severe immunodeficiency that characteristically develops in CTCL patients. The present findings thereby establish a novel link between SEs and immune dysregulation in CTCL, strengthening the rationale for antibiotic treatment of colonized patients with severe or progressive disease.

Enzymatic sialylation of IgA1 O-glycans: implications for studies of IgA nephropathy.

Patients with IgA nephropathy (IgAN) have elevated circulating levels of IgA1 with some O-glycans consisting of galactose (Gal)-deficient N-acetylgalactosamine (GalNAc) with or without N-acetylneuraminic acid (NeuAc). We have analyzed O-glycosylation heterogeneity of naturally asialo-IgA1 (Ale) myeloma protein that mimics Gal-deficient IgA1 (Gd-IgA1) of patients with IgAN, except that IgA1 O-glycans of IgAN patients are frequently sialylated. Specifically, serum IgA1 of healthy controls has more α2,3-sialylated O-glycans (NeuAc attached to Gal) than α2,6-sialylated O-glycans (NeuAc attached to GalNAc). As IgA1-producing cells from IgAN patients have an increased activity of α2,6-sialyltransferase (ST6GalNAc), we hypothesize that such activity may promote premature sialylation of GalNAc and, thus, production of Gd-IgA1, as sialylation of GalNAc prevents subsequent Gal attachment. Distribution of NeuAc in IgA1 O-glycans may play an important role in the pathogenesis of IgAN. To better understand biological functions of NeuAc in IgA1, we established protocols for enzymatic sialylation leading to α2,3- or α2,6-sialylation of IgA1 O-glycans. Sialylation of Gal-deficient asialo-IgA1 (Ale) myeloma protein by an ST6GalNAc enzyme generated sialylated IgA1 that mimics the Gal-deficient IgA1 glycoforms in patients with IgAN, characterized by α2,6-sialylated Gal-deficient GalNAc. In contrast, sialylation of the same myeloma protein by an α2,3-sialyltransferase yielded IgA1 typical for healthy controls, characterized by α2,3-sialylated Gal. The GalNAc-specific lectin from Helix aspersa (HAA) is used to measure levels of Gd-IgA1. We assessed HAA binding to IgA1 sialylated at Gal or GalNAc. As expected, α2,6-sialylation of IgA1 markedly decreased reactivity with HAA. Notably, α2,3-sialylation also decreased reactivity with HAA. Neuraminidase treatment recovered the original HAA reactivity in both instances. These results suggest that binding of a GalNAc-specific lectin is modulated by sialylation of GalNAc as well as Gal in the clustered IgA1 O-glycans. Thus, enzymatic sialylation offers a useful model to test the role of NeuAc in reactivities of the clustered O-glycans with lectins.

Bacterial toxins fuel disease progression in cutaneous T-cell lymphoma.

In patients with cutaneous T-cell lymphoma (CTCL) bacterial infections constitute a major clinical problem caused by compromised skin barrier and a progressive immunodeficiency. Indeed, the majority of patients with advanced disease die from infections with bacteria, e.g., Staphylococcus aureus. Bacterial toxins such as staphylococcal enterotoxins (SE) have long been suspected to be involved in the pathogenesis in CTCL. Here, we review links between bacterial infections and CTCL with focus on earlier studies addressing a direct role of SE on malignant T cells and recent data indicating novel indirect mechanisms involving SE- and cytokine-driven cross-talk between malignant- and non-malignant T cells.

Novel molecular method for identification of Streptococcus pneumoniae applicable to clinical microbiology and 16S rRNA sequence-based microbiome studies.

The close phylogenetic relationship of the important pathogen Streptococcus pneumoniae and several species of commensal streptococci, particularly Streptococcus mitis and Streptococcus pseudopneumoniae, and the recently demonstrated sharing of genes and phenotypic traits previously considered specific for S. pneumoniae hamper the exact identification of S. pneumoniae. Based on sequence analysis of 16S rRNA genes of a collection of 634 streptococcal strains, identified by multilocus sequence analysis, we detected a cytosine at position 203 present in all 440 strains of S. pneumoniae but replaced by an adenosine residue in all strains representing other species of mitis group streptococci. The S. pneumoniae-specific sequence signature could be demonstrated by sequence analysis or indirectly by restriction endonuclease digestion of a PCR amplicon covering the site. The S. pneumoniae-specific signature offers an inexpensive means for validation of the identity of clinical isolates and should be used as an integrated marker in the annotation procedure employed in 16S rRNA-based molecular studies of complex human microbiotas. This may avoid frequent misidentifications such as those we demonstrate to have occurred in previous reports and in reference sequence databases.

Multilocus sequence typing and phylogenetic analysis of Propionibacterium acnes.

Propionibacterium acnes is a commensal of human skin but is also implicated in the pathogenesis of acne vulgaris, in biofilm-associated infections of medical devices and endophthalmitis, and in infections of bone and dental root canals. Recent studies associate P. acnes with prostate cancer. As the species includes evolutionary lineages with distinct association with health and disease, there is a need for a high-resolution typing scheme. Recently, two multilocus sequence typing (MLST) schemes were reported, one based on nine and one based on seven housekeeping genes. In the present study, the two schemes were compared with reference to a phylogenetic tree based on 78 P. acnes genomes and their gene contents. Further support for a basically clonal population structure of P. acnes and a scenario of the global spread of epidemic clones of P. acnes was obtained. Compared to the Belfast scheme, the Aarhus MLST scheme (http://pacnes.mlst.net/), which is based on nine genes, offers significantly enhanced resolution and phylogenetic inferences more concordant with analyses based on a comprehensive sampling of the entire genomes, their gene contents, and their putative pathogenic potential.

Naturally occurring structural isomers in serum IgA1 o-glycosylation.

IgA is the most abundantly produced antibody and plays an important role in the mucosal immune system. Human IgA is represented by two isotypes, IgA1 and IgA2. The major structural difference between these two subclasses is the presence of nine potential sites of O-glycosylation in the hinge region between the first and second constant region domains of the heavy chain. Thr(225), Thr(228), Ser(230), Ser(232) and Thr(236) have been identified as the predominant sites of O-glycan attachment. The range and distribution of O-glycan chains at each site within the context of adjacent sites in this clustered region create a complex heterogeneity of surface epitopes that is incompletely defined. We previously described the analysis of IgA1 O-glycan heterogeneity by use of high resolution LC-MS and electron capture dissociation tandem MS to unambiguously localize all amino acid attachment sites in IgA1 (Ale) myeloma protein. Here, we report the identification and elucidation of IgA1 O-glycopeptide structural isomers that occur based on amino acid position of the attached glycans (positional isomers) and the structure of the O-glycan chains at individual sites (glycan isomers). These isomers are present in a model IgA1 (Mce1) myeloma protein and occur naturally in normal human serum IgA1. Variable O-glycan chains attached to Ser(230), Thr(233) or Thr(236) produce the predominant positional isomers, including O-glycans composed of a single GalNAc residue. These findings represent the first definitive identification of structural isomeric IgA1 O-glycoforms, define the single-site heterogeneity for all O-glycan sites in a single sample, and have implications for defining epitopes based on clustered O-glycan variability.

Clustered O-glycans of IgA1: defining macro- and microheterogeneity by use of electron capture/transfer dissociation.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) O-glycans, plays a pivotal role in the pathogenesis of the disease. It is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We have described the utility of activated ion-electron capture dissociation (AI-ECD) mass spectrometric analysis of IgA1 O-glycosylation. However, locating and characterizing the entire range of O-glycan attachment sites are analytically challenging due to the clustered serine and threonine residues in the HR of IgA1 heavy chain. To address this problem, we analyzed all glycoforms of the HR glycopeptides of a Gal-deficient IgA1 myeloma protein, mimicking the aberrant IgA1 in patients with IgAN, by use of a combination of IgA-specific proteases + trypsin and AI-ECD Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS). The IgA-specific proteases provided a variety of IgA1 HR fragments that allowed unambiguous localization of all O-glycosylation sites in the six most abundant glycoforms, including the sites deficient in Gal. Additionally, this protocol was adapted for on-line liquid chromatography (LC)-AI-ECD MS/MS and LC-electron transfer dissociation MS/MS analysis. Our results thus represent a new clinically relevant approach that requires ECD/electron transfer dissociation-type fragmentation to define the molecular events leading to pathogenesis of a chronic kidney disease. Furthermore, this work offers generally applicable principles for the analysis of clustered sites of O-glycosylation.

Important role for Toll-like receptor 9 in host defense against meningococcal sepsis.

Neisseria meningitidis is a leading cause of meningitis and sepsis. The pathogenesis of meningococcal disease is determined by both bacterial virulence factors and the host inflammatory response. Toll-like receptors (TLRs) are prominent activators of the inflammatory response, and TLR2, -4, and -9 have been reported to be involved in the host response to N. meningitidis. While TLR4 has been suggested to play an important role in early containment of infection, the roles of TLR2 and TLR9 in meningococcal disease are not well described. Using a model for meningococcal sepsis, we report that TLR9(-/-) mice displayed reduced survival and elevated levels of bacteremia compared to wild-type mice. In contrast, TLR2(-/-) mice controlled the infection in a manner comparable to that of wild-type mice. TLR9 deficiency was also associated with reduced bactericidal activity in vitro, which was accompanied by reduced production of nitric oxide by TLR9-deficient macrophages. Interestingly, TLR9(-/-) mice recruited more macrophages to the bloodstream than wild-type mice and produced elevated levels of cytokines at late time points during infection. At the cellular level, activation of signal transduction and induction of cytokine gene expression were independent of TLR2 or TLR9 in macrophages and conventional dendritic cells. In contrast, plasmacytoid dendritic cells relied entirely on TLR9 to induce these activities. Thus, our data demonstrate an important role for TLR9 in host defense against N. meningitidis.

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.

Analysis of O-glycan heterogeneity in IgA1 myeloma proteins by Fourier transform ion cyclotron resonance mass spectrometry: implications for IgA nephropathy.

IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis. In IgAN, IgA1 molecules with incompletely galactosylated O-linked glycans in the hinge region (HR) are present in mesangial immunodeposits and in circulating immune complexes. It is not known whether the galactose deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. We have previously demonstrated the first direct localization of multiple O-glycosylation sites on a single IgA1 myeloma protein by use of activated ion-electron capture dissociation (AI-ECD) Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry. Here, we report the analysis of IgA1 O-glycan heterogeneity by use of FT-ICR MS and liquid chromatography FT-ICR MS to obtain unbiased accurate mass profiles of IgA1 HR glycopeptides from three different IgA1 myeloma proteins. Additionally, we report the first AI-ECD fragmentation on an individual IgA1 O-glycopeptide from an IgA1 HR preparation that is reproducible for each IgA1 myeloma protein. These results suggest that future analysis of IgA1 HR from IgAN patients and normal healthy controls should be feasible.

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.

Reactivities of N-acetylgalactosamine-specific lectins with human IgA1 proteins.

Lectins are proteins with specificity of binding to certain monosaccharides or oligosaccharides. They can detect abnormal glycosylation patterns on immunoglobulins in patients with various chronic inflammatory diseases, including rheumatoid arthritis and IgA nephropathy (IgAN). However, lectins exhibit binding heterogeneity, depending on their source and methods of isolation. To characterize potential differences in recognition of terminal N-acetylgalactosamine (GalNAc) on IgA1, we evaluated the binding characteristics of several commercial preparations of GalNAc-specific lectins using a panel of IgA1 and, as controls, IgA2 and IgG myeloma proteins. These lectins originated from snails Helix aspersa (HAA) and Helix pomatia (HPA), and the plant Vicia villosa (VV). Only HAA and HPA bound exclusively to IgA1, with its O-linked glycans composed of GalNAc, galactose, and sialic acid. In contrast, VV reacted with sugars of both IgA subclasses and IgG, indicating that it also recognized N-linked glycans without GalNAc. Furthermore, HAA and HPA from several manufacturers differed in their ability to bind various IgA1 myeloma proteins and other GalNAc-containing glycoproteins in ELISA and Western blot. For serum samples from IgAN patients, HAA was the optimal lectin to study IgA1 glycosylation in ELISA and Western blot assays, including identification of the sites of attachment of the aberrant glycans. The galactose-deficient glycans were site-specific, localized mostly at Thr228 and/or Ser230. Because of the heterogeneity of GalNAc-specific lectins, they should be carefully characterized with appropriate substrates before undertaking any study.

Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates.

The aim of this study was to reinvestigate the relationships and the generic affiliations of the species Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis. The nicotinamide phosphoribosyltransferase gene (nadV) conferring V factor-independent growth was identified in Haemophilus aphrophilus. The gene encodes a polypeptide of 462 amino acids that shows 74.5 % amino acid sequence identity to the corresponding enzyme from Actinobacillus actinomycetemcomitans. Ten isolates of Haemophilus paraphrophilus all carried a nadV pseudogene. DNA from Haemophilus aphrophilus was able to transform Haemophilus paraphrophilus into the NAD-independent phenotype. The transformants carried a full-length nadV inserted in the former locus of the pseudogene. The DNA-DNA relatedness between the type strains of Haemophilus aphrophilus and Haemophilus paraphrophilus was 77 %. We conclude that the division into two species Haemophilus aphrophilus and Haemophilus paraphrophilus is not justified and that Haemophilus paraphrophilus should be considered a later heterotypic synonym of Haemophilus aphrophilus. Forty strains of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis were investigated by multilocus sequence analysis. The 40 strains form a monophyletic group clearly separate from other evolutionary lineages of the family Pasteurellaceae. We propose the transfer of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis to a new genus Aggregatibacter gen. nov. as Aggregatibacter actinomycetemcomitans comb. nov. (the type species; type strain ATCC 33384(T)=CCUG 13227(T)=CIP 52.106(T)=DSM 8324(T)=NCTC 9710(T)), Aggregatibacter aphrophilus comb. nov. (type strain ATCC 33389(T)=CCUG 3715(T)=CIP 70.73(T)=NCTC 5906(T)) and Aggregatibacter segnis comb. nov. (type strain HK316(T)=ATCC 33393(T)=CCUG 10787(T)=CCUG 12838(T)=CIP 103292(T)=NCTC 10977(T)). The species of the genus Aggregatibacter are independent of X factor and variably dependent on V factor for growth in vitro.

Host-derived pentapeptide affecting adhesion, proliferation, and local pH in biofilm communities composed of Streptococcus and Actinomyces species.

Salivary proline-rich proteins (PRPs) attach commensal Actinomyces and Streptococcus species to teeth. Here, gel filtration, mass spectrometry and Edman degradation were applied to show the release of a pentapeptide, RGRPQ, from PRP-1 upon proteolysis by Streptococcus gordonii. Moreover, synthetic RGRPQ and derivatives were used to investigate associated innate properties and responsible motifs. The RGRPQ peptide increased 2.5-fold the growth rate of S. gordonii via a Q-dependent sequence motif and selectively stimulated oral colonization of this organism in a rat model in vivo. In contrast, the growth of Streptococcus mutans, implicated in caries, was not affected. While the entire RGRPQ sequence was required to block sucrose-induced pH-decrease by S. gordonii and S. mutans, the N-terminal Arg residue mediated the pH increase (i.e., ammonia production) by S. gordonii alone (which exhibits Arg catabolism to ammonia). Strains of commensal viridans streptococci exhibited PRP degradation and Arg catabolism, whereas cariogenic species did not. The RGRPQ peptide mediated via a differential Q-dependent sequence motif, adhesion inhibition, and desorption of PRP-1-binding strains of A. naeslundii genospecies 2 (5 of 10 strains) but not of S. gordonii (n=5). The inhibitable A. naeslundii strains alone displayed the same binding profile as S. gordonii to hybrid peptides terminating in RGRPQ or GQSPQ, derived from the middle or C-terminal segments of PRP-1. The present findings indicate the presence of a host-bacterium interaction in which a host peptide released by bacterial proteolysis affects key properties in biofilm formation.

Differences in iron acquisition from human haemoglobin among strains of Actinobacillus actinomycetemcomitans.

To get a better insight into the physiology of the high-toxic JP2 clone of Actinobacillus actinomycetemcomitans serotype b, which is strongly associated with juvenile periodontitis in adolescents of African descent, the modes of iron acquisition in this clone were examined and compared to those of other strains of the species. None of the strains examined could utilize human transferrin as a source of iron. This was in accordance with the presence of a non-functional tbpA gene, which normally encodes the A subunit of the transferrin-binding-protein complex. Southern blot analysis indicated that functional duplications of tbpA were not present in the genome. Thus, A. actinomycetemcomitans seems to be in a process of evolution, in which iron acquisition from host transferrin is not essential as in many other members of the pasteurellaceae. All strains could utilize haem as a source of iron. All 11 A. actinomycetemcomitans strains examined harboured a single genomic sequence with homology to the hgpA gene encoding haemoglobin-binding protein A in Haemophilus influenzae. However, in all three strains belonging to the JP2 clone and in one serotype e strain hgpA was a pseudogene. Seven other strains possessed a functional hgpA gene which, according to insertion mutagenesis experiments, was responsible for the ability of these strains to utilize haemoglobin as a source of iron. Thus, the presence of an hgpA pseudogene and the inability to use human haemoglobin as an iron source discriminate the high-toxic JP2 clone from low-toxic serotype b strains and most other strains of A. actinomycetemcomitans.

Evolution of the paralogous hap and iga genes in Haemophilus influenzae: evidence for a conserved hap pseudogene associated with microcolony formation in the recently diverged Haemophilus aegyptius and H. influenzae biogroup aegyptius.

Certain non-capsulate strains belonging to the Haemophilus influenzae/Haemophilus aegyptius complex show unusually high pathogenicity, but the evolutionary origin of these virulent phenotypes, termed H. influenzae biogroup aegyptius, is as yet unknown. The aim of the present study was to elucidate the mechanisms of evolution of two paralogous genes, hap and iga, which encode the adhesion and penetration Hap protein and the IgA1 protease respectively. Partial sequencing of hap and iga genes in a comprehensive collection of strains belonging to the H. influenzae/H. aegyptius complex revealed considerable genetic polymorphism and pronounced mosaic-like patterns in both genes, but no evidence of intrastrain recombination between the two genes. A conserved hap pseudogene was present in all strains of H. aegyptius and H. influenzae biogroup aegyptius, each of which constituted distinct subpopulations as revealed by phylogenetic analysis. There was no evidence for a second, functional copy of the hap gene in these strains. The perturbed expression of the Hap serine protease appears to be associated with the formation of elongated bacterial cells growing in chains and a distinct colonization pattern on conjunctival cells, previously termed microcolony formation. The fact that individual hap pseudogenes differed from the ancestral sequence by zero to two positions within a 1.5 kb stretch suggests that the silencing event happened approximately 2000-11,000 years ago. Divergence of H. aegyptius and H. influenzae biogroup aegyptius occurred subsequent to this genetic event. The loss of Hap protein expression may be one of the genetic events that facilitated exploitation of the conjunctivae as a new niche.