Role of the Soluble Complement Regulator Factor H in Microbial Survival and Host Infection Susceptibility

The key player in human innate immune response is the complement system that attacks microbes and foreign particles invading human body. Complement cascade can be activated through three pathways, the classical, alternative, and lectin pathways. The classical and lectin pathways are activated by recognition of nonself structures while the alternative pathway is constantly in a slightly active stage and is triggered on surfaces that are missing self signals. Surface-deposited complement components act as opsonins for phagocytes while chemotactic and anaphylatoxic components released upon complement activation induce phagocyte recruitment, phagocytosis and the inflammatory response. The components of the late stage of the activation cascade form lytic transmembrane complexes on the surface of the target. Complement activation is regulated by host surface bound as well as fluid phase molecules that act on several steps during the cascade. Factor H (CFH) is an important fluid phase regulator. It is an elongated protein consisting of 20 domains and a common polymorphism Y402H in CFH is located in the domain seven. The same domain interacts with the surface exposed M protein of the pathogenic bacterium, Streptococcus pyogenes, and thereby inhibits the function of the complement system on the microbial surface. The aims of this thesis were to analyze the effects of CFH binding on microbial complement evasion in vivo and in vitro and to analyze how genetic variation in CFH affects S. pyogenes survival and host infection susceptibility. It was shown that Loa loa microfilariae isolated from patient blood carried host CFH and C4BP on themselves. The complement components deposited on microfilariae surface in vivo and in vitro were inactive indicating that the microfilariae evade complement attack using surface bound CFH and C4BP.Most of the S. pyogenes strains studied were found to bind CFH and a recombinant CFH fragment consisting of domains 5-7 (CFH5-7) in vitro. Binding of CFH and CFH5-7 to 38 studied S. pyogenes strains was variable and the binding affinity showed significant inverse relationship with formation of complement activation markers in serum. When a CFH5-7 fragment lacking the cofactor active domains was incubated with the CFH binding strains in an in vitro survival model the multiplication of the bacteria was significantly decreased showing that CFH binding via the domain seven is crucial for survival of S. pyogenes. CFH was isolated from human plasma obtained from donors that were genotyped homozygous for the Y402H polymorphism. The two allotypes bound differentially to the surface of the studied S. pyogenes strains and the diminished binding of the allotype CFH(402H) resulted in a significant decrease in multiplication of the bacteria in an in vitro survival assay. On the basis of these results it seems that the residue 402Y is crucial in S. pyogenes-CFH interaction and that the Y402H polymorphism affects opsonization and phagocytosis of the bacterium. The Y402H (C1277T) genotype was analyzed from patients with history of erysipelas or recurrent tonsillitis (n=487) and from 455 control subjects. Comparison of the allele and genotype frequencies between the patients and control subjects suggests that the polymorphism is associated with susceptibility to streptococcal infections. In conclusion, the results show that complement evasion by binding of complement regulators occurs both in vitro and in vivo and that within the S. pyogenes species CFH binding via the domain seven is a common way to evade complement attack and to enhance microbial survival in the host. It also seems that the CFH Y402H polymorphism affects S. pyogenes survival in vitro and this finding probably also has clinical relevance since the studied polymorphism suggests an association with host susceptibility to streptococcal infections erysipelas and recurrent tonsillitis.Komplementtijärjestelmä on keskeinen luontaiseen immuniteettiin kuuluva tekijä, joka toimii ensimmäisenä puolustuksena isäntään tunkeutuvia mikrobeja vastaan. Se voi aktivoitua sekä tunnistamalla vieraita rakenteita klassisen- tai lektiinitien kautta että ilman mitään spesifiä tunnistuskohdetta oikotieaktivaation kautta. Komplementtiaktivaation aikana pilkkoutuneista proteiinin paloista osa vapautuu plasmaan toimien houkuttimina ja aktivoijina luonnollisen immuniteetin puolustussoluille. Tietyillä kohteen pinnalle kiinnittyneillä proteiinien osilla on puolestaan kyky opsonoida eli kuorruttaa kohde, jotta fagosyytit voisivat tunnistaa sen. Aktivaatiokaskadin edetessä loppuun kohteen pinnalle voi muodostua solukalvon läpäiseviä komplekseja. Komplementin oikotie aktivoituu herkästi minkä tahansa kohteen pinnalla ja voi olla tuhoisa elimistön omillekin soluille ellei sitä säädellä tehokkaasti. Elimistömme omia rakenteita suojataan komplementin säätelijäproteiineilla, joita löytyy sekä plasmasta liukoisena että omien solujen solukalvoihin kiinnittyneinä. Faktori H(CFH) on plasmassa liukoisena esiintyvä pitkänomainen komplementin oikotieaktivaatiota säätelevä proteiini, johon kuuluu 20 samankaltaista osaa eli domeenia. Proteiinin seitsemännessä domeenissa sijaitse CFH:n polymorfia Y402H. M proteiini on A-streptokokkibakteerin (Streptococcus pyogenes)tärkeä virulenssitekijä, joka tarttuu CFH:n seitsemänteen domeeniin ja näin väistää komplementin tuhovaikutuksen. Tämän väitöskirjatyön tavoitteina oli selvittää kuinka CFH:n sitoutuminen mikrobin pinnalle edistää mikrobin komplementtijärjestelmän väistöä in vivo ja in vitro, ja vaikuttaako CFH:n geneettinen vaihtelu ihmispopulaatiossa yksilön infektioalttiuteen. Tutkimuksen tulokset osoittavat, että potilaan verestä eristetyt Loa loa sukkulamadon toukkamuodot eli mikrofilariat sitovat pinnalleen CFH ja C4BP säätelijäproteiineja in vivo ja in vitro. Lisäksi mikrobien pinnalla havaitut komplementin komponentit olivat pääosin inaktivoituja ilmeisesti näiden pinnalle sitoutuneiden sääteilijäproteiinien toimesta. Löydökset osoittavat, että säätelijäproteiinien läsnäolo Loa loa mikrofilarioiden pinnalla estää komplementin tuhovaikutusta mikrobia vastaan. Suurin osa tutkittavista S. pyogenes kannoista sitoi pinnalleen CFH:ta ja tämän rekombinanttifragmenttia, joka sisälsi CFH:n domeenit 5-7 (CFH5-7). Kun eri S. pyogenes kantojen aiheuttamaa komplementtiaktiivisuutta mitattiin seerumissa komplementtiaktivaatiota osoittavien markkerien avulla havaittiin, että kantojen aiheuttama komplementtiaktivaatio korreloi käänteisesti kantojen kykyyn sitoa CFH:ta. CFH5-7 fragmentin osoitettiin myös kilpailevan sitoutumispaikasta S. pyogenes bakteerin pinnalla kokopitkän CFH:n kanssa, ja kun viisi tutkittua S. pyogenes bakteerikantaa altistettiin CFH5-7 fragmentille, niiden lisääntyminen kokoveressä heikkeni merkitsevästi. Kokopitkät CFH proteiinit eristettiin Y402H polymorfian suhteen homotsygoottisiksi osoitettujen luovuttajien plasmasta. Sekä CFH5-7 fragmentin että kokopitkän CFH:n sitoutuminen tutkittujen S. pyogenes kantojen pinnalle oli heikompaa 402H proteiinilla kuin 402Y proteiinilla. Ex vivo mallissa bakteeri selviytyi heikommin allotyypin CFH(402H) suhteen homotsygoottiseksi osoitettujen luovuttajien veressä, johtuen ilmeisimmin CFH:n heikommasta sitoutumisesta bakteerin pinnalle. Kun verrattiin Y402H genotyyppi ja alleelifrekvenssejä ruusu- tai tonsillitti potilasaineistojen (n=487) ja kontrolliaineiston välillä havaittiin, että Y402H polymorfia voi mahdollisesti liittyä alttiuteen sairastua näihin S. pyogenes bakteerin aiheuttamiin infektioihin. Tämä väitöskirjatutkimus osoittaa, että mikrobit voivat sitoa pinnalleen komplementin säätelijäproteiineja in vitro ja in vivo ja, että CFH:n sitominen on S. pyogenes bakteerille oleellista komplementtivälitteisen tuhoutumisen välttämiseksi. CFH:n Y402H polymorfiasta johtuvat vaihtelut CFH:n sitoutumisvoimakkuuksissa S. pyogenes bakteerin pinnalle vaikuttavat ratkaisevasti siihen, kuinka mikrobi pystyy välttämään komplementin tuhovaikutuksen ja tätä kautta selviämään veressä. Tämä selittää saadut tulokset CFH:n Y402H polymorfian assosioitumisesta tiettyihin S. pyogenes infektioihin

CRP ja komplementti kudosvaurioiden tunnistuksessa ja tulehduksen säätelyssä

Vertaisarvioitu. English summary. Finnish summaryPeer reviewe

Mechanism of Borrelia immune evasion by FhbA-related proteins

Author summaryRelapsing fever and Lyme Disease are infectious diseases caused by borrelia bacteria. Relapsing fever occurs sporadically worldwide, whereas distribution of Lyme Disease is restricted to the Northern Hemisphere. Both infections are transmitted to humans by blood eating ticks or lice. These infections are often difficult to diagnose due to nonspecific symptoms. To be able to cause infection, borrelia must circumvent the human immune responses. Here we describe a mechanism, how borrelia bacteria protect themselves in the human host by utilizing host proteins. By using X-ray crystallography, we solved the structure of an outer membrane protein FhbA from a relapsing fever causing borreliae, Borrelia hermsii, in complex with human complement regulator factor H. FhbA has a unique alpha-helical fold that has not been reported earlier. The structure of the complex revealed how FhbA binds factor H in a very specific manner. Factor H bound to FhbA on the surface of borrelia protects bacteria from the complement system and lysis. Based on the structure, we performed structure-guided sequence database analysis, which suggests that similar proteins are present in all relapsing fever causing borrelia and possibly in some Lyme disease agents. Immune evasion facilitates survival of Borrelia, leading to infections like relapsing fever and Lyme disease. Important mechanism for complement evasion is acquisition of the main host complement inhibitor, factor H (FH). By determining the 2.2 angstrom crystal structure of Factor H binding protein A (FhbA) from Borrelia hermsii in complex with FH domains 19-20, combined with extensive mutagenesis, we identified the structural mechanism by which B. hermsii utilizes FhbA in immune evasion. Moreover, structure-guided sequence database analysis identified a new family of FhbA-related immune evasion molecules from Lyme disease and relapsing fever Borrelia. Conserved FH-binding mechanism within the FhbA-family was verified by analysis of a novel FH-binding protein from B. duttonii. By sequence analysis, we were able to group FH-binding proteins of Borrelia into four distinct phyletic types and identified novel putative FH-binding proteins. The conserved FH-binding mechanism of the FhbA-related proteins could aid in developing new approaches to inhibit virulence and complement resistance in Borrelia.Peer reviewe

Role of Pneumococcal NanA Neuraminidase Activity in Peripheral Blood

The most frequent form of hemolytic-uremic syndrome (HUS) is associated with infections caused by Shiga-like toxin-producing Enterohaemorrhagic Escherichia coli (STEC). In rarer cases HUS can be triggered by Streptococcus pneumoniae. While production of Shiga-like toxins explains STEC-HUS, the mechanisms of pneumococcal HUS are less well known. S. pneumoniae produces neuraminidases with activity against cell surface sialic acids that are critical for factor H-mediated complement regulation on cells and platelets. The aim of this study was to find out whether S. pneumoniae neuraminidase NanA could trigger complement activation and hemolysis in whole blood. We studied clinical S. pneumoniae isolates and two laboratory strains, a wild-type strain expressing NanA, and a NanA deletion mutant for their ability to remove sialic acids from various human cells and platelets. Red blood cell lysis and activation of complement was measured ex vivo by incubating whole blood with bacterial culture supernatants. We show here that NanA expressing S. pneumoniae strains and isolates are able to remove sialic acids from cells, and platelets. Removal of sialic acids by NanA increased complement activity in whole blood, while absence of NanA blocked complement triggering and hemolytic activity indicating that removal of sialic acids by NanA could potentially trigger pHUS.Peer reviewe

Fatal Babesiosis in Man, Finland, 2004

We report an unusual case of human babesiosis in Finland in a 53-year-old man with no history of splenectomy. He had a rudimentary spleen, coexisting Lyme borreliosis, exceptional dark streaks on his extremities, and subsequent disseminated aspergillosis. He was infected with Babesia divergens, which usually causes bovine babesiosis in Finland

Genotypic and phenotypic diversity of Lactobacillus rhamnosus clinical isolates, their comparison with strain GG and their recognition by complement system

Lactobacillus rhamnosus strains are ubiquitous in fermented foods, and in the human body where they are commensals naturally present in the normal microbiota composition of gut, vagina and skin. However, in some cases, Lactobacillus spp. have been implicated in bacteremia. The aim of the study was to examine the genomic and immunological properties of 16 clinical blood isolates of L. rhamnosus and to compare them to the well- studied L. rhamnosus probiotic strain GG. Blood cultures from bacteremic patients were collected at the Helsinki University Hospital laboratory in 2005-2011 and L. rhamnosus strains were isolated and characterized by genomic sequencing. The capacity of the L. rhamnosus strains to activate serum complement was studied using immunological assays for complement factor C3a and the terminal pathway complement complex (TCC). Binding of complement regulators factor H and C4bp was also determined using radioligand assays. Furthermore, the isolated strains were evaluated for their ability to aggregate platelets and to form biofilms in vitro. Genomic comparison between the clinical L. rhamnosus strains showed them to be clearly different from L. rhamnosus GG and to cluster in two distinct lineages. All L. rhamnosus strains activated complement in serum and none of them bound complement regulators. Four out of 16 clinical blood isolates induced platelet aggregation and/or formed more biofilms than L. rhamnosus GG, which did not display platelet aggregation activity nor showed strong biofilm formation. These findings suggest that clinical L. rhamnosus isolates show considerable heterogeneity but are clearly different from L. rhamnosus GG at the genomic level. All L. rhamnosus strains are still normally recognized by the human complement system.Peer reviewe

Staphylococcus aureus toxin LukSF dissociates from its membrane receptor target to enable renewed ligand sequestration

Staphylococcus aureus Panton-Valentine leukocidin is a pore-forming toxin targeting the human C5a receptor (hC5aR), enabling this pathogen to battle the immune response by destroying phagocytes through targeted lysis. The mechanisms that contribute to rapid cell lysis are largely unexplored. Here, we show that cell lysis may be enabled by a process of toxins targeting receptor clusters and present indirect evidence for receptor recycling that allows multiple toxin pores to be formed close together. With the use of live cell single-molecule super-resolution imaging, Forster resonance energy transfer and nanoscale total internal reflection fluorescence colocalization microscopy, we visualized toxin pore formation in the presence of its natural docking ligand. We demonstrate disassociation of hC5aR from toxin complexes and simultaneous binding of new ligands. This effect may free mobile receptors to amplify hyperinflammatory reactions in early stages of microbial infections and have implications for several other similar bicomponent toxins and the design of new antibiotics.Haapasalo, K., Wollman, A. J. M., de Haas, C. J. C., van Kessel, K. P. M., van Strijp, J. A. G., Leake, M. C. Staphylococcus aureus toxin LukSF dissociates from its membrane receptor target to enable renewed ligand sequestration.Peer reviewe

The Psoriasis Risk Allele HLA-C*06 : 02 Shows Evidence of Association with Chronic or Recurrent Streptococcal Tonsillitis

Pharyngeal tonsillitis is one of the most common upper respiratory tract infections, and group A streptococcus is the most important bacterial pathogen causing it. While most patients experience tonsillitis only rarely, a subset of patients suffers from recurrent or chronic tonsillitis or pharyngitis. The predisposing factors for recurring or chronic forms of this disease are not yet fully understood, but genetic predisposition has been suggested. A genetic association study using Illumina's Immunochip single-nucleotide polymorphism (SNP) array was performed to search for new genetic biomarkers in pharyngeal tonsillitis. More than 100,000 SNPs relevant to immune-mediated diseases were analyzed in a cohort of 95 patients subjected to tonsillectomy due to recurrent/chronic tonsillitis and 504 controls. Genetic association between the cases and controls showed strongest association with two peaks in the HLA locus (odds ratio [OR], 3.7 to 4.7; P = 4.9 x 10(-6) to 5.7 x 10(-6)). Further analysis with imputed classical HLA alleles suggested the known psoriasis risk allele HLA-C*06:02 as a risk factor for tonsillitis (P = 4.8 x 10(-4); OR, 2.3). In addition, the imputed HLA haplotype HLA-C*06:02/HLA-B*57:01, a reported risk haplotype in psoriasis, had the strongest risk for tonsillitis (P = 3.2 x 10(-4); OR, 6.5). These findings further support the previously reported link between streptococcal throat infections and psoriasis.Peer reviewe

Streptococcus pneumoniae pneumolysin and neuraminidase A convert high-density lipoproteins into pro-atherogenic particles

High-density lipoproteins (HDLs) are a group of different subpopulations of sialylated particles that have an essential role in the reverse cholesterol transport (RCT) pathway. Importantly, changes in the protein and lipid composition of HDLsmay lead to the formation of particles with reduced atheroprotective properties. Here, we show that Streptococcus pneumoniae pneumolysin (PLY) and neuraminidase A (NanA) impair HDL function by causing chemical and structural modifications of HDLs. The proteomic, lipidomic, cellular, and biochemical analysis revealed that PLY and NanA induce significant changes in sialic acid, protein, and lipid compositions of HDL. The modified HDL particles have reduced cholesterol acceptor potential from activated macrophages, elevated levels of malondialdehyde adducts, and show significantly increased complement activating capacity. These results suggest that accumulation of these modified HDL particles in the arterial intima may present a trigger for complement activation, inflammatory response, and thereby promote atherogenic disease progression.Peer reviewe

CRP gene variation affects early development of Alzheimer's disease-related plaques

Introduction We used the Tampere Autopsy Study (TASTY) series (n = 603, age 0-97 yrs), representing an unselected population outside institutions, to investigate the pathogenic involvement of inflammation in Alzheimer's disease-related lesions. Methods We studied senile plaque (SP), neurofibrillary tangles (NFT) and SP phenotype associations with 6 reported haplotype tagging single nucleotide polymorphisms (SNPs) in the CRP gene. CRP and Aβ immunohistochemistry was assessed using brain tissue microarrays. Results In multivariate analyses (age- and APOE-adjusted), non-neuritic SP were associated with the high-CRP TA-genotype (3.0% prevalence) of rs3091244 and CA-genotype (10.8%) of rs3093075 compared to common genotypes. Conversely, the low-CRP C allele (39.3%) of rs2794521 reduced the risk of harbouring early non-neuritic SP, compared to the TT genotype. CRP haplotype TAGCC (high) associated with non-neuritic SP, whereas haplotype CCGCC offered protection. TT genotypes (high) of rs3091244 and rs1130864 were associated with CRP staining. There were no associations between SNPs or haplotypes and NFT. CRP staining of the hippocampal CA1/2 region correlated with Aβ staining. Conclusions CRP gene variation affects early SP development in prodromal Alzheimer's disease, independent of APOE genotype.BioMed Central Open acces