Retrospective study of the epidemiological risk and serological diagnosis of human babesiosis in Asturias, Northwestern Spain.

BACKGROUND: Babesiosis is a globally growing tick-borne disease in humans. Severe babesiosis caused by Babesia divergens has been reported in two patients from Asturias (Northwestern Spain), suggesting an undetected risk for the disease. To analyze this risk, we retrospectively evaluated the seroprevalence of babesiosis in the Asturian population from 2015 through 2017, a period covering the intermediate years in which these two severe cases occurred. METHODS: Indirect fluorescent assay (IFA) and Western blot (WB) were performed to detect B. divergens IgG antibodies in 120 serum samples from Asturian patients infected with the tick-transmitted spirochete Borrelia burgdorferi sensu lato, a condition that indicates exposure to tick bites. RESULTS: This retrospective study confirmed a B. divergens seroprevalence rate of 39.2% according to IFA results. B. divergens incidence was 7.14 cases/100,000 population, exceeding previously reported seroprevalence rates. No differences in epidemiology and risk factors were found between patients infected solely with B. burgdorferi s.l. and those infected with B. burgdorferi s.l. and with IgG antibodies against B. divergens. This last group of patients lived in Central Asturias, had a milder clinical course and, according to WB results, developed different humoral responses against B. divergens. CONCLUSIONS: Babesia divergens parasites have circulated for several years in Asturias. Epidemiological evidence of babesiosis makes Asturias an emerging risk area for this zoonosis. Human babesiosis could also be relevant in other Spanish and European regions affected by borreliosis. Hence, the potential risk of babesiosis on human health in Asturias and other European forest regions needs to be addressed by the health authorities.

Reduced binding of apoE4 to complement factor H promotes amyloid-ß oligomerization and neuroinflammation.

The APOE4 variant of apolipoprotein E (apoE) is the most prevalent genetic risk allele associated with late-onset Alzheimer's disease (AD). ApoE interacts with complement regulator factor H (FH), but the role of this interaction in AD pathogenesis is unknown. Here we elucidate the mechanism by which isoform-specific binding of apoE to FH alters Aß1-42-mediated neurotoxicity and clearance. Flow cytometry and transcriptomic analysis reveal that apoE and FH reduce binding of Aß1-42 to complement receptor 3 (CR3) and subsequent phagocytosis by microglia which alters expression of genes involved in AD. Moreover, FH forms complement-resistant oligomers with apoE/Aß1-42 complexes and the formation of these complexes is isoform specific with apoE2 and apoE3 showing higher affinity to FH than apoE4. These FH/apoE complexes reduce Aß1-42 oligomerization and toxicity, and colocalize with complement activator C1q deposited on Aß plaques in the brain. These findings provide an important mechanistic insight into AD pathogenesis and explain how the strongest genetic risk factor for AD predisposes for neuroinflammation in the early stages of the disease pathology.

Mechanism of Borrelia immune evasion by FhbA-related proteins.

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 Å 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.

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 HDLs may 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.

Function and Dysfunction of Complement Factor H During Formation of Lipid-Rich Deposits.

Complement-mediated inflammation or dysregulation in lipid metabolism are associated with the pathogenesis of several diseases. These include age-related macular degeneration (AMD), C3 glomerulonephritis (C3GN), dense deposit disease (DDD), atherosclerosis, and Alzheimer's disease (AD). In all these diseases, formation of characteristic lipid-rich deposits is evident. Here, we will discuss molecular mechanisms whereby dysfunction of complement, and especially of its key regulator factor H, could be involved in lipid accumulation and related inflammation. The genetic associations to factor H polymorphisms, the role of factor H in the resolution of inflammation in lipid-rich deposits, modification of macrophage functions, and complement-mediated clearance of apoptotic and damaged cells indicate that the function of factor H is crucial in limiting inflammation in these diseases.

Streptococci and the complement system: interplay during infection, inflammation and autoimmunity.

Streptococci are a broad group of Gram-positive bacteria. This genus includes various human pathogens causing significant morbidity and mortality. Two of the most important human pathogens are Streptococcus pneumoniae (pneumococcus) and Streptococcus pyogenes (group A streptococcus or GAS). Streptococcal pathogens have evolved to express virulence factors that enable them to evade complement-mediated attack. These include factor H-binding M (S. pyogenes) and pneumococcal surface protein C (PspC) (S. pneumoniae) proteins. In addition, S. pyogenes and S. pneumoniae express cytolysins (streptolysin and pneumolysin), which are able to destroy host cells. Sometimes, the interplay between streptococci, the complement, and antistreptococcal immunity may lead to an excessive inflammatory response or autoimmune disease. Understanding the fundamental role of the complement system in microbial clearance and the bacterial escape mechanisms is of paramount importance for understanding microbial virulence, in general, and, the conversion of commensals to pathogens, more specifically. Such insights may help to identify novel antibiotic and vaccine targets in bacterial pathogens to counter their growing resistance to commonly used antibiotics.

Regulation of the Complement System by Pentraxins.

The functions of pentraxins, like C-reactive protein (CRP), serum amyloid protein P (SAP) and pentraxin-3 (PTX3), are to coordinate spatially and temporally targeted clearance of injured tissue components, to protect against infections and to regulate related inflammation together with the complement system. For this, pentraxins have a dual relationship with the complement system. Initially, after a focused binding to their targets, e.g., exposed phospholipids or cholesterol in the injured tissue area, or microbial components, the pentraxins activate complement by binding its first component C1q. However, the emerging inflammation needs to be limited to the target area. Therefore, pentraxins inhibit complement at the C3b stage to prevent excessive damage. The complement inhibitory functions of pentraxins are based on their ability to interact with complement inhibitors C4bp or factor H (FH). C4bp binds to SAP, while FH binds to both CRP and PTX3. FH promotes opsonophagocytosis through inactivation of C3b to iC3b, and inhibits AP activity thus preventing formation of the C5a anaphylatoxin and the complement membrane attack complex (MAC). Monitoring CRP levels gives important clinical information about the extent of tissue damage and severity of infections. CRP is a valuable marker for distinguishing bacterial infections from viral infections. Disturbances in the functions and interactions of pentraxins and complement are also involved in a number of human diseases. This review will summarize what is currently known about the FH family proteins and pentraxins that interact with FH. Furthermore, we will discuss diseases, where interactions between these molecules may play a role.

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.

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, Förster 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.

Complement Factor H and Apolipoprotein E Participate in Regulation of Inflammation in THP-1 Macrophages.

The alternative pathway (AP) of complement is constantly active in plasma and can easily be activated on self surfaces and trigger local inflammation. Host cells are protected from AP attack by Factor H (FH), the main AP regulator in plasma. Although complement is known to play a role in atherosclerosis, the mechanisms of its contribution are not fully understood. Since FH via its domains 5-7 binds apoliporotein E (apoE) and macrophages produce apoE we examined how FH could be involved in the antiatherogenic effects of apoE. We used blood peripheral monocytes and THP-1 monocyte/macrophage cells which were also loaded with acetylated low-density lipoprotein (LDL) to form foam cells. Binding of FH and apoE on these cells was analyzed by flow cytometry. High-density lipoprotein (HDL)-mediated cholesterol efflux of activated THP-1 cells was measured and transcriptomes of THP-1 cells using mRNA sequencing were determined. We found that binding of FH to human blood monocytes and cholesterol-loaded THP-1 macrophages increased apoE binding to these cells. Preincubation of fluorescent cholesterol labeled THP-1 macrophages in the presence of FH increased cholesterol efflux and cholesterol-loaded macrophages displayed reduced transcription of proinflammatory/proatherogenic factors and increased transcription of anti-inflammatory/anti-atherogenic factors. Further incubation of THP-1 cells with serum reduced C3b/iC3b deposition. Overall, our data indicate that apoE and FH interact with monocytic cells in a concerted action and this interaction reduces complement activation and inflammation in the atherosclerotic lesions. By this way FH may participate in mediating the beneficial effects of apoE in suppressing atherosclerotic lesion progression.

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 × 10-6 to 5.7 × 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 × 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 × 10-4; OR, 6.5). These findings further support the previously reported link between streptococcal throat infections and psoriasis.

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

[This corrects the article DOI: 10.1371/journal.pone.0176739.].

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.

Staphylococcal protein Ecb impairs complement receptor-1 mediated recognition of opsonized bacteria.

Staphyloccus aureus is a major human pathogen leading frequently to sepsis and soft tissue infections with abscesses. Multiple virulence factors including several immune modulating molecules contribute to its survival in the host. When S. aureus invades the human body, one of the first line defenses is the complement system, which opsonizes the bacteria with C3b and attract neutrophils by release of chemotactic peptides. Neutrophils express Complement receptor-1 [CR1, CD35) that interacts with the C3b-opsonized particles and thereby plays an important role in pathogen recognition by phagocytic cells. In this study we observed that a fraction of S. aureus culture supernatant prevented binding of C3b to neutrophils. This fraction consisted of S. aureus leukocidins and Efb. The C-terminus of Efb is known to bind C3b and shares significant sequence homology to the extracellular complement binding protein [Ecb). Here we show that S. aureus Ecb displays various mechanisms to block bacterial recognition by neutrophils. The presence of Ecb blocked direct interaction between soluble CR1 and C3b and reduced the cofactor activity of CR1 in proteolytic inactivation of C3b. Furthermore, Ecb could dose-dependently prevent recognition of C3b by cell-bound CR1 that lead to impaired phagocytosis of NHS-opsonized S. aureus. Phagocytosis was furthermore reduced in the presence of soluble CR1 [sCR1). These data indicate that the staphylococcal protein Ecb prevents recognition of C3b opsonized bacteria by neutrophil CR1 leading to impaired killing by phagocytosis and thereby contribute to immune evasion of S. aureus.

Complement Factor H Binds to Human Serum Apolipoprotein E and Mediates Complement Regulation on High Density Lipoprotein Particles.

The alternative pathway of complement is an important part of the innate immunity response against foreign particles invading the human body. To avoid damage to host cells, it needs to be efficiently down-regulated by plasma factor H (FH) as exemplified by various diseases caused by mutations in its domains 19-20 (FH19-20) and 5-7 (FH5-7). These regions are also the main interaction sites for microbial pathogens that bind host FH to evade complement attack. We previously showed that inhibition of FH binding by a recombinant FH5-7 construct impairs survival of FH binding pathogens in human blood. In this study we found that upon exposure to full blood, the addition of FH5-7 reduces survival of, surprisingly, also those microbes that are not able to bind FH. This effect was mediated by inhibition of complement regulation and subsequently enhanced neutrophil phagocytosis by FH5-7. We found that although FH5-7 does not reduce complement regulation in the actual fluid phase of plasma, it reduces regulation on HDL particles in plasma. Using affinity chromatography and mass spectrometry we revealed that FH interacts with serum apolipoprotein E (apoE) via FH5-7 domains. Furthermore, binding of FH5-7 to HDL was dependent on the concentration of apoE on the HDL particles. These findings explain why the addition of FH5-7 to plasma leads to excessive complement activation and phagocytosis of microbes in full anticoagulated blood. In conclusion, our data show how FH interacts with apoE molecules via domains 5-7 and regulates alternative pathway activation on plasma HDL particles.

Staphylococcal Ecb protein and host complement regulator factor H enhance functions of each other in bacterial immune evasion.

Staphylococcus aureus is a major human pathogen causing more than a tenth of all septicemia cases and often superficial and deep infections in various tissues. One of the immune evasion strategies of S. aureus is to secrete proteins that bind to the central complement opsonin C3b. One of these, extracellular complement binding protein (Ecb), is known to interfere directly with functions of C3b. Because C3b is also the target of the physiological plasma complement regulator, factor H (FH), we studied the effect of Ecb on the complement regulatory functions of FH. We show that Ecb enhances acquisition of FH from serum onto staphylococcal surfaces. Ecb and FH enhance mutual binding to C3b and also the function of each other in downregulating complement activation. Both Ecb and the C-terminal domains 19-20 of FH bind to the C3d part of C3b. We show that the mutual enhancing effect of Ecb and FH on binding to C3b depends on binding of the FH domain 19 to the C3d part of C3b next to the binding site of Ecb on C3d. Our results show that Ecb, FH, and C3b form a tripartite complex. Upon exposure of serum-sensitive Haemophilus influenzae to human serum, Ecb protected the bacteria, and this effect was enhanced by the addition of the C-terminal domains 19-20 of FH. This finding indicates that the tripartite complex formation could give additional protection to bacteria and that S. aureus is thereby able to use host FH and bacterial Ecb in a concerted action to eliminate C3b at the site of infection.

Acquisition of complement factor H is important for pathogenesis of Streptococcus pyogenes infections: evidence from bacterial in vitro survival and human genetic association.

Streptococcus pyogenes (or group A streptococcus [GAS]) is a major human pathogen causing infections, such as tonsillitis, erysipelas, and sepsis. Several GAS strains bind host complement regulator factor H (CFH) via its domain 7 and, thereby, evade complement attack and C3b-mediated opsonophagocytosis. Importance of CFH binding for survival of GAS has been poorly studied because removal of CFH from plasma or blood causes vigorous complement activation, and specific inhibitors of the interaction have not been available. In this study, we found that activation of human complement by different GAS strains (n = 38) correlated negatively with binding of CFH via its domains 5-7. The importance of acquisition of host CFH for survival of GAS in vitro was studied next by blocking the binding with recombinant CFH5-7 lacking the regulatory domains 1-4. Using this fragment in full human blood resulted in death or radically reduced multiplication of all of the studied CFH-binding GAS strains. To study the importance of CFH binding in vivo (i.e., for pathogenesis of streptococcal infections), we used our recent finding that GAS binding to CFH is diminished in vitro by polymorphism 402H, which is also associated with age-related macular degeneration. We showed that allele 402H is suggested to be associated with protection from erysipelas (n = 278) and streptococcal tonsillitis (n = 209) compared with controls (n = 455) (p < 0.05). Taken together, the bacterial in vitro survival data and human genetic association revealed that binding of CFH is important for pathogenesis of GAS infections and suggested that inhibition of CFH binding can be a novel therapeutic approach in GAS infections.

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.

Loa loa Microfilariae evade complement attack in vivo by acquiring regulatory proteins from host plasma.

Loa loa is a filarial nematode that infects humans. The adults live in subcutaneous tissues and produce microfilariae that live for several weeks in the blood circulation in order to be transmitted to another person via blood meals of a dipterian vector. As microfilariae live in continuous contact with plasma, it is obvious that they evade the complement system. We studied markers of complement activation and signs of complement regulation on Loa loa microfilariae in vivo. The microfilariae were isolated from anticoagulated blood samples of a Loa loa-infected Caucasian patient. C1q and some mannose-binding lectin but only a limited amount of C3b or C4b fragments and practically no C5 or C5b-9 were present on the microfilariae. The covalently microfilaria-bound C3 and C4 depositions were mainly inactive iC3b, C3c, and iC4b fragments indicating that microfilariae had regulated complement activation in vivo. Also, in vitro deposition of C3b onto the microfilariae upon serum exposure was limited. The patient-isolated microfilariae were found to carry the host complement regulators factor H and C4b-binding protein on the outermost layer, so called sheath. The microfilaria-bound factor H was functionally active. Binding of the complement regulators to the microfilariae was confirmed in vitro using (125)I-labeled factor H and C4b-binding protein. In conclusion, our study shows that Loa loa microfilariae block complement activation and acquire the host complement regulators factor H and C4b-binding protein in blood circulation. This is the first time that binding of complement regulators onto nonviral pathogens has been demonstrated to occur in humans in vivo.

Complement factor H allotype 402H is associated with increased C3b opsonization and phagocytosis of Streptococcus pyogenes.

The main virulence factor of group A streptococcus (GAS), M protein, binds plasma complement regulators factor H (FH) and FH-like protein 1 (FHL-1) leading to decreased opsonization. The M protein binding site on FH is within domain 7 in which also the age-related macular degeneration (AMD)-associated polymorphism Y402H is located. We studied if FH allotypes 402H and 402Y have different binding affinities to GAS. Plasma-derived FH allotype 402H and its recombinant fragment FH5-7(402H) showed decreased binding to several GAS strains. Growth of GAS in human blood taken from FH(402H) homozygous individuals was decreased when compared with blood taken from FH(402Y) homozygous individuals. The effect of the allotype 402H can be explained by combining the previous M protein mutagenesis data and the recently published crystal structure of FH6-8. In conclusion the data indicate that the AMD-associated allotype 402H leads to diminished binding of FH to GAS and increased opsonophagocytosis of the bacteria in blood. These results suggest that the homozygous presence of the allele 402H could be associated with decreased risk for severe GAS infections offering an explanation for the high frequency of the allele despite its association with visual impairment.