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Components of the extracellular matrix (ECM), when exposed to body fluids may promote local complement activation and inflammation. Pathologic complement activation at the glomerular basement membrane and at the Bruch's membrane is implicated in renal and eye diseases, respectively. Binding of soluble complement inhibitors to the ECM, including factor H (FH), is important to prevent excessive complement activation. Since the FH-related (FHR) proteins FHR1 and FHR5 are also implicated in these diseases, our aim was to study whether these FHRs can also bind to ECM components and affect local FH activity and complement activation. Both FH and the FHRs showed variable binding to ECM components. We identified laminin, fibromodulin, osteoadherin and PRELP as ligands of FHR1 and FHR5, and found that FHR1 bound to these ECM components through its C-terminal complement control protein (CCP) domains 4-5, whereas FHR5 bound via its middle region, CCPs 3-7. Aggrecan, biglycan and decorin did not bind FH, FHR1 and FHR5. FHR5 also bound to immobilized C3b, a model of surface-deposited C3b, via CCPs 3-7. By contrast, soluble C3, C3(H 2 O), and the C3 fragments C3b, iC3b and C3d bound to CCPs 8-9 of FHR5. Properdin, which was previously described to bind via CCPs 1-2 to FHR5, did not bind in its physiologically occurring serum forms in our assays. FHR1 and FHR5 inhibited the binding of FH to the identified ECM proteins in a dose-dependent manner, which resulted in reduced FH cofactor activity. Moreover, both FHR1 and FHR5 enhanced alternative complement pathway activation on immobilized ECM proteins when exposed to human serum, resulting in the increased deposition of C3-fragments, factor B and C5b-9. Thus, our results identify novel ECM ligands of FH family proteins and indicate that FHR1 and FHR5 are competitive inhibitors of FH on ECM and, when bound to these ligands, they may enhance local complement activation and promote inflammation under pathological conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Papp, Papp, Uzonyi, Cserhalmi, Csincsi, Szabó, Bánlaki, Ermert, Prohászka, Erdei, Ferreira, Blom and Józsi.)

Moraxella catarrhalis is a human-specific commensal of the respiratory tract and an opportunistic pathogen. It is one of the leading cause of otitis media in children and of acute exacerbations in patients with chronic obstructive pulmonary disease, resulting in significant morbidity and economic burden. Vaccines and new immunotherapeutic strategies to treat this emerging pathogen are needed. Complement is a key component of innate immunity that mediates the detection, response, and subsequent elimination of invading pathogens. Many pathogens including M. catarrhalis have evolved complement evasion mechanisms, which include the binding of human complement inhibitors such as C4b-binding protein (C4BP) and Factor H (FH). Inhibiting C4BP and FH acquisition by M. catarrhalis may provide a novel therapeutic avenue to treat infections. To achieve this, we created two chimeric proteins that combined the Moraxella-binding domains of C4BP and FH fused to human immunoglobulin Fcs: C4BP domains 1 and 2 and FH domains 6 and 7 fused to IgM and IgG Fc, respectively. As expected, FH6-7/IgG displaced FH from the bacterial surface while simultaneously activating complement via Fc-C1q interactions, together increasing pathogen elimination. C4BP1-2/IgM also increased serum killing of the bacteria through enhanced complement deposition, but did not displace C4BP from the surface of M. catarrhalis . These Fc fusion proteins could act as anti-infective immunotherapies. Many microbes bind the complement inhibitors C4BP and FH through the same domains as M. catarrhalis , therefore these Fc fusion proteins may be promising candidates as adjunctive therapy against many different drug-resistant pathogens., (Copyright © 2020 Laabei, Colineau, Bettoni, Maziarz, Ermert, Riesbeck, Ram and Blom.)

Streptococcus pyogenes is a major human pathogen that causes a variety of diseases ranging from mild skin and throat infections to fatal septicemia. In severe invasive infections, S. pyogenes encounters and interacts with components of the extracellular matrix (ECM), including small leucine rich-proteoglycans (SLRPs). In this study, we report a novel antimicrobial role played by SLRPs biglycan, decorin, fibromodulin and osteoadherin, specifically in promoting the eradication of S. pyogenes in a human sepsis model of infection. SLRPs can be released from the ECM and de novo synthesized by a number of cell types. We reveal that infection of human monocytes by S. pyogenes induces the expression of decorin. Furthermore, we show that the majority of genetically distinct and clinically relevant S. pyogenes isolates interact with SLRPs resulting in decreased survival in blood killing assays. Biglycan and decorin induce TLR2 and TLR4 signaling cascades resulting in secretion of proinflammatory and chemotactic molecules and recruitment of professional phagocytes. Surprisingly, SLRP-mediated elimination of S. pyogenes occurs independently of TLR activation. Our results indicate that SLRPs act in concert with human serum, enhancing deposition of complement activation fragments and the classical activator C1q on the bacterial surface, facilitating efficient microbial eradication. Addition of the complement C3 inhibitor compstatin significantly reverses SLRP-induced blood killing, confirming active complement as a key mediator in SLRP-mediated bacterial destruction. Taken together our results add to the functional repertoire of SLRPs, expanding to encompass their role in controlling bacterial infection., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: John Lambris is the founder of Amyndas Pharmaceuticals, which is developing complement inhibitors for therapeutic purposes. John Lambris is the inventor of patents or patent applications that describe the use of complement inhibitors for therapeutic purposes, some of which are developed by Amyndas Pharmaceuticals. John Lambris is also the inventor of the compstatin technology licensed to Apellis Pharmaceuticals [i.e., 4(1MeW)7 W/POT-4/APL-1 and PEGylated derivatives such as APL-2/pegcetacoplan]. The other authors have no financial conflicts of interest., (© 2020 The Authors.)

Gonorrhea is a sexually transmitted infection with 87 million new cases per year globally. Increasing antibiotic resistance has severely limited treatment options. A mechanism that Neisseria gonorrhoeae uses to evade complement attack is binding of the complement inhibitor C4b-binding protein (C4BP). We screened 107 porin B1a (PorB1a) and 83 PorB1b clinical isolates randomly selected from a Swedish strain collection over the last 10 years and noted that 96/107 (89.7%) PorB1a and 16/83 (19.3%) PorB1b bound C4BP; C4BP binding substantially correlated with the ability to evade complement-dependent killing (r = 0.78). We designed 2 chimeric proteins that fused C4BP domains to the backbone of IgG or IgM (C4BP-IgG; C4BP-IgM) with the aim of enhancing complement activation and killing of gonococci. Both proteins bound gonococci (KD C4BP-IgM = 2.4 nM; KD C4BP-IgG 980.7 nM), but only hexameric C4BP-IgM efficiently outcompeted heptameric C4BP from the bacterial surface, resulting in enhanced complement deposition and bacterial killing. Furthermore, C4BP-IgM substantially attenuated the duration and burden of colonization of 2 C4BP-binding gonococcal isolates but not a non-C4BP-binding strain in a mouse vaginal colonization model using human factor H/C4BP-transgenic mice. Our preclinical data present C4BP-IgM as an adjunct to conventional antimicrobials for the treatment of gonorrhea.

Pathogens that invade the human host are confronted by a multitude of defence mechanisms aimed at preventing colonization, dissemination and proliferation. The most frequent outcome of this interaction is microbial elimination, in which the complement system plays a major role. Complement, an essential feature of the innate immune machinery, rapidly identifies and marks pathogens for efficient removal. Consequently, this creates a selective pressure for microbes to evolve strategies to combat complement, permitting host colonization and access to resources. All successful pathogens have developed mechanisms to resist complement activity which are intimately aligned with their capacity to cause disease. In this review, we describe the successful methods various pathogens use to evade complement activation, shut down inflammatory signalling through complement, circumvent opsonisation and override terminal pathway lysis. This review summarizes how pathogens undermine innate immunity: 'The Hijackers Guide to Complement'., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Streptococcus pyogenes infects over 700 million people worldwide annually. Immune evasion strategies employed by the bacteria include binding of the complement inhibitors, C4b-binding protein (C4BP) and Factor H in a human-specific manner. We recently showed that human IgG increased C4BP binding to the bacterial surface, which promoted streptococcal immune evasion and increased mortality in mice. We sought to identify how IgG promotes C4BP binding to Protein H, a member of the M protein family. Dimerization of Protein H is pivotal for enhanced binding to human C4BP. First, we illustrated that Protein H, IgG, and C4BP formed a tripartite complex. Second, surface plasmon resonance revealed that Protein H binds IgG solely through Fc, but not Fab domains, and with high affinity (IgG-Protein H: K D = 0.4 nM; IgG-Fc-Protein H: K D ≤ 1.6 nM). Each IgG binds two Protein H molecules, while up to six molecules of Protein H bind one C4BP molecule. Third, interrupting Protein H dimerization either by raising temperature to 41°C or with a synthetic peptide prevented IgG-Protein H interactions. IgG-Fc fragments or monoclonal human IgG permitted maximal C4BP binding when used at concentrations from 0.1 to 10 mg/ml. In contrast, pooled human IgG enhanced C4BP binding at concentrations up to 1 mg/ml; decreased C4BP binding at 10 mg/ml occurred probably because of Fab-streptococcal interactions at these high IgG concentrations. Taken together, our data show how S. pyogenes exploits human IgG to evade complement and enhance its virulence. Elucidation of this mechanism could aid design of new therapeutics against S. pyogenes .

The human host has evolved elaborate protection mechanisms to prevent infection from the billions of microorganisms to which it host is exposed and is home. One of these systems, complement, is an evolutionary ancient arm of innate immunity essential for combatting bacterial infection. Complement permits the efficient labelling of bacteria with opsonins, supports phagocytosis, and facilitates phagocyte recruitment to the site of infection through the production of chemoattractants. However, it is by no means perfect, and certain organisms engage in an evolutionary arms race with the host where complement has become a major target to promote immune evasion. Streptococcus pyogenes is a major human pathogen that causes significant morbidity and mortality globally. S. pyogenes is also a member of an elite group of bacterial pathogens possessing a sophisticated arsenal of virulence determinants capable of interfering with complement. In this review, we focus on these complement evasins, their mechanism of action, and their importance in disease progression. Finally, we highlight new therapeutic options for fighting S. pyogenes, by interfering with one of its main mechanisms of complement evasion., (The Author(s). Published by S. Karger AG, Basel.)

The respiratory pathogen Moraxella catarrhalis is a human-specific commensal that frequently causes acute otitis media in children and stimulates acute exacerbations in chronic obstructive pulmonary disease patients. The exact molecular mechanisms defining host-pathogen interactions promoting pathogenesis are not clearly understood. Limited knowledge hampers vaccine and immunotherapeutic development required to treat this emerging pathogen. In this study, we reveal in detail a novel antibacterial role displayed by short leucine-rich proteoglycans (SLRPs) in concert with complement. We show that fibromodulin (FMOD), osteoadherin (OSAD), and biglycan (BGN) but not decorin (DCN) enhance serum killing of M. catarrhalis. Our results suggest that M. catarrhalis binding to SLRPs is a conserved feature, as the overwhelming majority of clinical and laboratory strains bound all four SLRPs. Furthermore, we resolve the binding mechanism responsible for this interaction and highlight the role of the ubiquitous surface protein (Usp) A2/A2H in mediating binding to host SLRPs. A conserved immune evasive strategy used by M. catarrhalis and other pathogens is the surface acquisition of host complement inhibitors such as C4b-binding protein (C4BP). We observed that FMOD, OSAD, and BGN competitively inhibit binding of C4BP to the surface of M. catarrhalis , resulting in increased C3b/iC3b deposition, membrane attack complex (MAC) formation, and subsequently decreased bacterial survival. Furthermore, both OSAD and BGN promote enhanced neutrophil killing in vitro, both in a complement-dependent and independent fashion. In summary, our results illustrate that SLRPs, FMOD, OSAD, and BGN portray complement-modulating activity enhancing M. catarrhalis killing, defining a new antibacterial role supplied by SLRPs., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Neutrophils are crucial for the human innate immunity and constitute the majority of leukocytes in circulation. Thus, blood neutrophil counts serve as a measure for the immune system's functionality. Hematological patients often have low neutrophil counts due to disease or chemotherapy. To increase neutrophil counts and thereby preventing infections in high-risk patients, recombinant G-CSF is widely used as adjunct therapy to stimulate the maturation of neutrophils. In addition, G-CSF is utilized to recruit stem cells (SCs) into the peripheral blood of SC donors. Still, the actual functionality of neutrophils resulting from G-CSF treatment remains insufficiently understood. We tested the ex vivo functionality of neutrophils isolated from blood of G-CSF-treated healthy SC donors. We quantified chemotaxis, oxidative burst, and phagocytosis before and after treatment and detected significantly reduced chemotactic activity upon G-CSF treatment. Similarly, in vitro treatment of previously untreated neutrophils with G-CSF led to reduced chemotactic activity. In addition, we revealed that this effect persists in the allogeneic SC recipients up to 4 weeks after neutrophil engraftment. Our data indicates that neutrophil quantity, as a sole measure of immunocompetence in high-risk patients should be considered cautiously as neutrophil functionality might be affected by the primary treatment.

Streptococcus pyogenes is an exclusively human pathogen that can provoke mild skin and throat infections but can also cause fatal septicemia. This gram-positive bacterium has developed several strategies to evade the human immune system, enabling S. pyogenes to survive in the host. These strategies include recruiting several human plasma proteins, such as the complement inhibitor, C4b-binding protein (C4BP), and human (hu)-IgG through its Fc region to the bacterial surface to evade immune recognition. We identified a novel virulence mechanism whereby IgG-enhanced binding of C4BP to five of 12 tested S. pyogenes strains expressed diverse M proteins that are important surface-expressed virulence factors. Importantly, all strains that bound C4BP in the absence of IgG bound more C4BP when IgG was present. Further studies with an M1 strain that additionally expressed protein H, also a member of the M protein family, revealed that binding of hu-IgG Fc to protein H increased the affinity of protein H for C4BP. Increased C4BP binding accentuated complement downregulation, resulting in diminished bacterial killing. Accordingly, mortality from S. pyogenes infection in hu-C4BP transgenic mice was increased when hu-IgG or its Fc portion alone was administered concomitantly. Electron microscopy analysis of human tissue samples with necrotizing fasciitis confirmed increased C4BP binding to S. pyogenes when IgG was present. Our findings provide evidence of a paradoxical function of hu-IgG bound through Fc to diverse S. pyogenes isolates that increases their virulence and may counteract the beneficial effects of IgG opsonization., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Bacteria can cause life-threatening infections, such as pneumonia, meningitis, or sepsis. Antibiotic therapy is a mainstay of treatment, although antimicrobial resistance has drastically increased over the years. Unfortunately, safe and effective vaccines against most pathogens have not yet been approved, and thus developing alternative treatments is important. We analyzed the efficiency of factor H (FH)6-7/Fc, a novel antibacterial immunotherapeutic protein against the Gram-positive bacterium Streptococcus pyogenes This protein is composed of two domains of complement inhibitor human FH (FH complement control protein modules 6 and 7) that bind to S. pyogenes , linked to the Fc region of IgG (FH6-7/Fc). FH6-7/Fc has previously been shown to enhance complement-dependent killing of, and facilitate bacterial clearance in, animal models of the Gram-negative pathogens Haemophilus influenzae and Neisseria meningitidis We hypothesized that activation of complement by FH6-7/Fc on the surface of Gram-positive bacteria such as S. pyogenes will enable professional phagocytes to eliminate the pathogen. We found that FH6-7/Fc alleviated S. pyogenes- induced sepsis in a transgenic mouse model expressing human FH ( S. pyogenes binds FH in a human-specific manner). Furthermore, FH6-7/Fc, which binds to protein H and selected M proteins, displaced FH from the bacterial surface, enhanced alternative pathway activation, and reduced bacterial blood burden by opsonophagocytosis in a C3-dependent manner in an ex vivo human whole-blood model. In conclusion, FH-Fc chimeric proteins could serve as adjunctive treatments against multidrug-resistant bacterial infections., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Background: Neutrophils are traditionally considered transcriptionally inactive. Compared to other immune cells, little is known about their transcriptional profile during interaction with pathogens., Methods: We analyzed the meta-transcriptome of the neutrophil-Candida albicans interplay and the transcriptome of C. albicans challenged with neutrophil extracellular traps (NETs) by RNA-Seq, considering yeast and hypha individually in each approach., Results: The neutrophil response to C. albicans yeast and hyphae was dominated by a morphotype-independent core response. However, 11 % of all differentially expressed genes were regulated in a specific manner when neutrophils encountered the hyphal form of C. albicans. While involving genes for transcriptional regulators, receptors, and cytokines, the neutrophil core response lacked typical antimicrobial effectors genes. Genes of the NOD-like receptor pathway, including NLRP3, were enriched. Neutrophil- and NET-provoked responses in C. albicans differed. At the same time, the Candida transcriptome upon neutrophil encounter and upon NET challenge included genes from various metabolic processes and indicate a mutual role of the regulators Tup1p, Efg1p, Hap43p, and Cap1p. Upon challenge with neutrophils and NETs, the overall Candida response was partially morphotype-specific. Yet again, actual oppositional regulation in yeasts and hyphae was only detected for the arginine metabolism in neutrophil-infecting C. albicans., Conclusions:  Taken together, our study provides a comprehensive and quantitative transcript profile of the neutrophil-C. albicans interaction. By considering the two major appearances of both, neutrophils and C. albicans, our study reveals yet undescribed insights into this medically relevant encounter. Hence, our findings will facilitate future research and potentially inspire novel therapy developments.

Respiratory tract infections are one of the leading causes of mortality worldwide urging better understanding of interactions between pathogens causing these infections and the host. Here we report that an extracellular matrix component proline/arginine-rich end leucine-rich repeat protein (PRELP) is a novel antibacterial component of innate immunity. We detected the presence of PRELP in human bronchoalveolar lavage fluid and showed that PRELP can be found in alveolar fluid, resident macrophages/monocytes, myofibroblasts, and the adventitia of blood vessels in lung tissue. PRELP specifically binds respiratory tract pathogens Moraxella catarrhalis , Haemophilus influenzae , and Streptococcus pneumoniae , but not other bacterial pathogens tested. We focused our study on M. catarrhalis and found that PRELP binds the majority of clinical isolates of M. catarrhalis ( n = 49) through interaction with the ubiquitous surface protein A2/A2H. M. catarrhalis usually resists complement-mediated serum killing by recruiting to its surface a complement inhibitor C4b-binding protein, which is also a ligand for PRELP. We found that PRELP competitively inhibits binding of C4b-binding protein to bacteria, which enhances membrane attack complex formation on M. catarrhalis and thus leads to increased serum sensitivity. Furthermore, PRELP enhances phagocytic killing of serum-opsonized M. catarrhalis by human neutrophils in vitro. Moreover, PRELP reduces Moraxella adherence to and invasion of human lung epithelial A549 cells. Taken together, PRELP enhances host innate immunity against M. catarrhalis through increasing complement-mediated attack, improving phagocytic killing activity of neutrophils, and preventing bacterial adherence to lung epithelial cells., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Periodontal disease is one of the most common inflammatory infectious diseases worldwide and it is associated with other syndromes, such as cardiovascular disease or rheumatoid arthritis. Recent advances in sequencing allowed for identification of novel periodontopathogens such as Gram-positive Filifactor alocis, but its virulence mechanisms remain largely unknown. We confirmed that F. alocis is a prevalent species in periodontitis patients, and we also observed strong correlation of this bacterium with clinical parameters, highlighting its role in the pathogenesis of the disease. Further, we found that preincubation of human serum with F. alocis resulted in abolished bactericidal activity and that F. alocis was surviving readily in full blood. We demonstrated that one of the key contributors to F. alocis complement resistance is a unique protein, FACIN (F. alocis complement inhibitor), which binds to C3, resulting in suppression of all complement pathways. Interestingly, FACIN is a nonclassical cell surface protein, a cytosolic enzyme acetylornithine transaminase, for which we now identified a moonlighting function. FACIN binds to C3 alone, but more importantly it also captures activated complement factor 3 within the complex with factor B, thereby locking in the convertase in an inactive state. Because of the indispensable role of alternative pathway convertase in amplifying complement cascades, its inhibition by FACIN results in a very potent downregulation of activated complement factor 3 opsonization on the pathogen surface, accompanied by reduction of downstream C5 cleavage., Competing Interests: The authors have no financial conflict of interest., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Helicobacter pylori is an important human pathogen and a common cause of peptic ulcers and gastric cancer. Despite H. pylori provoking strong innate and adaptive immune responses, the bacterium is able to successfully establish long-term infections. Vitronectin (Vn), a component of both the extracellular matrix and plasma, is involved in many physiological processes, including regulation of the complement system. The aim of this study was to define a receptor in H. pylori that binds Vn and determine the significance of the interaction for virulence. Surprisingly, by using proteomics, we found that the hydrogen peroxide-neutralizing enzyme catalase KatA is a major Vn-binding protein. Deletion of the katA gene in three different strains resulted in impaired binding of Vn. Recombinant KatA was generated and shown to bind with high affinity to a region between heparin-binding domain 2 and 3 of Vn that differs from previously characterised bacterial binding sites on the molecule. In terms of function, KatA protected H. pylori from complement-mediated killing in a Vn-dependent manner. Taken together, the virulence factor KatA is a Vn-binding protein that moonlights on the surface of H. pylori to promote bacterial evasion of host innate immunity.

Moraxella catarrhalis is a respiratory tract pathogen commonly causing otitis media in children and acute exacerbations in patients suffering from chronic obstructive pulmonary disease. Cartilage oligomeric matrix protein (COMP) functions as a structural component in cartilage, as well as a regulator of complement activity. Importantly, COMP is detected in resident macrophages and monocytes, alveolar fluid, and the endothelium of blood vessels in lung tissue. We show that the majority of clinical isolates of M. catarrhalis (n = 49), but not other tested bacterial pathogens, bind large amounts of COMP. COMP interacts directly with the ubiquitous surface protein A2 of M. catarrhalis. Binding of COMP correlates with survival of M. catarrhalis in human serum by inhibiting bactericidal activity of the complement membrane attack complex. Moreover, COMP inhibits phagocytic killing of M. catarrhalis by human neutrophils. We further observed that COMP reduces bacterial adhesion and uptake by human lung epithelial cells, thus protecting M. catarrhalis from intracellular killing by epithelial cells. Taken together, our findings uncover a novel mechanism that M. catarrhalis uses to evade host innate immunity., (Copyright © 2016 by The American Association of Immunologists, Inc.)

C4b-binding protein (C4BP) is best known as a potent soluble inhibitor of the classical and lectin pathways of the complement system. This large 500 kDa multimeric plasma glycoprotein is expressed mainly in the liver but also in lung and pancreas. It consists of several identical 75 kDa α-chains and often also one 40 kDa β-chain, both of which are mainly composed of complement control protein (CCP) domains. Structure-function studies revealed that one crucial binding site responsible for inhibition of complement is located to CCP1-3 of the α-chain. Binding of anticoagulant protein S to the CCP1 of the β-chain provides C4BP with the ability to strongly bind apoptotic and necrotic cells in order to prevent inflammation arising from activation of complement by these cells. Further, C4BP interacts strongly with various types of amyloid and enhances fibrillation of islet amyloid polypeptide secreted from pancreatic beta cells, which may attenuate pro-inflammatory and cytotoxic effects of this amyloid. Full deficiency of C4BP has not been identified but non-synonymous alterations in its sequence have been found in haemolytic uremic syndrome and recurrent pregnancy loss. Furthermore, C4BP is bound by several bacterial pathogens, notably Streptococcus pyogenes, which due to inhibition of complement and enhancement of bacterial adhesion to endothelial cells provides these bacteria with a survival advantage in the host. Thus, depending on the context, C4BP has a protective or detrimental role in the organism., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or 'double' tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the 'double' tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.

Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of (125)I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92-109, which specifically bound C4BP. Biacore was used to determine KD = 6 × 10(-7) M between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by S. pyogenes.

Candida albicans, the most commonly isolated human fungal pathogen, is able to grow as budding yeasts or filamentous forms, such as hyphae. The ability to switch morphology has been attributed a crucial role for the pathogenesis of C. albicans. To mimic disseminated candidiasis in humans, the mouse is the most widely used model organism. Neutrophils are essential immune cells to prevent opportunistic mycoses. To explore potential differences between the rodent infection model and the human host, we compared the interactions of C. albicans with neutrophil granulocytes from mice and humans. We revealed that murine neutrophils exhibited a significantly lower ability to kill C. albicans than their human counterparts. Strikingly, C. albicans yeast cells formed germ tubes upon internalization by murine neutrophils, eventually rupturing the neutrophil membrane and thereby, killing the phagocyte. On the contrary, growth and subsequent escape of C. albicans are blocked inside human neutrophils. According to our findings, this blockage in human neutrophils might be a result of higher levels of MPO activity and the presence of α-defensins. We therefore outline differences in antifungal immune defense between humans and mouse strains, which facilitates a more accurate interpretation of in vivo results.

The enteropathogen Yersinia pseudotuberculosis can survive in the harsh environment of lymphoid compartments that abounds in immune cells. This capacity is dependent on the plasmid-encoded Yersinia outer proteins (Yops) that are delivered into the host cell via a mechanism involving the Yersinia type III secretion system. We show that the virulence protein YopK has a role in the mechanism by which Y. pseudotuberculosis avoids the polymorphonuclear leukocyte or neutrophil (PMN) defense. A yopK mutant, which is attenuated in the mouse infection model, where it fails to cause systemic infection, was found to colonize Peyer's patches and mesenteric lymph nodes more rapidly than the wild-type strain. Further, in mice lacking PMNs, the yopK mutant caused full disease with systemic spread and typical symptoms. Analyses of effects on PMNs revealed that both the wild-type strain and the yopK mutant inhibited internalization and reactive oxygen species production, as well as neutrophil extracellular trap formation by PMNs. However, the wild-type strain effectively avoided induction of PMN death, whereas the mutant caused a necrosis-like PMN death. Taken together, our results indicate that YopK is required for the ability of Yersinia to resist the PMN defense, which is critical for the virulence of the pathogen. We suggest a mechanism whereby YopK functions to prevent unintended Yop delivery and thereby PMN disruption, resulting in necrosis-like cell death, which would enhance the inflammatory response favoring the host.

Proteasomes are the major enzyme complexes for non-lysosomal protein degradation in eukaryotic cells. Mammals express two sets of catalytic subunits: the constitutive subunits β1, β2 and β5 and the immunosubunits LMP2 (β1i), MECL-1 (β2i) and LMP7 (β5i). The LMP7-propeptide (proLMP7) is required for optimal maturation of LMP2/MECL-1-containing precursors to mature immunoproteasomes, but can also mediate efficient integration into mixed proteasomes containing β1 and β2. In contrast, the β5-propeptide (proβ5) has been suggested to promote preferential integration into β1/β2-containing precursors, consequently favouring the formation of constitutive proteasomes. Here, we show that proβ5 predominantly promotes integration into LMP2/MECL-1-containing precursors in IFNγ-stimulated, LMP7-deficient cells and infected LMP7-deficient mice. This demonstrates that proβ5 does not direct preferential integration into β1/β2-containing precursors, but instead promotes the formation of mixed LMP2/MECL-1/β5 proteasomes under inflammatory conditions. Moreover, the propeptides substantially differ in their capacity to promote proteasome maturation, with proLMP7 showing a significantly higher chaperone activity as compared to proβ5. Increased efficiency of proteasome maturation mediated by proLMP7 is required for optimal MHC class I cell surface expression and is equally important as the catalytic activity of immunoproteasomes. Intriguingly, induction of LMP7 by infection not only results in rapid exchange of constitutive by immunosubunits, as previously suggested, but also increases the total proteasome abundance within the infected tissue. Hence our data identify a novel LMP7-dependend mechanism to enhance the activity of the proteasome system in infection, which is based on the high chaperone activity of proLMP7 and relies on accelerated maturation of active proteasome complexes.

Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.

Neutrophil extracellular traps (NETs) play an important role in innate immunity to microbial infections. NETs have been described in several species, but the molecular details of NET formation and their role in infection has not been addressed, partly because we lack optimal experimental models. Here we describe tools to investigate NET formation in neutrophils isolated from mice. Upon in vitro stimulation of wild-type mouse neutrophils with PMA, we analyzed 3 important steps in the process of NET formation: reactive oxygen species (ROS) production, NET cell death and NET release. As expected, neutrophils from NADPH oxidase-deficient mice failed to produce ROS and did not die nor release NETs upon stimulation. We found that neutrophils from several mouse strains produced NETs with different efficiency and that NET formation correlated with the amount of ROS produced. Activation with Candida albicans also resulted in ROS production and NET cell death. The hyphal form of this fungus induced NETs more effectively than the yeast form. With this work, we provide tools to study in vitro NET assembly in the mouse system., (Copyright 2009 S. Karger AG, Basel.)

Neutrophils are professional phagocytes of the innate immune system that are essential to control bacterial and fungal infections. These cells engulf and kill invading microbes. Additionally, activated neutrophils are able to release neutrophil extracellular traps (NETs). These fibers consist of chromatin decorated with antimicrobial proteins to trap and kill microbes. Appropriate quantitative methods are required to understand the nature of interactions of neutrophils with pathogens. Here we present assays to measure killing mediated by phagocytosis, by NETs, by a combination of both, and by granular extract. As examples, we use Candida albicans for fungal and Shigella flexneri for bacterial pathogens.