The Emerging Role of Complement Lectin Pathway in Trypanosomatids: Molecular Bases in Activation, Genetic Deficiencies, Susceptibility to Infection, and Complement System-Based Therapeutics

The innate immune system is evolutionary and ancient and is the pivotal line of the host defense system to protect against invading pathogens and abnormal self-derived components. Cellular and molecular components are involved in recognition and effector mechanisms for a successful innate immune response. The complement lectin pathway (CLP) was discovered in 1990. These new components at the complement world are very efficient. Mannan-binding lectin (MBL) and ficolin not only recognize many molecular patterns of pathogens rapidly to activate complement but also display several strategies to evade innate immunity. Many studies have shown a relation between the deficit of complement factors and susceptibility to infection. The recently discovered CLP was shown to be important in host defense against protozoan microbes. Although the recognition of pathogen-associated molecular patterns by MBL and Ficolins reveal efficient complement activations, an increase in deficiency of complement factors and diversity of parasite strategies of immune evasion demonstrate the unsuccessful effort to control the infection. In the present paper, we will discuss basic aspects of complement activation, the structure of the lectin pathway components, genetic deficiency of complement factors, and new therapeutic opportunities to target the complement system to control infection

Ficolin-2 Levels and FCN2 Haplotypes Influence Hepatitis B Infection Outcome in Vietnamese Patients

Human Ficolin-2 (L-ficolins) encoded by FCN2 gene is a soluble serum protein that plays an important role in innate immunity and is mainly expressed in the liver. Ficolin-2 serum levels and FCN2 single nucleotide polymorphisms were associated to several infectious diseases. We initially screened the complete FCN2 gene in 48 healthy individuals of Vietnamese ethnicity. We genotyped a Vietnamese cohort comprising of 423 clinically classified hepatitis B virus patients and 303 controls for functional single nucleotide polymorphisms in the promoter region (-986G>A, -602G>A, -4A>G) and in exon 8 (+6424G>T) by real-time PCR and investigated the contribution of FCN2 genotypes and haplotypes to serum Ficolin-2 levels, viral load and liver enzyme levels. Haplotypes differed significantly between patients and controls (P = 0.002) and the haplotype AGGG was found frequently in controls in comparison to patients with hepatitis B virus and hepatocellular carcinoma (P = 0.0002 and P<0.0001) conferring a protective effect. Ficolin-2 levels differed significantly between patients and controls (p<0.0001). Patients with acute hepatitis B had higher serum Ficolin-2 levels compared to other patient groups and controls.The viral load was observed to be significantly distributed among the haplotypes (P = 0.04) and the AAAG haplotype contributed to higher Ficolin-2 levels and to viral load. Four novel single nucleotide polymorphisms in introns (-941G>T, -310G>A, +2363G>A, +4882G>A) and one synonymous mutation in exon 8 (+6485G>T) was observed. Strong linkage was found between the variant -986G>A and -4A>G. The very first study on Vietnamese cohort associates both Ficolin-2 serum levels and FCN2 haplotypes to hepatitis B virus infection and subsequent disease progression

Functional Analysis of Ficolin-3 Mediated Complement Activation

The recognition molecules of the lectin complement pathway are mannose-binding lectin and Ficolin -1, -2 and -3. Recently deficiency of Ficolin-3 was found to be associated with life threatening infections. Thus, we aimed to develop a functional method based on the ELISA platform for evaluating Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA) was acetylated (acBSA) and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition on acBSA were dependent only on Ficolin-3 in appropriate serum dilutions. Deposition of down stream complement components correlated highly significantly with the serum concentration of Ficolin-3 but not with Ficolin-2 in healthy donors. To make the assay robust for clinical use a chemical compound was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides the possibility to diagnose functional and genetic defects of Ficolin-3 and down stream components in the lectin complement pathway

Lectin-Dependent Enhancement of Ebola Virus Infection via Soluble and Transmembrane C-type Lectin Receptors

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes

Mechanisms of complement lectin pathway activation and resistance by trypanosomatid parasites

Studies in the past decade have demonstrated a crucial role for the complement lectin pathway in host defence against protozoan microbes. Recognition of pathogen surface molecules by mannan-binding lectin and ficolins revealed new mechanisms of innate immune defence and a diversity of parasite strategies of immune evasion. In the present review, we will discuss the current knowledge of: (1) the molecular mechanism of lectin pathway activation by trypanosomes; (2) the mechanisms of complement evasion by trypanosomes; and (3) host genetic deficiencies of complement lectin pathway factors that contribute to infection susceptibility and disease progression. This review will focus on trypanosomatids, the parasites that cause Chagas disease, leishmaniasis and sleeping sickness (African trypanosomiasis)

Emergence Of Extended-spectrum β-lactamase Producing Enterobacter Spp. In Patients With Bacteremia In A Tertiary Hospital In Southern Brazil

Background Extended-spectrum β-lactamases (ESBLs) are increasingly prevalent in Enterobacter spp., posing a challenge to the treatment of infections caused by this microorganism. The purpose of this retrospective study was to evaluate the prevalence, risk factors, and clinical outcomes of inpatients with bacteremia caused by ESBL and non ESBL-producing Enterobacter spp. in a tertiary hospital over the period 2004-2008. Methods The presence of blaCTX-M, blaTEM, blaSHV, and blaPER genes was detected by polymerase chain reaction (PCR) and nucleotide sequence analysis. Genetic similarity between strains was defined by pulsed-field gel electrophoresis (PFGE). Results Enterobacter spp. was identified in 205 of 4907 of the patients who had positive blood cultures during hospitalization. Of those cases, 41 (20%) were ESBL-producing Enterobacter spp. Nosocomial pneumonia was the main source of bacteremia caused by ESBL-producing Enterobacter spp. The presence of this microorganism was associated with longer hospital stays. The ESBL genes detected were: CTX-M-2 (23), CTX-M-59 (10), CTX-M-15 (1), SHV-12 (5), and PER-2 (2). While Enterobacter aerogenes strains showed mainly a clonal profile, Enterobacter cloacae strains were polyclonal. Conclusion Although no difference in clinical outcomes was observed between patients with infections by ESBL-producing and non-ESBL-producing strains, the detection of ESBL in Enterobacter spp. resulted in the change of antimicrobials in 75% of cases, having important implications in the decision-making regarding adequate antimicrobial therapy. © 2012 Elsevier España, S.L. All rights reserved.3228792Sanders, Jr.W.E., Sanders, C.C., Enterobacter spp.: Pathogens poised to flourish at the turn of the century (1997) Clin Microbiol Rev, 10, pp. 220-241Ho, P.L., Shek, R.H., Chow, K.H., Lai, W.M., Detection and characterization of extended-spectrum beta-lactamases among bloodstream isolates of Enterobacter spp. in Hong Kong, 2000-2002 (2005) J Antimicrob Chemother, 55, pp. 326-332Thomson, K.S., Extended-spectrum-β-lactamase, AmpC, and carbapenemase issues (2010) J Clin Microbiol, 48, pp. 1019-1025Rodríguez-Baño, J., Picón, E., Gijón, P., Hernández, J.R., Cisneros, J.M., Peña, C., Risk factors and prognosis of nosocomial bloodstream infections caused by extended-spectrum-beta-lactamase-producing Escherichia coli (2010) J Clin Microbiol, 48, pp. 1726-1731Schwaber, M.J., Carmeli, Y., Mortality and delay in effective therapy associated with extended-spectrum beta-lactamase production in Enterobacteriaceae bacteraemia: A systematic review and meta-analysis (2007) J Antimicrob Chemother, 60, pp. 913-920Tumbarello, M., Spanu, T., Di Bidino, R., Marchetti, M., Ruggeri, M., Trecarichi, E.M., Costs of bloodstream infections caused by Escherichia coli and influence of extended-spectrum-beta-lactamase production and inadequate initial antibiotic therapy (2010) Antimicrob Agents Chemother, 54, pp. 4085-4091Cheong, H.S., Ko, K.S., Kang, C.I., Chung, D.R., Peck, K.R., Song, J.H., Clinical significance of infections caused by extended-spectrum b-lactamase-producing Enterobacteriaceae Blood isolates with inducible AmpC b-lactamase (2012) Microbial Drug Resist, 18, pp. 446-452Towne, T.G., Lewis, J.S., Herrera, M., Wickes, B., Jorgensen, J.H., Detection of SHVtype extended-spectrum beta-lactamase in Enterobacter isolates (2010) J Clin Microbiol, 48, pp. 298-299(2011) Susceptibility TestingTwenty-First Informational Supplement M100-S21, , Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial CLSI, Wayne, PA, USAWoodford, N., Fagan, E.J., Ellington, M.J., Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum (beta)-lactamases (2006) J Antimicrob Chemother, 57, pp. 154-155Payne, J.P., Thomson, D.J., Molecular approaches for the detection and identification of β-lactamases (1998) Molecular Bacteriology: Protocols and Clinical Applications, pp. 495-512. , N. Woodford, A.P. Johnson, Humana Press Totowa, NJ, USABauernfeind, A., Stemplinger, I., Jungwirth, R., Mangold, P., Amann, S., Akalin, E., Characterization of beta-lactamase gene blaPER-2, which encodes an extended-spectrum class A beta-lactamase (1996) Antimicrob Agents Chemother, 40, pp. 616-620Kaufmann, M., Pulsed-field-gel-electrophoresis (1988) Molecular Bacteriology: Protocols and Clinical Applications, pp. 33-51. , N. Woodford, A.P. Johnson, Human Press NJGupta, N., Aparna, Choudhary, U., Garg, N., Arora, D.R., Enterobacter bacteremia (2003) J. Assoc Physicians India, 51, pp. 669-672Marchaim, D., Gottesman, T., Schwartz, O., Korem, M., Maor, Y., Rahav, G., National multicenter study of predictors and outcomes of bacteremia upon hospital admission caused by Enterobacteriaceae producing extended-spectrum beta-lactamases (2010) Antimicrob Agents Chemother, 54, pp. 5099-5104Pai, H., Hong, J.Y., Byeon, J.H., Kim, Y.K., Lee, H.J., High prevalence of extended-spectrum beta-lactamase-producing strains among blood isolates of Enterobacter spp collected in a tertiary hospital during an 8-year period and their antimicrobial susceptibility patterns (2004) Antimicrob Agents Chemother, 48, pp. 3159-3161García-Hernández, A., García-Vázquez, E., Gómez-Gómez, J., Canteras, M., Hernandez-Torres, A., Ruiz-Gomes, J., Predictor factors of ESBL versus non-ESBL Escherichia coli bacteraemia and influence of resistance on the mortality of the patients (2010) Med Clin (Barc), 136, pp. 56-60Marcos, M., Iñurrieta, A., Soriano, A., Martínez, J.A., Almela, M., Marco, F., Effect of antimicrobial therapy on mortality in 377 episodes of Enterobacter spp bacteraemia (2008) J Antimicrob Chemother, 62, pp. 397-403Bonnet, R., Dutour, C., Sampaio, J.L.M., Chanal, C., Sirot, D., Labia, R., Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp-240→Gly (2001) Antimicrob Agents Chemother, 45, pp. 2269-227

Reliable and rapid characterization of functional <it>FCN2</it> gene variants reveals diverse geographical patterns

<p>Abstract</p> <p>Background</p> <p>Ficolin-2 coded by <it>FCN2</it> gene is a soluble serum protein and an innate immune recognition element of the complement system. <it>FCN2</it> gene polymorphisms reveal distinct geographical patterns and are documented to alter serum ficolin levels and modulate disease susceptibility.</p> <p>Methods</p> <p>We employed a real-time PCR based on Fluorescence Resonance Energy Transfer (FRET) method to genotype four functional SNPs including <it>-986 G > A</it> (#rs3124952), <it>-602 G > A</it> (#rs3124953), <it>-4A > G</it> (#rs17514136) and <it>+6424 G > T</it> (#rs7851696) in the ficolin-2 (<it>FCN2)</it> gene. We characterized the <it>FCN2</it> variants in individuals representing Brazilian (n = 176), Nigerian (n = 180), Vietnamese (n = 172) and European Caucasian ethnicity (n = 165).</p> <p>Results</p> <p>We observed that the genotype distribution of three functional SNP variants (−<it>986 G > A, -602 G > A</it> and <it>-4A > G</it>) differ significantly between the populations investigated (<it>p</it> < 0.0001). The SNP variants were highly linked to each other and revealed significant population patterns. Also the distribution of haplotypes revealed distinct geographical patterns (<it>p</it> < 0.0001).</p> <p>Conclusions</p> <p>The observed distribution of the <it>FCN2</it> functional SNP variants may likely contribute to altered serum ficolin levels and this may depend on the different disease settings in world populations. To conclude, the use of FRET based real-time PCR especially for <it>FCN2</it> gene will benefit a larger scientific community who extensively depend on rapid, reliable method for <it>FCN2</it> genotyping.</p