MBL2 deficiency is associated with higher genomic bacterial loads during meningococcemia in young children.

Mannose binding lectin (MBL2) is a soluble pattern recognition receptor that is key to generating innate immune responses to invasive infection, including against the cardinal Gram-negative bacterium Neisseria meningitidis. Individuals homozygous or heterozygous for any of three variant alleles of MBL2 (O/O or A/O genotypes) have deficient concentrations of MBL2 in circulating blood, but previous studies linking MBL deficiency to susceptibility to meningococcal disease have not revealed a consistent association. We genotyped 741 patients with microbiologically-proven meningococcal disease and correlated MBL2 genotype with plasma bacterial load of N. meningitidis with blood samples taken during hospital admission. We show that individuals with genotypes compatible with MBL2 deficiency have higher measurable levels of bacterial plasma genomic load with the greatest effect seen in children <2 years of age. However, the overall impact of this is minor, because there was no evidence that such genotypes are more common in children with meningococcal disease compared with uninfected cohorts. The findings suggest that MBL2 supports innate immune defence against meningococcal disease in the early months of life, before acquired immunity is sufficiently robust for effective natural protection.

Mannose-binding lectin is present in human semen and modulates cellular adhesion of Neisseria gonorrhoeae in vitro.

Mannose-binding lectin (MBL) is an innate immune molecule present in blood and some mucosal tissues, which can influence microbial attachment and inflammatory responses of host cells during infection. In this study MBL was found to be present at a low concentration in semen samples in the range 1.2-24.9 ng/ml. Co-incubation of bacteria with semen resulted in the binding of MBL to the bacterial surface. Neisseria gonorrhoeae is a common cause of genitourinary infection. MBL bound to N. gonorrhoeae with strain-to-strain variation in the intensity of binding and nature of the bacterial receptor. Pretreatment with MBL concentrations similar to those found in human serum modulated the adhesion of N. gonorrhoeae strain FA1090 but not strain MS11 to epithelial cells. This effect was dose-dependent. This work demonstrates that MBL is present in human semen and modifies cellular responses to N. gonorrhoeae in a concentration-dependent manner.

Antibodies to mannose binding lectin in patients with systemic lupus erythematosus.

Deficiency of mannose binding lectin (MBL), a C-type lectin with structural similarities to C1q, has been shown to predispose to the development of systemic lupus erythematosus (SLE). Some patients have low serum MBL levels which cannot be explained by either structural gene mutations or promoter polymorphisms. The objective of this study was to detect the presence of autoantibodies against MBL and to evaluate their relationship to serum MBL levels. Anti-MBL antibodies of IgM and IgG classes from consecutive SLE patients (n = 135) and healthy subjects (n = 50) were measured by an in-house ELISA. Using the 90th percentile of controls as a cutoff, more SLE patients [23.7% (32/135)] were found to have IgG anti-MBL antibodies than normal controls [10.0% (5/50)] (P = 0.04). The same trend was observed when ethnicity was taken into account by analysing Caucasians alone (n = 90). IgM anti-MBL antibodies were only found in two SLE patients (2/22, 9.1%) who had no concomitant IgG anti-MBL antibodies. Serum levels of IgG anti-MBL antibodies were found to correlate with serum MBL levels (r = 0.55, P = 0.049). However, the levels of anti-MBL antibodies did not correlate with overall disease activity. Thus the production of anti-MBL antibodies is likely to be a specific antigen-driven process. Its role in lupus pathogenesis remains to be elucidated.

Anti-microbial activities of mannose-binding lectin.

Mannose-binding lectin (MBL; also known as mannan-binding lectin) is involved in first-line defence by binding to bacteria, viruses, protozoa and helminths through a pattern-recognition mode of detection and then initiating a range of host responses. Currently, we have been unable to extrapolate from what we know of the biochemistry of MBL binding to predict accurately the interaction of MBL with individual micro-organisms; even subtle surface alterations have been shown to have an extensive impact on MBL-mediated recognition of pathogens. MBL has a major protective effect through activation of the complement system via MBL-associated serine proteases (MASPs). This can cause the lysis of Gram-negative bacteria and also opsonize a wide spectrum of potential pathogens for phagocytosis. MBL may also influence phagocytosis in the absence of complement activation through an interaction with one or more collectin receptors. This may also be the basis for a direct effect of the protein on inflammatory responses. MBL can alter the function of microbial structures, such as gp120 of HIV, to prevent infection. The protein may also interact with the components of other cascade systems such as the clotting system, which will have a role in microbial pathogenesis. An understanding of these basic mechanisms will be vital if we are to use purified or recombinant MBL in therapeutic applications.

Mannose-binding lectin regulates the inflammatory response of human professional phagocytes to Neisseria meningitidis serogroup B.

The influence of the innate immune protein mannose-binding lectin (MBL) on the response of human phagocytes to Neisseria meningitidis was investigated. MBL increased the association of killed meningococci with neutrophils, monocytes, and macrophages by increasing the proportion of cells that recognized bacteria. MBL down-regulated the normal change in expression of the leukocyte adhesion molecules CD11b and CD62L. In an ex vivo model, the addition of MBL to the blood of MBL-deficient donors influenced the production of monocyte-derived inflammatory cytokines. The addition of high concentrations of MBL (>6 microg/mL) profoundly decreased the production of interleukin (IL)-6, IL-1beta, and tumor necrosis factor-alpha by monocytes in response to meningococci, whereas lower concentrations enhanced the production of IL-6 and IL-1beta. These results suggest that MBL not only is involved in complement activation but also is a potent regulator of inflammatory pathways and, as such, may affect the severity of meningococcal disease.

Mannose-binding lectin accelerates complement activation and increases serum killing of Neisseria meningitidis serogroup C.

The capacity for different lipo-oligosaccharide (LOS) sialylation patterns of Neisseria meningitidis serogroup C to influence the binding and function of the innate humoral component, mannose-binding lectin (MBL), was investigated. By use of flow cytometry and immunogold electron microscopy, a clinical isolate with reduced endogenous LOS sialylation was found to bind more MBL than did strains with higher endogenous sialylation. MBL binding was reduced but not ablated if the same strain was allowed to exogenously sialylate its LOS structures after incubation with cytidine-5'-monophospho-neuraminic acid. MBL binding led to an increased rate of complement activation, with enhanced deposition of the complement components C4 and C5b-9, and this correlated with an increase in bactericidal activity. LOS sialylation appears to be an important determinant of MBL binding to N. meningitidis and can modulate complement-dependent killing of the bacterium. These findings could explain the observed susceptibility to meningococcal disease of individuals genetically deficient in MBL.

The drug/metabolite transporter superfamily.

Previous work defined several families of secondary active transporters, including the prokaryotic small multidrug resistance (SMR) and rhamnose transporter (RhaT) families as well as the eukaryotic organellar triose phosphate transporter (TPT) and nucleotide-sugar transporter (NST) families. We show that these families as well as several other previously unrecognized families of established or putative secondary active transporters comprise a large ubiquitous superfamily found in bacteria, archaea and eukaryotes. We have designated it the drug/metabolite transporter (DMT) superfamily (transporter classification number 2.A.7) and have shown that it consists of 14 phylogenetic families, five of which include no functionally well-characterized members. The largest family in the DMT superfamily, the drug/metabolite exporter (DME) family, consists of over 100 sequenced members, several of which have been implicated in metabolite export. Each DMT family consists of proteins with a distinctive topology: four, five, nine or 10 putative transmembrane alpha helical spanners (TMSs) per polypeptide chain. The five TMS proteins include an N-terminal TMS lacking the four TMS proteins. The full-length proteins of 10 putative TMSs apparently arose by intragenic duplication of an element encoding a primordial five-TMS polypeptide. Sequenced members of the 14 families are tabulated and phylogenetic trees for all the families are presented. Sequence and topological analyses allow structural and functional predictions.

Mannose-binding lectin: targeting the microbial world for complement attack and opsonophagocytosis.

Mannose-binding lectin (MBL) is an important constituent of the innate immune system. This protein binds through multiple lectin domains to the repeating sugar arrays that decorate many microbial surfaces, and is then able to activate the complement system through a specific protease called MBL-associated protease-2. We have used flow cytometry to study both the binding of MBL to microorganisms and the subsequent activation of complement. For selected Gram-negative organisms, such as Salmonella and Neisseria, we have examined the relative roles of lipopolysaccharide (LPS) structure and capsule in determining binding and conclude that the LPS is of major importance. Our results from studies with several clinically relevant organisms also show that MBL binding detected by flow cytometry leads to measurable activation of purified C4, suggesting that the bound lectin is capable of initiating opsonophagocytosis and/or bacterial lysis. There is an increasing literature suggesting that MBL deficiency, which mainly results from three relatively common single point mutations in exon 1 of the gene, predisposes both to infection by extracellular pathogens and to autoimmune disease. In addition, the protein also modulates disease severity, at least in part through a complex, dose-dependent influence on cytokine production. The mechanisms and signalling pathways involved in such processes remain to be elucidated.

Differential recognition of obligate anaerobic bacteria by human mannose-binding lectin.

Deficiency of the innate, humoral immune component mannose-binding lectin (MBL) predisposes individuals to a variety of infections, but the importance of MBL in infection by anaerobes has not been addressed. The attachment of MBL to a wide range of anaerobic bacteria associated with human disease and colonization was surveyed. The results suggest that for the species we examined, resistance to MBL binding may be associated with organisms that are more commonly pathogenic and that MBL binding to some bacteria may be phase variable.

Mannose-binding lectin is a component of innate mucosal defense against Cryptosporidium parvum in AIDS.

BACKGROUND & AIMS: Nonimmune mechanisms of mucosal defense seem to be biologically important and might explain the observed variability in the course of enteric infection in immunodeficiency. Mannose-binding lectin (MBL) deficiency is associated with persistent diarrhea in children. We found that genetic determinants of MBL deficiency appear to predispose to cryptosporidiosis in patients with the acquired immunodeficiency syndrome (AIDS), and went on to study interactions of MBL and complement on Cryptosporidium parvum sporozoites. METHODS: This study involved cross-sectional study of MBL genotype and enteric infection in 72 Zambian AIDS patients with diarrhea, immunofluorescence analysis of MBL and C4 binding to C. parvum, and immunoblotting for MBL and complement in small intestinal fluid. RESULTS: Individuals homozygous for MBL structural gene mutations were at increased risk of cryptosporidiosis (odds ratio, 8.2; 95% confidence interval, 1. 5-42; P = 0.02). Lectin-mediated and concentration-dependent binding of purified MBL was detected on sporozoites but not oocysts, and MBL activated C4 on sporozoites. MBL, C3, C4, and albumin were detected in small intestinal fluid in half the patients tested, suggesting transudation of serum components into the enteropathic gut. CONCLUSIONS: The increased risk of cryptosporidiosis in MBL deficiency appears to include patients with AIDS. It may operate through MBL-mediated complement activation on sporozoites.

Restricted polymorphism of the mannose-binding lectin gene of indigenous Australians.

Mannose-binding lectin (MBL) is an important complement-activating protein of the human innate immune system. Deficiency of MBL is associated with an increased risk of various infections and arises from three structural gene mutations in exon 1 (variants B, C and D) and/or the presence of a low efficiency promoter. The C allele is found in sub-Saharan Africa whereas the B allele is found elsewhere, suggesting that these mutations occurred after the suggested hominid migration out of Africa [100-150 000 years before present (BP)]. Paradoxically, these alleles may have a selective advantage in protection against intracellular pathogens and occur at particularly high frequencies in sub-Saharan Africa (C variant) and South America (B variant). Since hominids reached Australia at least 50 000 years ago, a study of MBL polymorphisms in the indigenous population was of interest. Using heteroduplex technology we found a paucity of MBL structural gene mutations in two population groups from geographically distinct regions. Of 293 individuals tested, 289 were wild-type and four were heterozygous for either the B or D allele. In each individual with an MBL mutation the HLA haplotype profile suggested some Caucasian admixture. We also found a restricted range of MBL promoter haplotypes and the serum MBL levels were higher than those of any other ethnic group studied to date (median 3.07 microg/ml). Our data suggest that the B mutation probably arose between 50 000 and 20 000 BP. Its absence from the founder gene pool of indigenous Australians may also partly explain their vulnerability to intracellular infections such as tuberculosis.

The lipopolysaccharide structures of Salmonella enterica serovar Typhimurium and Neisseria gonorrhoeae determine the attachment of human mannose-binding lectin to intact organisms.

Mannose-binding lectin (MBL) is an important component of the innate immune system. It binds to the arrays of sugars commonly presented by microorganisms and activates the complement system independently of antibody. Despite detailed knowledge of the stereochemical basis of MBL binding, relatively little is known about how bacterial surface structures influence binding of the lectin. Using flow cytometry, we have measured the binding of MBL to a range of mutants of Salmonella enterica serovar Typhimurium and Neisseria gonorrhoeae which differ in the structure of expressed lipopolysaccharide (LPS). For both organisms, the possession of core LPS structures led to avid binding of MBL, which was abrogated by the addition of O antigen (Salmonella serovar Typhimurium) or sialic acid (N. gonorrhoeae). Truncation of the LPS within the core led to lower levels of MBL binding. It was not possible to predict the magnitude of MBL binding from the identity of the LPS terminal sugar alone, indicating that the three-dimensional disposition of LPS molecules is probably also of importance in determining MBL attachment. These results further support the hypothesis that LPS structure is a major determinant of MBL binding.

A broad-specificity multidrug efflux pump requiring a pair of homologous SMR-type proteins.

The Bacillus subtilis genome encodes seven homologues of the small multidrug resistance (SMR) family of drug efflux pumps. Six of these homologues are paired in three distinct operons, and coexpression in Escherichia coli of one such operon, ykkCD, but not expression of either ykkC or ykkD alone, gives rise to a broad specificity, multidrug-resistant phenotype including resistance to cationic, anionic, and neutral drugs.

Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition.

Mannose-binding lectin (MBL) is a collagenous serum lectin believed to be of importance in innate immunity. Genetically determined low levels of the protein are known to predispose to infections. In this study the binding of purified MBL to pathogens isolated from immunocompromised children was investigated by flow cytometry. Diverse Candida species, Aspergillus fumigatus, Staphylococcus aureus, and beta-hemolytic group A streptococci exhibited strong binding of MBL, whereas Escherichia coli, Klebsiella species, and Haemophilus influenzae type b were characterized by heterogeneous binding patterns. In contrast, beta-hemolytic group B streptococci, Streptococcus pneumoniae, and Staphylococcus epidermidis showed low levels of binding. Bound MBL was able to promote C4 deposition in a concentration-dependent manner. We conclude that MBL may be of importance in first-line immune defense against several important pathogens.

New glycoprotein-associated amino acid transporters.

The L-type amino acid transporter LAT1 has recently been identified as being a disulfide-linked "light chain" of the ubiquitously expressed glycoprotein 4F2hc/CD98. Several LAT1-related transporters have been identified, which share the same putative 12-transmembrane segment topology and also associate with the single transmembrane domain 4F2hc protein. They display differing amino acid substrate specificities, transport kinetics and localizations such as, for instance, y(+)LAT1 which is localized at the basolateral membrane of transporting epithelia, and the defect of which causes lysinuric protein intolerance. The b(0,+)AT transporter which associates with the 4F2hc-related rBAT protein to form the luminal high-affinity diamino acid transporter defective in cystinuria, belongs to the same family of glycoprotein-associated amino acid transporters (gpaATs). These glycoprotein-associated transporters function as amino acid exchangers. They extend the specificity range of vectorial amino acid transport when located in the same membrane as carriers that unidirectionally transport one of the exchanged substrates. gpaATs belong to a phylogenetic cluster within the amino acid/polyamine/choline (APC) superfamily of transporters. This cluster, which we designate the LAT family (named after its first vertebrate member), includes some members from nematodes, yeast and bacteria. The latter of these proteins presumably lack association with a second subunit. In this review, we focus on the animal members of the LAT cluster that form, together with some of the nematode members, the family of glycoprotein-associated amino acid transporters (gpaAT family).

Endothelial adhesion molecule expression and its inhibition by recombinant bactericidal/permeability-increasing protein are influenced by the capsulation and lipooligosaccharide structure of Neisseria meningitidis.

Vascular endothelial injury is responsible for many of the clinical manifestations of severe meningococcal disease. Binding and migration of activated host inflammatory cells is a central process in vascular damage. The expression and function of adhesion molecules regulate interactions between leukocytes and endothelial cells. Little is known about how meningococci directly influence these receptors. In this study we have explored the effect of Neisseria meningitidis on endothelial adhesion molecule expression and found this organism to be a potent inducer of the adhesion molecules CD62E, ICAM-1, and VCAM-1. Exposure of endothelium to a serogroup B strain of Neisseria meningitidis, B1940, and a range of isogenic mutants revealed that lipooligosaccharide (LOS) structure and capsulation influence the expression of adhesion molecules. Following only a brief exposure (15 min) to the bacteria, there were large differences in the capacity of the different mutants to induce vascular cell adhesion molecules, with the unencapsulated and truncated LOS strains being most potent (P < 0.05). Furthermore, the pattern of cell adhesion molecule expression was different with purified endotoxin from that with intact bacteria. Meningococci were more potent stimuli of CD62E expression than was endotoxin, whereas endotoxin was at least as effective as meningococci in inducing ICAM-1 and VCAM-1. The effect of bactericidal/permeability increasing protein (rBPI(21)), an antibacterial molecule with antiendotoxin properties, was also dependent on LOS structure. The strains which possessed a truncated or nonsialylated LOS, whether capsulated or not, were more sensitive to the inhibitory effects of rBPI(21). These findings could have important implications for the use of antiendotoxin therapy in meningococcal disease.

Phylogenetic characterization of novel transport protein families revealed by genome analyses.

As a result of recent genome sequencing projects as well as detailed biochemical, molecular genetic and physiological experimentation on representative transport proteins, we have come to realize that all organisms possess an extensive but limited array of transport protein types that allow the uptake of nutrients and excretion of toxic substances. These proteins fall into phylogenetic families that presumably reflect their evolutionary histories. Some of these families are restricted to a single phylogenetic group of organisms and may have arisen recently in evolutionary time while others are found ubiquitously and may be ancient. In this study we conduct systematic phylogenetic analyses of 26 families of transport systems that either had not been characterized previously or were in need of updating. Among the families analyzed are some that are bacterial-specific, others that are eukaryotic-specific, and others that are ubiquitous. They can function by either a channel-type or a carrier-type mechanism, and in the latter case, they are frequently energized by coupling solute transport to the flux of an ion down its electrochemical gradient. We tabulate the currently sequenced members of the 26 families analyzed, describe the properties of these families, and present partial multiple alignments, signature sequences and phylogenetic trees for them all.

The amino acid/auxin:proton symport permease family.

Amino acids and their derivatives are transported into and out of cells by a variety of permease types which comprise several distinct protein families. We here present a systematic analysis of a group of homologous transport proteins which together comprise the eukaryotic-specific amino acid/auxin permease (AAAP) family (TC #2. 18). In characterizing this family, we have (1) identified all sequenced members of the family, (2) aligned their sequences, (3) identified regions of striking conservation, (4) derived a family-specific signature sequence, and (5) proposed a topological model that appears to be applicable to all members of the family. We have also constructed AAAP family phylogenetic trees and dendrograms using six different programs that allow us to trace the evolutionary history of the family, estimate the relatedness of proteins from dissimilar organismal phyla, and evaluate the reliability of the different programs available for phylogenetic studies. The TREE and neighbor-joining programs gave fully consistent results while CLUSTAL W gave similar but non-identical results. Other programs gave less consistent results. The phylogenetic analyses reveal (1) that many plant AAAP family proteins arose recently by multiple gene duplication events that occurred within a single organism, (2) that some plant members of the family with strikingly different specificities diverged early in evolutionary history, and (3) that AAAP family proteins from fungi and animals diverged from the plant proteins long ago, possibly when animals, plants and fungi diverged from each other. The Neurospora protein nevertheless exhibits overlapping specificity with those found in plants. Preliminary evidence is presented suggesting that proteins of the AAAP family are distantly related to proteins of the large ubiquitous amino acid/polyamine/choline family (TC #2.3) as well as to those of two small bacterial amino acid transporter families, the ArAAP family (TC #2.42) and the STP family (TC #2.43).

The major facilitator superfamily.

In 1998 we updated earlier descriptions of the largest family of secondary transport carriers found in living organisms, the major facilitator superfamily (MFS). Seventeen families of transport proteins were shown to comprise this superfamily. We here report expansion of the MFS to include 29 established families as well as five probable families. Structural, functional, and mechanistic features of the constituent permeases are described, and each newly identified family is shown to exhibit specificity for a single class of substrates. Phylogenetic analyses define the evolutionary relationships of the members of each family to each other, and multiple alignments allow definition of family-specific signature sequences as well as all well-conserved sequence motifs. The work described serves to update previous publications and allows extrapolation of structural, functional and mechanistic information obtained with any one member of the superfamily to other members with limitations determined by the degrees of sequence divergence.

Activation of complement by mannose-binding lectin on isogenic mutants of Neisseria meningitidis serogroup B.

Mannose-binding lectin (MBL) is a serum protein that has been demonstrated to activate the classical complement pathway and to function directly as an opsonin. Although MBL deficiency is associated with a common opsonic defect and a predisposition to infection, the role of the protein in bacterial infection remains unclear. We have investigated MBL binding to Neisseria meningitidis serogroup B1940 and three isogenic mutants, and the subsequent activation of the two major isoforms of C4 (C4A and C4B) by an associated serine protease, MASP. The mutants lacked expression of the capsular polysaccharide (siaD-), the lipo-oligosaccharide (LOS) outer core that prevented LOS sialylation (cpsD-), or both capsule and LOS outer core (cps-). Using flow cytometry, it was possible to detect strong MBL binding to the cps- and cpsD- mutants over a wide range of concentrations. In contrast, minimal or no MBL binding was detected on the parent organism, with binding to siaD- only at higher MBL concentrations. C4 was activated and bound by mutants that had previously bound MBL/MASP, but there was no significant difference in the amounts of C4A and C4B bound. When sialic acid residues were removed from the parent organism by neuraminidase treatment, the binding of both MBL and C4 increased significantly. Our results suggest that MBL may bind to and activate complement on these encapsulated organisms, and the major determinants of these effects are the LOS structure and sialylation.