Kedarnath N. Sastry

Mannose-binding Lectin-deficient Mice Are Susceptible to Infection with Staphylococcus aureus

Gram-positive organisms like Staphylococcus aureus are a major cause of morbidity and mortality worldwide. Humoral response molecules together with phagocytes play a role in host responses to S. aureus. The mannose-binding lectin (MBL, also known as mannose-binding protein) is an oligomeric serum molecule that recognizes carbohydrates decorating a broad range of infectious agents including S. aureus. Circumstantial evidence in vitro and in vivo suggests that MBL plays a key role in first line host defense. We tested this contention directly in vivo by generating mice that were devoid of all MBL activity. We found that 100% of MBL-null mice died 48 h after exposure to an intravenous inoculation of S. aureus compared with 45% mortality in wild-type mice. Furthermore, we demonstrated that neutrophils and MBL are required to limit intraperitoneal infection with S. aureus. Our study provides direct evidence that MBL plays a key role in restricting the complications associated with S. aureus infection in mice and raises the idea that the MBL gene may act as a disease susceptibility gene against staphylococci infections in humans.

Pulmonary surfactant proteins A and D enhance neutrophil uptake of bacteria

The collectins are a class of collagenous lectin proteins present in serum and pulmonary secretions [pulmonary surfactant protein (SP) A and SP-D] that are believed to participate in innate immune responses to various pathogens. With the use of flow cytometric and fluorescent-microscopic assays, SP-A and SP-D were shown to increase calcium-dependent neutrophil uptake of Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus. Evidence is provided that the collectins enhanced bacterial uptake through a mechanism that involved both bacterial aggregation and direct actions on neutrophils. The degree of multimerization of SP-D preparations was a critical determinant of both aggregating activity and potency in enhancing bacterial uptake. The mechanisms of opsonizing activity of SP-D and SP-A differed in important respects from those of opsonizing antibodies. These results provide the first evidence that surfactant collectins may promote neutrophil-mediated clearance of bacteria in the lung independently of opsonizing antibody.

Enhanced Antiviral and Opsonic Activity of a Human Mannose-Binding Lectin and Surfactant Protein D Chimera

The carbohydrate recognition domains (CRDs) of human serum mannose-binding lectin (MBL) and pulmonary surfactant protein D (SP-D) have distinctive monosaccharide-binding properties, and their N-terminal and collagen domains have very different quaternary structures. We produced a chimeric protein containing the N terminus and collagen domain of human SP-D and the neck region and CRD of human MBL (SP-D/MBLneck+CRD) to create a novel human collectin. The chimera bound to influenza A virus (IAV), inhibited IAV hemagglutination activity and infectivity, and induced aggregation of viral particles to a much greater extent than MBL. Furthermore, SP-D/MBLneck+CRD caused much greater increases in neutrophil uptake of, and respiratory burst responses to, IAV than MBL. These results indicate that pathogen interactions mediated by the MBL CRD are strongly influenced by the N-terminal and collagen-domain backbone to which it is attached. The presence of the CRD of MBL in the chimera resulted in altered monosaccharide binding properties compared with SP-D. As a result, the chimera caused greater aggregation and neutralization of IAV than SP-D. Distinctive functional properties of collectin collagenous domains and CRDs can be exploited to generate novel human collectins with potential for therapy of influenza.

Characterization of murine mannose-binding protein genes Mbl1 and Mbl2 reveals features common to other collectin genes

Mannose-binding protein (MBP) is a member of a family of collagenous lectins (collectins), which are believed to play an important role in first-line host defense. In this study, the two genes encoding MBP in mice-Mbl1 and Mbl2-have been isolated and their exon-intron structure studied to understand their evolutionary relationship to the single human (MBL) and the two rat MBP genes. Mouse Mbl1 and Mbl2 have five and six exons, respectively. The structure of the mouse Mbl genes is similar to that of the rat and human MBP genes and shows homology to the other collectin genes, with the entire carbohydrate recognition domain being encoded in a single exon and all introns being in phase 1. The MBP encoded by mouse Mbl1 with three cysteines in the first coding exon, like the rat Mbl1 and human MBL, is capable of a higher degree of multimerization and has apparent ability to fix complement in the absence of antibody or C1q. However, the structural features of other exons, that is, the larger size of collagen domain region in the first coding exon (64 bp in Mbl2 vs 46 bp in Mbl1) and the smaller size of the exon encoding the trimerization domain (69 bp in Mbl2 vs 75 bp in Mbl1) reveal that the single human MBL gene is closely related to rodent Mbl2 rather than rodent Mbl1. The findings in this study suggest that in contrast to the evolution of another collectin gene-bovine surfactant protein-D-which duplicated in bovidae after divergence from humans, MBP gene most likely duplicated prior to human-roden divergence, and that the human homolog to Mbl1 was perhaps lost during evolution.

Regulation of the Mannan-Binding Lectin Pathway of Complement on Neisseria gonorrhoeae by C1-Inhibitor and α2-Macroglobulin

We examined complement activation by Neisseria gonorrhoeae via the mannan-binding lectin (MBL) pathway in normal human serum. Maximal binding of MBL complexed with MBL-associated serine proteases (MASPs) to N. gonorrhoeae was achieved at a concentration of 0.3 μg/ml. Preopsonization with MBL-MASP at concentrations as low as 0.03 μg/ml resulted in ∼60% killing of otherwise fully serum-resistant gonococci. However, MBL-depleted serum (MBLdS) reconstituted with MBL-MASP before incubation with organisms (postopsonization) failed to kill at a 100-fold higher concentration. Preopsonized organisms showed a 1.5-fold increase in C4, a 2.5-fold increase in C3b, and an ∼25-fold increase in factor Bb binding; enhanced C3b and factor Bb binding was classical pathway dependent. Preopsonization of bacteria with a mixture of pure C1-inhibitor and/or α2-macroglobulin added together with MBL-MASP, all at physiologic concentrations before adding MBLdS, totally reversed killing in 10% reconstituted serum. Reconstitution of MBLdS with supraphysiologic (24 μg/ml) concentrations of MBL-MASP partially overcame the effects of inhibitors (57% killing in 10% reconstituted serum). We also examined the effect of sialylation of gonococcal lipooligosaccharide (LOS) on MBL function. Partial sialylation of LOS did not decrease MBL or C4 binding but did decrease C3b binding by 50% and resulted in 80% survival in 10% serum (lacking bacteria-specific Abs) even when sialylated organisms were preopsonized with MBL. Full sialylation of LOS abolished MBL, C4, and C3b binding, resulting in 100% survival. Our studies indicate that MBL does not participate in complement activation on N. gonorrhoeae in the presence of “complete” serum that contains C1-inhibitor and α2-macroglobulin.

Somatic cell mapping of conglutinin (CGN1) to cattle syntenic group U29 and fluorescence in situ localization to Chromosome 28

A 260-bp genomic PstI fragment, which encodes a portion of the carbohydrate recognition domain, was used along with hybrid somatic cells to map the conglutinin gene (CGN1) to domestic cow (Bos taurus) syntenic group U29. In turn, a cosmid containing the entire bovine CGN1 was used with fluorescence in situ hybridization to sublocalize this gene to cattle chromosome (Chr) (BTA) 28 band 18. Since BTA 28 and several of the other small acrocentric autosomes of cattle are difficult to discriminate, we have also chromosomally sublocalized CGN1 to the p arm of the lone biarmed autosome of the gaur (Bos gaurus). The use of the gaur 2/28 Robertsonian as a marker chromosome and our assignment of CGN1 to BTA 28 should help resolve some of the nomenclatural questions involving this cattle chromosome.

The murine mannose-binding protein genes (Mbl1 and Mbl2) localize to Chromosomes 14 and 19

1Section of Genetics, Childrens Mercy Hospital/UMKC School of Medicine, Kansas City, Missouri 64108, USA 2Division of Biomedical Sciences, Mercer University School of Medicine, Macon, Georgia 31207, USA 3Division of Hematology/Oncology, Childrens Hospital and Harvard Medical School, Boston, Masschusetts 02115, USA 4Department of Pathology, Boston University School of Medicine, Boston, Massachusetts 02118, USA

Bovine conglutinin (BC) mRNA expressed in liver : cloning and characterization of the BC cDNA reveals strong homology to surfactant protein-D

Bovine conglutinin (BC) is a C-type lectin isolated from bovine serum that appears to play a role in first-line host defense. The BC cDNA was cloned from a bovine liver library and the nucleotide (nt) sequence of 1519 bp was determined. The longest open reading frame encoded a 20-amino-acid (aa) signal sequence and a mature protein of 351 aa. Analysis of the nt and deduced aa sequences revealed 87 and 78% identity, respectively, with the sequences of another vertebrate lectin: bovine surfactant protein-D (SP-D). Of interest, the expression of the BC mRNA, as determined by RNase protection assay, is restricted to liver, unlike bovine SP-D, a lung-surfactant protein.