Mapping of the natural resistance-associated macrophage protein 1 (NRAMP1) gene to pig chromosome 15

The natural resistance-associated macrophage protein 1 (NRAMP1) was mapped in the pig for study as a potential candidate gene in controlling pig resistance to Salmonella infection. Primers were designed from the pig cDNA to amplify a 1·6 kb fragment between exons 1 and 3. By using a pig-rodent somatic cell hybrid panel, NRAMP1 was mapped to pig chromosome 15 (SSC15) with 100% probability, and the regional assignment was SSC15q23-26 with 87% concordance. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker was developed by using the Hinf I enzyme and three alleles were identified from a population including 11 breeds. Linkage analysis confirmed the physical assignment by using the PiGMaP reference families. Pig NRAMP1 was linked to SSC15 markers S0088, S0149 and S0284 (LOD > 3). A small population study revealed large allele frequency differences among tested breeds. An A allele is only observed in dam (white) lines whereas a similar exclusivity of the C allele was seen in sire (colored) breeds.     

The natural resistance-associated macrophage protein and susceptibility to intracellular pathogens

Over 20 years ago it was recognised that murine susceptibility to several antigenically unrelated pathogens was influenced by a host genetic factor. Linkage studies suggested that Lsh, Ity, and Bcg, the leishmania-, salmonella-, and mycobacteria-susceptibility genes, may be one gene, located on mouse chromosome 1. A reverse genetics strategy identified a candidate gene, Nramp1, which was expressed only in reticuloendothelial cells. A single nonconservative amino acid substitution was found to correlate with the susceptibility genotype in 27 inbred mouse strains. The production of an Nramp1 gene-disrupted mouse and a transgenic mouse, which restored the resistance genotype, conclusively proved that Nramp1 is the Bcg/Lsh/Ity gene. The Nramp family includes genes expressed in both prokaryotic and eukaryotic species. These genes have provided clues to the possible function of Nramp1. The ubiquitously expressed gene Nramp2 is an Fe(2+) transporter and a mutation in this gene causes microcytic anaemia in mice and rats. The functions of Nramp1 and its human homologue, NRAMP1, remain unknown, though it is hypothesised that they may regulate the intraphagosomal concentration of Fe(2+) and/or other cations. The identification of polymorphisms in the human NRAMP1 gene has facilitated studies on the relevance of this gene to human mycobacterial susceptibility. NRAMP1 variant alleles are strongly associated with tuberculosis, indicating that this is an important mycobacterial-susceptibility gene in humans and confirming the usefulness of this mouse model in the study of human infectious disease susceptibility.     

Genetic and physical mapping of the natural resistance-associated macrophage protein 1 (NRAMP1) in chicken

The chicken natural resistance-associated macrophage protein 1 (NRAMP1) gene has been mapped by linkage analysis by use of a reference panel to develop the chicken molecular genetic linkage map and by fluorescence in situ hybridization. The chicken homolog of the murine Nramp1 gene was mapped to a linkage group located on Chromosome (Chr) 7q13, which includes three genes (CD28, NDUSF1, and EF1B) that have previously been mapped either to mouse Chr 1 or to human Chr 2q. Physical mapping by pulsed-field gel electrophoresis revealed that NRAMP1 is tightly linked to the villin gene and that the genomic organization (gene order and presence of CpG islands) of the chromosomal region carrying NRAMP1 is well conserved between the chicken and mammalian genomes. The regions on mouse Chr 1, human Chr 2q, and chicken Chr 7q that encompass NRAMP1 represent large conserved chromosomal segments between the mammalian and avian genomes. The chromosome mapping of the chicken NRAMP1 gene is a first step in determining its possible role in differential susceptibility to salmonellosis in this species.     

The natural resistance-associated macrophage protein 1 Slc11a1 (formerly Nramp1) and iron metabolism in macrophages

Slc11a1 (solute carrier family 11 member 1) (formerly Nramp1) modulation of iron metabolism in macrophages plays an important role in early phase macrophage activation, and therefore host innate immunity. This review focuses on the role of Nramp1 in intramacrophage iron metabolism, with emphasis on the two prevailing mechanisms of Nramp1 modulation of iron metabolism in macrophages.     

Determination of transmembrane topology of the Escherichia coli natural resistance-associated macrophage protein (Nramp) ortholog

The natural resistance-associated macrophage protein (Nramp) defines a conserved family of secondary metal transporters. Molecular evolutionary analysis of the Nramp family revealed the early duplication of an ancestral eukaryotic Nramp gene, which was likely derived from a bacterial ortholog and characterized as a proton-dependent manganese transporter MntH (Makui, H., Roig, E., Cole, S. T., Helmann, J. D., Gros, P., and Cellier, M. F. (2000) Mol. Microbiol. 35, 1065-1078). Escherichia coli MntH represents a model of choice to study structure function relationship in the Nramp protein family. Here, we report E. coli MntH transmembrane topology using a combination of in silico predictions, genetic fusion with cytoplasmic and periplasmic reporters, and MntH functional assays. Constructs of the secreted form of beta-lactamase (Blam) revealed extra loops between transmembrane domains 1/2, 5/6, 7/8, and 9/10, and placed the C terminus periplasmically; chloramphenicol acetyltransferase constructs indicated cytoplasmic loops 2/3, 6/7, 8/9, and 10/11. Two intra loops for which no data were produced (N terminus, intra loop 4/5) both display composition bias supporting their deduced localization. The extra loops 5/6 and 6/7 and periplasmic exposure of the C terminus were confirmed by targeted reporter insertion. Three of them preserved MntH function as measured by a disk assay of divalent metal uptake and a fluorescence assay of divalent metal-dependent proton transport, whereas a truncated form lacking transmembrane domain 11 was inactive. These results demonstrate that EcoliA is a type III integral membrane protein with 11 transmembrane domains transporting both divalent metal ions and protons.     

Role of toll-like receptor 2 and 4 gene polymorphisms in the development of allergic diseases with increased IgE levels

The aim of this research was to identify the role of the TLR2 (NP_003255.2) and TLR4 (NP_612564.1) gene polymorphisms with regard to the increase in the specific IgE production level and development of allergic diseases. Genotyping of three single nucleotide polymorphisms was performed using the polymerase chain reaction (PCR) method. The obtained results suggest that the most significant causative allergens are epidermal and household allergens (epidermis of cats, dogs, horse, D. farinae, and D. pteronyssinus). A relationship between the TLR2 (rs5743708) and TLR4 (rs4986790) polymorphisms and an increased production level of specific IgE in patients with allergic diseases was also observed.     

Natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts

Alphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter natural resistance-associated macrophage protein (NRAMP) as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were nonpermissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multipass membrane proteins for infection.     

Comparative fine maps of bovine toll-like receptor 4 and toll-like receptor 2 regions

Abstract Toll-like receptors are cell-surface receptors that activate innate and adaptive immune responses. We have used a 5000-rad, whole-genome radiation hybrid panel to map Toll-like receptor 4 (TLR4) to the distal end of bovine Chromosome (Chr) 8, and Toll-like receptor 2 (TLR2) to the proximal end of bovine Chr 17. To facilitate comparative mapping and contig construction, we have also used 5000- and 12,000-rad, whole-genome radiation hybrid panels to produce fine maps of the regions surrounding these genes in cattle. These fine maps triple the number of available markers in the TLR4 region and more than double the number of available markers in the TLR2 region. Comparative analyses show gene order conservation between the bovine Chr 8 region and human Chr 9, and between the bovine Chr 17 region and human Chr 4. In addition, the bovine Chr 8 region refines an evolutionary chromosomal breakpoint from a 10-megabase region to a 2.5-megabase region, and the bovine Chr 17 map suggests a new evolutionary chromosomal breakpoint.     

Structural characterisation and expression analysis of toll-like receptor 2 gene from catfish

Toll-like receptors (TLRs) are important components of innate immunity. They were found to recognise specific structures on pathogens termed pathogen-associated molecular patterns (PAMPs) and utilise conserved signaling pathways to activate pro-inflammatory cytokines and type-1 interferons. In spite of much understanding gained from the mammalian systems, many fish TLRs are unknown. Recent studies in Japanese flounder as well as in zebrafish suggested that the ligand binding and activation of inflammatory responses in fish may be different from and more complex than those found in mammals. In channel catfish, the major aquaculture species in the United States, only partial sequences of TLR3 and TLR5 were reported. As a part of efforts to characterise the innate immune components in channel catfish, here we cloned and sequenced both the cDNA and the gene for TLR2, a receptor believed mostly responsible for recognition of lipopeptides on the surface of most Gram-positive bacteria. However, expression analysis after infection with a Gram-negative bacterium, Edwardsiella ictaluri indicated that TLR2 was modestly down-regulated in the head kidney tissue of blue catfish, and with a similar pattern in the head kidney of channel catfish though the down-regulation in channel catfish was not statistically significant. In the spleen, an insignificant down-regulation was initially observed early after infection, with an increase of TLR expression later after infection. These results suggest the involvement of TLR2 in the responses after the bacterial infection. As LPS is believed to be the major PAMP for Gram-negative bacteria, additional research is warranted to determine the functions and mechanisms of TLR2 in infections of Gram-negative bacteria.     

Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling

Pattern recognition via Toll-like receptors (TLR) by antigen-presenting cells is an important element of innate immunity. We report that wild-type measles virus but not vaccine strains activate cells via both human and murine TLR2, and this is a property of the hemagglutinin (H) protein. The ability to activate cells via TLR2 by wild-type MV H protein is abolished by mutation of a single amino acid, asparagine at position 481 to tyrosine, as is found in attenuated strains, which is important for interaction with CD46, the receptor for these strains. TLR2 activation by MV wild-type H protein stimulates induction of proinflammatory cytokines such as interleukin-6 (IL-6) in human monocytic cells and surface expression of CD150, the receptor for all MV strains. Confirming the specificity of this interaction, wild-type H protein did not induce IL-6 release in macrophages from TLR2-/- mice. Thus, the unique property of MV wild-type strains to activate TLR2-dependent signals might essentially contribute not only to immune activation but also to viral spread and pathogenicity by upregulating the MV receptor on monocytes.     

In vitro response of the striped bass natural resistance-associated macrophage protein, Nramp, to LPS and Mycobacterium marinum exposure

Mycobacteriosis in Chesapeake Bay (USA) striped bass Morone saxatilis is an ongoing disease problem with important economic implications for a large commercial and recreational fishery. Additionally, striped bass serve as a reservoir of potential mycobacterial zoonoses. Recently, we described a striped bass gene homolog of the natural resistance-associated macrophage protein family (MsNramp), which is responsible for resistance to mycobacterial infections in mice. Striped bass MsNramp is strongly induced in peritoneal exudate cells (PE) in vivo after intraperitoneal injection with Mycobacterium spp. The purpose of the present study was to investigate short-term in vitro MsNramp expression and reactive oxygen intermediate (ROI) production in primary cultures of adherent PE after exposure to bacterial lipopolysaccharide (LPS), or live- or heat-killed (HK) Mycobacterium marinum. PE expressed significantly higher levels of MsNramp at 4 and 24 h post-treatment with live and HK M. marinum. MsNramp response to LPS was dose-dependent in these cells, with maximum expression at 4 h and 20 microg/ml LPS. Treatment of PE with LPS resulted in increased intracellular superoxide anion levels, whereas treatment with live M. marinum caused a significant depression. This study is the first report of induction of a teleost Nramp in vitro by mycobacteria, and supports findings of teleost Nramp induction by LPS.     

Studies on toll-like receptor stimuli responsiveness in HIV-1 and HIV-2 infections

Background: HIV-1 and HIV-2 are two related viruses with distinct clinical outcomes, where HIV-1 is more pathogenic and transmissible than HIV-2. The pathogenesis of both infections is influenced by the dysregulation and deterioration of the adaptive immune system. However, their effects on the responsiveness of innate immunity are less well known. Here, we report on toll-like receptor (TLR) stimuli responsiveness in HIV-1 or HIV-2 infections. Methods: Whole blood from 235 individuals living in Guinea-Bissau who were uninfected, infected with HIV-1, infected with HIV-2, and/or infected with HTLV-I, was stimulated with TLR7/8 and TLR9 agonists, R-848 and unmethylated CpG DNA. After TLR7/8 and TLR9 stimuli, the expression levels of IL-12 and IFN-α were related to gender, age, infection status, CD4⁺ T cell counts, and plasma viral load. Results: Defective TLR9 responsiveness was observed in the advanced disease stage, along with CD4⁺ T cell loss in both HIV-1 and HIV-2 infections. Moreover, TLR7/8 responsiveness was reduced in HIV-1 infected individuals compared with uninfected controls. Conclusions: Innate immunity responsiveness can be monitored by whole blood stimulation. Both advanced HIV-1 and HIV-2 infections may cause innate immunity dysregulation.     

Nasopharyngeal bacterial colonization and gene polymorphisms of mannose-binding lectin and toll-like receptors 2 and 4 in infants

Background

Human nasopharynx is often colonized by potentially pathogenic bacteria. Gene polymorphisms in mannose-binding lectin (MBL), toll-like receptor (TLR) 2 and TLR4 have been reported. The present study aimed to investigate possible association between nasopharyngeal bacterial colonization and gene polymorphisms of MBL, TLR2 and TLR4 in healthy infants.

Methodology/Principal Findings

From August 2008 to June 2010, 489 nasopharyngeal swabs and 412 blood samples were taken from 3-month-old healthy Finnish infants. Semi-quantitative culture was performed and pyrosequencing was used for detection of polymorphisms in MBL structural gene at codons 52, 54, and 57, TLR2 Arg753Gln and TLR4 Asp299Gly. Fifty-nine percent of subjects were culture positive for at least one of the four species: 11% for Streptococcus pneumoniae, 23% for Moraxella catarrhalis, 1% for Haemophilus influenzae and 25% for Staphylococcus aureus. Thirty-two percent of subjects had variant types in MBL, 5% had polymorphism of TLR2, and 18% had polymorphism of TLR4. Colonization rates of S. pneumoniae and S. aureus were significantly higher in infants with variant types of MBL than those with wild type (p = .011 and p = .024). Colonization rates of S. aureus and M. catarrhalis were significantly higher in infants with polymorphisms of TLR2 and of TLR4 than those without (p = .027 and p = .002).

Conclusions

Our study suggests that there is an association between nasopharyngeal bacterial colonization and genetic variation of MBL, TLR2 and TLR4 in young infants. This finding supports a role for these genetic variations in susceptibility of children to respiratory infections.     

Polymorphisms of the porcine dopamine beta-hydroxylase gene and their relation to reproduction and piglet survivability in an Iberian x Meishan F2 intercross

The goals of this study were to sequence and physically map the porcine dopaminebeta-hydroxylase (DBH) gene, as well as to perform an association study between polymorphisms of this gene and the reproductive performance and piglet survivability of F(2) pigs from an Iberian x Meishan cross. The porcine DBH gene was positioned by RH mapping near the telomere of chromosome 1q2.13, close to markers SSC10D08 and SW1301. Sequencing of DBH cDNAs from 10 pigs revealed the existence of six nucleotide polymorphisms, two of which led to non-synonymous amino acid substitutions within exon 3 at positions 463A>G and 616A>G that corresponded to Thr155Ala and Lys206Glu respectively. Three haplotypes segregated in an Iberian x Meishan population: DBH(X) (A(436)-A(616)), DBH(Y) (A(436)-G(616)) and DBH(Z) (G(436)-G(616)). The DBH haplotypes significantly affected rectal temperatures 1 h after birth (P = 0.002) and have a suggestive effect on the time to first colostrum intake (P = 0.019) and on birth weight (P = 0.019).