Evidence Supporting a Role for Mammalian Chitinases in Efficacy of Caspofungin against Experimental Aspergillosis in Immunocompromised Rats

Objectives:Caspofungin, currently used as salvage therapy for invasive pulmonary aspergillosis (IPA), strangely only causes morphological changes in fungal growth in vitro but does not inhibit the growth. In vivo it has good efficacy. Therefore the question arises how this in vivo activity is reached. Caspofungin is known to increase the amount of chitin in the fungal cell wall. Mammals produce two chitinases, chitotriosidase and AMCase, which can hydrolyse chitin. We hypothesized that the mammalian chitinases play a role in the in vivo efficacy of caspofungin.Methods:In order to determine the role of chitotriosidase and AMCase in IPA, both chitinases were measured in rats which did or did not receive caspofungin treatment. In order to understand the role of each chitinase in the breakdown of the caspofungin-exposed cells, we also exposed caspofungin treated fungi to recombinant enzymes in vitro.Results:IPA in immunocompromised rats caused a dramatic increase in chitinase activity. This increase in chitinase activity was still noted when rats were treated with caspofungin. In vitro, it was demonstrated that the action of both chitinases were needed to lyse the f

Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis

The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C173) of Gal-3 or lysine (K166) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial–host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization

<i>Arabidopsis</i> YAC restriction mapping

The approach of partial restriction mapping and vector hybridisation has been used to restriction map and align six yeast artificial chromosomes (YACs) corresponding to the top arm (~27.9 centiMorgans, cM) of Arabidopsis chromosome 5 and confirm the chimeric nature of a further four clones which map to this region. The restriction endonucleases Sma1 and Sfi1 which recognise rare-medium frequency sites in the Arabidopsis genome were used. This work has restriction mapped a 315 kb region that includes a number of genes implicated in floral development, namely PISTILLATA and TOUSLED, and a number of uncharacterised genes involved in male gametogenesis (e.g., Ms1 and Ms37). The information generated can be used to transcriptionally map genes to this contig and will provide data for the isolation of several uncharacterised floral development genes which lie in this region. This approach has demonstrated how large tracts of YAC DNA can be mapped and aligned to show the presence/absence of chimeric YAC clones and provide detailed restriction knowledge for a large genomic region to help facilitate the positional cloning of genes.Key words: yeast artificial chromosome, YAC, Arabidopsis thaliana, partial restriction mapping, floral development. </jats:p

Chitotriosidase deficiency is not associated with human hookworm infection in a Papua New Guinean population

AbstractHuman chitotriosidase (CHIT1) is a chitinolytic enzyme with suggested anti-fungal properties. Previous studies have suggested that chitotriosidase may also protect individuals against filarial nematode infections and malaria. A mutant allele, which renders chitotriosidase unstable and enzymatically inactive, is found at a frequency of >20% in Caucasians and other populations. This allele is found at much lower frequency in parts of West Africa where malarial and intestinal helminth infections are endemic. Here, we investigate whether there is a significant association between chitotriosidase genotype and the intensity of hookworm infection in 693 individuals from five villages in Papua New Guinea. Individuals were genotyped for chitotriosidase using a PCR-based assay. There was no association between CHIT1 genotype and the intensity of hookworm infection as determined by faecal egg counts. The frequency of the mutant allele was 0.251, very similar to that found in non-endemic countries. The extent of geographical variation in allele frequencies across worldwide populations was not high (Fst=0.11), and does not provide evidence for directional selection at this locus between different geographical areas. We conclude that the CHIT1 genotype does not play a crucial role in protection against hookworm infection. This does not correlate with a previous study that linked the mutant CHIT1 genotype to filariasis susceptibility. The possible reasons for this discrepancy are discussed

The Mla (Powdery Mildew) Resistance Cluster Is Associated With Three NBS-LRR Gene Families and Suppressed Recombination Within a 240-kb DNA Interval on Chromosome 5S (1HS) of Barley

Abstract Powdery mildew of barley, caused by Erysiphe graminis f. sp. hordei, is a model system for investigating the mechanism of gene-for-gene interaction between large-genome cereals and obligate-fungal pathogens. A large number of loci that confer resistance to this disease are located on the short arm of chromosome 5(1H). The Mla resistance-gene cluster is positioned near the telomeric end of this chromosome arm. AFLP-, RAPD-, and RFLP-derived markers were used to saturate the Mla region in a high-resolution recombinant population segregating for the (Mla6 + Mla14) and (Mla13 + Ml-Ru3) resistance specificities. These tightly linked genetic markers were used to identify and develop a physical contig of YAC and BAC clones spanning the Mla cluster. Three distinct NBS-LRR resistance-gene homologue (RGH) families were revealed via computational analysis of low-pass and BAC-end sequence data derived from Mla-spanning clones. Genetic and physical mapping delimited the Mla-associated, NBS-LRR gene families to a 240-kb interval. Recombination within the RGH families was at least 10-fold less frequent than between markers directly adjacent to the Mla cluster.</jats:p

Allelic Variation in TLR4 Is Linked to Susceptibility to Salmonella enterica Serovar Typhimurium Infection in Chickens

Toll-like receptor 4 (TLR4) is part of a group of evolutionarily conserved pattern recognition receptors involved in the activation of the immune system in response to various pathogens and in the innate defense against infection. We describe here the cloning and characterization of the avian orthologue of mammalian TLR4. Chicken TLR4 encodes a 843-amino-acid protein that contains a leucine-rich repeat extracellular domain, a short transmembrane domain typical of type I transmembrane proteins, and a Toll-interleukin-1R signaling domain characteristic of all TLR proteins. The chicken TLR4 protein shows 46% identity (64% similarity) to human TLR4 and 41% similarity to other TLR family members. Northern blot analysis reveals that TLR4 is expressed at approximately the same level in all tissues tested, including brain, thymus, kidney, intestine, muscle, liver, lung, bursa of Fabricius, heart, and spleen. The probe detected only one transcript of ca. 4.4 kb in length for all tissues except muscle where the size of TLR4 mRNA was ca. 9.6 kb. We have mapped TLR4 to microchromosome E41W17 in a region harboring the gene for tenascin C and known to be well conserved between the chicken and mammalian genomes. This region of the chicken genome was shown previously to harbor a Salmonella susceptibility locus. By using linkage analysis, TLR4 was shown to be linked to resistance to infection with Salmonella enterica serovar Typhimurium in chickens (likelihood ratio test of 10.2, P = 0.00138), suggesting a role of TLR4 in the host response of chickens to Salmonella infection

Grocott staining (A, D, G) and presence of AMCase (B, E, H) and chitotriosidase (C, F, I) in several rats.

<p>Panels A, B, and C show the lung of an uninfected rat. Panels D, E and F show the fungal focus in an infected, untreated rat. Panels G, H and I show the fungal focus in an infected, caspofungin treated rat. Original magnification ×400. All panels represent lungs on day 6 after inoculation. Slides were stained according to the described protocols. In Grocott staining (A, D, G), fungal hyphae are coloured black. Chitotriosidase- or AMCase-presenting cells are coloured red (B, C, E, F, H, I). In uninfected rats, normal morphology can be found in the lungs (A, B, C). In infected rats, normal morphology of alveoli is lost (D, E, F). Grocott staining shows many hyphae (D). An inflammatory response is found around the fungal focus, where chitotriosidase and AMCase are increasingly present (red zones) compared to an uninfected rat (E, F). After treatment with caspofungin, Grocott staining shows fungal material in all infected rats (G). AMCase bound fungal hyphae after treatment with caspofungin (H) and thus hyphae became visible. After treatment with caspofungin, chitotriosidase seemed to also bind the fungal cell wall and locate inside hyphal cells (I).</p