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

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

Chitinase activity and fungal load in immunocompromised rats inoculated with <i>A. fumigatus</i> conidia.

<p>Open squares: uninfected untreated rats; filled circles: infected untreated rats; grey triangles: infected rats, treated with caspofungin at 24 h post infection. Data are means of duplicates. Bars represent medians. For each group n≥4. A. Galactomannan (GM)-index, measured by Platelia assay. According to the manufacturer's manual, GM-index of <0,5 is considered negative; B, chitotriosidase activity, expressed in arbitrary units (a.u.); C, AMCase activity, expressed in arbitrary units (a.u.). * p<0.05; significant difference between the indicated groups.</p

<i>In vitro</i> binding of recombinant chitinases to <i>A. fumigatus</i> hyphae.

<p>Binding of recombinant chitotriosidase (A, B). Binding of recombinant chitotriosidase when incubated in combination with recombinant AMCase (C, D) and binding of recombinant AMCase (E, F). Panels A, C and E show unexposed hyphae. Panels B, D and F show caspofungin-exposed hyphae. A, B: Contrast and brightening were slightly modified in Photoshop due to the lack of colour. C, D, E, F: Photos were not modified in Photoshop. A–B Original magnification ×100. C–F Original magnification ×400. Slides were stained according to the described protocols. Binding of either recombinant enzyme is characterized by a red colour. Recombinant chitotriosidase did not bind to unexposed hyphae (A) or to caspofungin-exposed hyphae (B). When incubated with a combination of recombinant chitotriosidase and recombinant AMCase, recombinant chitotriosidase did bind to unexposed hyphae (arrow) and to conidial heads (C) and seemed to be taken up by the fungal cells after caspofungin exposure (D). Also the cell wall seemed to be lysed at several locations (arrows). Recombinant AMCase did bind to unexposed (E) and to caspofungin-exposed hyphae (F).</p

Calcofluor White staining of <i>in vitro</i> cultured <i>A. fumigatus</i> hyphae after incubation with recombinant chitotriosidase and recombinant AMCase.

<p>When hyphae were cultured on Sabauroud's agar (A), the cell wall remained regular and intact after incubation with the two recombinant chitinases. When hyphae were cultured on Sabauroud's agar with 1 mg/L caspofungin (B), the cell wall was irregular and disrupted after incubation with the two recombinant chitinases.</p