Ehrlichia minasensis sp. nov., isolated from the tick Rhipicephalus microplus.

Recently, we obtained a rickettsial isolate (Ehrlichia sp. UFMG-EVT) from the haemolymph of engorged Rhipicephalus microplus tick females. On the basis of maximum-likelihood phylogenetic analysis using 16S rRNA gene, groEL, dsb, gltA and trp36 sequences we showed that Ehrlichia sp. UFMG-EVT belongs to the α-Proteobacteria, family Anaplasmataceae, genus Ehrlichia. Ehrlichia sp. UFMG-EVT is a sister taxon of Ehrlichia canis with 16S rRNA gene, groEL, dsb, gltA and trp36 sequence similarities of 98.3 %, 97.2 %, 94.7 %, 94.3 % and 49.1 %, respectively. Ehrlichia sp. UFMG-EVT has been maintained in the laboratory by continuous passage in the IDE8 tick cell line where the ultrastructure was characterized using electron microscopy and was found to resemble that of E. canis, Ehrlichia muris and Ehrlichia chaffeensis, but not Ehrlichia ruminantium and Ehrlichia ewingii. We propose the name Ehrlichia minasensis sp. nov. for this bacterium to acknowledge the place from where it was initially isolated, Minas Gerais, Brazil. The type strain is strain Ehrlichia sp. UFMG-EVT ( = DSM 100393T = TCB-TBB-0018T).

Molecular and immunological characterization of three strains of Anaplasma marginale grown in cultured tick cells.

Anaplasma marginale is an economically important tick-borne pathogen of cattle that causes bovine anaplasmosis. A wide range of geographic strains of A. marginale have been isolated from cattle, several of which have been characterized using genomics and proteomics. While many of these strains have been propagated in tick lines, comparative analyses after propagation in tick cells have not been reported. The overall purpose of this research therefore was to compare the degree of conservation of selected genes after propagation in tick cell culture among A. marginale strains from the U.S. (the Virginia strain) and Brazil (UFMG1 and UFMG2 strains). The genes studied herein included those which encode the proteins HSP70 and SODB involved in heat shock and stress responses, respectively, and two genes that encode major surface proteins MSP4 and MSP5. Strain identities were first confirmed by sequencing the tandem repeats of the msp1a gene which encodes for the adhesin, MSP1a. The results of these studies demonstrated that the genes encoding for both stress response and heat shock proteins were highly conserved among the three A. marginale strains. Antibodies specific for MSP4, MSP5, SODB and HSP70 proteins were used to further characterize the A. marginale strains, and they reacted with all of these strains propagated in tick cell culture, providing further evidence for antigenic conservation. Although antigenic differences were not found among the three A. marginale strains, multi-locus sequence analysis (MLSA) performed with nucleotide sequences of these genes demonstrated that the A. marginale Brazilian and U.S. strains fall in different clades. These results showed that phylogenetically distant strains of A. marginale are antigenically conserved, even after several in vitro passages, supporting the use of some of the above conserved proteins as candidates for universal vaccines.

Characterization of the Aspergillus fumigatus chitosanase CsnB and evaluation of its potential use in serological diagnostics.

Aspergillus fumigatus is currently the major air-borne fungal pathogen as its asexual spores are distributed through the air. In severely immunocompromised patients, inhalation of these conidia can result in life-threatening infections. Invasive Aspergillosis, a major Aspergillus-associated disease, is associated with a high mortality reflecting short-comings in diagnostics and therapy. Current diagnostics largely rely on the serological detection of the galactomannan antigen. Detection of circulating antibodies is an alternative approach. In this study, we have characterized the chitosanase CsnB, a protein that was previously shown to be a major secreted A. fumigatus antigen and therefore a potential target for antibody-based diagnostics. To analyze the biological function of CsnB we have deleted the csnB gene and generated CsnB-specific antibodies. We found that A. fumigatus is able to grow on chitosan in a CsnB-dependent manner. During growth on chitosan elevated levels of CsnB are found in the supernatants indicating that chitosan triggers enhanced CsnB production. Unexpectedly we have found a similar activity for tartrate. Using recombinant proteins we analyzed antibody responses in patients at risk to develop invasive aspergillosis. We focussed this study on two antigens: CsnB and for comparison mitogillin, a secreted A. fumigatus ribotoxin. IgG responses were found to both proteins, but elevated antibody levels to CsnB and/or mitogillin showed no correlation to the results of the galactomannan antigen assay or clinical signs that are characteristic for fungal infections.

In vitro culture and structural differences in the major immunoreactive protein gp36 of geographically distant Ehrlichia canis isolates.

Ehrlichia canis, the etiologic agent of canine ehrlichiosis, is an obligate intracytoplasmic Gram-negative tick-borne bacterium belonging to the Anaplasmataceae family. E. canis is distributed worldwide and can cause serious and fatal infections in dogs. Among strains of E. canis, the 16S rRNA gene DNA sequences are highly conserved. Using this gene to genetically differentiate isolates is therefore difficult. As an alternative, the gene gp36, which encodes for a major immunoreactive protein in E. canis, has been successfully used to characterize the genetic diversity of this pathogen. The present study describes the isolation and continuous propagation of a Spanish and 2 South African isolates of E. canis in IDE8 tick cells. Subsequently, canine DH82 cell cultures were infected using initial bodies obtained from infected IDE8 cultures. It was possible to mimic the life cycle of E. canis in vitro by transferring infection from tick cells to canine cells and back again. To characterize these E. canis strains at the molecular level, the 16S rRNA and gp36 genes were amplified by PCR, sequenced, and aligned with corresponding sequences available in GenBank. All 16S rRNA sequences amplified in this study were identical to previously reported E. canis strains. Maximum likelihood analysis based on the gp36 amino acid sequences showed that the South African and Spanish strains fall into 2 well-defined phylogenetic clusters amongst other E. canis strains. The members of these 2 phylogenetic clusters shared 2 unique molecular properties in the gp36 amino acid sequences: (i) deletion of glycine 117 and (ii) the presence of an additional putative N-linked glycosylation site. We further show correlation between the putative secondary structure and the theoretical isoelectric point (pI) of the gp36 amino acid sequences. A putative role of gp36 as an adhesin in E. canis is discussed. Overall, we report the successful in vitro culture of 3 new E. canis strains which present different molecular properties in their gp36 sequences.

The two-component sensor kinase TcsC and its role in stress resistance of the human-pathogenic mold Aspergillus fumigatus.

Two-component signaling systems are widespread in bacteria, but also found in fungi. In this study, we have characterized TcsC, the only Group III two-component sensor kinase of Aspergillus fumigatus. TcsC is required for growth under hyperosmotic stress, but dispensable for normal growth, sporulation and conidial viability. A characteristic feature of the ΔtcsC mutant is its resistance to certain fungicides, like fludioxonil. Both hyperosmotic stress and treatment with fludioxonil result in a TcsC-dependent phosphorylation of SakA, the final MAP kinase in the high osmolarity glycerol (HOG) pathway, confirming a role for TcsC in this signaling pathway. In wild type cells fludioxonil induces a TcsC-dependent swelling and a complete, but reversible block of growth and cytokinesis. Several types of stress, such as hypoxia, exposure to farnesol or elevated concentrations of certain divalent cations, trigger a differentiation in A. fumigatus toward a "fluffy" growth phenotype resulting in white, dome-shaped colonies. The ΔtcsC mutant is clearly more susceptible to these morphogenetic changes suggesting that TcsC normally antagonizes this process. Although TcsC plays a role in the adaptation of A. fumigatus to hypoxia, it seems to be dispensable for virulence.

Studies on galactofuranose-containing glycostructures of the pathogenic mold Aspergillus fumigatus.

Galactofuranose is a hexose that is exclusively found in microbes and in particular in certain pathogenic species. In the mold Aspergillus fumigatus, it is the characteristic constituent of the cell wall component galactomannan. Detection of this carbohydrate is currently a widespread method used for diagnosis of systemic A. fumigatus infections. In this study, we raised and characterized 2 monoclonal antibodies that specifically react with galactofuranose-containing glycostructures. We investigated the distribution of surface-accessible galactomannan on different A. fumigatus morphotypes. We provide evidence that the antibodies recognize distinct antigens and are suitable to detect A. fumigatus hyphae in immunohistology. A mutant that is impaired in synthesis of galactofuranose stimulated a normal cytokine response in murine macrophages, which argues against galactomannan being a relevant PAMP, at least in mice. Purified galactomannan-specific monoclonal IgM L10-1 failed to inhibit the hyphal growth under in vitro conditions, but L10-1 binding to hyphae led to an enhanced deposition of the complement protein C1q. However, administration of purified L10-1 antibodies prior to infection was not able to protect mice. In conclusion, we have found no evidence for galactomannan being a relevant A. fumigatus PAMP and describe 2 novel galactomannan antibodies that might be valuable tools for the diagnosis of A. fumigatus infections and further analysis of the biological significance of galactomannan.

Characterisation of the CipC-like protein AFUA_5G09330 of the opportunistic human pathogenic mould Aspergillus fumigatus.

Aspergillus fumigatus is currently the major airborne fungal pathogen that menaces immunocompromised individuals. Germination of inhaled conidia is a hallmark of the early infection process, but little is known about the underlying mechanisms. The intention of our ongoing studies is the identification of A. fumigatus proteins that are differentially expressed during germination and may provide insights in the germination process. Using a proteomic approach, we identified AFUA_5G09330 as a major hyphal-specific protein. This result was confirmed using monoclonal antibodies generated in this study. AFUA_5G09330 belongs to a fungal-specific protein family. The eponymous CipC protein of A. nidulans has been shown to be induced by concanamycin A, and transcriptional data from Cryptococcus neoformans demonstrate a strong up-regulation of the expression of a homologous gene during infection. Our data provide evidence that AFUA_5G09330 is a monomeric, cytoplasmic protein. We found no evidence for an overexpression of AFUA_5G09330 induced by concanamycin A or other stress conditions. AFUA_5G09330 is exclusively found in the hyphal morphotype that enables an invasive growth of A. fumigatus during infection. Further studies are required to define the biological function of this hyphae-specific protein and its potential relevance for the pathogenicity of A. fumigatus.