Candida albicans Factor H Binding Molecule Hgt1p – A Low Glucose-Induced Transmembrane Protein Is Trafficked to the Cell Wall and Impairs Phagocytosis and Killing by Human Neutrophils

Complement is a tightly controlled arm of the innate immune system, facilitating phagocytosis and killing of invading pathogens. Factor H (FH) is the main fluid-phase inhibitor of the alternative pathway. Many pathogens can hijack FH from the host and protect themselves from complement-dependent killing. Candida albicans is a clinically important opportunistic yeast, expressing different FH binding molecules on its cell surface, which allow complement evasion. One such FH binding molecule is the transmembrane protein “High affinity glucose transporter 1” (Hgt1p), involved in glucose metabolism. This study demonstrated that Hgt1p transcription and expression is induced and highest at the low, but physiological glucose concentration of 0.1%. Thus, this concentration was used throughout the study. We also demonstrated the transport of Hgt1p to the fungal cell wall surface by vesicle trafficking and its release by exosomes containing Hgt1p integrated in the vesicular membrane. We corroborated Hgt1p as FH binding molecule. A polyclonal anti-Hgt1p antibody was created which interfered with the binding of FH, present in normal human serum to the fungal cell wall. A chimeric molecule consisting of FH domains 6 and 7 fused to human IgG1 Fc (FH6.7/Fc) even more comprehensively blocked FH binding, likely because FH6.7/Fc diverted FH away from fungal FH ligands other than Hgt1p. Reduced FH binding to the yeast was associated with a concomitant increase in C3b/iC3b deposition and resulted in significantly increased in vitro phagocytosis and killing by human neutrophils. In conclusion, Hgt1p also exhibits non-canonical functions such as binding FH after its export to the cell wall. Blocking Hgt1p-FH interactions may represent a tool to enhance complement activation on the fungal surface to promote phagocytosis and killing of C. albicans

Aspergillus fumigatus: un simple bioaerosol o un poderoso riesgo biológico?

Aspergillus fumigatus conidia constitute a common and widespread bioparticle of the air spectra. However, despite being innocuous to the majority of the healthy population, in immunocompromised patients, these bioparticles can cause a wide range of complaints ranging from mild allergenic reactions to severe disseminated diseases. One of the most devastating of Aspergillus-related diseases is invasive aspergillosis (IA) and despite all efforts, the morbidity and mortality remain unacceptably high. The poor outcomes of IA treatment are frequently associated with host status, delay of early diagnosis, and lack of adequate antifungal therapy, namely due to the resistance to azole therapy. The present work covers aspects of A. fumigatus biology and its role in allergy outset and in the development of invasive aspergillosis (IA). Additionally, topics of IA diagnosis and therapeutic approach are also reviewed while cell death is explored as a possible mechanism to the development of new therapeutical approaches.Los conidios de Aspergillus fumigatus son biopartículas comunes en el aire, que pueden causar una amplia variedad de patologías en pacientes inmunocomprometidos y inmunosupresos, que van desde las reacciones alérgicas leves a graves patologías. Una de las enfermedades más relevantes producidas por especies del género Aspergillus es la aspergilosis invasiva (AI), ya que la morbilidad y la mortalidad sigue siendo elevada, a pesar de todos los esfuerzos. Los escasos resultados al tratamiento utilizado en la AI, se asocian frecuentemente con estado del paciente (alteraciones metabólicas, malnutrición, deficiente respuesta inmunológica), retraso en el diagnóstico y falta de una terapia antifúngica adecuada, debido sobre todo a la resistencia terapéutica a la anfotericina B y a lostriazoles. El presente trabajo abarca aspectos de la biología de A. fumigatus y su papel en la alergia y desarrollo de la aspergilosis invasiva, además de temas de diagnóstico y enfoque terapéutico

<i>SNaPAfu</i>: A Novel Single Nucleotide Polymorphism Multiplex Assay for <i>Aspergillus fumigatus</i> Direct Detection, Identification and Genotyping in Clinical Specimens

<div><p>Objective</p><p>Early diagnosis of invasive aspergillosis is essential for positive patient outcome. Likewise genotyping of fungal isolates is desirable for outbreak control in clinical setting. We designed a molecular assay that combines detection, identification, and genotyping of <i>Aspergillus fumigatus</i> in a single reaction.</p><p>Methods</p><p>To this aim we combined 20 markers in a multiplex reaction and the results were seen following mini-sequencing readings. Pure culture extracts were firstly tested. Thereafter, <i>Aspergillus</i>-DNA samples obtained from clinical specimens of patients with possible, probable, or proven aspergillosis according to European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria.</p><p>Results</p><p>A new set of designed primers allowed multilocus sequence typing (MLST) gene amplification in a single multiplex reaction. The newly proposed <i>SNaPAfu</i> assay had a specificity of 100%, a sensitivity of 89% and detection limit of 1 ITS copy/mL (∼0.5 fg genomic <i>Aspergillus</i>-DNA/mL). The marker A49_F was detected in 89% of clinical samples. The <i>SNaPAfu</i> assay was accurately performed on clinical specimens using only 1% of DNA extract (total volume 50 µL) from 1 mL of used bronchoalveolar lavage.</p><p>Conclusions</p><p>The first highly sensitive and specific, time- and cost-economic multiplex assay was implemented that allows detection, identification, and genotyping of <i>A. fumigatus</i> strains in a single amplification followed by mini-sequencing reaction. The new test is suitable to clinical routine and will improve patient management.</p></div

The primers used in <i>SNaPAfu</i> assay.

#<p>Primer nomenclature and incorporated information on the targeting polymorphic position (e.g. A45_R means that a reverse primer was designed in the polymorphic position 45 of the gene ANXC4 of MLST panel).</p>§<p>Expected peak size in the electropherogram (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075968#pone-0075968-g002" target="_blank">Figure 2</a>); bp means base pairs.</p>*<p>Base expected in MLST and <i>SNaP</i> profiles; a complementary base is expected to be seen on the <i>SNaPAfu</i> electropherogram for primers designed for reverse sequence.</p

Clinical samples considered in this study and methods used for diagnosis of invasive aspergillosis (ARDS: Acute Respiratory Destress Syndrome; BAL: bronchoalveolar lavages; BS: skin biopsy samples; nm: not enough material to be tested; GM: galactomannan; PF-PCR: Pan-fungal PCF; (+) and (−) represent positive and negative results).

<p>Clinical samples considered in this study and methods used for diagnosis of invasive aspergillosis (ARDS: Acute Respiratory Destress Syndrome; BAL: bronchoalveolar lavages; BS: skin biopsy samples; nm: not enough material to be tested; GM: galactomannan; PF-PCR: Pan-fungal PCF; (+) and (−) represent positive and negative results).</p

<i>SNaPAfu</i> assay: A) position of each marker on the automated electropherogram; and B) example of an <i>Aspergillus fumigatus</i> profile (peaks: orange – ladder; blue – guanine; black – cytosine; green – adenine; red – thymine; the interpretation of such the peaks gives the electropherogram reading, then converted to the intermediate profile where the results of the markers with the reverse primer were converted in the complementary base – markers marked bold were converted); and C) the <i>SNaP</i> profile of the isolate presented above obtained according to <b>Table 2</b> (e.g. A45_R, A49_F, … Z198_F) to facilitate comparison with MLST data – markers are presented in alphabetic order; when markers are amplified using reverse primers (e.g. A45_R) the complementary base should be included in the <i>SNaP</i> profile.

<p><i>SNaPAfu</i> assay: A) position of each marker on the automated electropherogram; and B) example of an <i>Aspergillus fumigatus</i> profile (peaks: orange – ladder; blue – guanine; black – cytosine; green – adenine; red – thymine; the interpretation of such the peaks gives the electropherogram reading, then converted to the intermediate profile where the results of the markers with the reverse primer were converted in the complementary base – markers marked bold were converted); and C) the <i>SNaP</i> profile of the isolate presented above obtained according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075968#pone-0075968-t002" target="_blank"><b>Table 2</b></a> (e.g. A45_R, A49_F, … Z198_F) to facilitate comparison with MLST data – markers are presented in alphabetic order; when markers are amplified using reverse primers (e.g. A45_R) the complementary base should be included in the <i>SNaP</i> profile.</p

Primers used for MLST genotyping of <i>Aspergillus fumigatus</i> in a multiplex reaction.

*<p>Primer used for sequencing.</p