Aspergillus Collagen-Like Genes ( acl ): Identification, Sequence Polymorphism, and Assessment for PCR-Based Pathogen Detection

Abstract

fThe genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects.

Virulence determinants of the human pathogenic fungus Aspergillus fumigatus protect against soil amoeba predation

“…For a first molecular analysis, samples from uppermost layer of organic matter from various sites in three different middle European regions (Berlin and Jena, Germany and Povazska Bystrica, Slovakia) were used as source of genomic deoxyribonucleic acid (DNA). Aspergillus fumigatus and Dictyostelid ‐specific genes were amplified using primers homologous to collagenase‐like gene acl (Tuntevski et al ., ) and conserved regions of the 17s rRNA gene of D. discoideum respectively. Dictyostelids were detected in 22 of 32 sites (68%), while DNA from A. fumigatus was amplified from 15 samples (48%; Table ).…”

Reversible phospholipid nanogels for deoxyribonucleic acid fragment size determinations up to 1500 base pairs and integrated sample stacking

“…Unique sequences in Aspergillus collagenlike genes (acl) found in five pathogenic species were targeted by This separation additive optimized for larger DNA fragments estimated the size with a bias ranging from 0 to 6 bp (see Table 3), which represented a size accuracy within 2.5% of the true fragment size. Previously, these biomarkers were separated using a 40 cm long capillary and a 10% phospholipid preparation of [DMPC]/ [DHPC] equal to 2.5 at 30 C [29]. In that study, the amplicon size was estimated using three size standards of 150, 250, and 350 bp with a bias ranging from 1 to 7 bp, which represented a size accuracy within 2% of the true fragment size.…”

“…The DNA sequences were assembled and analyzed with the Lasergene 11 Genomics Suite (DNASTAR, Inc., Madison, WI) software, as previously described [28]. Isolation of genomic DNA and purification from fungal cultures, PCR primers (Eurofins Genomics, Huntsville, AL), amplification conditions, and verification of conserved amplicon sizes for acl-based detection of Aspergillus species were previously established [29].…”

Engineering specific chemical modification sites into a collagen‐like protein from Streptococcus pyogenes

“…This characteristic sequence is also found in a range of other proteins that also contain the triple‐helical motif . More recently, collagens have been identified in several nonanimal species, including bacteria, fungi, viruses, and phage . These proteins all show extended (Gly‐Xaa‐Yaa) n sequences and in selected instances have been clearly shown to fold into the triple‐helical structure …”