Atus, an opportunistic human mold pathogen that causes a lifethreatening infection

Atus, an opportunistic human mold pathogen that causes a lifethreatening infection known as invasive aspergillosis [16]. In this study, we characterized the A. fumigatus srgA gene, encoding a Sec4 homolog that was initially annotated in Aspergillus niger as secretionrelated GTPase A (SrgA) [17]. An A. fumigatus DsrgA mutant was constructed and shown to be associated with abnormal colonysec4 Homolog in A. fumigatusmorphology, attenuated conidiation, reduced hyphal growth, and hypersensitivity to environmental stress. However, there was surprising phenotypic POR8 heterogeneity among independent isolates of this mutant with respect to in vitro phenotypes and virulence, suggesting that the consequences of losing SrgA function is modified by the activation of different compensatory responses.has been described for Sec4 and related Sec proteins in Candida albicans [20,21]. This localization is consistent with the putative role for SrgA in the regulation of apical vesicle transport in filamentous fungi.Loss of SrgA Generates Phenotypic Heterogeneity in Colony MorphologyA DsrgA strain was constructed by replacing the entire srgA coding region with a phleomycin-resistance cassette. The expected deletion was identified by probing HindIII-digested genomic DNA with a srgA 59 flanking probe (probe A, Figure 2), revealing the loss of the wt 2.8 kb HindIII AKT inhibitor 2 cost fragment and the appearance of the expected 10.3 kb fragment. The DsrgA mutant showed surprising phenotypic heterogeneity when 18204824 plated for isolation on solid media, manifested by differences in colony size, the level of conidiation and colony sectoring (Figure 3A and 3B). Three distinct colonial morphologies were arbitrarily selected for further phenotypic analysis, using size and conidiation as a crude measure of individuality, hereafter referred to as DsrgA isolates A, B, and C (Figure 3C). Genotypic analysis by Southern blot, using a probe that is upstream of the srgA openreading frame (probe 18055761 B, Figure 2) confirmed that each DsrgA isolate lacked the srgA gene (Figure 3D). Moreover, no wt conidia were recovered by plating the mutant onto non-selective media, suggesting that the mutants are not heterokaryons that are protected by a small population of wt nuclei. The presence of the phleomycin resistance cassette, in the absence of any detectable srgA gene was also confirmed by PCR in each of the DsrgA isolates (data not shown). Together, these findings suggest that deletion of srgA generates phenotypic diversity in colony morphology, possibly due to the activation of compensatoryResults Identification of the Sec4 Homolog SrgA in A. fumigatusSrgA was previously identified in A. niger as one of five different secretion-related GTPases thought to be involved in mediating different stages of vesicle transport [17]. The corresponding gene in A. fumigatus (AFUA_4G04810), encodes a 206 amino acid protein in which multiple Rab-family motifs are found. Included within these shared motifs are the five “G box” sequences, which are present in all small GTPase families [18]. As shown in Figure 1A, there is high sequence homology within these G box motifs between A. fumigatus SrgA and other previously characterized fungal Sec4 proteins. Conservation within the G2 domain is particularly noteworthy, as this region is the effector domain, responsible for functional specificity within the Rab GTPase family [17]. Also contributing to Rab GTPase function are two conserved C-terminal cysteine residues, which are posttransl.Atus, an opportunistic human mold pathogen that causes a lifethreatening infection known as invasive aspergillosis [16]. In this study, we characterized the A. fumigatus srgA gene, encoding a Sec4 homolog that was initially annotated in Aspergillus niger as secretionrelated GTPase A (SrgA) [17]. An A. fumigatus DsrgA mutant was constructed and shown to be associated with abnormal colonysec4 Homolog in A. fumigatusmorphology, attenuated conidiation, reduced hyphal growth, and hypersensitivity to environmental stress. However, there was surprising phenotypic heterogeneity among independent isolates of this mutant with respect to in vitro phenotypes and virulence, suggesting that the consequences of losing SrgA function is modified by the activation of different compensatory responses.has been described for Sec4 and related Sec proteins in Candida albicans [20,21]. This localization is consistent with the putative role for SrgA in the regulation of apical vesicle transport in filamentous fungi.Loss of SrgA Generates Phenotypic Heterogeneity in Colony MorphologyA DsrgA strain was constructed by replacing the entire srgA coding region with a phleomycin-resistance cassette. The expected deletion was identified by probing HindIII-digested genomic DNA with a srgA 59 flanking probe (probe A, Figure 2), revealing the loss of the wt 2.8 kb HindIII fragment and the appearance of the expected 10.3 kb fragment. The DsrgA mutant showed surprising phenotypic heterogeneity when 18204824 plated for isolation on solid media, manifested by differences in colony size, the level of conidiation and colony sectoring (Figure 3A and 3B). Three distinct colonial morphologies were arbitrarily selected for further phenotypic analysis, using size and conidiation as a crude measure of individuality, hereafter referred to as DsrgA isolates A, B, and C (Figure 3C). Genotypic analysis by Southern blot, using a probe that is upstream of the srgA openreading frame (probe 18055761 B, Figure 2) confirmed that each DsrgA isolate lacked the srgA gene (Figure 3D). Moreover, no wt conidia were recovered by plating the mutant onto non-selective media, suggesting that the mutants are not heterokaryons that are protected by a small population of wt nuclei. The presence of the phleomycin resistance cassette, in the absence of any detectable srgA gene was also confirmed by PCR in each of the DsrgA isolates (data not shown). Together, these findings suggest that deletion of srgA generates phenotypic diversity in colony morphology, possibly due to the activation of compensatoryResults Identification of the Sec4 Homolog SrgA in A. fumigatusSrgA was previously identified in A. niger as one of five different secretion-related GTPases thought to be involved in mediating different stages of vesicle transport [17]. The corresponding gene in A. fumigatus (AFUA_4G04810), encodes a 206 amino acid protein in which multiple Rab-family motifs are found. Included within these shared motifs are the five “G box” sequences, which are present in all small GTPase families [18]. As shown in Figure 1A, there is high sequence homology within these G box motifs between A. fumigatus SrgA and other previously characterized fungal Sec4 proteins. Conservation within the G2 domain is particularly noteworthy, as this region is the effector domain, responsible for functional specificity within the Rab GTPase family [17]. Also contributing to Rab GTPase function are two conserved C-terminal cysteine residues, which are posttransl.