Developmental Growth Control Exerted via the Protein A Kinase Tpk2 in Ashbya gossypii.

Sporulation in Ashbya gossypii is induced by nutrient-limited conditions and leads to the formation of haploid spores. Using RNA-seq, we have determined a gene set induced upon sporulation, which bears considerable overlap with that of Saccharomyces cerevisiae but also contains A. gossypii-specific genes. Addition of cyclic AMP (cAMP) to nutrient-limited media blocks sporulation and represses the induction of sporulation specific genes. Deletion of the protein kinase A (PKA) catalytic subunits encoded by TPK1 and TPK2 showed reduced growth in tpk1 but enhanced growth in the tpk2 strain; however, both mutants sporulated well. Sporulation can be blocked by cAMP in tpk1 but not in tpk2 strains. Similarly, TPK2 acts at a second developmental switch promoting the break in spore dormancy. In S. cerevisiae, PKA phosphorylates and inhibits Msn2/4. The transcript profiles of the tpk1 and msn2/4 mutants were very similar to that of the wild type under sporulation conditions. However, deletion of the single A. gossypii MSN2/4 homolog generated a specific sporulation defect. We identified a set of genes involved in spore wall assembly that was downregulated in the msn2/4 mutant, particularly DIT2, suggesting that poor spore viability may be due to lysis of spores. Our results reveal specific functional differences between the two catalytic PKA subunits in A. gossypii and identified Tpk2 as the key A kinase that transduces developmental decisions of growth. Our data also suggest that Msn2/4 is involved only at a late step of sporulation in A. gossypii and is not a major regulator of IME1

Fungal model systems and the elucidation of pathogenicity determinants

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.Peer reviewedPublisher PD

Role of RIM101 for Sporulation at Alkaline pH in Ashbya gossypii

Microorganisms need to sense and adapt to fluctuations in the environmental pH. In fungal species, this response is mediated by the conserved pacC/RIM101 pathway. In Aspergillus nidulans, PacC activates alkaline-expressed genes and represses acid-controlled genes in response to alkaline pH and has important functions in regulating growth and conidia formation. In Saccharomyces cerevisiae, the PacC homolog Rim101 is required for adaptation to extracellular pH and to regulate transcription of IME1, the Initiator of MEiosis. S. cerevisiae rim101 mutants are defective in sporulation. In Ashbya gossypii, a filamentous fungus belonging to the family of Saccharomycetaceae, little is known about the role of pH in regulating growth and sporulation. Here, we deleted the AgRIM101 homolog (AFR190C). Our analyses show that Rim101 is important for growth and essential for sporulation at alkaline pH in A. gossypii. Acidic liquid sporulation media were alkalinized by sporulating strains, while the high pH of alkaline media (starting pH = 8.6) was reduced to a pH ~ 7.5 by these strains. However, Agrim101 mutants were unable to sporulate in alkaline media and failed to reduce the initial high pH, while they were capable of sporulation in acidic liquid media in which they increased the pH like the wild type

Fusobacterium necrophorum-PCR in pharyngotonsillitis - could the CT-value identify patients at risk for complications?

Background: Previously, we investigated tonsillar carriage of Fusobacterium necrophorum by PCR and found a high tonsillar carriage rate (21%) in asymptomatic 15-25 year olds, but the same age group is most commonly affected by severe F. necrophorum infections. Interestingly, we found Cycle threshold (Ct)-values in asymptomatic carriers to be high (median 29). Possibly, the Ct-value could differentiate between infection and tonsillar carriage, with bacterial load hypothetically being higher on infected tonsils. The purpose of this study was to investigate differences in F. necrophorum Ct-values in patients diagnosed with pharyngotonsillitis who did or did not develop complications. Methods: Patients with pharyngotonsillitis and positive F. necrophorum-PCR were enrolled from July 2016 - December 2020 in the Skåne Region, Sweden. Patients with prior complications or antibiotics (30 days) were excluded. Data was retrieved from registries and electronic charts. Patients were grouped by presence of any complication within 30 days, defined as a composite score of peritonsillar or pharyngeal abscess, otitis, sinusitis, sepsis or septic complications, chronic or recurrent tonsillitis (after 15-30 days) or hospitalization. Ct-values were presented with median and interquartile range (IQR) and compared with the Mann-Whitney U-test. Results: In total, 969 patients had pharyngotonsillitis and positive F. necrophorum-PCR. 29% developed complications. There was no difference in Ct-values between patients who did (median 21, IQR 19-25) or did not (median 21, IQR19-26) develop complications (p=0.51). Conclusion: In pharyngotonsillitis patients warranting extended work up for F. necrophorum, no difference in Ct-values between patients who did or did not develop complications was found. Most patients with pharyngotonsillitis had lower Ct-values than previously described in asymptomatic individuals, however factors such as degree of inflammation of tonsils, sampling technique and symptom duration were not accounted for

Legionella longbeachae wound infection: case report and review of reported Legionella wound infections

Extrapulmonary manifestations of infection with Legionella species, of which 24 may cause disease in humans, are very rare. Here, we describe a case of a 61-year-old woman with no history of immunosuppression presenting with pain and swelling of her index finger after a prick by rose thorns during gardening. Clinical examination showed fusiform swelling of the finger with mild redness, warmth, and fever. The blood sample revealed a normal white blood cell count and a slight increase in C-reactive protein. Intraoperative observation showed extensive infectious destruction of the tendon sheath, while the flexor tendons were spared. Conventional cultures were negative, while 16S rRNA PCR analysis identified Legionella longbeachae that also could be isolated on buffered charcoal yeast extract media. The patient was treated with oral levofloxacin for 13 days, and the infection healed quickly. The present case report, with a review of the literature, indicates that Legionella species wound infections may be underdiagnosed due to the requirement for specific media and diagnostic methods. It emphasizes the need for heightened awareness of these infections during history taking and clinical examination of patients presenting with cutaneous infections

Analysis of the cell wall integrity pathway of Ashbya gossypii.

Fungal cells are exposed to rapidly changing environmental conditions, in particular with regard to the osmotic potential. This requires constant remodeling of the cell wall and, therefore, the cell wall integrity (CWI) MAP-kinase pathway plays a major role in shaping the fungal cell wall to protect from adverse external stresses. To provide a comprehensive functional analysis of the Ashbya gossypii CWI pathway we generated a set of ten deletion mutants in conserved components including the cell surface sensors AgWSC1 and AgMID2, a putative Rho1-guanine nucleotide exchange factor, AgTUS1, the protein kinase C, AgPKC1, the MAP-kinases AgBCK1, AgMKK1 and AgMPK1, and transcription factors known to be involved in CWI signaling AgRLM1, AgSWI4 and AgSWI6. Deletion of AgPKC1 shows a severe growth defect with frequent tip cell lysis. Deletion of components of the MAP-kinase module generates a pronounced colony lysis phenotype in older regions of the mycelium. Cytoplasmic leakage was assayed using alkaline phosphatase and β-galactosidase release assays. This indicated that the lysis phenotypes of CWI pathway mutants may be useful to facilitate the isolation of riboflavin from A. gossypii. Remarkably, the Agwsc1 mutant showed a strong (up to 8-fold) increase of riboflavin in the growth medium compared to the parental strain

Fungal model systems and the elucidation of pathogenicity determinants

Fungi have the capacity to cause devastating diseases of both plants and animals, causing significant harvest losses that threaten food security and human mycoses with high mortality rates. As a consequence, there is a critical need to promote development of new antifungal drugs, which requires a comprehensive molecular knowledge of fungal pathogenesis. In this review, we critically evaluate current knowledge of seven fungal organisms used as major research models for fungal pathogenesis. These include pathogens of both animals and plants; Ashbya gossypii, Aspergillus fumigatus, Candida albicans, Fusarium oxysporum, Magnaporthe oryzae, Ustilago maydis and Zymoseptoria tritici. We present key insights into the virulence mechanisms deployed by each species and a comparative overview of key insights obtained from genomic analysis. We then consider current trends and future challenges associated with the study of fungal pathogenicity

Characterization of the Weimberg Pathway in Caulobacter crescentus

Caulobacter crescentus is a gram-negative bacterium that can utilize xylose as a substrate using the Weimberg pathway, which converts xylose to α-ketoglutarate in five steps without carbon loss. This is an interesting pathway for heterologous expression in other organisms in order to enable xylose utilization in biorefinery processes. C. crescentus was grown on xylose, arabinose and glucose, and maximum specific growth rates determined for the three substrates were 0.11 h−1, 0.05 h−1, and 0.15 h−1 respectively. Growth was found to be significantly inhibited at sugar concentration of 20 g L−1, shown primarily by an increased lag phase. Enzyme activity assays showed that the Weimberg pathway was active in cells grown, not only on xylose but also on arabinose. No activity was found for growth on glucose. Furthermore, substantial amounts of α-ketoglutarate—up to a yield of 0.4 g g−1—was excreted during growth on xylose, but no other extracellular intermediates in the Weimberg pathway were detected during growth on xylose. Apparently, C. crescentus is not well adapted for efficient growth on high xylose levels, and responds by an extended lag phase and secretion of α-ketoglutarate

Geographical differences in tonsillar carriage rates of Fusobacterium necrophorum – A cross-sectional study in Sweden and Zambia

Background: Previously, we investigated tonsillar carriage of Fusobacterium necrophorum by PCR and found a high tonsillar carriage rate (21%) in asymptomatic 15-25year olds, but the same age group is most commonly affected by severe F. necrophorum infections. Interestingly, we found Cycle threshold (Ct)-values in asymptomaticcarriers to be high (median 29). Possibly, the Ct-value could differentiate betweeninfection and tonsillar carriage, with bacterial load hypothetically being higher oninfected tonsils. The purpose of this study was to investigate differences in F. necrophorum Ct-values in patients diagnosed with pharyngotonsillitis who did or did notdevelop complications.Methods: Patients with pharyngotonsillitis and positive F. necrophorum-PCR were enrolled from July 2016 - December 2020 in the Skåne Region, Sweden. Patients with prior complications or antibiotics (30 days) were excluded. Data was retrieved from registries and electronic charts. Patients were grouped by presence of any complication within 30 days, defined as a composite score of peritonsillar or pharyngeal abscess, otitis, sinusitis, sepsis or septic complications, chronic or recurrent tonsillitis (after 15-30 days) or hospitalization. Ct-values were presented with median and interquartile range (IQR) and compared with the Mann-Whitney U-test. Results: In total, 969 patients had pharyngotonsillitis and positive F. necrophorum-PCR. 29% developed complications. There was no difference in Ct-values between patients who did (median 21, IQR 19-25) or did not (median 21, IQR19-26) develop complications (p=0.51). Conclusion: In pharyngotonsillitis patients warranting extended work up for F. necrophorum, no difference in Ct-values between patients who did or did not develop complications was found. Most patients with pharyngotonsillitis had lower Ct-values than previously described in asymptomatic individuals, however factors such as degree of inflammation of tonsils, sampling technique and symptom duration were not accounted for