LAMMER Kinase Governs the Expression and Cellular Localization of Gas2, a Key Regulator of Flocculation in Schizosaccharomyces pombe.

It was reported that LAMMER kinase in Schizosaccharomyces pombe plays an important role in cation-dependent and galactose-specific flocculation. Analogous to other flocculating yeasts, when cell wall extracts of the Δlkh1 strain were treated to the wild-type strain, it displayed flocculation. Gas2, a 1,3-ß-glucanosyl transferase, was isolated from the EDTA-extracted cell-surface proteins in the Δlkh1 strain. While disruption of the gas2+ gene was not lethal and reduced the flocculation activity of the ∆lkh1 strain, the expression of a secreted form of Gas2, in which the GPI anchor addition sequences had been removed, conferred the ability to flocculate upon the WT strain. The Gas2-mediated flocculation was strongly inhibited by galactose but not by glucose. Immunostaining analysis showed that the cell surface localization of Gas2 was crucial for the flocculation of fission yeast. In addition, we identified the regulation of mbx2+ expression by Lkh1 using RT-qPCR. Taken together, we found that Lkh1 induces asexual flocculation by regulating not only the localization of Gas2 but also the transcription of gas2+ through Mbx2.

LAMMER Kinase Modulates Cell Cycle by Phosphorylating the MBF Repressor, Yox1, in Schizosaccharomyces pombe.

Lkh1, a LAMMER kinase homolog in the fission yeast Schizosaccharomyces pombe, acts as a negative regulator of filamentous growth and flocculation. It is also involved in the response to oxidative stress. The lkh1-deletion mutant displays slower cell growth, shorter cell size, and abnormal DNA content compared to the wild type. These phenotypes suggest that Lkh1 controls cell size and cell cycle progression. When we performed microarray analysis using the lkh1-deletion mutant, we found that only four of the up-regulated genes in the lkh1-deletion were associated with the cell cycle. Interestingly, all of these genes are regulated by the Mlu1 cell cycle box binding factor (MBF), which is a transcription complex responsible for regulating the expression of cell cycle genes during the G1/S phase. Transcription analyses of the MBF-dependent cell-cycle genes, including negative feedback regulators, confirmed the up-regulation of these genes by the deletion of lkh1. Pull-down assay confirmed the interaction between Lkh1 and Yox1, which is a negative feedback regulator of MBF. This result supports the involvement of LAMMER kinase in cell cycle regulation by modulating MBF activity. In vitro kinase assay and NetPhosK 2.0 analysis with the Yox1T40,41A mutant allele revealed that T40 and T41 residues are the phosphorylation sites mediated by Lkh1. These sites affect the G1/S cell cycle progression of fission yeast by modulating the activity of the MBF complex.

Survival Factor A (SvfA) Contributes to Aspergillus nidulans Pathogenicity.

Survival factor A (SvfA) in Aspergillus nidulans plays multiple roles in growth and developmental processes. It is a candidate for a novel VeA-dependent protein involved in sexual development. VeA is a key developmental regulator in Aspergillus species that can interact with other velvet-family proteins and enter into the nucleus to function as a transcription factor. In yeast and fungi, SvfA-homologous proteins are required for survival under oxidative and cold-stress conditions. To assess the role of SvfA in virulence in A. nidulans, cell wall components, biofilm formation, and protease activity were evaluated in a svfA-gene-deletion or an AfsvfA-overexpressing strain. The svfA-deletion strain showed decreased production of ß-1,3-glucan in conidia, a cell wall pathogen-associated molecular pattern, with a decrease in gene expression for chitin synthases and ß-1,3-glucan synthase. The ability to form biofilms and produce proteases was reduced in the svfA-deletion strain. We hypothesized that the svfA-deletion strain was less virulent than the wild-type strain; therefore, we performed in vitro phagocytosis assays using alveolar macrophages and analyzed in vivo survival using two vertebrate animal models. While phagocytosis was reduced in mouse alveolar macrophages challenged with conidia from the svfA-deletion strain, the killing rate showed a significant increase with increased extracellular signal-regulated kinase ERK activation. The svfA-deletion conidia infection reduced host mortality in both T-cell-deficient zebrafish and chronic granulomatous disease mouse models. Taken together, these results indicate that SvfA plays a significant role in the pathogenicity of A. nidulans.

Different inhaled corticosteroid doses in triple therapy for chronic obstructive pulmonary disease: systematic review and Bayesian network meta-analysis.

A systematic review and Bayesian network meta-analysis is necessary to evaluate the efficacy and safety of triple therapy with different doses of inhaled corticosteroids (ICS) in stable chronic obstructive pulmonary disease (COPD). We selected 26 parallel randomized controlled trials (41,366 patients) comparing triple therapy with ICS/long-acting beta-agonist (LABA), LABA/long-acting muscarinic antagonist (LAMA), and LAMA in patients with stable COPD for ≥ 12 weeks from PubMed, EMBASE, the Cochrane Library, and clinical trial registries (search from inception to June 30, 2022). Triple therapy with high dose (HD)-ICS exhibited a lower risk of total exacerbation in pre-specified subgroups treated for ≥ 48 weeks than that with low dose (LD)-ICS (odds ratio [OR] = 0.66, 95% credible interval [CrI] = 0.52-0.94, low certainty of evidence) or medium dose (MD)-ICS (OR = 0.66, 95% CrI = 0.51-0.94, low certainty of evidence). Triple therapy with HD-ICS exhibited a lower risk of moderate-to-severe exacerbation in pre-specified subgroups with forced expiratory volume in 1 s < 65% (OR = 0.6, 95% CrI = 0.37-0.98, low certainty of evidence) or previous exacerbation history (OR = 0.6, 95% CrI = 0.36-0.999, very low certainty of evidence) than triple therapy with MD-ICS. Triple therapy with HD-ICS may reduce acute exacerbation in patients with COPD treated with other drug classes including triple therapy with LD- or MD-ICS or dual therapies.

The Gß-like Protein AfCpcB Affects Sexual Development, Response to Oxidative Stress and Phagocytosis by Alveolar Macrophages in Aspergillus fumigatus.

G-protein signaling is important for signal transduction, allowing various stimuli that are external to a cell to affect its internal molecules. In Aspergillus fumigatus, the roles of Gß-like protein CpcB on growth, asexual development, drug sensitivity, and virulence in a mouse model have been previously reported. To gain a deeper insight into Aspergillus fumigatus sexual development, the ΔAfcpcB strain was generated using the supermater AFB62 strain and crossed with AFIR928. This cross yields a decreased number of cleistothecia, including few ascospores. The sexual reproductive organ-specific transcriptional analysis using RNAs from the cleistothecia (sexual fruiting bodies) indicated that the CpcB is essential for the completion of sexual development by regulating the transcription of sexual genes, such as veA, steA, and vosA. The ΔAfcpcB strain revealed increased resistance to oxidative stress by regulating genes for catalase, peroxiredoxin, and ergosterol biosynthesis. The ΔAfcpcB strain showed decreased uptake by alveolar macrophages in vitro, decreased sensitivity to Congo red, decreased expression of cell wall genes, and increased expression of the hydrophobin genes. Taken together, these findings indicate that AfCpcB plays important roles in sexual development, phagocytosis by alveolar macrophages, biosynthesis of the cell wall, and oxidative stress response.

The LAMMER Kinase, LkhA, Affects Aspergillus fumigatus Pathogenicity by Modulating Reproduction and Biosynthesis of Cell Wall PAMPs.

The LAMMER kinase in eukaryotes is a well-conserved dual-specificity kinase. Aspergillus species cause a wide spectrum of diseases called aspergillosis in humans, depending on the underlying immune status of the host, such as allergy, aspergilloma, and invasive aspergillosis. Aspergillus fumigatus is the most common opportunistic fungal pathogen that causes invasive aspergillosis. Although LAMMER kinase has various functions in morphology, development, and cell cycle regulation in yeast and filamentous fungi, its function in A. fumigatus is not known. We performed molecular studies on the function of the A. fumigatus LAMMER kinase, AfLkhA, and reported its involvement in multiple cellular processes, including development and virulence. Deletion of AflkhA resulted in defects in colonial growth, production of conidia, and sexual development. Transcription and genetic analyses indicated that AfLkhA modulates the expression of key developmental regulatory genes. The AflkhA-deletion strain showed increased production of gliotoxins and protease activity. When conidia were challenged with alveolar macrophages, enodocytosis of conidia by macrophages was increased in the AflkhA-deletion strain, resulting from changes in expression of the cell wall genes and thus content of cell wall pathogen-associated molecular patterns, including ß-1,3-glucan and GM. While T cell-deficient zebrafish larvae were significantly susceptible to wild-type A. fumigatus infection, AflkhA-deletion conidia infection reduced host mortality. A. fumigatus AfLkhA is required for the establishment of virulence factors, including conidial production, mycotoxin synthesis, protease activity, and interaction with macrophages, which ultimately affect pathogenicity at the organismal level.

Mannitol-1-phosphate dehydrogenase, MpdA, is required for mannitol production in vegetative cells and involved in hyphal branching, heat resistance of conidia and sexual development in Aspergillus nidulans.

Aspergillus nidulans produces cleistothecia as sexual reproductive organs in a process affected by genetic and external factors. To gain a deeper insight into A. nidulans sexual development, we performed comparative proteome analyses based on the wild type developmental periods. We identified sexual development-specific proteins with a more than twofold increase in production during hypoxia or the sexual period compared to the asexual period. Among the sexual development-specific proteins analyzed by gene-deletion experiments and functional assays, MpdA, a putative mannitol-1-phosphate 5-dehydrogenase, plays multiple roles in growth and differentiation of A. nidulans. The most distinct mpdA-deletion phenotype was ascosporogenesis failure. Genetic mpdA deletion resulted in small cleistothecia with no functional ascospores. Transcriptional analyses indicated that MpdA modulates the expression of key development- and meiosis-regulatory genes during sexual development. The mpdA deletion increased hyphal branching and decreased conidial heat resistance. Mannitol production in conidia showed no difference, whereas it was decreased in mycelia and sexual cultures. Addition of mannitol during vegetative growth recovered the defects in conidial heat resistance and ascospore genesis. Taken together, these results indicate that MpdA plays an important role in sexual development, hyphal branching, and conidial heat resistance in Aspergillus nidulans.

Corrigendum: The LAMMER Kinase, LkhA, Affects Aspergillus fumigatus Pathogenicity by Modulating Reproduction and Biosynthesis of Cell Wall PAMPs.

[This corrects the article DOI: 10.3389/fcimb.2021.756206.].

Mutations in FAM50A suggest that Armfield XLID syndrome is a spliceosomopathy.

Intellectual disability (ID) is a heterogeneous clinical entity and includes an excess of males who harbor variants on the X-chromosome (XLID). We report rare FAM50A missense variants in the original Armfield XLID syndrome family localized in Xq28 and four additional unrelated males with overlapping features. Our fam50a knockout (KO) zebrafish model exhibits abnormal neurogenesis and craniofacial patterning, and in vivo complementation assays indicate that the patient-derived variants are hypomorphic. RNA sequencing analysis from fam50a KO zebrafish show dysregulation of the transcriptome, with augmented spliceosome mRNAs and depletion of transcripts involved in neurodevelopment. Zebrafish RNA-seq datasets show a preponderance of 3' alternative splicing events in fam50a KO, suggesting a role in the spliceosome C complex. These data are supported with transcriptomic signatures from cell lines derived from affected individuals and FAM50A protein-protein interaction data. In sum, Armfield XLID syndrome is a spliceosomopathy associated with aberrant mRNA processing during development.

The putative C2H2 transcription factor RocA is a novel regulator of development and secondary metabolism in Aspergillus nidulans.

Multiple transcriptional regulators play important roles in the coordination of developmental processes, including asexual and sexual development, and secondary metabolism in the filamentous fungus Aspergillus nidulans. In the present study, we characterized a novel putative C2H2-type transcription factor (TF), RocA, in relation to development and secondary metabolism. Deletion of rocA increased conidiation and caused defective sexual development. In contrast, the overexpression of rocA exerted opposite effects on both phenotypes. Additionally, nullifying rocA resulted in enhanced brlA expression and reduced nsdC expression, whereas its overexpression exerted the opposite effects. These results suggest that RocA functions as a negative regulator of asexual development by repressing the expression of brlA encoding a key asexual development activator, but as a positive regulator of sexual development by enhancing the expression of nsdC encoding a pivotal sexual development activator. Deletion of rocA increased the production of sterigmatocystin (ST), as well as the expression of its biosynthetic genes, aflR and stcU. Additionally, the expression of the biosynthetic genes for penicillin (PN), ipnA and acvA, and for terrequinone (TQ), tdiB and tdiE, was increased by rocA deletion. Thus, it appears that RocA functions as a negative transcriptional modulator of the secondary metabolic genes involved in ST, PN, and TQ biosynthesis. Taken together, we propose that RocA is a novel transcriptional regulator that may act either positively or negatively at multiple target genes necessary for asexual and sexual development and secondary metabolism.

Survival factor SvfA plays multiple roles in differentiation and is essential for completion of sexual development in Aspergillus nidulans.

The first member of the velvet family of proteins, VeA, regulates sexual development and secondary metabolism in the filamentous fungus Aspergillus nidulans. In our study, through comparative proteome analysis using wild type and veA-deletion strains, new putative regulators of sexual development were identified and functionally analyzed. Among these, SvfA, containing a yeast survival factor 1 domain, plays multiple roles in the growth and differentiation of A. nidulans. Deletion of the svfA gene resulted in increased sensitivity to oxidative and cold stress as in yeast. The svfA-deletion strain showed an increase in bi-polar germination and a decrease in radial growth rate. The deletion strain formed structurally abnormal conidiophores and thus produced lower amounts of conidiospores during asexual development. The svfA-deletion strain produced few Hülle cells and small cleistothecia with no ascospores, indicating the requirement of svfA for the completion of sexual development. Transcription and genetic analyses indicated that SvfA modulates the expression of key development regulatory genes. Western blot analysis revealed two forms of SvfA. The larger form showed sexual-specific and VeA-dependent production. Also, the deletion of svfA caused decreased ST (sterigmatocystin) production. We propose that SvfA is a novel central regulator of growth, differentiation and secondary metabolism in A. nidulans.

The LAMMER kinase is involved in morphogenesis and response to cell wall- and DNA-damaging stresses in Candida albicans.

Dual specificity LAMMER kinase has been reported to be conserved across species ranging from yeasts to animals and has multiple functions. Candida albicans undergoes dimorphic switching between yeast cells and hyphal growth forms as its key virulence factors. Deletion of KNS1, which encodes for LAMMER kinase in C. albicans, led to pseudohyphal growth on YPD media and defects in filamentous growth both on spider and YPD solid media containing 10% serum. These cells exhibited expanded central wrinkled regions and specifically reduced peripheral filaments. Among the several stresses tested, the kns1Δ strains showed sensitivity to cell-wall and DNA-replicative stress. Under fluorescent microscopy, an increase in chitin decomposition was observed near the bud necks and septa in kns1Δ cells. When the expression levels of genes for cell wall integrity (CWI) and the DNA repair mechanism were tested, the kns1 double-deletion cells showed abnormal patterns compared to wild-type cells; The transcript levels of genes for glycosylphosphatidylinositol (GPI)-anchored proteins were increased upon calcofluor white (CFW) treatment. Under DNA replicative stress, the expression of MluI-cell cycle box binding factor (MBF)-targeted genes, which are expressed during the G1/S transition in the cell cycle, was not increased in the kns1 double-deletion cells. This strain showed increased adhesion to the surface of an agar plate and zebrafish embryo. These results demonstrate that Kns1 is involved in dimorphic transition, cell wall integrity, response to DNA replicative stress, and adherence to the host cell surface in C. albicans.

The Dual-Specificity LAMMER Kinase Affects Stress-Response and Morphological Plasticity in Fungi.

The morphological plasticity of fungal pathogens has long been implicated in their virulence and is often influenced by extracellular factors. Complex signal transduction cascades are critical for sensing stresses imposed by external cues such as antifungal drugs, and for mediating appropriate cellular responses. Many of these signal transduction cascades are well-conserved and involve in the distinct morphogenetic processes during the life cycle of the pathogenic fungi. The dual-specificity LAMMER kinases are evolutionarily conserved across species ranging from yeasts to mammals and have multiple functions in various physiological processes; however, their functions in fungi are relatively unknown. In this review, we first describe the involvement of LAMMER kinases in cell surface changes, which often accompany alterations in growth pattern and differentiation. Then, we focus on the LAMMER kinase-dependent molecular machinery responsible for the stress responses and cell cycle regulation. Last, we discuss the possible cross-talk between LAMMER kinases and other signaling cascades, which integrates exogenous and host signals together with genetic factors to affect the morphological plasticity and virulence in fungi.

Expression of sexual genes in Aspergillus fumigatus homogeneous culture produced by vegetative mass mating.

There are presently no studies on the genes for sexual development of Aspergillus fumigatus in situ using mating culture, primarily because of challenging experimental conditions that require a significantly long period of induction and produce developmentally heterogenous culture, harboring very few sexual organs. In order to overcome these challenges, we developed an efficient and convenient procedure called 'vegetative mass mating (VeM)' for study at a molecular level. The VeM method enabled production of a developmentally homogenous A. fumigatus culture, harboring many sexual organs in a plate within a short period of two weeks. Feasibility of the use of VeM for functional study of genes during A. fumigatus sexual development was evaluated by analyzing the transcription pattern of genes involved in pheromone signal transduction and regulation of sexual development. Here, we present for the first time, an in situ expression pattern of sexual genes during the mating process, induced by the VeM method, which will enable and promote the sexual development study of A. fumigatus at the molecular level.

LAMMER Kinase Lkh1 Is an Upstream Regulator of Prk1-Mediated Non-Sexual Flocculation in Fission Yeast.

The cation-dependent galactose-specific flocculation activity of the Schizosaccharomyces pombe null mutant of lkh1 +, the gene encoding LAMMER kinase homolog, has previously been reported by our group. Here, we show that disruption of prk1 +, another flocculation associated regulatory kinase encoding gene, also resulted in cation-dependent galactose-specific flocculation. Deletion of prk1 increased the flocculation phenotype of the lkh1 + null mutant and its overexpression reversed the flocculation of cells caused by lkh1 deletion. Transcript levels of prk1 + were also decreased by lkh1 + deletion. Cumulatively, these results indicate that Lkh1 is one of the negative regulators acting upstream of Prk1, regulating non-sexual flocculation in fission yeast.

Clostridium kogasensis sp. nov., a novel member of the genus Clostridium, isolated from soil under a corroded gas pipeline.

Two bacterial strains, YHK0403(T) and YHK0508, isolated from soil under a corroded gas pipe line, were revealed as Gram-negative, obligately anaerobic, spore-forming and mesophilic bacteria. The cells were rod-shaped and motile by means of peritrichous flagella. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolates were members of the genus Clostridium and were the most closely related to Clostridium scatologenes KCTC 5588(T) (95.8% sequence similarity), followed by Clostridium magnum KCTC 15177(T) (95.8%), Clostridium drakei KCTC 5440(T) (95.7%) and Clostridium tyrobutyricum KCTC 5387(T) (94.9%). The G + C contents of the isolates were 29.6 mol%. Peptidoglycan in the cell wall was of the A1γ type with meso-diaminopimelic acid. The major polar lipid was diphosphatidylglycerol (DPG), and other minor lipids were revealed as phosphatidylglycerol (PG), phosphatidylethanolamine (PE), two unknown glycolipids (GL1 and GL2), an unknown aminoglycolipid (NGL), two unknown aminophospholipids (PN1 and PN2) and four unknown phospholipids (PL1 to PL4). Predominant fatty acids were C16:0 and C16:1cis9 DMA. The major end products from glucose fermentation were identified as butyrate (12.2 mmol) and acetate (9.8 mmol). Collectively, the results from a wide range of phenotypic tests, chemotaxonomic tests, and phylogenetic analysis indicated that the two isolates represent novel species of the genus Clostridium, for which the name Clostridium kogasensis sp. nov. (type strain, YHK0403(T) = KCTC 15258(T) = JCM 18719(T)) is proposed.

Depletion of ε-COP in the COPI Vesicular Coat Reduces Cleistothecium Production in Aspergillus nidulans.

We have previously isolated ε-COP, the α-COP interactor in COPI of Aspergillus nidulans, by yeast two-hybrid screening. To understand the function of ε-COP, the aneA (+) gene for ε-COP/AneA was deleted by homologous recombination using a gene-specific disruption cassette. Deletion of the ε-COP gene showed no detectable changes in vegetative growth or asexual development, but resulted in decrease in the production of the fruiting body, cleistothecium, under conditions favorable for sexual development. Unlike in the budding yeast Saccharomyces cerevisiae, in A. nidulans, over-expression of ε-COP did not rescue the thermo-sensitive growth defect of the α-COP mutant at 42℃. Together, these data show that ε-COP is not essential for viability, but it plays a role in fruiting body formation in A. nidulans.

Transcriptional regulation of fksA, a ß-1,3-glucan synthase gene, by the APSES protein StuA during Aspergillus nidulans development.

The temporal and spatial regulation of ß-1,3-glucan synthesis plays an important role in morphogenesis during fungal growth and development. Northern blot analysis showed that the transcription of fksA, the gene encoding ß-1,3-glucan synthase in Aspergillus nidulans, was cell-cycle-dependent and increased steadily over the duration of the vegetative period, but its overall expression during the asexual and sexual stages was fairly constant up until the time of transcription cessation. In an A. nidulans strain mutated in the eukaryotic bHLH-like APSES transcription factor stuA1, the transcriptional level of fksA, and consequently the content of alkali-insoluble cell wall ß-glucan, significantly increased at the conidial chain formation and maturation stage. Electrophoretic mobility shift assays revealed that StuA was bound to StREs (StuA Response Elements) on the fksA promoter region. Promoter analysis with sGFP-fusion constructs also indicated the negative regulation of fksA expression by StuA, especially during asexual development. Taken together, these data suggest that StuA plays an important role in cell wall biogenesis during the development of A. nidulans, by controlling the transcription level of fksA.

Luteimicrobium xylanilyticum sp. nov., isolated from the gut of a long-horned beetle, Massicus raddei.

A novel strain, designated W-15(T), was isolated from the gut of a long-horned beetle, Massicus raddei, collected in South Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strains belonged to the suborder Micrococcineae. Strain W-15(T) was most closely related to Luteimicrobium album RI148-Li105(T) (97.9 % similarity). Strain W-15(T) was Gram-stain-positive, rod- and coccus-shaped and non-motile. Growth was observed at 15-37 °C, at pH 4.5-8.5 and in the presence of 0-5.0 % NaCl. The cell-wall peptidoglycan of the strain was A4α (l-Lys-d-Ser-d-Asp). The major menaquinone present in this strain was MK-8 (H2) and the major cellular fatty acids were anteiso-C15 : 0, iso-C16 : 0, iso-C15 : 0 and anteiso-C17 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylinositol, an unknown lipid, an unknown phospholipid and an unknown phosphoglycolipid. The G+C content of genomic DNA of the strain was 73.8 mol%. On the basis of evidence from our polyphasic taxonomic study, strain W-15(T) is classified as representing a novel species in the suborder Micrococcineae, for which the name Luteimicrobium xylanilyticum sp. nov. is proposed. The type strain of this species is strain W-15(T) ( = KCTC 19882(T) = JCM 18090(T)).

Role of LAMMER Kinase in Cell Wall Biogenesis during Vegetative Growth of Aspergillus nidulans.

Depending on the acquisition of developmental competence, the expression of genes for ß-1,3-glucan synthase and chitin synthase was affected in different ways by Aspergillus nidulans LAMMER kinase. LAMMER kinase deletion, ΔlkhA, led to decrease in ß-1,3-glucan, but increase in chitin content. The ΔlkhA strain was also resistant to nikkomycin Z.