The sho1 sensor regulates growth, morphology, and oxidant adaptation in Aspergillus fumigatus but is not essential for development of invasive pulmonary aspergillosis

Aspergillus fumigatus is an important opportunistic fungal pathogen. This organism must be able to adapt to stress changes in the microenvironment during host invasion and systemic spread. The high-osmolarity-glycerol (HOG) mitogen-activated protein kinase (HOG-MAPK) signaling pathway plays an important role in regulating morphology, growth, and adaptation to stress and virulence in a number of fungal pathogens. The Sho1 adaptor protein is one important element of the two upstream branches of the HOG-MAPK pathway in Saccharomyces cerevisiae, a signal transduction cascade involved in adaptation to stress. We constructed a sho1 mutant of A. fumigatus, MA21. Both the growth and germination rates of the mutant were reduced, and the MA21 strain had an irregular hyphal morphology characterized by reduced production of phialides and conidia. This gene deletion mutant was sensitive to 2.5 mM hydrogen peroxide and 15 muM menadione, but it appeared to be minimally sensitive to diamide compared to the wild-type strain. In an immunosuppressed mouse model, the mutant was as virulent as the wild-type or complemented strains. These data support the idea that the loss of sho1, a highly conserved gene among fungi, regulates radial hyphal growth and delays germination of A. fumigatus conidia. In addition, the sho1 gene has a visible effect in the adaptation to oxidative stress in A. fumigatus similar to that in S. cerevisiae.     

An Os-1 family histidine kinase from a filamentous fungus confers fungicide-sensitivity to yeast

Three groups of fungicides (phenylpyrroles, dicarboximides, aromatic hydrocarbons) are effective against filamentous fungi. The target of these fungicides is the osmotic stress signal transduction pathway, which is dependent on the Os-1 family of two-component histidine kinases. These fungicides usually have no fungicidal effect on the yeast Saccharomyces cerevisiae. In this report, we found that expression of Hik1, an Os-1 orthologue from rice blast fungus, can confer fungicide-sensitivity to yeast. This requires both the histidine kinase and the response regulator domains of Hik1. Analysis of yeast mutants indicated that this sensitivity is Hog1- and Ssk1-dependent. In addition, our studies revealed an interaction between Hik1 and Ypd1. These observations suggest that Hik1 is a direct target of the fungicides or is a mediator of fungicide action and that the fungicidal effect is transmitted to the Hog1 pathway via Ypd1.     

MAPK Hog1 closes the S. cerevisiae glycerol channel Fps1 by phosphorylating and displacing its positive regulators

The aquaglyceroprin Fps1 is responsible for glycerol transport in yeast in response to changes in extracellular osmolarity. Control of Fps1 channel activity in response to hyperosmotic shock involves a redundant pair of regulators, Rgc1 (regulator of the glycerol channel 1) and Rgc2, and the MAPK Hog1 (high-osmolarity glycerol response 1). However, the mechanism by which these factors influence channel activity is unknown. We show that Rgc2 maintains Fps1 in the open channel state in the absence of osmotic stress by binding to its C-terminal cytoplasmic domain. This interaction involves a tripartite pleckstrin homology (PH) domain within Rgc2 and a partial PH domain within Fps1. Activation of Hog1 in response to hyperosmotic shock induces the rapid eviction of Rgc2 from Fps1 and consequent channel closure. Hog1 was recruited to the N-terminal cytoplasmic domain of Fps1, which it uses as a platform from which to multiply phosphorylate Rgc2. Thus, these results reveal the mechanism by which Hog1 regulates Fps1 in response to hyperosmotic shock.     

fos-1, a putative histidine kinase as a virulence factor for systemic aspergillosis.

In fungi, two-component histidine kinases have various functions including regulation of osmosensitivity, and of cell-wall assembly. Furthermore, one of these proteins, cos-1, has been shown to be important for virulence of Candida albicans. Recently, a putative histidine kinase, fos-1, has been isolated and partially characterized from Aspergillus fumigatus. Here we compare the virulence of a fos-1 deletion strain with that of the parental wild-type strain in a murine model of systemic aspergillosis. Our results show that the fos-1 deletion strain has significantly reduced virulence as compared with the parental wild-type strain. Thus, we propose that the fos-1 two-component histidine kinase is a virulence factor of A. fumigatus.     

Activation of the Hog1p kinase in Isc1p-deficient yeast cells is associated with mitochondrial dysfunction, oxidative stress sensitivity and premature aging

The Saccharomyces cerevisiae Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, plays a key role in mitochondrial function, oxidative stress resistance and chronological lifespan. Isc1p functions upstream of the ceramide-activated protein phosphatase Sit4p through the modulation of ceramide levels. Here, we show that both ceramide and loss of Isc1p lead to the activation of Hog1p, the MAPK of the high osmolarity glycerol (HOG) pathway that is functionally related to mammalian p38 and JNK. The hydrogen peroxide sensitivity and premature aging of isc1Δ cells was partially suppressed by HOG1 deletion. Notably, Hog1p activation mediated the mitochondrial dysfunction and catalase A deficiency associated with oxidative stress sensitivity and premature aging of isc1Δ cells. Downstream of Hog1p, Isc1p deficiency activated the cell wall integrity (CWI) pathway. Deletion of the SLT2 gene, which encodes for the MAPK of the CWI pathway, was lethal in isc1Δ cells and this mutant strain was hypersensitive to cell wall stress. However, the phenotypes of isc1Δ cells were not associated with cell wall defects. Our findings support a role for Hog1p in the regulation of mitochondrial function and suggest that constitutive activation of Hog1p is deleterious for isc1Δ cells under oxidative stress conditions and during chronological aging.     

Biochemical and genetic studies of a histidine regulatory mutant of Streptomyces coelicolor A3 (2)

The specific activities of three enzymes in a histidine regulatory mutant RF59 of Streptomyces coelicolor A3(2) resistant to the histidine analogue 1,2,4-triazolealanine (TRA) were measured and compared to the activity of the wild type strain. The first enzyme of the histidine pathway, phosphorybosyl-ATP-pyrophosphorylase (PR-ATP-pyrophosphorylase), of mutant RF59 and the wild type was sensitive to allosteric inhibition by L-histidine and hence feed-back inhibition was not affected by mutation, although the specific activity in the mutant was 2.9 fold higher than in the wild type. The other two enzymes coded by genes from the histidine operon were significantly derepressed. The enzyme D-erythroimidazoleglycerol phosphate dehydrase (IGP-dehydrase) in mutant RF59 had a 4.9 fold higher specific activity than in the wild type strain. The specific activity of the last enzyme of the pathway, histidinol-dehydrogenase (Hol-dehydrogenase), in the mutant was 4.7 fold derepressed compared to the wild type strain. The results of genetic crosses revealed the mapping of RF59 regulatory mutation between argA1 and cysD18 on S. coelicolor chromosome, suggesting that the mutant RF59 is a regulatory mutant unable to fully repress genes of the histidine pathway.     

烟曲霉pbs2基因功能初步探讨

目的 克隆烟曲霉pbs2基因,并构建烟曲霉pbs2基因突变株,了解该基因对烟曲霉形态学影响以及对渗透压、氧化压力物质敏感性的变化.方法 通过基因同源性比对,在烟曲霉基因组找出与酿酒酵母pbs2基因同源的序列,分析其结构并通过PCR扩增烟曲霉pbs2基因连同其上下游各约1.0 kb的DNA片段,将该片段重组到质粒pDHt/SK中形成PA,再通过PCR扩增pyrG作为筛选标记插入到PA的pbs2基因开放阅读框架(ORF)中,构建好pbs2基因敲除载体PB.将敲除载体转化到根瘤农杆菌,再利用ATMT法转化嘧啶营养缺陷株烟曲霉AF293.1,筛选出烟曲霉pbs2基因缺陷株△Pbs2.观察突变株和野生株AF293在基础培养基上的生长速度并测定二者对渗透压、氧化压力物质敏感性的变化.结果 经过SMART软件分析发现烟曲霉pbs2基因编码一种磷酸转移酶.在基础培养基上△pbs2生长速度同野生株间差异无统计学意义(P＞0.05),但在氧化压力和渗透压的信号传导中,△pbs2对氧化压力和渗透压的敏感性较野生株AF293敏感.结论 烟曲霉中pbs2基因不仅参与渗透压的传导,还参与氧化压力的传导,但只参与传导特定的氧化压力.