看護学部教官業績目録 ; 2003年1月～12月

Acinetobacter baumannii ATCC 19606 tolerates loss of lipopolysaccharide (LPS) caused by inactivation of early LPS pathway genes. However, mutations in pathway genes encoding steps downstream of LpxD have not been reported, implying that later biosynthetic steps may be essential for viability. Here, we determined if LpxH, the UDP-2,3-diacylglucosamine hydrolase that generates UMP-2,3-diacylglucosamine 1-phosphate (lipid X), was essential in A. baumannii ATCC 19606. Multiple attempts to disrupt lpxH on the genome were unsuccessful. When expression of LpxH was placed under control of an isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoter, the cells failed to grow under standard laboratory conditions without IPTG induction. Growth under LpxH depletion conditions (-IPTG) was rescued by chemical inhibition of LpxC, upstream of LpxH, indicating that toxic accumulation of LPS pathway intermediates underlies LpxH essentiality. Consistent with this, the levels of LpxH substrate (product of LpxD) and a C14:0(3-OH) acyl variant of the LpxD substrate had accumulated in cells that were depleted of LpxH causing a growth defect. Intriguingly, under these partial depletion conditions, there was also a smaller but reproducible accumulation of the downstream pathway intermediates disaccharide 1-monophosphate and lipid IVA suggesting a complex downstream response to LpxH depletion

HacA-Independent Functions of the ER Stress Sensor IreA Synergize with the Canonical UPR to Influence Virulence Traits in Aspergillus fumigatus

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacAi, or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireAΔ10. Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus

A Role for the Unfolded Protein Response (UPR) in Virulence and Antifungal Susceptibility in Aspergillus fumigatus

Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The ΔhacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37°C, but cell wall integrity was disrupted at 45°C, resulting in a dramatic loss in viability. The ΔhacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the ΔhacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy

The Aspergillus fumigatus metacaspases CasA and CasB facilitate growth under conditions of endoplasmic reticulum stress

We have examined the contribution of metacaspases to the growth and stress response of the opportu-nistic human mould pathogen, Aspergillus fumiga-tus, based on increasing evidence implicating the yeast metacaspase Yca1p in apoptotic-like pro-grammed cell death. Single metacaspase-deficient mutants were constructed by targeted disruption of each of the two metacaspase genes in A. fumigatus, casA and casB, and a metacaspase-deficient mutant, DcasA/DcasB, was constructed by disrupting both genes. Stationary phase cultures of wild-type A. fumigatus were associated with the appearance of typical markers of apoptosis, including elevated pro-teolytic activity against caspase substrates, phos-phatidylserine exposure on the outer leaflet of the membrane, and loss of viability. By contrast, phos-phatidylserine exposure was not observed in sta-tionary phase cultures of the DcasA/DcasB mutant, although caspase activity and viability was indi-stinguishable from wild type. The mutant retained wild-type virulence and showed no difference in sen-sitivity to a range of pro-apoptotic stimuli that have been reported to initiate yeast apoptosis. However, the DcasA/DcasB mutant showed a growth detriment in the presence of agents that disrupt endoplasmic reticulum homeostasis. These findings demonstrate that metacaspase activity in A. fumigatus contrib-utes to the apoptotic-like loss of membrane phos-pholipid asymmetry at stationary phase, and suggest that CasA and CasB have functions that support growth under conditions of endoplasmic reticulum stress

Divergent Protein Kinase A isoforms co-ordinately regulate conidial germination, carbohydrate metabolism and virulence in Aspergillus fumigatus

U ovom radu prikazan je postupak energetskog planiranja na primjeru Karlovačke županije. Poseban naglasak stavljen je na centralizirane toplinske sustave i njihov utjecaj na cijeli energetski sustav u vidu omogućavanja fleksibilnijeg vođenja sustava te veće implementacije obnovljivih izvora energije. U prvom dijelu rada dane su teoretske osnove kojima se nastojalo opisati centralizirane toplinske sustave, način njihova funkcioniranja te prednosti koje donose. Opisano je i trenutno stanje tih sustava u Hrvatskoj i Europskoj Uniji, kao i budući trendovi razvoja. Nadalje, dan je pregled najvažnijih zakonskih regulativa i direktiva na nacionalnoj i europskoj razini koje se odnose na centralizirane toplinske sustave te njihovu implementaciju. U drugom dijelu rada opisan je konkretan postupak energetskog planiranja Karlovačke županije. Za uspješno izvođenje tog postupka bilo je potrebno opisati trenutno stanje energetskog sustava županije te se upoznati s postojećim planovima razvoja. Prikazana su tri scenarija razvoja energetskog sustava. Prvi scenarij je razvijen prema principu „business as usual“ i on podrazumijeva nastavak postojećih trendova, dok preostala dva scenarija uključuju unaprjeđenje centraliziranih toplinskih sustava te visoku penetraciju obnovljivih izvora energije i centraliziranih toplinskih sustava. Izvršena je analiza potrošnje energije, kretanje uvoza i izvoza energije te emisija CO2 u različitim scenarijima.In this paper an example of energy planning for the Karlovac County has been demonstrated. District heating systems and their impact on the whole energy system in terms of more flexible control of the system and higher implementation of renewable energy sources have been emphasised. In the first part of the paper the theoretical background has been given with the purpose of describing district heating and cooling systems, principles of their operation and the benefits they bring. Current state of these systems in Croatia and European Union has been described, as well as the future development trends. Furthermore, an overview of the most important legislations and directives on the national and European level related to the district heating and cooling systems and their implementation has been given. In the second part of the paper the energy planning method for the Karlovac County has been described. For the successful application of this method, it was necessary to describe the current state of the energy system of the county, as well as get acquainted with the existing development plans. Three scenarios of the energy system development have been described. First scenario was developed by the „business as usual“ principle and it purports continuation of the existing trends, while the two other scenarios include district heating systems development and a high penetration of renewable energy sources and district heating systems. Energy consumption, energy import and export and CO2 emissions have been analysed in different scenarios

Unexpected Link between Metal Ion Deficiency and Autophagy in Aspergillus fumigatus▿ †

Autophagy is the major cellular pathway for bulk degradation of cytosolic material and is required to maintain viability under starvation conditions. To determine the contribution of autophagy to starvation stress responses in the filamentous fungus Aspergillus fumigatus, we disrupted the A. fumigatus atg1 gene, encoding a serine/threonine kinase required for autophagy. The ΔAfatg1 mutant showed abnormal conidiophore development and reduced conidiation, but the defect could be bypassed by increasing the nitrogen content of the medium. When transferred to starvation medium, wild-type hyphae were able to undergo a limited amount of growth, resulting in radial expansion of the colony. In contrast, the ΔAfatg1 mutant was unable to grow under these conditions. However, supplementation of the medium with metal ions rescued the ability of the ΔAfatg1 mutant to grow in the absence of a carbon or nitrogen source. Depleting the medium of cations by using EDTA was sufficient to induce autophagy in wild-type A. fumigatus, even in the presence of abundant carbon and nitrogen, and the ΔAfatg1 mutant was severely growth impaired under these conditions. These findings establish a role for autophagy in the recycling of internal nitrogen sources to support conidiophore development and suggest that autophagy also contributes to the recycling of essential metal ions to sustain hyphal growth when exogenous nutrients are scarce

LpxK is essential for growth of Acinetobacter baumannii ATCC 19606: relationship to toxic accumulation of lipid A pathway intermediates

Acinetobacter baumannii ATCC 19606 can grow without lipooligosaccharide (LOS). Lipid A, the major component of LOS, is therefore not essential in this species. Despite this, we previously reported that depletion of LpxH (the fourth enzyme in the lipid A biosynthetic pathway), prevented growth of this strain of A. baumannii due to the toxic accumulation of lipid A pathway intermediates. Here, we explored whether a similar phenomenon can occur with depletion of LpxK, a kinase that phosphorylates disaccharide 1-monophosphate (DSMP) at the 4'- position to yield lipid IVA. An A. baumannii ATCC 19606 derivative with LpxK expression under control of an isopropyl β-D-1-thiogalactopyranoside (IPTG) regulated expression system failed to grow without induction, indicating that LpxK is essential for growth. Light and electron microscopy of cells depleted of LpxK revealed morphological changes relating to the cell envelope, consistent with the notion of toxic accumulation of lipid A pathway intermediates disrupting cell membranes. Using a liquid chromatography-mass spectrometry (LCMS) approach, cellular accumulation of the detergent-like pathway intermediates DSMP and lipid X was shown. Toxic accumulation was further supported by restoration of growth upon chemical inhibition of LpxC (upstream of LpxK and the first committed step of lipid A biosynthesis) using CHIR-090. Interestingly, cerulenin, an inhibitor of fatty acid biosynthesis, and an acetyl-CoA carboxylase inhibitor, could also abrogate the requirement for LpxK expression. Intriguingly, growth rescue with these inhibitors was possible on cation-adjusted Mueller-Hinton agar, but not MacConkey agar. The latter contains outer membrane impermeable bile salts, suggesting that despite growth restoration, the cell membrane permeability barrier was not restored. Overall this indicates that LpxK is essential for growth of A. baumannii, since loss of LpxK causes accumulation of detergent-like pathway intermediates that inhibit cell growth

Impaired Ribosome Biogenesis Disrupts the Integration between Morphogenesis and Nuclear Duplication during the Germination of Aspergillus fumigatus▿

Aspergillus fumigatus is an important opportunistic fungal pathogen that is responsible for high mortality rates in the immunosuppressed population. CgrA, the A. fumigatus ortholog of a Saccharomyces cerevisiae nucleolar protein involved in ribosome biogenesis, contributes to the virulence of this fungus by supporting rapid growth at 37°C. To determine how CgrA affects ribosome biogenesis in A. fumigatus, polysome profile and ribosomal subunit analyses were performed on both wild-type A. fumigatus and a ΔcgrA mutant. The loss of CgrA was associated with a reduction in the level of 80S monosomes as well as an imbalance in the 60S:40S subunit ratio and the appearance of half-mer ribosomes. The gene expression profile in the ΔcgrA mutant revealed increased abundance of a subset of translational machinery mRNAs relative to the wild type, suggesting a potential compensatory response to CgrA deficiency. Although ΔcgrA conidia germinated normally at 22°C, they swelled excessively when incubated at 37°C and accumulated abnormally high numbers of nuclei. This hypernucleated phenotype could be replicated pharmacologically by germinating wild-type conidia under conditions of reductive stress. These findings indicate that the germination process is particularly vulnerable to global disruption of protein synthesis and suggest that CgrA is involved in both ribosome biogenesis and polarized cell growth in A. fumigatus

A pathway-directed positive growth restoration assay to facilitate the discovery of lipid A and fatty acid biosynthesis inhibitors in <i>Acinetobacter baumannii</i>

<div><p><i>Acinetobacter baumannii</i> ATCC 19606 can grow without lipooligosaccharide (LOS). Lack of LOS can result from disruption of the early lipid A biosynthetic pathway genes <i>lpxA</i>, <i>lpxC</i> or <i>lpxD</i>. Although LOS itself is not essential for growth of <i>A</i>. <i>baumannii</i> ATCC 19606, it was previously shown that depletion of the lipid A biosynthetic enzyme LpxK in cells inhibited growth due to the toxic accumulation of lipid A pathway intermediates. Growth of LpxK-depleted cells was restored by chemical inhibition of LOS biosynthesis using CHIR-090 (LpxC) and fatty acid biosynthesis using cerulenin (FabB/F) and pyridopyrimidine (acetyl-CoA-carboxylase). Here, we expand on this by showing that inhibition of enoyl-acyl carrier protein reductase (FabI), responsible for converting trans-2-enoyl-ACP into acyl-ACP during the fatty acid elongation cycle also restored growth during LpxK depletion. Inhibition of fatty acid biosynthesis during LpxK depletion rescued growth at 37°C, but not at 30°C, whereas rescue by LpxC inhibition was temperature independent. We exploited these observations to demonstrate proof of concept for a targeted medium-throughput growth restoration screening assay to identify small molecule inhibitors of LOS and fatty acid biosynthesis. The differential temperature dependence of fatty acid and LpxC inhibition provides a simple means by which to separate growth stimulating compounds by pathway. Targeted cell-based screening platforms such as this are important for faster identification of compounds inhibiting pathways of interest in antibacterial discovery for clinically relevant Gram-negative pathogens.</p></div