Sites Of Fungal Infection Research Articles

The granulocyte nucleus and lamin B receptor: avoiding the ovoid

The major human blood granulocyte, the neutrophil, is an essential component of the innate immunity system, emigrating from blood vessels and migrating through tight tissue spaces to the site of bacterial or fungal infection where they kill and phagocytose invading microbes. Since the late nineteenth century, it has been recognized that the human neutrophil nucleus is distinctly not ovoid as in other cell types, but possesses a lobulated (segmented) shape. This deformable nucleus enhances rapid migration. Recent studies have demonstrated that lamin B receptor (LBR) is necessary for the non-ovoid shape. LBR is an integral membrane protein of the nuclear envelope. A single dominant mutation in humans leads to neutrophils with hypolobulated nuclei (Pelger-Huet anomaly); homozygosity leads to ovoid granulocyte nuclei. Interestingly, LBR is also an enzyme involved in cholesterol metabolism. Homozygosity for null mutations is frequently lethal and associated with severe skeletal deformities. In addition to the necessity for LBR, formation of the mature granulocyte nucleus also depends upon lamin composition and microtubule integrity. These observations are part of a larger question on the relationships between nuclear shape and cellular function.

Imaging features of fungal infection in immuno-suppressed patients in a local ward outbreak

Purpose of studyTo examine the role of imaging in diagnosing and assessing fungal infections in paediatric patients undergoing chemotherapy in a facility, which had high fungal air contamination due to adjacent building construction work.Materials and methodNineteen patients aged five months to 12 years with various malignancies, mainly leukaemia, along with probable fungal infection were referred for imaging over a period of 12 months. The imaging findings from their CT and chest radiographs were reviewed by two radiologists and correlated with the clinical findings. Blood culture and/or biopsy of relevant lesions were performed for all patients.ResultsFungus was positively isolated in 11 out of 19 patients, but the remaining patients clinically had fungal infection. The most common species isolated was Candida sp. (five patients), followed by Aspergillus sp. The most common site of fungal infection was the lungs (10 out of 19 patients), where consolidation or cavitating nodules were seen on CT or the plain chest radiograph. One patient developed pulmonary artery aneurysm as a complication. The other sites affected were the intra-abdominal organs (liver, kidneys, and spleen) and the paranasal sinuses, shown on CT. Two patients with clinical evidence of infection and Candida sp. isolated from their blood, however, showed no abnormal findings on imaging.ConclusionEarly diagnosis of fungal infections in oncology patients undergoing chemotherapy is important, but diagnosis may be difficult through imaging because of the non-specific changes and the presence of abnormalities from the underlying disease. Even if a specific diagnosis cannot be reached, imaging is useful to monitor response to treatment and detect complications.

Targeted Gene Disruption of Glycerol-3-phosphate Dehydrogenase in Colletotrichum gloeosporioides Reveals Evidence That Glycerol Is a Significant Transferred Nutrient from Host Plant to Fungal Pathogen

Unidirectional transfer of nutrients from plant host to pathogen represents a most revealing aspect of the parasitic lifestyle of plant pathogens. Whereas much effort has been focused on sugars and amino acids, the identification of other significant metabolites is equally important for comprehensive characterization of metabolic interactions between plants and biotrophic fungal pathogens. Employing a strategy of targeted gene disruption, we generated a mutant strain (gpdhDelta) defective in glycerol-3-phosphate dehydrogenase in a hemibiotrophic plant pathogen, Colletotrichum gloeosporioides f.sp. malvae. The gpdhDelta strain had severe defects in carbon utilization as it could use neither glucose nor amino acids for sustained growth. Although the mutant mycelia were able to grow on potato dextrose agar medium, they displayed arrhythmicity in growth and failure to conidiate. The metabolic defect of gpdhDelta could be entirely ameliorated by glycerol in chemically defined minimal medium. Furthermore, glycerol was the one and only metabolite that could restore rhythmic growth and conidiation of gpdhDelta. Despite the profound defects in carbon source utilization, in planta the gpdhDelta strain exhibited normal pathogenicity, proceeded normally in its life cycle, and produced abundant conidia. Analysis of plant tissues at the peripheral zone of fungal infection sites revealed a time-dependent reduction in glycerol content. This study provides strong evidence for a role of glycerol as a significant transferred metabolite from plant to fungal pathogen.

Innovations in liposomal formulations for antimicrobial therapy

Failure of antimicrobial treatment is observed frequently in hospitalised patients. These patients are more susceptible to opportunistic infections and the course of infection is often more serious as these patients fail to develop an adequate inflammatory response. A possible way to improve antimicrobial treatment is to formulate drugs into liposomes. The purpose of the liposomal encapsulation is to improve control over the site, time and rate of drug delivery. This review describes patents claiming the use of liposomes for the delivery of antimicrobial agents and immunostimulants to sites of bacterial or fungal infections since 1998.

T‐DNA tagging of a pathogen inducible promoter in Arabidopsis thaliana

Summary Many events associated with the plant defence responses are regulated on the transcriptional level. Here we report the results of a promoter tagging approach to identify promoters that are induced upon pathogen attack in Arabidopsis thaliana. A line was identified in a T-DNA UidA tagged Arabidopsis library with induced GUS expression after Botrytis cinerea infection around the site of fungal infection. The upstream sequence was isolated and fused to the UidA gene and tested in transgenic Arabidopsis thaliana and Brassica napus plants. Promoter function was very similar to the expression pattern found in the original promoter tagged line. We found that the promoter sequence was located on Arabidopsis chromosome III and linked to a predicted open reading frame in the reverse orientation. The predicted gene codes for a putative receptor serine threonine protein kinase of 383 amino acids in size. The clone contains a protein kinase ATP binding region, a protein kinase active site, a region with similarity to motifs found in Alpha Isopropylmalate/homocitrate synthase enzymes and a putative leucine zipper motif. Analysis of the expression pattern of the gene using RT-PCR demonstrated that the putative receptor serine threonine protein kinase is up-regulated after Salicylic acid treatment and Botrytis infection.

Rds and Rih mediate hypersensitive cell death independent of gene-for-gene resistance to the oat crown rust pathogen Puccinia coronata f. sp. avenae.

The Pca crown rust resistance cluster in the diploid Avena genus confers gene-for-gene specificity to numerous isolates of Puccinia coronata f. sp. avenae. Recombination breakpoint analysis indicates that specificities conferred by the Pca cluster are controlled by at least five distinct genes, designated Pc81, Pc82, Pc83, Pc84, and Pc85. Avena plants with the appropriate genotype frequently respond to P. coronata by undergoing hypersensitive cell death at the sites of fungal infection. Autofluorescence of host cells in response to P. coronata occurs in plants that develop visible necrotic lesions but not in plants that lack this phenotype. Two newly described, non-Pc loci were shown to control hypersensitive cell death. Rds (resistance-dependent suppressor of cell death) suppresses the hypersensitive response (HR), but not the resistance, mediated by the Pc82 resistance gene. In contrast, Rih (resistance-independent hypersensitive cell death) confers HR in both resistant and susceptible plants. Linkage analysis indicates that Rds is unlinked to the Pca cluster, whereas Rih is tightly linked to it. These results indicate that multiple synchronous pathways affect the development of hypersensitive cell death and that HR is not essential for resistance to crown rust. Further characterization of these genes will clarify the relationship between plant disease resistance and localized hypersensitive cell death.

Compartmental pharmacokinetics and tissue distribution of the antifungal echinocandin lipopeptide micafungin (FK463) in rabbits.

The plasma pharmacokinetics and tissue distribution of the novel antifungal echinocandin-like lipopeptide micafungin (FK463) were investigated in healthy rabbits. Cohorts of three animals each received micafungin at 0.5, 1, and 2 mg/kg of body weight intravenously once daily for a total of 8 days. Serial plasma samples were collected on days 1 and 7, and tissue samples were obtained 30 min after the eighth dose. Drug concentrations were determined by validated high-performance liquid chromatographic methods. Plasma drug concentration data were fit to a two-compartment pharmacokinetic model, and pharmacokinetic parameters were estimated using weighted nonlinear least-square regression analysis. Micafungin demonstrated linear plasma pharmacokinetics without changes in total clearance and dose-normalized area under the concentration-time curve from 0 h to infinity. After administration of single doses to the rabbits, mean peak plasma drug concentrations ranged from 7.62 microg/ml at 0.5 mg/kg to 16.8 microg/ml at 2 mg/kg, the area under the concentration-time curve from 0 to 24 h ranged from 5.66 to 21.79 microg x h/ml, the apparent volume of distribution at steady state ranged from 0.296 to 0.343 liter/kg, and the elimination half-life ranged from 2.97 to 3.20 h, respectively. No significant changes in pharmacokinetic parameters and no accumulation was noted after multiple dosing. Mean tissue micafungin concentrations 30 min after the last of eight daily doses were highest in the lung (2.26 to 11.76 microg/g), liver (2.05 to 8.82 microg/g), spleen (1.87 to 9.05 microg/g), and kidney (1.40 to 6.12 microg/g). While micafungin was not detectable in cerebrospinal fluid, the concentration in brain tissue ranged from 0.08 to 0.18 microg/g. These findings indicate linear disposition of micafungin at dosages of 0.5 to 2 mg/kg and achievement of potentially therapeutic drug concentrations in plasma and tissues that are common sites of invasive fungal infections.

Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors.

Parsley WRKY proteins comprise a family of plant-specific zinc-finger-type factors implicated in the regulation of genes associated with pathogen defence. In vitro, these proteins bind specifically to functionally defined TGAC-containing W box promoter elements within the Pathogenesis-Related Class10 (PR-10) genes. Here we present in vivo data demonstrating that WRKY1 is a transcriptional activator mediating fungal elicitor-induced gene expression by binding to W box elements. In situ RNA hybridization revealed that the WRKY1 gene is rapidly and locally activated in parsley leaf tissue around fungal infection sites. Transient expression studies in parsley protoplasts showed that a specific arrangement of W box elements in the WRKY1 promoter itself is necessary and sufficient for early activation and that WRKY1 binds to such elements. Our results demonstrate that WRKY transcription factors play an important role in the regulation of early defence-response genes including regulation of WRKY1.

In vitro and in vivo antifungal activity of amphotericin B lipid complex: are phospholipases important?

Amphotericin B lipid complex for injection (ABLC) is a suspension of amphotericin B complexed with the lipids L-alpha-dimyristoylphosphatidylcholine (DMPC) and L-alpha-dimyristoylphosphatidylglycerol. ABLC is less toxic than amphotericin B deoxycholate (AmB-d), while it maintains the antifungal activity of AmB-d. Active amphotericin B can be released from ABLC by exogenously added (snake venom, bacteria, or Candida-derived) phospholipases or by phospholipases derived from activated mammalian vascular tissue (rat arteries). Such extracellular phospholipases are capable of hydrolyzing the major lipid in ABLC. Mutants of C. albicans that were resistant to ABLC but not AmB-d in vitro were deficient in extracellular phospholipase activity, as measured on egg yolk agar or as measured by their ability to hydrolyze DMPC in ABLC. ABLC was nevertheless effective in the treatment of experimental murine infections produced by these mutants. Isolates of Aspergillus species, apparently resistant to ABLC in vitro (but susceptible to AmB-d), were also susceptible to ABLC in vivo. We suggest that routine in vitro susceptibility tests with ABLC itself as the test material may not accurately predict the in vivo activity of ABLC and that the enhanced therapeutic index of ABLC relative to that of AmB-d in vivo may be due, in part, to the selective release of active amphotericin B from the complex at sites of fungal infection through the action of fungal or host cell-derived phospholipases.

Elemental micro-PIXE mapping of hypersensitive lesions in Lagenaria sphaerica (Cucurbitaceae) resistant to Sphaerotheca fuliginea (powdery mildew)

Genotypes of the Southern African cucurbit, Lagenaria sphaerica, that are resistant to powdery-mildew ( Sphaerotheca fuliginea) exhibit foliar hypersensitive (HS) lesions on inoculation with this fungal pathogen. Elemental distributions across radially symmetrical HS lesions, surrounding unlesioned leaf tissue and uninoculated leaf tissue, were obtained using the true elemental imaging system (Dynamic Analysis) of the NAC Van de Graaff nuclear microprobe. Raster scans of 3 MeV protons were complemented by simultaneous PIXE and BS point analyses. The composition of cellulose (C 6H 10O 5) was used as constant matrix composition for scans, and the sample thickness was found from BS spectra. Si and elements heavier than Ca contributed to matrix composition within HS lesions and the locally elevated Ca raised the limits of detection for some trace metals of interest. In comparison to uninoculated tissue, inoculated tissue was characterised by higher overall concentrations of all measured elements except Cu. Fully developed, 6 day-old HS lesions and the surrounding tissue could be divided into five zones, centred on the fungal infection site. Each zone was characterized by distinct local elemental distributions (either depletion, or accumulation to potentially phytotoxic levels).

Infection of the Migratory Grasshopper (Orthoptera: Acrididae) by Ingestion of the Entomopathogenic Fungus Beauveria bassiana

Infection of the migratory grasshopper. Melanoplus sanguinipes (F.). by ingestion of Beauveria bassiana (Balsamo) Vuillemin conidiospores was investigated. Laboratory bioassays were performed where grasshoppers were fed a wheat-bran formulation containing B. bassiana spores. Grasshopper mortality was directly related to the ingested spore dosages. Probit analysis estimated LD50 at 3.4 X 105 conidia per host. with 95% FL from 2.1 X 105 to 5.5 X 105. The LT50s for the top 3 dosage treatments where final mortality exceeded 50% were 9.6, 12.5. and 18.4 d for the respective dosages of 1.1 X 106, 5.4 X 106, and 1.0 X 107 conidia per host. The LT50 for the highest dosage (9.6 d) in the feeding bioassay was greater than that seen for insects immersed in a liquid inoculum but lower than for insects that had only their heads dipped. These data suggest that as a result of feeding. spores are brought to favorable infection sites within the head and mouthparts. The nature and sites of fungal infection by feeding were elucidated using epifluorescence and scanning electron microscopy. The exposed and internal mouthpart surfaces of the grasshopper were the predominant sites of spore attachment and germination. The presence of numerous hairs and cavities on these structures greatly facilitates fungal spore attachment and subsequent germination. It would appear that fungal invasion by way of the gut is an uncommon occurrence.

Rapid, transient, and highly localized induction of plastidial omega-3 fatty acid desaturase mRNA at fungal infection sites in Petroselinum crispum.

Parsley (Petroselinum crispum) plants and suspension-cultured cells have been used extensively for studies of non-host-resistance mechanisms in plant/pathogen interactions. We now show that treatment of cultured parsley cells with a defined peptide elicitor of fungal origin causes rapid and large changes in the levels of various unsaturated fatty acids. While linoleic acid decreased and linolenic acid increased steadily for several hours, comparatively sharp increases in oleic acid followed a biphasic time course. In contrast, the overall level of stearic acid remained unaffected. Using a PCR-based approach, a parsley cDNA was isolated sharing high sequence similarity with omega-3 fatty acid desaturases. Subsequent isolation and characterization of a full-length cDNA enabled its functional identification as a plastid-localized omega-3 fatty acid desaturase by complementation of the Arabidopsis thaliana fad7/8 double mutant which is low in trienoic fatty acids. omega-3 Fatty acid desaturase mRNA accumulated rapidly and transiently in elicitor-treated cultured parsley cells, protoplasts, and leaves, as well as highly localized around fungal infection sites in parsley leaf buds. These results indicate that unsaturated fatty acid metabolism is yet another component of the highly complex, transcriptionally regulated pathogen defense response in plants.

A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase.

We describe an aromatic alcohol dehydrogenase with properties indicating a novel type of function in the defense response of plants to pathogens. To obtain the enzyme free of contamination with possible isoforms, a parsley (Petroselinum crispum) cDNA comprising the entire coding region of the elicitor-responsive gene, ELI3, was expressed in Escherichia coli. In accord with large amino acid sequence similarities with established cinnamyl and benzyl alcohol dehydrogenases from other plants, the enzyme efficiently reduced various cinnamyl and benzyl aldehydes using NADPH as a co-substrate. Highest substrate affinities were observed for cinnamaldehyde, 4-coumaraldehyde and coniferaldehyde, whereas sinapaldehyde, one of the most efficient substrates of several previously analyzed cinnamyl alcohol dehydrogenases and a characteristic precursor molecule of angiosperm lignin, was not converted. A single form of ELI3 mRNA was strongly and rapidly induced in fungal elicitor-treated parsley cells. These results, together with earlier findings that the ELI3 gene is strongly activated both in elicitor-treated parsley cells and at fungal infection sites in parsley leaves, but not in lignifying tissue, suggest a specific role of this enzyme in pathogen defense-related phenylpropanoid metabolism.

Biphasic Temporal and Spatial Induction Patterns of Defense-Related mRNAs and Proteins in Fungus-Infected Parsley Leaves.

Previous experiments using in situ RNA hybridization have shown that the mRNAs encoding phenylalanine ammonia-lyase, 4-coumarate:coenzyme A ligase, and pathogenesis-related protein 1 accumulated transiently around fungal infection sites in parsley (Petroselinum crispum) leaf buds. These studies have now been extended by (a) analyzing different stages of the infection process and (b) monitoring the timing of appearance and the spatial distribution of the proteins as well as the corresponding mRNAs. An early and short period of mRNA induction throughout a large portion of the infected leaf was followed by a longer period, during which the mRNA levels remained high in a more localized area around the site of fungal penetration with sharp borders toward the surrounding tissue. This biphasic pattern of mRNA accumulation was followed after some delay by the same pattern of protein accumulation.

Gene activation by UV light, fungal elicitor or fungal infection in Petroselinum crispum is correlated with repression of cell cycle‐related genes

The effects of UV light or fungal elicitors on plant cells have so far been studied mostly with respect to defense-related gene activation. Here, an inverse correlation of these stimulatory effects with the activities of several cell cycle-related genes is demonstrated. Concomitant with the induction of flavonoid biosynthetic enzymes in UV-irradiated cell suspension cultures of parsley (Petroselinum crispum), total histone synthesis declined to about half the initial rate. A subclass of the histone H3 gene family was selected to demonstrate the close correlation of its expression with cell division, both in intact plants and cultured cells. Using RNA-blot and run-on transcription assays, it was shown that one arbitrarily selected subclass of each of the histone H2A, H2B, H3 and H4 gene families and of the genes encoding a p34cdc2 protein kinase and a mitotic cyclin were transcriptionally repressed in UV-irradiated as well as fungal elicitor-treated parsley cells. The timing and extent of repression differed between the two stimuli; the response to light was more transient and smaller in magnitude. These differential responses to light and elicitor were inversely correlated with the induction of phenylalanine ammonia-lyase, a key enzyme of phenylpropanoid metabolism. Essentially the same result was obtained with a defined oligopeptide elicitor, indicating that the same signaling pathway is responsible for defense-related gene activation and cell cycle-related gene repression. A temporary (UV light) or long-lasting (fungal elicitor) cessation of cell culture growth is most likely due to an arrest of cell division which may be a prerequisite for full commitment of the cells to transcriptional activation of full commitment of the cells to transcriptional activation of pathways involved in UV protection or pathogen defense. This conclusion is corroborated by the observation that the histone H3 mRNA level greatly declined around fungal infection sites in young parsley leaves.

Isolated Renal Mucormycosis Simulating a Renal Neoplasm in a Patient With the Acquired Immunodeficiency Syndrome

We report the case of a terminally ill patient with the acquired immunodeficiency syndrome who presented with a tender enlarged renal mass that simulated a renal neoplasm. A needle biopsy of the kidney revealed ischemic necrosis, vascular destruc tion, and fungal forms consistent with infection by the Zygomycetes family. Subse quently, autopsy confirmed this diagnosis and established that the kidney was the sole site of this fungal infection. Since fungal infections of the kidney simulating a renal tumor have not previously been described in acquired immunodeficiency syndrome, this report expands the list of lesions produced by opportunistic infections arising in a setting of human immunodeficiency virus-induced immunosuppression. Int J Surg Pathol 2(2):163-168, 1994

An ultrastructural study of tomato roots inoculated with pathogenic and non‐pathogenic necrotrophic fungi and a saprophytic fungus

Tomato cultivar Moneymaker was independently inoculated with Alternaria alternata, Cunninghamella elegans, Fusarium culmorum, F. oxysporum f.sp. lycopersici, F. oxysporum f.sp. pisi and Stromatinia gladioli and analysed ultrastructurally. The extent and amount of superficial fungal growth on tomato roots was similar but C. elegans, a saprophyte, was exceptional in that hyphae were not closely appressed to plant surfaces and did not adhere to plant cell walls.In general, the type of plant responses to fungal colonization and infection were similar in all of the interactions studied, with the exception of C. elegans which did not infect tomato root tissue. The failure to penetrate tomato roots by C. elegans may have been associated with the lack of hyphal adhesion to plant cell walls. Migration of cytoplasm and wall apposition/penetration papilla formation were regularly observed in tomato root tissue beneath appressed hyphae and at sites of fungal infection. Specific cellular reactions in the exodermis, namely the formation of wall ‘inclusions’ and appearance of ‘sensitive’ cells, indicated that exodermal cells were particularly responsive to fungal challenge. Fusarium oxysporum f.sp. lycopersici, a pathogen of tomato, invaded tomato root tissue more extensively than the other fungi inoculated onto tomato roots. Infection of tomato by the other fungi studied was variable, and the extent and success of fungal invasion was tentatively associated with their necrotrophic capability and typical host range.

Mycobacterium infection in pejerrey, Odonthestes bonariensis Cuvier & Valenciennes

Abstract. Mycobacterium infection was found in pejerrey, Odonthestes bonariensis Cuvier & Valenciennes, with and without saprolegniasis, in Japan. Many typical focal mycobacterial granulomas were found in the kidney, spleen, liver and pancreas, but they were not found in lateral musculature at sites of fungal infection, or in the intestine, ovary, heart and gills of infected fish. The isolated bacterium was photochromogenic and was identified as a rapidly growing species of the genus Mycobacterium.

BPF-1, a pathogen-induced DNA-binding protein involved in the plant defense response.

The mechanisms by which plants restrict the growth of pathogens include transient activation of numerous defense-related genes. Box P is a putative cis-acting element of a distinct group of such genes, including those encoding the enzyme phenylalanine ammonialyase (PAL). A DNA-binding activity to Box P was identified in nuclear extracts from cultured parsley cells and a cDNA encoding the protein BPF-1 (Box P-binding Factor) partially characterized. BPF-1 binds to this element with specificity similar to that of the binding activity in nuclear extracts. BPF-1 mRNA accumulates rapidly in elicitor-treated parsley cells and around fungal infection sites on parsley leaves. This accumulation is, at least partly, due to a rapid and transient increase in the transcription rate of BPF-1. Moreover, tight correlation between the relative amounts of BPF-1 and PAL mRNAs was observed in different organs of a parsley plant. These results are consistent with the hypothesis that BPF-1 is involved in disease resistance by modulating plant defense gene expression.

Temporal and spatial patterns of 1, 3‐β‐glucanase and chitinase induction in potato leaves infected by Phytophthora infestans

SummaryInfection of potato (Solarium tuberosum L.) leaves with the fungal pathogen Phytophthora infestans caused a similar, strong and coordinated induction of 1, 3‐β‐glucanases and chitinases in compatible (plant susceptible) and incompatible (plant resistant) interactions of two selected plant cultivars with appropriate races of the fungus. The temporal and spatial patterns of 1, 3‐β‐glucanase induction were studied in further detail by immunohistochemical and in‐situ hybridization methods. Accumulation of the protein was preceded by progressive activation of the corresponding gene, commencing near infection sites and spreading rapidly throughout the whole infected leaf as well as to adjacent, non‐infected leaves. Protein and mRNA distribution patterns were nearly identical in compatible and incompatible interactions. In comparison with 1, 3‐β‐glucanase mRNA, phenyl‐alanine ammonia‐lyase mRNA accumulated more rapidly and remained restricted to the vicinity of fungal infection sites, in addition to its constitutive occurrence in the vascular bundles. Even more rapid than any detectable mRNA induction was the accumulation of auto fluorescing material in plant cells immediately surrounding fungal structures, particularly and invariably in incompatible interactions and less frequently in compatible interactions. It is concluded that cultivar‐race‐specific resistance is established early in the interaction of potato leaves with P. infestans and hence the observed massive accumulation of 1, 3‐β‐glucanase and chitinase is presumably not involved in determining this specificity.