eIn Aspergillus nidulans, the AcuK and AcuM transcription factors form a complex that regulates gluconeogenesis. In Aspergillus fumigatus, AcuM governs gluconeogenesis and iron acquisition in vitro and virulence in immunosuppressed mice. However, the function of AcuK was previously unknown. Through in vitro studies, we found that A. fumigatus ⌬acuK single and ⌬acuK ⌬acuM double mutants had impaired gluconeogenesis and iron acquisition, similar to the ⌬acuM mutant. Also, the ⌬acuK, ⌬acuM, and ⌬acuK ⌬acuM mutants had similar virulence defects in mice. However, the ⌬acuK mutant had a milder defect in extracellular siderophore activity and induction of epithelial cell damage in vitro than did the ⌬acuM mutant. Moreover, overexpression of acuM in the ⌬acuK mutant altered expression of 3 genes and partially restored growth under iron-limited conditions, suggesting that AcuM can govern some genes independently of AcuK. Although the ⌬acuK and ⌬acuM mutants had very similar transcriptional profiles in vitro, their transcriptional profiles during murine pulmonary infection differed both from their in vitro profiles and from each other. While AcuK and AcuM governed the expression of only a few iron-responsive genes in vivo, they influenced the expression of other virulence-related genes, such as hexA and dvrA. Therefore, in A. fumigatus, while AcuK and AcuM likely function as part of the same complex, they can also function independently of each other. Furthermore, AcuK and AcuM have different target genes in vivo than in vitro, suggesting that in vivo infection stimulates unique transcriptional regulatory pathways in A. fumigatus. Aspergillus fumigatus causes a variety of diseases, including allergic bronchopulmonary aspergillosis, aspergilloma, invasive pulmonary aspergillosis, and hematogenously disseminated aspergillosis (1, 2). The incidence of invasive aspergillosis is rising due to the increasing number of immunocompromised patients, who are at risk for this disease (3, 4). Furthermore, because invasive aspergillosis remains difficult to diagnose and treat, this infection is still associated with significant mortality (5). One approach to developing new strategies to treat this frequently deadly infection is to target the factors that enable A. fumigatus to survive and proliferate within the host.Iron is an essential trace element for most living organisms, including pathogenic microorganisms. Consequently, sequestration of free iron by the host is a key factor for inhibiting the virulence of microbial pathogens (6-8). Furthermore, most successful pathogens have evolved mechanisms to obtain iron while inside the host. For example, A. fumigatus acquires iron from the host by both reductive iron assimilation and secretion of siderophores (9-11). Siderophore secretion is more important for growth within the host, because A. fumigatus mutants with defects in siderophore synthesis have dramatically attenuated virulence, whereas those with defects in reductive iron assimilation do not (11-13).Previously, we determined tha...

Talaromyces (Penicillium) marneffei is an important opportunistic fungal pathogen. It causes disseminated infection in immunocompromised patients especially in Southeast Asian countries. The pathogenicity of T. marneffei depends on the ability of the fungus to survive the killing process and replicate inside the macrophage. Major stresses inside the phagosome of macrophages are heat, oxidative substances and nutrient deprivation. The coping strategies of this pathogen with these stresses are under investigation. This paper summarizes factors relating to the stress responses that contribute to the intracellular survival of T. marneffei. These include molecules in the MAP signal transduction cascade, heat shock proteins, antioxidant enzymes and enzymes responsible in nutrient retrieval. There is speculation that the ability of T. marneffei to withstand these defenses plays an important role in its pathogenicity.

Purpose The PCR was compared with routine microbial studies for the detection of fungal pathogens in clinically suspected fungal keratitis. Methods A prospective nonrandomized study was undertaken at a tertiary eye care centre to evaluate 30 eyes of 30 patients with presumed fungal keratitis, both fresh and treated. Corneal scrapings were performed on each patient. The specimens were analysed by a semi-nested PCR assay using fungalspecific primers. PCR products were cloned and sequenced for identification, and compared with a conventional microbial work-up (smear and culture). Results Of the 30 samples, the PCR showed positivity in 93.3%, culture in 40%, and potassium hydroxide in 20%. Of the 28 PCR-positive cases, 12 were culture-positive and 16 were culture-negative. Two samples were both PCR and culture test negative. Culture-negative samples were PCR-positive in 16 of 18 (88.9%) cases. The PCR did not yield any false-negative findings in a culturepositive specimen. Both common and uncommon aetiologic fungi have been identified by DNA sequencing analysis. Conclusion The PCR was able to detect fungal DNA in a high proportion of culturenegative cases. Technical considerations of the PCR process include extraction of artifacts and amplification of non-pathogenic DNA. Nonetheless, our findings suggest that the PCR can be a useful adjunct to smear and culture in the rapid diagnosis of fungal keratitis, particularly in cases of failed detection from routine procedures.

Penicillium marneffei is a significant opportunistic fungal pathogen in Southeast Asia and its ability to survive inside the host macrophages is believed to be important in the establishment of infection. Previously, we isolated a gene encoding a catalase- peroxidase (cpeA) from P. marneffei and showed that the cpeA transcript is specifically upregulated during yeast phase growth at 37 °C. In this study, the cpeA transcript was found to be induced during the mycelium to yeast phase transition and during stress conditions induced by hydrogen peroxide treatment. Null mutation of cpeA reduced the fungal tolerance to hydrogen peroxide but not to heat stress. These results indicated that the CpeA plays a crucial role in this fungus' oxidative stress response. Western blot analysis demonstrated that the CpeA induced antibody production in P. marneffei-infected patients, including highly exposed-healthy people. This is the first report that the catalase-peroxidase possesses an immunogenic property in fungi.

Talaromyces marneffei is a dimorphic pathogenic fungus causing opportunistic infection in immunocompromised patients. It is a facultative intracellular pathogen and is usually found inside the host macrophages during infection. Alternative carbons and iron are the important nutrients associated with intracellular survival and pathogenesis of T. marneffei. This study reported the importance of the transcription factor AcuK in control of gluconeogenesis and iron acquisition in T. marneffei. Deletion of acuK gene in T. marneffei resulted in retardation of growth and germination in both mold and yeast phases. Microscopically, ΔacuK showed double nuclei hyphae. However, the yeast cells showed normal morphology. The ΔacuK failed to grow in iron-limiting conditions. Additionally, it could not grow in a medium containing gluconeogenic carbon sources. Moreover, ΔacuK showed higher susceptibility to macrophage killing than the wild type. These results demonstrated that AcuK controlled both iron acquisition and gluconeogenesis, and it could contribute to the pathogenicity of this fungus.