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1 Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
2 Department of Biological Sciences, Youngstown State University, Youngstown, OH, USA
Correspondence
Nongnuch Vanittanakom
drnongnuch{at}gmail.com
Received April 25, 2008
Accepted July 9, 2008
Penicillium marneffei is an intracellular dimorphic fungus that can cause a fatal disseminated disease in human immunodeficiency virus-infected patients. The factors that affect the pathogenicity of this fungus remain unclear. Here, we report the isolation and characterization of the gpdA cDNA and genomic clones encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in P. marneffei. Phylogenetic analysis of GAPDH amino acid sequences demonstrated the evolutionary relationship of P. marneffei to other fungi, including the intracellular pathogen Ajellomyces capsulatus. To assess the central importance of phagocytic cells in defence against P. marneffei infection, we used Northern blotting to investigate the response of the isocitrate lyase-encoding gene (acuD) and gpdA to nutrient deprivation inside macrophages. The results revealed that after macrophage internalization, the gene involved in the glyoxylate cycle, acuD, showed higher expression levels as early as 2 h from the start of co-incubation, and the differential expression could be observed again at 8 h after infection. In contrast, the expression of gpdA was downregulated in the yeast phase, as well as during macrophage infection after 2, 4 and 8 h of infection. The induction of P. marneffei acuD was shown to be coordinated with the downregulation of the glycolytic gpdA gene, implying that the cytoplasmic environment of macrophages is deficient in glucose and the glyoxylate pathway could be used by this pathogen to allow subsistence on two-carbon compounds within the host cell following its intracellular persistence.
Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
The GenBank/EMBL/DDBJ accession numbers for the cDNA and genomic DNA sequences of gpdA are EF987476 and EU000324, respectively.
Nucleotide and deduced amino acid sequences of the P. marneffei gpdA gene in this study are available as supplementary data with the online version of this paper.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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Analysis of the response of P. marneffei to macrophages provides a window into the alterations necessary for the organism to survive its first encounter with the immune system. Once inside the macrophage, the conidia of P. marneffei differentiate into the yeast form, which can pierce the macrophage, thus allowing it to resume proliferation (Vanittanakom et al., 2006; Cooper & McGinnis, 1997). Free yeast cells continue to grow and spread via a haematogenous route. Additionally, infected macrophages can aid in the systemic spread of P. marneffei throughout the host. This internalization and escape can be observed in an in vitro system in which P. marneffei and cultured macrophages are mixed. It is clear, however, that the morphogenetic change is only part of the response to phagocytosis. One method for analysing this encounter completely is to identify the alterations in transcription while P. marneffei undergoes phagocytosis.
Housekeeping enzymes are constitutively expressed in all organisms to perform essential metabolic functions for the purpose of survival. In recent years, a number of reports have suggested that some of these enzymes are multifunctional and may play a role in enhancing virulence for many pathogens (Pancholi & Chhatwal, 2003). For example, enzymes that are located on the surface of pathogens could interact with host components, whereas others may interact directly with the host cells to trigger signal transduction processes that enable colonization, invasion and persistence within the host.
Among the enzymes in the glycolytic pathway that may play a role in these processes is glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This enzyme catalyses the oxidative phosphorylation of glyceraldehyde-3-phosphate into 1, 3-biphosphoglycerate in the presence of nicotinamide adenine dinucleotide and inorganic phosphate. GAPDH appears to be a multifunctional protein found in several subcellular locations in eukaryotes, displaying functions unrelated to glycolysis, such as membrane transport and fusion, as well as nuclear RNA transport (McDonald & Moss, 1993; Singh & Green, 1993; Sirover, 1997, 1999). Also, in some pathogens, such as Candida albicans, Staphylococcus aureus, Staphylococcus epidermidis and Paracoccidioides brasiliensis, GAPDH has been found on the cell wall, where it may have various roles in host–pathogen interactions (Gil-Navarro et al., 1997; Gozalbo et al., 1998; Modun & Williams, 1999; Modun et al., 2000; Taylor & Heinrichs, 2002; Barbosa et al., 2004, 2006). GAPDH has long been thought of as the product of a housekeeping gene that exhibits invariant transcript levels. However, several studies have shown that the expression of the Paracoccidioides brasiliensis gapdh gene and the cognate protein are developmentally regulated in different growth phases of Paracoccidioides brasiliensis, with a higher expression in the parasitic yeast phase, thereby reinforcing possible new functions attributed to the enzyme (Barbosa et al., 2004, 2006). Until now, the GAPDH-encoding gene and its potential role in the infection process have not been characterized in P. marneffei.
Although little is known about the factors required for the intracellular persistence of P. marneffei, a study by Cánovas & Andrianopoulos (2006) indicated that the acuD gene, which encodes the glyoxylate bypass enzyme isocitrate lyase, is highly expressed in the yeast phase of this fungus. This observation suggests a link between carbon metabolism, cellular development and pathogenicity in this dimorphic fungal pathogen. The glyoxylate cycle allows two-carbon (C2) compounds to be used as carbon sources in gluconeogenesis. In fact, several lines of evidence indicate that intracellular pathogens may preferentially utilize C2 compounds, such as products of fatty-acid degradation, for energy production (Lorenz & Fink, 2001). Specifically, Mycobacterium tuberculosis, Candida albicans and Paracoccidioides brasiliensis are examples of other micro-organisms that respond to phagocytosis by inducing glyoxylate cycle genes, suggesting that their activity is required for persistence and survival within macrophages (Graham & Clark-Curtiss, 1999; Lorenz & Fink, 2001; Lorenz et al., 2004; Schnappinger et al., 2003; Barelle et al., 2006; Muñoz-Elías & McKinney, 2005; Derengowski et al., 2008). This metabolic shift also includes the downregulation of glycolytic genes.
In this study we have isolated and characterized cDNA and genomic clones, designated gpdA, encoding GAPDH in P. marneffei. Subsequently, we constructed a phylogenetic tree to assess evolutionary relationships among several fungal GAPDH-encoding genes. Also, we analysed the expression of gpdA in different developmental phases of P. marneffei. Finally, in order to evaluate the transcriptional status of genes related to other types of carbon metabolism, we investigated the expression of both gpdA and the glyoxylate cycle gene, acuD, after co-culture of P. marneffei conidia with murine macrophages. Our collective results suggest that the persistence of P. marneffei within host cells may rely upon the induction of the glyoxylate cycle and the concomitant downregulation of glycolysis.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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Isolation and characterization of P. marneffei gpdA. Genomic DNA was isolated from P. marneffei by a slight modification of a previously described method (Vanittanakom et al., 2002). A gpdA fragment was amplified using a previously described method (Thirach et al., 2007) with primers G1 (5'-ACHGGYGTYTTCACCACCA-3') and G2 (5'-ATGACCTTKCCDACRGCCT-3'). These primers were designed based upon the alignment of gpdA genes from several fungi. The PCR fragment was cloned, sequenced and used as a probe in screening a cDNA library (Pongpom et al., 2005) in order to obtain a full-length gpdA cDNA clone. Plaque hybridization screens were carried out using the ECL direct nucleic acid labelling and detection kit (Amersham Pharmacia Biotech). The complete genomic sequence encoding GAPDH was obtained by PCR amplification of P. marneffei genomic DNA. A 1400 bp PCR fragment was amplified by a sense primer (5'-CTATCACCGCAATCATGG-3') and the C-terminal by an antisense primer (5'-GAGCATACTGCGAAGAGTGG-3'). The amplified PCR product was purified, cloned and sequenced.
Analysis of sequenced data and phylogenetic tree construction. The cloned GAPDH-encoding gene from P. marneffei was analysed at both the nucleotide and deduced amino acid sequence levels. The software used in searching for homology/similarity included the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST), as well as DNA translation software (http://www.expasy.org/tools/dna.html). The GAPDH protein sequences of P. marneffei and 12 other fungal species were aligned for phylogenetic analysis using the neighbour-joining method from the MEGA3 program (Kumar et al., 2004).
Macrophage culture and recovery of P. marneffei cells after infection. The mouse macrophage cell line derived from a reticulum sarcoma, J774.1 (ATCC TIB-67), was used in this study. Cells were infected with P. marneffei conidia and fungal RNA extracted from both co-cultures and controls as previously described (Thirach et al., 2007). An aliquot of each RNA sample was checked for the absence of mouse DNA or RNA using murine gapdh-specific primers in a PCR or RT-PCR assay, respectively.
Differential expression of the P. marneffei acuD and gpdA genes in phase transition and during macrophage infection. Fifteen micrograms of total RNA extracted from cells at different phases (mould, conidia and yeast), as well as from conidia co-incubated with or without macrophages (2, 4 and 8 h), were separated by electrophoresis on a denaturing 1 % agarose gel using the NorthernMax-Gly system (Ambion), then transferred onto nylon membranes (Hybond-N+; Amersham) by capillary blotting. RNA was fixed to the membrane by UV cross-linking. To determine the differential expression of P. marneffei acuD and gpdA, the immobilized RNA was probed with a labelled PCR-generated DNA fragment of the acuD gene and reprobed with that of the GAPDH-encoding cDNA clone, consecutively. The acuD probe was generated by the PCR amplification of P. marneffei genomic DNA using primers that were designed from the nucleotide sequence (originally designated icl1) deposited in GenBank (accession number AF373018; forward primer, 5'-TATCATGGGTACCGACCTCCTTG-3'; reverse primer, 5'-AGATCGGCGAATGGTGCATAAGC-3'). Labelling, hybridization and detection of specific probes were carried out by following the directions from the ECL direct nucleic acid labelling and detection kit (Amersham).
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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Phylogenetic analysis
The analysis of GAPDH sequences has been shown to be useful for determining evolutionary relationships between different organisms (Ridder & Osiewacz, 1992) and among fungi (Smith, 1989; Barbosa et al., 2004). These associations may also reflect functional differences among the different taxa. The GAPDH protein sequences of P. marneffei and 12 other species were aligned for phylogenetic analysis using the neighbour-joining method. Based on the comparison of deduced GAPDH sequences it was possible to cluster the sequences in three clades comprising Eurotiomycetes, Dothideomycetes and Sordariomycetes. This analysis supported the classification of P. marneffei in the Eurotiomycetes clade, close to another dimorphic pathogen, Ajellomyces capsulatus. As expected, the pathogen Coccidioides immitis, another dimorphic fungus, as well as the monomorphic moulds Emericella nidulans and Aspergillus niger, occupied a position inside the same clade (Fig. 1a
). A comparison of the mosaic structure of P. marneffei gpdA and genes from other fungi was performed. Fig. 1(b) presents the positions of the introns in relation to the amino acid sequences of the GAPDH proteins. A comparison to other species in the Eurotiomycetes revealed that P. marneffei shared introns at positions 16/17, 20/21, 42/43, and 270/271, and the analysed Dothideomycetes yielded similar results, except for differences in the intron at position 16/17. The Sordariomycetes had the lowest number of introns (1–2), showing intron similarity to other clades at position 42 (Fig. 1a, b). Interestingly, the presence of well-defined clades comprising Eurotiomycetes, Dothideomycetes, and Sordariomycetes could be correlated with the patterns of introns in the cognate genes. Our data also confirmed the systemic relatedness of intron positions among the gpdA genes of the Eurotiomycetes examined.
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In our study, differential expression of gpdA was observed. The transcript of this gene was highly expressed in the mould phase (mycelial phase) of P. marneffei as compared to the yeast phase (Fig. 2). Conidia also possess higher levels of the gpdA transcript. These results indicate that gpdA may respond to nutritional limitation or some stage of the life cycle in the yeast form of P. marneffei. The function of P. marneffei GAPDH and its involvement in the response to nutrition in the yeast phase needs further investigation.
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In order to evaluate P. marneffei transcriptional response following co-culture with murine macrophages, expression analysis of the glyoxylate cycle and the glycolytic pathway, using acuD and gpdA as the respective markers, was undertaken by Northern blotting. The results revealed that after macrophage internalization of conidia, acuD had higher expression levels at as early as 2 h from the start of co-incubation when compared to cells grown in cell-free medium as a control, and the differential expression could be observed again at 8 h after infection (Fig. 2). This acuD expression pattern might be affected by the concentration of substrates inside macrophages. The induction of acuD observed following phagocytosis suggests a role of this metabolic pathway in P. marneffei adaptation inside macrophages, and provides further evidence of the state of nutritional deprivation inside the phagosome. Since this fungus is a facultative intracellular pathogen and glycolytic substrates are supposed to be absent or scarce in the phagolysosome environment (Schnappinger et al., 2003), the glyoxylate cycle may be required for the utilization of C2 compounds derived from fatty acids in energy production. The role of this pathway in the survival of micro-organisms within macrophages has been shown in pathogens such as M. tuberculosis and Candida albicans (Graham & Clark-Curtiss, 1999; Lorenz & Fink, 2001; Lorenz et al., 2004; Schnappinger et al., 2003; Barelle et al., 2006). In Paracoccidioides brasiliensis, induction of this cycle in response to macrophage microenvironments was shown to coordinate with the upregulation of the gluconeogenic phosphoenolpyruvate carboxykinase encoding-gene (pck) (Derengowski et al., 2008). Induction of the glyoxylate cycle upon phagocytosis has been described as an important pathogen adaptation to the glucose-poor environment within macrophages since it facilitates the assimilation of C2 compounds (Lorenz et al., 2004). This may indicate that the organism has identified simple carbon sources in this environment and is activating the pathways necessary to utilize them. An intriguing possibility is that substrates derived from the breakdown of fatty acids via β-oxidation, which results in acetyl-CoA, would require utilization of the glyoxylate cycle. If the fatty acid breakdown of host lipids or lipids from the micro-organism itself occurs within the phagolysosome, it might provide a carbon source accessible to the pathogen while it is inside the macrophage.
The macrophage phagosome is believed to be a poor source of glucose and amino acids (Fan et al., 2005; Lorenz et al., 2004). Such nutrient deprivation inside the macrophage induces a similar adaptive response from intracellular bacterial and fungal pathogens. Shortly following phagocytosis, Listeria monocytogenes and Candida albicans have a strong reduction in the expression of genes involved in glycolysis (Chatterjee et al., 2006; Lorenz et al., 2004). We show in this study that phagocytized P. marneffei cells also sense and respond to the glucose-depleted environment of the macrophage phagosome. Transcription of gpdA, the gene encoding a key enzyme (GAPDH) in the glycolysis pathway, was downregulated in internalized P. marneffei (Fig. 2). Similar results were found in Paracoccidioides brasiliensis (Tavares et al., 2007).
In summary, these data suggested that upon internalization by macrophages, P. marneffei responds to phagocytosis with the induction of an alternate carbon metabolism, and there is a dramatic downregulation of the P. marneffei glycolytic gene, gpdA. This alternate pathway involves induction of the glyoxylate cycle in response to nutrient deprivation inside the macrophages. More generally, discovering the role of the glyoxylate cycle in microbial virulence should refocus attention on the critical importance of basic metabolic pathways in the development of disease. If a pathogen is unable to synthesize the precursor nucleic and amino acids required for growth, it is unlikely to proliferate or persist. For this reason, elucidation of a pathogen's nutritional requirements in vivo, as well as the mechanism by which microbes acquire these nutrients once inside a host, is critical to the understanding of virulence and disease. Further investigation will evaluate the transcriptional status of genes related to other central carbon metabolic pathways, including gluconeogenesis in P. marneffei. Additional studies are planned to assess the relationship of these metabolic changes with regard to possible alterations of the host intracellular pH as part of the concerted survival and virulence mechanisms of P. marneffei.
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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