Thomas Nichterlein

Candida albicans Hyphal Formation and the Expression of the Efg1-Regulated Proteinases Sap4 to Sap6 Are Required for the Invasion of Parenchymal Organs

ABSTRACT The ability to change between yeast and hyphal cells (dimorphism) is known to be a virulence property of the human pathogen Candida albicans. The pathogenesis of disseminated candidosis involves adhesion and penetration of hyphal cells from a colonized mucosal site to internal organs. Parenchymal organs, such as the liver and pancreas, are invaded by C. albicans wild-type hyphal cells between 4 and 24 h after intraperitoneal (i.p.) infection of mice. In contrast, a hypha-deficient mutant lacking the transcription factor Efg1 was not able to invade or damage these organs. To investigate whether this was due to the inability to undergo the dimorphic transition or due to the lack of hypha-associated factors, we investigated the role of secreted aspartic proteinases during tissue invasion and their association with the different morphologies of C. albicans. Wild-type cells expressed a distinct pattern of SAP genes during i.p. infections. Within the first 72 h after infection, SAP1, SAP2, SAP4, SAP5, SAP6, and SAP9 were the most commonly expressed proteinase genes. Sap1 to Sap3 antigens were found on yeast and hyphal cells, while Sap4 to Sap6 antigens were predominantly found on hyphal cells in close contact with host cells, in particular, eosinophilic leukocytes. Mutants lacking EFG1 had either noticeably reduced or higher expressed levels of SAP4 to SAP6 transcripts in vitro depending on the culture conditions. During infection, efg1 mutants had a strongly reduced ability to produce hyphae, which was associated with reduced levels of SAP4 to SAP6 transcripts. Mutants lacking SAP1 to SAP3 had invasive properties indistinguishable from those of wild-type cells. In contrast, a triple mutant lacking SAP4 to SAP6 showed strongly reduced invasiveness but still produced hyphal cells. When the tissue damage of liver and pancreas caused by single sap4, sap5, and sap6 and double sap4 and -6, sap5 and -6, and sap4 and -5 double mutants was compared to the damage caused by wild-type cells, all mutants which lacked functional SAP6 showed significantly reduced tissue damage. These data demonstrate that strains which produce hyphal cells but lack hypha-associated proteinases, particularly that encoded by SAP6, are less invasive. In addition, it can be concluded that the reduced virulence of hypha-deficient mutants is not only due to the inability to form hyphae but also due to modified expression of the SAP genes normally associated with the hyphal morphology.

Stage-specific gene expression of Candida albicans in human blood

The pathogenic fungus Candida albicans commonly causes mucosal surface infections. In immunocompromised patients, C. albicans may penetrate into deeper tissue, enter the bloodstream and disseminate within the host causing life‐threatening systemic infections. In order to elucidate how C. albicans responds to the challenge of a blood environment, we analysed the transcription profile of C. albicans cells exposed to human blood using genomic arrays and a cDNA subtraction protocol. By combining data obtained with these two methods, we were able to identify unique sets of different fungal genes specifically expressed at different stages of this model that mimics bloodstream infections. By removing host cells and incubation in plasma, we were also able to identify several genes in which the expression level was significantly influenced by the presence of these cells. Differentially expressed genes included those that are involved in the general stress response, antioxidative response, glyoxylate cycle as well as putative virulence attributes. These data point to possible mechanisms by which C. albicans ensures survival in the hostile environment of the blood and how the fungus may escape the bloodstream as an essential step in its systemic dissemination.

Failure to induce organ-specific autoimmunity by breaking of tolerance: importance of the microenvironment

Peripheral tolerance is considered to be a safeguard against autoimmunity. Using a TCR‐transgenic mouse system displaying peripheral tolerance against a liver‐specific MHC class I Kb antigen, we investigated whether the breaking of tolerance would result in autoimmunity. Reversal of tolerance was achieved by simultaneous challenge with cells expressing the Kb autoantigen and IL‐2. Tolerance could not be broken with IL‐2 alone or when Kb‐ and IL‐2‐expressing cells were applied to different sites of the mice. However, despite the presence of activated autoreactive T cells that were able to reject Kb‐positive grafts no autoaggression against the Kb‐positive liver was observed. These results indicate that breaking of tolerance per se is not sufficient to cause liver‐specific autoimmunity. However, when in addition to breaking tolerance the mice were infected with a liver‐specific pathogen, autoaggression occurred. Thus, in this system at least two independent steps seem to be required for organ‐specific autoimmunity: reversal of peripheral tolerance resulting in functional activation of autoreactive T cells and conditioning of the liver microenvironment which enables the activated T cells to cause tissue damage.

Host-induced, stage-specific virulence gene activation in Candida albicans during infection.

An understanding of the complex interactions between pathogenic microbes and their host must include the identification of gene expression patterns during infection. To detect the activation of virulence genes in the opportunistic fungal pathogen Candida albicans in vivo by host signals, we devised a reporter system that is based on FLP‐mediated genetic recombination. The FLP gene, encoding the site‐specific recombinase FLP, was genetically modified for expression in C. albicans and fused to the promoter of the SAP2 gene that codes for one of the secreted aspartic proteinases, which are putative virulence factors of C. albicans. The SAP2P–FLP fusion was integrated into one of the SAP2 alleles in a strain that contained a deletable marker that conferred resistance to mycophenolic acid and was flanked by direct repeats of the FLP recognition target (FRT). Using this reporter system, a transient gene induction could be monitored at the level of single cells by the mycophenolic acid‐sensitive phenotype of the colonies generated from such cells after FLP‐mediated marker excision. In two mouse models of disseminated candidiasis, SAP2 expression was not observed in the initial phase of infection, but the SAP2 gene was strongly induced after dissemination into deep organs. In contrast, in a mouse model of oesophageal candidiasis in which dissemination into internal organs did not occur, no SAP2 expression was detected at any time. Our results support a role of the SAP2 gene in the late stages of an infection, after fungal spread into deep tissue. This new in vivo expression technology (IVET) for a human fungal pathogen allows the detection of virulence gene induction at different stages of an infection, and therefore provides clues about the role of these genes in the disease process.

CpG‐ODN‐induced inflammation is sufficient to cause T‐cell‐mediated autoaggression against hepatocytes

Autoimmune diseases are often associated with microbial infections. Molecular mimicry between microbial antigens and self‐epitopes has been suggested as a mechanism for breaking self‐toleranceand induction of autoimmunity. Since infections also cause inflammatory responses we explored the role of local inflammation in organ‐specific autoimmunity. For this purpose, transgenic mice were used expressing the MHC class I molecule Kb exclusively on hepatocytes. These mice exhibit Kb‐specific tolerance as exemplified by the acceptance of Kb+ grafts. Inflammatory reactions were induced by injection of immunostimulatory cytosine‐phosphorothioate‐guanine (CpG)‐rich oligodeoxynucleotides (ODN). Application of CpG‐ODN is sufficient to break tolerance in vivo, and to cause activation of Kb‐specific CD8+ T cells and subsequent autoaggression against hepatocytes. The CpG‐ODN‐induced inflammation appears to have two major effects. First, it causes infiltration of T cells into the liver parenchyma. Second, adhesion and costimulatory molecules are up‐regulated on hepatocytes so that the infiltrating CD8+ T cells encounter Kb on hepatocytes, which display an APC‐like phenotype, resulting in activation and tissue damage. Autoimmune hepatitis can be maintained for at least eight weeks by repeated application of CpG‐ODN but subsides after termination of the inflammatory stimulus, suggesting the requirement of additional factors for a self‐perpetuation of autoimmunity. These observations describe an additional pathway for the induction of autoimmunity, i.e. in the absence of microbial antigens inflammatory reactions alone can lead to infiltration of T cells into organs, resulting in breaking of tolerance and autoaggression. Moreover, the results provide evidence that T cell activation can take place not only in draining lymph nodes but also directly on parenchymal cells.

Host versus in vitro signals and intrastrain allelic differences in the expression of a Candida albicans virulence gene

The yeast Candida albicans is a harmless colonizer of mucosal surfaces in healthy people but can become a serious pathogen in immunocompromised patients, causing superficial as well as systemic infections. The evolution of gene families encoding pathogenicity‐related functions, like adhesins and secreted aspartic proteinases (Saps), which are differentially induced by host signals at various stages of colonization and infection, may have allowed C. albicans an optimal adaptation to many different host niches. We found that even the two alleles of a single gene can be differentially regulated in the diploid C. albicans. In the model strain SC5314, the in vitro expression of one of the two SAP2 alleles, SAP2‐1, depended on the presence of a functional SAP2‐2 allele. In contrast, inactivation of SAP2‐1 did not in‐fluence the expression of SAP2‐2. The proteinase encoded by the SAP2‐2 allele serves as a signal sensor and amplifier to enhance its own expression as well as to induce the SAP2‐1 allele to achieve maximal proteolytic activity under appropriate conditions. Using in vivo expression technology, we could demonstrate that the SAP2‐1 allele is significantly activated only in the late stages of systemic candidiasis in mice, whereas the SAP2‐2 allele is induced much earlier. The differential regulation of the two SAP2 alleles was due to differences in their pro‐moters, which contained a variable number of two pentameric nucleotide repeats. Mutations that re‐duced or increased the copy number of these repeats diminished the inducibility of the SAP2 promoter during infection but not in vitro, suggesting that the mutations affected interactions of regulatory factors that are necessary for SAP2 activation in vivo but dispensable for its induction in vitro. Therefore, the signals and signal transduction pathways that mediate SAP2 expression within certain host niches may differ from those that activate the gene in vitro. In addition to the generation of gene families whose members exhibit functional and regulatory diversification, C. albicans seems to use its diploid genome to create further variability and host adaptation by differential evolution of even the two alleles of a single gene.

Transcriptional Regulators Cph1p and Efg1p Mediate Activation of the Candida albicans Virulence Gene SAP5 during Infection

ABSTRACT The opportunistic fungal pathogen Candida albicans can cause superficial as well as systemic infections. Successful adaptation to the different host niches encountered during infection requires coordinated expression of various virulence traits, including the switch between yeast and hyphal growth forms and secretion of aspartic proteinases. Using an in vivo expression technology that is based on genetic recombination as a reporter of gene activation during experimental candidiasis in mice, we investigated whether two signal transduction pathways controlling hyphal growth, a mitogen-activated protein kinase cascade ending in the transcriptional activator Cph1p and a cyclic AMP-dependent regulatory pathway that involves the transcription factor Efg1p, also control expression of the SAP5 gene, which encodes one of the secreted aspartic proteinases and is induced by host signals soon after infection. Our results show that both transcriptional regulators are important for SAP5 activation in vivo. SAP5 expression was reduced in a cph1 mutant, although filamentous growth in infected tissue was not detectably impaired. SAP5 expression was also reduced, but not eliminated, in an efg1 null mutant, although this strain grew exclusively in the yeast form in infected tissue, demonstrating that in contrast to in vitro conditions, SAP5 activation during infection does not depend on growth of C. albicans in the hyphal form. In a cph1 efg1 double mutant, however, SAP5 expression in infected mice was almost completely eliminated, suggesting that the two signal transduction pathways are important for SAP5 expression in vivo. The avirulence of the cph1 efg1 mutant seemed to be caused not only by the inability to form hyphae but also by a loss of expression of additional virulence genes in the host.

Functional analysis of a vacuolar ABC transporter in wild-type Candida albicans reveals its involvement in virulence

ATP‐driven transport proteins belonging to the ATP‐binding cassette (ABC) superfamily perform important functions in cell metabolism and detoxification. Compounds can be actively transported across membranes, including the plasma membrane or organellar membranes. The vacuole is an important organelle in fungal cells required for compartmentalization of metabolites as well as toxic substances. Sequestration into the vacuole is often energy‐dependent. We present the first isolation and molecular analysis of a vacuolar ABC transporter gene in the opportunistic fungal pathogen Candida albicans. The protein encoded by the MLT1 gene is highly similar to Multiple Drug Resistance‐associated Protein (MRP)‐like transporters of yeast and higher organisms that form the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)/MRP subfamily of ABC transporters, a class of proteins so far not characterized in C. albicans. MLT1 expression is extensively growth phase‐regulated, and gene transcripts are inducible by metabolic poisons. Gene replacement mutants generated in wild‐type C. albicans with the dominant selection marker MPAR showed a profound reduction in virulence in a mouse peritonitis model that was reversed by complementation with an intact MLT1 gene. Hence, this report provides primary evidence for the involvement of vacuolar ABC transporters in fungal virulence.

Protein S-100B: a serum marker for ischemic and infectious injury of cerebral tissue.

Abstract The S-100B protein is released by injured astrocytes. After passage through a disintegrated blood-brain barrier (BBB) the molecule can be detected in the peripheral circulation. We investigated the association between the extent of brain injury and S-100B concentration in serum in cerebral injury caused by cerebral ischemia and cerebral fungal infection. Study I: The S-100B serum concentration was serially determined in 24 patients with ischemic stroke at 4, 8, 10, 24, 72 hours after the onset of symptoms. We observed that patients with brain lesions larger than 5 cm3 exhibited significantly increased serum levels of S-100B at 10, 24 and 72 hours compared to those with lesion volumes below 5 cm3. Furthermore, an association between S-100B serum concentration and neurological outcome was observed. Study II: In a mouse model of systemic fungal infection with Candida albicans we observed that serum levels of S-100B increased at day 1 after intravenous infection. At this time we could histologically demonstrate brain tissue injury by invading hyphae which had crossed the BBB. Furthermore, reactive astrogliosis was demonstrated by immunohistochemistry. On day 7 we found a significant decrease of S-100B serum level compared to day 1 and 4. This was associated with a demarcation of the fungi with leukocytes in brain tissue at this late phase of infection. No further invasion through the BBB was seen on day 7. In conclusion, serum levels of S-100B reflect the time course of tissue injury in cerebral ischemia and cerebral infection to a similar extent. Thus, S-100B may be a useful marker to assess cerebral tissue injury.

The Gut Origin of Bacterial Pancreatic Infection during Acute Experimental Pancreatitis in Rats

Background: Infections are frequent complications and determine clinical course and outcome in severe pancreatitis. A novel animal model was used to assess minimal transit time of bacterial translocation (BT) across the gut mucosa in vivo using green fluorescent protein-transfected Escherichia coli and intravital video microscopy. Methods: Three hours after induction of acute pancreatitis by i.p. injection of 40 µg/kg cerulein, 0.5 ml of a suspension of green fluorescent protein-transfected E. coli were injected into the lumen of a small bowel reservoir formed by ligature in anesthetized Wistar rats. Translocation of E. coli was assessed by intravital microscopy. Animals were sacrificed 5 h after induction of pancreatitis. Results: BT across the mucosa and into the muscularis propria took a mean ± SD of 36.4 ± 8 min and 80.9 ± 9.5 min, respectively, in sham animals. Pancreatitis resulted in a significantly shorter minimal transit time across the mucosa (16.4 ± 4.9 min, p = 0.007) and into the muscularis propria (47.7 ± 2.5 min, p = 0.001). E. coli were detected on frozen cross-sections and on bacteriological examination of pancreatic tissue in animals with acute pancreatitis but not in controls. Discussion: Intravital microscopy of fluorescent bacteria is a new approach towards studying BT in vivo. Minimal transit time of BT serves as a novel functional aspect of mucosal barrier function during acute pancreatitis. The observation of fluorescent bacteria translocating from the small bowel lumen into the pancreas provides substantial experimental proof for the gut-origin-hypothesis of infectious complications in pancreatitis.