Candida albicans causes both mucosal and disseminated infections, and its capacity to grow as both yeast and hyphae is a key virulence factor. Hyphal formation is a type of polarized growth, and members of the SR (serine-arginine) family of RNA-binding proteins influence polarized growth of both Saccharomyces cerevisiae and Aspergillus nidulans. Therefore, we investigated whether SR-like proteins affect filamentous growth and virulence of C. albicans. BLAST searches with S. cerevisiae SR-like protein Npl3 (ScNpl3) identified two C. albicans proteins: CaNpl3, an apparent ScNpl3 ortholog, and Slr1, another SR-like RNA-binding protein with no close S. cerevisiae ortholog. Whereas ScNpl3 was critical for growth, deletion of NPL3 in C. albicans resulted in few phenotypic changes. In contrast, the slr1Δ/Δ mutant had a reduced growth rate in vitro, decreased filamentation, and impaired capacity to damage epithelial and endothelial cells in vitro. Mice infected intravenously with the slr1Δ/Δ mutant strain had significantly prolonged survival compared to that of mice infected with the wild-type or slr1Δ/Δ mutant complemented with SLR1 (slr1Δ/Δ+SLR1) strain, without a concomitant decrease in kidney fungal burden. Histopathology, however, revealed differential localization of slr1Δ/Δ hyphal and yeast morphologies within the kidney. Mice infected with slr1Δ/Δ cells also had an increased brain fungal burden, which correlated with increased invasion of brain, but not umbilical vein, endothelial cells in vitro. The enhanced brain endothelial cell invasion was likely due to the increased surface exposure of the Als3 adhesin on slr1Δ/Δ cells. Our results indicate that Slr1 is an SR-like protein that influences C. albicans growth, filamentation, host cell interactions, and virulence.
In order to colonize the host and cause disease, Candida albicans must avoid being killed by host defense peptides. Previously, we determined that the regulatory protein Ssd1 governs antimicrobial peptide resistance in C. albicans. Here, we sought to identify additional genes whose products govern susceptibility to antimicrobial peptides. We discovered that a bcr1Δ/Δ mutant, like the ssd1Δ/Δ mutant, had increased susceptibility to the antimicrobial peptides, protamine, RP-1, and human β defensin-2. Homozygous deletion of BCR1 in the ssd1Δ/Δ mutant did not result in a further increase in antimicrobial peptide susceptibility. Exposure of the bcr1Δ/Δ and ssd1Δ/Δ mutants to RP-1 induced greater loss of mitochondrial membrane potential and increased plasma membrane permeability than with the control strains. Therefore, Bcr1 and Ssd1 govern antimicrobial peptide susceptibility and likely function in the same pathway. Furthermore, BCR1 mRNA expression was downregulated in the ssd1Δ/Δ mutant, and the forced expression of BCR1 in the ssd1Δ/Δ mutant partially restored antimicrobial peptide resistance. These results suggest that Bcr1 functions downstream of Ssd1. Interestingly, overexpression of 11 known Bcr1 target genes in the bcr1Δ/Δ mutant failed to restore antimicrobial peptide resistance, suggesting that other Bcr1 target genes are likely responsible for antimicrobial peptide resistance. Collectively, these results demonstrate that Bcr1 functions downstream of Ssd1 to govern antimicrobial peptide resistance by maintaining mitochondrial energetics and reducing membrane permeabilization.
In the human fungal pathogen Candida albicans, the CUG codon is translated 97% of the time as serine and 3% of the time as leucine, which potentially originates an array of proteins resulting from the translation of a single gene. Genes encoding cell surface proteins are enriched in CUG codons; thus, CUG mistranslation may influence the interactions of the organism with the host. To investigate this, we compared a C. albicans strain that misincorporates 28% of leucine at CUGs with a wild-type parental strain. The first strain displayed increased adherence to inert and host molecules. In addition, it was less susceptible to phagocytosis by murine macrophages, probably due to reduced exposure of cell surface β-glucans. To prove that these phenotypes occurred due to serine/leucine exchange, the C. albicans adhesin and invasin ALS3 was expressed in Saccharomyces cerevisiae in its two natural isoforms (Als3p-Leu and Als3p-Ser). The cells with heterologous expression of Als3p-Leu showed increased adherence to host substrates and flocculation. We propose that CUG mistranslation has been maintained during the evolution of C. albicans due to its potential to generate cell surface variability, which significantly alters fungus-host interactions.
The translation of genetic information into proteins is a highly accurate cellular process. In the human fungal pathogen Candida albicans, a unique mistranslation event involving the CUG codon occurs. The CUG codon is mainly translated as serine but can also be translated as leucine. Leucine and serine are two biochemically distinct amino acids, hydrophobic and hydrophilic, respectively. The increased rate of leucine incorporation at CUG decoding triggers C. albicans virulence attributes, such as morphogenesis, phenotypic switching, and adhesion. Here, we show that CUG mistranslation masks the fungal cell wall molecule β-glucan that is normally recognized by the host immune system, delaying its response. Furthermore, we demonstrate that two different proteins of the adhesin Als3 generated by CUG mistranslation confer increased hydrophobicity and adhesion ability on yeast cells. Thus, CUG mistranslation functions as a mechanism to create protein diversity with differential activities, constituting an advantage for a mainly asexual microorganism. This could explain its preservation during evolution.
Oropharyngeal candidiasis is a frequent cause of morbidity in patients with defects in cell-mediated immunity or saliva production. Animal models of this infection are important for studying disease pathogenesis and evaluating vaccines and antifungal therapies. Here we describe a simple murine model of oropharyngeal candidiasis. Mice are rendered susceptible to oral infection by injection with cortisone acetate and then inoculated by placing a swab saturated with Candida albicans sublingually. This process results in a reproducible level of infection, the histopathology of which mimics that of pseudomembranous oropharyngeal candidiasis in patients. Using this model, data are obtained after 5–9 days of work.
The opportunistic fungal pathogen, Candida albicans, enters the bloodstream and causes hematogenously disseminated infection in hospitalized patients. During the initiation of a hematogenously disseminated infection, endothelial cells are one of the first host cells to come in contact with C. albicans. Endothelial cells can significantly influence the local host response to C. albicans by expressing leukocyte adhesion molecules and pro-inflammatory cytokines. Thus, it is of interest to investigate the response of endothelial cells to C. albicans in vitro. We describe the use of real-time PCR and enzyme immunoassays to measure the effects of C. albicans on the endothelial cell production of E-selectin and tumor necrosis factor α in vitro.
Endothelial cell; Candida albicans; E-selectin; IL-8; EIA; real-time PCR
Biofilm formation by Candida albicans on medically implanted devices poses a significant clinical challenge. Here, we compared biofilm-associated gene expression in two clinical C. albicans isolates, SC5314 and WO-1, to identify shared gene regulatory responses that may be functionally relevant. Among the 62 genes most highly expressed in biofilms relative to planktonic (suspension-grown) cells, we were able to recover insertion mutations in 25 genes. Twenty mutants had altered biofilm-related properties, including cell substrate adherence, cell-cell signaling, and azole susceptibility. We focused on one of the most highly upregulated genes in our biofilm proles, RHR2, which specifies the glycerol biosynthetic enzyme glycerol-3-phosphatase. Glycerol is 5-fold-more abundant in biofilm cells than in planktonic cells, and an rhr2Δ/Δ strain accumulates 2-fold-less biofilm glycerol than does the wild type. Under in vitro conditions, the rhr2Δ/Δ mutant has reduced biofilm biomass and reduced adherence to silicone. The rhr2Δ/Δ mutant is also severely defective in biofilm formation in vivo in a rat catheter infection model. Expression profiling indicates that the rhr2Δ/Δ mutant has reduced expression of cell surface adhesin genes ALS1, ALS3, and HWP1, as well as many other biofilm-upregulated genes. Reduced adhesin expression may be the cause of the rhr2Δ/Δ mutant biofilm defect, because overexpression of ALS1, ALS3, or HWP1 restores biofilm formation ability to the mutant in vitro and in vivo. Our findings indicate that internal glycerol has a regulatory role in biofilm gene expression and that adhesin genes are among the main functional Rhr2-regulated genes.
Candida albicans is a major fungal pathogen, and infection can arise from the therapeutically intractable biofilms that it forms on medically implanted devices. It stands to reason that genes whose expression is induced during biofilm growth will function in the process, and our analysis of 25 such genes confirms that expectation. One gene is involved in synthesis of glycerol, a small metabolite that we find is abundant in biofilm cells. The impact of glycerol on biofilm formation is regulatory, not solely metabolic, because it is required for expression of numerous biofilm-associated genes. Restoration of expression of three of these genes that specify cell surface adhesins enables the glycerol-synthetic mutant to create a biofilm. Our findings emphasize the significance of metabolic pathways as therapeutic targets, because their disruption can have both physiological and regulatory consequences.
Candida spp. are the most common cause of mucosal and disseminated fungal infections in humans. Studies using mutant strains of mice have provided initial information about the roles of dectin-1, CARD9, and Th17 cytokines in the host defense against candidiasis. Recent technological advances have resulted in the identification of mutations in specific genes that predispose humans to develop candidal infection. The analysis of individuals with these mutations demonstrates that dectin-1 is critical for the host defense against vulvovaginal candidiasis and candidal colonization of the gastrointestinal tract. They also indicate that CARD9 is important for the preventing both mucosal and disseminated candidiasis, whereas the Th17 response is necessary for the defense against mucocutaneous candidiasis. This article reviews the recent studies of genetic defects in humans that result in an increased susceptibility to candidiasis and discusses how these studies provide new insight into the host defense against different types of candidal infections.
Dectin-1; CARD9; Th17; Candida; Host defense
Candida albicans is a causative agent of oropharyngeal candidiasis (OPC), a biofilm-like infection of the oral mucosa. Biofilm formation depends upon the C. albicans transcription factor Bcr1, and previous studies indicate that Bcr1 is required for OPC in a mouse model of infection. Here we have used a nanoString gene expression measurement platform to elucidate the role of Bcr1 in OPC-related gene expression. We chose for assays a panel of 134 genes that represent a range of morphogenetic and cell cycle functions as well as environmental and stress response pathways. We assayed gene expression in whole infected tongue samples. The results sketch a portrait of C. albicans gene expression in which numerous stress response pathways are activated during OPC. This one set of experiments identifies 64 new genes with significantly altered RNA levels during OPC, thus increasing substantially the number of known genes in this expression class. The bcr1Δ/Δ mutant had a much more limited gene expression defect during OPC infection than previously reported for in vitro growth conditions. Among major functional Bcr1 targets, we observed that ALS3 was Bcr1 dependent in vivo while HWP1 was not. We used null mutants and complemented strains to verify that Bcr1 and Hwp1 are required for OPC infection in this model. The role of Als3 is transient and mild, though significant. Our findings suggest that the versatility of C. albicans as a pathogen may reflect its ability to persist in the face of multiple stresses and underscore that transcriptional circuitry during infection may be distinct from that detailed during in vitro growth.
The pathogenesis of Staphylococcus aureus infective endocarditis (IE) is postulated to involve invasion and damage of endothelial cells (ECs). However, the precise relationships between S. aureus – EC interactions in vitro and IE virulence and treatment outcomes in vivo are poorly defined. Ten methicillin-resistant S. aureus (MRSA) clinical isolates previously tested for their virulence and vancomycin responsiveness in an experimental IE model were assessed in vitro for their hemolytic activity, protease production, and capacity to invade and damage ECs. There was a significant positive correlation between the in vitro EC damage caused by these MRSA strains and their virulence during experimental IE (in terms of bacterial densities in target tissues; P < 0.02). Importantly, higher EC damage was also significantly correlated with poor microbiologic response to vancomycin in the IE model (P < 0.001). Interestingly, the extent of EC damage was unrelated to a strain's ability to invade ECs, hemolytic activity and protease production, or β-toxin gene transcription. Inactivation of the agr locus in two MRSA strains caused ∼20% less damage as compared to the corresponding parental strains, indicating that a functional agr is required for maximal EC damage induction. Thus, MRSA-induced EC damage in vitro is a unique virulence phenotype that is independent of many other prototypical MRSA virulence factors, and may be a key biomarker for predicting MRSA virulence potential and antibiotic outcomes during endovascular infections.
The aim of the present study was to assess fluconazole pharmacokinetic measures in serum and cerebrospinal fluid (CSF); and the correlation of these measures with clinical outcomes of invasive fungal infections.
A randomized trial was conducted in HIV-infected patients receiving 3 different regimens of fluconazole plus amphotericin B (AmB) for the treatment of cryptococcal meningitis. Regimens included fluconazole 400 mg/day+AmB (AmB+Fluc400) or fluconazole 800 mg/day+AmB (AmB+Fluc800) (14 days followed by fluconazole alone at the randomized dose for 56 days); or AmB alone for 14 days followed by fluconazole 400 mg/day for 56 days. Serum (at 24 hours after dosing) and CSF samples were taken at Baseline and days 14 and 70 (serum only) for fluconazole measurement, using gas-liquid chromatography.
Sixty-four treated patients had fluconazole measurements; 11 in AmB group, 12 in AmB+Fluc400 group and 41 in AmB+Fluc800 group. Day 14 serum concentration geometric means were 24.7 mg/L for AmB+Fluc400 and 37.0 mg/L for AmB+Fluc800. Correspondingly, CSF concentration geometric means were 25.1 mg/L and 32.7 mg/L. Day 14 Serum and CSF concentrations were highly correlated for AmB+Fluc800 (p<0.001, r=0.873) and for AmB+Fluc400 (p=0.005, r=0.943). Increased Serum AUC appears associated with decreased mortality at day 70 (p=0.061, odds-ratio=2.19) as well as with increased study composite endpoint success at Days 42 and 70 (p=0.081, odds-ratio=2.25 and 0.058, 4.08; respectively).
High fluconazole dosage (800 mg/day) for the treatment of HIV-associated cryptococcal meningitis was associated with high serum and CSF fluconazole concentration. Overall, high serum and CSF concentration appear associated with increased survival and primary composite endpoint success.
Hyphal invasion of blood vessels is a prominent feature of invasive aspergillosis. During invasive pulmonary aspergillosis, Aspergillus fumigatus hyphae invade the abluminal endothelial cell surface, whereas they invade the luminal endothelial cell surface during hematogenous dissemination. We investigated the endothelial cell response to abluminal and luminal infection with A. fumigatus hyphae in vitro. We found that these hyphae invaded the abluminal endothelial cell surface without inducing the formation of endothelial cell pseudopods. Also, the internalized hyphae were surrounded by a loose network of microfilaments. In contrast, A. fumigatus hyphae invaded the luminal endothelial cell surface by inducing by the formation of endothelial cell pseudopods. These endocytosed hyphae were surrounded by a tight network of microfilaments. Abluminal infection induced greater E-selectin, IL-8, tissue factor, and TNF-α gene expression, but less endothelial cell damage than did luminal infection. Endothelial cell stimulation by infection of either surface was mediated by endothelial cell-derived TNF-α, and was not influenced by gliotoxin secreted by A. fumigatus. These differences in the endothelial cell response to abluminal versus luminal infection may contribute to differences in the pathogenesis of invasive versus hematogenously disseminated aspergillosis.
The fungus, Candida albicans interacts with epithelial cells in the human host both as a normal commensal and as an invasive pathogen. It has evolved multiple complementary mechanisms to adhere to epithelial cells. Adherent C. albicans cells can invade epithelial surfaces both by penetrating into individual epithelial cells, and by degrading inter-epithelial cell junctions and passing between epithelial cells. Invasion into epithelial cells is mediated by both induced endocytosis and active penetration, whereas degradation of epithelial cell junction proteins, such as E-cadherin occurs mainly via proteolysis by secreted aspartyl proteinases. C. albicans invasion of epithelial cells results in significant epithelial cell damage, which is probably induced by lytic enzymes, such as proteases and phospholipase secreted by the organism. Future challenges include identifying the epithelial cell targets of adhesins and invasins, and determining the mechanisms by which C. albicans actively penetrates epithelial cells and induces epithelial cell damage.
In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared to A. nidulans. The A. fumigatus ΔmedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The ΔmedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro-inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatus ΔmedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis.
Aspergillus fumigatus; conidiation; adherence; biofilm; virulence
Candida albicans is a fungal pathogen that causes severe disseminated infections that can be lethal in immunocompromised patients. Genetic factors are known to alter the initial susceptibility to and severity of C. albicans infection. We developed a next-generation computational genetic mapping program with advanced features to identify genetic factors affecting survival in a murine genetic model of hematogenous C. albicans infection. This computational tool was used to analyze the median survival data after inbred mouse strains were infected with C. albicans, which provides a useful experimental model for identification of host susceptibility factors. The computational analysis indicated that genetic variation within early classical complement pathway components (C1q, C1r, and C1s) could affect survival. Consistent with the computational results, serum C1 binding to this pathogen was strongly affected by C1rs alleles, as was survival of chromosome substitution strains. These results led to a combinatorial, conditional genetic model, involving an interaction between C5 and C1r/s alleles, which accurately predicted survival after infection. Beyond applicability to infectious disease, this information could increase our understanding of the genetic factors affecting susceptibility to autoimmune and neurodegenerative diseases.
A nine-year prospective study (2002–2010) on the prevalence of Candida dubliniensis among Candida bloodstream isolates is presented. The germ tube positive isolates were provisionally identified as C. dubliniensis by presence of fringed and rough colonies on sunflower seed agar. Subsequently, their identity was confirmed by Vitek2 Yeast identification system and/or by amplification and sequencing of the ITS region of rDNA. In all, 368 isolates were identified as C. dubliniensis; 67.1% came from respiratory specimens, 11.7% from oral swabs, 9.2% from urine, 3.8% from blood, 2.7% from vaginal swabs and 5.4% from other sources. All C. dubliniensis isolates tested by Etest were susceptible to voriconazole and amphotericin B. Resistance to fluconazole (≥8 µg/ml) was observed in 2.5% of C. dubliniensis isolates, 7 of which occurred between 2008–2010. Of note was the diagnosis of C. dubliniensis candidemia in 14 patients, 11 of them occurring between 2008–2010. None of the bloodstream isolate was resistant to fluconazole, while a solitary isolate showed increased MIC to 5-flucytosine (>32 µg/ml) and belonged to genotype 4. A review of literature since 1999 revealed 28 additional cases of C. dubliniensis candidemia, and 167 isolates identified from blood cultures since 1982. In conclusion, this study highlights a greater role of C. dubliniensis in bloodstream infections than hitherto recognized.
Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C. albicans, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis. Similar to C. albicans, C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging C. dubliniensis infections.
Relatively few transcription factors that govern the virulence of Aspergillus fumigatus are known. We constructed 11 A. fumigatus transcription factor mutants and screened them for altered virulence in Galleria mellonella larvae. We discovered that the zinc cluster transcription factor, AcuM, is essential for maximal virulence in this model, as well as in murine models of hematogenously disseminated and invasive pulmonary aspergillosis. Transcriptional profiling experiments suggested that AcuM suppresses sreA and induces hapX to stimulate expression of genes involved in both reductive iron assimilation and siderophore-mediated iron uptake. Consistent with these results, a ΔacuM mutant had reduced iron incorporation, decreased extracellular siderophore production, and impaired capacity to grow under iron-limited conditions. Interestingly, an Aspergillus nidulans ΔacuM mutant had normal extracellular siderophore production and growth under iron-limited conditions, indicating that AcuM does not govern iron acquisition in this organism. A. fumigatus AcuM also regulated genes involved in gluconeogenesis, and the ΔacuM mutant had impaired growth on gluconeogenic carbon sources. Deletion of sreA in the ΔacuM mutant restored iron uptake, extracellular siderophore production, and virulence, but not the defect in gluconeogenesis. Thus, AcuM represses SreA and thereby induces iron acquisition, a process that is essential for the maximal virulence of A. fumigatus.
During hematogenously disseminated disease, Candida albicans infects most organs, including the brain. We discovered that a C. albicans vps51Δ/Δ mutant had significantly increased tropism for the brain in the mouse model of disseminated disease. To investigate the mechanisms of this enhanced trafficking to the brain, we studied the interactions of wild-type C. albicans and the vps51Δ/Δ mutant with brain microvascular endothelial cells in vitro. These studies revealed that C. albicans invasion of brain endothelial cells is mediated by the fungal invasins, Als3 and Ssa1. Als3 binds to the gp96 heat shock protein, which is expressed on the surface of brain endothelial cells, but not human umbilical vein endothelial cells, whereas Ssa1 binds to a brain endothelial cell receptor other than gp96. The vps51Δ/Δ mutant has increased surface expression of Als3, which is a major cause of the increased capacity of this mutant to both invade brain endothelial cells in vitro and traffic to the brain in mice. Therefore, during disseminated disease, C. albicans traffics to and infects the brain by binding to gp96, a unique receptor that is expressed specifically on the surface of brain endothelial cells.
During hematogenously disseminated infection, the fungus Candida albicans is carried by the bloodstream to virtually all organs in the body, including the brain. C. albicans infection of the brain is a significant problem in premature infants with disseminated candidiasis. To infect the brain, C. albicans must adhere to and invade the endothelial cells that line cerebral blood vessels. These endothelial cells express unique proteins on their surface that are not expressed by endothelial cells of other vascular beds. Here, we show that C. albicans infects the brain by binding to gp96, a heat shock protein that is uniquely expressed on the surface of brain endothelial cells. Gp96 is bound by the C. albicans Als3 invasin, which induces the uptake of this organism by brain endothelial cells. The C. albicans Ssa1 invasin also mediates fungal uptake by brain endothelial cells, but does so by binding to a receptor other than gp96. Thus, during hematogenously disseminated infection, C. albicans traffics to and infects the brain by binding to gp96, a receptor that is expressed specifically on the surface of brain endothelial cells.
Candida albicans is part of the normal human flora, and it grows on mucosal surfaces in healthy individuals. In susceptible hosts, this organism can cause both mucosal and hematogenously disseminated disease. For C. albicans to persist in the host and induce disease, it must be able to adhere to biotic and abiotic surfaces, invade host cells, and obtain iron. The C. albicans hypha-specific surface protein Als3 is a member of the agglutinin-like sequence (Als) family of proteins and is important in all of these processes. Functioning as an adhesin, Als3 mediates attachment to epithelial cells, endothelial cells, and extracellular matrix proteins. It also plays an important role in biofilm formation on prosthetic surfaces, both alone and in mixed infection with Streptococcus gordonii. Als3 is one of two known C. albicans invasins. It binds to host cell receptors such as E-cadherin and N-cadherin and thereby induces host cells to endocytose the organism. Als3 also binds to host cell ferritin and enables C. albicans to utilize this protein as a source of iron. Because of its multiple functions and its high expression level in vivo, Als3 is a promising target for vaccines that induce protective cell-mediated and antibody responses. This review will summarize the multiple functions of this interesting and multifunctional protein.
Left ventricular (LV) dysfunction after successful cardiopulmonary resuscitation contributes to early death following resuscitation. Proinflammatory cytokines are known to depress myocardial function and TNF-alpha has been shown to increase after successful resuscitation. We hypothesized that blocking the effects of TNF-alpha with infliximab would prevent or minimize postresuscitation cardiac dysfunction.
Randomized, placebo-controlled comparative study.
Large animal research laboratory.
Twenty-eight anesthetized and instrumented domestic male swine (Yorkshire and Yorkshire/Hampshire mix, weight 35-45 kg).
Infusion of infliximab (5 mg/kg) or normal saline after resuscitation from VF cardiac arrest.
Measurements and Main Results
Hemodynamic variables, indices of LV function, and TNF-alpha were measured before and after 8 min of cardiac arrest during the early post-resuscitation period (3 hrs). Within 5 min of restoration of spontaneous circulation, 14 animals received infliximab, 5 mg/kg, infused over 30 min. Fourteen animals received an infusion of normal saline. Inotropes and vasopressors were not administered to either group following resuscitation. TNF-alpha increased following restoration of circulation and remained elevated throughout the observation period. Differences between groups were not significant. IL-1β concentration did not change significantly during the observation period in either study group. Mean arterial pressure and stroke work were significantly greater in the infliximab group within 30 mins of resuscitation and these differences were sustained throughout the 3 hr postresuscitation period. The effect of TNF-α blockade was evident only in animals with a significant increase (doubling) in plasma TNF-α at 30 minutes post-arrest.
TNF-α plays a role in post-arrest cardiac dysfunction and Infliximab may attenuate or prevent postresuscitation myocardial dysfunction when given immediately after resuscitation.
heart arrest; ventricular fibrillation; cardiopulmonary resuscitation; tumor-necrosis factor; interleukins; hemodynamics
The transcription factors that regulate Aspergillus fumigatus interactions with host cells and virulence are incompletely defined. We investigated the role of the putative C2H2 transcription factor DvrA in governing these processes. Although DvrA was identified by its limited homology to Candida albicans Bcr1, a ΔdvrA mutant strain of A. fumigatus had wild-type adherence to host constituents in vitro. However, it had increased capacity to damage both endothelial cells and a pulmonary epithelial cell line compared to the ability of the wild-type strain and a ΔdvrA::dvrA-complemented strain. This increase in damage required direct contact between the mutant and host cells. The ΔdvrA mutant also stimulated greater CCL20, interleukin-8, and tumor necrosis factor mRNA expression in a pulmonary epithelial cell line compared to levels induced by the control strains. Also, it was resistant to nikkomycin Z, suggesting an altered cell wall composition. As predicted by these in vitro results, the ΔdvrA mutant had increased virulence and stimulated a greater pulmonary inflammatory response than the wild-type strain and ΔdvrA::dvrA-complemented strains in the nonneutropenic mouse model of invasive pulmonary aspergillosis. These results indicate that DvrA influences A. fumigatus virulence as well as its capacity to damage host cells and stimulate a proinflammatory response.
The protein phosphatase calcineurin is a key mediator of virulence and antifungal susceptibility of multiple fungal pathogens including Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, and has clinical potential as a therapeutic target to increase the efficacy of the current antifungal armamentarium. Despite the importance of this signaling pathway, few components of the calcineurin-signaling pathway are known in C. albicans. Here we identified and analyzed additional components of the C. albicans calcineurin cascade, including the RCN1 (Regulator of Calcineurin1), MID1, and CCH1 genes, which mediate calcineurin functions in other species. When heterologous expressed in S. cerevisiae, C. albicans Rcn1 inhibited calcineurin function. Although rcn1/rcn1, mid1/mid1, and cch1/cch1 mutant strains share some phenotypes with calcineurin mutants, they do not completely recapitulate the phenotypes of a calcineurin mutant strain. These studies extend our understanding of the C. albicans calcineurin signaling cascade and its host-niche specific role in virulence.
Candida albicans; calcineurin; Rcn1; Mid1; Cch1; fungal pathogenesis; fluconazole
Aspergillus fumigatus is a pathogenic mold which causes invasive, often fatal, pulmonary disease in immunocompromised individuals. Recently, proteins involved in the biosynthesis of trehalose have been linked with virulence in other pathogenic fungi. We found that the trehalose content increased during the developmental life cycle of A. fumigatus, throughout which putative trehalose synthase genes tpsA and tpsB were significantly expressed. The trehalose content of A. fumigatus hyphae also increased after heat shock but not in response to other stressors. This increase in trehalose directly correlated with an increase in expression of tpsB but not tpsA. However, deletion of both tpsA and tpsB was required to block trehalose accumulation during development and heat shock. The ΔtpsAB double mutant had delayed germination at 37°C, suggesting a developmental defect. At 50°C, the majority of ΔtpsAB spores were found to be nonviable, and those that were viable had severely delayed germination, growth, and subsequent sporulation. ΔtpsAB spores were also susceptible to oxidative stress. Surprisingly, the ΔtpsAB double mutant was hypervirulent in a murine model of invasive aspergillosis, and this increased virulence was associated with alterations in the cell wall and resistance to macrophage phagocytosis. Thus, while trehalose biosynthesis is required for a number of biological processes that both promote and inhibit virulence, in A. fumigatus the predominant effect is a reduction in pathogenicity. This finding contrasts sharply with those for other fungi, in which trehalose biosynthesis acts to enhance virulence.
Candida albicans Ssa1 and Ssa2 are members of the HSP70 family of heat shock proteins that are expressed on the cell surface and function as receptors for antimicrobial peptides such as histatins. We investigated the role of Ssa1 and Ssa2 in mediating pathogenic host cell interactions and virulence. A C. albicans ssa1Δ/Δ mutant had attenuated virulence in murine models of disseminated and oropharyngeal candidiasis, whereas an ssa2Δ/Δ mutant did not. In vitro studies revealed that the ssa1Δ/Δ mutant caused markedly less damage to endothelial cells and oral epithelial cell lines. Also, the ssa1Δ/Δ mutant had defective binding to endothelial cell N-cadherin and epithelial cell E-cadherin, receptors that mediate host cell endocytosis of C. albicans. As a result, this mutant had impaired capacity to induce its own endocytosis by endothelial cells and oral epithelial cells. Latex beads coated with recombinant Ssa1 were avidly endocytosed by both endothelial cells and oral epithelial cells, demonstrating that Ssa1 is sufficient to induce host cell endocytosis. These results indicate that Ssa1 is a novel invasin that binds to host cell cadherins, induces host cell endocytosis, and is critical for C. albicans to cause maximal damage to host cells and induce disseminated and oropharyngeal disease.
The fungus Candida albicans can proliferate in the mouth, causing oropharyngeal candidiasis. In other patients, it can enter the bloodstream and spread throughout the body, resulting in hematogenously disseminated candidiasis. Fungal invasion of host cells is a key feature of both types of infection. One mechanism by which C. albicans invades both the epithelial cell lining of the oropharynx and the endothelial cell lining of the blood vessels is by inducing its own uptake. This uptake is induced in part by the binding of the C. albicans invasin Als3 to host cell proteins, which include N- and E-cadherin. Here we show that C. albicans Ssa1, a member of the 70 kDa heat shock protein family, is expressed on the surface of C. albicans where it functions as an invasin. The key role of Ssa1 in host cell invasion is illustrated by the reduced capacity of an ssa1Δ/Δ null mutant to induce its own uptake by epithelial and endothelial cells in vitro, and by the significantly attenuated virulence of this mutant in mouse models of oropharyngeal candidiasis and disseminated candidiasis. Thus, Ssa1 is the second identified invasin of C. albicans.