Staphylococcal biofilm exopolysaccharide protects against Caenorhabditis elegans immune defenses

Staphylococcus epidermidis and Staphylococcus aureus are leading causes of hospital-acquired infections that have become increasingly difficult to treat due to the prevalence of antibiotic resistance in these organisms. The ability of staphylococci to produce biofilm is an important virulence mechanism that allows bacteria both to adhere to living and artificial surfaces and to resist host immune factors and antibiotics. Here, we show that the icaADBC locus, which synthesizes the biofilm-associated polysaccharide intercellular adhesin (PIA) in staphylococci, is required for the formation of a lethal S. epidermidis infection in the intestine of the model nematode Caenorhabditis elegans. Susceptibility to S. epidermidis infection is influenced by mutation of the C. elegans PMK-1 p38 mitogen-activated protein (MAP) kinase or DAF-2 insulin-signaling pathways. Loss of PIA production abrogates nematocidal activity and leads to reduced bacterial accumulation in the C. elegans intestine, while overexpression of the icaADBC locus in S. aureus augments virulence towards nematodes. PIA-producing S. epidermidis has a significant survival advantage over ica-deficient S. epidermidis within the intestinal tract of wild-type C. elegans, but not in immunocompromised nematodes harboring a loss-of-function mutation in the p38 MAP kinase pathway gene sek-1. Moreover, sek-1 and pmk-1 mutants are equally sensitive to wild-type and icaADBC-deficient S. epidermidis. These results suggest that biofilm exopolysaccharide enhances virulence by playing an immunoprotective role during colonization of the C. elegans intestine. These studies demonstrate that C. elegans can serve as a simple animal model for studying host-pathogen interactions involving staphylococcal biofilm exopolysaccharide and suggest that the protective activity of biofilm matrix represents an ancient conserved function for resisting predation.     

Candida albicans Hyphal Formation and Virulence Assessed Using a Caenorhabditis elegans Infection Model

Candida albicans colonizes the human gastrointestinal tract and can cause life-threatening systemic infection in susceptible hosts. We study here C. albicans virulence determinants using the nematode Caenorhabditis elegans in a pathogenesis system that models candidiasis. The yeast form of C. albicans is ingested into the C. elegans digestive tract. In liquid media, the yeast cells then undergo morphological change to form hyphae, which results in aggressive tissue destruction and death of the nematode. Several lines of evidence demonstrate that hyphal formation is critical for C. albicans pathogenesis in C. elegans. First, two yeast species unable to form hyphae (Debaryomyces hansenii and Candida lusitaniae) were less virulent than C. albicans in the C. elegans assay. Second, three C. albicans mutant strains compromised in their ability to form hyphae (efg1Δ/efg1Δ, flo8Δ/flo8Δ, and cph1Δ/cph1Δ efg1Δ/efg1Δ) were dramatically attenuated for virulence. Third, the conditional tet-NRG1 strain, which enables the external manipulation of morphogenesis in vivo, was more virulent toward C. elegans when the assay was conducted under conditions that permit hyphal growth. Finally, we demonstrate the utility of the C. elegans assay in a screen for C. albicans virulence determinants, which identified several genes important for both hyphal formation in vivo and the killing of C. elegans, including the recently described CAS5 and ADA2 genes. These studies in a C. elegans-C. albicans infection model provide insights into the virulence mechanisms of an important human pathogen.Candida albicans is the most common human fungal pathogen; however, our knowledge of its virulence mechanisms is incomplete, and our best antifungal agents are often ineffective in treating severe candidiasis (3). Infections with Candida species account for 70 to 90% of all invasive mycoses (32) and can be associated with devastating consequences, particularly in intensive care units where mortality rates reach 40% (24, 34). The drug resistance of pathogenic fungi exacerbates this problem and often limits therapeutic options (35). The identification of virulence pathways that can be targeted with novel antifungal therapies is urgently needed (31, 38, 46).One approach to understand the genetic mechanisms of virulence is to use invertebrates, such as the nematode Caenorhabditis elegans, as model hosts (43). Studies of C. elegans infection with Pseudomonas aeruginosa and Cryptococcus neoformans, for example, have led to the identification of evolutionarily conserved mechanisms of host immunity and microbial virulence (1, 21, 50). However, efforts to design an accurate nonmammalian model of C. albicans pathogenesis have been stymied, in part because it has been difficult to capture the role of Candida dimorphism in these systems.Morphogenesis in C. albicans is intricately related to pathogenesis and thus has been intensively studied. C. albicans hyphae are important for tissue destruction and host invasion (3). As such, C. albicans mutants and non-albicans Candida species that are unable to form true hyphae are attenuated for virulence (3, 37). However, C. albicans yeast cells also have virulence attributes (4, 33) that are likely involved in dissemination of the fungus through the bloodstream, and the establishment of infection at distant sites. To date, genetic screens to identify the determinants of Candida morphology have been conducted in vitro. Determining the role of these genes in virulence has traditionally involved separate and often laborious studies in mammals. Therefore, an expedient system to study morphogenesis of C. albicans in vivo and accurately model pathogenesis would offer many important advantages.Here, we study C. albicans pathogenesis using the invertebrate host C. elegans. C. albicans yeast cells are ingested into the gastrointestinal tract. In liquid media, the yeast cells form hyphae, which results in an aggressive infection that ultimately kills the nematode. Fungal hyphae destroy worm tissues and pierce the collagenous cuticle of the animal, a phenotype that is easily visible using a dissecting microscope. By studying mutants and genetically engineered C. albicans strains, we show that hyphal formation is required for full virulence in this system. Finally, we illustrate the utility of the C. elegans-C. albicans infection assay in a screen for genes involved in Candida morphogenesis and virulence.     

Identification of Antifungal Compounds Active against Candida albicans Using an Improved High-Throughput Caenorhabditis elegans Assay

Candida albicans, the most common human pathogenic fungus, can establish a persistent lethal infection in the intestine of the microscopic nematode Caenorhabditis elegans. The C. elegans–C. albicans infection model was previously adapted to screen for antifungal compounds. Modifications to this screen have been made to facilitate a high-throughput assay including co-inoculation of nematodes with C. albicans and instrumentation allowing precise dispensing of worms into assay wells, eliminating two labor-intensive steps. This high-throughput method was utilized to screen a library of 3,228 compounds represented by 1,948 bioactive compounds and 1,280 small molecules derived via diversity-oriented synthesis. Nineteen compounds were identified that conferred an increase in C. elegans survival, including most known antifungal compounds within the chemical library. In addition to seven clinically used antifungal compounds, twelve compounds were identified which are not primarily used as antifungal agents, including three immunosuppressive drugs. This assay also allowed the assessment of the relative minimal inhibitory concentration, the effective concentration in vivo, and the toxicity of the compound in a single assay.     

Resveratrol lacks antifungal activity against Candida albicans

The putative candicidal activity of resveratrol is currently a matter of controversy. Here, the antifungal activity as well as the antioxidant response of resveratrol against Candida albicans, have been tested in a set of strains with a well-established genetic background At the doses usually employed in antifungal tests (10-40 μg/ml), resveratrol has no effect on the exponential growth of the C. albicans CAI.4 strain, a tenfold increase (400 μg/ml) was required in order to record a certain degree of cell killing, which was negligible in comparison with the strong antifungal effect caused by the addition of amphotericin B (5 μg/ml). An identical pattern was recorded in the prototrophic strains of C. albicans SC5314 and RM-100, whereas the oxidative sensitive trehalose-deficient mutant (tps1/tps1 strain) was totally refractory to the presence of resveratrol. In turn, the serum-induced yeast-to-hypha transition remained unaffected upon addition of different concentrations of resveratrol. Determination of endogenous trehalose and catalase activity, two antioxidant markers in C. albicans; revealed no significant changes in their basal contents induced by resveratrol. Collectively, our results seem to dismiss a main antifungal role as well as the therapeutic application of resveratrol against the infections caused by C. albicans.     

Concanavalin-A induces IL-17 production during the course of Candida albicans infection

In a previous study, our group verified that 100% of mice survived to a lethal dose of Candida albicans following pretreatment with concanavalin-A (Con-A) for 3 days. This work proposed to investigate whether treatment could mediate an adaptative immune response involving T(H) 17 cells. A significant increase in IL-17 levels at 6 h postinfection was observed and was maintained up to 18 h in the Con-A group, whereas in control mice, a reduction in this cytokine was verified. In addition, T(H) 17 cells develop in the presence of TGF-β, IL-1 β, and IL-6 that were increased significantly 2 h postinfection in Con-A-treated mice. Macrophages were involved in the process, engulfing greater numbers of yeast cells, and were activated through TNF-α and interferon-γ produced at significant levels at 2 h postinfection. A significant increase in IL-12 levels was also observed at 2 h postinfection. Thus, activated macrophages were probably more capable of killing and processing Candida antigens, signalizing an adaptative immune response. Macrophages from controls did not prevent yeast-to-hyphae transition and were partially destroyed, as shown in scanning microscopy. These results suggest that treatment with Con-A facilitated the triggering of T(H) 17 and T(H) 1 responses via IL-17 and IFN-γ production, leading to the resolution of C. albicans infection.     

Evaluation of antifungal pharmacodynamic characteristics of AmBisome against Candida albicans

A liposomal formulation of Amphotericin B (AmBisome), with small unilamellar vesicles containing amphotericin B, shows characteristic pharmacokinetics as liposomes, and in consequence, has different pharmacological activity and toxicity from amphotericin B deoxycholate (Fungizone). In this study, we evaluated the antifungal pharmacodynamic characteristics of AmBisome against Candida albicans using the in vitro time-kill method and murine systemic infection model. A time-kill study indicated that the in vitro fungicidal activities of AmBisome and Fungizone against C. albicans ATCC 90029 increased with increasing drug concentration. For in vivo experiments, leucopenic mice were infected intravenously with the isolate 4 hr prior to the start of therapy. The infected mice were treated for 24 hr with twelve dosing regimens of AmBisome administered at 8-, 12-, 24-hr dosing intervals. Correlation analysis between the fungal burden in the kidney after 24 hr of therapy and each pharmacokinetic/pharmacodynamic parameter showed that the peak level/MIC ratio was the best predictive parameter of the in vivo outcome of AmBisome. These results suggest that AmBisome, as well as Fungizone, has concentration-dependent antifungal activity. Furthermore, since AmBisome can safely achieve higher concentrations in serum than Fungizone, AmBisome is thought to have superior potency to Fungizone against fungal infections.     

Synthetic arylquinuclidine derivatives exhibit antifungal activity against Candida albicans, Candida tropicalis and Candida parapsilopsis

Background

Severely burned patients may develop life-threatening nosocomial infections due to Pseudomonas aeruginosa, which can exhibit a high-level of resistance to antimicrobial drugs and has a propensity to cause nosocomial outbreaks. Antiseptic and topical antimicrobial compounds constitute major resources for burns care but in vitro testing of their activity is not performed in practice.

Results

In our burn unit, a P. aeruginosa clone multiresistant to antibiotics colonized or infected 26 patients over a 2-year period. This resident clone was characterized by PCR based on ERIC sequences. We investigated the susceptibility of the resident clone to silver sulphadiazine and to the main topical antimicrobial agents currently used in the burn unit. We proposed an optimized diffusion assay used for comparative analysis of P. aeruginosa strains. The resident clone displayed lower susceptibility to silver sulphadiazine and cerium silver sulphadiazine than strains unrelated to the resident clone in the unit or unrelated to the burn unit.

Conclusions

The diffusion assay developed herein detects differences in behaviour against antimicrobials between tested strains and a reference population. The method could be proposed for use in semi-routine practice of medical microbiology.     

Hydrogen peroxide induces hyphal differentiation in Candida albicans

In this study, we demonstrate that hyphal differentiation is induced by the subtoxic concentration of exogenous H₂O₂ in Candida albicans. This finding is confirmed by the changing intracellular concentration of H₂O₂. In order to induce the same level of differentiation, low concentrations of exogenous H₂O₂ are required for the null mutants of the thiol-specific antioxidant and catalase, while higher concentrations are needed for cells treated with ascorbic acid, an antioxidant chemical.     

Toll-like receptor-2 is essential in murine defenses against Candida albicans infections

In this work, we studied the role of toll-like receptor-2 (TLR2) in murine defenses against Candida albicans. TLR2-deficient mice experimentally infected intraperitoneally (i.p.) or intravenously (i.v.) in vivo had very significant impaired survival compared with that of control mice. In vitro production of TNF-alpha and macrophage inhibitory protein-2 (MIP-2) by macrophages from TLR2-/- mice in response to yeasts and hyphae of C. albicans were significantly lower (80% and 40%, respectively; P <0.05) than production by macrophages from wild-type mice. This impaired production of TNF-alpha and MIP-2 probably contributed to the 41% decreased recruitment of neutrophils to the peritoneal cavity of i.p. infected TLR2-/- mice. In contrast, in vitro phagocytosis of yeasts and production of reactive oxygen intermediates (ROI) were not affected in macrophages from TLR2-/- animals. Our data indicate that TLR2 plays a major role in the response of macrophages to C. albicans, triggering cytokine and chemokine expression, and it is essential for in vivo protection against infection.     

Coprisin-induced antifungal effects in Candida albicans correlate with apoptotic mechanisms

Coprisin is a 43-mer defensin-like peptide from the dung beetle, Copris tripartitus. Here, we investigated the induction of apoptosis by coprisin in Candida albicans cells. Coprisin exerted antifungal and fungicidal activity without any hemolytic effect. Confocal microscopy indicated that coprisin accumulated in the nucleus of cells. The membrane studies, 1,6-diphenyl-1,3,5-hexatriene, calcein-leakage, and giant unilamellar vesicle assays, confirmed that coprisin did not disrupt the fungal plasma membrane at all. Moreover, the activity of coprisin was energy- and salt-dependent. Next, we investigated whether coprisin induced apoptosis in C. albicans. Annexin V-FITC staining and TUNEL assay showed that coprisin was involved with both the early and the late stages of apoptosis. Coprisin also increased the intracellular reactive oxygen species level, and hydroxyl radicals were included at high levels among the species. The effect of thiourea as a hydroxyl radical scavenger further confirmed the existence of the hydroxyl radicals. Furthermore, coprisin induced mitochondrial membrane potential dysfunction, cytochrome c release, and activation of metacaspases. In summary, this study suggests that coprisin could be a model molecule for a large family of novel antimicrobial peptides possessing apoptotic activity.     

Studies on Shigella boydii infection in Caenorhabditis elegans and bioinformatics analysis of immune regulatory protein interactions

Shigella boydii causes bacillary dysentery or shigellosis and generates a significant burden in the developing nations. S. boydii-mediated infection assays were performed at both physiological and molecular levels using Caenorhabditis elegans as a host. Continuous exposure of worms to S. boydii showed a reduced life span indicating the pathogenicity of Shigella. Quantitative Real-Time PCR analysis was performed to analyze the expression and regulation of host specific candidate-antimicrobial genes (clec-60, clec-87, lys-7), which were expressed significantly during early infection, but weakened during the latter hours. Increased mortality of mutant RB1285 by S. boydii and Shigella flexneri indicated the role of lys-7 during Shigella infection. Protein-protein interactions (PPIs) database was used to analyze the interaction of immune proteins in both C. elegans and humans. In addition, the expression and regulation were revealed about immune genes (clec-61, clec-62, clec-63, F54D5.3 and ZK1320.2), which encode several intermediate immune protein partners (CLEC-61, CLEC-62, CLEC-63, F54D5.3, ZK1320.2, W03D2.6 and THN-2) that interact with LYS-7 and CLEC-60 and were found to play a role in C. elegans immune defense against S. boydii infections. Similarly, the immune genes that are specific to the human defense system, which encode IGHV4-39, A2M, LTF, and CD79A, were predicted to be expressed with LYZ and MBL2, thus indicating their regulation during Shigella infections. Our results using the lowest eukaryotic model system and human database indicated that the major players involved in immunity-related processes appear to be common in cases of Shigella sp. mediated immune responses. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction.     

新疆地区白念珠菌基因型分析及其体外药物敏感性研究

目的研究新疆地区汉族和维吾尔族患者来源的50株白念珠菌的基因型及其对两性霉素B、5-氟胞嘧啶、米卡芬净、伊曲康唑、氟康唑和咪康唑的体外敏感性。方法采用PCR法扩增白念珠菌rDNA 25S的Ⅰ类内含子包含区,根据扩增产物的大小判断基因型(A型为450 bp,B型为840 bp,C型为450 bp和840 bp)。采用CLSI M27-A液基微量稀释法测定50株白念珠菌对上述6种抗真菌药的体外敏感性。结果 50株菌分为3种基因型:A型30株,B型和C型各10株。所有菌株对两性霉素B、5-氟胞嘧啶、米卡芬净和咪康唑的MIC值较低,MIC范围依次为0.25～0.5μg/mL,0.125～0.5μg/mL,≤0.03μg/mL,0.25～8μg/mL;对伊曲康唑和氟康唑的MIC值较高,MIC范围分别为0.25～8μg/mL,0.5～64μg/mL。B型和C型对5-氟胞嘧啶的MIC值均为0.125μg/mL,对伊曲康唑和氟康唑的耐药率分别为84%、70%。不同族别来源的菌株基因型比较无显著差异(P>0.05),不同基因型菌株的抗真菌药物敏感性比较也无显著差异(P>0.05)。结论新疆地区白念珠菌分A,B,C三种基因型。汉...     

Synthesis of Sordaricin analogues as potent antifungal agents against Candida albicans

Sordaricin derivatives possessing a cyclohexane ring appendage attached via an ether, thioether, amine, oxime, ester or amide linkage were synthesized and their antifungal activity was evaluated in vitro. Compounds containing a thioether bond or an oxime bond as a linkage exhibited potent MICs (< or = 0.125 microg/mL) against four Candida albicans strains including azole-low-susceptible strains. They were also active (MIC < or = 0.125 microg/mL) against Candida glabrata. Their in vivo efficacy was confirmed in a murine intravenous infection model with Candida albicans.     

The synergistic antifungal activity of resveratrol with azoles against Candida albicans

Candida albicans is one of the most common clinical pathogenic microorganisms and it is becoming a serious health threat, particularly to immunocompromised populations. Drug resistance of Candida species has also frequently emerged, and combination therapy for fungal infections has attracted considerable attention. In this study, we established the Qinling Mountains myxobacterial secondary metabolites library and a synergic assay in combination with ketoconazole against C. albicans was introduced for metabolites screening. Two active compounds with synergic anticandidal activities were obtained, which were identified as trans-resveratrol and cis-resveratrol. According to our study, resveratrol can reduce the dosage to 1/64 of ketoconazole as well as itraconazole. Furthermore, synergistic anticandidal activity of resveratrol combined with azoles was verified against a panel of clinical C. albicans isolates, and the combination strategy enhanced the azoles susceptibility of three fluconazole-resistant isolates. These findings suggest that resveratrol enhances the efficacy of azoles and provides a promising application in therapy of C. albicans infection.     

Differential inhibition of Candida albicans CYP51 with azole antifungal stereoisomers

Azole antifungal compounds are important in agriculture and in the treatment of mycotic infection The target enzyme, sterol 14α-demethylase (CYP51), is inhibited through binding of triazole N-4 to the haem of this P450, as a sixth ligand together with the N-1 substituent groups interacting in some way with the apoprotein. Here we use Saccharomyces cerevisiae expression systems for the target enzyme of Candida albicans to investigate binding of enantiomers of the azole antifungal compounds SCH39304 and tetraconazole. A molecular model produced previously provided qualitative explanations for these differences. Interaction of the azole antifungal aromatic group with Phe-233 or -235 may cause the higher activity for (R)-tetraconazole while inactivity of the (SS)-enantiomer of SCH39304 was predicted to result from incompatibility of the hydrophilic sulfonyl moiety when located into the hydrophobic pocket of the active site.     

Physical stress primes the immune response of Galleria mellonella larvae to infection by Candida albicans

Larvae of the greater wax moth (Galleria mellonella) that had been subjected to physical stress by shaking in cupped hands for 2 min showed reduced susceptibility to infection by Candida albicans when infected 24 h after the stress event. Physically stressed larvae demonstrated an increase in haemocyte density and elevated mRNA levels of galiomicin and an inducible metalloproteinase inhibitor (IMPI) but not transferrin or gallerimycin. In contrast, previous work has demonstrated that microbial priming of larvae resulted in the induction of all four genes. Examination of the expression of proteins in the insect haemolymph using 2D electrophoresis and MALDI TOF analysis revealed an increase in the intensity of a number of peptides showing some similarities with proteins associated with the insect immune response to infection. This study demonstrates that non-lethal physical stress primes the immune response of G. mellonella and this is mediated by elevated haemocyte numbers, increased mRNA levels of genes coding for two antimicrobial peptides and the appearance of novel peptides in the haemolymph. This work demonstrates that physical priming increases the insect immune response but the mechanism of this priming is different to that induced by low level exposure to microbial pathogens.