Virulence of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis in reconstituted human tissue models

Candida parapsilosis is an increasingly important human pathogen. To study the interactions of C. parapsilosis with human tissues, we evaluated the effects of the CBS 604 type strain and three different clinical isolates on reconstituted human oral epithelial and epidermal tissues. The newly described species Candida orthopsilosis and Candida metapsilosis were also examined in these models. Microscopy of reconstituted tissues infected with yeast cells revealed severe attenuation, morphological changes and cellular damage. C. orthopsilosis caused damage similar to C. parapsilosis isolates, whereas C. metapsilosis was less virulent. To further quantitate tissue damage, we measured lactate dehydrogenase (LDH) in the culture supernatant. The relative LDH measurements correlated with our histopathological observations. We also examined the effect of the lipase inhibitor Ebelactone B and proteinase inhibitor Pepstatin A, to establish the utility of this model for studying factors of C. parapsilosis virulence. Both Ebelactone B and Pepstatin A reduced the destruction of epidermal and epithelial tissues. Our data show that reconstituted human tissues are extremely useful for modeling host interactions with C. parapsilosis and for studying fungal virulence factors.     

Candida albicans and Candida parapsilosis Rapidly Up-Regulate Galectin-3 Secretion by Human Gingival Epithelial Cells

Galectin-3 is a β-galactoside-binding C-type lectin that plays an important role in innate immunity. The purpose of this study was to determine whether Candida albicans and Candida parapsilosis up-regulate galectin-3 secretion by human gingival epithelial cells and gingival fibroblasts. Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts were incubated in the presence or absence of C. albicans or C. parapsilosis without serum. Levels of secreted human galectin-3 in culture supernatants were measured by enzyme-linked immunosorbent assay. We also pretreated Ca9-22 cells with cytochalasin D (an actin polymerization inhibitor), ALLN (a calpain inhibitor) and LY294002 [a phosphatidylinositol-3 kinase (PI3K) inhibitor] to determine whether the up-regulation of galectin-3 secretion was mediated by cytoskeletal changes, protease activity, or PI3K signaling. Galectin-3 secretion was significantly and rapidly up-regulated by live C. albicans and C. parapsilosis, as well as heat-killed C. albicans. In addition, cytochalasin D, LY294002 and ALLN did not inhibit the up-regulation in galectin-3 secretion. These results suggest that both live and heat-killed C. albicans and C. parapsilosis may increase the activity of the innate immune system and invasion by other microorganisms via up-regulation of galectin-3 secretion.     

Adherence and receptor relationships of Candida albicans.

The cell surface of Candida albicans is composed of a variety of polysaccharides such as glucan, chitin, and mannan. The first two components primarily provide structure, while the mannan, often covalently linked to protein, constitutes the major antigen of the organism. Mannoproteins also have enzymatic activity (acid protease) and ligand-receptor functions. The complement receptors of C. albicans appear to be mannoproteins that are required for the adherence of the organism to endothelial cells. This is certainly true of the CR3-like protein of C. albicans. Proof that the CR3 is the Candida receptor for endothelial cells is derived from two observations. First, mutants lacking CR3 activity are less adherent in vitro and, in fact, less virulent. Second, the ligand recognized by the CR3 receptor (C3bi) as well as anti-CR3 antibodies blocks adherence of the organism to endothelial cells. The CR2 of C. albicans appears to promote the adherence of the organism to plastic substrates. Unlike the CR2 of mammalian cells, the Candida CR2 recognizes ligands containing the RGD sequence of amino acids in addition to the C3d ligand, which does not contain the RGD sequence. There is uncertainty as to whether the Candida CR2 and CR3 are, in fact, different proteins. A mannoprotein has also been described as the adhesin for epithelial cells. In this case, the receptor has a lectinlike activity and recognizes fucose- or glucosamine-containing glycoproteins of epithelial cells, depending on the strain of C. albicans. The oligosaccharide component of the receptor is probably not involved in ligand recognition and may serve to stabilize the receptor. However, the oligosaccharide factor 6 epitope of mannan may also provide adhesin activity in the recognition of epithelial cells. Mannoproteins can be extracted from cells by a number of reagents. Zymolyase, for instance, tends to remove structural mannoproteins, which contain relatively little protein and are linked to glucan. Reagents such as dithiothreitol, on the other hand, tend to extract mannoproteins containing higher amounts of protein that appear to have receptor function. The mannoproteins of C. albicans are dynamically expressed and may be growth phase and growth form specific.     

白色念珠菌分泌型天冬氨酸蛋白酶在口腔扁平苔藓患者唾液中的表达

研究白色念珠菌分泌型天冬氨酸蛋白酶(SAP)家族在扁平苔藓患者唾液中的表达,进而探讨白色念珠菌病与扁平苔藓的关系。选取白色念珠菌携带者、白色念珠菌病患者、携带白色念珠菌的扁平苔藓患者、伴有白色念珠菌病的扁平苔藓患者各20例,念珠菌培养阴性的扁平苔藓患者10例。从唾液标本中直接提取RNA,进行RT-PCR。白色念珠菌携带者唾液中有SAP2、SAP4-6、SAP7的不同表达,携带白色念珠菌的口腔扁平苔藓患者唾液中有SAP2、SAP4-6、SAP7、SAP8、SAP9的不同表达,所有携带者中均未表现SAP1、SAP3和SAP10的阳性扩增。白色念珠菌患病者及伴有白色念珠菌病的口腔扁平苔藓患者唾液中有SAP1-10的不同表达。念珠菌培养阴性的扁平苔藓患者均无SAP1-10的阳性扩增。SAP9与扁平苔藓密切相关。     

The role of galectin‐3 in phagocytosis of Candida albicans and Candida parapsilosis by human neutrophils

Candida albicans causes the majority of invasive candidiasis in immunocompromised adults while Candida parapsilosis is a leading cause of neonatal candidiasis. While much work has focused on how the immune system recognizes and responds to C. albicans, less is known about host interaction with C. parapsilosis. This study investigates the human neutrophil phagocytic response to these species. Neutrophils underwent phagocytosis of C. parapsilosis yeast and C. albicans hyphae much more efficiently than C. albicans yeast. Treatment of neutrophils with a galectin‐3 (gal3) blocking antibody inhibited phagocytosis of C. parapsilosis yeast and C. albicans hyphae, but not C. albicans yeast. The majority of neutrophil gal3 was expressed intracellularly and was secreted from neutrophils after treatment with C. parapsilosis mannan. When neutrophils were treated with exogenous gal3, phagocytosis of both C. albicans and C. parapsilosis yeast increased. Exposure of neutrophils to C. parapsilosis yeast increased phagocytosis of C. albicans yeast and was inhibited by gal3 blocking antibody. Taken together, these data indicate that gal3 secreted from neutrophils may act as a pro‐inflammatory autocrine/paracrine signal in neutrophil phagocytosis and suggest that gal3 has a unique role in neutrophil response to C. parapsilosis yeast and C. albicans hyphae distinct from C. albicans yeast.     

基于iTRAQ技术分析五倍子作用白念珠菌后的差异蛋白表达

为了探讨五倍子抗白念珠菌的作用机制,提取4 μg/mL五倍子提取液作用后的白念珠菌（SC5314）总蛋白,采用同位素标记相对和绝对定量（isobaric tags for relative and absolute quantitation,iTRAQ）蛋白质组学技术、液相色谱串联质谱（LC-MS/MS）技术分析和鉴定差异表达的蛋白质,并对差异表达蛋白进行生物信息学分析。经LC-MS/MS鉴定出3 721种蛋白质,其中,差异表达蛋白104种,包括57种表达上调蛋白和47种表达下调蛋白。通过生物信息学分析发现,上述差异蛋白参与了氧化还原反应、过氧化氢分解代谢以及能量代谢等生物学过程。研究表明,五倍子抗白念珠菌的作用机制可能是通过抑制氧化磷酸化,进而影响菌体能量代谢和物质生成,最终导致细胞结构与功能的改变。     

Adherence of Candida albicans and Candida dubliniensis to buccal and vaginal cells

Twenty-seven Candida albicans strains and 26 Candida dubliniensis strains, isolated from HIV patients, were tested for their adherence to buccal and vaginal epithelial cells. Both species showed important levels of adhesion to buccal and vaginal epithelial cells, although C. albicans showed the highest levels of adhesion. These results suggest that both Candida species are well adapted, in terms of adhesion capability, to the oral and vaginal environment.     

Adherence and biofilm formation of non-Candida albicans Candida species

Most cases of candidosis have been attributed to Candida albicans, but recently non-C. albicans Candida species have been identified as frequent human pathogens. Candida pathogenicity has been attributed to several factors, including adhesion to medical devices and/or host cells, biofilm formation, and secretion of hydrolytic enzymes (proteases, phospholipases and haemolysins). Although 'new'Candida species are emerging, there is still a lack of information about their pathogenicity. This review discusses recent advances in our knowledge of Candida glabrata, Candida parapsilosis and Candida tropicalis virulence factors, specifically those of adhesion and biofilm formation, which are key components in Candida pathogenicity.     

In Vitro Effect of Amphotericin B on Candida albicans,Candida glabrata and Candida parapsilosis Biofilm Formation

Candida spp. biofilm is considered highly resistant to conventional antifungals. The aim of this study was to investigate the in vitro effect of amphotericin B on Candida spp. biofilms at different stages of maturation. We investigated the activity of amphotericin B against 78 clinical isolates of Candida spp., representing three species, growing as planktonic and sessile cells, by a widely accepted broth microdilution method. The in vitro effect on sessile cell viability was evaluated by MTT reduction assay. All examined strains were susceptible to amphotericin B when grown as free-living cells. At the early stages of biofilm maturation 96.7–100.0 % strains, depending on species, displayed amphotericin B sessile minimal inhibitory concentration (SMIC) ≤1 μg/mL. Mature Candida spp. biofilm of 32.1–90.0 % strains displayed amphotericin B SMIC ≤1 μg/mL. Based on these results, amphotericin B displays species- and strain-depending activity against Candida spp. biofilms.     

Adherence of Pseudomonas aeruginosa and Candida albicans to urinary catheters

In this study, the in vitro adherence capabilities of Pseudomonas aeruginosa and Candida albicans clinical isolates to urinary catheters were investigated. Quantitative analysis was performed by colony-forming unit counts and scanning electron microscopy. Results demonstrated that the adherence of P. aeruginosa to urinary catheters was enhanced in the presence of C. albicans, while C. albicans adherence was not significantly affected. Further investigations are warranted to fully understand the pathogenic potential of their interaction in order to aid in the design of novel strategies for the prevention and treatment of catheter-related UTIs.     

Heterologous Expression of Candida albicans Cell Wall-Associated Adhesins in Saccharomyces cerevisiae Reveals Differential Specificities in Adherence and Biofilm Formation and in Binding Oral Streptococcus gordonii

Colonization and infection of the human host by opportunistic pathogen Candida albicans derive from an ability of this fungus to colonize mucosal tissues and prosthetic devices within the polymicrobial communities present. To determine the functions of C. albicans cell wall proteins in interactions with host or bacterial molecules, Saccharomyces cerevisiae was utilized as a surrogate host to express C. albicans cell wall proteins Als3p, Eap1p, Hwp1p, and Rbt1p. Salivary pellicle and fibrinogen were identified as novel substrata for Als3p and Hwp1p, while only Als3p mediated adherence of S. cerevisiae to basement membrane collagen type IV. Parental S. cerevisiae cells failed to form biofilms on salivary pellicle, polystyrene, or silicone, but cells expressing Als3p or Hwp1p exhibited significant attachment to each surface. Virulence factor Rbt1p also conferred lower-level binding to salivary pellicle and polystyrene. S. cerevisiae cells expressing Eap1p formed robust biofilms upon polystyrene surfaces but not salivary pellicle. Proteins Als3p and Eap1p, and to a lesser degree Hwp1p, conferred upon S. cerevisiae the ability to bind cells of the oral primary colonizing bacterium Streptococcus gordonii. These interactions, which occurred independently of amyloid aggregate formation, provide the first examples of specific C. albicans surface proteins serving as receptors for bacterial adhesins. Streptococcus gordonii did not bind parental S. cerevisiae or cells expressing Rbt1p. Taken collectively, these data suggest that a network of cell wall proteins comprising Als3p, Hwp1p, and Eap1p, with complementary adhesive functions, promotes interactions of C. albicans with host and bacterial molecules, thus leading to effective colonization within polymicrobial communities.Candida albicans is a pleiomorphic fungus found on mucosal surfaces of the gastrointestinal and genitourinary tracts, skin, and oral cavity (2). As an opportunistic pathogen, C. albicans can form potentially lethal fungal masses in the kidney, heart, and brain upon gaining access to the bloodstream (4), and invasive fungal infections are becoming increasingly problematic in the clinical setting (34). Candida species are now the third most common cause of nosocomial bloodstream infections. In the United States alone there are an estimated 70,000 cases per year of disseminated candidiasis (34), with an associated health care cost of $2 billion to $4 billion/year (44, 45). C. albicans is also responsible for >90% of oral fungal diseases derived from polymicrobial biofilms, and ≤90% of HIV-infected individuals suffer from oral candidiasis, which may progress to advanced esophageal candidiasis (10).C. albicans can colonize a wide variety of sites within the host in addition to mucosal tissues, such as catheters, stents, surgical implants, and dentures. This ability can be attributed, at least in part, to the large number of proteins expressed on the candidal cell surface, which mediate adhesion to a range of substrata. Cell wall proteins (CWPs) in C. albicans also play a critical role in biofilm formation. Within the host, Candida species are frequently found as part of polymicrobial biofilms, in which antagonistic, synergistic, and mutualistic interactions among microbes significantly influence composition of the community microflora (17). This is particularly pertinent for colonization of the oral cavity, where up to 100 different microbial species may be isolated from a single site at any given time. To successfully colonize the host and cause disease, C. albicans must therefore not only attach directly to host tissues or medical devices but also navigate interactions with a diverse microflora to ensure the availability of suitable binding sites, nutrients, and growth conditions.It has been shown that C. albicans coaggregates (coadheres) strongly with Streptococcus bacteria indigenous to the human oral cavity such as Streptococcus gordonii and Streptococcus sanguinis (13, 18). These bacteria are pioneer colonizers of oral cavity surfaces, and it is hypothesized that interactions with these streptococci may promote oral carriage and persistence of C. albicans, thereby supporting candidal reservoirs for opportunistic infections following disruption of the oral ecology. Previous work by Holmes et al. (13, 14) identified Streptococcus gordonii cell wall-associated polypeptides SspA, SspB, and CshA, together with linear cell wall phosphopolysaccharides, as potential targets for C. albicans binding streptococcal cells. However, the reciprocal receptors on the surface of C. albicans recognized by streptococci have yet to be identified.This work utilizes Saccharomyces cerevisiae, which does not bind streptococci, as a heterologous host for expression and identification of candidal surface proteins targeted by Streptococcus gordonii. Four surface proteins were selected that had been previously implicated in C. albicans colonization and pathogenesis: Als3p, Eap1p, Hwp1p, and Rbt1p. Als3p (comprehensively reviewed by Hoyer et al. [15]), Hwp1p (29, 40), and Eap1p (20, 22) are associated with mediating interactions of C. albicans with host epithelial cells and with biofilm formation in catheter models. Expression of Als3p or Hwp1p has been shown to be hypha specific, while Eap1p is expressed by each morphological form (16, 20, 41). Rbt1p shares 43% sequence identity with Hwp1p and has been associated with virulence in mouse and rabbit models of C. albicans infection (6). Using a recombinase-based Gateway cloning system (Invitrogen), each of the C. albicans proteins was expressed on the surface of S. cerevisiae. Their functional properties in adherence and biofilm formation were determined, and proteins Als3p and Eap1p were identified as potential Streptococcus gordonii receptors on the surface of C. albicans.     

Secreted aspartic proteases of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida lusitaniae. Inhibition with peptidomimetic inhibitors.

The frequency of Candida infections has increased in recent years and it has been accompanied by a significant rise in morbidity and mortality. The secretion of aspartic proteases by Candida spp. was demonstrated to be one of the virulence determinants. Candida albicans is classified as the major human pathogen in the genus Candida. However, other species of this genus have been found to cause an increasing number of candidiases. We isolated secreted aspartic proteases (Saps) of C. albicans (Sap2p), C. tropicalis (Sapt1p), C. parapsilosis (Sapp1p), and C. lusitaniae (Saplp) from culture media. All the isolated proteases were N-terminally sequenced. Their specific proteolytic activities and sensitivity to series of peptidomimetic inhibitors modified in the type of scissile bond replacement as well as in the N- and C-termini were analyzed. The most divergent substrate specificity was observed for the Sap of C. tropicalis. The specificity of Sap of C. lusitaniae is most closely related to that of Sap of C. parapsilosis. We designed and prepared an inhibitor containing phenylstatine isoster that was equipotent towards all four proteases within the range of 10-10-10-9 M. The HIV-1 protease inhibitors ritonavir, saquinavir, indinavir, and nelfinavir were also tested for the inhibition of four Saps. Only ritonavir and saquinavir inhibited Sap2p, Sapt1p, Sapp1p, and Saplp in micromolar concentrations.     

Virulence and pathogenicity of a Candida albicans mutant with reduced filamentation

Deletion of DNA polymerase eta (Rad30/Polη) in pathogenic yeast Candida albicans is known to reduce filamentation induced by serum, ultraviolet, and cisplatin. Because nonfilamentous C. albicans is widely accepted as avirulent form, here we explored the virulence and pathogenicity of a rad30Δ strain of C. albicans in cell‐based and animal systems. Flow cytometry of cocultured fungal and differentiated macrophage cells revealed that comparatively higher percentage of macrophages was associated with the wild‐type than rad30Δ cells. In contrast, higher number of Polη‐deficient C. albicans adhered per macrophage membrane. Imaging flow cytometry showed that the wild‐type C. albicans developed hyphae after phagocytosis that caused necrotic death of macrophages to evade their clearance. Conversely, phagosomes kill the fungal cells as estimated by increased metacaspase activity in wild‐type C. albicans. Despite the morphological differences, both wild‐type and rad30? C. albicans were virulent with a varying degree of pathogenicity in mice models. Notably, mice with Th1 immunity were comparatively less susceptible to systemic fungal infection than Th2 type. Thus, our study clearly suggests that the modes of interaction of morphologically different C. albicans strains with the host immune cells are diverged, and host genetic background and several other attributing factors of the fungus could additionally determine their virulence.     

Aquaporin Expression and Freeze Tolerance in Candida albicans

Aquaporins are members of the major intrinsic protein superfamily of integral membrane proteins which enable the transport of water, glycerol, and other solutes across membranes in various organisms. In microorganisms, the physiological role of aquaporins is not yet defined. We found a clear correlation between expression of the Candida albicans aquaporin-encoding gene AQY1 and freeze tolerance. A connection with the function for the aquaporin in the natural environment of C. albicans is, however, not obvious.     

Virulence and pathogenicity of Candida albicans is enhanced in biofilms containing oral bacteria

This study examined the influence of bacteria on the virulence and pathogenicity of candidal biofilms. Mature biofilms (Candida albicans-only, bacteria-only, C. albicans with bacteria) were generated on acrylic and either analysed directly, or used to infect a reconstituted human oral epithelium (RHOE). Analyses included Candida hyphae enumeration and assessment of Candida virulence gene expression. Lactate dehydrogenase (LDH) activity and Candida tissue invasion following biofilm infection of the RHOE were also measured. Candida hyphae were more prevalent (p < 0.05) in acrylic biofilms also containing bacteria, with genes encoding secreted aspartyl-proteinases (SAP4/SAP6) and hyphal-wall protein (HWP1) up-regulated (p < 0.05). Candida adhesin genes (ALS3/EPA1), SAP6 and HWP1 were up-regulated in mixed-species biofilm infections of RHOE. Multi-species infections exhibited higher hyphal proportions (p < 0.05), up-regulation of IL-18, higher LDH activity and tissue invasion. As the presence of bacteria in acrylic biofilms promoted Candida virulence, consideration should be given to the bacterial component when managing denture biofilm associated candidoses.