Signaling through Syk or CARD9 Mediates Species-Specific Anti-Candida Protection in Bone Marrow Chimeric Mice.

The spleen tyrosine kinase (Syk) and the downstream adaptor protein CARD9 are crucial signaling molecules in antimicrobial immunity. Candida parapsilosis is an emerging fungal pathogen with a high incidence in neonates, while Candida albicans is the most common agent of candidiasis. While signaling through Syk/CARD9 promotes protective host mechanisms in response to C. albicans, its function in immunity against C. parapsilosis remains unclear. Here, we generated Syk-/- and CARD9-/- bone marrow chimeric mice to study the role of Syk/CARD9 signaling in immune responses to C. parapsilosis compared to C. albicans. We demonstrate various functions of this pathway (e.g., phagocytosis, phagosome acidification, and killing) in Candida-challenged, bone marrow-derived macrophages with differential involvement of Syk and CARD9 along with species-specific differences in cytokine production. We report that Syk-/- or CARD9-/- chimeras rapidly display high susceptibility to C. albicans, while C. parapsilosis infection exacerbates over a prolonged period in these animals. Thus, our results establish that Syk and CARD9 contribute to systemic resistance to C. parapsilosis and C. albicans differently. Additionally, we confirm prior studies but also detail new insights into the fundamental roles of both proteins in immunity against C. albicans. Our data further suggest that Syk has a more prominent influence on anti-Candida immunity than CARD9. Therefore, this study reinforces the Syk/CARD9 pathway as a potential target for anti-Candida immune therapy. IMPORTANCE While C. albicans remains the most clinically significant Candida species, C. parapsilosis is an emerging pathogen with increased affinity to neonates. Syk/CARD9 signaling is crucial in immunity to C. albicans, but its role in in vivo responses to other pathogenic Candida species is largely unexplored. We used mice with hematopoietic systems deficient in Syk or CARD9 to comparatively study the function of these proteins in anti-Candida immunity. We demonstrate that Syk/CARD9 signaling has a protective role against C. parapsilosis differently than against C. albicans. Thus, this study is the first to reveal that Syk can exert immune responses during systemic Candida infections species specifically. Additionally, Syk-dependent immunity to a nonalbicans Candida species in an in vivo murine model has not been reported previously. We highlight that the contribution of Syk and CARD9 to fungal infections are not identical and underline this pathway as a promising immune-therapeutic target to fight Candida infections.

Investigation of OCH1 in the Virulence of Candida parapsilosis Using a New Neonatal Mouse Model.

Candida parapsilosis is an opportunistic human fungal pathogen that poses a serious threat to low birth weight neonates, particularly at intensive care units. In premature infants, the distinct immune responses to Candida infections are not well understood. Although several in vivo models exist to study systemic candidiasis, only a few are available to investigate dissemination in newborns. In addition, the majority of related studies apply intraperitoneal infection rather than intravenous inoculation of murine infants that may be less efficient when studying systemic invasion. In this study, we describe a novel and conveniently applicable intravenous neonatal mouse model to monitor systemic C. parapsilosis infection. Using the currently developed model, we aimed to analyze the pathogenic properties of different C. parapsilosis strains. We infected 2 days-old BALB/c mouse pups via the external facial vein with different doses of C. parapsilosis strains. Homogenous dissemination of yeast cells was found in the spleen, kidney, liver and brain of infected newborn mice. Colonization of harvested organs was also confirmed by histological examinations. Fungal burdens in newborn mice showed a difference for two isolates of C. parapsilosis. C. parapsilosis CLIB infection resulted in higher colonization of the spleen, kidney and liver of neonatal mice compared to the C. parapsilosis GA1 strain at day 2 after the infection. In a comprehensive study with the adult mice infection, we also presented the attenuated virulence of a C. parapsilosis cell wall mutant (OCH1) in this model. Significantly less och1Δ/Δ null mutant cells were recovered from the spleen, kidney and liver of newborn mice compared to the wild type strain. When investigating the cytokine response of neonatal mice to C. parapsilosis infection, we found elevated TNFα, KC, and IL-1ß expression levels in all organs examined when compared to the uninfected control. Furthermore, all three measured cytokines showed a significantly elevated expression when newborn mice were infected with och1Δ/Δ cells compared to the wild type strain. This result further supported the inclusion of OCH1 in C. parapsilosis pathogenicity. To our current knowledge, this is the first study that uses a mice neonatal intravenous infection model to investigate C. parapsilosis infection.

Specific pathways mediating inflammasome activation by Candida parapsilosis.

Candida albicans and C. parapsilosis are human pathogens causing severe infections. The NLRP3 inflammasome plays a crucial role in host defence against C. albicans, but it has been previously unknown whether C. parapsilosis activates this complex. Here we show that C. parapsilosis induces caspase-1 activation and interleukin-1ß (IL-1ß) secretion in THP-1, as well as primary, human macrophages. IL-1ß secretion was dependent on NLRP3, K+-efflux, TLR4, IRAK, Syk, caspase-1, caspase-8 and NADPH-oxidase. Importantly, while C. albicans induced robust IL-1ß release after 4 h, C. parapsilosis was not able to stimulate the production of IL-1ß after this short incubation period. We also found that C. parapsilosis was phagocytosed to a lesser extent, and induced significantly lower ROS production and lysosomal cathepsin B release compared to C. albicans, suggesting that the low extent of inflammasome activation by C. parapsilosis may result from a delay in the so-called "signal 2". In conclusion, this is the first study to examine the molecular pathways responsible for the IL-1ß production in response to a non-albicans Candida species, and these results enhance our understanding about the immune response against C. parapsilosis.

Candida parapsilosis Secreted Lipase as an Important Virulence Factor.

The prevalence of Candida parapsilosis, an opportunistic human pathogenic fungal species, is increasing at an alarming rate in the hospital environment. Patients at risk for C. parapsilosis infection include those with immunosuppression, such as individuals with cancer, AIDS, and low birth weight premature neonates as well as patients that had undergone abdominal surgery. Neonatal candidiasis caused by C. parapsilosis has been widely reported across the globe. Various reports have shown that, compared to other Candida species, certain C. parapsilosis clinical isolates were less susceptible to antifungals such as amphotericin B, fluconazole, and caspofungin. In addition, some studies have even reported multi-echinocandin or multi-azole resistant strains of C. parapsilosis. C. parapsilosis has several virulence factors that contribute to its capacity for host invasion and among these factors extracellular lipases have a major role in pathogenesis. In this review we have collected all the recent relevant studies that confirm the involvement of secreted lipases in C. parapsilosis pathogenesis, using both in vitro and in vivo models of infection. Of particular note, an available lipase deficient C. parapsilosis strain has been utilized to demonstrate that the lack of secreted lipases decreased virulence, reduced tissue damage, and was less able to survive within phagocytes or mice compared to the wild type. Since fungal secreted lipases have different characteristics than lipolytic enzymes present in humans, C. parapsilosis extracellular lipases may be potential targets for the development of novel antifungal drugs.

Analysis of oral yeast microflora in patients with oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most common form of oral cancer, in this study, the association between OSCC and oral yeast carriage was investigated. FINDINGS: 20 patients having OSCC as well as 40 healthy controls were tested for the presence of yeasts in the oral cavity. Fungal burdens were examined by colony forming unit determinations, while the different yeast genera in patient samples were identified by matrix-associated laser desorption/ionization-time of flight-mass spectrometry. We found that the level of oral yeast carriage was significantly higher in patients with OSCC that was accompanied by a higher diversity of yeasts in the oral cavity of these patients. We also examined the extracellular enzyme production of isolated Candida spp.; however, we found that there was no association between the lipase/protease producing capacity of Candida strains and the higher colonisation rate of neoplastic epithelium. CONCLUSIONS: In conclusion, our results corroborate the findings of previous studies regarding the association between oral yeast carriage and epithelial carcinoma.

Role of Protein Glycosylation in Candida parapsilosis Cell Wall Integrity and Host Interaction.

Candida parapsilosis is an important, emerging opportunistic fungal pathogen. Highly mannosylated fungal cell wall proteins are initial contact points with host immune systems. In Candida albicans, Och1 is a Golgi α1,6-mannosyltransferase that plays a key role in the elaboration of the N-linked mannan outer chain. Here, we disrupted C. parapsilosis OCH1 to gain insights into the contribution of N-linked mannosylation to cell fitness and to interactions with immune cells. Loss of Och1 in C. parapsilosis resulted in cellular aggregation, failure of morphogenesis, enhanced susceptibility to cell wall perturbing agents and defects in wall composition. We removed the cell wall O-linked mannans by ß-elimination, and assessed the relevance of mannans during interaction with human monocytes. Results indicated that O-linked mannans are important for IL-1ß stimulation in a dectin-1 and TLR4-dependent pathway; whereas both, N- and O-linked mannans are equally important ligands for TNFα and IL-6 stimulation, but neither is involved in IL-10 production. Furthermore, mice infected with C. parapsilosis och1Δ null mutant cells had significantly lower fungal burdens compared to wild-type (WT)-challenged counterparts. Therefore, our data are the first to demonstrate that C. parapsilosis N- and O-linked mannans have different roles in host interactions than those reported for C. albicans.

Candida parapsilosis produces prostaglandins from exogenous arachidonic acid and OLE2 is not required for their synthesis.

Prostaglandins are C20 fatty acid metabolites with diverse biological functions. In mammalian cells, prostaglandins are produced from arachidonic acid (AA) via cyclooxygenases (COX1 and COX2). Although fungi do not possess cyclooxygenase homologues, several pathogenic species are able to produce prostaglandins from host-derived arachidonic acid. In this study, we characterized the prostaglandin profile of the emerging human pathogen Candida parapsilosis with HPLC-MS and compared it to that of C. albicans. We found that both species synthesized prostaglandins (mainly PGD2 and PGE2) from exogenous AA. Furthermore, as OLE2 has been associated with prostaglandin synthesis in C. albicans, we generated homozygous OLE2 deletion mutants in C. parapsilosis and examined their PGE2 production. However, the PGE2 production of the OLE2 KO strain was similar to that of wild type (WT), indicating that OLE2 is not required for prostaglandin synthesis in C. parapsilosis. Interestingly, analyses of the fatty acid composition of WT and OLE2 KO cells by gas chromatography (GC) highlighted the accumulation of palmitoleic and oleic acid in the OLE2 deletion mutant. The OLE2 KO cells were killed more efficiently by human monocytes-derived macrophages (MDMs) as well as induced higher interleukin-10 (IL-10) secretion, indicating that OLE2 affects the virulence of C. parapsilosis. Taken together, these results contribute to the better understanding of fatty acid biosynthesis pathways in C. parapsilosis.

Kinetic studies of Candida parapsilosis phagocytosis by macrophages and detection of intracellular survival mechanisms.

Even though the number of Candida infections due to non-albicans species like C. parapsilosis has been increasing, little is known about their pathomechanisms. Certain aspects of C. parapsilosis and host interactions have already been investigated; however we lack information about the innate cellular responses toward this species. The aim of our project was to dissect and compare the phagocytosis of C. parapsilosis to C. albicans and to another Candida species C. glabrata by murine and human macrophages by live cell video microscopy. We broke down the phagocytic process into three stages: macrophage migration, engulfment of fungal cells and host cell killing after the uptake. Our results showed increased macrophage migration toward C. parapsilosis and we observed differences during the engulfment processes when comparing the three species. The engulfment time of C. parapsilosis was comparable to that of C. albicans regardless of the pseudohypha length and spatial orientation relative to phagocytes, while the rate of host cell killing and the overall uptake regarding C. parapsilosis showed similarities mainly with C. glabrata. Furthermore, we observed difference between human and murine phagocytes in the uptake of C. parapsilosis. UV-treatment of fungal cells had varied effects on phagocytosis dependent upon which Candida strain was used. Besides statistical analysis, live cell imaging videos showed that this species similarly to the other two also has the ability to survive in host cells via the following mechanisms: yeast replication, and pseudohypha growth inside of phagocytes, exocytosis of fungal cells and also abortion of host cell mitosis following the uptake. According to our knowledge this is the first study that provides a thorough examination of C. parapsilosis phagocytosis and reports intracellular survival mechanisms associated with this species.

Secreted Candida parapsilosis lipase modulates the immune response of primary human macrophages.

Candida parapsilosis is an important opportunistic pathogen with increasing prevalence. Extracellular lipases have been shown to play an important role in the virulence of pathogenic Candida species. However, studying the role of secreted lipase in C. albicans is challenging due to the lack of a mutant strain deficient in all 10 lipase genes. In contrast, we have previously constructed a lipase mutant C. parapsilosis strain lacking both CpLIP1 and CpLIP2, and shown that it has significantly decreased virulence in various infection models, and is killed more efficiently by mouse macrophages. In the present study, we compared the response of human peripheral blood monocyte-derived macrophages to a wild type (wt) as well as a lipase-deficient (lip(-/-)) C. parapsilosis strain that has been previously established in our lab. Although macrophages phagocytosed both strains with similar efficiency, lipase mutants were killed more efficiently according to fluorescent microscopic analysis. The more efficient killing of lip(-/-) cells was confirmed by CFU-determinations. Phagocytosis of wt and lip(-/-)C. parapsilosis was also examined by flow cytometry, revealing that both strains were internelized to the similar extent by macrophages. Additionally, quantitative imaging analysis revealed that the rate of phagolysosome fusion was higher in case of lip(-/-)C. parapsilosis. Interestingly, macrophages stimulated with lip(-/-)C. parapsilosis showed at least 1.5-fold higher expression of TNFα, IL-1ß, IL-6, IL-8, and PTGS-2 after 12 h compared with those infected with wt C. parapsilosis, as determined by qRT-PCR. Furthermore, the lip(-/-)C. parapsilosis strain induced significantly higher TNFα, IL-1ß, IL-6, and IL-10 protein production in macrophages after 24 h compared with the wt strain. These findings confirm the role of fungal lipases as important virulence factors during C. parapsilosis infection.

Transcriptome profile of the murine macrophage cell response to Candida parapsilosis.

Candida parapsilosis is a human fungal pathogen with increasing global significance. Understanding how macrophages respond to C. parapsilosis at the molecular level will facilitate the development of novel therapeutic paradigms. The complex response of murine macrophages to infection with C. parapsilosis was investigated at the level of gene expression using an Agilent mouse microarray. We identified 155 and 511 differentially regulated genes at 3 and 8h post-infection, respectively. Most of the upregulated genes encoded molecules involved in immune response and inflammation, transcription, signaling, apoptosis, cell cycle, electron transport and cell adhesion. Typical of the classically activated macrophages, there was significant upregulation of genes coordinating the production of inflammatory cytokines such as TNF, IL-1 and IL-15. Further, we used both primary murine macrophages and macrophages differentiated from human peripheral mononuclear cells to confirm the upregulation of the TNF-receptor family member TNFRSF9 that is associated with Th1 T-helper cell responses. Additionally, the microarray data indicate significant differences between the response to C. parapsilosis infection and that of C. albicans.

The role of pancreatic ductal secretion in protection against acute pancreatitis in mice*.

OBJECTIVES: A common potentially fatal disease of the pancreas is acute pancreatitis, for which there is no treatment. Most studies of this disorder focus on the damage to acinar cells since they are assumed to be the primary target of multiple stressors affecting the pancreas. However, increasing evidence suggests that the ducts may also have a crucial role in induction of the disease. To test this hypothesis, we sought to determine the specific role of the duct in the induction of acute pancreatitis using well-established disease models and mice with deletion of the Na/H exchanger regulatory factor-1 that have selectively impaired ductal function. DESIGN: Randomized animal study. SETTING: Animal research laboratory. SUBJECTS: Wild-type and Na/H exchanger regulatory factor-1 knockout mice. INTERVENTIONS: Acute necrotizing pancreatitis was induced by i.p. administration of cerulein or by intraductal administration of sodium taurocholate. The pancreatic expression of Na/H exchanger regulatory factor-1 and cystic fibrosis transmembrane conductance regulator (a key player in the control of ductal secretion) was analyzed by immunohistochemistry. In vivo pancreatic ductal secretion was studied in anesthetized mice. Functions of pancreatic acinar and ductal cells as well as inflammatory cells were analyzed in vitro. MEASUREMENTS AND MAIN RESULTS: Deletion of Na/H exchanger regulatory factor-1 resulted in gross mislocalization of cystic fibrosis transmembrane conductance regulator, causing marked reduction in pancreatic ductal fluid and bicarbonate secretion. Importantly, deletion of Na/H exchanger regulatory factor-1 had no deleterious effect on functions of acinar and inflammatory cells. Deletion of Na/H exchanger regulatory factor-1, which specifically impaired ductal function, increased the severity of acute pancreatitis in the two mouse models tested. CONCLUSIONS: Our findings provide the first direct evidence for the crucial role of ductal secretion in protecting the pancreas from acute pancreatitis and strongly suggest that improved ductal function should be an important modality in prevention and treatment of the disease.

Characterization of virulence properties in the C. parapsilosis sensu lato species.

The C. parapsilosis sensu lato group involves three closely related species, C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis. Although their overall clinical importance is dramatically increasing, there are few studies regarding the virulence properties of the species of the psilosis complex. In this study, we tested 63 C. parapsilosis sensu stricto, 12 C. metapsilosis and 18 C. orthopsilosis isolates for the ability to produce extracellular proteases, secrete lipases and form pseudohyphae. Significant differences were noted between species, with the C. metapsilosis strains failing to secrete lipase or to produce pseudohyphae. Nine different clinical isolates each of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis were co-cultured with immortalized murine or primary human macrophages. C. parapsilosis sensu stricto isolates showed a significantly higher resistance to killing by primary human macrophages compared to C. orthopsilosis and C. metapsilosis isolates. In contrast, the killing of isolates by J774.2 mouse macrophages did not differ significantly between species. However, C. parapsilosis sensu stricto isolates induced the most damage to murine and human macrophages, and C. metapsilosis strains were the least toxic. Furthermore, strains that produced lipase or pseudohyphae were most resistant to macrophage-mediated killing and produced the most cellular damage. Finally, we used 9 isolates of each of the C. parapsilosis sensus lato species to examine their impact on the survival of Galleriamellonella larvae. The mortality rate of G. mellonella larvae infected with C. metapsilosis isolates was significantly lower than those infected with C. parapsilosis sensu stricto or C. orthopsilosis strains. Taken together, our findings demonstrate that C. metapsilosis is indeed the least virulent member of the psilosis group, and also highlight the importance of pseudohyphae and secreted lipases during fungal-host interactions.

Candida albicans and Candida parapsilosis induce different T-cell responses in human peripheral blood mononuclear cells.

Candida parapsilosis is the third most frequent cause of candidemia. Despite its clinical importance, little is known about the human immunological response to C. parapsilosis. In this study, we compared the cytokine responses evoked by Candida albicans and C. parapsilosis. C. parapsilosis-stimulated human peripheral blood mononuclear cells (PBMCs) produced similar quantities of tumor necrosis factor α and interleukin 6 and slightly lower amounts of interleukin 1ß, compared with C. albicans-stimulated cells. PBMCs stimulated with C. parapsilosis displayed a skewed T-helper cell response, producing more interleukin 10 and less interferon γ than cells stimulated with C. albicans. Notably, C. parapsilosis induced much less interleukin 17 and interleukin 22 production as compared to C. albicans. Inhibition of the 3 classical mitogen-activated protein kinases (p38 kinase, ERK, and JNK) revealed kinase-dependent differences in reductions in cytokine production by the 2 Candida species. Decreased cytokine production after inhibition of dectin 1 revealed that this receptor plays a major role in the recognition of both C. albicans and C. parapsilosis. These data improve understanding of the immune response triggered by C. parapsilosis, a first step for the future design of immunotherapeutic strategies for these infections.

A new beta-glucosidase gene from the zygomycete fungus Rhizomucor miehei.

In this study, a beta-glucosidase coding gene (bgl) of the zygomycete fungus Rhizomucor miehei has been cloned and characterized. The gene comprises a total of 2,826 bp including the coding sequence of a 717 amino acids length putative protein and 10 introns dispersed in the whole coding region. The putative N-and C-terminal catalytic domains (aa 68 to aa 274 and aa 358-601, respectively) were identified; the two domains are connected with a 84-amino-acids linker. The catalytic region showed an extensive sequence homology with other fungal beta-glucosidases classified as family 3 glycoside hydrolases. The isolated Rhizomucor gene was expressed in the related fungus Mucor circinelloides. Transformant Mucor strains maintained the introduced plasmid in an autoreplicative manner and showed significantly higher cellobiase activity than the recipient strain.

Changing views on the goal of reproductive genetic counselling in Hungary.

Over the last few decades, the goal of genetic counselling has been interpreted as giving value-neutral information about the genetic risk, the genetic disorder, and screening, diagnostic or treatment possibilities in order to promote the autonomous decision-making of counsellees. Recently, however, the theoretical possibility and the practical necessity of this non-directive approach have been questioned, and redefinition of the objective of genetic counselling is required. In our paper, we intend to contribute to this clarification process by critically examining the views of Hungarian genetic counsellors on the objective of genetic counselling, and by exploring the expectations of the counsellees. Our ethical analysis has revealed that counsellors supported divergent counselling goals, such as preventing diseases by giving direct recommendations, promoting the autonomous decision-making of clients by providing value-neutral information and facilitating careful deliberation by actively taking part in the resolution. The self-administered survey conducted among 170 counsellees has discovered that the majority of the respondents expected detailed information (98%), psychological support (68%), the counsellor's help in making decisions (68%), and the possibility of control over the resolution process (92%). The need to tailor help to individual clients has also been demonstrated, since a direct relationship was found between success in decision-making and whether unanswered questions remained or the calming effect of the consultation was felt. Of the methods proposed by the counsellors, the interpretive counselling approach promised fulfilment of the clients' wishes and respect for the accepted ethical norms; thus, teaching of this approach should be taken into consideration during the development of the post-graduate training curriculum for counsellors.

Interpretation in reproductive genetic counseling: a methodological framework.

In case of genetic risk, parents are often faced with reproductive decisions affecting their life essentially, so it is advisable to pursue careful deliberation. For this reason, the genetic counselor is expected to help the counselee make well-informed and well-considered decisions, which requires the understanding of the patient as an individual. To reach emphatic understanding, physicians can use the results of the Gadamerian theory of interpretation that contains the idea -- as it has been summarized by V. Arnason -- that four aspects of openness are necessary to fully understand the other, such as openness to oneself, to the other, to the subject matter and to tradition. In our paper, we are applying the four-openness model of interpretation to genetic consultation, and we argue that during counseling double interpretation takes place: the physician interprets the patient, and the patient interprets the physician. Double interpretation leads to the clarification of those factors which influence the patient's decision-making: the counselor's attitude and prejudices, the counselee's values and needs, the medical, social, and moral implications of the genetic disease, and the social expectations. By adopting the theory of interpretation, counselors can also advance the provision of emotional support patients need in hard situations.

[Ethical aspects of disclosing information on prenatal screening for Down's syndrome]. / A tájékoztatás legfontosabb etikai vonatkozásai a Down-szindróma praenatalis szurése során.

Giving detailed information on prenatal screening for Down's syndrome is considered as paramount since this medical procedure intends to enhance the patient's self-governance in reproductive issues. Not only the respect for autonomy, but also the increased maternal anxiety and the reproductive decisions following the positive test result demand from the genetic professional to offer the test through genetic counselling. The counsellor's awareness about the expectations of pregnant women and the clarification of her own attitude concerning the screening can contribute to the effectiveness of counselling. The content of information embraces the technical aspects of screening and its consequences, like the description of Down's syndrome, the method of screening, the way of risk assessment, the detection rate, the false positive and false negative test results, the diagnostic procedures, and the termination of pregnancy. Written information leaflets should be completed by personal communication as the combination of these two forms has proved to be the most useful. The process of consultation is influenced by the communication skill of the genetic professional and the information seeking activity of the patient, so doctors should be trained to communicate better and patients should be encouraged to get more information about the screening.