Impact of Candida albicans hyphal wall protein 1 (HWP1) genotype on biofilm production and fungal susceptibility to microglial cells

The hyphal wall protein 1 (HWP1) gene of Candida albicans encodes for a fungal cell wall protein, required for hyphal development and yeast adhesion to epithelial cells; yet, its role in pathogenesis remains largely unknown. In the present study, we analyzed two C. albicans laboratory strains, the DAY286 (HWP1/HWP1) and the null mutant FJS24 (hwp1/hwp1) and six clinical isolates [3 harbouring the homozygous HWP1 gene (HWP1/HWP1) and 3 the heterologous gene (HWP1/hwp1)]. Biofilm production, fungal HWP1 mRNA levels and ultrastructural morphology were investigated; also, the susceptibility of these strains to microglial cells was evaluated, in terms of fungal damage and immune cell-mediated secretory response. When comparing the two laboratory strains, biofilm was produced to a similar extent independently on the genetic background, while the susceptibility to microglial cell-mediated damage was higher in the hwp1/hwp1 mutant than in the HWP1/HWP1 counterpart. Also, transmission electron microscopy revealed differences between the two in terms of abundance in surface adhesin-like structures, fungal cell wall shape and intracellular granules. When comparing the clinical isolates grouped according to their HWP1 genotype, reduced biofilm production and increased susceptibility to microglial cell-mediated damage occurred in the HWP1/hwp1 isolates with respect to the HWP1/HWP1 counterparts; furthermore, upon exposure to microglial cells, the HWP1/HWP1 isolates, but not the HWP1/hwp1 counterpart, showed enhanced HWP1 mRNA levels. Finally, both laboratory and clinical isolates exhibited reduced ability to stimulate TNFα and nitric oxide production by microglial cells in the case of heterozygous or null mutant HWP1 genotype. Overall, these data indicate that C. albicans HWP1 genotype influences pathogen morphological structure as well as its interaction with microglial cells, while fungal biofilm production results unaffected, thus arguing on its role as virulence factor that directly affects host mediated defences

Candida metapsilosis as the least virulent member of the 'C. parapsilosis' complex

Results of recent molecular studies have provided evidence of three distinct species within the Candida parapsilosis complex, namely Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis. While there are initial data pertaining to the virulence of these Candida species with respect to reconstituted epidermal and oral epithelial tissues, there have been no studies, as of yet, on their interaction with immune cells. Employing an in vitro infection model using microglial cells, we investigated the pathogenetic potential of different isolates of each of these three species. We show that C. metapsilosis isolates are more susceptible to microglia-mediated antifungal activity, as compared with those of C. parapsilosis and C. orthopsilosis. Interestingly, C. metapsilosis isolates are also phagocytosed to a lower extent, but the yeast-containing phagosomes exhibit the highest degree of acidification in comparison with the phagosomes containing C. parapsilosis or C. orthopsilosis. Furthermore, when assessing microglia secretory response to infection, comparable high levels of MIP-1\u3b1 and little or no TNF-\u3b1 production are observed with all of these Candida species. Finally, unlike C. metapsilosis infected cells, microglial cells infected with C. parapsilosis and C. orthopsilosis release high and time-dependent levels of lactate dehydrogenase (LDH). Overall, these findings point to C. metapsilosis as the least virulent member of the \u2018C. parapsilosis\u2019 complex

Growth of Epstein-Barr Virus on monocytes and its effects on immune functions.

EBV is the etiologic agent of infectious mononucleosis. It is also associated with different malignancies and other diseases including emophagocytic syndrome, a severe disorder in which erithrocytes are phagocytated by macrophages and B cells. Moreover, EBV has been postulated to be involved in the pathogenesis of multiple sclerosis. We investigated the ability of EBV to infect a monocytic human cell line (THP-1) in vitro and the effects of virus growth on the monocytic immune functions. The EBV chronically infected B cell line B95.8, stimulated with phorbol and butyrrate, was used as a source of virus. The growth of EBV on THP-1 virus was documented by IFA for viral antigens and quantification of viral DNA by RealTime PCR TaqMan. EBV infected THP-1 disclosed augmented phagocytosis of Candida albicans as assessed by a phagocytosis assay performed with a double fluorescence staining which allows to distinguish internalized fungi from those not ingested. Moreover, EBV infected THP-1 also displayed an increased ability to kill cells of a human oligodendrocytes line (MO3.1). Clones of chronically infected THP-1 cells have been obtained which are presently being characterized.Our results, showing alteration of monocytic immune functions induced by EBVinfection, support a pathogenetic role of this virus in emophagocytic syndrome and in multiple sclerosis

Beretti F., Cermelli C., Cenacchi V., Orsi C., Blasi E., Portolani M.

Microglia activation occurs during brain injury, ischemia and several neurological disorders. Moreover, microglial cells are highly dynamic structures also during the “resting” state in vivo, releasing neurotrophic factors and/or pro- and anti-inflammatory cytokines. Recently, we obtained a transmissible cytotoxic activity (TCA) from the cerebrospinal fluid of a patient with brain ischemia. We demonstrated that this TCA is associated with one or 2 protein(s), that supposedly underwent misfolding, and causes apoptotic cytotoxicity in a variety of cell lines. In this work, we studied microglia response to this TCA. The murine brain macrophage cell line RR4 was stimulated with TCA. Its response was evaluated as phagocytosis and antifungal activity against C. Albicans, and as secretion pattern, measuring MIP-1and TNF- by commercial sandwich ELISAs, nitric oxide (NO) by Griess reaction and phosphorylation of p38 by Fast Activated Cell-based ELISA.Microglia stimulated with TCA showed an increase in phagocytosis of C. albicans. On the contrary, the macrophage capability to kill the ingested fungi was diminished. The analysis of soluble factors secreted by microglial cells in response to TCA demonstrated an increase in MIP-1TNF- NO and an activation of p38 MAP kinase. These results suggest an initial microglia activation induced by TCA leading to an increase in candida ingestion not followed by killing of the fungus. Activation of p38 MAP kinase could suggest the induction of a signaling cascade leading to TCA production which in turn could cause failure in the antifungal activity. Moreover, MIP-1a, NO and TNF-a secretion by microglia cells and induction of apoptosis could provide an in vitro model for the cell events associated with brain ischemia

Gene expression profiling on monocytes displaying Herpes Simplex virus 1-induced dysregulation of antifungal defences.

BACKGROUND: Epstein Barr Virus (EBV) is responsible of many oncologic and non-oncologic diseases, including hemophagocytic syndrome in which red blood cells are phagocytosed by macrophages and B cells. EBV is reported to infect mainly B lymphocytes and epithelial cells; virus can be detected also in rare monocytes/macrophages, but its efficient replication in these cells has not been demonstrated.MATERIALS AND METHODS: EBV was obtained from P3RH1 cells stimulated with phorbol and butirate. A human monocytic cell line (THP-1) and an EBV negative B cell line (BJA-B) were infected with P3HR1- EBV strain: virus growth was monitored by IFA with a monoclonal antibody and by Real-Time PCR TaqMan. The effects of virus replication on phagocytosis of Candida albicans were evaluated by means of a double stain immunofluorescence assay. Intracellular killing of phagocytosed candida cells was measured by the colony forming unit inhibition assay.RESULTS AND CONCLUSION EBV can productively infect THP-1 monocyte cell line. EBV infection results in a significant increase in macrophage phagocytic activity versus Candida albicans as well as alteration of intracellular killing. Also BJA-B cells were stimulated in their phagocytic activity by EBV infectio

Clinical performance of a commercial real-time PCR assay for Aspergillus DNA detection in serum samples from high-risk patients: comparison with a galactomannan enzyme immunoassay

We investigated the clinical performance of a polymerase chain reaction (PCR)-based commercial platform, the Myconostica MycAssay™ Aspergillus (MAP), for fungal DNA detection in the serum of patients at risk of invasive aspergillosis (IA). Sixty-four hospitalized patients were prospectively enrolled and a total of 71 different episodes were investigated (30 episodes were clinically/microbiologically classified as IA and 41 as control episodes). When MAP was compared to the galactomannan (GM) assay, no significant differences were found in terms of sensitivity (46.7 % vs. 50.0 %), specificity (97.6 % vs. 95.1 %), positive predictive value (PPV) (93.3 % vs. 88.2 %), and negative predictive value (NPV) (71.4 % vs. 72.2 %). The corresponding areas under the curve (AUC) of the receiver operating characteristic (ROC) curves were also superimposable. Overall, because of the good agreement between the two assays and considering the high specificity and PPV of the MAP, we suggest the use of this PCR-based platform as a second-level examination for the evaluation of clinically undefined cases where culture or GM have provided positive results

HSV-1 induces dysregulation of monocyte anticandida functions

Clinical cases of double infections by fungi and viruses are increasing, especially in immunocompromised hosts. To date, the biomolecular events characterizing the outcome of polymicrobic diseases remain poorly investigated: little is known on the mutual interactions occurring between pathogens and on their concomitant, either synergistic or antagonistic, effects. In order to investigate the interplay occurring between pathogens in the course of double infections, we set up an in vitro model in which the monocytic cell line THP-1, was infected with HSV-1 and then exposed to Candida albicans. The effects of HSV-1 infection on macrophages were measured as capability to alter macrophage-mediated effector functions, namely phagocytosis and killing of Candida, and as gene and protein expression by FACS and RNA microarrays. Phagocytosis of Candida by THP-1 cells was significantly increased when macrophages were infected with HSV-1. Conversely, antifungal activity was impaired in HSV-1 infected macrophages at 6 hours after infection with Candida. FACS analysis of protein expression revealed a significant downregulation of TLR2 and TLR4, important molecules involved in fungi recognition and increase in the number of apoptotic cells. Gene expression profile disclosed a huge decrease in gene presence in HSV-1 infected macrophage (40% of gene presence in uninfected cells vs 20% in infected cells). The analysis of gene clusters showed a downregulation of genes involved in opsonized phagocytosis and intracellular killing, of many adhesion molecules and of TLR2; on the contrary, several lectin receptor genes (involved in Candida adhesion and phagocytosis) and apoptosis genes were significantly up-regulated

Candida albicans biofilm can retain and release Human Herpes simplex virus type 1 in vitro

Microorganisms universally attach to surfaces and produce extracellular polymeric saccharides (EPS), resulting in the formation of a biofilm. Biofilms can pose a serious problem for public health because of the increased resistance of biofilm-associated organisms to antimicrobial agents and the potential for these organisms to cause infections in patients with indwelling medical devices. Moreover, involvement of enteric viruses with a variety of biofilms has been reported, although very little is known about this phenomenon. The presence of some pathogenic viruses in water biofilms underlines the ability of viruses to attach and cling to biofilms retaining their infectivity. No information is available so far on interactions between pathogenic viruses and Candida albicans biofilm. This biofilm is responsible for severe device-related disseminated infections causing invasive candidemias with a very high rate of mortality. The aim of this in vitro study was to ascertain whether Herpes Simplex Virus type 1 (HSV-1) can be encompassed in Candida biofilm produced in cell culture plates and/or on silicone and PVC catheters. HSV-1 was added to mature biofilms and the amount of infectious virus embedded in biofilm matrix detached by washing and energetic scratching was titrated on VERO cells 24-48 h later. Experiments with planktonic Candida were carried out in parallel, as well as in the absence of Candida. According to our results, free virus particles of HSV-1, as well as HSV-1 infected cells, remain embedded in Candida biofilm on tissue cell culture plates as well as on both types of catheter with a significantly higher load than in the presence of planktonic Candida or in the negative controls. These results provide the first evidence that infectious viruses, after being entrapped in Candida biofilms, can retain their infectivity and be released posing a health risk for patients with implanted medical devices. Interactions between HSV-1 embedded in Candida biofilm and disinfectants as well as neutralizing antibodies and drugs are discussed

Effects of different mouthwashes on Candida albicans adhesion, susceptibility to phagocytic cells and capacity to elicit pro-inflammatory cytokine response

Introduction. Oral candidiasis is a frequent opportunistic fungal infection, occurring especially in susceptible individuals. This pathology, mainly associated with Candida albicans species, may be prevented by a good oral hygiene, including the daily use of toothbrush and mouthwashes (MoWs). Among several virulence factors, C. albicans has the ability to adhere to epithelial surfaces, to avoid phagocytosis and/or intracellular killing and to elicit proinflammatory cytokines production. We have previously demonstrated that both C. albicans hyphal development and biofilm formation/persistence are affected by MoWs, provided that they contain chlorhexidine digluconate. Therefore, in this study we aim to expand our knowledge on MoWs effects by investigating the behaviour of MoWs-treated C. albicans, in terms of adhesion to both abiotic and biotic surfaces, susceptibility to phagocytosis and capacity to elicit pro-inflammatory immune responses. Materials and methods. C. albicans SC5314 and 6 commercial MoWs have been employed: 4 with and 2 without chlorhexidine digluconate (CHX), a component known to have antibacterial and antifungal activity. Adhesion was assessed by a bioluminescent strain of C. albicans SC5314; MoWs-treated and PBS-treated fungal cells were incubated in 96-well plates containing or not a monolayer of TR-146 oral epithelial cell line; after 60 min, plates were washed and the residual bioluminescent signal recorded. Susceptibility to phagocytosis was assessed by exposing MoWs-treated and PBS-treated C. albicans to phagocytic cell line BV2 (effector:target=1:2). Following 24 hours incubation of TR-146 cells with MoWs-treated and PBS-treated C. albicans, cytokine levels in supernatants were measured. Results. Adhesion of MoWs-treated C. albicans to abiotic surfaces was significantly lower than PBS-treated Candida. Adhesion of MoWs-treated C. albicans to TR-146 cells was significantly lower than PBS-treated Candida, in all but MoW 4. No differences could be highlighted in terms of susceptibility to phagocytosis (percent phagocytic cells and phagocytosis index) between MoWs-treated and PBS-treated Candida. On the contrary, significantly higher acidic phagolysosomes percentages were recorded from Candida treated with 4 out of 6 MoWs, with respect to PBS-treated fungi. Finally, Candida pretreatment with 4 out of 6 MoWs and 5 out of 6 MoWs impaired the production of IL-1alpha and IL-1beta respectively. Discussion and conclusions. C. albicans adhesion, susceptibility to phagocytosis and capacity to elicit pro-inflammatory cytokine response are affected by MoWs, especially those containing CHX. Thus, special attention should be used when choosing MoWs whether prevention and/or treatment of Candida-associated oral pathologies was intended

POLIMORFISMO DEL GENE HWP1 IN CANDIDA ALBICANS: POSSIBILE RUOLO NELLA PRODUZIONE DI BIOFILM E NELL’INTERAZIONE CON IL MACROFAGO

Introduzione: Candida albicans (Ca) è frequentemente responsabile di candidosi profonde, soprattutto nei portatori di cateteri od altri dispositivi medici, sulla cui superficie spesso si riscontra la comparsa di biofilm (1). La formazione di biofilm, a cui si associa la maggior resistenza di Ca ai farmaci antifungini così come l’aumentata virulenza (2), procede secondo una complessa serie di eventi, finemente controllati che coinvolgono numerosi geni tra cui HWP1 (Hyphal Wall Protein 1), codificante per una proteina essenziale per la crescita ifale. Il presente studio intende valutare il ruolo del biofilm ed in particolare del genopito HWP1 nell’interazione in vitro tra Ca e macrofago. Materiali e metodi: isolati clinici di Ca recentemente identificati come eterozigoti ed omozigoti (3 per ciascun assetto genetico) per il gene HWP1 (3), sono stati studiati in vitro per a) capacità di produrre biofilm, in presenza o assenza di siero fetale bovino e/o di CO2, e b) suscettibilità alle cellule microgliali BV2 (4); le indagini hanno comportato la quantificazione della produzione di biofilm mediante colorazione con cristal violetto o XTT, e valutazione della reattività del macrofago mediante dosaggio nei surnatanti di citochine, lattato deidrogenasi, ecc. Risultati: i ceppi saggiati hanno dimostrato a) aumentata produzione di biofilm in presenza di siero e di CO2 in tutti i ceppi, ma in modo più evidente negli isolati omozigoti; b) maggiore resistenza verso la microglia, nel caso dei ceppi omozigoti per HWP1 e c) produzione più elevata di TNFα in macrofagi esposti ai ceppi omozigoti. Conclusioni: questi dati indicano che la produzione di biofilm conferisce a Ca una maggiore resistenza alla reattività della cellula microgliale; la differenza osservata tra ceppi genotipicamente diversi per HWP1 suggerisce un ruolo importante di questo gene, oltre che nella crescita delle ife, anche nella interazione con le cellule dell’immunità innata. (1) Douglas, L. J. (2003). Trends Microbiol 11: 30–36 (2) Katragkou A et al. (2010). JID, 15:1941-1949 (3) Borghi et al. (2011). 21st ECCMID, Milano (4) Blasi E et al. (1991). J Neuroimmunol 32: 49-5