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. 2001 Dec;104(4):455–461. doi: 10.1046/j.1365-2567.2001.01328.x

Human immunodeficiency virus type 1 Tat binds to Candida albicans, inducing hyphae but augmenting phagocytosis in vitro

Andreas Gruber *, Claudia P Lell *, Cornelia Speth *, Heribert Stoiber *, Cornelia Lass-Flörl *, Anja Sonneborn †, Joachim F Ernst †, Manfred P Dierich *, Reinhard Würzner *
PMCID: PMC1783331  PMID: 11899432

Abstract

Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein, binds through its RGD-motif to human integrin receptors. Candida albicans, the commonest cause of mucosal candidiasis in subjects infected with HIV-1, also possesses RGD-binding capacity. The present study reveals that Tat binds to C. albicans but not to C. tropicalis. Tat binding was markedly reduced by laminin and to a lesser extent by a complement C3 peptide containing the RGD motif, but not by a control peptide. The outgrowth of C. albicans was accelerated following binding of Tat, but phagocytosis of opsonized C. albicans was also increased after Tat binding. Thus, Tat binding promotes fungal virulence by inducing hyphae but may also reduce it by augmenting phagocytosis. The net effect of Tat in vivo is difficult to judge but in view of the many disease-promoting effects of Tat we propose that accelerating the formation of hyphae dominates over the augmentation of phagocytosis.

Introduction

Human immunodeficiency virus type 1 (HIV-1) encodes six regulatory or accessory proteins (Tat, Rev, Vif, Vpr, Vpu and Nef) that are not found in other classes of retroviruses.1 The HIV-1 transactivating protein, Tat, which is synthesized at both early and late stages of the viral replication cycle, is essential for viral replication.2,3 The interaction of Tat with the Tat activation region stem–loop RNA structure (located at the 5′ terminus of viral mRNAs) and with cellular factors is required for transactivation.1 Despite its nuclear localization and function, Tat is secreted by infected cells.4 Tat contains an RGD sequence, highly conserved among HIV-1 isolates,5 which is generally believed to be implicated in cell adherence. Extracellular Tat has been shown to bind to α5β1, αvβ3 and αvβ5 integrins,6,7 modulating cell proliferation4,8–11 (including induction of apoptosis; see refs. 9 and 10), and to act as a chemoattractant for monocytes and dendritic cells.12,13

Opportunistic infections represent a common complication during HIV pathogenesis and do not only indicate, but also lead to, a progression of acquired immune deficiency syndrome (AIDS)14,15 and to a reduction in the survival time of HIV-infected subjects.16,17 Oral candidiasis is one of the most common AIDS-defining fungal opportunistic infections in HIV-1 positive subjects.18 Recently, we discovered the binding of HIV-1 glycoprotein (gp)160/gp41 to Candida albicans19 and its enhancing effect on candidal virulence.20 It was proposed that both viral (HIV) and fungal (Candida) disease may be augmented by this direct interaction.20

Adhesion to host tissue represents an important fungal virulence factor as it resembles the critical first step in the pathogenesis of Candida infection (reviewed in ref. 21), and is probably mediated by fungal cell wall mannoproteins with structural, immunological and functional integrin-like features.22–24 In particular, these moieties are able to bind to multiple host proteins containing an RGD motif (reviewed in ref. 25).

The aim of the present study was to investigate a possible binding of HIV-1 Tat to C. albicans and to determine its impact on the fungus itself as well as on certain components of the host immune system.

Materials and Methods

Organisms and culture conditions

The following organisms were used: C. albicans CBS 5982 (Centraal Bureau voor Schimmelcultures, Baarn, the Netherlands); C. albicans SC5314;26 C. tropicalis ATCC 13803 (American Type Culture Collection, Manassas, VA); C. albicans isolate KH827/96 (a clinical isolate from a transplant patient at the University Hospital of Innsbruck, Innsbruck, Austria); and C. albicans isolate K8 (a clinical isolate from a female patient with recurrent vulvovaginal candidiasis at the University Hospital of Bratislava, Slovakia). The fungi were initially grown on Sabouraud dextrose agar plates (Oxoid, Basingstoke, UK) and then stored at 4°. The organisms were inoculated into RPMI-1640 (HyClone, Cramlington, UK) at an initial concentration of 1 × 106 cells/ml and used directly for yeast morphology, or incubated for 20 hr at 30° for yeast-hyphae transition of C. albicans.

Proteins and peptides

HIV-1IIIB Tat expressed in Escherichia coli was provided by Dr J. Raina (Medical Research Council AIDS-Directed Programme, Potters Bar, UK). HIV-1SF2 gp120, expressed in Chinese hamster ovary cells, was supplied by Dr K. Steimer, Chiron Corporation (AIDS Research and Reference Reagent Programme, Division of AIDS, NIAID, NIH, Bethesda, MD). Laminin was purchased from Sigma (St. Louis, MO). Synthetic peptides from human complement component C3 (huC3-RGD: MILEICTRYRGDQDA) and from guinea-pig C3 (pigC3-LGD: MILGICTRYLGDQDA) were obtained from genXpress (Maria Wörth, Austria).

Antibodies

Monoclonal mouse anti-Tat antibody ID9D5, recognizing an epitope outside the RGD-containing region, was provided by Dr D. Helland and Dr A. M. Szilvay (Medical Research Council AIDS-Directed Programme). Polyclonal anti-gp120 (DV-012, from sheep) was generated by Dr M. Phelan (AIDS Research and Reference Reagent Programme). Polyclonal rabbit anti-human C3d complement antibody was purchased from Dako (Glostrup, Denmark). Terminal complement complex (TCC) formation was measured by using the neoepitope-specific monoclonal mouse antibody WU 13–15.27 Detection of the these antibodies was performed by using fluorescein isothiocyanate (FITC)-conjugated antibodies (Dako).

Binding and inhibition studies

For binding studies, cells of the strain under investigation were incubated for 1 hr at 4° with HIV-1 Tat. The amount of bound Tat on cells with yeast morphology was determined by cell cytometry (FACScan; Becton-Dickinson, Heidelberg, Germany). Per sample, 5 × 103 events were recorded and analysed using the Lysis II software (Becton-Dickinson). In binding-inhibition assays, cells with yeast morphology were incubated with the respective protein or peptide prior to Tat incubation. In addition, both yeast and hyphae of Candida were assessed for their Tat-binding capacity by indirect immunofluorescence using an incident-light fluorescence microscope (Axioplan; Carl Zeiss, Oberkochen, Germany).

Assessment of morphology changes

The morphology and growth of C. albicans pretreated with (or without) HIV-1 proteins were investigated by measuring its elongation, as recently described.20 Briefly, 1 × 106 yeast cells/ml of RPMI-1640 were incubated with or without HIV-1 proteins for 1 hr at 4°. Following a washing step, cells were inoculated into microwell plates (Greiner, Kremsmünster, Austria) and incubated at 30°. The morphology of the organisms was determined microscopically at the time-points indicated; a micrometer was used for length measurement of 50 organisms per sample. Appropriate measures were taken to minimize observer bias: the observer was blinded to the culture conditions and the 50 organisms counted were selected at random from a representative section.

Preparation of monocytes and polymorphonuclear leucocytes (PMNL)

Human monocytes and PMNL were isolated using both standard methods and methods described previously.20 Briefly, buffy coats were diluted 1:2 in phosphate-buffered saline (PBS), layered over Ficoll–Paque (Pharmacia, Uppsala, Sweden), and centrifuged at 650 g for 30 min at 20°. Monocytes were separated from other peripheral blood mononuclear cells by adherence to plastic (Tissue culture plastic; Costar, Cambridge, UK). PMNL were freed from remaining erythrocytes by two 30-second cycles of hypotonic lysis with distilled water.

Phagocytosis

Phagocytosis of viable C. albicans yeast cells by monocytes and PMNL was assessed as described previously.20 Yeast cells were treated with or without HIV-1 Tat, opsonized with 10% (vol/vol) human serum (pooled from three healthy donors) for 30 min at 37° and finally labelled with 250 µm fluorescein 5-isothiocyanate (FITC isomer 1; Sigma) in PBS (pH 8·0) for 30 min at room temperature. Serum supplemented with 10 mm EDTA served as a control. FITC-labelled organisms were then incubated with monocytes and PMNL, respectively, at a ratio of 10:1, for 30 min at 37° under gentle agitation. The assay was stopped by adding ice-cold PBS containing 4% (vol/vol) formaldehyde and 0·02% (wt/vol) EDTA. Phagocytic activity was assessed using an incident-light fluorescence microscope. One-hundred PMNLs were assessed for determination of ingested fluorescent C. albicans. The observer was blinded to the culture conditions.

Statistics

Statistical significance was determined using the Student's t-test. A P-value of < 0·05 was considered significant.

Results

Binding of HIV-1 Tat to C. albicans and C. tropicalis

HIV-1 Tat bound to yeast cells of C. albicans CBS 5982 in a dose-dependent manner, as detected by the monoclonal anti-Tat antibody, ID9D5 (Fig. 1). Binding was first detected at 0·001 µm Tat, and was significant at 0·01 µm Tat (P < 0·01) when compared to yeast cells incubated with anti-Tat antibody in the absence of Tat. No binding of HIV-1 gp120 to C. albicans was detected using recombinant gp120 and the polyclonal anti-gp120 antibody DV-012 (Fig. 1), confirming previous results.19 Tat also bound to clinical strains of C. albicans, but not to the pathogenic yeast C. tropicalis (Fig. 2).

Figure 1.

Figure 1

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Binding of Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein, to Candida albicans. Yeast cells of C. albicans (CBS 5982) were incubated at the indicated concentrations with the HIV-1 proteins Tat or glycoprotein (gp)120. Bound protein was detected by using ID9D5, an anti-Tat specific monoclonal antibody, or DV-012, an anti-gp120 specific polyclonal antibody, and secondary fluorescein isothiocyanate (FITC)-conjugated antibody. The percentage of fluorescent C. albicans was determined by flow cytometry. Each point represents the mean and standard deviation of data obtained from three replicate experiments.

Figure 2.

Figure 2

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Binding of Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein, to different yeast strains. Tat binding (thick line) to yeast cells of the respective strains was detected as described in legend to Figure 1. As a control, yeast cells were incubated with buffer instead of Tat followed by addition of ID9D5, an anti-Tat specific monoclonal antibody, and then secondary fluorescein isothiocyanate (FITC)-conjugated antibody (thin line). Percentages shown represent the increase in mean fluorescence intensity after Tat treatment as compared to control treatment. Data shown are representative of experiments performed at least in duplicate.

Inhibition, by different ligands, of Tat binding to C. albicans

Binding of HIV-1 Tat (0·1 µm) was observed by indirect immunofluorescence both on yeast cells and on elongated forms, predominantly pseudohyphae, of C. albicans (CBS 5982; Fig. 3a). Laminin, a ligand known to bind to C. albicans,28,29 markedly blocked Tat binding to C. albicans when used at equimolar concentrations (≈ 50%; Fig. 3b). Direct immunofluorescence microscopy even suggested an almost abolished binding of Tat to C. albicans (Fig. 3a), which was indeed accomplished by a 10-fold molar excess of laminin over Tat (Fig. 3b). Furthermore, an RGD-containing peptide of human complement component C3 (huC3-RGD) also reduced, in a dose-dependent manner, the amount of Tat bound to C. albicans. Another peptide derived from guinea-pig C3 (pigC3-LGD), sharing 86·7% amino acid sequence homology with huC3-RGD but containing an LGD instead of an RGD motif, did not significantly alter the binding of Tat to C. albicans (Fig. 3b).

Figure 3.

Figure 3

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Binding of Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein, to Candida albicans, and inhibition of Tat binding. (a) Binding of 0·1 µm Tat to yeast cells (A) and (B) and outgrown forms, predominantly pseudohyphae (C) and (D), and inhibition of Tat binding by 0·1 µm laminin, respectively (E), (F), (G) and (H), was studied by indirect immunofluorescence, as described in the legend to Figure 1. (A), (C), (E), (G) fluorescence microscopy; (B), (D), (F), (H) light microscopy. (b) Quantitative determination (using flow cytometry) of the inhibition of Tat binding on C. albicans (CBS 5982) yeast cells by different ligands. Each value represents the mean and standard deviation of at least two experiments; significance is indicated (Student's t-test, *P < 0·05; **P < 0·01; ***P < 0·001).

Effect of HIV-1 proteins on the morphology and growth of C. albicans

The growth behaviour of C. albicans (CBS 5982), in the presence or absence of HIV proteins, was assessed by measuring its outgrowth. Cells with yeast morphology were used that germinated after 2 hr and developed pseudohyphae and hyphae with increasing length during the following 4 hr. HIV-1 Tat (0·1 µm) slightly enhanced germination and significantly amplified the elongation of C. albicans after 4 hr and 6 hr (both P < 0·02; Fig. 4). In contrast, outgrowth was not significantly altered by treatment of C. albicans with HIV-1 gp120 (Fig. 4), as shown previously.20

Figure 4.

Figure 4

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The effect of Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein, on Candida albicans elongation. C. albicans (CBS 5982) yeast cells, incubated with or without 0·1 µm Tat or 0·1 µm glycoprotein (gp)120, were measured at the time-points indicated. Each point represents the mean and standard deviation of four experiments.

Phagocytosis, by monocytes and by PMNL, of C. albicans pretreated with HIV-1 Tat

Following an earlier hypothesis that RGD-binding molecules on C. albicans may interfere with its engulfment by phagocytic cells,30 the effect of Tat binding to C. albicans was studied further. Approximately 56% of serum-opsonized FITC-labelled viable C. albicans (CBS 5982) were phagocytosed by monocytes (Fig. 5). Treatment of C. albicans with EDTA serum significantly reduced phagocytosis. Binding of 0·1 µm Tat on C. albicans prior to serum opsonization increased monocyte phagocytic activity by ≈28% (P < 0·01). No significant effect was observed at a concentration of 0·01 µm Tat.

Figure 5.

Figure 5

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Phagocytosis by monocytes and by polymorphonuclear leucocytes (PMNL) of opsonized Candida albicans pretreated with Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein. Prior to phagocytosis, C. albicans (CBS 5982) yeast cells were preincubated with or without different concentrations of Tat. Thereafter, they were incubated with normal human; serum supplemented with 10 mm EDTA served as a control. Each value represents the mean and standard deviation of at least four experiments; significance is indicated (Student's t-test, *P < 0·05; **P < 0·01; ***P < 0·001).

In addition, phagocytic activity of PMNL was not markedly altered after treatment of C. albicans with 0·1 µm Tat prior to serum opsonization (≈ 9% increase, P < 0·01; Fig. 5).

Furthermore, it was investigated whether the effect of Tat on phagocytosis was caused by a possible interference of Tat with opsonization of C. albicans. Therefore, deposition of human C3d and formation of TCC on yeast cells was determined (Table 1). No significant differences concerning deposition of C3 fragments or TCC formation were seen after treatment of C. albicans with HIV-1 Tat when compared to untreated, but opsonized, yeast cells (Table 1).

Table 1.

Effect of Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein, on complement deposition on the surface of Candida albicans

C3d TCC
– 310 ± 13 78 ± 5
10 mm EDTA 47 ± 4*** 32 ± 9**
0·01 µm Tat 282 ± 33 86 ± 10
0·1 µm Tat 307 ± 29 76 ± 8
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Deposition of C3d, as well as formation of terminal complement complex (TCC) on the surface of C. albicans (CBS 5982), was determined by flow cytometry. Treatment of yeast cells was performed as described in legend to Fig. 5. Each value represents the mean ± standard deviation of the mean fluorescence intensity; data were obtained from at least two replicate experiments.

Significance (Student's t-test) is indicated:

**

P < 0·01;

***

P < 0·001.

Discussion

HIV-1 Tat was shown to bind to C. albicans. Binding was not restricted to oval yeast cells, pseudohyphal or hyphal morphology. Furthermore, Tat bound to both the laboratory strain and clinical isolates of C. albicans, but not to C. tropicalis. Whether the latter observation is related to distinct mechanisms of epithelial adhesion that have been previously elaborated for C. albicans and C. tropicalis31,32 warrants further study. Tat binding to C. albicans yeast cells was markedly inhibited by laminin and only slightly reduced by an RGD-containing C3 peptide. However, binding of Tat to C. albicans yeast cells was not affected by a homologous peptide where the RGD tripeptide was replaced by LGD. Thus, the RGD sequence in the C-terminal portion of Tat may partially, but not exclusively, participate in the binding of the viral protein to C. albicans. Sequence alignments, performed using the program clustal w,33 on protein sequences extracted from the GenBank database34 revealed that laminin does not only share an RGD motif with Tat but also a basic region, not present in fibrin or the RGD peptides used, which may be involved in binding. It is clearly possible that different Tat domains are involved in outgrowth and phagocytosis, and further studies, e.g. using synthetic Tat peptides, may allow the mapping of these functions to the corresponding domains. Several integrin-like and non-integrin surface molecules of C. albicans have been shown to bind laminin (reviewed in ref. 25). One or several of these surface molecules may therefore accomplish Tat binding to C. albicans. Pathogens causing HIV-related illnesses such as Pneumocystis carinii pneumonia, Mycobacterium avium disease or pulmonary Aspergillus infection, all possess laminin binding molecules on their surface35–37 and thus may also interact with HIV-1 Tat. This hypothesis is currently being investigated. Interestingly, Tat not only bound to C. albicans but even promoted its elongation, a process that will probably add to the virulence of this fungal pathogen.38–40

In order to investigate the molecular basis of this transition in response to Tat, the expression of CPH1 and EFG1 genes, known to be involved in inducing a yeast-to-hyphae transition in response to environmental changes,41,42 was evaluated. These represent important candidate genes, although filamentation of Candida is still possible in efg1/cph1 double mutants.43 After exposure of the Tat protein to C. albicans cells during hyphal induction, a markedly lower level of CPH1 mRNA was detected compared to the untreated fungal cells, whereas the expression of the EFG1 gene remained unchanged, i.e. both genes were apparently not activated (data not shown). The CPH1 pathway is activated by a signalling pathway involving mitogen-activated kinases mediating phosphorylation of intracellular proteins. This, and the fact that the HIV-1 Tat protein increases serine phosphorylation44 in human cells, led to an investigation of phosphorylation of intracellular proteins upon Tat binding. Interestingly, Tat induced a delayed but enhanced phosphorylation of intracellular serine proteins which may, at least in part, be responsible for the observed induction of fungal elongation (data not shown). Further molecular studies are in progress.

In contrast to promoting fungal virulence by augmenting outgrowth, Tat binding may also result in an enhanced clearance of C. albicans by the immune system, as C. albicans, incubated with Tat prior to serum opsonization, was phagocytosed by monocytes to a higher extent than yeast cells opsonized with serum only. This effect was less pronounced when PMNL were analysed. However, Tat binding enhanced neither the subsequent deposition of C3 fragments nor the formation of the TCC on the surface of C. albicans. This disproves the hypothesis that an increased opsonization, caused by prior binding of Tat, is responsible for the enhanced engulfment of yeast cells by the two different phagocytic cell populations. In contrast, the enhanced phagocytosis may, at least partially, originate from the chemotactic property of Tat.12,13,45

There is mounting evidence that lentiviruses, including HIV, can be transmitted via the oral mucosa46 and that cells within the mucosa are productively infected with HIV.47 Local inflammation in the oral cavity may further promote viral replication and thus increase the exposure of Candida to viral products, as discussed previously.20 It has been shown that the level of HIV-1 RNA, not the number of CD4 T cells, is predictive of oropharygeal Candida colonization.48 Furthermore, patients at all stages of HIV infection, compared to uninfected controls, can still mount substantial local immune responses to oral opportunistic infections, such as C. albicans.49 These findings challenge the view that weakened mucosal immunity alone is responsible for the prevalence of fungal infections in HIV-infected subjects.

A main question which has to be addressed is whether the concentrations used in this study can actually be reached in vivo. This appears to be feasible, although not systemically as the Tat concentrations in HIV-1-positive sera are regularly in the nanomolar range.10 However, it is conceivable that the in vivo Tat concentrations are much higher (comparable to the ones used in this study) in the vicinity of the oral mucosa, as discussed above.

The net effect of Tat in vivo is difficult to judge. Augmented chemotactic and phagocytic properties of Tat may be abolished by its ability to induce an increased fungal outgrowth (this study) and to markedly abrogate lymphocyte proliferation in response to Candida.50 Thus, soluble and Candida-bound Tat may be in part responsible for the rise of oral Candida in number51 and virulence52,53 early in HIV infection, although there are still normal numbers of CD4+ lymphocytes.54 Furthermore, the fungicidal activity of monocytes and neutrophils is impaired during HIV infection.55 Thus, increased uptake of C. albicans by phagocytic cells may not eliminate the former but rather, either directly56 or indirectly via immune activation,14,15,57 enhance the expression of HIV and thereby further undermine the immune system. In particular, gp120 reduces the antifungal capacity of monocytes,58 whereas gp160/gp41 promotes fungal virulence.20 Thus, the multiple interactions between HIV and Candida may add to the pathogenesis of HIV infection.59

In conclusion, HIV-1 Tat binds to C. albicans via laminin-binding moieties and may thereby affect fungal virulence by promoting elongation. Whether it also improves antifungal host defence in vivo, by enhancing the uptake of serum-opsonized Candida by monocytes and neutrophils, appears unlikely in view of the many HIV disease-promoting effects of Tat.

Acknowledgments

We thank Dr Xiaojie Zhu (Innsbruck, Austria) for her help; Dr J. Raina, Dr D. Helland, Dr A. M. Szilvay and the Medical Research Council AIDS-Directed Programme, Potters Bar, UK, for providing HIV-1 Tat and monoclonal antibody ID9D5; Dr K. Steimer (Chiron Corporation); Dr M. Phelan, and the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, MD, USA, for bestowing HIV-1 gp120 and polyclonal antibody DV-012; and Dr H. Bujdáková (Bratislava, Slovakia) for providing clinical isolates of C. albicans. We are grateful to the German Society of Immunology and the Dr Kurt-and-Irmgard-Meister foundation for kindly supporting A. Gruber. The project was funded by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (P13551-MOB to R.W.) and the Ludwig Boltzmann-Gesellschaft, Austria.

Abbreviations

AIDS

acquired immune deficiency syndrome

C. albicans

Candida albicans

gp

glycoprotein

HIV

human immunodeficiency virus

PMNL

polymorphonuclear leucocytes

TCC

terminal complement complex

References

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