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Penicillium marneffei is a dimorphic pathogenic fungus which causes human infection in Southeast Asia, particularly in Thailand (2, 14), Hong Kong (16), and southern China (11). Until relatively recently, the incidence of human infection with P. marneffei was very low (4, 8); however, with the arrival of the AIDS epidemic, the number of cases occurring in immunocompromised individuals has risen dramatically (2, 14). Infection is presumed to originate in the lungs after inhalation of the airborne conidia, which are sufficiently small to reach the alveoli. To date, very little information has been made available on the mechanisms underlying the pathogenesis of P. marneffei infections, and the factors influencing the initiation of infection in the lung have not been investigated.

Extracellular matrix proteins have been implicated in the attachment of a variety of pathogens to both host tissues and cells (5, 7, 10, 12, 13). Of particular interest in this area has been the identification of a laminin-binding protein on the surface of Aspergillus fumigatus conidia (1, 6, 15). The receptor appears to be a sialic acid-specific lectin (1), and the interaction between receptor and laminin is saturable and specific (1, 6). Laminin is an extracellular matrix glycoprotein which is present in basement membranes, and in the lung this glycoprotein can be exposed after tissue damage resulting either from inflammatory processes or from the lytic activity of bacterial toxins or drugs. Given that P. marneffei infections are hypothesized to arise in a manner similar to A. fumigatus infections (after inhalation of conidia), it is possible that P. marneffei conidia also utilize a laminin-binding mechanism.

P. marneffei ATCC 200051 was grown in the mycelial phase on Sabouraud dextrose agar slopes at 30°C. Conidia were obtained from 8-day-old cultures by flooding the agar slopes with 10 ml of sterile distilled water containing 0.05% (vol/vol) Tween 20 and by scraping the aerial mycelium. The resulting suspension was then filtered successively through three layers of glass wool and centrifuged (13,000 × g, 2 min). Afterwards, the pellet was resuspended in sterile phosphate-buffered saline (PBS) (0.01 M, pH 7.4) and washed twice more, and conidia were quantified. For the immunofluorescence assay, suspensions of mycelial scraping were not filtered through glass wool but were instead washed three times in sterile PBS.

Suspensions of mycelial scrapings containing in excess of 10⁷ conidia/ml were washed once in sterile PBS and then resuspended in 250 µl of sterile PBS containing laminin (derived from Engelbreth-Holm-Swarm mouse sarcoma [Sigma Chemical Co., Poole, Dorset, United Kingdom]) at a concentration of 500 µg/ml. After incubation for 3 h at 37°C, the suspensions were washed three times in PBS, resuspended in 250 µl of rabbit anti-laminin antibody (Dako Ltd., High Wycombe, United Kingdom) at an initial concentration of 1 mg/ml and diluted 1:10 in PBS plus 1% bovine serum albumin (BSA), and incubated for 1 h at 37°C. The suspensions were then washed and resuspended in fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin antibody (Jackson Immunochemicals, West Grove, Pa.) (at an initial concentration of 1 mg/ml and diluted 1:20) in PBS plus 1% BSA. Finally, the suspensions were washed again and examined by immunofluorescence microscopy. Negative controls consisted of suspensions incubated in the absence of laminin, anti-laminin antibody, and FITC-conjugated goat anti-rabbit immunoglobulin (all replaced with PBS).

Adherence assays were performed essentially as described by Coulot et al. (3). Briefly, laminin was immobilized on 96-well microtiter plates (Maxisorp; Nunc A/S, Kamstrup, Denmark) by incubating 100 µl of a range of different laminin concentrations (from 0.1 µg/ml to 500 µg/ml) at 4°C overnight in PBS in each well (3) (a standard laminin concentration of 100 µg/ml was used in all subsequent experiments). Plates were then washed and blocked, and conidia of P. marneffei (100 µl per well at 10⁶ conidia per ml) were added to each. Nonadherent cells were removed by washing, and the numbers of adherent conidia were assessed as described previously (3). Control wells were incubated in PBS but in the absence of laminin. Results, expressed as the number of adherent conidia in 10 fields, are presented as the means of triplicate counts performed three times (with standard deviations included). Statistical analysis was performed by using the Student t test.

The effect of preincubation (30 min at 37°C) with the following reagents on the adherence of conidia to immobilized laminin was then determined: soluble laminin, Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR) peptides, BSA (all at 1 mg/ml), glucose (200 mM), galactose (200 mM), mannose (200 mM), asialomucin (200 µg/ml), mucin (200 µg/ml), and N-acetylneuraminic acid (NANA) (200 mM) (all values shown are final concentrations; all reagents were from Sigma and all were made up in PBS). Conidia preincubated in PBS only were used as a positive control.

In some experiments, rabbit anti-laminin antibody (at an initial concentration of 1 mg/ml and at a final dilution of either 1:50 or 1:100 in PBS) was added to wells coated with immobilized laminin, together with P. marneffei conidia, and the adherence was quantified as described above.

The strong immunofluorescence labelling observed on the surface of P. marneffei conidia clearly demonstrated their interaction with laminin (Fig. 1a and b). Labelling of individual conidia tended to be uniform with no obvious spatial localization. Phialides, the bottle-shaped structures which give rise to conidia, were also strongly fluorescent (Fig. 1c and d). However, the hyphal elements distal to phialides were nonfluorescent, as were all hyphal elements (Fig. 1a, b, c, and d). Fluorescence was dependent on the previous interaction of the cells with laminin and its appropriate recognition, since no reactivity was evident when the cells were incubated in the absence of laminin (data not shown).

View larger version (72K): [in this window] [in a new window]   FIG. 1.   Immunofluorescence identification of the binding of laminin. (a and b) Phase-contrast and immunofluorescence microscopy of a conidial chain incubated with laminin, anti-laminin antibody, and FITC-labelled conjugate. Note that uppermost conidia is labelled less intensely than others in chain. Negatively staining hyphae are indicated with arrows. (c and d) Phase-contrast and immunofluorescence microscopy of phialides incubated as described above. Negatively staining distal hyphae are indicated with arrows. Bars, 10 µm.

Conidia of 8-day-old cultures were incubated with immobilized laminin at increasing concentrations ranging from 0.1 to 500 µg/ml, and conidial counts demonstrated that adherence increased progressively and then reached a plateau at a concentration of 100 µg/ml (data not shown). Approximately 10% of the 10⁵ conidia added to the wells remained attached to the plates after washing. Controls in which no laminin was present on the bottom of wells demonstrated little or no attachment (in subsequent data, these control values have been subtracted from experimental values and are thus not shown).

When conidia were preincubated with soluble laminin prior to addition to laminin-coated wells, inhibition of adherence was almost complete (Fig. 2) compared to the PBS control. In contrast, preincubation with the tripeptide RGD and the peptide YIGSR, BSA, glucose, galactose, and mannose had no inhibitory effect on conidial adherence (data not shown). Asialomucin also demonstrated no effect (Fig. 2). Conversely, preincubation with mucin had an approximately 25% inhibitory effect, while preincubation with NANA had a pronounced inhibitory effect (Fig. 2). Incubation of conidia with rabbit anti-laminin antibody in wells coated with immobilized laminin resulted in a pronounced inhibition of conidial adhesion (Fig. 2). Antibody dilutions of 1:50 and 1:100 were equally effective.

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Specificity of attachment of P. marneffei conidia to immobilized laminin. Wells were coated with a 100-µg/ml laminin solution, and conidia were allowed to adhere after preincubation in PBS (A), soluble laminin (1 mg/ml) (B), asialomucin (200 µg/ml) (C), mucin (200 µg/ml) (D), and NANA (200 mM) (E). Conidia were also coincubated in the presence of anti-laminin antibody (1:50) (F). The values in the presence of soluble laminin, NANA, and anti-laminin antibody were significantly different from the value with PBS (P < 0.01). Values for preincubation with BSA, glucose, galactose, mannose, RGD tripeptide, and peptide YIGSR were not statistically different from control values in the absence of any inhibitor (data not shown).

Conceptually, it would seem likely that attachment of P. marneffei conidia to the bronchioalveolar epithelium is a crucial step in the establishment of initial infection. The ability to adhere to epithelial cells may represent a means by which conidia avoid entrapment by respiratory tract mucus and removal by the action of ciliary cells. An obvious model for the adherence of P. marneffei conidia has been provided in recent times by studies elucidating the adherence of A. fumigatus conidia to various extracellular matrix components (1, 3, 6, 15).

Immunofluorescence labelling clearly demonstrated the presence of laminin binding sites on the surface of P. marneffei conidia and on the surface of phialides, from which conidia are produced. The observed absence of hyphal labelling is broadly analogous to the lack of extracellular matrix protein receptors on the surface of A. fumigatus mycelia (6).

There would appear to be at least a degree of specificity in the interaction between P. marneffei conidia and laminin, since both soluble laminin and a specific immune serum (rabbit anti-laminin polyclonal antisera) were capable of inhibiting the adherence of conidia to immobilized laminin. In contrast, no inhibition was observed in the presence of an equimolar concentration of BSA.

When tested in the laminin adherence assay, various monosaccharides demonstrated no inhibitory effect. However, there was complete inhibition of adherence when conidia were preincubated with NANA. Bovine submaxillary mucin also demonstrated some inhibition of adherence, whereas asialomucin was unable to cause inhibition. The former is rich in terminal sialic acid residues, whereas the latter represents its desialylated form. Taken together, these results are indicative of the presence on the conidial surface of a lectin which binds laminin via terminal sialic acid residues on the carbohydrate chains of this glycoprotein. It is important to note, however, that at least part of the inhibitory effect resulting from the pretreatment with NANA may be due to a pH shift generated by the latter. Laminin is known to be heavily glycosylated with carbohydrate chains bearing terminal sialic acid residues (1, 9), and this recognition system would appear to be directly analogous to that recently elucidated for laminin binding by A. fumigatus conidia (1). These recognition systems appear to be distinct from the A. fumigatus fibronectin binding mechanism which is inhibited by the peptide RGD (6). It is of note that the latter had no effect on the attachment of P. marneffei conidia to immobilized laminin. Further studies on the interaction described in this report will involve the identification and full characterization of the receptor(s) involved in this sialic acid-dependent process.