Environmental Stress and Encystment of Pneumocystis carinii

The structural elements and molecular events associated with encystment of Pneumocystis carinii are poorly understood. The cyst form of P. carinii is present in the lungs of infected hosts in low numbers as compared to the trophozoite; as few as 1/100 of the total organism number in severely infected animals are cysts [3]. It is not known if the cyst form is infectious or the degree to which it participates in eliciting immune responses from the host. Studies using drugs, that specifically prevent formation or destabilization of the cyst wall result in lower numbers of both trophozoites and cysts, suggesting that the cyst is an integral life cycle stage of P. carinii [1,4]. The environmental or organism signals that trigger encystment of P. carinii are unknown. Cysts appear late in the infection [3], suggesting that damage done to the host pulmonary system may create an inhospitable environment or reduce nutritional resources for the trophozoite, thereby triggering formation of the more environmental resistant and quiescent form, the cyst. This study sought to determine if a specific source of stress in the trophozoites’ environment is associated with encystment. MATERIALS AND METHODS Female Sprague Dawley rats (colony 202) were obtained from Harlan (Indianapolis, Ind.) and housed in an AAALAC-approved facility. Rats were immunosuppressed with dexamethasone (Dex) (0.36 mg/liter) in their drinking water for seven days. Then P. carinii organisms were introduced into rat lungs by transtracheal inoculation as previously described [2]. Broncheoalveolar lavage (BAL) fluid collection was performed as described previously [3]. Briefly, the trachea was exposed by blunt dissection. The thoracic cavity was opened by incisions through the ribcage and removing the ribcage to allow full expansion of the lungs during lavage. A 14-gauge Angiocath (Becton Dickinson, Infusion Therapy Systems, Sandy, UT) with a 10-mL syringe was inserted into the trachea, and the lungs were lavaged with aliquots of sterile, pyrogen-free saline solution at 378C, pH 7.3. The saline was recovered from the lung by withdrawing the plunger of the syringe in a deliberate manner. In some cases, only 5 mL of lavage fluid was instilled and recovered. Some lavage samples for pH determination were obtained from animals that were incubated at 48C for 24 hr after cardiac exsanguination. In these cases, lavage was performed as described above, with special attention to slow instillation and removal of lavage fluid to account for the increased friability of the tissue. Severity of P. carinii infection was determined by scoring numbers of organisms on histochemically stained impression smears of lung tissue using the semi-logarithmic scale of Barlett et al. [2]. The smears were stained with Wright’s-Giemsa stain for both trophozoite and cysts and with Grocott methenamine-silver nitrate stain for cyst forms [2]. For culture experiments, human embryonic lung (HEL 299) cells of fibroblast morphology (CCL-137; ATCC, Manassas, VA) were grown as monolayers on 24-well culture dishes using minimum essential medium (MEM) with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, 10% fetal calf serum (FCS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Prior to inoculation of monolayers with P. carinii, the pH of the MEM was adjusted using 1 N HCl to 7.4, 7.3, 7.2, 7.1, 7.0, or 6.9. P. carinii samples were diluted to ten trophozoites per 10003 field to seed wells in the different pH media. Every other day, four individual wells were mixed using a Pasteur pipet, and 10-lL aliquots were