Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection

Abstract

Background: Pneumocystis carinii causes pneumonia in immunocompromised patients with a high morbidity and mortality rate, but the interaction between this organism and the host cell is not well understood. The purpose of this research was to study the response of host cells to P. carinii infection on a molecular level.

Results: The technique of mRNA differential display was used to detect genes whose expression may be affected by P. carinii infection. The nucleotide sequence of one differentially displayed DNA fragment was found to be identical to that of the rat mitochondrial ATPase 6 gene, which is a subunit of the F0F1-ATP synthase complex. A four-fold increase in expression of this gene was verified by Northern blot analysis of total RNA extracted from P. carinii-infected rat lung versus that from mock-infected rat lung. Localization of the cells containing ATPase 6 mRNA was accomplished by in situ hybridization. In sections of non-infected rat lung, these cells were found lining the distal parts of the respiratory tree and in apical areas of the alveoli. Histological location of these cells suggested that they were Clara cells and type II pneumocytes. This hypothesis was confirmed by co-localizing the mRNAs for ATPase 6 and surfactant protein B (SP-B) to the same cells by two-color fluorescent in situ hybridization.

Conclusions: The ATPase 6 gene is over expressed during P. carinii infection, and type II pneumocytes and Clara cells are the cell types responsible for this over-expression.

Figure 1

Products of mRNA Differential Display. Samples from mock-infected (M) and P. carinii-infected (I) cells were run in adjacent lanes on a 6% denaturing polyacrylamide gel. These PCR reactions contained the "A" anchored primer (H-T₁₁A) and one of the arbitrary primers (AP4, AP6, or AP8). The arrows indicate differentially displayed products A4II, A6I, and A8I.

Figure 2

Northern Hybridizations Using the Cloned ATPase 6 Gene Fragment as Probe. Eight μg each of total RNAs derived from non-infected rat lung (N) and P. carinii-infected rat lung (I) were electrophoresed on an agarose gel containing formaldehyde, transferred to Nytran Plus membrane, and probed with a fragment of the 18S rRNA gene (control probe) and the mitochondrial ATPase 6 gene fragment.

Figure 3

in situ Hybridizations Using Digoxigenin-labeled Riboprobes for ATPase 6 (A) and SP-B (B) with Colorimetric Detection. Three micrometer-thick sections of dexamethasone-suppressed non-infected rat lung (NRL) and Pc-infected rat lung (PcIRL) were hybridized with ATPase 6 or SP-B probes. Left-side panels are representative sections probed with sense probes (S). Right-side panels show representative sections probed with anti-sense probes (AS). Arrows indicate cells that show the blue precipitate (indicating ATPase 6 expression) in the section of NRL probed with AS probe.

Figure 4

Double-probe Fluorescent in situ Hybridization Using Digoxigenin-labeled ATPase 6 and Fluorescein-labeled SP-B Riboprobes. Three micrometer-thick sections of normal rat lung (NRL) (A and G) and Pc-infected rat lung (PcIRL) (B and H) were processed for double-probe fluorescent in situ hybridization. Panels A (NRL) and B (PcIRL) are composite images of sections reacted with a mixture of anti-sense digoxigenin-labeled ATPase 6 and fluorescein-labeled SP-B probes. Panels C and E are split images of the composite A, and panels D and F are split images of the composite B, showing concurrence of red (ATPase 6) and green (SP-B) signal in the same cells. Panels G (NRL) and H (PcIRL) are composite images of sections reacted with both ATPase 6 and SP-B sense probes showing very little background signal. Arrows in panel B indicate the punctate DAPI-stained (blue) nuclei of P. carinii organisms.