Differences in the BAL proteome after Klebsiella pneumoniae infection in wild type and SP-A-/- mice

Abstract

Background: Surfactant protein-A (SP-A) has been shown to play a variety of roles related to lung host defense function. Mice lacking SP-A are more susceptible to infection than wild type C57BL/6 mice. We studied bronchoalveolar lavage (BAL) protein expression in wild type and SP-A-/- mice infected with Klebsiella pneumoniae by 2D-DIGE.

Methods: Mice were infected intratracheally with K. pneumoniae and after 4 and 24 hours they were subject to BAL. Cell-free BAL was analyzed by 2D-DIGE on two-dimensional gels with pH ranges of 4-7 and 7-11. Under baseline conditions and at 4 and 24 hr post-infection BAL was compared between untreated and infected wild type and SP-A-/- mice. Sixty proteins identified by mass spectrometry were categorized as host defense, redox regulation, and protein metabolism/modification.

Results: We found: 1) ~75% of 32 host defense proteins were lower in uninfected SP-A-/- vs wild type, suggesting increased susceptibility to infection or oxidative injury; 2) At 4 hr post-infection > 2/3 of identified proteins were higher in SP-A-/- than wild type mice, almost the exact opposite of untreated mice; 3) At 24 hr post-infection some proteins continued increasing, but many returned to baseline; 4) In infected wild type mice significant changes occurred in 13 of 60 proteins, with 12 of 13 increasing, vs on 4 significant changes in SP-A-/- mice. Infection response patterns between strains demonstrated both commonalities and differences. In several cases changes between 4 and 24 hr followed different patterns between strains.

Conclusions: These indicate that SP-A plays a key role in regulating the BAL proteome, functioning indirectly to regulate lung host defense function, possibly via the macrophage. In the absence of SP-A baseline levels of many host defense molecules are lower. However, many of these indirect deficits in SP-A-/- mice are rapidly compensated for during infection, indicating that SP-A also has a direct role on host defense against K. pneumoniae that may be instrumental in determining clinical course.

Figure 1

BAL total protein concentration. The total protein concentration of cell-free BAL fluid was determined. The total amount of fluid recovered during BAL (~90% of instilled volume) did not differ significantly among groups. The histogram depicts mean protein concentration values (n = 4/group) and standard deviations are indicated by error bars. Values of WT mice that differ significantly (p < 0.05) from baseline by t-tests are indicated by an asterisk (*) and values of SP-A-/- mice that differ from baseline values are indicated with a pound sign (#).

Figure 2

Reference gels. The reference gels for two-dimensional separations in pH 4-7 gels (A) and pH 7-11 gels (B) are shown. Protein spots that have been identified by MALDI-ToF/ToF are circled and numbered and all proteins are named in Table 1. Note that in cases where multiple isoforms are circled, the identity of all isoforms has been confirmed by MALDI-ToF/ToF.

Figure 3

Network analysis of BAL proteome changes under baseline conditions. Changes in levels of expression between WT and SP-A-/- mice under baseline conditions and at 4 hr after infection (Figure 4) were analyzed using the Ingenuity Pathway program focusing on pathways related to lung disease (shown as Function (Fx) in olive green squares). Proteins undergoing significant changes are indicated in bold. Changing proteins that were not included in the pathway are shown in the inset in the lower right corner. The relationship between symbol color and relative levels of expression is shown in the upper left corner. Unshaded proteins in the pathway were not identified in our gels. The proteins included in the analysis and the Ingenuity abbreviation are listed below (see also Table 1): Cytosolic malate dehydrogenase: MDH1; Ferritin light chain 1: FTL; Gamma-actin: ACTG1; Gelsolin: GSN; Glutathione S-transferase, alpha 3: GSTA3; Glutathione S-transferase, alpha 4: GSTA4; Glutathione S-transferase, mu 1: GSTM5; Glutathione S-transferase, omega 1: GSTO1; Glutathione S-transferase, Ya chain (GST class-alpha): GSTA5; Haptoglobin: HP; Hemoglobin subunit alpha: HBA2; Isocitrate dehydrogenase: IDH1; Keratin complex 1, acidic, gene 10: KRT10; Kpnb1 protein b: KPNB1; Lactate dehydrogenase 2, B chain: LDHB; Lysozyme 2:; LYZ; Murinoglobulin-1 precursor: MUG1; Myosin heavy chain IIB: MYH4; Peroxiredoxin 1: PRDX1; Peroxiredoxin 6: PRDX6; Pregnancy zone protein: PZP; Prothrombin precursor: F2; Pulmonary surfactant associated protein A precursor: SFTPA1; Rho GDP dissociation inhibitor (GDI) alpha: ARHGDIA; SEC14-like 3: SEC14L3; Selenium binding protein 1: SELENBP1; Toll-like receptor 13 precursor: TLR13; Transferrin: TF; Tyrosine-3-monooxygenase/tryptophan 5-monooxygenase activation protein, Epsilon polypeptide: YWHAE

Figure 4

Network analysis of BAL proteome changes at 4 hours after infection. Changes in levels of expression between WT and SP-A-/- mice under baseline conditions (Figure 3) and at 4 hr after infection (Figure 4) were analyzed using the Ingenuity Pathway program. A description of the figure and the abbreviations used are the same as those in the legend for Figure 3.