Host-pathogen interactions of Actinobacillus pleuropneumoniae with porcine lung and tracheal epithelial cells

Abstract

Host-pathogen interactions are of great importance in understanding the pathogenesis of infectious microorganisms. We developed in vitro models to study the host-pathogen interactions of porcine respiratory tract pathogens using two immortalized epithelial cell lines, namely, the newborn pig trachea (NPTr) and St. Jude porcine lung (SJPL) cell lines. We first studied the interactions of Actinobacillus pleuropneumoniae, an important swine pathogen, using these models. Under conditions where cytotoxicity was absent or low, we showed that A. pleuropneumoniae adheres to both cell lines, stimulating the induction of NF-kappaB. The NPTr cells consequently secrete interleukin 8, while the SJPL cells do not, since they are deprived of the NF-kappaB p65 subunit. Cell death ultimately occurs by necrosis, not apoptosis. The transcriptomic profile of A. pleuropneumoniae was determined after contact with the porcine lung epithelial cells by using DNA microarrays. Genes such as tadB and rcpA, members of a putative adhesin locus, and a gene whose product has high homology to the Hsf autotransporter adhesin of Haemophilus influenzae were upregulated, as were the genes pgaBC, involved in biofilm biosynthesis, while capsular polysaccharide-associated genes were downregulated. The in vitro models also proved to be efficient with other swine pathogens, such as Actinobacillus suis, Haemophilus parasuis, and Pasteurella multocida. Our results demonstrate that interactions of A. pleuropneumoniae with host epithelial cells seem to involve complex cross talk which results in regulation of various bacterial genes, including some coding for putative adhesins. Furthermore, our data demonstrate the potential of these in vitro models in studying the host-pathogen interactions of other porcine respiratory tract pathogens.

FIG. 1.

SJPL (filled bars) and NPTr (empty bars) cells were assessed for cytotoxicity following an infection with A. pleuropneumoniae strain S4074 at an MOI of 10:1.

FIG. 2.

NPTr (a and c) or SJPL (b and d) cells stained with Giemsa stain in the presence (c and d) or absence (a and b) of A. pleuropneumoniae S4074, seen through a Leica DMR microscope at an original magnification of ×1,000.

FIG. 3.

Adherence of A. pleuropneumoniae S4074 to SJPL (filled bars) or NPTr (empty bars) cells from 1 to 3 h.

FIG. 4.

EMSA (A) or supershift assay (B) performed on nuclear proteins of SJPL and NPTr cells following an incubation (30 to 180 min) with A. pleuropneumoniae S4074 or no treatment for the control (Nil). For controls, proteins were incubated with specific oligonucleotides (S) and nonspecific oligonucleotides (NS). The probe alone was also loaded on the gel (P). For the supershift assay (B), proteins were incubated with p50 antibodies (p50), p65 antibodies (p65), or no antibodies (-). Arrows demonstrate the subunit band shifts (B).

FIG. 5.

Production of IL-8 by NPTr cells following an induction with heat-killed A. pleuropneumoniae S4074 (▪) or when not stimulated (▴).

FIG. 6.

Adherence of 12 members of the Pasteurellaceae to the SJPL (filled bars) or NPTr (empty bars) cell line after 3 h of incubation. The strains include A. pleuropneumoniae serotype 1 S4074 and FMV91-6514, A. pleuropneumoniae serotype 5b L20 and 05-6501, A. pleuropneumoniae serotype 5a 05-4817, A. pleuropneumoniae serotype 7 WF83 and 05-3695, H. parasuis serotype 5 Nagasaki and 29755, A. suis serotype O2/K2 H91-0380, and P. multocida capsular type A 88-761 and capsular type D 1703. Asterisks represent statistical differences (P < 0.05) in adherence of the given strain between the two cell lines.

FIG. 7.

Invasion by A. pleuropneumoniae S4074 (▪), H. parasuis Nagasaki (▴), and H. parasuis 29755 (•) of SJPL (full line) or NPTr (dashed line) cells from 1 h to 3 h.