C3 promotes clearance of Klebsiella pneumoniae by A549 epithelial cells.

The airway epithelium represents a primary site for contact between microbes and their hosts. To assess the role of complement in this event, we studied the interaction between the A549 cell line derived from human alveolar epithelial cells and a major nosocomial pathogen, Klebsiella pneumoniae, in the presence of serum. In vitro, we found that C3 opsonization of poorly encapsulated K. pneumoniae clinical isolates and an unencapsulated mutant enhanced dramatically bacterial internalization by A549 epithelial cells compared to highly encapsulated clinical isolates. Local complement components (either present in the human bronchoalveolar lavage or produced by A549 epithelial cells) were sufficient to opsonize K. pneumoniae. CD46 could competitively inhibit the internalization of K. pneumoniae by the epithelial cells, suggesting that CD46 is a receptor for the binding of complement-opsonized K. pneumoniae to these cells. We observed that poorly encapsulated strains appeared into the alveolar epithelial cells in vivo but that (by contrast) they were completely avirulent in a mouse model of pneumonia compared to the highly encapsulated strains. Our results show that bacterial opsonization by complement enhances the internalization of the avirulent microorganisms by nonphagocytic cells such as A549 epithelial cells and allows an efficient innate defense.

Role of the htrA gene in Klebsiella pneumoniae virulence.

We recently described the use of mini-Tn5 to generate complement-sensitive mutants derived from a complement-resistant Klebsiella pneumoniae clinical isolate deficient in the lipopolysaccharide O side chain. One mutant with a reduced capacity to survive in nonimmune human sera carried the transposon inserted in the htrA gene. We cloned and sequenced the gene and predicted from the deduced amino acid sequence that the putative HtrA homolog contains structural features similar to those of previously described HtrA proteins. To investigate the biological functions and the role of the htrA gene in the virulence of K. pneumoniae, we constructed an isogenic mutant by insertion-duplication mutagenesis. Characterization of the mutant showed that it had greater sensitivity to temperature (50 degrees C) and oxidative stress (H(2)O(2)) than the parent strain. Furthermore, the htrA mutant produced less capsule, bound more molecules of complement component C3, and was more sensitive to complement and whole-blood killing than was the parent strain. Finally, disruption of the htrA gene in a virulent K. pneumoniae strain caused a reduction of its virulence in a mice model. Our results indicate that the htrA gene plays an important role in the virulence of K. pneumoniae.

Molecular analysis of the contribution of the capsular polysaccharide and the lipopolysaccharide O side chain to the virulence of Klebsiella pneumoniae in a murine model of pneumonia.

Klebsiella pneumoniae is a common cause of gram-negative bacterial nosocomial pneumonia. Two surface polysaccharides, lipopolysaccharide (LPS) O side chain and capsular polysaccharide (CPS), are critical for the microorganism in causing sepsis, but little is known about their role in pneumonia. To investigate their contribution in the pathogenesis of K. pneumoniae pneumonia, we characterized the host response to bacterial challenge with a highly virulent clinical isolate or with isogenic insertion-duplication mutants deficient in CPS or LPS O side chain in a murine model of pneumonia. Animals challenged intratracheally with the wild-type or LPS O side chain-deficient strain developed pneumonia and became bacteremic before death. Extensive lung lesions as well as pleuritis, vasculitis, and edema were observed by histopathological examination, and polymorphonuclear infiltration was also demonstrated. In contrast, none of the animals challenged with the unencapsulated strain developed pneumonia or bacteremia. Examination of tissue from this group did not identify lung lesions, and none of the infected animals died. Analysis of the early host defense mechanisms that contributed to the clearance of the unencapsulated mutant showed that the levels of C3 deposited on the unencapsulated mutant surface were threefold higher than those for the wild-type and LPS O side chain-deficient strains. Furthermore, phagocytosis of the unencapsulated mutant by human alveolar macrophages (AM) was more efficient than that of the wild-type and LPS O side chain-deficient strains. We conclude that CPS, but not LPS O side chain, is essential for Klebsiella pneumonia because it modulates the deposition of C3 and protects the microorganisms against human AM phagocytosis.