Abstract
Klebsiella pneumoniae, an important nosocomial pathogen, causes suppurative infection, pneumonia, urinary tract infection and septicemia in humans. To gain a further understanding of its pathogenesis, a novel in vivo expression technology (IVET) was performed to identify K. pneumoniae CG43 genes that are specifically expressed during infection of BALB/c mice. The IVET employed a UDP-glucose pyrophosphorylase (galU)-deficient mutant of K. pneumoniae which is incapable of utilizing galactose and synthesizing capsular polysaccharide, as demonstrated by its low virulence to BALB/c mice and a white nonmucoid colony morphology on MacConkey-galactose agar. By using a copy of promoterless galU gene as the reporter, IVE promoters that render the galU mutant virulent while maintaining the white nonmucoid colony phenotype could be identified. A total of 20 IVE genes were obtained through the in vivo selection. Five of them have been identified previously as virulence-associated genes in other pathogens, while another five with characterized functions are involved in regulation and transportation of nutrient uptake, biosynthesis of isoprenoids, and protein folding. No known functions have been attributed to the other 10 sequences. Two of the 20 IVE genes were found to turn on under iron deprivation, whereas the expression of another five genes was activated in the presence of paraquat, a superoxide generator. Sequence comparison revealed that the iucA promoter, which drives the expression of aerobactin upon iron deprivation in vivo, might be carried on a mobile region similar to the SHI-2 pathogenicity island of Shigella flexneri. Using the iucA promoter as a probe, we have isolated and sequence determined an 18,732-bp long region, which acts as a mobile genetic element that carries two virulence determinants for Klebsiella infection, the iron-acquisition capacity (encoded by iucABCD iutA) and the mucoidy of capsule (enhanced by rmpA2). The rmpA2 locus, which encodes an activator for the production of capsular polysaccharide (CPS), was subjected to further characterization. We constructed an rmpA2 deletion mutant and two luxAB transcriptional fusions, each containing a putative promoter region of the K. pneumoniae K2 cps genes. Activity of these two Pcps::luxAB constructs was enhanced in the presence of multicopy rmpA2. The use of DNA electrophoretic mobility shift assay further demonstrated that RmpA2 protein directly interacts with the K2 cps promoters. The stability of RmpA2 protein was significantly enhanced in the strain deficient in ATP-dependent Lon protease. RmpA2 represses its own gene expression. Therefore, the enhancement of K2 CPS synthesis in K. pneumoniae CG43 by RmpA2 can be attributed to its transcriptional activation of K2 cps genes, and the expression level of rmpA2 itself is autoregulated and under the control of Lon protease.