Acinetobacter baumannii is an important nosocomial pathogen that causes a high morbidity and mortality rate in infected patients, but pathogenic mechanisms of this microorganism regarding the secretion and delivery of virulence factors to host cells have not been characterized. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs) that play a role in the delivery of virulence factors to host cells. A. baumannii has been shown to secrete OMVs when cultured in vitro, but the role of OMVs in A. baumannii pathogenesis is not well elucidated. In the present study, we evaluated the secretion and delivery of virulence factors of A. baumannii to host cells via the OMVs and assessed the cytotoxic activity of outer membrane protein A (AbOmpA) packaged in the OMVs. A. baumannii ATCC 19606T secreted OMVs during in vivo infection as well as in vitro cultures. Potential virulence factors, including AbOmpA and tissue-degrading enzymes, were associated with A. baumannii OMVs. A. baumannii OMVs interacted with lipid rafts in the plasma membranes and then delivered virulence factors to host cells. The OMVs from A. baumannii ATCC 19606T induced apoptosis of host cells, whereas this effect was not detected in the OMVs from the ΔompA mutant, thereby reflecting AbOmpA-dependent host cell death. The N-terminal region of AbOmpA22-170 was responsible for host cell death. In conclusion, the OMV-mediated delivery of virulence factors to host cells may well contribute to pathogenesis during A. baumannii infection.
Bacteremia is a common systemic disease caused by Acinetobacter baumannii, an important hospital-acquired pathogen among critically ill patients. The complement system is central to innate immune defense against invading bacteria in the blood. The present study investigated the susceptibility of clinical A. baumannii isolates to normal human sera (NHS), and determined the resistance mechanism of A. baumannii against complement-mediated lysis. The survival of A. baumannii isolates from bacteremic patients was significantly decreased in undiluted NHS, but they were resistant to 40% NHS. The alternative complement pathway was responsible for the direct killing of bacteria. The main regulator of the alternative complement pathway, factor H, bound to the surface of live A. baumannii treated with NHS. Factor H interacted with the outer membrane proteins with molecular sizes of 38 (AbOmpA), 32, and 24 kDa. The isogenic AbOmpA(-) mutant was highly susceptible to NHS in comparison with the wild-type A. baumannii strain, suggesting that AbOmpA was an important complement regulator-acquiring surface protein. These results indicate that A. baumannii evades complement attack through the acquisition of factor H to their surface.
Runx2 is a master transcription factor for chondrocyte and osteoblast differentiation and bone formation. However expression of Runx2 (by RT-PCR), has been reported in non-skeletal tissues such as breast, T cells and testis. To better define Runx2 activity in non-skeletal tissues, we examined transgenic (Tg) mice expressing LacZ gene under control of 3.0 kb (3 Kb Tg) or 1.0 kb (1 Kb Tg) of the Runx2 distal (P1) promoter, Runx2 LacZ knock-in (Runx2 +/LacZ ) and Runx2/P1 LacZ knock-in (Runx2/P +/LacZ ). In the Runx2 3 Kb Tg mouse, β-galactosidase (β-gal) expression appeared in various non-skeletal tissues including testis, skin, adrenal gland and brain. β-gal expression from both 3 Kb and 1 Kb Tg, reflecting activity of the Runx2 promoter, was readily detectable in seminiferous tubules of the testis and the epididymis. At the single cell level, β-gal was detected in spermatids and mature sperms not in sertoli or Leydig cells. We also detected a positive signal from the Runx2 +/LacZ and Runx2/P1 +/LacZ mice. Indeed, Runx2 expression was observed in isolated mature sperms, which was confirmed by RT-PCR and western blot analysis. Runx2, however, was not related to sex determination and sperm motility. Runx2 mediated β-gal activity is also found robustly in the hippocampus and frontal lobe of the brain in Runx2 +/LacZ . Collectively, these results indicate that Runx2 is expressed in several non-skeletal tissues particularly sperms of testis and hippocampus of brain. It suggests that Runx2 may play an important role in male reproductive organ testis and brain.
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