Häse Claudia C
Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis, OR 97331, USA.
FEMS Microbiol Lett. 2003 Oct 10;227(1):65-71. doi: 10.1016/S0378-1097(03)00649-9.
Vibrio cholerae is known to secrete a large number of proteins into the extracellular milieu, including the important virulence factor cholera toxin (CT). However, one of the most abundant proteins found in V. cholerae supernatants is the zinc-metalloprotease HA/protease (HAP). Whereas efficient protein secretion in Escherichia coli requires ATP hydrolysis and the proton motive force (pmf), little is known about the energy requirements for protein secretion in V. cholerae. To analyze some of the energy requirements for protein secretion in V. cholerae, HAP accumulation in culture supernatants following growth in the presence of various ionophores was assayed. Extracellular production of HAP was strongly reduced in the presence of monensin, an artificial Na(+)/H(+) antiporter that collapses the DeltapNa(+) across the membrane without affecting Deltapsi, whereas the protonophore CCCP had no significant effect on the extracellular accumulation of HAP. In contrast, extracellular protease production in Pseudomonas aeruginosa was affected by CCCP, but not monensin. Furthermore, extracellular protease production of V. cholerae, but not P. aeruginosa, was increased in increasing amounts of NaCl in the culture medium. Together these results indicate that the V. cholerae HAP requires an intact sodium motive force (smf) for its efficient translocation across the membranes, whereas extracellular protease production by P. aeruginosa requires only pmf. As the entry of some bacteriophage genomes has been reported to require pmf, the effects of ionophores on the efficiency of tranduction of V. cholerae by the CTXPhi phage were analyzed. CTXPhi transduction was strongly affected by CCCP, but not monensin, suggesting that phage entry requires pmf but not smf. Understanding the energy requirements for these potentially important virulence aspects of pathogens might lead to novel intervention strategies.
已知霍乱弧菌会向细胞外环境分泌大量蛋白质，其中包括重要的毒力因子霍乱毒素（CT）。然而，在霍乱弧菌培养上清液中发现的最丰富的蛋白质之一是锌金属蛋白酶HA/蛋白酶（HAP）。虽然在大肠杆菌中高效的蛋白质分泌需要ATP水解和质子动力（pmf），但对于霍乱弧菌中蛋白质分泌的能量需求却知之甚少。为了分析霍乱弧菌中蛋白质分泌的一些能量需求，检测了在各种离子载体存在下生长后培养上清液中HAP的积累情况。在莫能菌素存在的情况下，HAP的细胞外产生显著减少，莫能菌素是一种人工Na(+)/H(+)反向转运体，它会破坏跨膜的ΔpNa(+)而不影响ΔΨ，而质子载体CCCP对HAP的细胞外积累没有显著影响。相比之下，铜绿假单胞菌的细胞外蛋白酶产生受到CCCP的影响，但不受莫能菌素的影响。此外，培养基中NaCl含量增加时，霍乱弧菌的细胞外蛋白酶产生增加，而铜绿假单胞菌则不然。这些结果共同表明，霍乱弧菌HAP高效转运穿过膜需要完整的钠动力（smf），而铜绿假单胞菌的细胞外蛋白酶产生仅需要pmf。由于据报道一些噬菌体基因组的进入需要pmf，因此分析了离子载体对CTXPhi噬菌体转导霍乱弧菌效率的影响。CTXPhi转导受到CCCP的强烈影响，但不受莫能菌素的影响，这表明噬菌体进入需要pmf而不是smf。了解病原体这些潜在重要毒力方面的能量需求可能会带来新的干预策略。
