Next to production of virulence fac

Next to production of virulence factors and their associa-tion to the cell, it is also necessary to look at degradation of virulence factors during cultivation. Taxonomically, B. pertus-sis is classified as protease negative, since gelatin plates show no liquefaction around colonies. However, degradation of FHA has been reported [8] as well as the proteolytic degrada-tion of subunits 1 and 5 of PT [9]. The PheeAsp sequence that is recognized by the B. pertussis protease described by Cyr et al. [10] occurs 7 times in FHA, once in PT, once in PRN and twice in the fimC protein. The effect of B. pertussis’ pro-tease during cultivations has not been studied before, but may have quite an impact on the virulence factor content and may complicate the interpretation of experimental results.In conclusion, for a number of culture parameters the effect on virulence factor expression and their association to the cell is not properly quantified for production processes. In addi-tion, the effect of proteolytic activity during the cultivation is unknown. Therefore, the aim of this paper is to properly quantify the effect of a number of parameters on virulence fac-tor expression, their association to the cell, and the effect of these parameters on proteolytic activity. From Fig. 1C it is clear that if PT is formed, all other virulence factors are also expressed. Therefore, PT production is used as a measure for virulence factor production.As a starting medium, the chemically defined medium de-veloped by Thalen et al. [11] was used rather than any of the complex production media or the chemically defined Stainer Scholte medium [12]. The latter media lead to the for-mation of large amounts of ammonium and poly-hydroxybuty-rate, which can influence virulence factor production. In the medium developed by Thalen et al. [11], only minor amounts of these compounds are formed. Furthermore, growth on this medium is far more reproducible than growth on a complex production medium. On the basis of the data generated, it should become clear what the impact of each single parameter is on B. pertussis’ virulence factor production. The impli-cations of the effects found for the different parameters (Table 1) for the improvement of existing production processes will be discussed.