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Chapter 3 : Analysis of Acid Phosphatase and Esterase/Lipase Mutants of

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Analysis of Acid Phosphatase and Esterase/Lipase Mutants of , Page 1 of 2

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Abstract:

The identification of a gene in offered new insight into the molecular pathogenesis of legionellosis. Three mutants, NU254, NU255, and NU256, were selected from the screening and further studied. Supernatants from these mutants contained minimal acid phosphatase activity, while possessing normal levels of other -dependent exoproteins. To determine if the map gene was necessary for intracellular replication of , macrophage-like U937 cells and amoebae were infected at multiplicity of infection of 0.1 and 1, respectively. The authors have demonstrated that has two acid phosphatases that can be differentiated by their different sensitivity to tartrate, and while the tartrate-sensitive Map seems not to be essential for intracellular replication, the role of the tartrate-resistant phosphatase has yet to be determined. The gene that was interrupted by the transposon was determined, and a basic local alignment search tool (BLAST) analysis of the sequence showed that the putative protein shared some homology to lipase enzymes. Supernatants from the mutant presented a defect in its ability to hydrolyze p-nitrophenyl (PNP) palmitate (indication of esterase/lipase) and in the liberation of free fatty acid from phosphatidylcholine (indication of phospholipase A).

Citation: Aragon V, Kurtz S, Cianciotto N, McClain M, Engleberg N. 2002. Analysis of Acid Phosphatase and Esterase/Lipase Mutants of , p 18-21. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch3

Key Concept Ranking

Type II Secretion System
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Acid Phosphatase
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Figures

Image of FIGURE 1
FIGURE 1

Intracellular infection by wild-type and mutant U937 cells (top panel) and amoebae (bottom panel) were infected at a multiplicity of infection (MOI) of 0.1 and 1, respectively, with wild-type 130b (●) or lipase/esterase mutant AA407 (□). The number of bacteria in each well was quantitated at 0, 24, 48, and 72 h by plating aliquots on buffered charcoal-yeast extract (BCYE) agar. Results represent the mean (± standard deviation) of triplicate wells and are representative of two independent experiments.

Citation: Aragon V, Kurtz S, Cianciotto N, McClain M, Engleberg N. 2002. Analysis of Acid Phosphatase and Esterase/Lipase Mutants of , p 18-21. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch3
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Image of FIGURE 2
FIGURE 2

Enzymatic activities secreted by strains. Late-log phase supernatants of wild-type 130b (black bars) and AA407 (white bars) were tested for their ability to hydrolyze PNPpalmitate (A), phosphatidylcholine (B), PNPcaprylate (C), PNPphosphate (D), and PNPphosphorylcholine (E). Bars represent the mean (± standard deviation) of the activity found in three cultures and are representative of the results obtained in two independent experiments.

Citation: Aragon V, Kurtz S, Cianciotto N, McClain M, Engleberg N. 2002. Analysis of Acid Phosphatase and Esterase/Lipase Mutants of , p 18-21. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch3
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References

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