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. 2008 May 9;283(19):12909–12918. doi: 10.1074/jbc.M801006200

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Copyright © 2008, The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 7. — A, immunoblot of trichloroacetic acid-precipitated culture supernatants of derivatives of L. pneumophila 130b. A band corresponding to Lpg1905 was detected in all strains carrying pMIP expressing Lpg1905 and mutant derivatives. Lane 1, L. pneumophila 130b (pMIP). Lane 2, L. pneumophila lpg1905::km (pMIP). Lane 3, L. pneumophila lpg1905::km (pMIP:lpg1905). Lane 4, L. pneumophila lpg1905::km (pMIP:lpg1905R122A). Lane 5, L. pneumophila lpg1905::km (pMIP:lpg1905N168A). Lane 6, L. pneumophila lpg1905:: km (pMIP:lpg1905Q193A). Lane 7, L. pneumophila lpg1905::km (pMIP: lpg1905W384A). Lane 8, L. pneumophila lpg1905::km (pMIP:lpg1905E159A). B, effect on bacterial replication in THP-1 macrophages of complementing the lpg1905 mutant derivative of L. pneumophila 130b with an expression plasmid expressing Lpg1905 and mutated derivatives. The results are expressed as log₁₀ colony-forming units present in the extracellular medium and associated with cells at specific time points after inoculation (means + standard deviation of at least three independent experiments from duplicate wells). The strains tested were L. pneumophila 130b (pMIP) (•), lpg1905::km (pMIP) (▪), lpg1905::km (pMIP:lpg1905) (▴), lpg1905::km (pMIP:lpg1905E159A) (⋄), lpg1905::km (pMIP:lpg1905R122A) (♦), lpg1905::km (pMIP:lpg1905N168A) (○), lpg1905::km (pMIP:lpg1905Q193A) (□), and lpg1905::km (pMIP:lpg1905W384A) (▵). C, competitive pulmonary infection of A/J mice with L. pneumophila 130b and mutant derivatives at 72 h. The results are expressed as the CI of the mutant (test) strain for each mouse. The horizontal bar indicates the average CI. The lpg1905::km mutant derivative was tested in competition with L. pneumophila 130b (mutant, •). Strains tested in a 1:1 mixed infection with L. pneumophila 130b (pMIP) were lpg1905::km (pMIP, ▪), lpg1905::km (pMIP:lpg1905,) (Lpg1905, ▴), lpg1905::km (pMIP:lpg1905E159A) (E159A, ▾), lpg1905::km (pMIP:lpg1905R122A) (R122A, ⋄), lpg1905::km (pMIP:lpg1905N168A) (N168A, ○), lpg1905::km (pMIP:lpg1905Q193A) (Q193A, □), and lpg1905::km (pMIP:lpg1905W384A) (W384A, ▵). The average CI for each strain was: CI = 0.18 (lpg1905::km mutant), CI = 0.014 (pMIP); CI = 0.526 (Lpg1905), CI = 0.152 (E159A); CI = 0.021 (R122A); CI = 0.024 (N168A); CI = 0.003 (Q193A); and CI = 0.003 (W384A). The CI values of all strains carrying mutant derivatives of Lpg1905 expressed from pMIP were significantly lower than that observed for lpg1905::km (pMIP:lpg1905)(p < 0.05, unpaired two-tailed t test).