In vitro secretion kinetics of proteins from Legionella pneumophila in comparison to proteins from non-pneumophila species

Abstract

It has been shown that the loss of PilD, a prepilin peptidase necessary for type IV pilus biogenesis and establishment of the type II secretion apparatus is associated with loss of virulence in Legionella pneumophila. L. pneumophila is the species most frequently associated with Legionnaires' disease, but virulence factors unique to this species are not known, so the secretion kinetics of several pilD-dependent enzyme activities, including protease, acid phosphatase, phospholipase A (PLA) and lysophospholipase A (LPLA), of L. pneumophila and non-pneumophila species were compared during growth in BYE broth. Enzyme activity appeared during mid-exponential growth phase and reached maximal levels on entry into stationary growth phase. None of the enzyme activities were unique to L. pneumophila and it did not exclusively secrete the highest amounts of the hydrolytic proteins. However, the timing of PLA and LPLA secretion in L. pneumophila differed compared to other species. PLA activity was secreted prior to LPLA activity in L. pneumophila, which may lead to an accumulation of the cytotoxic agent lysophosphatidylcholine (LPC). In addition to L. pneumophila, several other Legionella species, including Legionella steigerwaltii and Legionella gormanii, were able to enrich for LPC due to a very potent PLA activity accompanied by only moderate LPLA activity. These species, in contrast to L. pneumophila, have not been shown to multiply within monocytic host cells. Thus none of the secreted enzymic activities investigated were unique to L. pneumophila, nor were they secreted at high concentrations. However, the timing of PLA and LPLA secretion may contribute to pathogenicity.

The Global Regulatory Proteins LetA and RpoS Control Phospholipase A, Lysophospholipase A, Acyltransferase, and Other Hydrolytic Activities of Legionella pneumophila JR32

“…L. pneumophila possesses several secreted and cellassociated lipolytic activities: PLA, LPLA, GCAT, and lipase activity (4,5,10,22,24,26,27) (Table 1). In order to examine the role of the global regulator proteins RpoS and LetA with regard to the lipolytic activities, we tested late logarithmic (OD 660 of 2.0) and mid-logarithmic (OD 660 of 1.0) culture supernatants and cell lysates of the L. pneumophila JR32 wildtype strain and its isogenic rpoS and letA mutants with different substrates for lipolytic activities.…”

“…For the determination of phosphatase activity, 50 l L. pneumophila culture supernatant or cell lysate (diluted at 1:2 with 40 mM Tris-HCl (pH 7.5, 25°C) was incubated with the same volume of substrate mixture, containing 10 mM p-nitrophenylphosphate (p-NPP) (Sigma-Aldrich, Taufkirchen, Germany), 6 mM NaN 3 , 1% (vol/vol) Triton X-100 in 40 mM Tris-HCl at 37°C and with continuous agitation at 170 rpm for 1 h as previously described (24). Subsequently the release of p-NP was measured optically at a wavelength of 405 nm and compared to p-NP (Sigma-Aldrich, Taufkirchen, Germany) standard solutions.…”

“…For the detection of protease activity, 50 l L. pneumophila culture supernatant was incubated with the double volume of substrate mixture, containing 1.33% (wt/vol) azocasein (Sigma-Aldrich, Taufkirchen, Germany) in 40 mM Tris-HCl, pH 7.5 (25°C) at 37°C and with continuous agitation at 170 rpm for 1 h as previously described (24). After treatment as described by Prestidge (56), samples were measured at a wavelength of 420 nm and compared to a reaction product of a protease standard solution (Bacillus polymyxa protease, Sigma-Aldrich, Taufkirchen, Germany; concentrations used in experiment: 0.1, 0.25, and 0.5 U/mg).…”

“…Since secreted lipase and protease activities in other bacteria are regulated by GacA (LetA) and RpoS, and since secreted hydrolytic activities of L. pneumophila are expressed at peak levels during late logarithmic growth phase and entry into stationary phase (24), it is possible that the L. pneumophila enzymes are regulated in a similar manner. L. pneumophila possesses a range of secreted and cell-associated hydrolytic activities: phospholipase A (PLA), lysophospholipase A (LPLA), glycerophospholipid: cholesterol acyltransferase (GCAT), lipase/esterase, protease, phosphatase, p-nitrophenylphosphorylcholine (p-NPPC) hydrolase, and RNase.…”

Copper (II) sulfate charring for high sensitivity on-plate fluorescent detection of lipids and sterols: quantitative analyses of the composition of functional secretory vesicles

“…Later improvements to the technique included the use of cupric sulfate in place of cupric acetate [2,3] to address shortcomings in detection-specifically with regard to the differential sensitivity to saturated and unsaturated lipids [4,5]. The technique has since been largely unchanged and remains a routine, widely used method for detecting lipids on TLC plates [6][7][8][9].…”