The bacterium Xenorhabdus nematophila is a virulent insect pathogen. Part of its pathogenicity is due to impairing cellular immunity by blocking biosynthesis of eicosanoids, the major recognized signal transduction system in insect cellular immunity. X. nematophila inhibits the first step in eicosanoid biosynthesis, phospholipase A2 (PLA2). Here we report that the bacterium inhibits PLA2 from two insect immune tissues, hemocytes and fat body, as well as PLA2s selected to represent a wide range of organisms, including prokaryotes, insects, reptiles, and mammals. Our finding on a bacterial in-hibitor of PLA2 activity contributes new insight into the chemical ecology of microbe-host interactions, which usually involve actions rather than inhibitors of PLA2s

Kim, Yonggyun

Park, Youngjin

Stanley, David

English

UNL | Libraries

University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln
Faculty Publications: Department of Entomology Entomology, Department of
2004
The Bacterium Xenorhabdus nematophila Inhibits
Phospholipases A2 from Insect, Prokaryote, and
Vertebrate Sources
Youngjin Park
University of Nebraska-Lincoln
Yonggyun Kim
Andong National University, hosanna@andong.ac.kr
David Stanley
University of Nebraska-Lincoln, dstanley1@unl.edu
Follow this and additional works at: http://digitalcommons.unl.edu/entomologyfacpub
Part of the Entomology Commons
This Article is brought to you for free and open access by the Entomology, Department of at DigitalCommons@University of Nebraska - Lincoln. It has
been accepted for inclusion in Faculty Publications: Department of Entomology by an authorized administrator of DigitalCommons@University of
Nebraska - Lincoln.
Park, Youngjin; Kim, Yonggyun; and Stanley, David, "The Bacterium Xenorhabdus nematophila Inhibits Phospholipases A2 from Insect,
Prokaryote, and Vertebrate Sources" (2004). Faculty Publications: Department of Entomology. 452.
http://digitalcommons.unl.edu/entomologyfacpub/452
 
 
 
Published in Naturwissenschaften 91 (2004), pp. 371–373; doi: 10.1007/s00114-004-0548-2. 
Copyright © 2004 Springer-Verlag. Used by permission. 
Submitted February 17, 2004; accepted June 19, 2004; published online July 24, 2004. 
 
 
The Bacterium Xenorhabdus nematophila 
Inhibits Phospholipases A2 from 
Insect, Prokaryote, and Vertebrate Sources 
 
 
Youngjin Park,1 Yonggyun Kim,2 and David Stanley1 
 
1. Insect Biochemical Physiology Laboratory, University of Nebraska, Lincoln, NE, 68583-0816, USA 
2. School of Bioresource Sciences, Andong National University, 760-749 Andong, South Korea 
 
Corresponding author – David Stanley, email dstanley1@unl.edu 
 
Abstract 
The bacterium Xenorhabdus nematophila is a virulent insect pathogen. Part of its pathogenicity is due 
to impairing cellular immunity by blocking biosynthesis of eicosanoids, the major recognized signal 
transduction system in insect cellular immunity. X. nematophila inhibits the first step in eicosanoid 
biosynthesis, phospholipase A2 (PLA2). Here we report that the bacterium inhibits PLA2 from two 
insect immune tissues, hemocytes and fat body, as well as PLA2s selected to represent a wide range 
of organisms, including prokaryotes, insects, reptiles, and mammals. Our finding on a bacterial in-
hibitor of PLA2 activity contributes new insight into the chemical ecology of microbe-host interac-
tions, which usually involve actions rather than inhibitors of PLA2s. 
 
Introduction 
 
Insect innate immune systems are categorized into humoral (induced biosynthesis of anti-
bacterial proteins) and cellular (direct interactions between invaders and circulating he-
mocytes) immune functions (Stanley 2004). While these systems provide effective protec-
tion from many invaders, some microbes are very potent insect pathogens because they 
are able to overcome or evade innate immune reactions. The Gram-negative enterobacte-
rium Xenorhabdus nematophila is an intestinal symbiont of the insect pathogenic nematode 
Steinernema carpocapsae. X. nematophila enters insects with its host nematode, which defe-
cates X. nematophila cells into the insect hemolymph, where they multiply and quickly kill 
PA R K ,  K I M ,  A N D  S T A N L E Y ,  N A T U R W I S S E N S C H A F T E N  9 1  ( 2 0 0 4 )  
2 
the insect. The freshly killed insect becomes an ideal microenvironment for nematode re-
production and development, from which a new generation of nematodes emerges in 
search of other hosts (Park and Kim 2000). 
X. nematophila is an extremely virulent insect pathogen. Infections of less than 40 bacte-
rial cells are lethal to tobacco hornworms, Manduca sexta (Forst et al. 1997) and beet 
armyworms, Spodoptera exigua (Park et al. 1999). Part of the virulence is due to bacterial 
factors which inhibit insect immune reactions to infection. X. nematophila is cytotoxic to 
insect hemocytes and inhibits prophenoloxidase activation (Dunphy and Webster 1991). 
More recently, Park and Kim (2000; Park et al. 2003) indicated that X. nematophila impairs 
cellular immune reactions by inhibiting biosynthesis of prostaglandins and other eico-
sanoids—signal moieties responsible for mobilizing insect cellular immunity (Stanley 
2004). 
Eicosanoid biosynthesis begins with action of a PLA2, which hydrolyzes arachidonic 
acid from cellular phospholipids (Six and Dennis 2000). In insect systems, bacterial infec-
tion stimulates increased hemocytic PLA2 activity (Tunaz et al. 2003). We recently learned 
that factors in X. nematophila culture medium directly inhibit insect hemocytic PLA2 activ-
ity (Park et al. 2004). PLA2s make up an enzyme superfamily of extraordinarily broad bio-
logical significance, including digestion, pathophysiology, phospholipid remodeling, cell 
signaling, and down-regulating cell signals (for example by hydrolyzing platelet-activating 
factor; Six and Dennis 2000). PLA2s also act in organismal-level interactions, such as venom 
toxicity, microbial virulence, and host defense. Given the broad biological significance of 
these enzymes, microbes which produce PLA2 inhibitors have potential to influence a wide 
range of PLA2-mediated interspecific relationships. To consider this, we tested the idea 
that the X. nematophila factors responsible for inhibiting insect hemocyte PLA2s also inhibit 
PLA2s from other sources. 
 
Materials and methods 
 
Radioactive phosphatidylcholine [1-palmitol, 2-arachidonyl (arachidonyl-1-14C), 1.9 GBq/ 
mmol] was purchased from DuPont New England Nuclear (Wilmington, Delaware). An-
alytical grade organic solvents were from Fisher (Fair Lawn, New Jersey). Arachidonic 
acid, EGTA and PLA2s from Streptomyces violaceoruber, porcine pancreas, and from snake 
Naja mossambica and honey bee Apis mellifera venoms were purchased from Sigma (St. 
Louis, Missouri). 
Tobacco hornworms, Manduca sexta, were reared from eggs (surface sterilized in 0.525% 
bleach) in individual, sealed 60-ml portion cups charged with sterilized culture medium 
(Park et al. 2003). Early fifth-stadium hornworms were used in all experiments. 
The insect pathogenic bacterium, Xenorhabdus nematophila (strain K1), was cultured in 
tryptic soy agar (Difco, USA) at 28°C for 48 h. Bacteria were cultured in 25 ml of media in 
50-ml flasks in a rotary shaker at 100 rpm and used in stationary phase. After washing the 
bacterial cells three times with sterile phosphate-buffered saline (10 mM phosphate buffer 
with 0.8% NaCl, pH 7.4), the cells were suspended in 1 ml of PBS as already described 
(Park et al. 1999). The suspended cells (10 μl; 108 cfu/ml) were used to assess the influence 
of bacterial cells on fat-body PLA2 activity. 
PA R K ,  K I M ,  A N D  S T A N L E Y ,  N A T U R W I S S E N S C H A F T E N  9 1  ( 2 0 0 4 )  
3 
The X. nematophila PLA2-inhibitory factors were prepared from overnight cultures (suf-
ficient for inhibitory factor secretion) in the same conditions as described above. The cul-
ture medium was centrifuged for 30 min at 10,000 g. The cell-free supernatant was mixed 
with an equal volume of butanol, then separated into organic and aqueous fractions. The 
organic fractions from three extraction steps were combined and dried on a rotary evapo-
rator at 40°C for 5 min. The organic extract was taken up in 1 ml of 50% EtOH; 10-μl ali-
quots were used to assess the influence of the organic extract on fat-body PLA2 activity. 
To fractionate the organic extract, the butanol was applied to a silica gel column (30 cm 
× 1 cm column, 70–230 mesh gel, Merck, Germany) and eluted in 20 steps (50 ml/step) with 
a linear concentration gradient of chloroform/methanol from 99:1 to 0:100 (v/v). Each frac-
tion was dried, taken up in 1 ml of 50% ethanol, and 10-μl aliquots were assayed for the 
presence PLA2-inhibitory activity. PLA2-inhibitory activity was obtained in fractions 15 
and 17 and these were designated ORG-1 and ORG-2 for the experiments reported here. 
Determination of PLA2 activities followed the protocols used by Tunaz et al. (2003) in 
which the release of radiolabeled arachidonic acid from phosphatidylcholine [1-palmitol, 
2-arachidonyl (arachidonyl-1-14C)] substrate was monitored by TLC. The PLA2 assay was 
first optimized with respect to protein (100 mg optimal) and substrate concentrations (0.05 
μCi optimal), reaction time (30 min optimal) and the optimal parameters were used in 
routine assays. 
 
Results 
 
Suspensions of X. nematophila and the organic extract prepared from the bacterial culture 
medium severely inhibited PLA2 activity in tobacco hornworm fat-body preparations (see 
fig. 1; F = 46.35, df = 2,6, P = 0.0002). We recorded approximately 15 pmol/mg per min of 
PLA2 activity in control preparations, which was reduced to approximately one-third that 
value in the presence of bacterial or organic extract preparations. After fractionating the 
organic extract into 20 fractions, we found that PLA2 activity was severely curtailed in re-
actions conducted in the presence of ORG-1 and ORG-2. In addition to the inhibition of the 
fat-body PLA2 activity, PLA2 activity from four other sources was severely inhibited in 
reactions conducted in the presence of ORG-2 (see fig. 2; F = 118.13, df = 7,16, P = 0.0001). 
These PLA2 sources represent secretory PLA2s from a mammal (porcine pancreas), a reptile 
venom (snake, N. mossambica), an insect venom (honey bee, A. mellifera), and a prokaryote 
(bacterium, S. vilaceoruber). 
  
PA R K ,  K I M ,  A N D  S T A N L E Y ,  N A T U R W I S S E N S C H A F T E N  9 1  ( 2 0 0 4 )  
4 
 
 
Figure 1. Inhibition of M. sexta fat-body PLA2 activity in reactions carried out in the pres-
ence of X. nematophila (XN), or the organic extract of the bacterial culture medium (ORG), 
or two fractions prepared from ORG (ORG-1 and ORG-2). Each histogram bar (solid fill 
for controls and unfilled for experimental treatments) displays mean PLA2 activity 
(pmol/mg protein per min); the error bars represent 1 SD. Bars with the same superscript 
letter are not significantly different from each other (LSD, P < 0.05). 
 
 
 
Figure 2. The influence of ORG-2 on PLA2 activity of commercial enzymes prepared from 
porcine pancreas (PP), honey bee venom (HB), Streptomyces vilaceoruber (SV), and Naja 
mossambica (NM). Each histogram bar (solid fill for controls and unfilled for reactions con-
ducted in the presence of ORG-2) displays mean PLA2 activity (pmol/mg protein per min); 
the error bars represent 1 SD. Bars with the same superscript letter are not significantly 
different from each other (LSD, P < 0.05). 
PA R K ,  K I M ,  A N D  S T A N L E Y ,  N A T U R W I S S E N S C H A F T E N  9 1  ( 2 0 0 4 )  
5 
Discussion 
 
The bacterium X. nematophila secretes PLA2-inhibitory factors extractable from the culture 
medium into organic solvents. Our preliminary isolation work indicates the presence of 
two organic soluble factors. These factors inhibit the tobacco hornworm hemocytic PLA2 
(Park et al. 2004), the hornworm fat-body PLA2, and secretory PLA2s from porcine pan-
creas, the bacterium S. vilaceoruber, and venoms from the honey bee, A. mellifera, and the 
spitting cobra, N. mosambica. The X. nematophila factors are inhibitory to PLA2s from a wide 
phylogenetic range of organisms, including prokaryotes, arthropods, reptiles, and insects. 
The significance of this finding lies in the recognition that PLA2s act in many organismal 
interactions. The primary function of the X. nematophila PLA2-inhibitory factors seems to 
be impeding the biosynthesis of eicosanoids and thereby impairing insect cellular immune 
reactions to the presence of the bacterium. Impairment of host immune reactions may also 
be adaptive for the bacterium’s symbiotic partner, the nematode, which could otherwise 
be isolated and killed by hemocytic encapsulation reactions. Beyond this, microbes pro-
duce and secrete many chemicals which act against other microbes. PLA2s number among 
microbial products that can act against other microbes and also exert their toxic influences 
on hosts. Because the nematodes grow better in insect cadavers in which most of the living 
bacteria are X. nematophila (Forst et al. 1997), we suppose the X. nematophila PLA2-inhibitory 
factors also serve in reducing the numbers of other microbes. 
Part of the virulence of many microbes, including viruses (Canaan et al. 2004), fungi 
(Nakashima et al. 2003), and bacteria (Phillips et al. 2003), involves secretion of toxic mi-
crobial PLA2s; however, the details of PLA2-mediated microbe-host interactions can be 
complex. At least some toxic PLA2s are thought to exert their influence via receptor-mediated 
events (Cupillard et al. 1999) and via direct cell damage. A new variation on this theme 
was recently discovered. A PLA2 from the pathogenic bacterium Pseudomonas aeruginosa 
(the cytotoxin ExoU) requires the presence of host cell factors for activation (Phillips et al. 
2003). Hence, the pathogenicity expressed via ExoU requires cooperation with the injured 
host. While microbial PLA2s can be toxic, mammalian hosts also produce and secrete PLA2s 
which are bactericidal via hydrolysis of the outer membrane of Gram-positive bacteria 
(Dubouix et al. 2003). We infer that a great deal of new information will emerge on PLA2-
mediated microbe-host interactions. The idea that a bacterial species produces and secretes 
PLA2-inhibitory factors would be seen as still another variation on the theme. 
Finally, we suspect that secretion of broad-spectrum PLA2 inhibitors will not be re-
stricted to representatives of other Xenorhabdus and closely related Photorhabdus species. 
 
Acknowledgments – This is paper number 14554 of the Nebraska Agricultural Research Division 
(Project NEB-17-054). Support is acknowledged from NSF Grant No. 0201376 and by the postdoctoral 
fellowship program of the Korean Science and Engineering Foundation (KOSEF). All experiments 
reported in this paper were conducted in strict accordance with current laws in the United States of 
America. 
  
PA R K ,  K I M ,  A N D  S T A N L E Y ,  N A T U R W I S S E N S C H A F T E N  9 1  ( 2 0 0 4 )  
6 
References 
 
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Group IB and Group IIA secreted phospholipases A2 are natural ligands of the mouse 180-kDa 
M-type receptor. J Biol Chem 274:7043–7051. 
Dubouix A, Campanac C, Fauvel J, Simon MF, Salles JP, Roques C, Chap H, Marty N (2003) Bacteri-
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Dunphy GB, Webster JM (1991) Antihemocytic surface components of Xenorhabdus nematophilus var. 
dutki and their modification by serum of nonimmune larvae of Galleria mellonella. J Invertebr 
Pathol 58:40–51. 
Forst S, Dowds B, Boemare N, Stackebrandt E (1997) Xenorhabdus and Photorhabdus spp.: bugs that 
kill bugs. Annu Rev Microbiol 51:47–72. 
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Secretory phospholipases A2 induce neurite outgrowth in PC12 cells. Biochem J 376:655–666. 
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the symbiotic bacteria to the entomopathogenic nematode, Steinernema carpocapsae. J Insect Phys-
iol 46:1469–1476. 
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with Steinernema carpocapsae in Korea. J Asia-Pacific Entomol 2:105–111. 
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sexta. J Invertebr Pathol. DOI: 10.1016/ j.jip.2004.05.002. 
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and characterization. Biochim Biophys Acta 1488:1–19. 
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The Bacterium \u3ci\u3eXenorhabdus nematophila\u3c/i\u3e Inhibits Phospholipases A\u3csub\u3e2\u3c/sub\u3e from Insect, Prokaryote, and Vertebrate Sources

DigitalCommons@University of Nebraska

University of Nebraska - LincolnDigitalCommons@University of Nebraska - LincolnFaculty Publications: Department of Entomology Entomology, Department of2004The Bacterium Xenorhabdus nematophila InhibitsPhospholipases A2 from Insect, Prokaryote, andVertebrate SourcesYoungjin ParkUniversity of Nebraska-LincolnYonggyun KimAndong National University, hosanna@andong.ac.krDavid StanleyUniversity of Nebraska-Lincoln, dstanley1@unl.eduFollow this and additional works at: http://digitalcommons.unl.edu/entomologyfacpubPart of the Entomology CommonsThis Article is brought to you for free and open access by the Entomology, Department of at DigitalCommons@University of Nebraska - Lincoln. It hasbeen accepted for inclusion in Faculty Publications: Department of Entomology by an authorized administrator of DigitalCommons@University ofNebraska - Lincoln.Park, Youngjin; Kim, Yonggyun; and Stanley, David, "The Bacterium Xenorhabdus nematophila Inhibits Phospholipases A2 from Insect,Prokaryote, and Vertebrate Sources" (2004). Faculty Publications: Department of Entomology. 452.http://digitalcommons.unl.edu/entomologyfacpub/452   Published in Naturwissenschaften 91 (2004), pp. 371–373; doi: 10.1007/s00114-004-0548-2. Copyright © 2004 Springer-Verlag. Used by permission. Submitted February 17, 2004; accepted June 19, 2004; published online July 24, 2004.   The Bacterium Xenorhabdus nematophila Inhibits Phospholipases A2 from Insect, Prokaryote, and Vertebrate Sources   Youngjin Park,1 Yonggyun Kim,2 and David Stanley1  1. Insect Biochemical Physiology Laboratory, University of Nebraska, Lincoln, NE, 68583-0816, USA 2. School of Bioresource Sciences, Andong National University, 760-749 Andong, South Korea  Corresponding author – David Stanley, email dstanley1@unl.edu  Abstract The bacterium Xenorhabdus nematophila is a virulent insect pathogen. Part of its pathogenicity is due to impairing cellular immunity by blocking biosynthesis of eicosanoids, the major recognized signal transduction system in insect cellular immunity. X. nematophila inhibits the first step in eicosanoid biosynthesis, phospholipase A2 (PLA2). Here we report that the bacterium inhibits PLA2 from two insect immune tissues, hemocytes and fat body, as well as PLA2s selected to represent a wide range of organisms, including prokaryotes, insects, reptiles, and mammals. Our finding on a bacterial in-hibitor of PLA2 activity contributes new insight into the chemical ecology of microbe-host interac-tions, which usually involve actions rather than inhibitors of PLA2s.  Introduction  Insect innate immune systems are categorized into humoral (induced biosynthesis of anti-bacterial proteins) and cellular (direct interactions between invaders and circulating he-mocytes) immune functions (Stanley 2004). While these systems provide effective protec-tion from many invaders, some microbes are very potent insect pathogens because they are able to overcome or evade innate immune reactions. The Gram-negative enterobacte-rium Xenorhabdus nematophila is an intestinal symbiont of the insect pathogenic nematode Steinernema carpocapsae. X. nematophila enters insects with its host nematode, which defe-cates X. nematophila cells into the insect hemolymph, where they multiply and quickly kill PA R K ,  K I M ,  A N D  S T A N L E Y ,  N A T U R W I S S E N S C H A F T E N  9 1  ( 2 0 0 4 )  2 the insect. The freshly killed insect becomes an ideal microenvironment for nematode re-production and development, from which a new generation of nematodes emerges in search of other hosts (Park and Kim 2000). X. nematophila is an extremely virulent insect pathogen. Infections of less than 40 bacte-rial cells are lethal to tobacco hornworms, Manduca sexta (Forst et al. 1997) and beet armyworms, Spodoptera exigua (Park et al. 1999). Part of the virulence is due to bacterial factors which inhibit insect immune reactions to infection. X. nematophila is cytotoxic to insect hemocytes and inhibits prophenoloxidase activation (Dunphy and Webster 1991). More recently, Park and Kim (2000; Park et al. 2003) indicated that X. nematophila impairs cellular immune reactions by inhibiting biosynthesis of prostaglandins and other eico-sanoids—signal moieties responsible for mobilizing insect cellular immunity (Stanley 2004). Eicosanoid biosynthesis begins with action of a PLA2, which hydrolyzes arachidonic acid from cellular phospholipids (Six and Dennis 2000). In insect systems, bacterial infec-tion stimulates increased hemocytic PLA2 activity (Tunaz et al. 2003). We recently learned that factors in X. nematophila culture medium directly inhibit insect hemocytic PLA2 activ-ity (Park et al. 2004). PLA2s make up an enzyme superfamily of extraordinarily broad bio-logical significance, including digestion, pathophysiology, phospholipid remodeling, cell signaling, and down-regulating cell signals (for example by hydrolyzing platelet-activating factor; Six and Dennis 2000). PLA2s also act in organismal-level interactions, such as venom toxicity, microbial virulence, and host defense. Given the broad biological significance of these enzymes, microbes which produce PLA2 inhibitors have potential to influence a wide range of PLA2-mediated interspecific relationships. To consider this, we tested the idea that the X. nematophila factors responsible for inhibiting insect hemocyte PLA2s also inhibit PLA2s from other sources.  Materials and methods  Radioactive phosphatidylcholine [1-palmitol, 2-arachidonyl (arachidonyl-1-14C), 1.9 GBq/ mmol] was purchased from DuPont New England Nuclear (Wilmington, Delaware). An-alytical grade organic solvents were from Fisher (Fair Lawn, New Jersey). Arachidonic acid, EGTA and PLA2s from Streptomyces violaceoruber, porcine pancreas, and from snake Naja mossambica and honey bee Apis mellifera venoms were purchased from Sigma (St. Louis, Missouri). Tobacco hornworms, Manduca sexta, were reared from eggs (surface sterilized in 0.525% bleach) in individual, sealed 60-ml portion cups charged with sterilized culture medium (Park et al. 2003). Early fifth-stadium hornworms were used in all experiments. The insect pathogenic bacterium, Xenorhabdus nematophila (strain K1), was cultured in tryptic soy agar (Difco, USA) at 28°C for 48 h. Bacteria were cultured in 25 ml of media in 50-ml flasks in a rotary shaker at 100 rpm and used in stationary phase. After washing the bacterial cells three times with sterile phosphate-buffered saline (10 mM phosphate buffer with 0.8% NaCl, pH 7.4), the cells were suspended in 1 ml of PBS as already described (Park et al. 1999). The suspended cells (10 μl; 108 cfu/ml) were used to assess the influence of bacterial cells on fat-body PLA2 activity. PA R K ,  K I M ,  A N D  S T A N L E Y ,  N A T U R W I S S E N S C H A F T E N  9 1  ( 2 0 0 4 )  3 The X. nematophila PLA2-inhibitory factors were prepared from overnight cultures (suf-ficient for inhibitory factor secretion) in the same conditions as described above. The cul-ture medium was centrifuged for 30 min at 10,000 g. The cell-free supernatant was mixed with an equal volume of butanol, then separated into organic and aqueous fractions. The organic fractions from three extraction steps were combined and dried on a rotary evapo-rator at 40°C for 5 min. The organic extract was taken up in 1 ml of 50% EtOH; 10-μl ali-quots were used to assess the influence of the organic extract on fat-body PLA2 activity. To fractionate the organic extract, the butanol was applied to a silica gel column (30 cm × 1 cm column, 70–230 mesh gel, Merck, Germany) and eluted in 20 steps (50 ml/step) with a linear concentration gradient of chloroform/methanol from 99:1 to 0:100 (v/v). Each frac-tion was dried, taken up in 1 ml of 50% ethanol, and 10-μl aliquots were assayed for the presence PLA2-inhibitory activity. PLA2-inhibitory activity was obtained in fractions 15 and 17 and these were designated ORG-1 and ORG-2 for the experiments reported here. Determination of PLA2 activities followed the protocols used by Tunaz et al. (2003) in which the release of radiolabeled arachidonic acid from phosphatidylcholine [1-palmitol, 2-arachidonyl (arachidonyl-1-14C)] substrate was monitored by TLC. The PLA2 assay was first optimized with respect to protein (100 mg optimal) and substrate concentrations (0.05 μCi optimal), reaction time (30 min optimal) and the optimal parameters were used in routine assays.  Results  Suspensions of X. nematophila and the organic extract prepared from the bacterial culture medium severely inhibited PLA2 activity in tobacco hornworm fat-body preparations (see fig. 1; F = 46.35, df = 2,6, P = 0.0002). We recorded approximately 15 pmol/mg per min of PLA2 activity in control preparations, which was reduced to approximately one-third that value in the presence of bacterial or organic extract preparations. After fractionating the organic extract into 20 fractions, we found that PLA2 activity was severely curtailed in re-actions conducted in the presence of ORG-1 and ORG-2. In addition to the inhibition of the fat-body PLA2 activity, PLA2 activity from four other sources was severely inhibited in reactions conducted in the presence of ORG-2 (see fig. 2; F = 118.13, df = 7,16, P = 0.0001). These PLA2 sources represent secretory PLA2s from a mammal (porcine pancreas), a reptile venom (snake, N. mossambica), an insect venom (honey bee, A. mellifera), and a prokaryote (bacterium, S. vilaceoruber).   PA R K ,  K I M ,  A N D  S T A N L E Y ,  N A T U R W I S S E N S C H A F T E N  9 1  ( 2 0 0 4 )  4   Figure 1. Inhibition of M. sexta fat-body PLA2 activity in reactions carried out in the pres-ence of X. nematophila (XN), or the organic extract of the bacterial culture medium (ORG), or two fractions prepared from ORG (ORG-1 and ORG-2). Each histogram bar (solid fill for controls and unfilled for experimental treatments) displays mean PLA2 activity (pmol/mg protein per min); the error bars represent 1 SD. Bars with the same superscript letter are not significantly different from each other (LSD, P < 0.05).    Figure 2. The influence of ORG-2 on PLA2 activity of commercial enzymes prepared from porcine pancreas (PP), honey bee venom (HB), Streptomyces vilaceoruber (SV), and Naja mossambica (NM). Each histogram bar (solid fill for controls and unfilled for reactions con-ducted in the presence of ORG-2) displays mean PLA2 activity (pmol/mg protein per min); the error bars represent 1 SD. Bars with the same superscript letter are not significantly different from each other (LSD, P < 0.05). PA R K ,  K I M ,  A N D  S T A N L E Y ,  N A T U R W I S S E N S C H A F T E N  9 1  ( 2 0 0 4 )  5 Discussion  The bacterium X. nematophila secretes PLA2-inhibitory factors extractable from the culture medium into organic solvents. Our preliminary isolation work indicates the presence of two organic soluble factors. These factors inhibit the tobacco hornworm hemocytic PLA2 (Park et al. 2004), the hornworm fat-body PLA2, and secretory PLA2s from porcine pan-creas, the bacterium S. vilaceoruber, and venoms from the honey bee, A. mellifera, and the spitting cobra, N. mosambica. The X. nematophila factors are inhibitory to PLA2s from a wide phylogenetic range of organisms, including prokaryotes, arthropods, reptiles, and insects. The significance of this finding lies in the recognition that PLA2s act in many organismal interactions. The primary function of the X. nematophila PLA2-inhibitory factors seems to be impeding the biosynthesis of eicosanoids and thereby impairing insect cellular immune reactions to the presence of the bacterium. Impairment of host immune reactions may also be adaptive for the bacterium’s symbiotic partner, the nematode, which could otherwise be isolated and killed by hemocytic encapsulation reactions. Beyond this, microbes pro-duce and secrete many chemicals which act against other microbes. PLA2s number among microbial products that can act against other microbes and also exert their toxic influences on hosts. Because the nematodes grow better in insect cadavers in which most of the living bacteria are X. nematophila (Forst et al. 1997), we suppose the X. nematophila PLA2-inhibitory factors also serve in reducing the numbers of other microbes. Part of the virulence of many microbes, including viruses (Canaan et al. 2004), fungi (Nakashima et al. 2003), and bacteria (Phillips et al. 2003), involves secretion of toxic mi-crobial PLA2s; however, the details of PLA2-mediated microbe-host interactions can be complex. At least some toxic PLA2s are thought to exert their influence via receptor-mediated events (Cupillard et al. 1999) and via direct cell damage. A new variation on this theme was recently discovered. A PLA2 from the pathogenic bacterium Pseudomonas aeruginosa (the cytotoxin ExoU) requires the presence of host cell factors for activation (Phillips et al. 2003). Hence, the pathogenicity expressed via ExoU requires cooperation with the injured host. While microbial PLA2s can be toxic, mammalian hosts also produce and secrete PLA2s which are bactericidal via hydrolysis of the outer membrane of Gram-positive bacteria (Dubouix et al. 2003). We infer that a great deal of new information will emerge on PLA2-mediated microbe-host interactions. The idea that a bacterial species produces and secretes PLA2-inhibitory factors would be seen as still another variation on the theme. Finally, we suspect that secretion of broad-spectrum PLA2 inhibitors will not be re-stricted to representatives of other Xenorhabdus and closely related Photorhabdus species.  Acknowledgments – This is paper number 14554 of the Nebraska Agricultural Research Division (Project NEB-17-054). Support is acknowledged from NSF Grant No. 0201376 and by the postdoctoral fellowship program of the Korean Science and Engineering Foundation (KOSEF). All experiments reported in this paper were conducted in strict accordance with current laws in the United States of America.   PA R K ,  K I M ,  A N D  S T A N L E Y ,  N A T U R W I S S E N S C H A F T E N  9 1  ( 2 0 0 4 )  6 References  Canaan S, Zadori Z, Ghomashchi F, Bollinger J, Sadilek M, Moreau ME, Tijssen P, Gelb MH (2004) Interfacial enzymology of parvovirus phospholipases A2. J Biol Chem 279:14502–14508. Cupillard L, Mulherkar R, Gomez N, Kadam S, Valentin E, Lazdunski M, Lambeau G (1999) Both Group IB and Group IIA secreted phospholipases A2 are natural ligands of the mouse 180-kDa M-type receptor. J Biol Chem 274:7043–7051. 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Park Y, Kim Y, Yi Y (1999) Identification and characterization of a symbiotic bacterium associated with Steinernema carpocapsae in Korea. J Asia-Pacific Entomol 2:105–111. Park Y, Kim Y, Putnam SM, Stanley DW (2003) The bacterium Xenorhabdus nematophilus depresses nodulation reactions to infection by inhibiting eicosanoid biosynthesis in tobacco hornworms, Manduca sexta. Arch Insect Biochem Physiol 52:71–80. Park Y, Kim Y, Tunaz H, Stanley DW (2004) An entomopathogenic bacterium, Xenorhabdus nema-tophila, inhibits hemocytic phospholipase A2 (PLA2) activity in tobacco hornworms, Manduca sexta. J Invertebr Pathol. DOI: 10.1016/ j.jip.2004.05.002. Phillips RM, Six DA, Dennis EA, Ghosh P (2003) In vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors. J Biol Chem 278:41326–41332. Six DA, Dennis EA (2000) The expanding superfamily of phospholipase A2 enzymes: classification and characterization. Biochim Biophys Acta 1488:1–19. Stanley DW (2004) Prostaglandins and other eicosanoids. In: Gilbert LI, Iatrou K, Gill S (eds) Com-prehensive insect physiology, biochemistry, pharmacology and molecular biology. Elsevier, Ox-ford. Tunaz H, Park Y, Buyukguzel K, Bedick JC, Nor Aliza AR, Stanley DW (2003) Eicosanoids in insect immunity: bacterial infection stimulates hemocytic phospholipase A2 activity in tobacco horn-worms. Arch Insect Biochem Physiol 52:1–6. 

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The Bacterium \u3ci\u3eXenorhabdus nematophila\u3c/i\u3e Inhibits Phospholipases A\u3csub\u3e2\u3c/sub\u3e from Insect, Prokaryote, and Vertebrate Sources

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