Colloquium

Plants and animals share functionally commonbacterial virulence factorsLaurence G. Rahme*†‡, Frederick M. Ausubel§, Hui Cao†‡, Eliana Drenkard§, Boyan C. Goumnerov†‡, Gee W. Lau†‡,Shalina Mahajan-Miklos§¶, Julia Plotnikova§, Man-Wah Tan§i, John Tsongalis‡, Cynthia L. Walendziewicz‡,and Ronald G. Tompkins†‡

†Department of Surgery, Harvard Medical School, ‡Department of Surgery, Massachusetts General Hospital and Shriner’s Burn Institute, Boston, MA 02114;§Department of Genetics, Harvard Medical School, and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114; andiSociety of Fellows, Harvard University, 78 Mount Auburn Street, Cambridge, MA 02138

By exploiting the ability of Pseudomonas aeruginosa to infect avariety of vertebrate and nonvertebrate hosts, we have developedmodel systems that use plants and nematodes as adjuncts tomammalian models to help elucidate the molecular basis of P.aeruginosa pathogenesis. Our studies reveal a remarkable degreeof conservation in the virulence mechanisms used by P. aeruginosato infect hosts of divergent evolutionary origins.

Bacterial pathogens infect a wide variety of evolutionarydistinct hosts, including both lower and higher eukaryotes.

In all of these cases, the pathogen must have the ability torecognize, become associated with, exploit the nutrient reservesof, and combat the defense responses of its specific host. Toaccomplish these tasks, pathogens use an extensive arsenal ofvirulence-related factors.

Many pathogens cause disease in a single or limited numberof host species as a consequence of a long coevolutionary history.However, the interactions between host and pathogen that limithost range and determine host resistance or susceptibility arepoorly understood. Although many bacterial virulence compo-nents are thought to be host-specific, numerous studies havedemonstrated the existence of what appear to be universalvirulence mechanisms used by diverse bacterial species (1).Similarly, recent work has revealed common features underlyinghost defense responses against pathogens in plants, insects, andmammals (2). Thus, some of the underlying virulence mecha-nisms of pathogens, as well as the host defenses against them, arelikely to have ancient evolutionary origins preserved acrossphylogeny.

There remains a great deal to learn about the molecular natureof antagonistic encounters between pathogenic bacteria andtheir hosts. Despite our limited knowledge, significant advanceshave been made in developing techniques that facilitate ourunderstanding of virulence mechanisms and the critical role ofthe host during pathogenesis (3). Our laboratories have devel-oped a technique, which we refer to as multihost pathogenesis,to study pathogens that cause disease in both vertebrate andnonvertebrate hosts (4–8).

Epidemiological studies carried out in the 1970s suggested thatclinical isolates of Pseudomonas aeruginosa might be capable ofcausing disease in plants (9, 10). Based on this premise, wedeveloped a model pathogenesis system by using a humanclinical isolate of P. aeruginosa, strain UCBPP-PA14 (PA14) thatelicits disease in plants, nematodes, insects, and mice (4–7, 11).P. aeruginosa is the most common causative organism of sepsisin burned patients and the leading cause of pulmonary infectionsand mortality in cystic fibrosis patients (12). In addition, thisimportant human opportunistic pathogen infects injured, immu-nodeficient, or otherwise compromised individuals (13). Thepathophysiology of infections caused by P. aeruginosa is complex

as shown by the clinical diversity of diseases associated with thisorganism and the multiplicity of virulence factors it produces.Although a natural soil inhabitant, P. aeruginosa is versatile in itsmetabolic potential, which allows it to survive in a variety ofnatural and hospital environments. It appears that the combi-nation of environmental persistence, versatility in virulencemechanisms, and multiple virulence factors allows P. aeruginosato be effective both as an opportunistic human pathogen and asa plant pathogen.

None of the currently available animal models fully mimics allaspects of any single human disease caused by P. aeruginosa.Moreover, it is not practical to use any current vertebrate animalmodel to systematically screen for pathogenicity-related P.aeruginosa mutants. This latter problem has become acutelyapparent as the Pseudomonas Genome Project nears comple-tion, and the relevance of thousands of P. aeruginosa genes tohuman pathogenesis needs to be determined. In contrast tovertebrate animal models, host-pathogen interaction modelsthat use nonvertebrate hosts permit genomewide screening forP. aeruginosa virulence factors required for pathogenesis. Ifthere is substantial overlap in the virulence factors required forpathogenesis in invertebrate and mammalian hosts, large-scalescreening in invertebrate hosts can become an efficient methodfor identifying previously unknown virulence-related genes rel-evant to human pathogenesis.

Using P. aeruginosa strain PA14, we provided direct evi-dence that P. aeruginosa uses a shared subset of virulencefactors to elicit disease in both plants (Arabidopsis thaliana)and animals (mice) (4, 11). More recently, we extended theArabidopsis-P. aeruginosa model to additional nonvertebratehosts, including the nematode Caenorhabditis elegans (7, 14,15) and the insects Galleria melonella (8) and Drosophilamelanogaster (S.M.-M., G.W.L., L. Perkins, F.M.A., andL.G.R., unpublished data).

This paper summarizes the use of a plant pathogenesis modelto identify previously unknown virulence factors and highlightsthe remarkable conservation in the virulence mechanisms usedby P. aeruginosa to infect evolutionary divergent hosts.

P. aeruginosa PathogenesisDespite quite detailed knowledge about some of the extracel-lular proteins and several surface-associated components syn-thesized by P. aeruginosa, our understanding of P. aeruginosa

This paper was presented at the National Academy of Sciences colloquium ‘‘Virulence andDefense in Host-Pathogen Interactions: Common Features Between Plants and Animals,’’held December 9–11, 1999, at the Arnold and Mabel Beckman Center in Irvine, CA.

*To whom reprint requests should be addressed. E-mail: rahme@frodo.mgh.harvard.edu.

¶Present address: Micrbobia, Inc., One Kendall Square, Building 1400W, Suite 1418, Cam-bridge, MA 02139.

PNAS u August 1, 2000 u vol. 97 u no. 16 u 8815–8821

COLL

OQ

UIU

M

Dow

nloa

ded

by g

uest

on

May

11,

202

0

infections is only rudimentary. Much remains to be learnedabout individual virulence factors essential for pathogenicity andthe mechanism by which these factors work together duringpathogenesis.

P. aeruginosa virulence factors facilitate tissue invasion andsystemic spread and include pili and flagella, endotoxins, exotoxins,vascular permeability factors, and a variety of excreted enzymes(16). P. aeruginosa proteases degrade a variety of host proteins andhave a direct destructive effect on skin tissue (17, 18). Elastase(LasB), a potent metalloprotease with broad substrate specificity,degrades host proteins, such as elastin, collagen, transferrin, Ig, andsome of the components of complement (19). The LasB elastaseacts in concert with both LasA protease and alkaline protease tocause efficient elastolysis, which is required for the tissue damageassociated with P. aeruginosa pathogenesis (20). The P. aeruginosaalginate capsule plays an essential role in chronic pulmonaryinfection in cystic fibrosis patients (21, 22). Finally, the P. aeruginosalipopolysaccharide also has been shown to be an important viru-lence factor (23).

The synthesis of several P. aeruginosa-secreted virulencefactors is transcriptionally regulated by environmental stimuli(24–26). For example, phospholipase C is regulated by theavailability of phosphate (27, 28), whereas exotoxin A andelastase are regulated by the concentration of iron in the growthmedium (29–32). Moreover, the production of a large number ofexoproducts, including elastase, alkaline protease, the LasAprotease, hemolysin, pyocyanin, and rhamnolipids is cell density-dependent and is regulated by the so-called quorum-sensingcascade (33). The P. aeruginosa quorum-sensing cascade ismediated by low molecular mass homoserine lactones that aresynthesized by the products of the lasI and rhlI genes. Theconcentration of these homoserine lactones is monitored by theproducts of the lasR and rhlR genes, which serve as globaltranscriptional activators of a variety of exoproducts relevant forP. aeruginosa pathogenesis. In addition to homoserine lactones,it recently has been shown that 2-heptyl-3-hydroxy-4-quinolonealso serves as an intracellular signal molecule involved in theactivation of pathogeneicity-related factors (34). This moleculebelongs to the 4-quinolone chemical family, best known for theantibiotic activity of many of its members.

In addition to regulating the production of a variety of specificvirulence-related factors, the quorum-sensing cascade as well asother cell-to-cell signaling mechanisms are involved in thedifferentiation of P. aeruginosa biofilms (35, 36), matrix-enclosedbacterial populations that attach to each other and to biotic, orabiotic surfaces (37). P. aeruginosa is protected from adverseenvironmental conditions and from antibacterial agents whilegrowing in this matrix-enclosed structure that has been impli-cated as a contributing factor in the persistence and severity ofP. aeruginosa infections. It is thought that when nutrient condi-tions become limiting, bacteria are shed from the biofilm andenter the planktonic (free-living) phase (37). In this manner,cells are able to colonize new habitats and form new biofilms.

Another quorum-sensing regulated virulence factor is pyocy-anin, a blue-green-colored phenazine (33, 38). This secondarymetabolite has antimicrobial activity against several species ofbacteria, fungi, and protozoa, a quality attributed to its redox-active potential (39). Although little is known about the natureof the enzymes that catalyze the formation of pyocyanin in P.aeruginosa, the conversion of chorismate to anthranilate isthought to be a key step in the pathway that is most likelycatalyzed by the anthranilate synthetase encoded by the phnAand phnB genes (40). Even though pyocyanin-induced freeradical production appears to be responsible for much of itsantimicrobial activity (41), the role of pyocyanin in P. aeruginosa-associated tissue injury is less clear.

Development of the Arabidopsis-P. aeruginosa ModelAs described briefly above, we developed the Arabidopsis-P.aeruginosa pathogenesis model based on the P. aeruginosa strainPA14. This was accomplished as follows. A collection of 75 P.aeruginosa strains, of which 30 were human isolates, wasscreened for its ability to elicit disease on leaves of at least fourdifferent Arabidopsis ecotypes (4). Several strains elicited dis-ease characterized by varying degrees of soft-rot symptoms in allfour ecotypes (wild varieties) tested. Importantly, two strains,PA14 (a human isolate) and UCBPP-PA29 (a plant isolate),caused severe soft-rot symptom in some, but not all of theecotypes tested. The finding that these two P. aeruginosa strainsexhibit ecotype specificity is typical of plant pathogen interac-

Fig. 1. Macroscopic and microscopic symptoms elicited by P. aeruginosa strain UCBPP-PA14 infiltrated into Arabidopsis leaves. (A) The upper part of anArabidopsis leaf (ecotype LL-O) was infiltrated with bacterial suspensions at a titer of 103 cfuycm2 and photographed 2 days postinfection. A chlorotic zone (whitearrow) surrounds the soft-rot symptoms. (B and C) Arabidopsis leaves infiltrated with bacterial suspension and stained with trypan blue 2 days postinfection.Whole leaves were examined by using a Zeiss Axioskop and photographed. Bacterial movement was observed along leaf veins (arrows indicate vessel parenchymafilled with bacteria). Bacteria are absent in xylem vessels (arrowheads).

8816 u www.pnas.org Rahme et al.

Dow

nloa

ded

by g

uest

on

May

11,

202

0

tions and reflects the selection of plant accessions (ecotypes)that are resistant to particular strains (races) of a pathogen.These observations support the notion that P. aeruginosa is anatural plant pathogen in the wild.

Fig. 1A illustrates the symptomatology observed on a suscep-tible 6-week-old Arabidopsis plant when the upper part of a leafis inoculated with P. aeruginosa strain PA14. The severe symp-toms elicited by PA14 include a water-soaked reaction zone andchlorosis (yellowing). Soft-rot symptoms begin as small water-soaked lesions, which enlarge rapidly in diameter. Completemaceration and collapse of the infiltrated leaf occurs 4–5 daysafter infection. As shown in Fig. 1B, trypan blue staining ofinfected leaves reveals the presence of P. aeruginosa in theintercellular spaces of the leaf as well as bacterial movementalong the veins and bacterial proliferation in vessel parenchymaand companion cells. No bacteria are detected in xylem. Fastpropagation of P. aeruginosa in the vessel parenchyma is theprelude to systemic infection and maceration of the entire plant.As illustrated in Fig. 2, detached leaves of Arabidopsis exposedto PA14 culture suspension show that P. aeruginosa cells attachto the leaf epidermis and congregate at the stomal openingswhere they gain entry to the leaf interior.

Similar to other phytopathogenic bacteria, P. aeruginosastrains that elicit disease symptoms are also able to proliferate in

Arabidopsis leaves. Table 1 lists the maximal levels of growthreached by several P. aeruginosa strains and PA14 mutants at thefourth day postinfection. The degree of proliferation was cor-related with the severity of disease symptoms. In each case,reduced symptoms were associated with reduced bacterialcounts in leaves (Table 1; ref. 4).

In our efforts to identify additional plant species susceptibleto P. aeruginosa infection, we found that soft-rot symptoms alsowere observed when PA14 was inoculated into the midrib oflettuce leaf stem (Fig. 3B). In addition to PA14, lettuce issusceptible to the well-characterized P. aeruginosa strains PAKand PA01. In later stages of infection, all three P. aeruginosastrains invade the entire midrib of lettuce leaves, resulting incomplete maceration and collapse of the tissue (Fig. 3B).

Pathogenicity of PA14 in a Mouse Burn ModelWe primarily have used a mouse full-thickness skin burn modelto assess PA14 pathogenicity in a mammalian host (42). Severalfactors facilitate the colonization and establishment of thebacteria in this model. First, the inoculum is delivered in a scaldeschar induced on the abdominal surface of an anesthetizedmouse. This method provides the bacteria with necrotic tissue asnutrients and with protection from the active components of theimmune system, because the circulation in this area is impeded.Second, it is well known that burn trauma itself induces ageneralized decrease in the efficiency of the immune system,which aids in the development of systemic infections and sepsis.The first signs of developing infection and sepsis can be observedclinically and histopathologically 12–24 h postinoculation. Mor-tality studies were conducted to assess the presence of systemicinfection (Table 1). Tissue biopsies from the site of inoculationwere obtained and compared to assess local invasiveness. Aspresented in Table 1, strains PA14 and PA29 as well the P.aeruginosa human isolates PA01 and PAK all proliferated andinvaded the muscle underlying the eschar, but strain PA14resulted in a higher mortality than the other strains. Animalsusually succumbed to the infection 36–48 h postinoculation.

We conducted a series of morphological studies to furtherinvestigate the dynamics of PA14 infections in the burned mousemodel. Specimens from control animals and from burned andinoculated animals were processed by standard histologic tech-niques. Inoculated mice were killed at varying time intervals andhistopathologic examination of parenchymal organs was under-taken. The findings revealed that a local inflammatory responsedeveloped during the first 24 h at the site of inoculation and thatsystemic infection, including multiple organ involvement, oc-curred between 24 and 36 h. Histologic sections removed fromlivers and kidneys 36 h postinoculation revealed massive perivas-cular bacterial invasion of the organs and associated tissue

Fig. 2. P. aeruginosa entering the mesophylic compartment of an Arabi-dopsis leaf through a stomatal opening. Detached Arabidopsis leaves weresoaked in a bacterial suspension at a titer of 103 cfuyml. At 24 h postinfection,the leaves were fixed in osmium tetroxide, followed by dehydration in etha-nol. The leaves then were coated in Technics Sputter Coater Hummer II andsubsequently examined and photographed by using an Amray (Bedford, MA)1000 scanning electron microscope. Bacteria are shown to be primarily con-centrated on the surface of guard cells, above the stoma.

Table 1. Animal mortality and proliferation studies of P. aeruginosa strains and isogenicUCBPP-PA14 mutants in Arabidopsis and in a mouse burned model

P. aeruginosa strain cfuycm2 leaf area*Mean titer 6 SD in biopsies

adjacent to burn†

% Mousemortality‡

UCBPP-PA14 2.6 3 107 6 2.0 3 107 6.0 3 107 6 2.1 3 107 77UCBPP-PA29 2.7 3 107 6 1.3 3 107 8.2 3 107 6 2.0 3 107 5PAK 6.0 3 104 6 3.0 3 104 6.0 3 107 6 1.2 3 107 20PAO1 8.0 3 105 6 2.7 3 105 4.0 3 107 6 1.8 3 107 25UCBPP-PA14 plcS 1.1 3 105 6 0.5 3 105 6.7 3 104 6 7.5 3 104 40UCBPP-PA14 toxA 1.5 3 106 6 1.0 3 106 1.4 3 108 6 1.7 3 108 40UCBPP-PA14 gacA 6.0 3 103 6 2.1 3 103 NT 0

For both quantitation and mortality studies, mice were injected with 5 3 104 cfuyml. NT, not determined.*Means of four samples 6 SD of maximum bacterial counts obtained at 5 days postinfection with 103 cells.†No viable bacterial cells were retrieved from the underlying rectus abdominus muscle immediately after bacterialinjection or in animals that received a sham injury in other studies.

‡The number of animals that died of sepsis was monitored each day for 10 days.

Rahme et al. PNAS u August 1, 2000 u vol. 97 u no. 16 u 8817

COLL

OQ

UIU

M

Dow

nloa

ded

by g

uest

on

May

11,

202

0

necrosis (Fig. 4). These studies demonstrate the ability of PA14to overcome the immune defense mechanisms in the host andestablish a generalized infection.

Shared Virulence Factors in Plant and Animal PathogenesisThe relevance of the Arabidopsis-P. aeruginosa model tomammalian pathogenesis initially was demonstrated by testingwhether two P. aeruginosa virulence factors, exotoxin A andphosopholipase C, thought to inf luence tissue invasion andsystemic spread in mammals (4), also play a role in Arabidopsispathogenesis. Exotoxin A (toxA) is a potent mammalianprotein synthesis inhibitor (43). Phospholipase C (plcS) pref-erentially degrades phospholipids found in eukaryotic cellmembranes (44), which leads to a systemic inf lammatoryresponse (45). Conversely, a known virulence determinantimportant for plant pathogenesis in Pseudomonas syringae,gacA, was tested for its role in mouse pathogenesis. GacAencodes a transcriptional activator of genes encoding extra-cellular products involved in pathogenicity (46). Isogenic PA14plcS, toxA, and gacA mutants were constructed by markerexchange, and the resulting mutants were tested for pathoge-nicity in the Arabidopsis leaf infiltration assay and in theburned mouse model. Decreased pathogenicity was observedin both hosts with all three of the isogenic PA14 mutants.Unlike the wild-type PA14, none of the mutants caused

maceration and collapse of Arabidopsis leaves, and the growthof the mutants was significantly reduced (4). Similarly, all threemutants exhibited significantly lower mortality than the wild-type strain in the mouse model. Consistent with its known rolein pathogenesis, the plcS mutant exhibited reduced ability toproliferate and invade the tissue adjacent to the burn area(Table 1). Subsequent to these experiments, it was shown thatGacA functions upstream of lasI-lasR and rhlI-rhlR quorum-sensing regulatory systems (47) to regulate virulence geneexpression (33) and type 4 pilus-mediated twitching motility inP. aeruginosa (48).

Use of Nonvertebrate Hosts to Identify Virulence-AssociatedGenes Involved in Mammalian PathogenesisThe observation that P. aeruginosa uses common virulence genesto infect both animals and plants led us to theorize thatpreviously unknown virulence determinants required for P.aeruginosa pathogenesis in animals could be identified by screen-ing randomly mutagenized PA14 clones for ones that exhibited

Fig. 3. (A) Strategy used to screen for P. aeruginosa virulence-associatedgenes. (B) Midribs of lettuce leaves were inoculated with 10 ml of a bacterialsuspension at a titer of 103 cfuyml and photographed 4 days postinfection.Bottom leaf was inoculated with the wild-type strain PA14 (black arrowhead)and top leaf was inoculated with the PA14-toxA mutant. Brown spots (whitearrowheads) on the midrib of the top leaf reveal inoculation sites.

Fig. 4. Histologic section of mouse liver and kidney taken 36 h postinocu-lation with P. aeruginosa strain PA14. (A) Massive perivascular bacterialinfiltration and associated necrosis of the surrounding hepatocytes (arrow)are visible in the liver section. Brown and Hopps (B&H) Gram stain at 340magnification. (B) Section of kidney from the same animal showing largebacterial infiltrates within the tubules of the renal medulla (arrow). B&H Gramstain at 340 magnification.

8818 u www.pnas.org Rahme et al.

Dow

nloa

ded

by g

uest

on

May

11,

202

0

decreased virulence in plants. This experimental approach over-comes a major limitation imposed by an animal model, that is,the large number of animals required to undertake a systematicgenetic screen for the identification of virulence-related genes.

P. aeruginosa strain PA14 was mutagenized with transposonTnphoA, and 2,500 prototrophic mutants were screened forimpaired virulence in a lettuce stem assay. In these high-throughput screens, we substituted Arabidopsis with lettucebecause several mutants could be tested on one lettuce midrib by‘‘tooth-picking’’ single colonies directly from plates that containthe mutant clones (Fig. 3; ref. 5). Following the methodologydepicted in Fig. 3, the virulence-attenuated mutants identifiedsubsequently were tested for their ability to elicit disease symp-toms and proliferate in Arabidopsis leaves. As summarized inTable 2, in addition to toxA and plcS a total of 11 virulence-related genes were identified among the 2,500 prototrophsscreened that elicited null, weak, or moderate rotting symptomson lettuce and Arabidopsis plants (Table 2, group I). Importantly,the reduced ability of the mutants to cause disease symptomscorrelated with reduced bacterial counts in leaves (5).

In addition to demonstrating its ability to cause disease inplants and mice, we have shown that PA14 kills C. elegans whenit is presented to the nematodes as a food source (15). In low-saltmedium, PA14 kills worms over a period of 2–3 days (slowkilling) by an infection-like process that correlates with theaccumulation of bacteria in the worm intestine (14). Alterna-tively, PA14 grown in a high-salt, rich medium kills worms within4–24 h (fast killing) by a toxin-mediated process (7). Fast andslow killing appear to be mechanistically distinct because most P.aeruginosa mutants that are attenuated in slow killing are notattenuated in fast killing and vice versa. Altogether, 13 P.aeruginosa genes have been identified that when mutated exhibitreduced virulence in the C. elegans slow or fast killing assays. Ofthese 13 genes, nine also are required for maximum patho-

genicity in the Arabidopsis leaf infiltration assay and are listed inTable 2 (group II genes; gacA was identified in both the plant andnematode screens).

Eight of the 20 P. aeruginosa genes listed in Table 2 that wereidentified by screening in plants and nematodes do not corre-spond to previously identified proteins from other species (5–7).Remarkably, at least 15 of these 20 genes are required forpathogenesis in the burned mouse model.

What Types of Virulence Mechanisms Are ConservedThrough Evolution?The ‘‘multihost’’ pathogenesis screens performed in our labo-ratories identified a variety of virulence-associated genes thatencode proteins involved in transcriptional control, posttran-scriptional control, eff lux systems, biosynthetic enzymes in-volved in phenazine production, toxins, and proteins of unknownfunction. Table 2 highlights the striking conservation in thevirulence mechanisms used by P. aeruginosa to infect plants,nematodes, and mammals. It is remarkable that at least 15 of theP. aeruginosa mutants isolated in either a plant or a nematodescreen were found to be required for full virulence in the burnedmouse model.

One virulence-related factor found to play a critical role inpathogenesis in plants, nematodes, and mice is the periplasmicdisulfide bond-forming enzyme encoded by the dsbA gene (49),whose function is likely to affect several periplasmic virulence-related proteins. An important role for dsbA in pathogenesis hasbeen described in several human pathogens, including Shigellaflexneri (50) and Vibrio cholera (51), and in the bacterial phy-topathogen Erwinia chrysanthemi (52).

Three virulence-related genes identified in our screen, hrpM,gacS, and gacA, previously had been shown by other researchersto be important in phytopathogenesis. However, our studiesfound that these genes also play an important role in mammalian

Table 2. P. aeruginosa mutants causing decreased virulence in plants

StrainSymptoms elicited

in Arabidopsis*% Mousemortality† Gene identity and comments

PA14 Severe 100 Wild type

Group I mutants isolated from plant screens

33C7 None 0 Unknown‡

1D7 Weak 50 gacA, quorum-sensing regulator, two-component system lemA-gacA25A12 Weak 87 UnknownplcS Moderate 40 plcS, degrades phospholipids of eukaryotic membranestoxA Moderate 40 toxA, inhibits eukaryotic protein synthesis33A9 Moderate 0 Unknown, absent in PA01, reduced motility, increased surface attachment25F1 Moderate 20 Homologue of Chlorobium tepidum orfT34H4 Moderate 33 Unknown, contains a bipartite nuclear localization signalpho34B12 Moderate 56 Unknown, phenazine regulator, contains a HTH motifpho15 Moderate 62 dsbA, periplasmic S-S forming enzyme16G12 Moderate 100 Unknown

Group II mutants isolated from C. elegans screens

36A4 None 0 Homologue of P. syringae hrpM50E12 Weak 0 Homologue of A. vinelandii PstP protein, required for accumulation of poly-b-hydroxybutyrate48D9 Weak 50 Homologue of lemA, sensor of the two-component system lemA-gacA41C1 Weak 81 Homologue of putative E. coli integral membrane protein AefA23A2 Weak 85 mexA, non-ATPase membrane efflux pump41A5 Weak 100 Unknown6A6, 3E8 Moderate 18 phzB, phenazine biosynthesis12A1 Moderate 50 lasR, quorum-sensing regulator8C12 Moderate 63 Unknown

*Symptoms observed 4–5 days postinfection. None, no symptoms; weak, localized water soaking and chlorosis of tissue circumscribing the inoculation site;moderate, moderate water-soaking and chlorosis with most of the tissue softened around the inoculation site at 2–3 days postinfection.

†Mice were injected with '5 3 105 cells.‡Unknown, BLASTX analysis yielded no encoded proteins with significant homology.

Rahme et al. PNAS u August 1, 2000 u vol. 97 u no. 16 u 8819

COLL

OQ

UIU

M

Dow

nloa

ded

by g

uest

on

May

11,

202

0

pathogenesis. The three genes also are required for effectivekilling of C. elegans (6, 7). The hrpM gene of P. syringae pv.syringae was identified as a homologue of the Escherichia colimdoH gene, which is part of an operon involved in the synthesisof membrane-derived oligosaccharides (MDO) (53). AlthoughMDO have been found to have different functions in a varietyof Gram-negative bacteria, the role of MDO in bacterial patho-genesis is not well understood. Mutation in the hrpM locus of theplant pathogen P. siryngae pv. syringae abolishes both the devel-opment of disease symptoms on host plants as well as thehypersensitivity response in nonhost plants (54). The GacSprotein is a sensor kinase of a two-component bacterial family ofregulators (55). GacA, the cognate response regulator of GacS,initially was identified as a global regulator of secondary me-tabolites in P. fluorescens (56, 57). It has been shown thatmutations in both the gacA and gacS genes in P. siryngae lead todecreased lesion formation in beans and no production of thetoxin syringomycin (46, 55, 58).

The requirement of the lasR, gacS, and gacA gene products forpathogenicity in plants, nematodes, and mice provides evidencethat quorum sensing and regulated export of proteins are generalfeatures of pathogenesis for all three hosts. The lasR, gacS, andgacA genes are present in numerous plant and animal bacterialpathogens as well as in saprophytes. It is likely that LasR, GacS,and GacA initially served as master regulators, enabling ances-tral Gram-negative organisms to adapt to their environment.Subsequently, these proteins evolved to regulate a variety ofgenes that allowed prokaryotes to invade and establish theirpresence in eukaryotic hosts.

Another class of conserved virulence factors important inplant and nematode pathogenesis is the genes involved multidrugefflux pumps. The mexA gene corresponding to mutant 23A2encodes a component of a multidrug efflux pump; it recently hasbeen shown to be involved in active efflux of P. aeruginosaautoinducers that are not freely diffusable (59). However, mu-tant 23A2 was only marginally compromised in the mouse burnmodel. Further studies using a lower inoculum need to beconducted to ascertain its role as a virulence factor in mamma-lian pathogenesis. An additional protein relevant to plant patho-genesis, but not to mammalian pathogenesis, is a homologue ofthe putative E. coli integral membrane protein AefA (Proposite:PS01246). The function of this protein is yet unknown.

Other known proteins identified in our screens, but notpreviously shown to be involved in pathogenesis, include a PtsPhomologue of A. vinelandii required for poly-b-hydroxybutyrateaccumulation. The ptsP gene is predicted to encode enzyme INtr,a presumptive transcriptional regulator of RpoN-dependentoperons (60).

One of the novel virulence factors identified in the plantscreen (Pho34B12) affects hemolytic and elastolytic activity aswell as pyocyanin production (refs. 5 and 7; H.C. and L.G.R.,unpublished work), all of which are under quorum-sensingregulation. The protein encoded by pho34B12 contains a helix–turn–helix DNA binding motif similar to that found in the LysRfamily of transcriptional regulators (ref. 5; H.C. and L.G.R.,

unpublished work). This class of proteins includes regulatorsinvolved in both mammalian and plant pathogenesis (1).

Our multihost pathogenesis study results indicate that phena-zines are an important class of virulence-related effector mol-ecules. Despite intensive in vitro analyses of phenazines, thephysiological significance of their role in P. aeruginosa patho-genesis in mammals has been controversial (61). Before ourstudies, there had been no demonstration of their role in vivo.Mutants 3E8 and 6A6 correspond to the previously identifiedphzB gene in P. fluorescens and phzY gene in P. aureofaciens. BothphzB and phzY are present in operons known to regulateproduction of phenazine-1-carboxylate (62). Additional effectormolecules that play a role in multihost pathogenesis include theToxA protein and the previously unidentified virulence geneencoding the 34H4 protein. Current work indicates that 34H4contains a bi-partite nuclear localization signal required fortranslocation of the protein into the nucleus of mammalian cells(G.W.L. and L.G.R., unpublished work).

A common phenotype of at least two of the mutants, 33A9 and3E8, is reduced motility and altered surface attachment ability(Table 2). Mutant 3E8 exhibits reduced attachment to abioticsurfaces, such as polyvinilchloride plastic surfaces (ref. 63;S.M.-M. and F.M.A., unpublished work) whereas 33A9 exhibitedincreased surface attachment ability (E.D., G. O’Toole, F.M.A.,and L.G.R., unpublished work). Bacterial adhesion is an essen-tial step in biofilm formation and consequently, in bacterialvirulence. No significant similarity to any known genes wasfound in the gene corresponding to mutant 33A9. Interestingly,DNA sequence analysis of the region containing the 33A9 generevealed that this gene is not present in the recently sequencedgenome of the P. aeruginosa strain PAO1.

ConclusionsIn summary, we can draw the following conclusions from ourstudies of P. aeruginosa multihost pathogenesis. First, the varietyof virulence-associated genes described in our experimentsindicates that the multihost strategy has few limitations withregard to the categories of virulence-related functions that canbe identified. Second, the fact that most of the genes identifiedwere not previously known to be involved in pathogenesis-related functions demonstrates that the multihost strategy isparticularly efficient in identifying novel P. aeruginosa virulencefactors. Third, and perhaps most importantly, even though manypathogens cause disease in a single or limited number of hostspecies these studies provide strong evidence that there existsseveral universal bacterial virulence mechanisms highly con-served across phylogeny.

Although some of the virulence-associated genes identifiedhave homologues in other pathogenic bacteria, the exact role ofthese genes in pathogenesis remains unclear in most cases. Theuse of genetically tractable host systems, such as plants, nema-todes, and insects, will generate important information abouthost responses and lead to a better understanding of thefundamental molecular mechanisms that underlie bacterialpathogenesis.

1. Finlay, B. B. & Falkow, S. (1997) Microbiol. Mol. Biol. Rev. 61, 136–169.2. Kopp, E. B. & Medzhitov, R. (1999) Curr. Opin. Immunol. 11, 13–18.3. Strauss, E. J. & Falkow, S. (1997) Science 276, 707–711.4. Rahme, L. G., Stevens, E. J., Wolfort, S. F., Shao, J., Tompkins, R. G. &

Ausubel, F. M. (1995) Science 268, 1899–1902.5. Rahme, L. G., Tan, M.-W., Le, L., Wong, S. M., Tompkins, R. G.,

Calderwood, S. B. & Ausubel, F. M. (1997) Proc. Natl. Acad. Sci. USA 94,13245–13250.

6. Tan, M.-W., Rahme, L. G., Sternberg, J. A., Tompkins, R. G. & Ausubel, F. M.(1999) Proc. Natl. Acad. Sci. USA 96, 2408–2413.

7. Mahajan-Miklos, S., Tan, M.-W., Rahme, L. G. & Ausubel, F. M. (1999) Cell96, 47–56.

8. Jander, G., Rahme, L. G. & Ausubel, F. M. (2000) J. Bacteriol. 182, 3843–3845.

9. Kominos, S. D., Copeland, C. E., Grosiak, B. & Postic, B. (1972) Appl.Microbiol. 24, 567–570.

10. Cho, J. J., Schroth, M. N., Kominos, S. D. & Green, S. K. (1975) Phytopathology65, 425–431.

11. Finlay, B. B. (1999) Cell 96, 315–318.12. Doring, D. (1993) in Pseudomonas aeruginosa as an Opportunistic Pathogen, ed.

Campa, M. (Plenum, New York), pp. 245–273.13. Wood, R. E. (1976) Hosp. Prac. 11, 91–100.14. Tan, M.-W., Mahajan-Miklos, S. & Ausubel, F. M. (1999) Proc. Natl. Acad. Sci.

USA 96, 715–720.15. Tan, M.-W. & Ausubel, F. M. (2000) Curr. Opin. Microbiol. 3, 29–34.16. Fink, R. B., Jr., ed. (1993) in Pseudomonas aeruginosa the Opportunist:

Pathogenesis and Disease (CRC, Boca Raton, FL), pp. 1–5.

8820 u www.pnas.org Rahme et al.

Dow

nloa

ded

by g

uest

on

May

11,

202

0

17. Kawaharajo, K., Homma, J. Y., Aoyama, Y., Okada, K. & Morihara, K. (1975)Jpn. J. Exp. Med. 45, 79–88.

18. Holder, I. A. & Neely, A. N. (1991) Antib. Chemother. 44, 99–105.19. Galloway, D. R. (1991) Mol. Microbiol. 5, 2315–2321.20. Peters, J. E. & Galloway, D. R. (1990) J. Bacteriol. 172, 2236–2240.21. Ohman, D. E., Cryz, S. J. & Iglewski, B. H. (1980) J. Bacteriol. 142,

836–842.22. Zielinski, N. A., DeVault, J. D., Roychoudhury, S., May, T. B., Kimbara, K.,

Kato, J., Shinabarger, D., Kitano, K., Berry, A., Misra, T. & Chakrabarty,A. M. (1990) in Pseudomonas: Biotransformations, Pathogenesis, and EvolvingBiotechnology, eds. Silver, S., Chakrabarty, A. M., Iglewski, B. & Kaplan, S.(Am. Soc. Microbiol., Washington, DC), pp. 15–27.

23. Pitt, T. L. (1989) Antibiot. Chemother. 42, 1–7.24. Nicas, T. I. & Iglewski, B. H. (1986) J. Clin. Microbiol. 23, 967–969.25. Jones, S., Yu, B., Bainton, N. J., Birdsall, M., Bycroft, B. W., Chhabra, S. R.,

Cox, A. J. R., Golby, P., Reeves, P. J., Stephens, S., et al. (1993) EMBO J. 12,2477–2482.

26. Passador, L., Cook, J. M., Gambello, M. J., Rust, L. & Iglewski, B. H. (1993)Science 260, 1127–1130.

27. Pritchard, A. E. & Vasil, M. L. (1986) J. Bacteriol. 176, 291–298.28. Shortridge, V. D., Lazdunski, A. & Vasil, M. L. (1992) Mol. Microbiol. 6,

863–871.29. Grant, C. C. R. & Vasil, M. L. (1986) J. Bacteriol. 168, 1112–1119.30. Lory, S. (1986) J. Bacteriol. 168, 1451–1456.31. Frank, D. W., West, S. E. H. & Iglewski, B. H. (1990) in Molecular Basis of

Bacterial Pathogenesis, eds. Iglewski, B. H. & Clark, V. L. (Academic, SanDiego), Vol. 11, pp. 427–455.

32. Iglewski, B. H., Rust, L. & Bever, R. A. (1990) in Pseudomonas: Biotransfor-mations, Pathogenesis, and Evolving Biotechnology, eds. Silver, S., Chakrabarty,A. M., Iglewski, B. & Kaplan, S. (Am. Soc. Microbiol., Washington, DC), pp.36–43.

33. Pesci, E. C. & Iglewski, B. H. (1997) Trends Microbiol. 4, 132–134.34. Pesci, E. C., Milbank, J. B. J., Pearson, J. P., McKnight, S., Kende, A. S.,

Greenberg, E. P. & Iglewski, B. H. (1999) Proc. Natl. Acad. Sci. USA 96,11229–11234.

35. McLean, R. J. C., Whiteley, M., Stickler, D. J. & Fuqua, W. C. (1997) FEMSMicrobiol. Lett. 154, 259–263.

36. Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W.& Greenberg, E. P. (1998) Science 280, 295–298.

37. Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R. & Lappin-Scott, H. M. (1995) Annu. Rev. Microbiol. 49, 711–745.

38. Fuqua, W. C., Winans, S. C. & Greenberg, E. P. (1994) J. Bacteriol. 176, 269–275.

39. Sorensen, R. U., Klinger, J. D., Cash, H. A., Chase, P. A. & Dearborn, D. G.(1983) Infect. Immun. 41, 321–330.

40. Essar, D. W., Eberly, L., Hadero, A. & Crawford, I. P. (1990) J. Bacteriol. 172,884–900.

41. Hassan, H. M. & Fridovich, I. (1980) J. Bacteriol. 141, 1556–1163.42. Stevens, E. J., Ryan, C. M., Friedberg, J. S., Barnhill, R. L., Yarmush, M. L.

& Tompkins, R. G. (1994) J. Burn Care Rehabil. 15, 232–235.43. Iglewski, B. H. & Kabat, D. (1975) Proc. Natl. Acad. Sci. USA 72, 2284–2288.44. Berka, R. M. & Vasil, M. L. (1982) J. Bacteriol. 152, 239–245.45. Besterman, J. M., Duronio, V. & Cuatrecasas, P. (1986) Proc. Natl. Acad. Sci.

USA 83, 6785–6789.46. Rich, J. J., Kinscherf, T. G., Kitten, T. & Willis, D. K. (1994) J. Bacteriol. 176,

7468–7475.47. Reimmann, C., Beyeler, M., Latifi, A., Winteler, H., Foglino, M., Lazdunski,

A. & Haas, D. (1997) Mol. Microbiol. 24, 309–319.48. Glessner, A., Smith, R. S., Iglewski, B. H. & Robinson, J. B. (1999) J. Bacteriol.

181, 1623–1629.49. Bardwell, J. C. A., McGovern, K. & Beckwith, J. (1991) Cell 67, 581–589.50. Watarai, M., Tobe, T., Yoshikawa, M. & Sasakawa, C. (1995) Proc. Natl. Acad.

Sci. USA 92, 4927–4931.51. Peek, J. A. & Taylor, R. K. (1992) Proc. Natl. Acad. Sci. USA 89, 6210–6214.52. Shevchik, V. E., Bortoli-German, I., Robert-Baudouy, J., Robinet, S., Barras,

F. & Condemine, G. (1995) Mol. Microbiol. 16, 745–753.53. Loubens, I., Debarbieux, L., Bohin, A., Lacroix, J.-M. & Bohin, J.-P. (1993)

Mol. Microbiol. 10, 329–340.54. Anderson, D. M. & Mills, D. (1985) Phytopathology 75, 104–108.55. Hrabak, E. M. & Willis, D. K. (1992) J. Bacteriol. 174, 3011–3020.56. Laville, J., Voisard, C., Keel, C., Maurhofer, M., Defago, G. & Haas, D. (1992)

Proc. Natl. Acad. Sci. USA 89, 1562–1566.57. Gaffney, T. D., Lam, S. T., Ligon, J., Gates, K., Frazelle, A., DiMaio, J., Hill,

S., Goodwin, S., Torkewitz, N., Allshouse, A. M., et al. (1994) Mol. Plant-Microbe Interact. 7, 455–463.

58. Hrabak, E. M. & Willis, D. K. (1993) Mol. Plant-Microbe Interact. 6, 368–375.59. Pearson, J. P., Van Delden, C. & Iglewski, B. H. (1999) J. Bacteriol 181, 1203–1210.60. Reizer, J., Reizer, A., Merrick, M. J., Plunkett, G., Rose, D. J. & Saier, M. H. J.

(1996) Gene 181, 103–108.61. Sorensen, R. U. & Joseph, F. J. (1993) in Pseudomonas aeruginosa as an

Opportunistic Pathogen, eds. Campa, M., Bendenelli, M. & Friedman, H.(Plenum, New York), pp. 43–57.

62. Mavrodi, D. V., Ksenzenko, V. N., Bonsall, R. F., Cook, R. J., Boronin, A. M.& Thomashow, L. S. (1998) J. Bacteriol. 180, 2541–2548.

63. O’Toole, G. A. & Kolter, R. (1998) Mol. Microbiol. 28, 449–461.

Rahme et al. PNAS u August 1, 2000 u vol. 97 u no. 16 u 8821

COLL

OQ

UIU

M

Dow

nloa

ded

by g

uest

on

May

11,

202

0