C Ommon M Echanisms for P Athogens of P Lants and a Nimals

9 Jul 2001 17:23 AR AR138-11.tex AR138-11.sgm ARv2(2001/05/10)P1: GJB

Annu. Rev. Phytopathol. 2001. 39:259–84Copyright c© 2001 by Annual Reviews. All rights reserved

COMMON MECHANISMS FOR PATHOGENS

OF PLANTS AND ANIMALS

Hui Cao, Regina L. Baldini, and Laurence G. RahmeDepartment of Surgery, Harvard Medical School, Massachusetts General Hospitaland Shriner’s Burn Hospital, Boston, Massachusetts 02114;e-mail: [email protected]

Key Words microbial pathogenesis,Pseudomonas aeruginosa, Bulkholderiacepacia, Erwinia spp., common virulence factors

■ Abstract The vast evolutionary gulf between plants and animals—in terms ofstructure, composition, and many environmental factors—would seem to preclude thepossibility that these organisms could act as receptive hosts to the same microorga-nism. However, some pathogens are capable of establishing themselves and thrivingin members of both the plant and animal kingdoms. The identification of functionallyconserved virulence mechanisms required to infect hosts of divergent evolutionaryorigins demonstrates the remarkable conservation in some of the underlying virulencemechanisms of pathogenesis and is changing researchers’ thinking about the evolutionof microbial pathogenesis.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260GRAM-NEGATIVE PATHOGENS THAT INFECTBOTH PLANTS AND HUMANS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260Erwinia spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261Burkholderia cepacia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261Pseudomonas aeruginosa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

THE COMMON PLAYERS OF BACTERIA IN PLANTAND ANIMAL PATHOGENESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262Shared Transcriptional Regulators of the Regulator Classin Plant and Animal Pathogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

“Controllers”: Another Member of the Regulator Class. . . . . . . . . . . . . . . . . . . . . . 268Effector Class. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

VIRULENCE FACTORS SPECIFIC FOR PATHOGENESISIN PLANT HOSTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

COMMON FEATURES IN THE INNATE DEFENSE RESPONSE OFPLANTS, INSECTS, AND MAMMALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

0066-4286/01/0901-0259$14.00 259


260 CAO ¥ BALDINI ¥ RAHME

INTRODUCTION

Since the beginning of the past century, or earlier, various strains of the Gram-negative pathogensPseudomonas aeruginosa, Burkholderia cepacia, andErwiniaspp. have been recognized as capable of infecting plants and humans (16, 35, 47,134). Today, a large body of evidence demonstrates the existence of universalpathogenic mechanisms used by diverse bacterial pathogens and the parallels inthe key features underlying host defense responses against pathogens in plants,invertebrates, and mammalian hosts (reviewed in 21, 27, 57, 68, 91, 116). Thesestudies imply that some of the underlying virulence mechanisms of pathogenesisand the host defenses against them are likely to have ancient evolutionary originsand may be conserved across phylogenies. Although a lot is still to be learned aboutthe complexity of host-pathogen interactions, significant advances have been madethrough the development of techniques that facilitate our understanding of viru-lence mechanisms and the critical role of the host during pathogenesis (reviewedin 137). In particular, the availability of the genome sequences of a number ofpathogenic organisms and those of their hosts recently released (1, 83) and soon tobe determined, provide the opportunity for exciting and challenging scientific ad-vances. These developments will undoubtedly revolutionize researchers’ thinking.However, even though the postgenomic era represents a biological revolution, westill have to determine the biological functions of the sequenced genes, assign viru-lence and defense functions to them, and define their role in disease development.The identification of a shared subset of virulence factors required to elicit diseasein plants, insects, nematodes, and mammals allows high-throughput screens tobe performed in which bacterial mutants with reduced virulence in these diversehosts can be isolated (61, 86, 116, 140, 141). In this review we discuss virulencefactors that are conserved in evolution and mechanisms of pathogenesis identifiedin the Gram-negative multihost pathogenP. aeruginosaand also present in otherbacterial pathogens of plants or humans.

GRAM-NEGATIVE PATHOGENS THAT INFECTBOTH PLANTS AND HUMANS

Numerous clinical reports have presented evidence of the role of various strainsof Erwinia spp.,B. cepacia, andP. aeruginosain nosocomial infections (11, 48,63). However, in the past 30 years, the frequency, etiology, and epidemiologyof nosocomial infections have changed with the advancement of medical care,particularly with respect to the increasing number of hospitalized patients re-quiring intensive care. Nevertheless, bothP. aeruginosaandB. cepaciastill re-ceive continuous attention due to their importance as nosocomial pathogens andin particular as the cause of respiratory tract infections in patients with cysticfibrosis.


COMMON MECHANISMS FOR PATHOGENS 261

Erwinia spp.

Strains ofErwinia spp. cause important plant diseases and they belong to thefamily of Enterobacteriaceae, a major group of much significance for human andanimal hygiene.E. carotovorais a well-characterized plant pathogen that causessoft rot in a variety of plant hosts (reviewed in 7). HumanErwinia isolates havebeen recovered from wounds, abscesses, skin, sputum, ear infections, and mostoften in mixed cultures with other bacterial species of known human pathogenicpotential. However, as mentioned above, better medical care and use of antibioticshave largely eliminated the threat of infections caused byErwinia spp.

Burkholderia cepacia

Isolates, now designated asB. cepacia, were first described in the 1950s as thecausal agent of soft rot ofAlliumspp., called sour skin of onions (16).B. cepaciaisa member ofBurkholderiaspecies that are common inhabitants of the rhizosphereof important crop plants and are considered beneficial microorganisms becauseof their ability to control plant diseases caused by nematodes and fungi (52, 104).Various environmental isolates ofB. cepaciahave extraordinary degradative abili-ties. However,B. cepaciais also recognized as a life-threatening pathogen amongseveral groups of immunocompromised patients. Therefore, considerable efforthas been expended to distinguish between clinical and environmental isolates ofB. cepaciawith regard to their virulence potentials. Recent studies by Melnikovet al (92) have shown that clinical and environmental isolates ofB. cepaciaex-hibit different cytotoxicities toward macrophages and mast cells. In addition, plant,clinical, or environmental isolates were found to have multiple replicons, with theclinical strains carrying three or four replicons (150).

Pseudomonas aeruginosa

AlthoughP. aeruginosahas long been a recognized human opportunistic pathogen,a number of epidemiological studies reported that human isolates ofP. aeruginosahave been found to elicit disease in a variety of plants (31, 71, 128).P. aeruginosais the most common causative organism of sepsis in burn patients and the leadingcause of pulmonary infections and mortality in cystic fibrosis (CF) patients (28).In addition, this important human opportunistic pathogen infects injured, immuno-deficient, or otherwise compromised individuals (152). The pathophysiology ofinfections due toP. aeruginosais complex, as is evidenced by the clinical diversityof diseases associated with this organism and by the multiplicity of virulencefactors it produces. Although a natural soil inhabitant, it is versatile in its metabolicpotential, allowing it to survive in a number of natural and hospital environments.Apparently, the combination of environmental persistence, versatility in virulencemechanisms, and multiple virulence factors allowsP. aeruginosato be effectiveas both a human opportunistic pathogen and a plant pathogen.



Taking advantage of the versatility of this pathogen, we used aP. aeruginosahuman isolate strain UCBPP-PA14 (referred to as PA14 hereafter), and we providedthe first evidence thatP. aeruginosautilizes a shared subset of virulence factorsto elicit disease in both plants and animals (117). In this system, the PA14 strainis able to cause soft rot disease in lettuce andArabidopsisplants at the least,as well as to be infectious for and cause lethality in mice (116). Over the pastfew years, this system was extended to include additional genetically tractablenonmammalian hosts such as nematodes (86, 141) and insects (61; G.W. Lau &L.G. Rahme, unpublished information). The establishment of this “multihost”pathogenesis system allowed us to perform a high-throughput screen in whichP. aeruginosamutants with reduced virulence in at least one of the hosts of thissystem (plants, insects or nematodes) and mice are identified. The remarkablefinding of this high-throughput multihost screen approach is that the majority ofmutants identified as relevant in at least one of the nonvertebrate hosts of themultihost system were found to be relevant to mammalian pathogenesis. Thisdemonstrates the striking conservation of the virulence mechanisms used byP.aeruginosato infect hosts of divergent evolutionary origins. The following sectionsdiscuss, in detail, with the exception of the type III secretion system, a numberof common factors of pathogenesis in both plant and animal hosts, based on thestudies with theP. aeruginosa-multihost system. The type III secretion system hasbeen extensively reviewed elsewhere (22, 58, 99).

THE COMMON PLAYERS OF BACTERIA IN PLANTAND ANIMAL PATHOGENESIS

The high-throughput multihost screen has identified multiple virulence-associatedgenes encoding proteins involved in a variety of functions and unidentified proteinsfrom other species (116). The virulence genes identified from either a plant or anematode screen and that are required for infection in both plant and animal hostscan be categorized into two major classes. One class consists of regulatory factorsthat control a number of target genes, many of which are virulence-associated. Wedesignate this class as Regulator Class, and it contains transcriptional regulatorsand genes that have pleiotropic effect. The other class consists of effector proteins,each of which performs a distinct function in pathogenesis. This class is named asEffector Class. The following sections describe in detail several genes included inTable 1 that belong to these two classes.

Shared Transcriptional Regulators of the Regulator Classin Plant and Animal Pathogenesis

An increasing number of transcriptional regulators are found to belong to quorumsensing regulatory systems. Quorum sensing is the bacterial cell-cell communica-tion mechanism by which bacteria coordinate the expression of a number of target



TABLE 1 Common virulence genes identified from UCBPP-PA14-multihost system

Symptomselicited in % Mortality Gene identity

Strain Arabidopsisa in miceb and comments Classc

PA14 Severe 100 Wild-type

48D9 Weak 50 Homologue oflemA, Rsensor of the two-component systemlemA-gacA

ID7 Weak 50 gacA, quorum sensing Rregulator, two-componentsystemlemA-gacA

12A1 Moderate 50 lasR, quorum sensing regulator R

pho34B12 Moderate 56 Novel; LysR-like transcriptional Rregulator and named asmvfR

rpoN Moderate 60 rpoN, sigma factor 54 R

pho15 Moderate 62 dsbA, periplasmic S-S R∗

forming enzyme

mucD Moderate 45 mucD, homologue ofdegP R∗

plcS Moderate 40 plcS, degrades phospholipids of Eeukaryotic membranes

toxA Moderate 40 toxA, inhibits eukaryotic Eprotein synthesis

3E8 Moderate 18 Homologue ofphzB, involved Ein phenazine biosynthesis

36A4 None 0 Homologue ofP. syringae hrpM E

34H4 Moderate 33 Novel; contains a bi-partite Enuclear localization signal

aSymptoms observed 4–5 days postinfection. None, no symptoms; weak, localized water soaking and chlorosis oftissue circumscribing the inoculation site; moderate, moderate water-soaking and chlorosis with most of the tissuesoftened around the inoculation site at 2–3 days postinfection.bMice were injected with∼5 × 105 cells.cR: Regulator Class; R∗: Controller genes of Regulator Class; E: Effector Class.

genes through cell density. This complex regulatory mechanism of gene expressionis utilized by both Gram-negative and Gram-positive bacteria, and these bacteriaare usually associated with plants or animals in one way or another [e.g. symbioticor pathogenic (25)]. The quorum sensing system is enormously advantageous forbacteria because it allows for improved adaptation to the environment, in termsof better utilization of nutrient supply and protection from deleterious conditions.Since quorum sensing plays an essential role in the interaction of bacteria with their



environment, it is, not surprisingly, one of the common mechanisms for pathogen-esis in both plant and animal hosts. Although the individual target genes under thecontrol of quorum sensing systems may differ among different bacterial species,the mechanism of regulation is, however, very well conserved by the detectionof cell density. The significance of this tight cell-density dependent regulation inpathogenesis could be explained by noting that factors that may trigger host de-fense response are not expressed until bacterial cell population has reached a levelthat can overwhelm the host.

THE GacS-GacA TWO-COMPONENT REGULATOR SYSTEM First, we discuss a few in-teresting transcriptional regulators of the Regulator Class. One of them is the globalregulatorgacA. In fluorescent Pseudomonads,gacAis part of the two-componentsystem,gacS-gacA, in which gacSencodes the transmembrane cognate sensorandgacAencodes the response regulator that belongs to the FixJ family (76). Instrains ofP. fluorescens(38, 76, 126), phytopathogensP. syringae(121),P. viridi-flava(80, 82), andP. marginalis(81),gacAregulates the production of antibiotics,toxins, and some lytic exoenzymes. These toxins and extracellular enzymes areimportant pathogenicity factors.E. carotovorastrains with mutations in the ho-mologues ofgacAexhibit significantly reduced virulence in plant infection anddecreased production of extracellular plant cell wall–degrading enzymes (32, 33).In P. aeruginosa, gacApartially controls the production of pyocyanin, cyanide,and lipase (118, 119) and functions upstream of quorum sensing regulatory sys-tems (119) to regulate the expression of a number of virulence genes (110) andtype-IV pilus-mediated twitching motility (44). Using the PA14-multihost system,mutations in eithergacSor gacA led to dramatic reduction in virulence in bothplant and animal hosts (116). Homologues ofgacAhave also been identified inEscherichia coli(95) and in the human pathogenSalmonella typhimurium. ThesirA gene ofS. typhimurium, found to be the homologue ofgacA, is required forinvasion and virulence (64).

THE AUTOINDUCER/REGULATOR OF QUORUM SENSING The quorum sensing sys-tems of Gram-negative bacteria consist of two components: the signaling moleculeor so-called autoinducer, and the transcriptional activator (R protein) that autoin-ducers bind to. Autoinducers are N-acyl homoserine lactones (AHLs) that aresecreted, and the level of these signaling molecules in the environment is di-rectly related to the bacterial population density. When sufficient bacterial cellsare present and autoinducer concentration reaches a certain threshold level, the au-toinducer molecules will bind to the transcriptional activator R proteins to activateor repress the expression of a number of target genes.

Specific genes for the autoinducer/transcriptional regulator of quorum sensingsystems have been identified in bacterial strains that are pathogens for both plantsand animals, includingB. cepacia, Erwinia spp., andP. aeruginosa. A quorumsensing system,cepI/cepR, has been identified inB. cepacia(79). This systempositively regulates protease production and negatively controls the synthesis of



siderophore. The addition ofP. aeruginosa-spent media toB. cepaciaculturessignificantly increases both siderophore synthesis and protease production inB.cepacia(79). Because, in many instances, CF patients colonized withB. cepaciaare also infected byP. aeruginosa, it is speculated thatB. cepaciamay utilizethe exogenous AHLs produced byP. aeruginosato initiate infection. Similarly,quorum sensing systems have been found inErwinia spp., includingE. caro-tovora(expI/expRandcarI/carR) (5, 19, 65, 90, 114),E. chrysanthemi(expI/expR)(98, 120),E. stewartii (esaI/esaR) (8), andE. agglomerans(eagI/eagR) (139).The expI/expRsystem ofE. carotovorawas found to positively regulate theexpression of tissue-macerating enzymes, including pectin lyases, polygalactur-onase, cellulase, and proteases. Mutation inexpI was shown to significantly re-duce the production of the tissue-degrading enzymes and the mutant was unable tomacerate plant tissues (65, 114). Similarly, inE. chrysanthemi, the quorum sensingsystemexpI/expRwas shown to positively regulate production of pectinases, oneof the major virulence factors in soft-rot causingErwinia spp. (120). A secondquorum sensing system,carI/carR of E. carotovora, was shown to control thesynthesis of the broad-spectrum antibiotic carbapenem (5, 19, 90). The coordina-tion of antibiotic synthesis with the production of tissue-macerating enzymes usingquorum sensing may serve as a strategy forE. carotovora(or any other bacteriapossessing similar systems, includingP. aeruginosa) to counteract the competitionfrom other microorganisms during infection.

In P. aeruginosa, two quorum sensing systems have been identified as relevantin both plants and animals (117, 118, 141). One is thelasI/lasRsystem, wherelasI encodes a synthase for the synthesis of autoinducer N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) andlasR encodes the transcriptionalactivator (105, 107). The binding of 3-oxo-C12-HSL to LasR activates theprotein and the complex will positively regulate the expression oflasB, lasA, aprA,andtoxA (40, 41, 107, 144). Elastase (lasB), a potent metalloprotease with broadsubstrate specificity, can degrade host proteins such as elastin, collagen, transferrin,immunoglobulin, and some complement components (reviewed in 39). The LasBelastase acts in concert with both LasA protease and alkaline protease (aprA)to result in efficient elastolysis required for the tissue damage associated withP. aeruginosapathogenesis (112). Exotoxin A (toxA) is a potent proteinsynthesis inhibitor (59), long been considered an important virulence factor ofP. aeruginosa. The lasI/lasRsystem also regulates the type II secretion pathwaycomponentxcp genes (17). The type II pathway is crucial for the secretion of anumber of virulence-related enzymes, including elastase, alkaline protease, andphospholipase C.

The other quorum sensing system identified inP. aeruginosaas relevant inboth plants and animals is therhlI/rhlR system, whererhlI encodes a synthasefor the synthesis of autoinducer N-butyryl-L-homoserine lactone (C4-HSL) andRhlR is a transcription activator (14, 108). Primarily, therhlI/rhlR system regulatesthe expression ofrhlAB, required for the production of rhamnolipid (101, 102),stationary-phase sigma factor RpoS (74), and the production of secondary



metabolites pyocyanin and cyanide (75, 151). The link between the two quorumsensing systems is demonstrated by the fact that thelasI/lasRsystem positivelyregulates the expression of bothrhlI andrhlR (74, 111).

Recently, quorum sensing has been found to participate in the regulation ofbiofilm formation, another important factor in pathogenesis (24). Biofilm is a modeof growth in which bacterial cells are encased in a polysaccharide matrix attachedto a solid surface. Microscopic studies of biofilm revealed that it contains dis-tinct mushroom and stalk-like structures with intervening water channels to allownutrient uptake and waste discharge. During infection, bacteria within a biofilmare usually more resistant to host defenses including phagocytes, antibodies, andcomplement.

Interestingly, in the animal pathogens enterohemorrhagicEscherichia coli(EHEC) and enteropathogenicE.coli (EPEC), quorum sensing is utilized to reg-ulate expression of the type III secretion systems (132). Both EHEC and EPECcause attaching and effacing lesions on intestinal epithelia. A pathogenicity is-land, called the locus of enterocyte effacement (LEE), which encodes a type IIIsecretion system and other products involved in lesion formation, is believed toplay a major role in pathogenesis (30, 89). The majority of the LEE-encodedgenes were found to be regulated by quorum sensing. Type III secretion systemshave long been considered one of the conserved mechanisms for pathogenesisin different hosts. The association of quorum sensing with the type III secre-tion system as demonstrated in EHEC and EPEC prompts the speculation thatthese two “common” systems for pathogenesis may be associated, both with eachother and with the regulation of virulence in a variety of pathogenic bacteria.Whether a similar regulation takes place in plant pathogenic bacteria has yet to bedemonstrated.

It appears that the majority of the genes regulated by the quorum sensing systemsare virulence related, as mentioned above. The important roles that the quorumsensing systems play in pathogenesis is further demonstrated by a fewin vivostudies. In a neonatal mouse model of pneumonia,P. aeruginosastrains harboringmutations inlasR, lasI, rhlI , or mutations in bothlasI and rhlI genes exhibitedsignificantly reduced virulence when compared to the wild-type parental strain,with the doubleI mutant being the least virulent (106, 142). In a thermal injurymouse model, strains with mutations inlasR, lasI, rhlI , or in bothrhlI and lasIwere found to have significantly reduced virulencein vivothan the wild-type strain(124, 125, 140). The mutant strains caused less mortality in the burned mice andfewer bacterial cells were recovered from skins, spleens, and livers (125). In thestrain with mutations in bothlasIandrhlI , complementation withlasI, rhlI , or bothlasI andrhlI , on a multicopy plasmid resulted in both increased virulencein vivoand greater ability to spread within the burned skin of infected mice (125). In a studyinvolving CF patients, sputum samples from the lungs of these patients infectedwith P. aeruginosawere analyzed for the expression oflasR, lasA, lasB, andtoxA.A coordinated expression pattern oflasA, lasB, andtoxA was established and itwas found to correlate with the expression oflasR(136).



If we consider the utilization of quorum sensing systems as a common mecha-nism in pathogenesis in plant and animal hosts, it does not necessarily mean thatevery single gene regulated by quorum sensing is among the shared pathogenicityfactors for divergent hosts. To conclude that a specific gene is a common viru-lence factor in both plants and animals, we should be able to demonstrate that themutation in this gene causes reduced virulence in both plant and animal hosts.For instance,toxA of P. aeruginosais regulated by the quorum sensing systemlasI/lasR. We showed that the PA14 mutant strain with mutation in thetoxAgenedid not cause maceration and collapse of the leaves and that its growth was signifi-cantly reduced inArabidopsisand lettuce (117). Similarly, in the mouse model,the mutant exhibited significantly lower mortality than the wild-type strain (117).Therefore,toxA of P. aeruginosacan be considered a common virulence fac-tor for pathogenesis in both plants and animals. However, forlasA, lasB, andaprA genes, also regulated by quorum sensing, no such conclusion can be drawnbecause of the lack of direct evidence demonstrating their role in plant patho-genesis. These proteases could be required for pathogenesis in plant and ani-mal hosts because they may facilitate the entry and dissemination of the bacteriaby degrading host proteins that form part of the physical barrier and defensesystems. The generation of mutations at these loci and testing of these mutantstrains in both plant and animal hosts would certainly be necessary if thesespeculations are to be verified.

LysR-LIKE TRANSCRIPTIONAL REGULATOR MvfR (MULTIPLE VIRULENCE FACTOR REG-

ULATOR) The third transcriptional regulator is a novel gene, and we have iden-tified its function based on a series of genetic and biochemical experiments (H.Cao, J. Tsongalis, B. Goumnerov & L.G. Rahme, unpublished information). ThemvfRgene was found to encode a transcriptional regulator that belongs to theLysR family. LysR-like transcriptional regulators have been found in both Gram-negative and Gram-positive bacteria, including bacteria that are pathogenic inplant or animal hosts. A number of LysR-like transcriptional regulators have beenshown to regulate the expression of virulence-related genes required for patho-genesis in both plant and animal hosts (36). InP. aeruginosa, it was shown thatMvfR positively regulates the hemolytic and elastolytic activities as well as py-ocyanin production (86, 118). It is also closely associated with quorum sensingsystems by regulating the production of autoinducer 3-oxo-C12-HSL (H. Cao &L.G. Rahme, unpublished).

RpoN In Gram-negative bacteria, the alternate sigma factorσ 54 (RpoN) plays animportant role in adaptation to the environment by activating a variety of genesin response to external stimuli (2). In various bacteria,σ 54 is required for theexpression of the enzymatic pathways responsible for nitrogen utilization, dicar-boxylate transport, xylene degradation, and hydrogen utilization (2). A number ofstudies have shown thatσ 54 is also involved in the regulation of virulence-relatedfactors in both plant and animal pathogens. In the plant pathogenP. syringae,



σ 54 positively regulates thehrp gene expression and the biosynthesis of theP. sy-ringae toxin, coronatine (53, 54). Recent studies have shown that the mutation inthe generpoN that encodes forσ 54 significantly impairs the ability ofP. syringaeto cause infection inArabidopsis(53, 54).σ 54 was also shown to regulate thecapsular expression in the human pathogenKlebsiella pneumoniae4. In P. aerug-inosa, σ 54 participates in the regulation of pilin, flagellin, and alginate synthesis(46, 145, 153). The PA14-multihost system mutation at therpoN locus results ina drastic reduction in virulence in both plant and animal hosts, indicating thatσ 54

is another common regulator required for pathogenesis in different hosts (E.L.Hendrickson, L.G. Rahme & F.M. Ausubel, unpublished information).

“Controllers”: Another Member of the Regulator Class

In addition to transcriptional regulators in the Regulator Class, there are genes thatare not transcription factors but are required for the proper functioning of a numberof bacterial proteins and thus we name them “controllers.”

DsbA One such controller gene isdsbA. This gene was first identified inE. coliand was shown to encode a periplasmic disulfide bond-forming enzyme (6) whoserole is likely to affect the appropriate folding (and thus the function) of a numberof periplasmic and secreted proteins. An important role fordsbAin pathogenesishas been described in several human pathogens, includingShigella flexneri(147)andVibrio cholera(109), and in the phytopathogensErwinia chrysanthemi(129),Erwinia carotovora(146), andP. syringae(70). Mutations in thedsbAgene ofE.chrysanthemiandE. carotovoraresulted in a severe defect in secretion of pectatelyase, the major tissue-macerating enzyme and virulence factor of soft-rot causingErwinia spp. InP. syringaepv. tomatoDC3000 strain, a mutation indsbAgenecaused reduced virulence inArabidopsisand tomato. It also exhibits pleiotropiceffect affecting type III secretion, motility, and colony morphology (70). InB.cepacia, a mutation in thedsbAgene contributes to a significant reduction in pro-duction of extracellular proteases and alkaline phosphatase (51). ThedsbAmutantstrain also exhibited a defect in motility and an increase in sensitivity to metal ions(e.g. Cd2+, Zn2+) and to a number of antibiotics (51). InP. aeruginosastrain PA14,the mutation indsbAgene (pho15) resulted in a significant reduction in virulenceboth in infection of lettuce andArabidopsisas well as mortality in mice (118). Thepleiotropic effect ofdsbAon pathogenesis is probably due to its capability to affectthe function of a number of virulence-associated proteins (including extracellularproteases, toxins, and type III secretion apparatus components) by controlling theproper folding of these proteins through disulfide bond formation.

DegP Another such gene isdegP. In E. coli, degP(htrA) was shown to encode aperiplasmic protein that acts as a chaperone to ensure stability of other periplasmicand membrane proteins at normal growth temperatures and switches to act asa serine protease to remove any improperly folded proteins when temperatures



are elevated (133). Therefore,degPplays the essential role of controlling thestability and turnover of those periplasmic and membrane-associated proteins inboth physiological conditions and under heat or oxidative stresses (69, 131, 133). Inthe phytopathogenP. syringaepv.maculicolastrain 4326, thedegPgene is requiredfor full virulence in Arabidopsis(135). In P. aeruginosa, two genes have beenidentified as showing significant homology todegP—one isalgWand the other ismucD(12). These two genes exhibit overlapping functions as mutations in bothof them resulted in increased sensitivity to elevated temperatures and to oxidativeradicals including hydrogen peroxide. The mutations also cause conversion tomucoidy. However, these two genes have distinct features since mutation ofmucDdid not increase sensitivity to the oxidative radical generator paraquat, whereas themutation ofalgW did. ThemucDgene is under the transcriptional regulation ofalgU, whereas there is no evidence showing thatalgWis (12). In theP. aeruginosa-PA14-multihost system, a mutation inmucDwas found to cause reduced virulencein plant, insects, nematodes, and mice (P. Yorgey, L.G. Rahme & F.M. Ausubel,unpublished information). Based on its essential roles in heat shock response andresistance to oxidative stress, it could be speculated thatdegPmay play a criticalrole in the protection of bacteria from host defense responses that involve oxidativeburst.

The members of the Regulator Class discussed in this section are present inmany plant and animal bacterial pathogens as well as in saprophytes. Thus, wecould speculate that these regulatory systems initially served as master regulators,enabling ancestral prokaryotic organisms to adapt to their environment. Later,with the appearance of eukaryotes, these regulators may have evolved to regulatea variety of genes that allowed prokaryotes to invade and colonize the eukaryotichosts.

Effector Class

In contrast to the Regulator Class where genes are controlling a variety of down-stream virulence-associated genes, genes in the Effector Class do not normally reg-ulate other genes, instead, their products perform defined functions that directlycontribute to pathogenesis. In this class, we discuss a few members, includinggenes coding for factors for adhesion/biofilm formation, enzymes (phospholipaseC), toxins (toxA), and genes implicated in the biosynthesis of phenazines (py-ocyanin).

PHOSPHOLIPASE C Bacterial phospholipase C is capable of degrading eukaryoticcell membrane component phospholipids. InP. aeruginosa, phospholipase C hasbeen demonstrated to be an important pathogenicity factor in bothin vitro andin vivo studies. Together with lipase, it exhibits severe pathological effects onplatelets, granulocytes, and monocytes (10, 72). It was shown that phospholipaseC causes the release of inflammatory mediators from human granulocytes andmonocytesin vitro (72). The phospholipase C ofP. aeruginosawas found to play



an important role in a chronic rat lung infection model (73). Mutation at the locusencoding phospholipase C was shown to cause reduction in virulence in a thermalinjury mouse model (103, 130). In the PA14-multihost model system, it was shownfor the first time that a mutation at the phospholipase C locus (plcS) resulted inreduction in the virulence of the bacteria in plant hosts as well (117). Unlikeanimal systems, little is known about the possible role that phospholipase C playsduring the infection of plant hosts (117). Thus, further studies of the function ofphospholipase C during the pathogenesis of plant hosts should reveal more detailsabout common mechanism of pathogenesis.

EXOTOXIN A Another effector shown to be a common pathogenesis factor in bothplants and animals istoxA, which encodes theP. aeruginosatoxin, exotoxin A. Inthe PA14-multihost model system, a mutation intoxAcaused a dramatic reductionin virulence ofP. aeruginosain both plant and animal hosts (117). Exotoxin Ais an ADP-ribosyltransferase that targets eukaryotic translation elongation factorII. Therefore, it acts as a potent inhibitor of protein synthesis in eukaryotic cells.Since protein synthesis is one of the housekeeping functions of an eukaryotic cell,the mechanism of exotoxin A’s function may explain its broad-spectrum effect inpathogenesis.

PHENAZINES Other effector genes are those involved in the biosynthesis of phenaz-ines, a group of secondary metabolites that exhibit antimicrobial activity againstseveral species of bacteria, fungi, and protozoa (reviewed in 37, 110). This charac-teristic of phenazines has been widely employed in biocontrol—where the presenceof phenazine-producing Pseudomonads in the plant rhizosphere would protect theplant from infection by certain fungal strains. Blue-colored pyocyanin is a majorspecies of phenazine identified inP. aeruginosa. Despite its function as an anti-biotic, the role of pyocyanin in the pathogenesis ofP. aeruginosais less clear. Anumber ofin vitro studies have shown that pyocyanin interferes with the func-tioning of human platelet cells, endothelial cells, and leukocytes, as well as af-fects human ciliary beat and baboon pulmonary mucociliary clearance. However,there was noin vivo evidence to clearly associate pyocyanin with pathogenesisprior to our studies using the PA14-multihost system. In our studies, we isolatedfive mutants that are deficient in production of pyocyanin in addition to exhibit-ing reduced virulence in both plant and animal hosts (H. Cao, J. Tsongalis &L.G. Rahme, unpublished information). Although the exact role of each gene inthe synthesis of pyocyanin remains to be determined, they showed homology toknown enzymes. One of them,3E8(86), was shown to be the homologue ofphzBof P. fluorescens2–79 (88) andP. aureofaciens30–84 (113).phzBis part of thephzABCDEFGoperon that is an essential component in the biosynthetic pathwayof phenazines, of which pyocyanin is a member (88, 113). In addition to thephzoperon, thephnABoperon identified by Essar et al (34) has been shown to playan important role in pyocyanin biosynthesis. ThephnABoperon encodes anthrani-late synthase that converts chorismate to anthranilate, leading to the synthesis of



pyocyanin. Using the PA14-multihost system, we showed that mutation in thephnABoperon reduced virulence in both plant and animal hosts (L.G. Rahme,unpublished information).

HrpM HOMOLOGUE Membrane-derived oligosaccharides (MDO) or periplasmicglucans have been found in a variety of Gram-negative bacteria and diverse func-tions (though poorly understood) have been associated with them, including theirroles in virulence, adaptation in hypoosmotic environments, and cell signaling (67).In E. coli, themdoGHoperon is required for the synthesis of MDO.mdoGen-codes a 56-kDa periplasmic protein whose function remains unknown, andmdoHencodes a 97-kDa cytoplasmic membrane-spanning protein that is necessary fornormal glucosyl transferase activity (84). InP. syringae, a locus namedhrpM wasfound to be a homologue ofmdoH. The mutation at thehrpM locus resulted inreduced virulence ofP. syringaein host plants and a reduced ability to cause ahypersensitive response (HR) in nonhost plants (96). AlthoughhrpM mutationsevidently affect delivery ofavr effectors to the plant host (the mutants do not elicitthe HR or cause disease), they do not interfere with the secretion of an extracel-lular effector, namely hairpin ofP. syringaepv. phaseolicola(A.P. Tampakaki &N.J. Panopoulos, unpublished). Using the PA14-multihost system, we isolated alocus,36A4, that encodes a homologue ofhrpM. The mutation at this locus signi-ficantly reduced the virulence ofP. aeruginosain both plant and animal hosts (86).Though MDO has been identified in other human pathogens includingSalmonellaandKlebsiella, no evidence has been found to associate MDO with pathogenesisin animals.

THE 34H4 LOCUS The locus34H4contains one of the novel genes identified withthe PA14-multihost system that are required for pathogenesis in both plant and an-imal hosts. Although a homology search has not identified similarity to any knowngenes, a profile scan revealed the presence of a putative bipartite eukaryotic nuclearlocalization signal (NLS) (G.W. Lau & L.G. Rahme, unpublished information).Our data indicated that the 34H4 protein translocates into the nuclei of host cells.Though the exact function of 34H4 remains to be determined, it represents aninteresting type of bacterial virulence factor in that it interacts with eukaryoticcells by targeting their nuclei. This in itself also constitutes part of the commonmechanism for pathogenesis in different hosts.

The multihost-pathogenesis system revealed a large number of the virulence-associated genes that encode for unknown proteins not yet identified in otherspecies (reviewed in 116). Further characterization of these genes will determinethe class to which they belong. Based on our findings, many of these novel geneswill likely encode for proteins present in other Gram-negative organisms thatare relevant to pathogenesis. Therefore, they may not be restricted to being onlycommon virulence factors inP. aeruginosa—they could be part of the universalcommon mechanisms for pathogenesis of many different pathogenic bacteria inboth plant and animal hosts.



ADHESION/SURFACE ATTACHMENT Adhering or attaching to the host tissue surfacenormally constitutes the first step of a bacterial infection. Therefore, adhesion orattachment should be considered as part of the common mechanisms for bacterialpathogenesis in different hosts. For instance, Figure 1 shows the attachment ofP.aeruginosastrain PA14 cells toArabidopsisecotype Ll-0 leaf surfaces, includingbiofilm-like structure formed on trichomes (Figure 1a), congregation at the stom-atal openings (Figure 1b) and invasion of plant parenchyma vessel cells (115, 116).Interestingly, the polar attachment of PA14 cells to the cell wall ofArabidopsisparenchyma vessel cells, illustrated in Figure 1c, is comparable to the adhesion ofP. aeruginosacells to mouse tracheal cells presented in a review by Goldberg &Pier (45). Since we have not identified any genes from our PA14-multihost systemthat are involved solely in the adhesion process, we will discuss a few well-studiedfactors for adhesion and what it is known about their role in pathogenesis in plantand animal hosts.

ADHESINS The type-IV pili are filaments found at the poles of a variety of bac-terial pathogens. In tomato plants,Pseudomonas syringaepv. tomatotype-IV pilimutants achieved lower population sizes in field experiments, and non-piliatedP. syringaepv. tomatomutants were more readily washed from leaves in lab-oratory experiments, suggesting a role for pili in the colonization of plants byphytopathogenic bacteria (55). Motility in the plant-associatedPseudomonas sy-ringae is also related to a better fitness for epiphytic conditions (55). Type IVpilus is the most studied adhesin ofPseudomonas aeruginosa, and it accounts for90% of its capability to adhere to human lung cells in culture (49). Comolli et al(20) have shown that non-piliated mutants ofP. aeruginosacause less epithelialcell damagein vitro and have decreased virulence in a mouse model of acutepneumonia.

Duarte et al (29) demonstrated that two strains ofE. chrysanthemicould adhereand cause oxidative stress and death to mammalian cells in culture. An envelopeprotein immunologically related to enteropathogenicE. coli intimin is expressedat higher levels whenE. chrysanthemicells are grown in the presence of mam-malian cells, but more studies are needed to confirm the role of this intimin-likeprotein in the phenomena analyzed by these authors and in the plant pathogenesismodel.

FLAGELLA Flagella are essential for swimming and chemotaxis ofPseudomonas,as well as for the initial steps in biofilm development (100). They are also importantin the first steps of infection, since nonflagellated strains are impaired in virulence,although the strains found in chronic infections in CF lungs are no longer flag-ellated. It has been suggested that the flagellum is important for the bacteria toreach the epithelial cells but, as the infection is established, its high immunogenic-ity and binding to macrophages and PMN leukocytes may promote the growthof variants lacking flagella, better adapted to the CF lung environment (87). Mu-tants of the plant-growth stimulatingPseudomonas fluorescenswithout flagella



are no longer capable of colonizing potato roots (26). Flagellin of an incompatiblestrain ofPseudomonas avenaeacts as an elicitor of hypersensitive response incultured rice cells. Although the purified protein induces cell death, it occurredto a lower extent than when bacterial whole cells or cell extracts were used, in-dicating that additional factors are involved in this process (18). A screening fornonvirulent mutants of the plant pathogenErwinia carotovorasubsp.atrosepticaresulted in one non-motile clone that could be complemented by a DNA fragmentcontaining genes involved in flagella biosynthesis and similar to the Spa proteinsof the human pathogenShigella flexneri, which are involved in the surface presen-tation of antigens, indicating a common strategy in plant and animal pathogens(97).

LIPOPOLYSACCHARIDES (LPS) AND EXOPOLYSACCHARIDES (EPS) Lipopolysaccha-rides (LPS), which are amphipathic components of the outer membrane of Gram-negative bacteria, are involved in the adherence of several bacterial pathogensto their hosts (60). Smooth, wild-type LPS strains ofPseudomonas aeruginosaare usually more virulent than rough isogenic mutants in various animal modelsystems (122). An LPS rough mutant ofRalstonia(Pseudomonas) solanacearumis nonvirulent, whereas the smooth parental strain B1 is able to cause disease inthe plant host. It was also shown that purified LPS from B1 could bind tobaccomesophyll cell walls (148). Two mutants ofR. solanacearumwith altered LPSare not able to survive in planta, and do not cause the symptoms observed for thewild-type parental strain (143). A gene cluster namedopsfrom R. solanacearumis involved in both LPS and exopolysaccharide (EPS) biosynthesis (66). Com-plementation studies have shown that restoration of LPS biosynthesis alone didnot restore the virulence phenotype, indicating that EPS is also important for fullvirulence (66).

VIRULENCE FACTORS SPECIFIC FOR PATHOGENESISIN PLANT HOSTS

Because of the strategy employed in our studies with the PA14-multihost system,in addition to those common virulence factors, we identified genes that are re-quired for virulence in plant hosts but not in animal hosts. In addition to severalpreviously unknown genes, we identified an interesting pattern in three genes withknown functions, all of which are associated with antibiotic resistance (one ofthem ismexA, which encodes a non-ATPase membrane efflux pump). This findingmay be explained if we consider the fact that plants normally produce a vari-ety of phenolic compounds (e.g. phytoalexins) with antimicrobial activities andwhich are part of the defense response; hence, antibiotic resistance-related genesmay contribute to the survival of pathogens in a plant host. The identification ofplant-specific virulence factors clearly indicates an ability of bacteria to distin-guish between different hosts. Although this is not the major theme of this review,



we would like to point out that understanding host-specific virulence factors inaddition to those common pathogenesis mechanisms should facilitate the ultimateelucidation of bacterial pathogenesis in any specific type of hosts used in thescreen.

COMMON FEATURES IN THE INNATE DEFENSE RESPONSEOF PLANTS, INSECTS, AND MAMMALS

Although host defense responses against pathogens are outside of the scope ofthis review, we briefly discuss some common features of defense responses sharedamong plants, insects, and mammals.

Recent studies have uncovered remarkable conservation of innate defense mech-anisms among plants, insects, and mammals that point to a common ancestry ofthe systems. Both animals and plants respond to bacterial infection by activatinga membrane-generated oxidative burst, utilizing NADPH as the source of elec-trons (28). Remarkably, the generation of oxygen radicals by NADPH oxidase inboth plants and animals appears to utilize the same multicomponent complex con-sisting of a membrane-associated flavocytochrome and three cytosolic activatingcomponents: p47phox, p67phox, and the small GTPase referred to as Rac (85). Stud-ies conducted by Hassanain et al (50) have shown that plant Rac proteins inducesuperoxide production in mammalian cells. These studies demonstrate the remark-able structural and functional conservation of Rho/Rac proteins between plant andanimal kingdoms during evolution. Moreover, the signal transduction pathwaysleading from an oxidative burst to the activation of defense-related genes may beconserved between plants and animals. In the mammalian innate immune response,reactive oxygen species activate the transcription factors NF-κB (127) and AP-1(93, 138).

Another important finding is that plant proteins involved in the activation ofthe plant defense response resemble theDrosophilaToll receptor and its humanhomologs, the Toll-like receptors (TLRs) (123). The plant defense response ismediated by so-called disease resistance orR genes (27).R genes are abundant,comprise a limited number of structurally distinct families, and confer resistanceto viruses, bacteria, fungi, nematodes, and aphids (9, 23, 94). The most preva-lent R gene family shows homology with the cytoplasmic domains of the Tolland mammalian interleukin-1 receptor (IL-1R) (77, 149). TLR2 and TLR4 func-tion in innate immunity to activate microbial defense pathways to trigger inflam-matory responses after recognition of conserved molecular structures of distinctmicrobial pathogen classes (62). Interestingly, TLR-inflammatory-mediated re-sponses resemble plant defense responses, including the production of antimi-crobial active oxygen species, nitric oxide, and ultimately cell death (3, 13). Inmammalian cells, IL-1R is involved in transduction of a pathogen-generated sig-nal that results in the translocation of NF-κB from the cytoplasm to the nucleus(43).



In addition to the above, plants, invertebrates, and vertebrates all produce aclass of antimicrobial peptides called defensins in a pathogen-inducible manner(15, 42, 56). Defensins from all of these species are folded similarly, which suggeststhat they may predate the point of evolutionary divergence between animals andplants. It has been shown that the components of the regulatory cascade that leadto the synthesis of defensins inDrosophilahave homologues in the cascade thatmediates the cytokine-induced activation of NF-κB in mammals. This observationfurther supports the notion that host defenses in higher eukaryotes involve a highlyconserved regulatory pathway mediated by a set of interacting homologous proteindomains present in proteins found in all eukaryotes (78).

CONCLUSION

In this review, we have examined in detail several components that belong tothe common mechanisms for bacterial pathogenesis in both plant and animalhosts, largely based on the studies performed with the PA14-multihost system. Ingeneral, these common virulence factors fall into two classes: one whose memberscontribute to virulence by regulating or controlling a number of virulence-relatedgenes (Regulator Class) and the other whose members exert effects directly by per-forming a defined virulence-related function (Effector Class). The majority of thesecommon virulence factors are widely conserved across different bacterial species,including the GacS-GacA two-component regulatory system, quorum sensing sys-tem, adhesion-related factors (type IV pili, flagella and LPS), sigma factorσ 54,dsbA, degP, plcS, andhrpM. Since the number of mutants screened to date is farfrom saturating the screen for identification of any possible virulence-associatedgene in the genome ofP. aeruginosa, we believe that more common factors forpathogenesis in addition to those discussed in this review will be identified byscreening additional PA14 mutant libraries.

To date, we have not been able to identify any studies performed in eitherErwinia spp. orB. cepaciausing bacteria-multihost system similar to theP. aerug-inosasystem. Therefore, to establish a similar bacteria-multihost system as inP.aeruginosa, it would be necessary to identify a particularErwinia spp. orB. cepa-cia strain that is infectious in both plant and animal hosts. This would not onlyconfirm what we have found in theP. aeruginosasystem, but also would com-plement what cannot be achieved in theP. aeruginosasystem because of possiblelimitations. Overall, the identification of functionally conserved bacterial virulencefactors should lead to both a better understanding of the fundamental mechanismsof bacterial pathogenesis and of the complexity of host-pathogen interactions ingeneral.

ACKNOWLEDGMENT

We would like to thank Drs. F.M. Ausubel and N.J. Panopoulos for allowing us toinclude in this review previously unpublished data.



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11 Jul 2001 13:51 AR AR138-11-COLOR.tex AR138-11-COLOR.SGM ARv2(2001/05/10)P1: FUI

Figure 1 Attachment ofPseudomonas aeruginosaPA14 strain to the surface struc-tures of Arabidopsis ecotype Ll-0 leaf. (a) Clustering of PA14 cells on trichome;(b) congregating of PA14 cells at stomatal opening; (c) attaching of PA14 cells on thecell wall of a parenchyma vessel cell. Arrowheads point to the holes in the plant cellwall with a diameter similar to that of the bacteria. b, PA14 cells; Bar= 1µm.

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