Accumulating evidence from a number of studies strongly suggests that proteins orthologous to those involved in type IV pili (Tfp) assembly and function are required for Francisella pathogenicity. However, the molecular mechanisms by which the components exert their influence on virulence remain poorly understood. Owing to the conservation and promiscuity of Tfp biogenesis machineries, expression of Tfp pilins in heterologous species has been used successfully to analyse organelle structure–function relationships. In this study we expressed a number of Francisella pilin genes in the Tfp-expressing pathogen Neisseria gonorrhoeae lacking its endogenous pilin subunit. Two gene products, the orthologous PilA proteins from Francisella tularensis subspecies tularensis and novicida, were capable of restoring the expression of Tfp-like appendages that were shown to be dependent upon the neisserial Tfp biogenesis machinery for surface localization. Expression of Francisella PilA pilins also partially restored competence for natural transformation in N. gonorrhoeae. This phenotype was not complemented by expression of the PulG and XcpT proteins, which are equivalent components of the related type II protein secretion system. Taken together, these findings provide compelling, although indirect, evidence of the potential for Francisella PilA proteins to express functional Tfp.
AasF.,
WolfgangM.,
FryeS.,
DunhamS.,
LøvoldC.,
KoomeyM.2002; Competence for natural transformation in Neisseria gonorrhoeae: components of DNA binding and uptake linked to type IV pilus expression. Mol Microbiol 46:749–760
AasF.,
Egge-JacobsenW.,
Winther-LarsenH.,
LøvoldC.,
HitchenP.,
DellA.,
KoomeyM.2006; Neisseria gonorrhoeae type IV pili undergo multisite, hierarchical modifications with phosphoethanolamine and phosphocholine requiring an enzyme structurally related to lipopolysaccharide phosphoethanolamine transferases. J Biol Chem 281:27712–27723
AasF.,
Winther-LarsenH.,
WolfgangM.,
FryeS.,
LøvoldC.,
RoosN.,
van PuttenJ.,
KoomeyM.2007b; Substitutions in the N-terminal alpha helical spine of Neisseria gonorrhoeae pilin affect Type IV pilus assembly, dynamics and associated functions. Mol Microbiol 63:69–85
AlmR.,
MattickJ.1996; Identification of two genes with prepilin-like leader sequences involved in type 4 fimbrial biogenesis in Pseudomonas aeruginosa
. J Bacteriol 178:3809–3817
AlmR.,
BoderoA.,
FreeP.,
MattickJ.1996a; Identification of a novel gene, pilZ, essential for type 4 fimbrial biogenesis in Pseudomonas aeruginosa
. J Bacteriol 178:46–53
AlmR.,
HallinanJ.,
WatsonA.,
MattickJ.1996b; Fimbrial biogenesis genes of Pseudomonas aeruginosa: pilW and pilX increase the similarity of type 4 fimbriae to the GSP protein-secretion systems and pilY1 encodes a gonococcal PilC homologue. Mol Microbiol 22:161–173
CarbonnelleE.,
HelaineS.,
NassifX.,
PelicicV.2006; A systematic genetic analysis in Neisseria meningitidis defines the Pil proteins required for assembly, functionality, stabilization and export of type IV pili. Mol Microbiol 61:1510–1522
ChakrabortyS.,
MonfettM.,
MaierT.,
BenachJ.,
FrankD.,
ThanassiD.2008; Type IV pili in Francisella tularensis: roles of pilF and pilT in fiber assembly, host cell adherence, and virulence. Infect Immun 76:2852–2861
DennisD.,
InglesbyT.,
HendersonD.,
BartlettJ. G.,
AscherM. S.,
EitzenE.,
FineA. D.,
FriedlanderA. M.,
HauerJ.other authors2001; Tularemia as a biological weapon: medical and public health management. JAMA 285:2763–2773
DurandE.,
BernadacA.,
BallG.,
LazdunskiA.,
SturgisJ.,
FillouxA.2003; Type II protein secretion in Pseudomonas aeruginosa: the pseudopilus is a multifibrillar and adhesive structure. J Bacteriol 185:2749–2758
EllemanT.,
HoyneP.,
StewartD.,
McKernN.,
PetersonJ.1986; Expression of pili from Bacteroides nodosus in Pseudomonas aeruginosa
. J Bacteriol 168:574–580
ForslundA.,
KuoppaK.,
SvenssonK.,
SalomonssonE.,
JohanssonA.,
ByströmM.,
OystonP. C.,
MichellS. L.,
TitballR. W.other authors2006; Direct repeat-mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis
. Mol Microbiol 59:1818–1830
FullnerK.,
MekalanosJ.1999; Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae
. Infect Immun 67:1393–1404
HeggeF.,
HitchenP.,
AasF.,
KristiansenH.,
LøvoldC.,
Egge-JacobsenW.,
PanicoM.,
LeongW. Y.,
BullV.other authors2004; Unique modifications with phosphocholine and phosphoethanolamine define alternate antigenic forms of Neisseria gonorrhoeae type IV pili. Proc Natl Acad Sci U S A 101:10798–10803
HelaineS.,
DyerD.,
NassifX.,
PelicicV.,
ForestK.2007; 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili. Proc Natl Acad Sci U S A 104:15888–15893
HoyneP.,
HaasR.,
MeyerT.,
DaviesJ.,
EllemanT.1992; Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa
. J Bacteriol 174:7321–7327
KirnT.,
BoseN.,
TaylorR.2003; Secretion of a soluble colonization factor by the TCP type 4 pilus biogenesis pathway in Vibrio cholerae
. Mol Microbiol 49:81–92
LongC.,
HayesS.,
van PuttenJ.,
HarveyH.,
ApicellaM.,
SeifertH.2001; Modulation of gonococcal piliation by regulatable transcription of pilE
. J Bacteriol 183:1600–1609
LuH.,
MotleyS.,
LoryS.1997; Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion. Mol Microbiol 25:247–259
MartinP.,
WatsonA.,
McCaulT.,
MattickJ.1995; Characterization of a five-gene cluster required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa
. Mol Microbiol 16:497–508
ParkH.,
WolfgangM.,
KoomeyM.2002; Modification of type IV pilus-associated epithelial cell adherence and multicellular behavior by the PilU protein of Neisseria gonorrhoeae
. Infect Immun 70:3891–3903
PeabodyC.,
ChungY.,
YenM.,
Vidal-IngigliardiD.,
PugsleyA.,
SaierM. J.2003; Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella. Microbiology 149:3051–3072
SandströmG.,
TärnvikA.,
Wolf-WatzH.,
LöfgrenS.1984; Antigen from Francisella tularensis: nonidentity between determinants participating in cell-mediated and humoral reactions. Infect Immun 45:101–106
SauvonnetN.,
GounonP.,
PugsleyA.2000; PpdD type IV pilin of Escherichia coli K-12 can be assembled into pili in Pseudomonas aeruginosa
. J Bacteriol 182:848–854
StromM.,
NunnD.,
LoryS.1993; A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. Proc Natl Acad Sci U S A 90:2404–2408
TønjumT.,
KoomeyM.1997; The pilus colonization factor of pathogenic neisserial species: organelle biogenesis and structure/function relationships – a review. Gene 192:155–163
VignonG.,
KöhlerR.,
LarquetE.,
GirouxS.,
PrévostM.,
RouxP.,
PugsleyA.2003; Type IV-like pili formed by the type II secreton: specificity, composition, bundling, polar localization and surface presentation of peptides. J Bacteriol 185:3416–3428
VikA.,
AasF.,
AnonsenJ.,
BilsboroughS.,
SchneiderA.,
Egge-JacobsenW.,
KoomeyM.2009; Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae
. Proc Natl Acad Sci U S A 106:4447–4452
WhippM.,
DavisJ.,
LumG.,
de BoerJ.,
ZhouY.,
BeardenS.,
PetersenJ.,
ChuM.,
HoggG.2003; Characterization of a novicida-like subspecies of Francisella tularensis isolated in Australia. J Med Microbiol 52:839–842
WhitchurchC.,
HobbsM.,
LivingstonS.,
KrishnapillaiV.,
MattickJ.1991; Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria. Gene 101:33–44
Winther-LarsenH.,
HeggeF.,
WolfgangM.,
HayesS.,
van PuttenJ.,
KoomeyM.2001; Neisseria gonorrhoeae PilV, a type IV pilus-associated protein essential to human epithelial cell adherence. Proc Natl Acad Sci U S A 98:15276–15281
Winther-LarsenH.,
WolfgangM.,
DunhamS.,
van PuttenJ.,
DorwardD.,
LøvoldC.,
AasF.,
KoomeyM.2005; A conserved set of pilin-like molecules controls type IV pilus dynamics and organelle-associated functions in Neisseria gonorrhoeae
. Mol Microbiol 56:903–917
Winther-LarsenH.,
WolfgangM.,
van PuttenJ.,
RoosN.,
AasF.,
Egge-JacobsenW.,
MaierB.,
KoomeyM.2007; Pseudomonas aeruginosa Type IV pilus expression in Neisseria gonorrhoeae: effects of pilin subunit composition on function and organelle dynamics. J Bacteriol 189:6676–6685
WolfgangM.,
LauerP.,
ParkH.,
BrossayL.,
HébertJ.,
KoomeyM.1998a; PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae
. Mol Microbiol 29:321–330
WolfgangM.,
ParkH.,
HayesS.,
van PuttenJ.,
KoomeyM.1998b; Suppression of an absolute defect in type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae
. Proc Natl Acad Sci U S A 95:14973–14978
WolfgangM.,
van PuttenJ.,
HayesS.,
KoomeyM.1999; The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation. Mol Microbiol 31:1345–1357
WolfgangM.,
van PuttenJ.,
HayesS.,
DorwardD.,
KoomeyM.2000; Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili. EMBO J 19:6408–6418
ZogajX.,
ChakrabortyS.,
LiuJ.,
ThanassiD.,
KloseK.2008; Characterization of the Francisella tularensis subsp. novicida type IV pilus. Microbiology 154:2139–2150