2 : effects of pilin subunit composition on function accepted · 3 gram negative species of...

1

Pseudomonas aeruginosa Type IV pilus expression in Neisseria 1

gonorrhoeae: effects of pilin subunit composition on function 2

and organelle dynamics 3

4

Hanne C. Winther-Larsen1,2*

, Matthew C. Wolfgang3, Jos PM. van Putten

4, Norbert 5

Roos2, Finn Erik Aas

1,2, Wolfgang M. Egge-Jacobsen

1,2, Berenike Maier

5,6, and Michael 6

Koomey1,2

. 7

8

1Centre for Molecular Biology and Neuroscience and

2Department of Molecular 9

Biosciences, University of Oslo, 0316 Oslo, Norway; 3Department of Microbiology and 10

Immunology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, 11

USA; 4Department of Infectious Diseases and Immunology, Utrecht University, NL-3584 12

CL, Utrecht, The Netherlands; 5Department of Biological Sciences, Columbia University, 13

New York, NY 10027, USA and 6

Present address; Westfälische Wilhelms-Universität 14

Münster, Institut für Allgemeine Zoologie und Genetik, 48149 Münster, Germany. 15

16

* For correspondence: E - mail: [email protected], Tel. (+47) 22 85 41 38; Fax 17

(+47) 22 85 60 41. Department of Molecular Biosciences, University of Oslo, P.O.Box 18

1041 Blindern, 0317 Oslo, Norway 19

Running title: P. aeruginosa Type IV pilus expression in N. gonorrhoeae 20

Key words: PilT, PilC, minor pilins, adherence, transformability, pilus retraction, PilA, 21

bacteriophage, molecular recognition 22

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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00407-07 JB Accepts, published online ahead of print on 15 June 2007

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ABSTRACT 1

Type IV pili (Tfp) play central roles in the expression of many phenotypes including 2

motility, multicellular behaviour, sensitive to bacteriophages, natural genetic 3

transformation, and adherence. In Neisseria gonorrhoeae, these properties require 4

ancillary proteins that act in conjunction with Tfp expression and influence organelle 5

dynamics. Here, the intrinsic contributions of the pilin protein itself to Tfp dynamics and 6

associated phenotypes were examined by expressing the Pseudomonas aeruginosa 7

PilAPAK

pilin subunit in N. gonorrhoeae. We show that although PilAPAK

pilin can be 8

readily assembled into Tfp in this background, steady state levels of purifiable fibers are 9

dramatically reduced relative those of endogenous pili. This defect is due to aberrant Tfp 10

dynamics as it is suppressed in the absence of the PilT pilus retraction ATPase. 11

Functionally, PilAPAK

pilin complements gonococcal adherence for human epithelial cells 12

but only in a pilT background and this property remains dependent on coexpression of 13

both the PilC adhesin and the PilV pilin-like protein. As P. aeruginosa pilin only 14

moderately supports neisserial sequence–specific transformation despite its assembly 15

proficiency, these results together suggest that PilAPAK

pilin functions suboptimally in 16

this environment. This appears to be due to diminished compatibility with resident 17

proteins essential for Tfp function and dynamics. Despite this, PilAPAK

pili support 18

retractile force generation in this background equivalent to that reported for endogenous 19

pili. Furthermore, PilAPAK

pili are both necessary and sufficient for bacteriophage PO4 20

binding although the strain remains phage resistant. Together, these findings have 21

significant implications for Tfp biology in both N. gonorrhoeae and P. aeruginosa. 22

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INTRODUCTION 1

Type IV pili (Tfp) are proteinaceous surface structures found ubiquitously in 2

Gram negative species of medical, environmental and ecological importance. Tfp are 3

defined by their shared structural, biochemical, and morphological features and a highly 4

conserved biogenesis pathway (26) . In line with these conserved features, trans-species 5

complementation of various Tfp biogenesis mutants has been achieved in a number of 6

cases. Trans-species complementation was first documented when the pilin subunit 7

protein from Dichelobacter nodosus was expressed as Tfp in Pseudomonas aeruginosa 8

(11). D. nodosus Tfp purified from this heterologous source engenders immunity to ovine 9

foot rot and recombinant D. nodosus Tfp remains in use today as a vaccine (12). This 10

same methodology was subsequently exploited with success in a number of studies 11

involving the expression of pilins from Moraxella bovis (3), Neisseria gonorrhoeae (20), 12

Escherichia coli (33) and Pseudomonas syringae (29) as Tfp in P. aeruginosa. Similar 13

gene swapping and complementation studies of the related type II secretion systems also 14

revealed relaxed specificity with heterologous expression of pilin-like proteins of the 15

PulG family resulting in formation of pseudopilus structures analogous to Tfp (10, 36) . 16

Strains expressing heterologous pilin subunits provide unique opportunities to 17

examine relationships between Tfp, accessory factors and associated phenotypes. The 18

latter include colonization of biotic and abiotic surfaces, motility across solid surfaces, 19

multicellularity, susceptibility to bacteriophage infection as well as horizontal gene 20

transfer via transformation and conjugation. However these phenotypes are not 21

universally seen in Tfp expressing species. This may reflect constraints associated with 22

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structural features unique to particular pilin subunits or the requirement for ancillary 1

factors that act in concert with an intact Tfp biogenesis pathway to impart function. Tfp 2

pilin subunits from PAK and PAO strains of P. aeruginosa pilin have been shown to 3

complement pilin null mutants of Pseudomonas stutzeri in DNA uptake required for 4

transformation (16) as does the P. aeruginosa PAK pilin subunit when expressed in N. 5

gonorrhoeae (2). It is important to note that competence for natural transformation has 6

not been documented in P. aeruginosa strains. As DNA uptake in both these backgrounds 7

requires an intact Tfp biogenesis pathway and a pilin subunit capable of being assembled, 8

it is presumed that heterologous pili are expressed in these cases but this has yet to be 9

demonstrated. Heterospecific complementation of type II secretion defects likewise 10

suggest that PulG type pseudopilins function by virtue of their abilities to oligomerize 11

into pseudopilus polymers (10, 36). In both the Tfp associated competence and secretion 12

systems, functionality is not attributable to the unique structural features of individual 13

pilins but rather correlates best with the assembly proficiencies of the subunits. 14

Numerous bacteriophages utilize pili as a primary receptor to infect cells. 15

Infection here involves binding of phage particles to the sides or tip of the pilus and 16

retraction of the pilus which brings the phage into contact with a second, cell surface 17

associated receptor. Phage components governing molecular recognition of pili have been 18

well defined in a number of systems. For example, minor coat proteins of the g3p family 19

act as pilus receptors in many filamentous phages (8, 18, 19). The structural features and 20

components of pili that participate in these interactions have yet to be defined in any 21

system. With regard to Tfp / bacteriophage systems, strain P. aeruginosa PAK and the 22

pilus-dependent bacteriophage PO4 have been utilized extensively. In these works, 23

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expression of a remarkably diverse group of exogenous Tfp pilin subunits in P. 1

aeruginosa PAK backgrounds (lacking endogenous pilin - pilA) have been reported to 2

restore twitching motility (indicative of pilus retraction) and productive PO4 infection 3

(29, 37). Similarly, three distinct pilin genes were reported to restore PO4 sensitivity in a 4

pilA strain of P. stutzeri (16). Based on the reported relaxed specificity of PO4 phage vis 5

a vis subunit composition, some have surmised that a “minor” pilus associated protein 6

might actually be serving as the primary receptor (29). Alternatively, PO4 phage might 7

recognize a degenerate motif widely distributed in Tfp polymers or pilin subunits. To 8

further cloud this issue, one study reported that P. aeruginosa strain PAO is readily 9

susceptible to PO4 (5) while another concluded that strain PAO only became PO4 10

sensitive when complemented by the PAK pilin gene (37). The molecular contribution of 11

Tfp to phage binding and recognition in P. aeruginosa, or for that matter in any Tfp 12

expressing species, thus remain a matter of controversy. 13

Another issue of interest in Tfp biology relates to the mechanisms by which pili 14

promote adherence to mammalian tissue. Tfp mediated adherence of N. gonorrhoeae to 15

human epithelial cells requires the coordinated expression of PilC, which has intrinsic 16

adhesin properties, and six pilin-like proteins that appear to impact on adherence by 17

influencing PilC activity or trafficking (39, 40, 42). Each of these proteins co-purify with 18

Tfp and all but the PilV pilin-like protein impact on Tfp dynamics by promoting 19

extension/polymerization events in the presence of the PilT pilus retraction ATPase. In 20

contrast, Tfp-mediated epithelial cell adherence exhibited by Pseudomonas aeruginosa 21

PAK is thought to require a receptor binding domain located within residues 128–144 of 22

the C-terminal region of the PilA pilin subunit itself (21). This receptor binding domain is 23

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only exposed at the tip of the pilus (23). To date, no study has directly examined 1

adherence phenotypes related to heterologous expression of PilA as Tfp. 2

In an effort to delineate the potential contributions of pilin subunit structure and 3

chemistry to Tfp associated phenotypes, we undertook a systematic assessment of 4

phenotypes imparted by expression of P. aeruginosa PilAPAK

pilin in N. gonorrhoeae. 5

6

MATERIALS AND METHODS 7

Bacterial strains and plasmids. Gonococcal strains used in this study are 8

described in Table 1. Antibiotics were used at concentrations previously described for 9

selection of gonococcal transformants (40). E. coli HB101 was used for plasmid 10

propagation. Isolation and purification of plasmid DNA were performed using Qiagen 11

columns according to the manufacturers specifications (Qiagen, Chatsworth, CA). The 12

plasmid pPilE::cat (17) was used to inactivate the wildtype pilE locus creating the strain 13

KS45. The pilTind, pilC2 phase-off variant mutant (KS56) was isolated according to the 14

same procedure described earlier (42). 15

16

Characterization of twitching motility. Twitching motility was assessed by 17

observation of cells at the periphery of colonies using a Stereozoom 7 (Bausch and 18

Lomb) stereomicroscope as described as well as by the slide culture method in which 19

cells are inoculated onto Gc agar slices on microscope slides, covered with a coverslip 20

and visualized under a Zeiss phase microscope using the 40× objective (41). 21

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1

Tfp retraction assay. For retraction experiments, 3-µm silica beads 2

(Polysciences) were coated with poly(L-lysine) and adsorbed to glass coverslides by 3

centrifugation. 2 µm carboxylated latex beads (Polysciences) were added without further 4

treatment to a suspension of gonococci, mounted them on a microscope slide, and sealed 5

them. The optical tweezers system including calibration methods and the determination 6

of the velocity vs. force curve has been described previously (25). 7

8

Epithelial cell bacterial adherence. Primary cultures of human corneal epithelial 9

cells were established (35) and the adherence assays of gonococcal strains to the human 10

corneal epithelial cells were performed as described (39). 11

12

Pilus purification. Pili were purified by the ammonium sulphate procedure as 13

previously described (41) except that the bacterial cells were collected from two heavily 14

streaked Petri dishes and suspended in 1 ml of 0.15 M ethanolamine, pH 10.5, and the 15

centrifugation time lengths were reduced to 5 min. 16

17

SDS/PAGE, immunoblotting and staining. Procedures for SDS/PAGE, 18

immunoblotting and coomassie staining have previously been described (15) and silver 19

staining SDS/PAGE gels were performed according to standard techniques. PilA, PilE, 20

PilC and PilV were detected by immunoblotting of pilus preparations and whole cell 21

lysates using specific rabbit polyclonal antibodies and alkaline phosphatase-coupled goat 22

anti-rabbit antibodies (Tago). The PilA specific sera was a gift from E. Gotschlich 23

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(Rockefeller University, NY) and the PilC specific sera was a gift from A.-B. Jonsson 1

(Uppsala University, Uppsala). The PilV- and PilE-specific sera were previously 2

described (1, 39). 3

4

Immunofluorescence microscopy. This was performed as described (40) except 5

that the gonococcal strains were grown up to OD550 = 0.2 before fixing them on poly(L-6

lysine) coated glass coverslips. The gonococcal pili were labeled using rabbit serum 7

raised against purified Tfp from strain N400 (9), and the PAK pili were labeled with PilA 8

specific antiserum. Images shown correspond to fields that are representative of the 9

overall observations. 10

11

Immunogold and transmission electron microscopy. Sample grids were 12

prepared by touching carbon-coated Formvar copper grids to individual bacterial colonies 13

grown on GC agar (18 h, 37°C, 5 % CO2) and fixed with 0.5 % glutaraldehyde in PBS, 14

pH 7.4 for 5 min. Grids were washed three times on drops of PBS and subsequently 15

negatively stained with 0.5 % ammonium molybdate in water for 5 min before viewed in 16

a Philips CM100 transmission electron microscope. For immunogold labeling the grids 17

with fixed bacteria were first blocked with 0.5 % newborn calf serum (NCS) and then 18

incubated with rabbit anti-pilin PilA specific antibody (dilution 1:500) for 10 min at room 19

temperature. After washing 4 times on drops of PBS, the grids were incubated with gold-20

conjugated Protein A (5 nm). After three rinses on drops of PBS followed by three rinses 21

on drops of water, the grids were stained 4 minutes with uranyl acetate. The same 22

procedure was repeated using rabbit antisera specific for PilE; lots 2-66 and 904 (dilution 23

1:100), lot T36 (1:10) and lot T40 (1:20). The T36 and T40 antibodies specifically 24

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recognizing PilE epitopes exposed at the tips of the pilus structure (14). The PilE 904 1

antibodies were made against purified N. gonorrhoeae pilin subunit from the strain N400. 2

For the double immunogold labeling the grids with N. gonorrhoeae were incubated with 3

PilA specific antisera (dilution 1:400 for 30 min), rinsed 3 x 5 min on drops of PBS 4

before incubation with gold-conjugated Protein A (10 nm gold for 30 min). The grids 5

were washed 3 x 5 min on drops of PBS, then blocked by incubation on free Protein A 6

for 15 min and rinsed 3 x 5 min on drops of PBS before being incubated with the rabbit 7

anti-PilE specific antibodies (dilution 1:400) for 30 min at room temp. This was followed 8

by 3 x 5 min washes with PBS and then incubated with gold-conjugated Protein A (5 nm 9

gold for 30 min) before rinsing 3 x 5 min on drops of PBS followed by 2 x 5 min washes 10

on drops of dH2O. Finally, the grids were stained for 10 min with 2% uranyl acetate. The 11

procedure was repeated by changing the PilE and PilA specific antiserum incubation 12

steps. As an experimental control to exclude the possibility of cross-reactivity, the 13

incubation step with the second primary antibody was replaced with incubation on drops 14

of PBS. 15

16

Interactions of phage PO4 with Tfp. Standard methods were used for phage 17

PO4 preparation and phage titration (5, 6). Electron microscopy was used to study PO4 18

virons absorbed to N. gonorrhoeae cells by a method derived from (4). N. gonorrhoeae 19

cells grown overnight on GC plates (37ºC, 5% CO2) were resuspended in GC broth to 2 x 20

108 cells/ml and mixed with an equal volume of phage PO4 at 6x10

9 PFU/ml. After 21

gentle shaking at 37ºC for 10 min, bacteria were subjected to negatively stained and 22

immunogold labeling for electron microscopy. 23

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1

Sample Preparation for Intact Protein MS. Isolated PilA protein was rinsed 2

and washed with a methanol/chloroform precipitation procedure as described (38). 3

Briefly, one hundred µl of the aqueous PilA solution at 2-3 mg/ml protein was diluted 1:3 4

(v/v) with methanol and mixed briefly. Both 100 µl CHCl3 and 200 µl of water were 5

added consecutively, and were followed each time by a mixing step. Phase separation was 6

achieved by centrifugation (4,000 × g, for 8 min), yielding precipitated PilA at the 7

interface. The upper methanol/water phase was removed, and 400 µl methanol was 8

added. After mixing, the pellet was recovered by centrifugation (13,000 × g, 8 min). The 9

pellet was dried for 5 min in the inverted tube before dissolving the sample in 50 µl of 10

water/70% formic acid/acetonitrile 3:1:3 (v/v/v). Samples were subjected immediately to 11

mass spectrometric analyses or frozen at -80°C. 12

Infusional MS Analysis of Intact Protein. All data were acquired on a 13

quadrupole time-of-flight mass spectrometer (Q-Tof micro, Micromass, Manchester,

UK) 14

equipped with the standard Z-spray ESI source. Sample solutions were infused into the 15

ESI source at a flow rate of 5 µl/min using a syringe pump (Cole-Parmer Instrument 16

Company, Model SP 100i, Vernon Hills, USA). The source block temperature was 17

maintained at 80°C. Nitrogen was used as both desolvation and nebulizing gas with flow 18

rates of 300 and 20 l/h, respectively. Mass spectrometric analyses were performed in the 19

electrospray positive mode with the following parameter settings (parameter names as 20

used in the MassLynx NT software, version 3.5): capillary voltage, 3000V; sample cone 21

voltage 25V; extraction cone voltage 4.3V; ion energy 3V; and collision energy 10eV. 22

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Mass spectral resolution was typically 4000. The MS Survey was obtained in a mass 1

range from 150 m/z and 1700 m/z. Mass calibration in a mass range of 100-2200 m/z was 2

performed using the ES tune mix solution from Agilent (Agilent, Palo Alto, CA, USA). 3

The MS spectra were analyzed using the MassLynx software (version 3.5). For 4

deconvolution, spectra were processed with the MaxEnt1 program of the MassLynx 5

software. 6

7

8

RESULTS 9

P. aeruginosa Tfp expression in N. gonorrhoeae. We previously demonstrated 10

the ability of PilAPAK

pilin to partially complement competence for natural 11

transformation in a N. gonorrhoeae strain defective in expression of endogenous PilE (2). 12

PilAPAK

pilin was expressed in this background as a pilE translational fusion such that 13

following processing by prepilin peptidase, the mature polypeptide was entirely P. 14

aeruginosa derived. This approach also insured levels of expression equivalent to that of 15

endogenous pilin. When examined in whole cell lysates, levels of heterologous pilin were 16

indistinguishable from that of endogenous PilE (Fig. 1). In contrast, levels of PilAPAK

17

pilin in purifiable Tfp were dramatically reduced relative to that of PilE pilin. This effect 18

was not related to an inhibitory effect of PilAPAK

pilin on the integrity of the biogenesis 19

machinery since levels of purifiable PilE remained unaltered in a strain co-expressing 20

PilAPAK

. Furthermore, levels of PilAPAK

pilin in the shear-recoverable fraction were 21

nearly equivalent to that of PilE when tested in a mutant background lacking the PilT 22

retraction ATPase. Thus, the failure to recover high levels of PilAPAK

pili here is related 23

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to distorted Tfp dynamics associated with organelle retraction rather than an intrinsic 1

inability of PilAPAK

pilin to polymerize into Tfp in an N. gonorrhoeae background. 2

Immunofluoresence microscopy provided further insights into heterospecific 3

PilAPAK

pilus expression (Fig. 2). In contrast to the bundles of short, lateral aggregates 4

seen for endogenous Tfp, pili in the strain solely expressing PilAPAK

pilin were detected 5

primarily as long individual filaments together with a lower abundance of short fibers. 6

Moreover, levels of PilAPAK

pilus antigen seen by this technique were similar to that seen 7

for endogenous pilus antigen. Co-expression of endogenous PilE in this background 8

resulted in a dramatic increase in the amount of PilAPAK

pilus antigen detected. Thus, 9

PilE stimulates the levels of PilAPAK

antigen seen using this technique. In both these 10

instances, there was a clear discrepancy between levels of PilAPAK

pili seen by 11

purification yield versus the immunofluoresence assay. Expression of PilAPAK

pilin alone 12

in a pilT background led to a strong increase in the levels of immunoreactive pili. 13

Evidence for a unique tip structure on P. aeruginosa Tfp in N. gonorrhoeae. 14

Immunoelectron microscopy confirmed the specificities of the PilE and PilA derived sera 15

seen in the immunofluoresence assays (Fig. 3). Additionally, differential labeling using 16

sequential treatment with coupled 10 and 5 nm gold particles showed two serologically 17

distinct pilus populations on each cell. There was no evidence for polymer heterogeneity 18

vis a vis PilE and PilAPAK

. Nonetheless, a significant proportion of PilAPAK

pili 19

(approximately 50% on average) stood out due to the absence of immunolabeling at their 20

free ends (Fig. 4). In those instances where it was possible to orient and track filaments 21

back to cells, it was clear that such ends corresponded to the distal pilus end. These non-22

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reactive segments were additionally noteworthy due to their relatively constant contour 1

length. In addition, non-reactive tip structures were strongly associated with concurrent 2

PilE expression as they were undetectable in its absence. A simple explanation for this 3

phenomenon might then be that the tip structures were comprised of PilE. Efforts to test 4

this were equivocal due to the low valency with which PilE antibodies reacted with 5

homologous pili. We also investigated this by using antibodies previously published to 6

bind specifically to the tip of PilE pili (14), but no binding was detected (results not 7

shown). Regardless of the basis for these findings, this is to our knowledge the first 8

direct evidence in any Tfp system for a distinct, filament tip structure. 9

Characterization of Tfp retraction – associated events in N. gonorrhoeae 10

expressing P. aeruginosa PilAPAK

. As dynamic polymers, Tfp undergo rounds of 11

extension and retraction modeled as pilin subunit polymerization and depolymerization 12

(43). To assess if PilAPAK

pili were capable of undergoing retraction in N. gonorrhoeae, 13

strains were examined microscopically for the expression of twitching motility by 14

examining cell movement at the periphery of colonies as well as by the slide culture 15

method. Zones of “crawling”cells moving in jerky fashion relative to one another were 16

readily detectable in the PilAPAK

pilin background and this movement was abolished in a 17

pilT background. These phenotypes in the PilAPAK

pilin background were 18

indistinguishable from those seen for wildtype control strain (data not shown). 19

We next characterized the kinetics and force generation of PilAPAK

pilus 20

retraction using laser tweezers (Fig. 5). The method has been described previously (25). 21

In short, single cells of N. gonorrhoeae were immobilized at the surface of a microscope 22

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cover slide. Subsequently, a 2µm latex bead was trapped in the laser tweezers and placed 1

in close vicinity of the bacterium. When a pilus bound to the bead and retracted the 2

displacement of the bead was used as a measure of pilus displacement as a function of 3

time as a function of the optical restoring force acting on the bead. It was found that pili 4

retracted at a frequency of 0.16 ± 0.03 retractions/sec, i.e. on average a pilus would bind 5

to the bead and retract once in 6 ± 1 seconds. The retraction velocity was constant at v = 6

1600 ± 100 nm/sec at forces below 40pN. At forces > 40pN the velocity decreased and 7

the velocity was 270 ± 30 nm/sec at 100pN, i.e. the velocity decreased by a factor of ~6 8

from 0pN and 100pN. Force-dependent elongation of pili was not observed with this 9

strain. 10

Characterization of human epithelial cell adherence mediated by P. 11

aeruginosa Tfp in N. gonorrhoeae. Current models invoke that human epithelial 12

adherence mediated by PAK pili requires a pilin subunit receptor binding domain 13

localized to residues 128 to144 (21). To directly test this hypothesis, we first examined 14

the interaction of the strain solely expressing PilAPAK

pilin and found that it did not 15

adhere and was indistinguishable from a mutant devoid of pilin subunit expression (Fig. 16

6). As this finding might be due to the low steady state levels of pili expressed in this 17

background, its pilT derivative was examined and found to adhere well with an average 18

of more than 100 bacteria/epithelial cell being observed. We then examined the influence 19

of PilC, the N. gonorrhoeae Tfp-associated epithelial cell adhesin, and the PilV pilin-like 20

protein on this phenotype. Although PilC is also required for high, steady state Tfp levels, 21

this role in organelle expression is dispensable in a pilT background. As shown in Fig. 7, 22

levels of purifiable PilAPAK

Tfp were undiminished in the pilC, pilT and pilT, pilV 23

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backgrounds. Nonetheless, epithelial adherence was significantly diminished in these 1

backgrounds (i.e. to the level seen for a pilin null mutant) (Fig. 6). Despite the presence 2

of the PilAPAK

subunit, epithelial cell adherence proficiency was dependent on PilC and 3

PilV. Moreover like the situation in the homologous PilE expressing background (39), 4

PilC was critical to the ability of PilV to co-purify with Tfp while levels of co-purifying 5

PilC were only moderately influenced by PilV (Fig. 7). Additionally, levels of co-6

purifying PilV in the PilAPAK

background never achieved those seen in the corresponding 7

PilE background (Fig. 7, lanes 2 vs. 8). 8

P. aeruginosa PilAPAK

pilin in its assembled form is both necessary and 9

sufficient for bacteriophage PO4 binding. The capacity of heterologous Tfp to function 10

as primary bacteriophage receptors has primarily been assessed by productive infection 11

as seen by plaque formation or growth inhibition. As N. gonorrhoeae strains expressing 12

PAK pili showed no discernible phenotypes following exposure to high titer PO4 phage 13

stocks (not shown), Tfp-phage interactions were assessed directly by TEM using a strain 14

expressing solely exogenous pilin. Using this approach, PAK pili were clearly decorated 15

with PO4 phage in a manner indistinguishable from that reported for strain P. aeruginosa 16

PAK (Fig. 8, (5)). Specifically, phage particles were distributed tail–first along PAK pili 17

filaments and these interactions appeared to be mediated by a direct interaction between 18

pili and phage tail fibers. To further confirm the specificity of these events, strains 19

coexpressing endogenous and PAK pili were first exposed to phage and then 20

immunolabeled with PAK specific antibodies. As shown in Fig. 8E, PO4 phage were 21

exclusively associated with PAK pili. We conclude that PilAPAK

pilin in its assembled 22

form is both necessary and sufficient for PO4 recognition. Additionally, the resistance of 23

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N. gonorrhoeae strains expressing PAK pili to productive PO4 infection must result from 1

a block subsequent to phage binding its primary receptor. 2

Intact mass analysis of P. aeruginosa PAK pilin. Endogenous PilE pilin 3

undergoes unique post translational modifications including glycosylation and covalent 4

addition of the phosphoforms phosphethanolamine and phosphocholine (17). To assess 5

the status of PilAPAK

pilin in N. gonorrhoeae, electrospray ionization MS of the intact 6

protein was performed. Following deconvolution of the data signals, this revealed one 7

well-defined peak at m/z 14,993 consistent with the mass values predicted from PilD - 8

proteolytically processed but otherwise unmodified protein (Supplementary Fig. S1). 9

This finding was further corroborated by analysis of intact PilAPAK

pilin derived from P. 10

aeruginosa which yielded an identical value. Thus, PilAPAK

pilin does not undergo 11

covalent post translational modifications in either background. Therefore, the phenotypes 12

seen for PAK expressing N. gonorrhoeae strains are not influenced by additional or 13

alternative modifications in subunit structure and chemistry. 14

15

DISCUSSION 16

Prior studies of Tfp functions utilizing expression of heterologous pilin subunits 17

have focused on a limited set of phenotypes comprised of twitching motility, phage 18

sensitivity and competence for natural transformation. Our findings using N. gonorrhoeae 19

expressing P. aeruginosa PilAPAK

pilin provide new insight into the contributions of 20

subunit constitution to Tfp function. Most notably, PilAPAK

complemented a pilE mutant 21

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in adherence for human primary corneal epithelial cells and this property required the 1

PilC adhesin and the PilV pilin-like protein. Thus, the pilin subunit itself is not the main 2

determinant of adherence. Rather in its polymeric form, it appears to act in a generic 3

fashion so as to support the display of the PilC adhesin. In line with this, PilC and PilV 4

were recoverable in the sheared Tfp fraction from the PilAPAK

background. Moreover, 5

like the case with endogenous pilin (39), Tfp-associated PilC levels were moderately 6

reduced in a pilV background and PilV levels were reduced in the pilC background. 7

Together with the PilC co-purification data, immuno EM localization studies suggest that 8

PilC is directly associated with Tfp (31). For such a model to be true here, PilC would 9

have to associate directly or indirectly with the heterologous Tfp structure. However, we 10

cannot rule out that PilA derived Tfp or the process of Tfp assembly and extrusion in 11

itself supports PilC function independent of its direct association with Tfp. The data also 12

reveal signs that adherence proficiency imparted by heterologous Tfp is not equivalent to 13

that seen in the wildtype background. Notably, adherence imparted by PilAPAK

was only 14

detected in a pilT mutant. This failure to detect epithelial cell binding in the wildtype 15

background could be ascribed to low, steady state Tfp levels. Even when this defect was 16

suppressed in the pilT background, levels of adherence never reached those seen in the 17

wildtype strain. This may in part be attributed to the non-autoagglutinating phenotype 18

exhibited by the complemented strain as bacterial - bacterial aggregation contributes to 19

overall adherence proficiency (27). 20

The requirement for PilC and PilV for PilAPAK

pilin - mediated adherence was 21

surprising given the prevailing dogma that P. aeruginosa Tfp - mediated adherence to 22

human epithelial cell requires a receptor binding domain located within the C-terminal 23

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region of pilin (21). Clearly, adherence mediated by such an intrinsic domain is not 1

manifest in the gonococcal background. The in vivo relevance of the PilA binding domain 2

and its putative asialoGM1 glycosphingolipid receptor (23) has recently been drawn into 3

question (13). Assuming that there is in fact an intrinsic PilA receptor binding domain, 4

we surmise that it is not properly exposed in the gonococcal background. 5

Our previous work documented the ability of PilAPAK

pilin to complement DNA 6

binding and uptake required for natural transformability (2). However, the frequency of 7

transformation supported by PilA was only 2.5% of that seen in the wild-type 8

background. Many studies have shown that strains expressing very low, steady levels of 9

endogenous Tfp (resulting from reduced levels of pilin expression or pilE alleles partially 10

defective in assembly) retain high level transformability and such strains express far 11

lower levels of Tfp than those seen here for the PilAPAK

pilin (1, 24, 30). The reduced 12

activity of PilAPAK

vis a vis transformation seems therefore unrelated to a quantitative 13

defect in Tfp expression. Rather, the data imply a degree of functional incompatibility 14

between PilAPAK

itself or PilAPAK

Tfp and other components involved in DNA binding 15

and uptake. It is important to note here that overexpression of the pilin-like ComP protein 16

enhanced DNA uptake and transformability over a 100-fold in this background while no 17

increase in levels of Tfp were seen (2). Thus, PilAPAK

appears defective in supporting 18

ComP function or a ComP dependent pathway in a manner unrelated to its assembly 19

proficiency. 20

In addition to the sub-optimal human cell adherence and transformation 21

phenotypes associated with PilAPAK

, other findings suggest diminished compatibility 22

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between the exogenous pilin and endogenous machinery. Most notably, the levels of 1

purifiable PilAPAK

Tfp were remarkably low in a wildtype background but in the absence 2

of the PilT retraction ATPase were nearly equivalent to those of endogenous Tfp. This 3

PilT - mediated effect was specific for exogenous Tfp as levels of endogenous Tfp 4

expressed simultaneously were not similarly diminished. Thus, this phenomenon 5

involved a specific rather than a general perturbation of Tfp dynamics in the cell. Steady-6

state Tfp levels are partly dictated by the relative frequencies of extension/polymerization 7

event initiation versus the frequencies with which extension/polymerization events are 8

terminated by retraction/disassembly (mediated by PilT) (40). It appears then that 9

PilAPAK

pilus assembly sites are disproportionally susceptible to attack by the PilT 10

associated retraction machinery. This phenotype is reminiscent of that seen in N. 11

gonorrhoeae null mutants lacking so called effectors of pilus homeostasis (40, 42, 43). 12

These factors act by promoting extension/polymerization events in the presence of PilT 13

and include PilC and all of the five pilin-like proteins encoded within the pilH-L locus. 14

Therefore, we propose that PilAPAK

may interact sub-optimally with these resident 15

effector proteins. Like PilC, the PilH, I, J, K and L proteins are each required for both 16

human cell adherence and transformation competence (40). Therefore, the disparate 17

phenotypes seen in the PilAPAK

background might also reflect diminished functionality of 18

these components. 19

Simultaneous expression of endogenous pilin and P. aeruginosa PilAPAK

at 20

identical levels revealed previously unseen interactions. First, PilA pili were demarcated 21

by a tip structure whose presence was PilE - dependent. The simplest explanation could 22

be that this tip structure is comprised of components requiring PilE for localization to this 23

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site but further studies are required to assess these possibilities. Whatever its nature, the 1

presence of the tip element is not essential to Tfp - associated functions. Secondly, PilE 2

influenced the length distributions of PilAPAK

pilus filaments seen by immunofluoresence 3

with a heterogeneous mixture of pilus lengths being seen in its presence and only long, 4

single filaments seen in its absence. This differential effect was only seen in the presence 5

of PilT suggesting that the action of PilE was in some way influenced by pilus retraction. 6

In contrast, PilE had no effect on the levels of purifiable PilAPAK

pili which were 7

profoundly reduced relative to those of endogenous Tfp. These discordant results seen for 8

PilAPAK

Tfp levels in wildtype backgrounds using the purification versus direct 9

immunodetections methods are difficult to reconcile. Obviously, the cultivation 10

conditions are clearly different as Tfp are purified from strains propagated on agar 11

medium while immunodetection required shorter times of propagation in liquid medium 12

on cover slips. The use of poly(L-lysine) coated cover slips here might be a factor as Tfp 13

undergoing extension and retraction might be irreversibly trapped or captured externally 14

so as to prevent retraction or otherwise distort organelle dynamics. By way of example, 15

treatment of P. aeruginosa PAO1 with pilus - specific RNA phage has been demonstrated 16

to stimulate pilus formation by an unknown mechanism (7). 17

With regard to twitching motility, our results were not unexpected as other studies 18

have documented the ability of diverse pilin subunits to form Tfp and support this 19

property in P. aeruginosa. Here, we showed that replacement of PilE by PilAPAK

does not 20

significantly impact force generation by Tfp retraction. Like pilus retraction with 21

endogenous Tfp (25), the velocity is constant at forces < 40pN and at higher forces, the 22

velocity decreases but pili were still able to retract against 100pN. However, the average 23

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retraction velocity at low forces was increased as compared to the wild type. This 1

observation suggests that the maximum force is likely determined by a molecular motor 2

in or near the membrane and that the structure and/or composition of the pilin subunit 3

may influence the speed of pilus retraction. 4

Despite the vast number of instances in which bacteriophages are known to 5

initiate infection by adsorption to pili, the pilus component involved has yet to be 6

unambiguously identified in any system. In this study, we demonstrated that expression 7

of P. aeruginosa PilA as Tfp in N. gonorrhoeae was both necessary and sufficient to 8

engender binding of bacteriophage PO4. It follows then that a specific PO4 receptor is 9

encompassed within PilA or within a PilA oligomer. Nonetheless, other species 10

expressing pilin subunits structurally distinct from PilAPAK

exhibit PO4 sensitivity (in a 11

Tfp dependent fashion) and it has been reported that expression of any one of diverse set 12

of Tfp pilin subunits in a P. aeruginosa PAK pilA background restored PO4 sensitivity 13

(29, 37). It is formally possible then that in some strains either a functional redundancy of 14

PO4 receptors exists or that PO4 exhibits relaxed specificity for Tfp organelles in some 15

(but not all) instances. It is perhaps worth noting here that some filamentous phages are 16

able to infect F- E. coli at frequencies of approximately 10

-6 and infectivity can be 17

dramatically increased by treatment that perturbs the outer membrane. (32). There thus is 18

clear precedence for the ability to bypass the requirement for the primary pilus receptor 19

albeit with reduced frequency. Further studies examining the direct interactions between 20

phage and Tfp as detailed here and elsewhere (19) should help clarify these issues. 21

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In summary, this systematic analysis of phenotypes imparted by heterologous 1

pilin subunit expression in N. gonorrhoeae provides new insights into the correlations 2

between Tfp expression, structure and associated functions. 3

4

ACKNOWLEDGEMENTS 5

6

We thank E. C. Gotschlich (Rockefeller University, NY, US), J. Tainer (The 7

Scripps Research Institute, La Jolla, USA) and A.-B. Jonsson (Uppsala University, 8

Uppsala, Sweden) for the gifts of antibodies. We also thank S. Lory (Harvard Medical 9

School, MA, US) for providing bacteriophage PO4 and the Cornea Bank Amsterdam for 10

providing eye tissue. This work was supported by funds from EMBO short-term 11

fellowship ASTF 139.00-02 (H. C. W-L.) and the Research Council of Norway 12

Functional Genomics initiative (FUGE) directed through The Consortium of Advanced 13

Microbial Sciences and Technologies (CAMST). 14 ACCEPTED


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4

5

6

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FIGURE LEGENDS 1

2

Figure 1. Characterization of PilE and PilA expression in N. gonorrhoeae using 3

whole cells lysates and purified pili. Silver stained (A, E) or coomassie stained (D) SDS 4

PAGE gels loaded with either whole cell lysates (A) or purified pili (E, D). 5

Immunoblotting of whole cell lysates (B and C) or purified pili (F and G) by using anti-6

PilE specific antibodies (B and F) or anti-PilA specific antibodies (C and G). Lane; 1, 7

N401 (wildtype); 2, KS48 (iga::pilA); 3, KS49 (iga::pilA, pilT); 4, MW24 (pilE); 5, 8

KS50 (pilE, iga::pilA); 6, KS51 (pilE, iga::pilA, pilT). Note: polyclonal antibodies to 9

PilA cross react with PilE due to a shared epitope encompassed within the highly 10

conserved amino-termini of the proteins (28). 11

12

Figure 2. Tfp characterization in gonococcal strains using immunofluorescence. 13

Gonococcal cells (green) and Tfp (red) are detected using indirect immunofluorescence. 14

Anti-PilE specific antibodies (2-66) were used in the two leftmost columns while anti-15

PilA specific antibodies were used for the two rightmost columns. Wt (N400), pilE 16

(KS45); iga::pilA (KS48), iga::pilA, pilT (KS49); iga::pilA, pilE (KS52); iga::pilA, pilE, 17

pilT (KS53). 18

19

Figure 3. Piliation of wildtype and mutant gonococcal strains analysed by 20

immunogold labeling and transmission electron microscopy. (A, B) Negatively 21

stained and 5 nm immunogold labeled Tfp using anti-PilA specific antibodies. (C, D) 22

Negatively stained and sequentially immunogold-labeled Tfp fibers using anti-PilE 23

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specific antibodies (10 nm gold particles) followed by anti-PilA specific antibodies (5 nm 1

gold particles). (E, F) Negatively stained and sequentially immunogold labeling of Tfp 2

using anti-PilA specific antibodies (10 nm gold particles) followed by anti-PilE specific 3

antibodies (5 nm gold particles). The strain used was KS49 (iga::pilA, pilT). 4

5

Figure 4. Evidence for a unique tip structure on P. aeruginosa Tfp in N. 6

gonorrhoeae. Negative staining and immunogold transmission electron microscopy 7

using anti-PilA specific antibodies (5 nm gold particles). Lower left panel was only 8

labeled with anti-PilA specific antibodies and not with gold particles. Space bar = 200 9

nm. Strain KS48 (iga::pilA), KS49 (iga::pilA, pilT), KS52 (iga::pilA, pilE,) and KS53 10

(iga::pilA, pilE, pilT). 11

12

Figure 5. Tfp associated force generation for strains expressing PilAPAK

pili. 13

Velocity-versus-force relationship for pilus retraction for N. gonorrhoeae expressing PilA 14

pili KS52 (pilE, iga::pilA). 15

16

Figure 6. Adherence of wild type and mutant gonococcal strains to human corneal 17

epithelial cells. (A) Adherent cells are stained with crystal violet. All panels are shown at 18

the same level of magnification. Adherence was also quantitated by determining the 19

average numbers of adherent bacteria per cell (see supplementary data, Table S1). (B) 20

Indirect immunofluorescenese on gonococcal Tfp using rabbit antibodies specific for 21

PilA followed by Alexa red (594 nm) conjugated goat anti-rabbit IgG antibodies (red). 22

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Gonococci were detected using fluorescent-labeled monoclonal antibodies (green). Wt 1

(N400), pilE (KS45); iga::pilA (KS48), iga::pilA, pilT (KS49); iga::pilA, pilE (KS52); 2

iga::pilA, pilE, pilT (KS53); iga::pilA, pilC, pilE, pilT (KS58); iga::pilA, pilE, pilT, pilV 3

(KS55). 4

5

Figure 7. Quantitative analysis of PilC and PilV in gonococcal strains. Immunoblot of 6

purified pili (A, B) or whole cell lysates (D, E) using rabbit antibodies specific for PilC 7

(A, D) or PilV (B, E). Coomassie stained SDS/PAGE gel showing relative amounts of 8

PilE and PilA in purified pili (C). Bottom panel; Lanes 1, KS46 (iga::pilE, pilE); 2, KS53 9

(iga::pilA, pilE, pilT); 3, KS58 (pilT, pilC2off, iga::pilA, pilE); 4, KS56 (pilT, pilC2off); 10

5, MW7 (pilT, pilC1, pilC2); 6, KS55 (pilT, pilV, iga::pilA, pilE); 7, GV6 (pilT, pilV); 8 11

KS47 (iga::pilE, pilE, pilT). 12

13

Figure 8. PilAPAK

pilin is necessary and sufficient for bacteriophage PO4 binding to 14

pili in N. gonorrhoeae. The bacteriophage PO4 bind tail first along PilA pili. Negative 15

staining (A, B, C) and immunogold transmission electron microscopy using anti-PilA 16

specific antibodies (5 nm gold particles) (D, E). Space bar = 200 nm. Strain used; KS49 17

(iga::pilA, pilT). PO4 do not bind to PilE pili (seen as unlabelled bundles in E). 18

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Table 1. List of bacterial strains. 1 2 Strains Parental strain Relevant genotype

a References

VD300 MS11 (22)

N400 VD300 recA6(tetM) (34)

N401 N400 recA6(kan) (41)

MW4 N401 pilTind (41)

MW7 N401 pilTind, pilC1::ermC, pilC2::cat (41)

MW24 N401 pilEind (41)

GE101 VD300 pilEind (43)

GE107 GE101 pilEind, iga::pilA (2)

GE200 N400 iga::pilE (27)

GE45 MW4 iga::pilE, pilTind (1)

KS45b N400 pilE::cat This work

KS46 GE200 iga::pilE, pilE::cat This work

KS47 GE45 iga::pilE, pilE::cat, pilTind This work

KS48 N401 iga::pilA This work

KS49 MW4 iga::pilA, pilTind This work

KS50 MW24 pilEind, iga::pilA This work

KS51 KS50 pilEind, iga::pilA, pilT::cat This work

KS52 KS48 pilE::cat, iga::pilA This work

KS53 KS49 pilE::cat, iga::pilA, pilTind This work

GV6 MW4 pilTind, pilVfs (39)

KS54 GV6 pilTind, pilVfs, iga::pilA This work

KS55 KS54 pilTind, pilVfs, iga::pilA, pilE::cat This work

KS56 MW4 pilTind, pilC2off This work

KS57 KS56 pilTind, pilC2off, iga::pilA This work

KS58 KS57 pilTind, pilC2off, iga::pilA, pilE::cat This work

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a The genotypes are indicated here with antibiotic resistance markers and other relevant 1

genotypes although they are mentioned only as simple mutant alleles throughout the rest 2

of the manuscript. 3

b Construction of the plasmid used to transform N400 to make strain KS45 is described in 4

(17). 5

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2 : effects of pilin subunit composition on function accepted · 3 gram negative species of...

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