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Posttranslational Modification of Pili upon Cell Contact Triggers N. meningitidis Dissemination

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Science  11 Feb 2011:
Vol. 331, Issue 6018, pp. 778-782
DOI: 10.1126/science.1200729

Abstract

The Gram-negative bacterium Neisseria meningitidis asymptomatically colonizes the throat of 10 to 30% of the human population, but throat colonization can also act as the port of entry to the blood (septicemia) and then the brain (meningitis). Colonization is mediated by filamentous organelles referred to as type IV pili, which allow the formation of bacterial aggregates associated with host cells. We found that proliferation of N. meningitidis in contact with host cells increased the transcription of a bacterial gene encoding a transferase that adds phosphoglycerol onto type IV pili. This unusual posttranslational modification specifically released type IV pili-dependent contacts between bacteria. In turn, this regulated detachment process allowed propagation of the bacterium to new colonization sites and also migration across the epithelium, a prerequisite for dissemination and invasive disease.

The Gram-negative bacterium Neisseria meningitidis is a leading cause of septicemia and meningitis in humans (1). Initially, individual bacteria adhere to the nasopharynx epithelium via their type IV pili, a filamentous organelle common to numerous pathogenic bacterial species (2). In the following hours, bacteria proliferate on the cellular surface in tight three-dimensional aggregates termed microcolonies. The formation of these aggregates results from homotypic, type IV pili–mediated contacts between the bacteria themselves and contacts between bacteria and the host cell plasma membrane. Contacts with host cells are enhanced by the formation of bacteria-induced plasma membrane protrusions (3). After this proliferation phase, individual bacteria are thought to detach from the microcolonies, leading to propagation to new hosts and dissemination throughout the body in case of invasive infection. Understanding the molecular mechanisms underlying the life cycle of N. meningitidis is a key step toward identification of prevention and treatment strategies of meningococcemia. The major component of Neisseria spp. type IV pili (PilE or pilin) is modified with phosphocholine (PC), phosphoethanolamine (PE), or phosphoglycerol (PG) (46). We wanted to determine the impact of these unusual posttranslational modifications (PTM) on the pathogenesis of N. meningitidis.

A whole-protein mass spectrometry approach was chosen to determine the phosphorylation state of type IV pili (7, 8). Analysis of purified pili from the well-characterized 8013 strain (9) grown on solid medium (10) yielded a main peak with a mass of 17,491 daltons and a minor secondary peak with a mass of 17,645 daltons (Fig. 1A) corresponding to the addition of one PG (154 daltons, fig. S1A). Analysis of purified pili from strains carrying point mutations substituting conserved serine residues 69 and 93 of the PilE protein with alanines (Fig. 1, B and C) showed that all pilin subunits were modified with PG on Ser69 (17,491 daltons), whereas only about 15% of pilin subunits were also modified on Ser93 (17,645 dalton). The NMV_0885 gene (ortholog of NMA1705 and NMB1508) was a good candidate to carry out this activity because it is part of the cluster of orthologous group (COG) titled phosphoglycerol transferase and related proteins (COG1368, fig. S1C) (11). Analysis of type IV pili purified from a strain carrying a deletion in the NMV_0885 gene revealed a single peak of 17,337 daltons that corresponds to pilin without any PG (Fig. 1D), demonstrating that this gene is responsible for the transfer of PG onto the pilin. We thus named the transferase PptB (pilin phosphotransferase B).

Fig. 1

Modification of the pilin subunit with PG by the PptB transferase. Whole-protein mass spectrometry analysis of type IV pili purified from the wild-type strain (WT) (A), from a strain harboring a S69A mutation in the pilE gene (B), from a strain harboring a S93A mutation in the pilE gene (C), and from a mutant in the NMV_885 gene (∆pptB) (D).

The pptB gene was previously described as a member of a group of 16 N. meningitidis genes containing a two-component system-regulated promoter referred to as CREN for contact regulatory element of Neisseria (1214). Transcription of pptB increased two- to threefold over a period of 4 hours after adhesion to epithelial cells (12), suggesting that modification of type IV pili with PG could be triggered upon contact with host cells. To test this possibility, we expressed the pptB gene under the transcriptional control of the isopropyl-β-d-thiogalactopyranoside (IPTG)–inducible lac promoter to mimic the threefold induction found on host cells (Fig. 2A). In the presence of inducer, the peak corresponding to two PG modifications (17,645 daltons) became the most abundant form (Fig. 2, B and C, and fig. S2). Substitution of Ser93 into an alanine (S93A) (Fig. 2D) indicated that Ser93 is the main phosphorylation site upon induction of pptB, whereas Ser69 phosphorylation level remained constant. The expression level of the pptB gene thus determines the phosphorylation level of the pilin subunits on Ser93.

Fig. 2

Effect of the transcription level of the pptB gene on the glycerophosphorylation of the pilin subunit. The pptB gene was placed under the control of the IPTG-inducible lac promoter to mimic the induction of the pptB gene when bacteria are growing in contact with host cells. (A) Determination of the level of transcription by real-time reverse-transcription polymerase chain reaction of the pptBind construct in the presence of different amounts of IPTG relative to the wild-type strain cultivated in suspension. The dotted line indicates the level of transcription reached after 4 hours of contact with host cells as previously reported with the same strain and on the same cells (12). For further experiments, a concentration of 0.1 mM was used to mimic the effect of cell contact. The graph represents the mean ± SEM of three independent experiments. (B) Whole-protein mass spectrometry analysis of the pilin subunit purified from the pptBind strain in absence of IPTG and therefore with low amounts of PptB (pptBind – IPTG). (C) Effect of increased pptB level after induction of the pptBind construct with 0.1 mM IPTG on pilin mass (pptBind + IPTG). (D) Analysis of the pilin subunit containing the S93A substitution in the pptBind strain in presence of 0.1 mM IPTG (pptBind S93A + IPTG). (E) Molecular modeling of the N. meningitidis pilin monomer with the PTMs found when bacteria are grown in suspension: GATDH (glyceramido tri-deoxy hexose) sugar modification on Ser63 (yellow) and glycerophosphate on Ser69 (magenta). The structure is displayed with the Pymol software (www.pymol.org). (F) Positively charged environment of Ser93 on the structure of PilE corresponding to the area outlined in (E). A patch of five lysine (K) residues indicated in blue surround Ser93 in red. (G) Predicted structure of the pilin modified with PG on Ser93.

We used two-dimensional gel electrophoresis followed by immunoblot to demonstrate increased modification of pilin with the negatively charged PG while bacteria were proliferating in contact with host cells. Analysis of PilE from bacteria growing in suspension displayed a major spot with an isoelectric point around 6 (fig. S3). Upon incubation with host cells, spots corresponding to acidic forms appeared after 2 and 4 hours. Pilin isoelectric point changes did not occur in the pilES93A-expressing strain. Thus, after pptB transcriptional increase upon contact with host cells, the isoelectric point of a significant proportion of the major pilin subunit became more acidic after modification of Ser93.

To gain insight into the potential impact of this modification, we modeled the three-dimensional structure of the pilin from N. meningitidis with and without PG on Ser93 based on the known N. gonorrhoeae pilin structure and energy minimization in the context of the pilus fiber (Fig. 2E). Ser93 is surrounded by five lysine residues (Fig. 2F), a positively charged patch postulated to be important in type IV pili function (15). Modification of Ser93 with PG introduces a negative charge carried by the PG group protruding from the pilus structure (Fig. 2G and fig. S4). Modeling of bundles of four antiparallel pili fibers showed that the average interaction energy was largely favorable (60 kcal difference) for the pili with no modification on Ser93 when compared with the same structure with a PG on Ser93 (Fig. 3, A to C). Thus, modification of Ser93 with PG would strongly destabilize fiber interaction, suggesting that this PTM could have important consequences for type IV pili function.

Fig. 3

Effect of pilin glycerophosphorylation on the aggregative functions of type IV pili. (A to C) Molecular modeling of pili fibers interacting with each other in a bundle. We modeled a regular antiparallel bundle involving four pili fibers by a combination of systematic and random search in the distance between pili, the rotation, and the tilt of the pilus from the overall bundle axis and analyzed them in terms of their interaction energy and the geometry. Each pilus fiber contains 20 monomers of the pilin subunit. (A) Side view of the bundle formed by pilin subunits displaying a PG only on Ser69. The PG modifications are displayed as spheres, phosphate atoms in blue and carbon atoms in green. (B) Top view of the structure shown in (A), showing the close contacts between the fibers. (C) Top view of the unstable bundle that would be formed by the pilin modified with PG on both Ser69 and Ser93. (D) Effect of increased pilin phosphorylation on the organization of pilus fibers analyzed by transmission electron microscopy for the WT strain, the ∆pptB strain, and the pptBind strain with and without 0.1 mM IPTG. Wide arrows indicate thick bundles of fibers, and narrow arrows indicate thin fibers likely to be individual pili (6 to 8 nm). Insets represent a higher magnification of pili fibers. (E to G) The ability of bacteria to form aggregates was determined by a technique based on direct observation by fluorescence microscopy. The percentage of bacteria engaged in an aggregate relative to the total amount of bacteria in the same volume is indicated. Four independent experiments each done in triplicate were performed; error bars denote mean ± SEM. Statistical analyses were done with Student’s t test; n.s., nonsignificant; *P < 0.05. (E) Aggregation was compared between the WT strain, the nonpiliated pilE strain, the ∆pptB strain, and the pilES93A strain. (F) Aggregation was determined in the pptBind strain in the presence of different amounts of IPTG. (G) Effect on aggregation of increased pptB expression in a mutant expressing the PilE protein with an alanine on position 93 (pptBindPilES93A).

We addressed the functional role of this PTM in key steps of N. meningitidis pathogenesis. Pilin modification with PG did not have any effect on the amount of type IV pili present on the bacterial surface (fig. S5, A and B). In contrast, as predicted by molecular modeling, ultrastructural analysis by negative staining showed that increased pilin modification with PG blocked the formation of bundles of pili (Fig. 3D). In the wild-type strain, pili bundles were commonly about 30 nm wide, thus containing several 6- to 8-nm-wide individual fibers (Fig. 3D). Upon transcriptional induction of the pptB gene, only thin fibers having the expected size of individual pili could be found (Fig. 3D and fig. S5C). Increased modification of pilin with PG thus blocks bundle formation.

Type IV pili bundle formation and N. meningitidis aggregation are linked (16). pptB gene deletion or the S93A substitution led to increased aggregate formation in suspension (Fig. 3E) and consistently increased transcription of the pptB gene abrogated bacterial aggregation (Fig. 3F). The effect of increased pptB transcription on aggregation was rescued by the S93A point mutation (Fig. 3G). Increased modification of Ser93 with PG thus strongly reduces type IV pili–dependent bacterial aggregation by introducing a negative charge at this site (fig. S6B). This anti-aggregative effect appeared to overcome the pro-aggregative activity of the minor pilin PilX (fig. S6, C and D).

The effect of pilin modification with PG on adhesion to epithelial cells was evaluated. The first step of adhesion, which is the contact of individual bacteria with the cell surface (Fig. 4A), occurred independently of the level of glycerophosphorylation (Fig. 4B). Bacterial microcolonies formed by the strains affected in pilin modification with PG did not appear morphologically different from the wild-type multilayered microcolonies (Fig. 4C). Furthermore, pilin glycerophosphorylation had little effect on the total number of cell-associated bacteria after 6 hours (Fig. 4D). To investigate the effect of increased pilin glycerophosphorylation on detachment, we determined the number of bacteria disengaging from microcolonies over time. A laminar flow chamber was used as a tool to progressively collect detaching bacteria (Fig. 4E and fig. S7A). Whereas the number of wild-type bacteria released from the infected monolayer slowly increased with time after 3 hours of infection, detachment of the ∆pptB mutant was impaired (1.5 × 106 versus 3.4 × 105 bacteria per ml at 7 hours). About 20 to 30% of bacteria adhering at 6 hours detached in the following hours of infection. The increase in pilin glycerophosphorylation thus favors the release of a proportion of individual bacteria from the microcolonies that has little impact on the number of adhering bacteria.

Fig. 4

Effect of pilin glycerophosphorylation on the interaction with host cells. (A) Initial adhesion occurs with individual or small groups of bacteria as viewed by scanning electron microscopy (arrows point to a diplococcus). (B) Initial binding of individual bacteria to the cellular surface was determined by taking advantage of a flow chamber assay. In the presence of low-intensity flow (0.05 dynes/cm2), adhering green fluorescent protein–expressing bacteria can be easily distinguished from bacteria in suspension that follow the liquid movement. Results are indicated as the percentage of adhesion relative to the WT strain. (C) After initial adhesion, bacteria proliferate and form large bacterial aggregates known as microcolonies. (D) Adhesive properties of the different strains to epithelial cells in static conditions were determined by standard dilution plating assays after 6 hours of infection. The transcription of the pptB gene was induced in the pptBind strain with 0.1 mM IPTG after initial adhesion. (E) The number of bacteria detaching from microcolonies adhering to host cells was determined for the wild type and the ∆pptB mutant over time by collecting bacteria coming out of a flow chamber submitted to low-intensity flow (0.15 dynes/cm2). The experiment was performed in parallel for the WT strain (solid circles) and for the ∆pptB mutant (open circles). Results for the nonadherent ∆pilC1 strain are also included (squares). (F) The ability of the different strains to transmigrate across an epithelial cell monolayer for a period of 1.5 hours was determined after a 4.5-hour infection period. Results are presented as a percentage relative to wild type. Three independent experiments each done in triplicate were performed; in (B) and (F), error bars denote mean of three experiments ± SEM; in (D) and (E), representative experiments are shown. Statistical analyses were done with Student’s t test; *P < 0.05.

Bacteria released from microcolonies are more likely to cross the epithelium (17), and we tested the ability of the ΔpptB mutant to transmigrate across an epithelial cell monolayer (Fig. 4F and fig. S7). After 6 hours, the ∆pptB strain had crossed the monolayer 20-fold less efficiently than the wild-type strain, indicating a defect as strong as the nonpiliated pilE strain. The strain expressing a point mutation on Ser93 of the pilin exhibited a similar defect, showing that the effect of the pptB deletion was mediated by the modification of pilin on Ser93. PG modification of Ser93 is thus necessary for efficient transmigration across an epithelial barrier.

After several rounds of N. meningitidis division on the host cell surface, transcription of the pptB gene increases, the major pilin becomes increasingly modified with PG, bacteria lose their aggregative properties, and a small proportion of bacteria are released from the colonies (fig. S8). At the molecular level, pilin glycerophosphorylation on Ser93 introduces a steric hindrance and a negative charge in the center of a positively charge patch on pili surface, leading to an inhibitory effect on pili bundling and thus blocking bacterial aggregation. Overall, this process has several potential selective advantages for the bacterium, including transmission to new hosts and colonization of new sites in the same host, thus avoiding nutrient exhaustion and possibly favoring escape from the local immune surveillance. Through a regulated posttranslational modification, N. meningitidis adopts a multiply-and-run strategy, presumably selected and fine-tuned through evolution as a propagation mechanism, key for survival of the bacterium in nature. Because selection of this property in the context of the commensal lifestyle of the bacteria also favors transmigration across the epithelium, it is likely to affect human health by favoring invasive infections.

Supporting Online Material

www.sciencemag.org/cgi/content/full/331/6018/778/DC1

Materials and Methods

Figs. S1 to S8

References

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Authors thank M.-C. Prévost, S. Guadagnini, D. Lankar, J.-L. Beretti, M. Dupuis, G. Guilbaud for help with experiments and A.-M. Lennon, E. Camerer, S. Silva, and K. Melican for critical reading of the manuscript. This work was supported by Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche (including Avenir program), and Agence Nationale de la Recherche grants PCV08_319010 and FRM INE20100118195.
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