Neisseria meningitidis filamentous phage MDA promotes colonisation by selecting hyperadhesive pili variants

Domain: biology
Published: 2025-12-20
Editorial outcome: major_revision

Abstract
Filamentous phages are non-lytic phages mutually beneficial to their bacterial hosts. In Neisseria meningitidis, the filamentous phage MDA is associated with invasive diseases thanks to its key role in the formation of biofilm during epithelium colonisation. The infection model for filamentous phages has been defined for phages Ff and CTX. These phages bind to the tips of bacterial pili before being translocated into the periplasm of their hosts. The aim of this study is to investigate the relationship between filamentous phage infection and type IV pili, using the bacterium Neisseria meningitidis and the bacteriophage MDA as model organisms. We show that MDAΦ rather binds to type IV pili along their entire length with preferential binding to positively charged variants of the major fibre-forming pilin, demonstrating a role for antigenic variation in phage infection. Strikingly, bacteria expressing the more positively charged pilin are also the most adhesive, suggesting that MDAΦ primarily target the most adhesive bacteria. Finally, we show that adhesion to human cells is sufficient to amplify the phage-positive meningococcal population. Overall, this study reveals how a filamentous phage can target hyperadhesive bacterial variants and promote their selection, thereby establishing a link between phage infection and bacterial colonisation.

Introduction
The contribution of phages to the virulence of their bacterial hosts has been studied mainly for Caudovirales, which represent 96% of the known phages1. In contrast, little is known about inoviruses, a family of bacteriophages carrying a single-stranded circular DNA with unique morphology and infectious cycle characteristics, to which the filamentous phage subfamily belongs. However, filamentous phages are found in several major pathogenic species2. The first filamentous phage of this family was described in Escherichia coli in the 1960s by several groups3–6. A second well-known filamentous phage, hosted by Vibrio cholerae, was discovered by Matthew K. Waldor and John J. Mekalanos. This phage, called CTXΦ, encodes the cholera toxin genes, which promotes the dissemination of CTX-carrying V. cholerae7. The main feature is the absence of bacteria lysis during the infectious cycle8. The Pf4 phage of Pseudomonas aeruginosa is another example. It remains inactive until the bacteria perceive environmental signals (such as oxidative stress), then Pf4 genes are among the most induced genes in P. aeruginosa biofilms where Pf4 filamentous phages contribute to biofilms as a structural components and as a physical barrier to antibiotics and as an important immune modulator during infection9. Finally, filamentous phages have been described in Neisseria species and in particular in Neisseria meningitidis where the filamentous phage named MDA for Meningococcal Disease Associated phage, also known as Nf1 phage10, plays a key role in biofilm formation and is associated with virulence11–14. Other filamentous bacteriophages contribute to the virulence of many bacteria in a variety of ways2.

Filamentous phages that infect diderm bacteria must cross both the outer and inner membranes to infect their target. Although the cell surface receptors are unknown for the majority of filamentous phages described so far, a role for retractable bacterial pili has been suspected for their entry, such as Ff and related phages that use the sexual/conjugative pilus F of E. coli2,15. Early observations of f1 phages by electron microscopy suggested that the phages bind to the tip of the pilus15. This was confirmed experimentally for CTXΦ, in which the phage adsorption protein pIII interacted directly with the pilus tip-subunit TcpB16. In the case of N. meningitidis, we have previously shown that MDA phage entry into the host requires retractable type IV pili17, but the bacterial proteins involved in the MDAΦ-pili interaction are not known.

Thanks to pili retraction, the phage is thought to cross the bacterium’s outer membrane through the pilus secretin and then enter the periplasm, where, as previously described, the Ff or CTX phages interact with the TolQRA system for the disassembly and entry of the single-stranded DNA viral genome into the cytosol. In the absence of the TolQR complex, the homologous ExbBD complex can perform phage uptake with TolA18,19. Overall, our knowledge of the molecular mechanisms by which filamentous phages interact with their associated pilus and enter bacteria is incomplete for most filamentous phages and remains to be determined.

N. meningitidis is a commensal bacterium, commonly present asymptomatically in the human nasopharynx. Under certain circumstances, the bacterium can enter the bloodstream and cause sepsis and/or meningitis after crossing the blood-brain barrier20. Survival in the extracellular fluid is dependent on a small number of virulence factors such as the capsular polysaccharide, the iron chelation systems and factor H binding protein21,22. In addition, type IV pili are essential for blood vessel colonisation, a key step in the pathogenesis of N. meningitidis23–26. Other bacterial attributes, such as Opa proteins, are likely to play an essential role in nasopharyngeal colonisation27. While few virulence factors are associated with meningococcal invasiveness, no virulence factor provides a clear explanation for why certain strains of N. meningitidis are responsible for outbreaks of invasive infection. One exception is the presence of an 8kb island in the genome of N. meningitidis invasive isolates, the meningococcal filamentous MDA prophage10,12–14,17. The MDA prophage may be found up to 4 times per genome, with partial heterogeneity in these sequences suggesting that a strain can be re-infected by the same phage or by a phage from another strain28,29. Previous results have suggested that MDA phages enhance epithelial cell colonisation by promoting bacterial-bacterial interactions and increasing bacterial biomass in the nasopharynx11, which in turn should increase the frequency of bacterial dissemination in the bloodstream and/or the spread of bacteria within the human population.

The dissemination of the MDA phage is likely to be associated with the hijacking of the type IV pilus machinery, a feature that appears to be shared with most other inoviruses such as CTX and Pf4. Type IV pili are highly dynamic filaments that generate tensive forces during cycles of elongation and retraction, powered by a dedicated elongation motor called PilF and retraction motor called PilT30. Filament biogenesis relies on a complex multi-protein machinery that, in N. meningitidis, contains 15 proteins essential for biogenesis (PilC1 and C2, D, E, F, G, H, I, J, K, M, N, O, P, Q, W), PilE being the pili-forming pilin and PilQ the secretin through which the pilus crosses the outer membrane. As determined by cryo-electron tomography, the type IV pili biogenesis machinery is a multilayered structure spanning the entire cell envelope31,32. Meningococcal strains express either a class I or a class II pilE gene33. Class I pilE are subject to antigenic variation and type IV pili are therefore considered to be one of the most variable structures on the meningococcal surface. This pilE gene consists of a constant region and a variable domain that can vary by recombination with one of the eight pilS silent cassettes. In addition, in the early 1990s, it was shown that antigenic variation in PilE plays an important role in cell adhesion to the host and escape from the immune response34,35. Conversely, the class II pilins do not undergo antigenic variation and form a class of conserved pilin subunits36. In addition, the PilE protein is highly post-translationally modified (glycosylation and addition of phosphoglycerol)37. Finally, type IV pili have been implicated in numerous functions including bacterial interaction with human cells, DNA uptake, twitching motility and bacterial-bacterial interactions34,38–40.

Here, we use N. meningitidis as a model to dissect the molecular mechanism of the interaction between filamentous phages and type IV pili, and how this shapes the bacterial population. Unlike most filamentous phages, we demonstrate that the type IV pili tip adhesin is not involved in infection. Conversely, we observe the binding of MDAΦ to type IV pili along their fibre, an interaction that took advantages of the positive charges carried by certain PilE variants that are also strongly associated with type IV pili-mediated adhesion, suggesting a key role for antigenic variation in MDAΦ infection. Finally, we show that adhesion to human cells is sufficient to allow enrichment of a meningococcal population with phage-positive bacteria, demonstrating the association between adhesion and the presence of the MDAΦ, two features associated with the invasiveness of N. meningitidis.

Results
MDAΦ infection is dependent on retractable type IV pili and specific pilE sequences
Interaction of inoviruses with their host bacteria and subsequent entry into the cytosol is thought to be highly pili-dependent. In the case of MDAΦ, we have previously shown that both the major pilin PilE and the retraction ATPase PilT are required for phage transduction, whereas other minor pilins of the fibre (PilV, ComP, and PilX) are not required17. To gain further insight into the pili-phage interaction, we extended our previous screen by assessing the efficacy of phage transduction in a comprehensive library of individual mutants of the type IV pili machinery, including proteins involved in fibre assembly or in the machinery itself, (PilD, F, G, P, Q, T, T2, U, W, Z and TfpC, TsaP and NMA0415) and proteins that compose the fibre (PilE, PilV, PilX, ComP) or the tip (PilH, I, J, K, C1, and C2) (Fig. 1a–d). Transduction efficiency was quantified in a strain deleted for the MDA prophage (ZΔMDA) and using a purified MDA phage carrying a spectinomycin resistance gene (orf6-aadA1) (MDA (orf6-aadA1) Φ) for colony forming unit (CFU) quantification (Fig. 1a). Note that the ZΔMDA strain is derived from a clinical isolate that has not been selected for a specific pilE sequence - as for the NEM8013 2C4.3 strain (clone 12)41 so it corresponds to a mix of pilE sequences. Transduction was compared with transformation efficiency (Fig. 1b) and pili quantity (Fig. 1c). The pili quantity was estimated by immunostaining of the pili mutants introduced into a ZΔMDA pilE expressing strain34. This strain expresses type IV pili, which can be recognised by the 20D9 antibody42. Transformation and pili quantity are two proxies for the expression of functional type IV pili43. Transduction was impaired in mutants with low pili quantity and low transformation efficiency, as well as in the ΔpilT mutant, confirming that the phage only infects strains expressing fully retractable pili. The contribution of the proteins thought to form the pilus tip (PilH, I, J, K) is difficult to interpret as their respective mutants were very poorly piliated. However, the infection efficiency of the four individual mutants was higher than that of the non-piliated ΔpilE mutant, suggesting that these proteins have a minor role in phage infection.

Finally, we studied PilC1 and PilC2, two proteins that are thought to be located at the tip of the type IV pilus based on homology with other type IV pilus models44–46. The ΔpilC1/C2 double mutant is barely piliated and does not support efficient phage transduction. Conversely, both ΔpilC1 and ΔpilC2 single mutants were piliated and fully transduced by the phage (Fig. 1a).

Finally, we investigated the role of PilE in phage entry and transduction. We took advantage of the antigenic variation of the class I pilE gene and analysed the transduction efficiency of MDA (orf6-aadA1) Φ depending on pilE sequence. We took advantage of previous constructs available in our team in which pilE and pilE variant sequences were fused to a kanamycin resistance gene to allow their selection34. We therefore constructed two phage-receptive strains with the two different pilE variant sequences34. The two strains differ in the variable part of PilE and especially in the D-region between the two conserved C-terminal cysteine residues (C120 and C152 for PilE SA) (Supplementary Fig. 1a–c). We then evaluated the transduction efficiency in these two strains compared to the parental strain ZΔMDA. The MDA (orf6-aadA1) Φ transduction efficiencies differ among the pilE SA, pilE SB, and ZΔMDA strains. The pilE SA strain exhibits a significantly reduced phage transduction rate compared to ZΔMDA (Fig. 1e, p=0.0006). The transduction rate of pilE SB is also lower than that of pilE SA, although this difference is not statistically significant (p=0.0505). These findings suggest a potential role of the pilE sequence in modulating transduction efficiency. Finally, we investigated the role of PilE post-translational modification in phage transduction (Supplementary Fig. 1d). Mutations in the two genes encoding the glycosyltransferases PglA and PglC or in the gene encoding pilin phosphotransferase B (PptB) have little effect on phage transduction.

PilS loci are a reservoir of pilE sequences compatible with MDAΦ infection
Our results above suggested that PilE variants may have different efficiencies for infection by MDAΦ. However, the Fig. 1e raised the question of whether transduced meningococci from the PilE SA-expressing strains still express this PilE variant or whether the phage has infected bacteria expressing another PilE variant that is present in the population due to antigenic variation despite the use of the kanamycin resistance gene in frame with pilE SA. To answer this question, we first sequenced the pilE gene of 10 independent clones of the ZΔMDA pilE SA strains (Fig. 2a, b). Seven clones expressed the PilE SA sequence or a closely related sequence (98% identity) and three clones had more than five different mutations compared with the PilE SA sequence (Fig. 2b). We then transduced the ZΔMDA pilE SA strain with MDA (orf6-aadA1) Φ, selected the transduced population on spectinomycin and sequenced 10 independent clones again (Fig. 2a, c). After transduction, none of the 10 sequences obtained before transduction were recovered (Fig. 2b, c). Five of the 10 sequences were identical and were designated PilE SD. This sequence carried a D-region completely different from that of PilE SA, as well as several other variations between G58 and D113 of PilE SA (Fig. 2c, d and Supplementary Fig. 2a, b). Interestingly, the pilE SD sequence is closely related to the pilS3 silent locus of the parental strain (Fig. 2d and Supplementary Fig. 2a, b), suggesting that antigenic variation of PilE still occurs despite the addition of the kanamycin resistance gene in frame with pilE. To confirm that antigenic variation in the ZΔMDA pilE SA strain helps explain the level of infection by MDA (orf6-aadA1) Φ, we constructed two mutS+ ΔrecA mutant ZΔMDA strains expressing either PilE SA or PilE SD variants, and we infected both strains with MDA (orf6-aadA1) Φ. Deletion of recA is sufficient to abolish the homologous recombination required for antigenic variation47. Meanwhile, the inactivated copy of the mutS gene in the ZΔMDA strain was reverted to WT (mutS+) to limit the spontaneous appearance of mutants29. The strain was subsequently designated ZΔMDA mutS+ΔrecA. Transduction rate was determined as above and compared to the initial ZΔMDA strain (Fig. 2f). It should be noted that phage infection is independent of RecA. We recovered very few colonies with the ZΔMDA mutS+ΔrecA pilE SA strain, whereas the ZΔMDA mutS+ΔrecA pilE SD strain was transduced as efficiently as the initial ZΔMDA strain. We then confirmed that the efficiency of transformation was in the same range between the pilE SA and the pilE SD in ZΔMDA mutS+ strains - the recA deletion preventing homologous recombination - (Fig. 2g). While the transformation frequency was decreased in both strains compared to the parental strain, we observed no statistical differences between strains expressing pilE SA or pilE SD. Overall, these results suggest that: (i) some variants of PilE appear to be more susceptible to infection; (ii) MDAΦ is able to specifically infect a low-abundance clone expressing a specific PilE variant.

The type IV pili tip is not essential for MDAΦ entry and transduction
The near absence of phage infection in the ZΔMDA mutS+ΔrecA pilE SA strain suggests that the pili tip is not the receptor for MDAΦ. To rule out this hypothesis, we decided to further investigate the role of the PilC proteins. The two PilC proteins are bimodular proteins with a similar C-terminal domain that is thought to be anchored at the pilus tip based on the Myxococcus xanthus model45. The N-terminal (N-ter) domain is involved in bacterial adhesion and differs between PilC1 and PilC248,49 (Fig. 3a). Since the ΔpilC1/C2 double mutant strain is apparently barely piliated, we sought to complement a pilC null strain for the expression of recombinant PilC1 mutants of decreasing size (Fig. 3a) and to evaluate transduction and transformation in a strain expressing the PilE SD variant (Fig. 3b, c). Deletion of the amino acids from 3 to 555 in the N-terminal part of PilC1 did not affect transduction or transformation frequency, supporting the idea that the N-terminal domain of PilC is not recognised by the MDAΦ and is not required for pili biogenesis. Increasing deletion to 3–843 residues reduced both the transduction and the transformation frequencies of the resulting strain. Interestingly, the truncated protein PilC1Δ3-843 allowed transduction and transformation at a level close to 10% of the control (although not statistically significant for transduction) while truncated PilC1Δ4-977 has lost both and transduction and transformation competency (Fig. 3b, c). This suggests that PilC1Δ3-843 and PilC1Δ4-977 do not complement for piliation and cannot be included in our analysis. This supports the view that PilC does not play a role in MDAΦ entry. In a complementary approach, we investigated a possible interaction between the MDAΦ ORF6 (Supplementary Fig. 3a) and proteins of the pilus tip using the bacterial two-hybrid system. Based on structure and sequence homology with proteins pIII of M13 and CTXΦ pIII of CTXΦ, ORF6 was previously annotated as a phage adsorption protein, and we showed in a prior work that ORF6 is required for MDAΦ transduction17. In the existing model, the adsorption protein should interact with the pili tip to cross the outer membrane. We therefore assessed the interaction of ORF6 with the N-terminal domain of PilC1 or PilC2, with the C-terminal domain of PilC1 and with the globular heads of the pilins PilH, I, J, and K (Supplementary Fig. 3b). No positive result was observed, suggesting that there is no interaction between ORF6 and the N-terminal domain of PilC1, assuming that the proteins are folded properly. Considering that expression of the first 843 amino acids of PilC is not required for phage transduction (see Fig. 3b), our results suggest that entry of the MDAΦ into meningococci relies mostly on a mechanism independent of an interaction between ORF6 and the pilus tip proteins PilC1/2, PilH, PilI, PilJ, and PilK.

The MDAΦ binds to the pilus filament
Considering the role of antigenic variation in MDAΦ transduction, we reasoned that the MDA phages should interact directly with the pilus fibre rather than the tip. The assembly of ORF4 and PilE proteins in filaments makes a two-hybrid assay between them inappropriate. To visualise the possible interaction between a MDAΦ and a pilus, we first performed transmission electron microscopy (TEM) on whole bacteria incubated with phages. To prevent phage internalisation and to facilitate observation of the phage-pili interaction, we used a strain mutated for pili retraction and expressing the PilE SD variant. The ZΔMDA ΔpilT pilE SD strain was thus incubated with purified bacteriophages, washed to remove free bacteriophages and processed for immunogold against the major coat protein ORF4 and TEM (Fig. 4a). Labelled phages were indeed found along the entire length of the pili bundles. Labelling of phage-free bacteria showed no staining (Fig. 4a). We next assessed MDAΦ binding to pili by quantifying phage precipitation by bacteria expressing either the PilE SA or PilE SD variant. As above, we used strains mutated for pili retraction. We incubated purified MDA (orf6-aadA1) Φ with the strains ZΔMDA ΔpilT pilE SA or ZΔMDA ΔpilT pilE SD and a pili-deficient strain (ZΔMDA ΔpilE) as a negative control (Fig. 4b, c). Bacteria were then washed and immobilised on glass slides before immunostaining with anti-ORF4 antibodies. The aera of ORF4 labelling was analysed using ImageJ and normalised to that of bacteria stained with DAPI (Fig. 4b). As expected, phages did not bind to the non-piliated ZΔMDA ΔpilE strain. The remaining signal for the non-piliated ZΔMDA ΔpilE strain likely corresponds to background and non-specific labelling. However, there is a significant difference in phage precipitation depending on the pilE sequence. MDA phages bound to the PilE SA variant at reduced levels, whereas they bound to the strain specifically expressing the PilE SD variant (Fig. 4b, c). To better visualise the phage-pili interaction, we have decided to perform high-resolution STED (Stimulated Emission Depletion) microscopy on precipitated bacteria. However, as the PilE SD variant was not recognised by our anti-PilE 20D9 antibody, we had to derive a new strain with phage binding and transduction properties whose pili were labelled by our antibody. To this end, we followed the same strategy as that used to obtain the PilE SD strain. We transduced the ZΔMDA strain expressing the PilE SB variant (Fig. 1e and Supplementary Fig. 1c) and selected transduced derivatives. We have selected one new pili variant, named PilE SE, which was favourable for phage entry and recognised by the 20D9 antibody (Supplementary Fig. 4a, b). We then incubated purified MDA (orf6::aph3’) Φ with the strain ZΔMDA ΔpilT pilE SE. The bacteria were washed and immobilised on glass slides before immunostaining with anti-ORF4 and anti-pili 20D9 antibodies. The slides were then processed for high-resolution STED microscopy (Fig. 4d and Supplementary Fig. 4a). Pili staining is shown as dotted lines, which we had previously observed using high-resolution microscopy with the anti-pili 20D9 antibody43,48, and most of the phage staining appears to be associated with pili along their entire length. We also quantified phage retention by ΔpilT strains expressing either the PilE SB or PilE SE variant and the amount of pili around the strains as in Fig. 4b (Supplementary Fig. 4d, e). The phage retention is the ratio of phage surface labelling to bacterial surface labelling after phage precipitation (see materials and method). As expected, the strain expressing the PilE SE variant appeared to retain significantly more phage than the variant expressing PilE SB (p=0.0006). Interestingly, the ZΔMDA ΔpilT pilE SB strain produces more pili than the ZΔMDA ΔpilT pilE SE strain, but the amount of pili around these two variants is not significantly different (p=0.5162). Finally, to confirm the absence of a role for ORF6 in the interaction between the phage capsid and pili, we used a phage preparation produced from a strain deleted for orf617 (MDA (orf6::aph3’) Φ). We incubated the purified MDA (orf6::aph3’) Φ preparation with the strain ZΔMDA ΔpilT pilE SD as above and used the pili-deficient strain (ZΔMDA ΔpilE) as a negative control (Supplementary Fig. 5). We observed no significant difference between the retention of the MDA (orf6-aadA1) Φ and the MDA (orf6::aph3’) Φ on pili. This confirms that ORF6 does not play a role in the primary interaction between the phage and the pili. Taken together, these results suggest that, in contrast to what has been shown for other filamentous phages, MDAΦ preferentially interacts with pili along their fibres rather than at their tips, an association that strongly correlates with the transduction rate.

MDAΦ transduction is associated with positively charged pili
The differences between the PilE SA and PilE SD variants are essentially differences in amino acids in the D-region, with amino acids having different charges, resulting in a change in the isoelectric point of the pilin (Fig. 5a and Supplementary Fig. 2b). Interestingly, the coat of MDAΦ, consisting of the major coat protein ORF4, has recently been modelled by cryo-EM50. This model showed that the MDAΦ surface was predominantly negatively charged. We therefore reasoned that the interaction between MDAΦ and pili might be facilitated by opposite charges on the negatively charged MDAΦ coat and positively charged pili such as those displaying the PilE SD variant. To test this hypothesis, we first selected four clones expressing different PilE variants from the ZΔMDA strain. These variants were selected on the basis of amino acid charge and were named PilE Z1, PilE Z2, PilE Z3, and PilE Z4, with PilE Z1 being the most positively charged and PilE Z4 the most negatively charged (Fig. 5a and Supplementary Fig. 2b). We then modelled in silico the pilus fibre of these strains and the structure of PilE SA and PilE SD using Alphafold and the cryo-EM reconstruction of the N. meningitidis type IV pilus (5KUA; https://www.rcsb.org/structure/5kua). The Fig. 5a shows the difference in charged amino acids exposed for the six PilE variants studied. PilE SD has a majority of positively charged amino acids on its surface, while PilE SA has a majority of negatively charged amino acids, with the other four strains falling between these two extremes. We then assessed the frequency of MDA (orf6-aadA1) Φ transduction in these ZΔMDA clones that predominantly express pilE Z1, pilE Z2, pilE Z3, or pilE Z4 (Fig. 5b). We observed a correlation between pili charge and phage transduction: bacteria with positively charged pili were better transduced by phages than those expressing negatively charged pili.

We further examined the differences between the amino acid sequences of PilE SA and PilE SD, focusing on the charged amino acids (Supplementary Fig. 2b). We decided to focus on position 70-71 and the D-region. We therefore constructed a ZΔMDA mutS+ ΔrecA pilE SA mutant in which we exchanged A70 D71 with S70 K71 of PilE SD and/or the D-region with that of PilE SD, and vice versa between PilE SD and PilE SA (Fig. 5c). The structure of these mutants was then modelled using Alphafold to visualise charged amino acids (Supplementary Fig. 6a, b). We then assessed the MDA (orf6-aadA1) Φ transduction rate in all these mutants (Fig. 5d). The exchange between the D-regions dramatically increased (PilE SA to PilE SD) or decreased (PilE SD to PilE SA) the transduction rate, highlighting the role of this region. Modifications at positions 70-71 had little effect on the pilin-exposed charges, and therefore had little effect on the transduction rate. These data support our hypothesis that the interaction between MDAΦ and type IV pili benefits from a positively charged pilus.

Adhesion and transduction both determine the selection of the same pilS cassette
Both MDAΦ and pili are associated with the invasiveness of N. meningitidis. We therefore sought to determine whether adhesion to human cells could select for a PilE variant that favours transduction by MDAΦ or whether strains transduced by MDAΦ could select for a more adhesive PilE variant. We have previously shown that transduction of MDA (orf6-aadA1) Φ in a ZΔMDA pilE SA strain, after spectinomycin selection, resulted in a population expressing predominantly the PilE SD variant (Fig. 2e), which appeared to correspond to an antigenic variation achieved between the pilE gene and the pilS3 cassette (Fig. 2d and Supplementary Fig. 2a, b). To assess the proportion of pilS3/pilE variant in an adhesive or transduced population, we designed a qPCR assay with one primer specific to the pilS3 sequence and one primer specific to the constant region of pilE Z4 (Supplementary Fig. 2b). A second pair of primers targeting pilT was used as a control. We validated this method on DNA samples from two ZΔMDA ΔrecA strains (which avoid antigenic variation) expressing PilE SD or PilE Z4 corresponding to an antigenic variation achieved between the pilE gene and the pilS8 cassette (Supplementary Fig. 2a, b). As expected, we obtained a proportion of pilE S3 positive bacteria close to 100% for the ZΔMDA ΔrecA pilE SD strain, and a proportion of pilE S3 positive bacteria of 0.01% for the ZΔMDA ΔrecA pilE Z4 strain (Supplementary Fig. 7a). We then examined the proportion of bacteria carrying a recombination of the pilE gene with the pilS3 locus in the ZΔMDA pilE Z4 strain. Only 0.1% of the population was pilE S3 positive (Supplementary Fig. 7b). This strain was further used to assess the proportion of bacteria expressing the PilE S3 variant after adhesion to endothelial or epithelial cells and then the subsequent transduction by MDA (orf6-aadA1) Φ. In a first experiment (Fig. 6a), we collected ZΔMDA pilE Z4 bacteria adherent to FaDu epithelial cells or EA.Hy926 endothelial cells or after incubation in DMEM, and we quantified both the proportion of bacteria expressing PilE S3 and the frequency of transduction. The proportion of bacteria expressing PilE S3 increased after adhesion to epithelial and endothelial cells (Fig. 6b) from 0.13% of the population to almost 1% and 0.85% respectively. Similarly, adhesion to epithelial and endothelial cells increased the transduction rate by almost fourfold (Fig. 6c, p=0.0226 and p=0.0084, respectively). In a second experiment (Fig. 6d), we transduced the ZΔMDA pilE Z4 strain with MDA (orf6-aadA1) Φ and quantified the adhesion of the resulting population. The initial ZΔMDA pilE Z4 strain was incubated in transduction media without phage and kept as a control. The population infected by the phage and selected on spectinomycin became predominantly pilE S3 positive (68% of the total population), whereas it remained at 0.18% in the control population (Fig. 6e). After transduction, the spectinomycin-resistant population was 10.6 times more adhesive to epithelial cells and 4.7 times more adhesive to endothelial cells than the initial population (Fig. 6f, g, p<0.0001 and p<0.0001 respectively). Therefore, there is a strong link between transduction and adhesion by selection of a specific pilE variant.

Enrichment of phage-infected bacteria during adhesion
Finally, we asked whether adhesion to human cells would be sufficient to increase the proportion of phage-containing bacteria in a given bacterial population. To answer this question, we brought a ZΔMDA pilE Z4 population into contact with purified MDA (orf6-aadA1) Φ without further antibiotic selection. Bacteria were then incubated on epithelial and endothelial cells or incubated in DMEM as a control. After 3h, the cells were washed, adhesive bacteria were recovered and the proportion of phage-containing bacteria was assessed by serial dilution on agar plates containing spectinomycin and CFU counting (Fig. 7a). As a control, we confirmed that the growth and adhesion of the two bacterial populations - phage infected or not - were similar (Supplementary Fig. 7c). Bacterial adhesion to epithelial or endothelial cells was sufficient to increase the MDA-carrying population by 2.1 and 2.4 times, respectively, compared to the total population (Fig. 7b, p=0.0189 and p=0.0116 respectively). This result highlights the mutual benefit of the MDAΦ-meningococcus interaction in promoting both phage propagation and N. meningitidis colonisation (Fig. 7c).

Methods
Bacterial strains and culture media
The N. meningitidis strains used in this study are listed in Supplementary Data 1 and are derived from Z5463, a naturally transformable serogroup A strain isolated from the throat of a patient with meningitis in the Gambia in 198354. N. meningitidis mutants used in this work are listed in the Supplementary Data 1. It should be noted that the ZΔMDA strain was constructed from the wild-type Z5463 strain. The initial mutant strain was clonal (one colony was selected), but underwent several passages to eliminate the circular cytoplasmic form of MDAΦ. Considering that the frequency of antigenic variation in N. meningitidis is 1 to 2×10−3, the resulting strain expresses several different PilE sequences. E. coli strain DH5α was used for DNA cloning and plasmid propagation. E. coli Oxi-Blue55 was used for two-hybrid assay. E. coli Oxi-Blue, derived from the Oxi-BTH strain, facilitates the correct folding of proteins formed by disulphide bonds within its cytoplasm.

The N. meningitidis strains were grown at 37°C in 5% CO2 on Brain Heart Infusion Agar (BHI Agar, Condalab) plate containing 5% of heat-inactivated horse serum (Gibco), or in gonococcal base (GCB) agar (Difco) plates containing 12μM FeSO4 and Kellogg’s supplements56 or in BHI liquid medium (Condalab) or in GC-liquid medium [1.5% proteose peptone (Difco); 0.4% KH2PO4, 0.1% K2HPO4, 0.1% NaCl], sometimes with 12μM FeSO4 and Kellogg’s supplements. E. coli was grown at 30°C or 37°C on Lysogeny Broth (LB) agar or in LB liquid medium. For antibiotic selection of E. coli strains, kanamycin (Km) was used at a concentration of 50µg/ml, erythromycin (Em) at 150µg/ml, and ticarcillin (Tic) at 100µg/ml. To select strains derived from N. meningitidis, Km was used at a concentration of 200μg/ml, spectinomycin (Sp) at 75μg/ml, Em at 3μg/ml, chloramphenicol (Cm) at 5μg/ml, nalidixic acid (Nal) at 1µg/ml, and tetracyclin (Tet) at 2.5µg/ml.

Cell lines and culture conditions
The pharynx carcinoma-derived epithelial cell line (FaDu; ATCC #HTB-43) and the human umbilical vein cell line (EA.hy926; ATCC #CRL-2922) were obtained from the American Type Culture Collection (ATCC). The FaDu and EA.hy926 cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% decomplemented foetal calf serum (FCS; Gibco). Cells were grown at 37°C in a humidified incubator under 5% CO2.

Phage preparation, quantification, and transduction assay
Phage preparation. Phage preparation was performed as previously described using two different strains: ZS2 strain is a derivative of strain Z5463 to which a Sp resistance gene (aadA1) has been added in the MDA prophage (MDA (orf6-aadA1) Φ), just after orf6, without promoter, and Z5463 orf6::aph3’ strain, previously described17, derivative from Z5463 with prophage gene orf6 replaced by a kanamycin resistance cassette without promoter (MDA (orf6::aph3’) Φ). Bacteria were pelleted from 200 ml of an overnight culture in BHI liquid medium containing Sp at 200µg/ml (for ZS2) or Km at 300µg/ml (for Z5463 orf6::aph3’). After filtration at 0.45µm, the supernatant was treated with DNase I (25µg/ml) for 1h at 20°C. Phage was precipitated by the addition of 0.5M NaCl and 10% polyethyleneglycol 6000 and incubated overnight at 4°C. The phage was then pelleted by centrifugation at 10,000×g for 30min and resuspended in PBS 1X (with CaCl2 and MgCl2; Gibco). The phage suspension was centrifuged at 200×g for 10min, and the supernatant was collected to remove insoluble particles. It was then filtered through a 0.45µm filter to prevent contamination. The purified phage preparation was used directly for immunogold labelling, immunofluorescence, or bacterial transduction assays.

qPCR quantification of phage concentration
Phage concentration was quantified by qPCR using two primer pairs: one targeting the phage genome (ORF6-F/R for MDA (orf6-aadA1) Φ or ORF4-F/R for MDA (orf6::aph3’) Φ), and another targeting the Glucose-6-phosphate dehydrogenase (G6PD-F/R) to assess total bacterial chromosome number. qPCR was performed with Luna Universal qPCR Master Mix (NEB) on a QuantStudio 1 (Applied Biosystems), run in triplicates. Cycling conditions were 95°C for 1min, followed by 40 cycles of 95°C for 15s and 60°C for 30s. A standard curve was generated using genomic DNA from strain Z5463 Δorf1, assumed to contain a single phage copy per genome. The resulting standard equations enabled quantification of circular phage DNA, the relative amount of phage is obtained by Number ORF6/ORF4 - Number G6PD. After quantification, the average phage quantity is estimated to be 2×107 phage/µl.

Transduction assay
Recipient strains were grown overnight on agar plates and resuspended at an OD600 of 1 in transduction liquid medium [containing GC-liquid medium, 12μM FeSO4, Kellogg’s supplements, 2.5mM MgCl2, 2.5mM MgSO4, and 25µg/ml DNase I]. Two hundred microliters of the bacterial suspension (corresponding to 2×108 bacteria) were aliquoted into a 24-well plate and mixed with 30µl of the MDA phage preparation. This corresponds approximately to a bacteria:phage ratio of 1:3. The mix was incubated for 15min at 37°C with shaking and 15min without shaking, and 800µl of transduction liquid medium was added to each well. Plates were incubated for a further 2h at 37°C without shaking. If necessary, bacteria were plated on agar plates with or without Sp and stored the next day at −80°C in BHI/15% glycerol.

To determine the transduction rates, bacteria were diluted and plated on agar plates with or without Sp before counting of CFU the next day. The transduction rate was calculated by dividing the number of CFU obtained on Sp-containing agar plates by that on antibiotic-free agar plates. The efficacy of the phage preparation was verified by transducing 30µl of the phage preparation into the ZΔMDA strain and was validated if the transduction rate was greater than 10−3. If no bacteria were obtained at the least diluted point on the agar plates, a CFU of 0.9 was arbitrarily assigned and the point was marked as below the detection limit. A detection threshold corresponding to the frequency of spontaneous resistance to spectinomycin was experimentally calculated using the ZΔMDA strain (3.2×10−8±4×10−8–SD; n=3 experiments in duplicate).

Transformation of N. meningitidis
Strains of interest were grown overnight on BHI agar plates and resuspended to OD600 = 1 in liquid transformation medium [containing GC-liquid medium, 12μM FeSO4, Kellogg’s supplements, MgCl2 at 2.5mM, and MgSO4 at 2.5mM]. Five µl of PCR product or 150ng of plasmid or three µl of genomic DNA were added to 200µl of this bacterial suspension and incubated for 30min at 37°C, 5% CO2 with shaking. Bacteria were then grown for 2h after the addition of 800µl of liquid transformation medium. Transformants were selected on BHI agar plates containing corresponding the appropriate selective antibiotic.

To determine the transformation frequency, bacteria were resuspended to OD600 = 0.1 in step 1 transformation media [containing GC-liquid medium, MgSO4 at 5mM]. One thousand ng of genomic DNA carrying a Nal-resistant cassette was added to 200µl of this bacterial suspension and incubated for 30min at 37°C, 5% CO2 with shaking. Bacteria were then grown without agitation for 2h after the addition of 800µl of step 2 transformation liquid medium [containing 12μM FeSO4 and Kellogg’s supplements except L-Glutamine]. This experiment has been described previously30. After incubation, bacteria were diluted and plated on agar plates with and without Nal before CFU counts the next day. The transformation frequency was calculated by dividing the number of CFU obtained on Nal-containing agar plates by the number of CFU obtained on antibiotic-free agar plates. If no bacteria were obtained at the least diluted point on the agar plates, a CFU of 0.9 was arbitrarily assigned, and the point was marked as below the detection limit.

PCR amplification, plasmid construction and mutant generation
PCR amplification and purification. Sequence amplifications for construction and sequencing were conducted using Q5 High Fidelity DNA polymerase (New England Biolabs) and the oligonucleotides listed in Supplementary Data 2. All oligonucleotides were purchased from Integrated DNA Technologies (eu.idtdna.com). After amplification, PCR products amplified from plasmids were incubated for 1h at 37°C with DpnI enzyme (NEB) to digest the methylated plasmid backbones. PCR products were purified using Monarch DNA Gel Extraction kit (New England Biolabs) or Monarch PCR & DNA Cleanup Kit (New England Biolabs). All mutants were verified by PCR amplification using OneTaq DNA Polymerase (New England Biolabs) on bacterial genomic DNA extracted using the Wizard Genomic DNA purification kit (Promega) according to the manufacturer’s instructions.

Plasmid construction and mutant generation
All plasmids used in this work are listed in the Supplementary Data 3. ZΔMDA pilE SA and ZΔMDA pilE SB. The pilE SA and pilE SB alleles fused to the Km resistance gene and the flanking regions (≈250pb upstream and downstream) were amplified by PCR from the genomic DNA of strains NEM8013 pilE SA and -pilE SB with primers 5′_pilE_Fw and 3′_pilE_Rv34 and used for transformation and allelic exchange in ZΔMDA. Bacteria were then isolated on Km, and pilE sequences were verified by Sanger sequencing. The same methodology was used to construct ZΔMDA pilE SD and ZΔMDA pilE SE with PCR amplification from the genomic DNA of transduced clonal strains obtained respectively from transduction of ZΔMDA pilE SA and ZΔMDA pilE SB.

Obtention of mutS+ derivatives. We took advantage of the plasmid containing the Z2491 mutS+ allele with a Sp resistance cassette inserted after the gene, leaving a downstream intergenic sequence at the 3′ end, allowing the allelic exchange to be performed (as described by Omer et al.29). The Sp resistance gene was swapped with a Cm resistance gene by PCR mutagenesis using appropriate primers (Supplementary Data 2). The ZΔMDA mutS+ strain was obtained by transformation of a vector containing the mutS+ allele with Cm resistance downstream of the gene and allelic exchange. Bacteria were then isolated on Cm and verified by PCR.

ZΔMDA mutS+ ΔrecA -pilE SA and -pilE SD and derived mutants. The pilE SA and pilE SD alleles fused to the Km resistance gene and their flanking regions (≈250pb upstream and downstream) were cloned into the pMiniT 2.0 vector as above and using primers 5′_pilE_Fw and 3′_pilE_Rv. The resulting plasmids (namely pMiniT_pilE SA-KmR and pMiniT_pilE SD-KmR) were sequenced and used as templates for site-directed PCR mutagenesis using the appropriate primers (Supplementary Data 2). PCR products corresponding to the pilE loci were obtained from wild-type and mutagenised pMiniT_pilE SA-KmR or pMiniT_pilE SD-KmR, using primers 5′_pilE_Fw and 3′_pilE_Rv and were introduced into N. meningitidis strain ZΔMDA mutS+ by transformation and allelic exchange. Bacteria were then isolated on Km plates, and pilE sequences were verified by Sanger sequencing. To obtain ZΔMDA mutS+ ΔrecA -pilE SA, -pilE SD or other derivatives variants of pilE, a genomic DNA from ZΔMDA ΔrecA was introduced into strains ZΔMDA mutS -pilE SA, -pilE SD or other derivatives variants of pilE by transformation and allelic exchange. Bacteria were isolated on Tet plates, and pilE sequences were verified by Sanger sequencing and recA deletion was verified by PCR.

ZΔMDA pilE SB. The pilE SB allele was cloned into pMiniT resulting in plasmid pMiniT_pilE SB-KmR. The Km resistance cassette fused to pilE gene was swapped with a Sp resistance gene by PCR-based mutagenesis using appropriate primers (Supplementary Data 2). The resulting pilE variant was introduced into strain ZΔMDA at the locus of pilE SB by transformation. Cloning and transformation strategies were the same as described for pMiniT_pilE SA-KmR.

pNM99Ptet-pilC1 and -pilC2 and derived plasmids. The pilC1 and pilC2 genes from strain Z5463 were cloned into the pMiniT 2.0 vector as described above. To avoid phase variation, the poly(G) sequences present in the two pilC signal peptide sequences were swapped with an amino acids identical alternative sequence using PCR mutagenesis with the primers C1.Z_polyG_Fw/Rv and C2.Z_polyG_Fw/Rv. The resulting plasmids, pMiniT- pilC1fixed (Z5463) and pMiniT- pilC2fixed (Z5463), were sequenced and used as a matrix for cloning of pilC1 and pilC2 into pNM99 57, where gene expression is under the control of a tet promoter (primers pNM99_TET_Fw/Rv, pilC1_Z5463_pNM99_Fw/Rv, pilC2_Z5463_pNM99_Fw/Rv). Deletion mutants of pilC1 were obtained by PCR mutagenesis using the appropriate primers directly on the pNM99 -pilC1 plasmid. Mutations were verified by Sanger sequencing.

ZΔMDA ΔpilC1 ΔpilC2 complemented for PilC1 and PilC2 and derived mutants. Markerless null mutants were constructed as described by Wuckelt et al.57. Briefly, the gene of interest was first swapped by allelic exchange with the APG cassette carrying a Km resistance marker (aphA-3) and two counter-selectable marker genes (pheS* and galK). The markerless null mutant was then obtained by removing the APG cassette by allelic exchange using a PCR product of the 5′ and 3′ regions flanking the gene of interest. Using this strategy, we first obtained a ZΔMDA ΔpilC2 mutant and we inserted the complementation construct from the pNM99 -pilC1 and -pilC2 plasmids (see above) by transformation and allelic exchange. Complemented bacteria were selected on Em plates. We then obtained the ΔpilC1 mutant using the same strategy as for the ΔpilC2 mutant. The PilC expression, required for transformation, was allowed by the addition of anhydrotetracycline (20ng/ml). The primers used are described in the Supplementary Data 2.

Transposon mutants (ΔpilC1, ΔpilD, ΔpilE, ΔpilF, ΔpilG, ΔpilH, ΔpilI, ΔpilJ, ΔpilK, ΔpilQ, ΔpilT, ΔpilT2, ΔpilU, ΔpilV, ΔpilW, ΔpilX, ΔpilZ, ΔtsaP, ΔpglA, ΔpglC) and other deletion mutants (ΔpilP, ΔpilC2, ΔNMA0415, ΔtfpC, ΔpptB, ΔpilC1/C2, ΔpilT, ΔrecA). We took advantage of mutants originally generated in strain NEM8013 by in vitro transposition58. Regions of interest were amplified by PCR on the genomic DNA and then introduced into ZΔMDA by transformation of purified PCR products and allelic exchange. Bacteria were then isolated on Km plates, and mutants were verified by PCR. Other deletion mutants (except for the markerless mutants ΔpilC1 and ΔpilC2, see above) were obtained by inserting an antibiotic resistance cassette in place of the target gene (from START to STOP codons, except for pilC2 where the last 537 nucleotides were retained) by allelic exchange after transformation into the strain ZΔMDA. The PCR product carrying the cassette flanked by regions upstream and downstream of the target gene was obtained by overlap PCR using primers listed in Supplementary Data 2. All transformants were selected on BHI agar plates containing a selective antibiotic. Mutations in the piliation machinery and pilus fiber genes were also introduced into strain ZΔMDA pilE SA by transformation with genomic DNA from the corresponding ZΔMDA mutant, followed by allelic exchange.

Two-hybrid system. The Bacterial Adenylate Cyclase Two-Hybrid System Kit (BACTH System Kit) from Euromedex was used. BACTH plasmids were designed to detect adenylate cyclase-fused protein expression. A 6His-tag was added to pKNT25/pKT25 and a FLAG-tag was added to pUT18/pUT18C. Divergent primers carrying sequences for the tag halves were designed to anneal just after the ATG start codon or just before the stop codon of the free extremity of the adenylate cyclase subunits. The PCR products form the complete tag upon plasmid ligation (same ligation method as for pilE mutagenesis). Then, to add inserts to the BACTH vector backbone, complementary primers were designed to overlap on 15–30bp, and the two PCR products (insert and vector backbone) were purified and assembled using the NEBuilder HiFi DNA Assembly kit (NEB). The plasmids corresponding to tolA3 in pUT18C, gIII in pKT25, orf6 in pKNT25, pUT18, pKT25, pUT18C, pilC1 in pKNT25, pUT18, pilC2 in pKNT25, pUT18, pilC1 in pKT25, pUT18C, pilH in pKT25, pUT18C, pilI in pKT25, pUT18C, pilJ in pKT25, pUT18C, pilK in pKT25, pUT18C are listed in Supplementary Data 3.

Bacterial adenylate cyclase two-hybrid assay
Competent Oxi-Blue E. coli were co-transformed with 10ng each of two recombinant plasmids encoding T18 and T25, respectively, Flag and 6His tagged. Transformed bacteria were plates on LB-agar plates supplemented with ampicillin and kanamycin. For each co-transformants, 3 clones were inoculated in duplicate (2×3 clones) into LB wells with Tic, Km, and IPTG 0.5mM (isopropyl-β-D thiogalactopyranoside). Plates were incubated for 24h at 37°C with shaking, then 5µl of each culture were plated in duplicate on LB agar plates supplemented with Tic, Km, IPTG 0.5mM and X-gal 40µg/ml (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Plates were incubated at 30°C, and the colour of the colonies was assessed after 24 to 48h.

Adhesion to human cells
The 24-well plates were seeded with EA.Hy926 or FaDu cells and grown to confluence. Bacterial strains grown overnight on BHI agar plates were adjusted to OD600 = 0.05 in DMEM/10% FCS and cultured for 2h at 37°C with agitation and 5% CO2. The number of bacteria in the inoculum was estimated and cells were infected with 5×106 bacteria (Fig. 6a–g) or 1×108 bacteria (Fig. 7a, b). The exact number of CFU was determined by serial dilution and enumeration of CFU. After 1h (Fig. 6d) or 3h (Figs. 6a and 7a and Supplementary Fig. 7) of contact, unbound bacteria were removed by six washes with 500µl of PBS and the cells were detached by mechanical action in 500µl DMEM/10% FCS. If necessary, bacteria were plated on BHI agar plates and stored the next day at −80°C in BHI/15% glycerol. To determine the adhesion frequency, adherent bacteria were diluted and spread on agar plates before counting of CFU the next day. The adhesive frequency was obtained by dividing the number of CFU of adherent bacteria by the number of CFU of the inoculum.

Bacterial preparation prior to immunolabeling for pilus quantification
In a 24-well plate, 2×10⁷ bacteria were deposited onto coverslips precoated with D-polylysine the day before. The plate was centrifuged at 2500×g for 1min and then incubated at 37°C for 40min. The medium was removed and the wells were washed once with PBS before adding 500µl of PBS 1X/4% PFA for 20min. Finally, the wells were washed with PBS, and the coverslips were immunolabelled.

Phage precipitation
Bacterial strains grown overnight on BHI agar plates were adjusted to OD600 = 0.05 in transduction liquid medium without DNase I and incubated at 37°C for 2h. Approximately 2×108 bacteria were transferred to a 2ml tube. One hundred µl of 107 MDA (orf6-aadA1) phage/µl solution or MDA (orf6::aph3’) phage/µl solution was added to the bacteria, and the tubes were filled up to 1.4 ml with transduction liquid medium (without DNase I). The mix was incubated for 30min at 37°C, then centrifuged at 400×g for 4min, 800µl of supernatant was removed and centrifuged again for 3min (to pellet bacteria that have settled to the sides of the tubes). The supernatant was removed and the pellet was resuspended in 800µl PBS 1X in a 1.5ml tube. The suspension was centrifuged again at 400×g for 4min, 600µl of supernatant was removed, centrifuged again for 3min, and finally the pellet was resuspended in 100µl of PBS 1X/4% PFA (paraformaldehyde; Life Technologies). Twenty µl of bacteria in PBS/4% PFA were spread on a slide and dried for 2h before immunolabelling.

Phage/pili labelling and quantification
The slides or coverslips were washed with PBS/0.1M NH4Cl for 2min and incubated with PBS/4% BSA for 5min. MDA (orf6-aadA1) Φ and MDA (orf6::aph3’) Φ were stained with the rabbit anti-ORF4 antibody used at 1/100 dilution. For tests with strains expressing PilE SB and PilE SE, pili were stained with mouse anti-PilE antibody 20D9, used at 1/1000 dilution. Secondary antibodies were goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Molecular Probes) used at 1/400 dilution, and goat anti-mouse antibody conjugated with Alexa Fluor 546. Bacteria were detected by DNA staining using 100ng/ml DAPI (49,6-Diamidine-29-phenylindole dihydrochloride). Coverslips were mounted in Mowiol. Images were captured using a Zeiss Apotome fluorescence microscope and images were collected and processed using the ZEN (ZEISS Efficient Navigation) software. MDA binding to bacteria and pili quantity were estimated by immunofluorescence as the ratio of phage surface or pili surface labelling to bacterial surface labelling estimated by staining bacterial DNA with DAPI (arb. units: arbitrary unit).

Stimulated-emission-depletion (STED) microscopy
Bacteria with or without bound phages in PBS/4% PFA were spread on 22-mm high-precision cover glasses with thickness of 170μm, (No. 1.5H, Marienfield) and dried for 2h. The slides were then washed with PBS/0.1M NH4Cl for 2min and blocked with 4% BSA–PBS for 10min, labelled with monoclonal anti-PilE antibody 20D9 (1:1000 in 4% BSA–PBS) and monoclonal anti-ORF4 antibody (1:100 in 4% BSA–PBS) and then incubated with goat anti-mouse Alexa Fluor 514 secondary antibody fragments (1:1000) and goat anti-rabbit ATTO 550 secondary antibody fragments (1:500). Bacteria were detected by DNA staining with 100ng/ml DAPI. Slides were mounted with ProLong Gold antifade reagent (Invitrogen), refractive index: 1.47.

Images were captured on a Leica TCS SP8 STED microscope, controlled by LASX software, equipped with a 660nm depletion laser and a 100x NA 1.4 objective. Images were acquired using a gated hybrid detector.

The images were then deconvolved using the Huygens software with the ‘Deconvolution Express, standard profile’ parameter.

Electron microscopy: negative staining and immunogold-labelling
Bacteria in PBS/4% PFA with and without co-precipitated MDAΦ were adsorbed onto Carbon/Formvar-coated 200-square-mesh copper grids (Euromedex, Souffelweyersheim, France) for 15min. The grids were then washed twice for 1min with PBS and placed sequentially on drops of the following reagents at room temperature: PBS/0.1M glycine (10min), PBS/1% BSA (bovine serum albumin)/5% normal goat serum (15min), and then on a drop of anti-ORF4 N-terminal antibody diluted 1/50 in PBS/1% BSA (60min). After three washes in PBS, the grids were placed on a drop of Fab2’ goat anti-rabbit conjugated to 10nm diameter gold (Aurion, Wageningen, The Netherlands) diluted 1/40 in PBS/1% BSA for 60min. The grids were then washed three times in PBS (1min), fixed in PBS/1% glutaraldehyde (5min), washed twice in PBS and twice in distilled water. The grids were then treated with 1% uranyl acetate, air-dried and viewed. Images were acquired using a Jeol 1400 TEM (Jeol, Croissy-sur-Seine, France) operating at 120keV and equipped with a RIO CMOS camera (Ametek SAS, Elancourt, France).

Quantification of the proportion of pilS3 D-region by qPCR
We estimated the proportion of bacteria with the pilS3 D-region sequence at the pilE locus by qPCR. One pair of primers was designed to anneal to the pilE locus. One primer was designed in the pilS3 D-region and one primer on the conserved upstream part of the pilE gene (pilS3-F/R). The second pair of primers (pilT-F/R) was designed to anneal to the pilT locus, allowing the quantification of the total number of chromosomes. qPCR was performed on genomic DNA using Luna Universal qPCR Master Mix (NEB). All samples were performed in three independent experiments. The cycling conditions were as follows: 95°C for 1min, and 40 cycles of 95°C for 15s and 60°C for 40s on QuantStudio 1 (Applied Biosystems). For the standard curve, genomic DNA from ZΔMDA mutS+ ΔrecA pilE SD (considered to contain 100% of bacteria expressing pilE D-region S3) was used. Serial dilutions of this DNA were amplified and a standard curve equation was recorded for each primer pair and used to quantify the samples. For each quantification, DNA from pilE clonal and pilE clonal strains, consisting of 100% and 0% of bacteria expressing pilE D-region S3, respectively, were used as positive and negative controls.

Bioinformatics
Pilus models were generated by running the amino acid sequences of PilE, PilC1, PilC2 in Alphafold 3 (https://golgi.sandbox.google.com) with general parameters. Several generated PilE models were aligned to subunits of the biological assembly 5KUA using ChimeraX version: 1.7.1. The structures were coloured using the coulombic electrostatic potential command of ChimeraX with the default colouring range. Folding structures of PilC1 and PilC2 were also obtained by running the amino acid sequences in Alphafold 3 with general parameters.

Sequence alignments were performed using Clustal-omega (1.2.4) (https://www.ebi.ac.uk/jdispatcher/msa/clustalo). Isoelectric points of PilE sequences were determined from their PilE amino acid sequences using the tool iep 45/5.0.0.1 in Galaxy France.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 8.02. Taking into account the number of replicates, the normality of the distribution for the whole datasets was assessed using QQ plots. When necessary, data were log transformed. Homogeneity of variance was tested using the Brown–Forsythe F-test. If the distribution of the data conformed to the law of normality and the variance of the groups was homogeneous, multiple comparison analyses were assessed using a one-way ANOVA with multiple comparisons test and appropriate correction, and data were expressed as mean±SEM or median ±95% confidence interval (relevant p-values were reported in the figures). In the case of different variances, a Brown–Forsythe and Welch ANOVA test was performed. A two-way ANOVA with Sidak correction was performed in the Supplementary Fig. 7. The H0 hypothesis was rejected at a significance level of p≤0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Mouville et al is a creatively devised study designed to investigate the role of a filamentous\nbacteriophage in meningococcal biology. It is a clear extension of the research groups previous work on the meningococcal\nMDA phage biology. In some of this groups previous work, they have shown that the MDA bacteriophage is able to infect\ndifferent N. meningitidis strains, using the type IV pili as a receptor, and that infection requires PilE and PilT\n(https://doi.org/10.1099/mic.0.000215). In this study, they explore how changes in PilE sequence impact the ability of the\nphage to infect the meningococcus and propose mechanisms by which the changes in infectivity could impact bacterial\nvirulence. Their data clearly demonstrate how antigenic variants of PilE can be selected for using the MDA phage and that\nthese variants are better able to adhere to epithelial and endothelial cells.\nGeneral comments\nWhile the data is presented in a logical fashion, because of how the transduction data is presented (normalized to\nZ5463ΔMDA), it is difficult to interpret the some of the data. Figure 1a-d clearly demonstrates how various pilin-associated\nproteins impact transduction frequency. However, Imhaus and Duménil (https://doi.org/10.15252/embj.201488031)\npreviously showed that a meningococcus has about 6 pili per bacterium, and a pilX mutant has a three-fold reduction in pili.\nThis is similar to the change in quantity that the authors report in figure 1c . On line 168, the authors state “The ∆pilC1/C2\ndouble mutant is barely piliated”. However, their data indicates that there is only about a 3 fold reduction.\nThis is not barely piliated (see also line 227).\nIn figure 1e, the authors do not define the PilE sequence of ZΔMDA (line 179). This should be in supplemental figure 1).\nSupplemental figure 2 does not have this sequence, or pilEsb, and this also needs to be added here. In figure 1, they do not\ndefine the limit of detection for a transduction experiment? If PilE is truly required for transduction, it would seem that the\ntransductants identified in figure 1 A for PilE represent the mutation frequency to spectinomycin resistance. Is this true?\nOtherwise, it suggests that PilE is not required for transduction but enhances transduction. If one assumes that PilE\nexpression is needed for transduction, and the starting concentration of bacteria in their experiments is 109 CFU/ml (about\nan OD of 0.6), then the efficiency of transduction in this figure is 10-5. This becomes important when trying to interpret the\ndata in Figure 6 (see below).\nIn figure 2, panel b, when the authors sequenced 10 random colonies, 50% underwent phase variation. This is much higher\nthan that that has been reported for the meningococcus (doi: 10.1128/JB.00465-18). How do the authors explain this\nextraordinarily high rate?\nIn figure 3, it would be useful to know how the truncated PilC proteins are impacting surface charge. If there is no change,\nthis would strengthen their argument in the data they present in figure 5.\nIn figure 5, the authors have assumed that small changes in pilin, which change the PI of pilin, impact the surface charge of\nthe bacteria. It is possible that other phase variable surface molecules are also changing (Opa as an example)? The authors\nneed to demonstrate that the amino acid changes are contributing to surface charge changes, perhaps by measuring the\nZeta potential of the various strains.\nIn figure 6, panel B it would appear that pilEs3 represents about 0.1% of the starting inoculum. After incubating with cells\nthere is about a 10 fold enrichment for selecting the spontaneous phase variant. Howevewr, in figure 3 above the change is\n50%. How do the authors explain this discrepancy? In panel D, given the low percentage of pilEs3, and the low transduction\nefficiency, it is not surprising that virtually all of the transduced bacteria are pilEs3 positive (about 4 logs). What is surprising\nis that there is only a 1 log increase in adherence. Doesn’t this argue that the presence of the phage interferes with\nadherence?\nBille et al have previously show that phage expression promotes bacterial aggregation\n(https://doi.org/10.1371/journal.ppat.1006495). In figure 7, if the presence of phage promotes bacterial aggregation, such,\nadhering bacteria containing phage would be represented by microcolonies that would be larger than those lacking the\nphage. One might explain the modest increase in the presence of phage in adhering bacteria, as seen in figure 7 panel B as\nsimply the result of this. Do the authors have confocal data to show that the control and adherent data are producing the\nsame size microcolonies on cells?.\nIn the discussion, the authors try to link pilin expression with virulence. It is hard for this reviewer to understand the logic.\nThis relationship may exist the first time a bacterium becomes infected with MDA, but subsequent replication in a host would\nallow for antigenic variation where the strain no longer expresses the PilE needed for phage infection. Unless the strain is\nconstantly being reinfected with MDA, how would phage infection be linked to virulence. Do the authors have any indication\nof this?\nSpecific comments\nLine 162. that the phage only fits into strains expressing fully retractable pili. I think the authors mean “that the phage only\ninfects strains expressing fully retractable pili.”\nLine 215. Overall, these results suggest that MDAΦ: (i) infects meningococci expressing a specific PilE variant. This really\nisn’t correct. The data really says (in agreement with their overall interpretation) that some variants appear to be more\nsusceptible to infection.\nIn discussing the data in figure 2, the authors have missed an opportunity to use their data to conclude that phage infection is\nrecA independent\nLine 410. The DNA sequence of strain Z5463 is known: hence its pilS variability is also known.\nOn line 411-12, they indicate. “This allows us to observe the preferential integration of phages into the genome of N.\nmeningitidis expressing high pI PilE variants, although this variant is expressed by a minority of bacteria. It is unclear what\nthe highlighted phrase means.\nIn figure 4b, the data for each replicate needs to be visualized.\nIn figure 5, it would be helpful if the legend stated that ZΔMDA was expressing pilESD\nIn figure 6, panels B and D. Were the PCRs performed on the three independent experiments or 3 times on the same\nexperiment.\nIn supplemental figure 2 it would be helpful to have the DNA sequence of the PilE z1-4 strains.\nIn supplemental figure 4, the colors of the first three columns are white. Legend needs to explain this.\nIn the table of strains, the construction of the ΔMDA strain has1 the wrong reference. It should be J Exp Med (2005) 201 (12):\n1905–1913.", "reviewer_2": "A filamentous phage promotes meningococcal colonization by selecting hyperadhesive pili variants linking phage infection\nto bacterial virulence\nThis is a manuscript showing that MDA phage aids in selection of N. meningitidis variants with superior adhesion to the host\ncells and thereby is bound to increase virulence during the infection of a host. This is an interesting but not novel concept\nwhen it comes to filamentous phages and their role in virulence of their hosts.\nThe manuscript is generally very well written, however important filamentous bacteriophage biology published findings that\nare relevant to this manuscript has been missed, both from the recent years and from the past.\nThere are a few methodological gaps in the manuscript and some experimental findings that require clarification, proper\ncontrols or statistical analyses.\nMajor comments\nL233-234 “PilC1Δ3-843 allowed transduction and transformation at a level equivalent to 10% of the control (Fig. 3b,c).”\nIsn’t this an indication that the C domain of PilC may be involved in binding of MDA?\nL242 Supplementary Fig. 3b:\nThis figure describes bacterial adenylate cyclase two-component assay which is here used to rule out the interactions of\nOrf6 with minor pilins. Unfortunately there is no appropriate positive control showing that the Orf6 fusions used in this\nexperiment have folded into a functional form. This is crucial because the type of two-hybrid system used in this experiment\nmeans that the proteins have to fold correctly in the cytoplasm. Given that the tested proteins fold in the periplasm prior to\nassembly into the phage/pili, and have S-S bridges that are required for their correct folding (which do not form in the\ncytoplasm), it is quite possible that none of the pilins or Orf6 in this experiment are folded into functional domains. In the\nabsence of Orf6 and pilin positive control interactions, negative results shown in this figure cannot discern between failed\ninteractions and misfolding of one or both partners.\nThe only positive control is pIII from M13 phage (M13 pIII - TolA interaction gives the only positive signal in the figure). Why\nwas MDA Orf6 interaction with -N.m. TolA not tested in this setup? Why were no interactions with pilE tested, given the\nmanuscript is focused on showing that PilE is the key receptor for MDA?\nL250-251 + Fig. 4. “we first performed transmission electron microscopy (TEM) on whole bacteria\nincubated with phages “\nThe TEM shows MDA longitudinally associated with the pilus, through pVIII-pilE interactions. This may be a secondary step\nafter the initial binding of Orf6 to either pilE or minor pilins, or both, and may strongly contribute to the overall binding.\nCompetition studies using purified MDA Orf6 or MDA phage that has truncated pIII (without the host-binding domains) are\nrequired to resolve the pairing i.e. does pIII first interact with -pilC(C-terminal domain), then pVIII interacts with pilE? or Orf6-\npilE concurrently with pVIII-pilE?\nL251-257/ Fig. 4a; Supplementary Fig. 4: Why is the negative control shown in supplementary figure? Side by side\ncomparison would be more impactful. Was there any statistical analysis of gold bead pilus-association done on multiple\ncells? This would strengthen the validity of observations.\nFig. 6-7: Q - why are the values shown in 6c and 6f-g relative (% relative to the value of the control sample), while absolute\nvalue is shown in 7b?\nL461 “10% NaCl and 20% polyethylene glycol 6000” – Is this the stock concentration or the final concentration? This is way\ntoo high as a final concentration for precipitation (normally it is 0.5 M NaCl, 5% PEG for filamentous phages). If only one\nround of PEG precipitation was done and no further purification steps were performed, the samples would be highly\ncontaminated with host proteins and cellular debris or vesicles such as OMVs.\n462-463 “resuspended in PBS 1X (with CaCl2 and MgCl2; Gibco) and used directly”\nIn the presence of divalent cations OM fragments or vesicles will not be soluble. Was the insoluble pellet removed from the\nphage stock by centrifugation, to eliminate OMVs?\nL467-468 “Two hundred microliters of the bacterial suspension were aliquoted into a 24-well plate and mixed with 30 μl of\nthe MDA phage preparation.”\nWere the phage stocks titrated? How many phages were used per transduction? What was the m.o.i.? Were there any\nattempts to quantify phages by densitometry or spectrophotometry? This is also essential to ensure that the same amount of\nphages is always used between different experiments. See Waldor and Mekalanos 1996\nhttps://www.science.org/doi/10.1126/science.272.5270.1910 (Table 1) and publications thereafter.\nSame below:\nL475-479 “The efficacy of the phage preparation was verified by transducing 30 μl of the phage preparation into the ZΔMDA\nstrain and validated if the transduction frequency was greater than 10-3. If no bacteria were obtained at the least diluted point\non the agar plates, a CFU of 0.9 was arbitrarily assigned and the point was marked as below the detection limit.\nSame comment as above: how many phages were used, i.e. what was the m.o.i.?\nL607-609 Quantification of pili by western blotting:\nWhat was used to plot a standard curve here? Standard should be serial dilutions of a purified pilin subunit quantified by a\ncolorimetric of other absolute protein quantification method. Serial dilutions of quantified pilin subunits should be run on the\nsame SDS-PAGE gel along with the pili samples, transferred and used to construct a standard curve so that the extracted\npilin bands in the westerns can be correctly quantified.\nL612 “Phage precipitation” method\nThis looks like a protocol for detecting bacteria-associated vs. free phages. The assay commences by mixing phage and\nbacteria at a huge excess of phages (10E11 phages per 2x10E8; i.e. m.o.i. 500). Since the efficiency of transduction is well\nbelow 100% based on data in the manuscript (albeit absolute numbers are mostly missing), this assay where there is huge\nexcess of phages over the host bacteria is puzzling.\nIt is good to see that the phages were quantified before use in this experiment. Method of quantification, however, was not\ndescribed. Titer vs. quantity of the phages seem to be strongly dependent on the strain, hence it is really important to\nmeasure the number of phage particles (quantity of phage) in addition to titration based on host infection.\nGiven the very crude MDA phage purification method is described earlier in the methods section, which results in phage\npreparations containing large amount of contaminating proteins derived from the host (L451-463; also see a comment\nabove), it would not be advisable to use simple spectroscopy to quantify phages. Instead, quantification of phage DNA\nwould be a much more accurate measure of the number of phages per volume of the sample. Methodology for phage DNA\nquantification is well-established in the phage literature (densitometry or qPCR).\nMinor comments\nL35-36 F pilus is not a type IV pilus. Rewrite the sentence:\nThe paradigm of filamentous phage infection has been defined for the phages Ff and CTX, where binding to the tips of\nbacterial type IV pili is a means of infecting their bacterial host\nL42-44 Taken together, this study reveals how the propagation strategy of a filamentous phage, aimed at selecting the best\ncoloniser as a host, can promote the selection of hyperadhesive bacterial variants, linking phage infection to bacterial\nvirulence.\nConvoluted sentence. Simplify.\nL62-63 The first filamentous phage of this family was described by Peter H. Hofschneider in Escherichia coli in the 1960s\nand named M13 (Ray et al., 1966).\nThis is not the first report, look for earlier references for Ff\nHofschneider PH. 1963. Untersuchungen uber kleine E. coli K 12 bakteriophagen 1 und 2 mitteilung. Z Naturforsch Pt B 18:\n203–210. doi:10.1515/znb-1963-0306\nLoeb T. 1960. Isolation of a bacteriophage specific for the F+ and Hfr mating types of Escherichia coli K-12. Science 131:\n932–933. doi:10.1126/science.131.3404.932\nMarvin DA, Hoffmann-Berling H. 1963. A Fibrous DNA phage (Fd) and a spherical RNA phage (Fr) specific for male strains\nof E. coli ii. Physical characteristics. Z Naturforsch B 18: 884–893. doi:10.1515/znb-1963-1106\nL63 replace “famous” with “well-known”\nL64 replace “CTX” with “CTXϕ”\nL66 “Their main features are the mutually beneficial trait conferred to their host”\nFf are not beneficial to the host. They slow the cell cycle and cause envelope stress. Rewrite.\nL68-75 Paragraph reads as if only Pf4, CTX and MDA filamentous phages have been reported to have an effect of bacterial\nhost. However, there are a number of filamentous phages that infect Pseudomonas, Vibrio and plant pathogenic bacteria\nsuch as Xanthomonas etc. that have been reported to have impact on bacterial virulence.\nL76-L77 “Almost all filamentous phages described so far infect diderm bacteria and must therefore cross both the outer and\ninner membranes to infect their target.”\nThis is no longer correct. There are over 10,000 filamentous phage-derived prophages the current sequence databases.\nMany gram-positive bacteria host filamentous phages, as well as some archaea. See Roux S, et al. 2019. Cryptic inoviruses\nrevealed as pervasive in bacteria and archaea across Earth’s biomes. Nat Microbiol 4: 1895–1906. doi:10.1038/s41564-\n019-0510-x (and the follow-up articles).\nRewrite: Filamentous phages that infect diderm bacteria must cross both the outer and inner membranes to infect their target.\nL80 “such as Ff and related phages that use the sexual/conjugative pilus F of E. coli (Hay and Lithgow, 2019).”\nCited paper is a general review on Ff phages. There are much more appropriate primary research references for this. Use\nmore appropriate references.\nL86-87 “pilus porin”\nType IV pilus assembly system does not have a porin. Its outer membrane component pilQ is a channel of the secretin family\nthat is unrelated to porins.\nReplace with the correct term.\nL118-119", "reviewer_comment": "same as above. PilQ is not porin, it is a channel of the secretin family.\nL138 – comment: binding of filamentous phages to the tip of the pilus is not a paradigm. For example, filamentous phages\ninfect unpiliated bacteria (e.g. Yersinia pestis filamentous phage CUS-1–\nhttps://www.sciencedirect.com/science/article/pii/S0042682210005064)\nL160-161 “Transduction was impaired in mutants with low pili quantity and low transformation efficiency, with the exception\nof the ΔpilT mutant, confirming …”\nUnclear sentence. It could be read as if transduction is not impaired in ΔpilT mutants. Rewrite.\nL165 “these proteins are poorly involved in phage transduction.”\nRewrite: “these proteins have a minor role in phage transduction”\nAdditional comment: Transduction is used as an assay for MDA infection, so can be referred to as infection.\nL203-204 “To confirm that antigenic variation in the ZΔMDA pilESA strain is sufficient to explain transduction by MDAΦ, we\nconstructed…”\nL220-222 “The near absence of phage infection in the ZΔMDA mutS+ΔrecA pilESA strain seems to disqualify the pili tip as\nan MDAΦ receptor, although it is considered to be the receptor for filamentous phages f1 and CTX (Jacobson, 1972;\nGutierrez-Rodarte et al., 2019).”\nClumsy sentence (“disqualify”); repetition the statement about the pilus tip is unnecessary. Rewrite.\n229-230 “the first 555 amino acids of PilC1”\nIt cannot be “first”; it can only be “N-terminal” 555 amino acids. However according to the figure, deletion is from residue 3 to\n555, inclusive. Rewrite.\n231-232 “Deletion of more than the first 555 amino acids reduced …”\nChange to\nIncreasing deletion to 3-843 residues reduced …\nThroughout: Subscripts and superscripts were not included in chemical formulae: MgCl2, MgSO4 – check and correct\nthroughout.", "reviewer_3": "Summary: In this study, the authors build upon their past work into the role of MDAɸ in N. meningitidis colonization and\nbiology. The authors confirm and expand their earlier data that Tfp biogenesis is critical for MDA phage transduction and that\nMDAɸ uses its capsid protein to adhere to PilE along the length of the Tfp. The authors find that MDAɸ preferentially binds\nto and infects bacteria expressing positively charged PilE variants and that phage transduction not only enhances host\ncolonization but host colonization also enhances phage transduction. This work adds important knowledge to the critically\nunderstudied filamentous phage literature and represents one of the first (to our knowledge) connections between Tfp\nantigenic variation and phage infection. However, the authors need to explain better the effect of the deletion of each Tfp\ncomponent on pilus assembly and morphology.\nMajor points:\n1. All of the PilE variants (Figure 5) have higher transduction efficiencies than pilESA. How representative is the reduced\ncharge of pilESA in primary isolates?\n2. The authors should list or discuss the T4p morphology of all the mutants; PilE quantity alone does not indicate piliation.\nDoing this will clarify the connection between pilus biogenesis and phage transduction. For instance, some of the pilin\ndeletions (i.e., pilW) appear to have an intermediate transduction phenotype, which could be due to altered piliation.\n3. Others have shown that tfpC deletion in N. meningitidis and N. gonorrhoeae affects Tfp biogenesis, pilin quantity, and\ntransformation efficiency (Muir et al. Nature Communications, 2020; Hu et al. mBio, 2020). In this study, a ΔtfpC mutant\ndisplayed no difference in transformation efficiency and pilin quantity. The authors should discuss this discrepancy.\n4. While they show that PilE is critical for phage transduction, the authors should be careful not to deemphasize the potential\nrole of the other Tfp components in this process. For example, PilQ may also be important for MDA phage transduction, but\nits loss also results in non-piliated bacteria. Thus, the loss of piliation could be confounding the effect of pilQ deletion in\nphage transduction.\nMinor Points:\n1. Line 58: Caudovirales should be italicized.\n2. Line 66: Please clarify the “mutually beneficial trait”.\n3. Line 69: Please provide examples of the environmental signals that trigger phage gene induction.\n4. Line 140: Please choose a different word from “benefits” or clarify that the phage benefits from the positive charge.\n5. Figure 1: List or discuss the piliation states of each mutant.\n6. Figure 1: Reconfigure the graphs so that the transduction, transformation, and pilin quantities are all next to each other for\neach gene deletion. The colors of each bar could be different for each measurement.\n7. Figure 1 A-C needs statistics.\n8. Lines 205-206: this statement needs a citation.\n9. Line 215: Re-write this for “better infects certain variant”, as the phage can still infect other pilE variants, just at lower\nefficiency\n10. Line 228: Why was PilC1 chosen instead of pilC2?\n11. Please speculate why a pilE deletion retains some transduction and transformation efficiency.\n12. Line 265: it looks like some phage did bind to PilESA, just at reduced levels\n13. Supplemental Figure 4e: What is the MDA binding to in the ΔpilE strain? It is unclear how they calculated binding. This\nshould be clarified in lines 280-282\n14. Supplemental Figure 3: all these experiments test Orf6 binding to the pilus fiber proteins. Why was Orf4 not tested, as\nthis protein was shown to be responsible for PilE binding?\n15. Figure 5b: clarify that the reference strain (ZΔMDA) carries the PilESD variant. It is unclear from this graph.\n16. Line 318-319: Why 70-71 in D region was analyzed is unclear. Are these charges not on the surface? Please justify.\n17. Figure 6B: The % positive and negative bacteria with the pilES3 variant should be separate bars. This graph is\ndeceiving because it appears that % pilES3 positive bacteria are at 50%, when it is only 1%.\n18. Figure 6: Clarify that for figures A-C, transduction occurs after colonization, while for figures D-F, transduction occurs\nbefore colonization\n19. Figure 6c should have the same Y-axis scale as Figures 6f-g.\n20. Can you comment on how widespread the MDA phage is in N. meningitidis?\n21. Figure 7C: This image suggests that the MDA phage improves transmission, but the data do not necessarily support this.\n22. Can MDA phage bind the Class II PilE and infect?\n23. Is it possible that only a few pili are necessary? Some of these mutants may appear non-piliated but may have a few pili\ncapable of facilitating transduction.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "In figure 1e, the authors do not define the PilE sequence of ZΔMDA (line 179). This should be in\nsupplemental figure 1.\nSupplemental figure 2 does not have this sequence, or pilEsb, and this also needs to be added here.\nWe agree with Reviewer #1. However, the pilE gene of ZΔMDA cannot be determined, since\nthe strain corresponds to a mix of pilE sequences, with no dominant one having been\ndetermined.\nI do not understand this response. The strain has been manipulated in the laboratory to lack phage. This would\nindicate a selection event. Therefore, even if the parent from the original case was diverse, the mutant should be\nclonal. This information is needed to understand the relationship between the parent and the mutant pilis that they\nare analyzing. What if the PilD region of the parent is the same as PilESA or PillE SB?\nWe have added this information to line 127.\nIn figure 1, they do not define the limit of detection for a transduction experiment? If PilE is truly required for transduction, it\nwould seem that the transductants identified in figure 1a for PilE represent the mutation frequency to spectinomycin\nresistance. Is this true?\nWe thank the reviewer for this comment. Indeed, it is known that filamentous phages interact\nwith pili, allowing them to be pulled in by the pili secretin (Hay et al., EMBO reports 2019\nhttps://doi.org/10.15252/embr.201847427).\nThis reference does not show that the phage interact with the gonococcal pilin secretin\nHere, we observed that transduction in the ΔpilE mutant is 10,000 times lower than in the wild type. The same is true for the\nΔpilQ mutant. Therefore, we can assume that any pili-defective mutant will have a transduction frequency below the\ndetection threshold, and that colonies growing in these mutants will be spectinomycin-resistant colonies due to spontaneous\nmutation (note that spectinomycin resistance is due to a mutation in the 16S rRNA: https://doi.org/10.1128/aac.44.5.1365-\n1366.200). We have therefore added the background threshold to the figure 1.\nThe establishment of background is based on a series of assumptions. If they are transductants, they should have\nthe spec cassette. They should use this to determine the detection level.\nIn figure 2, panel b, when the authors sequenced 10 random colonies, 50% underwent phase variation.\nThis is much higher than that that has been reported for the meningococcus (doi: 10.1128/JB.00465-\n18). How do the authors explain this extraordinarily high rate?\nWe agree with Reviewer #1 and were also surprised. However, this rate must be considered\nin relation to the number of generations that have passed since the PilESA variant was\nintroduced to the original strain (at least two overnight cultures on plates). We observed the\nsame variant rate with a PilESB variant.\nThis suggests that the starting cultures had mixed genotypyes. Furthermore, the strain Z5463 has a mutS mutation\n(Omer et al., PLosOne 2011, doi.org/10.1371/journal.pone.0017145), which may explain the\nhigh rate of punctual mutations observed in three strains of the ten strains. If the authors explanation is correct, why did\nthey see the same pilin mutations and not a variety?.\nIn the discussion, the authors try to link phage expression with virulence. It is hard for this reviewer to\nunderstand the logic. This relationship may exist the first time a bacterium becomes infected with MDA, but subsequent\nreplication in a host would allow for antigenic variation where the strain no longer expresses the PilE needed for phage\ninfection. Unless the strain is constantly being reinfected with MDA, how would phage infection be linked to virulence. Do\nthe authors have any indication of this?\nThe reviewer #1 raises an important question. The presence of an average of two to four prophages per N. meningitidis\nstrain indicates that prophages result from reinfection rather than duplication (Kawai et al., DNA Research 2005", "reviewer_doi": "10.1093/gbe/evaa023) and thus bacteria should be constantly\nreinfected.\nWe now discuss this point in the text (lines 409-411).\nThese references do not really address the question. One of the references suggests that phage acquisition is by\ntransformation.", "reviewer_2": "Authors have taken some steps to improve the quality of writing and data, however manuscript requires more work on both\nfronts.\nThe most critical issue is that number of MDAphi used in binding and transduction experiments is not given. Rather it was\ngenerally stated that purified phage numbers were \"10E7 per microliter at average\". This gives quite a low phage:bacteria\nratio. Given that most binding assay results are given in relative values, the veracity of findings is hard to gauge.", "reviewer_3": "The authors have responded well to the previous three reviews."}, "author_response": "RESPONSES TO REFEREES\nWe would like to thank the reviewers for their positive feedback on our work and for their comments. As\nrequested by the editor, we have addressed the reviewers' concerns and modified the manuscript and\nfigures in the revised version accordingly. These changes are presented point-by-point below.\nFollowing editorial requests, we have reduced the size of the abstract and proposed a shorter title:\n“The meningococcal disease-associated phage promotes meningococcal colonisation by selecting\nhyperadhesive pili variants”.\nLine numbers correspond to the document « Mouville_et_al_manuscript_Marked-Up »\nReviewer #1 (Remarks to the Author):\nWhile the data is presented in a logical fashion, because of how the transduction data is presented\n(normalized to Z5463ΔMDA), it is difficult to interpret the some of the data. Figure 1a-d clearly\ndemonstrates how various pilin-associated proteins impact transduction frequency. However, Imhaus\nand Duménil (https://doi.org/10.15252/embj.201488031) previously showed that a meningococcus has\nabout 6 pili per bacterium, and a pilX mutant has a three-fold reduction in pili. This is similar to the\nchange in quantity that the authors report in figure 1c. On line 168, the authors state “The ∆pilC1/C2\ndouble mutant is barely piliated”. However, their data indicates that there is only about a 3-fold reduction.\nThis is not barely piliated (see also line 227).\nWe understand that our method of quantifying pili was misleading for the three reviewers. It is\nbased on a method of pili extraction and purification that was previously published in PNAS in\n2021 (https://doi.org/10.1073/pnas.2109364118). We agree that the sensitivity of this assay is\nvery low. Consequently, the background signal appeared to be at levels up to 5% of those\nobserved in the wild type. As suggested by reviewer #1, we have proposed a new strategy for\npili quantification based on imaging of assembled pili: Imhaus and Duménil\n(https://doi.org/10.15252/embj.201488031). Unlike the method proposed by Imhaus et al., we\ndid not quantify the number of pili, which requires the observation of isolated bacteria, but\nrather the total surface area of pili detected by immunofluorescence, normalised to the surface\narea of bacterial DNA detected by DAPI. Using this strategy, we demonstrate that the\npilC1/pilC2 double mutant exhibits piliation similar to that observed in pilE or pilQ deficient\nmutants – which was expected. Figure 1c and its legend have been modified accordingly (lines\n739-755). The material and method used to construct the strains used for immunofluorescence\nand the quantification itself have been added (lines 582-585 and 642-646).\nIn figure 1e, the authors do not define the PilE sequence of ZΔMDA (line 179). This should be in\nsupplemental figure 1.\nSupplemental figure 2 does not have this sequence, or pilEsb, and this also needs to be added here.\nWe agree with Reviewer #1. However, the pilE gene of Z∆MDA cannot be determined, since\nthe strain corresponds to a mix of pilE sequences, with no dominant one having been\ndetermined. We have added this information to line 127.\nWe have also added the nucleic and protein sequences of pilE to supp Fig 2a and 2b.\nSB\nIn figure 1, they do not define the limit of detection for a transduction experiment? If PilE is truly required\nfor transduction, it would seem that the transductants identified in figure 1a for PilE represent the\nmutation frequency to spectinomycin resistance. Is this true?\nWe thank the reviewer for this comment. Indeed, it is known that filamentous phages interact\nwith pili, allowing them to be pulled in by the pili secretin (Hay et al., EMBO reports 2019\nhttps://doi.org/10.15252/embr.201847427). Here, we observed that transduction in the ∆pilE\nmutant is 10,000 times lower than in the wild type. The same is true for the ∆pilQ mutant.\nTherefore, we can assume that any pili-defective mutant will have a transduction frequency\nbelow the detection threshold, and that colonies growing in these mutants will be\nspectinomycin-resistant colonies due to spontaneous mutation (note that spectinomycin\nresistance is due to a mutation in the 16S rRNA: https://doi.org/10.1128/aac.44.5.1365-\n1366.200). We have therefore added the background threshold to the figure 1.\nOtherwise, it suggests that PilE is not required for transduction but enhances transduction. If one\nassumes that PilE expression is needed for transduction, and the starting concentration of bacteria in\ntheir experiments is 109 CFU/ml (about an OD of 0.6), then the efficiency?? of transduction in this figure\nis 10-5. This becomes important when trying to interpret the data in Figure 6 (see below).\nIndeed, we did not mention the quantity of phage per µl added for transduction experiment in\nthe original manuscript. The transduction frequency was mentioned in line 483: “The efficacy\nof the phage preparation was verified by transducing 30 μl of the phage preparation into the\nZΔMDA strain and was validated if the transduction frequency was greater than 10-3\", but in\nthe absence of phage quantity this sentence was misleading. We have now added the number\nof phages per µl of phage preparation (assessed by qPCR) (lines 461-470). Considering this\nvalue (2.107), we can assume that the transduction efficiency is 10-3 when 30 μl of phages are\nmixed with 200 µl of bacteria resuspended at an OD of 1 in transduction liquid medium\n600\n(M.O.I. = 3).\nIn figure 2, panel b, when the authors sequenced 10 random colonies, 50% underwent phase variation.\nThis is much higher than that that has been reported for the meningococcus (doi: 10.1128/JB.00465-\n18). How do the authors explain this extraordinarily high rate?\nWe agree with Reviewer #1 and were also surprised. However, this rate must be considered\nin relation to the number of generations that have passed since the PilE variant was\nSA\nintroduced to the original strain (at least two overnight cultures on plates). We observed the\nsame variant rate with a PilE variant. Furthermore, the strain Z5463 has a mutS mutation\nSB\n(Omer et al., PLosOne 2011, doi.org/10.1371/journal.pone.0017145), which may explain the\nhigh rate of punctual mutations observed in three strains of the ten strains.\nIn figure 3, it would be useful to know how the truncated PilC proteins are impacting surface charge. If\nthere is no change, this would strengthen their argument in the data they present in figure 5.\nWe agree with Reviewer #1. The T4aP model propose that one PilC/PilY1 protein is exposed\nat the tip of each pilus (Treuner-Lange et al. Nature Communications 2020"}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed all of my concerns"}, "author_response": ""}]

Decision letter