Hyperinvasive and quinolone-resistant meningococci cc4821 achieved immune evasion through capsule switching

Abstract

Background

Neisseria meningitidis stands as one of the predominant etiological agents of bacterial meningitis worldwide. Sequence type (ST) 4821 clonal complex (cc4821), notably linked to the hyperinvasive and quinolone-resistant clone China^{CC4821−R1−C/B}, has emerged and caused the epidemiological shift from serogroup A (MenA, mostly cc5 and cc1) to MenC in China since 2003 and the subsequent introduction of MenA + C vaccination. However, the evolution path of capsule in MenB China^{CC4821−R1−C/B} under the vaccine-induced selective pressure remains unclear. For phylogenetic analysis, nucleotide sequences of the capsular polysaccharide synthesis (cps) gene cluster were extracted from MenC and MenB China^{CC4821−R1−C/B} genomes in the Neisseria PubMLST Database. Recombination analyses were conducted using software MEGA and RDP. The capsule switching from MenC to MenB in China^{CC4821−R1−C/B} was conducted via natural transformation.

Results

Phylogenetic analysis of the cps loci revealed that recombination events probably occurred in region A. Among MenB China^{CC4821−R1−C/B} isolates, isolate Nm119 (B: P1.7-2,14: F3-3: ST-9455 [cc4821]) was selected as the representative strain. Through sequence analysis, a potential donor strain (2061370; B: P1.19-1,15 − 11: F3-7: ST-269 [cc269]) was identified from 80 candidate isolates, with a 5,527-bp recombination sequence spanning the region between ctrG and cssA in region A. To further validate capsule switching from MenC to MenB, we performed biological experiments using a MenC China^{CC4821−R1−C/B} isolate (Nm044; C: P1.7-2,14: F3-3: ST-4821 [cc4821]) as a recipient strain. The recombination sequence identified in the transformant (Nm044Nm119_T20) was only 414 bp larger than the sequence predicted by the in silico analysis. Functional analysis of the transformant revealed that its capsular polysaccharide expression and serum survival capacity were comparable to those of the donor strain. The capsule switching pattern, exemplified by Nm119, could serve as a model for MenB China^{CC4821−R1−C/B}. Through MenC→MenB capsule switching, China^{CC4821−R1−C/B} isolates have gained the capacity to evade immunity induced by MenA + C vaccines.

Conclusions

The potential capsule switch from MenC→MenB in China^{CC4821−R1−C/B} isolates has been confirmed through both sequence-based analyses and molecular biological experiments. The MenB capsule likely originated from a cc269 strain. Given the potential of capsule switching events to compromise vaccine efficacy, enhanced surveillance of capsule variation in cc4821 isolates is warranted under current immunization strategies in China.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-026-05194-2.

Background

Neisseria meningitidis (Nm), the causative agent of invasive meningococcal disease (IMD), has been listed as one of the four pathogens in Defeating Meningitis by 2030: A Global Road Map by the World Health Organization (WHO), as meningitis remained a much-feared disease worldwide with a high fatality rate and a propensity to cause epidemics and presented a major challenge for health systems, economies, and society [1]. Between 1990 and 2010, over 1.2 million IMD cases were estimated to occur annually, leading to approximately 135,000 deaths each year and survivors often with severe neurological sequelae [2, 3]. In 2019, IMD caused an estimated 141,000 deaths globally [4, 5]. Over the past decade, 184 IMD cases have been reported in 97 publications between January 2011 and March 2023, which highlighted adolescents as the most affected age group with 47% of cases presenting as meningitis, 37% as sepsis, and 14.6% leading to death [6]. IMD’s mortality rates remain significant, such as 12% in Romania from 2001 to 2024, 14.3% in Argentina in 2022, and 10.3% in the USA in 2023 [7–9]. Nm is classified into at least 12 serogroups based on the composition of the different capsular polysaccharides, with six serogroups—A, B, C, W, X, and Y—responsible for most IMD cases globally [10]. In recent years, serogroup B Nm (MenB) has become predominant across various age groups in Europe, North America, Australia, and other regions worldwide [11]. Molecular epidemiological studies have provided critical insights into specific clonal complexes associated with IMD, which are found rare in asymptomatic carriers [12]. Among these, the ST-269 clonal complex (cc269) primarlily linked to MenB, has demonstrated significant expansion since the early 21st century. For instance, cc269 was responsible for 69.4% of IMD cases in Canada between 2009 and 2013 [13], and more than 30% of IMD cases in England and Wales between 2000 and 2008 [14].

In China, prior to the epidemic of cc4821 MenC isolates, cc5 MenA isolates were predominant in 1965–1973, followed by a shift to cc1 MenA isolates during 1974–1975. The introduction of the MenA meningococcal polysaccharide vaccine (MPV) in 1980 marked a significant tuning point [15], leading to a decline in IMD cases. Two decades later, following the IMD outbreaks in Anhui Province in 2003 [16], cc4821 (genomically equivalent to lineage L44 [17]) MenC strain was initially identified, marking a transition from the previously dominant MenA (mostly cc5 and cc1) to MenC (cc4821) [18]. After the introduction of MenA + C vaccines in 2005, the national immunization schedule in China was updated to include a four-dose regimen for IMD prevention, including two doses of MenA univalent MPV for children aged 6–18 months and another two doses of MenA + C bivalent MPV (1 dose for children at 3 years and 1 dose for children at 6 years) [19]. Subsequently, MenB isolates began to rise in prevalence, gradually surpassing MenC as the dominant circulating strains [16, 20]. Within the global cc4821 population, four distinct sublineages have been identified, among which L44.1 was identical to China^{CC4821−R1−C/B} clone— a novel hyperinvasive and quinolone-resistant clone [15, 18]. This clone exhibits not only resistance to quinolones but also emergent resistance to other antibiotics, such as penicillin and cefotaxime [18, 21]. Furthermore, it possesses high-virulence characteristics, enhancing its ability to adhere to and invade host cells [22]. Several studies have reported the presence of both MenC and MenB isolates within China^{CC4821−R1−C/B} clone [16, 18, 23, 24], but the MenB isolates emerged later than the MenC isolates, suggesting a potential capsule switching event during the dissemination of this clone.

Capsule switching in Nm occurs through horizontal gene transfer of capsular polysaccharide synthesis (cps) genes between meningococci of different serogroups. Capsular polysaccharides (CPSs) are critical virulence factors and serve as the basis for the serogroup classification of Nm. Moreover, CPSs are essential antigenic components of several meningococcal vaccines [25]. The cps gene cluster responsible for CPS biosynthesis and transport is structurally organized into six functional regions designated D-A-C-E-D’-B [10]. Region A contains genes that exhibit significant variability across different serogroups. Among these, cssA-C are responsible for sialic capsule synthesis. The polysialyltransferase genes are termed csc and csb in MenC and MenB respectively, and ctrG facilitates the surface translocation of sialic acid capsules, while the O-acetyltransferase specific to the MenC strain is encoded by cssE. Regions B (ctrE and ctrF) and C (ctrA-D) encompass genes essential for the transportation and translocation of capsule polymers to the bacterial surface. Regions D and D’ contain an intact and truncated remnant copy of a gene encoding UDP-galactose epimerase, designated galE1 and galE2, respectively. Additionally, region E contains a gene designated tex, a homologue of a transcript factor, although its biological function remains uncharacterized [10]. Although capsule switching events among various serogroups of Nm have been identified through in silico sequence analysis [26–29], their occurrence and functional implication remain unvalidated by experimental biological assays.

Additionally, a genomic analysis of cc4821 isolates conducted by the Chinese Center for Disease Control and Prevention (China CDC) revealed capsule switching between MenC and MenB [23]. The hypothesis that MenB isolates emerged from MenC isolates within the China^{CC4821−R1−C/B} via capsule switching offers a plausible explanation for the observed temporal and serogroup distribution patterns. However, further investigations are necessary to elucidate the specific donor strains and the corresponding recombination breakpoints. In this study, we leveraged comprehensive genomic data from the PubMLST database to analyze China^{CC4821−R1−C/B} genomes, with the aim of identifying potential donor strains for MenB and characterizing the associated recombination breakpoints. These in silico predictions were subsequently validated through natural transformation experiments, to clarify the critical details of capsule switching from MenC to MenB within Nm under the selective pressure imposed by MenA + C vaccines. Given the absence of MenB meningococcal vaccines in China, the increasing prevalence of cc4821 MenB isolates presents a significant challenge to current vaccination strategies. By systematically addressing these research objectives, this study will advance our understanding of meningococcal immune evasion and provide theoretical support for future surveillance and immunoprevention efforts against Nm.

Materials and methods

Safety considerations

Nm is classified as a Biosafety level 2 (BSL-2) pathogen. Consequently, all procedures were conducted under BSL-2 conditions, in accordance with the Laboratory Biosafety Manual, 4th edition [30]. It is mandatory for all personnel to be immunized with MenACWY meningococcal vaccines, given that no MenB vaccines are licensed in mainland China. In case infection following any exposure to Nm occurs, prompt systemic antibiotic treatment is requested to cure the disease.

Genomic sequences of quinolone-resistant clone China^{CC4821−R1−C/B}

China^{CC4821−R1−C/B} is identical to cc4821 L44.1 sublineage. A total of 105 China^{CC4821−R1−C/B} isolates were identified as belonging to L44.1 in the Neisseria PubMLST Database based on the L44 core genome multi-locus sequence typing (cgMLST) scheme (1,699 loci) specific for cc4821 isolates. This collection comprised 23 MenB isolates (21.9%, 23/105) and 82 MenC isolates (78.1%, 82/105) (Supplementary Table 1). The corresponding gene sequences and allele designations were confirmed on the PubMLST website (www.pubmlst.org/neisseria, last accessed in April 2025). Genome sequences of the isolates belonging to this clone were also downloaded for gene alignment, recombination breakpoint analysis, and phylogenetic analysis. Among the 105 isolates, 053442 was isolated from the Anhui MenC outbreaks in China in 2004 and served as a reference strain for the study of the cc4821 lineage, representing the China^{CC4821−R1−C/B} clone [31]. Four additional reference strains utilized in previous capsule switching analyses [27, 32], including FAM18 (C: P1.5,2: F1-30: ST-11 [cc11]), 8013 (C: P1.21,26- 2: F1-5: ST-177 [cc18]), H44/76 (B: P1.7,16: F3-3: ST-32 [cc32]), and MC58 (B: P1.7,16-2: F1-5: ST-74 [cc32]), were also incorporated in the study (Supplementary Table 2). FAM18, the first genome sequence for the disease-causing serogroup C meningococci, was reported in the United States in 1983 [33]; and 8013, its complete genome sequence is the basis of a biological resource (NeMeSys) for Nm systematic functional analysis, and was reported in France in 1989 [34]; they were both served as reference strains for MenC isolates. H44/76, a widely used serogroup B laboratory strain, was reported in 1976 [35]; and MC58, with its complete sequence being the basis of designing vaccines against MenB isolates [36], was reported in 1983; both were reference strains for MenB isolates.

Phylogenetic analysis

Phylogenetic analysis of the cps gene clusters was conducted on the Neisseria PubMLST website [37]. Genome sequences and corresponding data of China^{CC4821−R1−C/B} isolates were retrieved by querying their PubMLST IDs (Supplementary Table 1). The genome comparator tool was employed to perform the phylogenetic analysis within the database. The analyzed regions and their respective gene compositions were as follows: Region D (rfbC, rfbA, rfbB, and galE1), region A (ctrG, cssE, csc, csb, cssC, cssB, cssA, and igr_up_NEIS0055), region C (ctrA, ctrB, ctrC, and ctrD), region E (tex, NEIS2910, and NEIS2854), region D’ (galE2, rfbB2, rfBA2, and rfbC2), region B (ctrE and ctrF) [10]. Regions D and D’ were included in phylogenetic analysis only when all genomic sequences were complete rather than draft, as it is difficult to distinguish the two regions using draft genome sequences. The resulting phylogenetic trees were visualized using Split Tree (version 4.0) [38].

Identification of donor strain and recombination breakpoints

Potential donor strains and recombination events were analyzed according to the following criteria. A strain was regarded as a candidate donor if it met all of the following conditions [27]: (1) The matching length was at least 2 kb; (2) The difference between the donor and the recombination sequence was less than 1 single nucleotide polymorphism (SNP) per 1000 base pairs; (3) The established recombination gene alleles were present in the majority of the presumed donor strains within the Neisseria PubMLST database.

Recombination breakpoints were determined through sequence alignment, recombination events prediction, and phylogenetic analysis in the software MEGA (version 11) [39]. The analysis proceeded sequentially from the 5’ to 3’ direction, and a MEG format file was generated for downstream analyses, conducted using the Recombination Detection Program (RDP; version 4.56) to examine recombination events [40]. Potential recombination breakpoints were identified using any of the seven algorithms, including RDP, Chimaera, Bootscan, 3Seq, GENECONV, MaxChi, and SiScan. Flanking sequences adjacent to the recombination sequences were aligned, and a UPGMA tree was constructed to analyze the evolutionary relationships among the sequences of potential donor strains, recombination strains, and the reference strains.

Serum bactericidal antibody assay

To screen sera containing bactericidal antibodies specific to MenC isolates rather than MenB isolates, we measured the bactericidal antibody titers in six serum samples from healthy individuals, which were previously tested at the Shanghai Municipal Center for Disease Control and Prevention (CDC) and showed bactericidal effects against the MenC vaccine strain C11 (Supplementary Table 3). The experiment procedure followed an established protocol [41], utilizing the MenC China^{CC4821−R1−C/B} isolate Nm044 (C: P1.7-2,14: F3-3: ST-4821 [cc4821]) and the MenB China^{CC4821−R1−C/B} isolate Nm119 (B: P1.7-2,14: F3-3: ST-9455 [cc4821]) as target strains (Supplementary Table 4). Approximately 20 colonies of freshly inoculated strains were plated for confluent growth onto Columbia horse blood agar plates (CHBA) (Thermofisher, USA), and incubated at 37 ℃ with 5% CO₂ for four hours. The bacteria were then suspended in Hank’s Balanced Salts (without Ca²⁺or Mg²⁺) (Gibco, USA) supplemented with 0.1% glucose, adjusting the suspension to 6–10 × 10⁴ CFU/ml. The bacterial suspension was mixed with active rabbit complement (Dynal, USA) at a ratio of 1:1, and 20 µl of the mixture was added to each well of a round-bottom 96-well microplate containing 20 µl of serially twofold diluted serum. This mixture was incubated at 37 ℃ without CO₂ for one hour. Subsequently, 10 µl of the reaction mixture from each well was plated onto a CHBA plate using a multichannel pipette and incubated overnight at 37 ℃ with 5% CO₂. The number of colonies on each CHBA plate was counted using a colony counter, and serum bactericidal antibody (SBA) titers were defined as the highest dilution of serum resulting in ≥ 50% bacterial killing.

Transformation experiments

To simulate horizontal gene transfer via homologous recombination, the MenC China^{CC4821−R1−C/B} strain, Nm044, was selected as the recipient for natural transformation, while Nm119 served as the donor, providing chromosomal DNA containing the MenB cps sequence. Following the optimization of a previously described protocol for natural transformation [42], the chromosome DNA fragment from Nm119 was used to transfer the MenB cps nucleotides into Nm044. The QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) was used to purify the chromosomal DNA of the donor strain Nm119. After overnight incubation on CHBA, Nm044 was mixed with the donor chromosome DNA (500 ng) in a nutrient broth containing BBL IsoVitaleX (BD, Sparks, MD, USA) and peptone (Oxoid, Thermofisher, USA). This mixture was incubated at 37 °C with 5% CO₂ for 5 to 6 h. Subsequently, serum MH2020013, which showed a bactericidal antibody specifically against Nm044 (MenC) rather than Nm119 (MenB), was added to the mixture. Following a one-hour incubation, the mixture was plated onto CHBA and cultured at 37 °C with 5% CO₂ for 24 to 48 h. A total of 137 colonies were formed, from which only one MenB transformant (Nm044Nm119_T20) was successfully screened and subjected to genomic sequencing using the PacBio platform for further confirmation and downstream studies.

Serum agglutination assay

To evaluate the expression of CPS in the transformant, we employed Nm antiserum specific to MenB and MenC (BD, USA) and conducted a slide agglutination test. The bacterial suspension of Nm was mixed with the antiserum on a glass slide following the manufacturer’s instructions, and the agglutination reaction was observed against a black background.

Whole-genome sequencing

DNA from Nm119 and the transformant was purified using the QIAamp DNA Mini Kit. The purified genomic DNA samples were sheared into 15–20 kb segments utilizing g-Tube technology (Covaris, Woburn, Massachusetts, USA), and were subsequently used to generate SMRT Bell libraries with a SMRTbell prep kit 3.0 (Pacbio, USA). Following library construction, the single-stranded DNA templates were annealed and polymerase-bound at the base of the fixed zero-mode waveguides for subsequent sequencing. Paired-end indexed reads ranging from 5 kb -15 kb were generated on a PacBio Sequel platform. The genome sequence data were assembled using ABySS (version 2.0.2), CANU, and GapCloser (version 1.12) [43–45].

Capsule quantification

The phenol-sulfuric acid method was utilized to determine the concentration of CPS in Nm, according to the manufacturer’s instructions of the Polysaccharide Content Measurement Kit (Saint-Bio, China). Briefly, Nm was incubated in 4 ml GC media supplemented with 1% Isovitalex Enrichment (BD, USA) till OD₆₀₀ reached 1.0. The bacterial suspension was washed with 80% ethanol, resuspended in 2 ml of distilled water, and heated at 100 °C for 5 min to obtain the polysaccharide detection solution. For the assay, 100 µl of the detection solution was slowly mixed with 250 µl concentrated sulfuric acid, with distilled water serving as a blank and glucose as a standard. The mixture was then heated at 95 °C for 20 min, and absorbance was measured at 488 nm.

Survival in human serum assay

Both the transformant and clinical strains (1 × 10⁵ CFU/ml) were incubated in 1 ml PBS with 5% normal human serum at 37 °C under shaking condition [46]. The survival percentage of Nm was determined by performing serial dilution and plating on CHBA after 2-hour incubation.

Detection of CPS after transformation experiments

The CPS of MenB and MenC was characterized using reverse transcription PCR, periodate-resorcinol microassay, and nuclear magnetic resonance (NMR) spectroscopy. Detailed procedures for these methodologies are provided in Supplementary information.

Statistics

t-test was used for analyzing statistical differences between two independent samples. All experiments were performed at least three independent replicates, and data are presented as mean ± standard deviation. P < 0.05 was considered statistically significant.

Results

Phylogenetic analysis of the quinolone-resistant clone China^{CC4821−R1−C/B}

Based on the L44 cgMLST scheme, all 448 cc4821 isolates retrieved from the Neisseria PubMLST database were classified into four sublineages, with the L44.1 sublineage corresponding to the China^{CC4821−R1−C/B} clone (Supplementary Fig. 1). An allele-based phylogenetic analysis was conducted on the cps gene cluster sequences from 105 China^{CC4821−R1−C/B} isolates, alongside general reference strains for MenB (MC58 and H44/76) and MenC (FAM18 and 8013). Most clinical MenB isolates within the China^{CC4821−R1−C/B} clone exhibited unique cps characteristics, and did not share uniform traits in regions B, C, and E, with the exception of region A (Fig. 1). This observation suggests the occurrence of potential recombination events specifically in region A.

Fig. 1.

Phylogenetic analysis of capsular polysaccharide synthesis (cps) regions (A, C, E, and B) in China^{CC4821−R1−C/B}. The phylogenetic analysis was conducted according to the allelic number of cps gene in regions A, C, E, and B, which included 105 China^{CC4821−R1−C/B} isolates and the general MenB (MC58 and H44/76) and MenC (FAM18 and 8013) reference strains. Black circles, MenC isolates; red squares, MenB isolates; blue arrow, targeting the isolate. The function of the separate cps region was depicted below. Region A, capsule synthesis; region C, capsule transport; region B, capsule translocation; region E, unknown

Potential origin of the cps of MenB isolates within the China^{CC4821−R1−C/B}

To investigate the origin of MenB isolates within the China^{CC4821−R1−C/B} clone, we retrieved and aligned the genomic sequences of all available MenB cc4821 isolates. Comparative analysis revealed that all MenB isolates shared a homologous fragment located between the csb and ctrA gene with isolate Nm119 (Supplementary Fig. 2). Based on these findings and the availability of its complete genome sequence, Nm119 was selected for subsequent in-depth analysis.

To identify potential donor strains for Nm119 within the Neisseria PubMLST Database, specific screening criteria were established based on the alleles of Nm119 within region A, specifically targeting csb (NEIS2161_1), cssC (NEIS0052_2), cssB (NEIS0053_1), and cssA (NEIS0054_3). This approach yielded 80 candidate isolates (Supplementary Table 5), of which 24 isolates (30%) belonged to cc269 and 16 isolates (20%) were assigned to cc32, both exhibiting high sequence similarity. Based on the third criterion for donor strain screening—the recombination gene alleles should be present in the majority of the presumed donor strains within the Neisseria PubMLST database—cc269 isolates were considered more likely to be potential donor of the cps of Nm119. Considering that the first MenC China^{CC4821−R1−C/B} isolate 053442 was identified in 2004, whereas Nm119 was collected in 2011 [10], an isolate similar to 2061370 (B: P1.19-1,15-11: F3-7: ST-269 [cc269]; PubMLST id 100462) [47], which was isolated in the Netherlands between 2004 and 2011 (in 2006), was hypothesized to be a potential donor of the cps sequence for Nm119. Consequently, 2061370 was selected for further analysis. Subsequently, a sequence-based phylogenetic analysis was performed to identify the region where recombination events were likely to have occurred. As shown in Supplementary Fig. 3, Nm119, H44/76, and 2061370 formed a distinct phylogenetic cluster within region A, separated from other reference strains. Additionally, Nm119 and 053442 clustered together in regions D, CED’, and B, suggesting that a recombination event may have occurred specifically within region A.

Analysis of capsular polysaccharide synthesis (cps) locus on Nm119

The full-length cps coding region in strain 053442 spans 25.6 kb and is organized sequentially into regions D-A-C-E-D’-B (Fig. 2). Comparative sequence analysis revealed that within region A, Nm119 and its potential donor strain 2061370 exhibited a high degree of sequence similarity (99.9%). In contrast, regions D and C-E-D’ displayed distinct sequences between the two isolates. Based on this pattern, we hypothesized that a genetic recombination event has occurred specifically within region A, introducing a recombination sequence containing the genes csb, cssC, cssB, and cssA into Nm119.

Fig. 2.

Analysis of gene loci in the capsular polysaccharide synthesis (cps) encoding region among five isolates. The isolates include 2061370, Nm119, FAM18, 8013, and 053442. The position of the gene locus was according to the sequence of 053442. The same alleles are represented in the same color. Blue-grey represents region D, brown represents region A (csb is in red), blue represents region C, yellow represents region E, purple represents region D’, and green represents region B. The depths of the color depict different alleles in a certain region. The arrow depicts gene orientation

Analysis of recombination breakpoints

Given the indication of a potential recombination event within region A, we aimed to precisely localize the recombination breakpoints in Nm119. To achieve this, we performed a multiple sequence alignment of contig sequences containing the cps coding region from five isolates: 2061370, Nm119, FAM18, 8013, and 053442. This analysis identified a 5,527-bp segment in Nm119 that likely originated from 2061370. The upstream recombination breakpoint was mapped to position 379 bp within the ctrG gene, while the downstream recombination breakpoint was located 13 bp downstream of the cssA gene (Fig. 3a-c). In addition, the recombination event was validated using the RDP software (Fig. 3d). Subsequent phylogenetic analysis of the recombination sequence confirmed that the 5,527 bp sequence in Nm119 is highly similar to its counterpart in 2061370, whereas the flanking sequences in Nm119 showed significant divergence from those in 2061370 (Fig. 3e).

Fig. 3.

Recombination events and breakpoints in the cps of Nm119. a The upstream recombination breakpoint in Nm119 is located at 379 bp within the ctrG gene, with the reference strain being Nm119. b The recombination sequence 484 bp away from the upstream recombination breakpoint. c The downstream recombination breakpoint in Nm119 is located 13 bp downstream of the cssA gene. d The RDP visualization provides a clear representation of the recombination sequences within the recombination event, with region A of cps locus marked the position of recombination breakpoints. Different coloured bars represent different isolates, with the black bar representing the unknown isolate. The length of the bar does not indicate the length of the sequence. e The phylogenetic analysis of the recombination sequences reveals a high degree of similarity between Nm119 and 2061370, while the sequences flanking the recombination sequences are different from 2061370, with the cinerous arrows corresponding to the locations of recombination breakpoints

Experimental verification of capsule switching

Based on sequence analysis, it was hypothesized that Nm119 evolved through capsule switching from a MenC isolate within the China^{CC4821−R1−C/B}. To experimentally verify this mechanism, we selected the MenC China^{CC4821−R1−C/B} isolate, Nm044 (C: P1.7-2,14: F3-3: ST-4821 [cc4821]) as the recipient for natural transformation, with Nm119 serving as the donor to provide its MenB cps sequence. During the capsule switching experiment, only a single transformant, designated Nm044Nm119_T20, was screened with a serum (MH2020013) exhibiting bactericidal activity specific to MenC rather than MenB isolates. The transformant (Nm044Nm119_T20) was subsequently subjected to PacBio sequencing. Analysis of the resulting genome sequence predicted its molecular characterization to be B: P1.7-2,14: F3-3: ST-4821 (cc4821). Notably, the csb gene sequence (NEIS2161_1) was identical to that of the donor strain Nm119, while all other molecular characteristics matched those of the recipient strain Nm044. Alignment of the cps sequence and its flanking regions among Nm044Nm119_T20 (transformant), Nm044 (recipient), and Nm119 (donor) confirmed the occurrence of recombination events. The specific recombination breakpoints and corresponding sequences were identified. The upstream recombination breakpoint was located 36 bp upstream of the ctrG gene, at an intergenic region, while the downstream recombination breakpoint was positioned 13 bp downstream of the cssA gene (Fig. 4a-c). Additionally, comparison between the theoretically predicted recombination sequence and the experimentally obtained fragment revealed that they share identical segments encompassing the csb, cssC, cssB, and cssA genes. The recombination sequence obtained from the capsule switching experiment was found to be only 414 bp longer than the theoretically predicted recombination sequence, with the sequence difference localized within the ctrG gene (Fig. 4d). These results indicated that the actual capsule switching process closely matched the in silico prediction. Furthermore, sequence analysis of the transformant from the capsule switching experiments confirmed that no additional recombination events occurred in other genomic regions, including the lgtA-E gene cluster responsible for synthesizing the α-chain extension of lipooligosaccharide (LOS) [48, 49].

Fig. 4.

Recombination events and breakpoints in capsule switching experiments. a After capsule switching, the upstream recombination breakpoint is located 36 bp upstream of the ctrG gene, with the reference strain being Nm119. b The recombination sequence 973 bp away from the upstream recombination breakpoint. c After capsule switching, the downstream recombination breakpoint is located 13 bp downstream of the cssA gene. d Alignment of theoretical and actual recombination sequences, with the blue rectangle representing the difference. e After capsule switching, the transformant (Nm044Nm119_T20) showed a positive reaction with serogroup B anti-serum in the slide agglutination test. f The concentrations of CPS in Nm119 and Nm044Nm119_T20 were both decreased compared to Nm044. g Nm119 and Nm044Nm119_T20 showed decreased serum survival rates compared to Nm044. Nm044Nm119_T20 (transformant), Nm044 (recipient), Nm119 (donor). Nm, Neisseria meningitidis. **, P < 0.01; ***, P < 0.001; were calculated by t-test with Nm044 as the control

Finally, to confirm the expression of the recombination CPS, we performed a serum agglutination test on the transformant Nm044Nm119_T20 and identified its serogroup as MenB (Fig. 4e). Similar to the donor strain Nm119, the transformant exhibited significantly lower CPS concentration and serum survival rate compared to the recipient Nm044 (Fig. 4f & g). Additionally, reverse transcription PCR was employed to assess the expression of serogroup-specific genes in the transformant (Supplementary Table 6). Both Nm119 and Nm044Nm119_T20 demonstrated expression of csb, and no expression of csc (Supplementary Fig. 4a&b). In the periodate-resorcinol microassay, the concentrations of sialic acid (N-acetylneuraminic acid) in Nm119 and Nm044Nm119_T20 were lower than those in Nm044 (Supplementary Fig. 4c). Furthermore, NMR spectroscopy confirmed the presence of O-acetylation peaks at 2.03–2.10 ppm in Nm044, whereas these signals were absent in both Nm119 and the transformant (Supplementary Fig. 5). This finding was consistent with the previous report that O-acetylation was present in MenC CPS but absent in MenB CPS [50].

Conservation of the recombination sequence in Nm119 among MenB isolates within the China^{CC4821−R1−C/B} clone

To assess the recombination patterns of MenB isolates within the China^{CC4821−R1−C/B} clone from 2005 to 2021, we performed a comparative sequence analysis of the 5,527-bp recombination region. The majority of MenB isolates (81.8%, 18/22) exhibited highly similar profiles (≤ 5 SNPs) to Nm119, with the exception of Nm468, Nm511, 431210, and JX2005-119, each of which had an incomplete sequence. Phylogenetic analysis of the recombination sequences revealed a single cluster closely related to Nm119 (Fig. 5). These findings indicted that the capsule switching pattern observed in Nm119 could explain the emergency of the mainstream MenB isolates within the China^{CC4821−R1−C/B} clone.

Fig. 5.

Summary of recombination sequences in MenB isolates within the China^{CC4821−R1−C/B} clone. a Graphic depiction of sequence identities in 22 MenB China^{CC4821−R1−C/B} isolates relative to the 5,527-bp recombination sequence of Nm119. b Maximum likelihood tree depicting phylogenetic relationship between the 5,527-bp recombination sequences from 23 MenB isolates. Orange box, genes with ≤ 5 nucleotide differences; purple box, genes ≥ 6 nucleotide differences; MenB, serogroup B meningococcus

Discussion

This study employed sequence-based analysis and biological experiments to demonstrate that the quinolone-resistant clone China^{CC4821−R1−C/B} has the capacity to conduct a MenC→MenB capsule switching to survive and evade immune pressure induced by scheduled MenA + C meningococcal vaccination. Furthermore, we identified a potential donor strain, 2061370, which belonged to cc269 and was isolated in 2006 in the Netherlands. The predicted recombination sequence was validated through natural transformation experiments, confirming that the recombination cps gene cluster expresses MenB CPS. The resulting transformant exhibited CPS production and serum survival rates comparable to those of the donor strain. Notably, this study provides the first experimental validation of meningococcal capsule switching, bridging a critical theoretical gap in our understanding of this evolutionary mechanism.

Nm119 is a MenB isolate belonging to the China^{CC4821−R1−C/B} clone, which was predominantly composed of MenC isolates but has shown an increasing prevalence of MenB isolates in recent years [18]. The China^{CC4821−R1−C/B} clone, which was first identified in 2004, initially featured serogroup C and has been a key driver in shifting the predominant meningococcal serogroup from MenA to MenC in China [16]. Notably, the introduction of MenA + C meningococcal vaccines into China’s national immunization program in 2005 coincided with the initial detection of MenB cc4821 isolates within the China^{CC4821−R1−C/B} clone [20]. This temporal correlation suggests that under the selective pressure exerted by MenA + C vaccination, MenC→MenB capsule switching may have occurred within the cc4821 lineages. Capsule switching has been documented in several studies through sequence analysis, including: cc5 from MenA to MenC in China [32]; cc573 from MenY to MenB in Canada [51]; cc11 from MenC to MenW and from MenC to MenB [27, 29]; ST-14273 from MenB to MenX in 2020 [28]; cc2057 from MenB to MenY [26]; and cc7 from MenA to MenX in 2022 [52]. Notably, over half of those capsule switching events were temporally associated with the implementation of meningococcal vaccines, suggesting that capsule switching might represent an adaptive strategy for immune evasion in Nm. As a hyperinvasive and quinolone-resistant clone, China^{CC4821−R1−C/B} has recently acquired resistance to penicillin and cefotaxime [21]. The observed capsule switching from MenC to MenB within this clone likely further enhances its ability to evade the bactericidal antibodies generated by the MenA + C vaccines [53]. Given the current unavailability of MenB meningococcal vaccines in Mainland China, both multidrug resistance and capsule-switching ability pose significant challenges to the treatment and prevention of cc4821 meningococcal infections.

The recombination sequence identified in this study was located between the ctrG and cssA genes, which are situated within region A of the cps locus. Recombination events in region A have been frequently associated with capsule switching, as evidence by documented cases such as cc5 A→C in China [32], cc11 C→W [27], and ST-7 A→X [54, 55]. These findings collectively highlight region A as a genomic hotspot for capsule switching phenomenon. Furthermore, the upstream recombination breakpoint was located within the ctrG gene, which encodes a protein essential for coupling the capsule assembly complex to the membrane transport machinery, thereby facilitating the surface expression of CPS [56]. The downstream recombination breakpoint was found in an intergenic region adjacent to the cssA gene. This gene encodes UDP-N-acetylglucosamine 2-epimerase, an enzyme that catalyzes the conversion of UDP-N-acetyl-α-d-glucosamine into N-acetyl-d-mannosamine and UDP—the initial step in sialic acid biosynthesis. The enzyme UDP-N-acetylglucosamine 2-epimerase is crucial both for the biosynthesis of the (α2→9)-linked polysialic acid capsule in MenC isolates and for lipooligosaccharide sialylation [57]. In our study, the transformant exhibited reduced CPS production compared to the recipient strain, a phenotype may be associated with the recombination event occurring between ctrG and cssA, where several genes critical for CPS biosynthesis reside [10]. The recombination led to CPS expression levels in the transformant that closely resembled that of the donor strain, with both strains producing lower CPS amounts than the recipient strain.

This study identified isolate 2061370 and closely related isolates, which belonged to cc269, as potential donor strains of the cps nucleotides in the MenB China^{CC4821−R1−C/B} isolate Nm119. Till now, only three cc269 isolates from China have been deposited in the PubMLST database, including B153 (1985; ST-5657; no genome data), NmNx17 (1985; B: P1.12-1,13: F4-3: ST-5657 [cc269]), and 651679 (2016; ST-12798; no genome data). Comparative analysis of the cps sequences between Nm119 and NmNx17 revealed high similarity within the fragment from ctrG to cssA (Supplementary Table 7), suggesting that 2061370-like strains may have had opportunity for genetic exchange with cc4821 isolates, thereby providing a recombinant cps sequence. While multiple capsule switching events have been documented in previous studies [26–29, 32, 51], none have been biologically validated. In this study, we employed a previously established natural transformation platform, which was originally developed to investigate horizontal gene transfer of quinolone and penicillin resistance between commensal Neisseria and Nm isolates [21, 58, 59], to experimentally confirm capsule switching. Using serum with bactericidal activity specific to MenC but not MenB isolates, we successfully selected the transformant and obtained direct evidence for capsule switching from MenC to MenB within the China^{CC4821−R1−C/B} isolates. Furthermore, the size and the genomic location of the experimentally identified recombination sequence closely matched theorical predictions, with only a 414-bp difference, thereby supporting the reliability of sequence-based inference for capsule switching events. The serogroup of the transformant was confirmed to be MenB, indicating that the switched CPS was expressed normally. These results directly demonstrate the capacity of China^{CC4821−R1−C/B} isolates to switch their serogroups. The present study provides comprehensive experimental and genomic data on capsule switching, thereby advancing the theoretical framework underlying Nm capsule switching mechanisms.

This study has a few limitations. Due to the unavailability of a cc269 stain in our research, we selected a cc4821 MenB isolate (Nm119), rather than a cc269 isolate similar to 2061370, to provide the recombination sequence in the capsule switching experiment. However, it is possible that the recombination sequence in Nm119 originated from a cc269 strain and appeared to be highly conserved during the capsule switching process, which is the focus of our experiment investigation. Furthermore, we performed a comparative sequence analysis of the 5,527-bp recombination sequence across all available cc269 MenB isolates. The results revealed that half of the MenB isolates (50.1%, 745/1,486) exhibited highly similar genetic profiles (≤ 5 SNPs) to Nm119 (Supplementary Fig. 6). Although the primary aim of the capsule switching experiment was to demonstrate the ability of China^{CC4821−R1−C/B} clone to alter its serogroup by acquiring the csb gene from MenB isolates, a cc269 donor strain may be a better donor than the cc4821 MenB isolate Nm119 to understand the mechanism of MenC→MenB capsule switching. Several factors that may influence the experimental outcomes warrant consideration. The selection of transformants relied on a serum bacterial assay designed to mimics selective pressure exerted by MenA + C meningococcal vaccines. The recombination breakpoints might vary when using different strains of Nm, potentially altering the recombination sequence accordingly. In addition, recombination efficiency might be influenced by the degree of sequence similarity and the presence or absence of DNA uptake sequence (DUS) during these events. Capsule switching is believed to be facilitated by high DNA sequence similarity between the involved serogroups [60]. Moreover, DUS plays a critical role in natural transformation, as these multicopy sequences are essential for DNA uptake and integration [61]. Utilizing a purified 5,527-bp recombination sequence rather than a purified genomic sequence from Nm119 could strengthen the hypothesis presented in our research. Finally, some MenB isolates within the China^{CC4821−R1−C/B} clone could not be fully characterized due to the lack of complete genomic data.

Conclusions

In summary, this study represents the first integrated theoretical and experimental validation of MenC→MenB capsule switching within the China^{CC4821−R1−C/B} clone, establishing a novel framework for investigating this evolutionary mechanism. We identified a potential donor strain for the cps sequences in a representative MenB China^{CC4821−R1−C/B} isolate and elucidated the genetic basis underlying the capsule switching of MenB. Further research should focus on assessing the virulence, immunogenicity, and other phenotypic characteristics of the capsule-switched strains through in vivo and in vitro studies, which will further advance our understanding of meningococcal capsule switching. Given the persistent selective pressure from vaccination and the demonstrated capability of Nm to evade the immune system, continuous surveillance for the increase of MenB isolates and new capsule switching events is necessary in China, as well as the introduction of MenB vaccines.

Authors’ contributions

MC, QG, and MZ conceived and designed the experiments. JH, YJ, PL, and YS performed all experiments. JH, PL, and MC analyzed the data. MC supervised the study and wrote the paper. JH and QG revised the paper. All authors have read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (82272381, 81872909, 82373918, and 82472322). The funders had no role in the study design, data collection, and interpretation, or the decision to submit the work for publication.

Data availability

All the assembled genomes sequenced in this study were submitted to the Neisseria PubMLST Database and assigned with corresponding PubMLST ID listed in Supplementary Table 3. The PubMLST ID of the MenB transformant Nm044Nm119_T20 is 128688.

Declarations

Ethics approval and consent to participate

The study involving serum from healthy persons was reviewed and approved by the Ethics Committee of Minhang Hospital, Fudan University (036–01 K), and conducted following the Helsinki Declaration of 2013 [62], with informed consent obtained from the healthy persons before sample collection.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.