Deletion of the Meningococcal fetA Gene Used for Antigen Sequence Typing of Invasive and Commensal Isolates from Germany: Frequencies and Mechanisms

Antigen sequence typing (ST) of FetA is part of the moleculartyping scheme of Neisseria meningitidis. Among invasive meningococcalisolates from 2,201 patients in Germany, we identified 11 strainslacking the fetA gene because of deletions mediated by repeatarrays flanking the gene, i.e., Correia elements, repeat sequence13 (RS13), and duplicated RS3. Geographic mapping and multilocusST of invasive isolates revealed that fetA deletion was a sporadicevent without genetic fixation. Among 821 carrier strains, 12strains lacked fetA, suggesting that fetA is maintained duringasymptomatic carriage. Interestingly, most of these isolatesbelonged to the multilocus ST-35 clonal complex (cc). ST-35cc strains and the recently published ST-192 strains from BurkinaFaso may benefit from loss of fetA, but their infrequent occurrenceamong invasive isolates currently does not affect fetA antigenST.

INTRODUCTION

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INTRODUCTION

Antigen sequence typing (ST) is now widely used for highly discriminatoryand precise typing of Neisseria meningitidis (8). Finetypingdata are used to monitor epidemiological changes over time andspace and to unravel the plethora of circulating antigen typesfor the sake of subcapsular vaccine development. We recentlydemonstrated the benefits of including FetA antigen typing inthe typing regime at the German National Reference Laboratoryfor Meningococci (5). With regard to the investigation of infectionsin 1,616 patients, the addition of FetA antigen typing increasedthe number of finetypes 2.3-fold compared to the use of serogroupingin conjunction with PorA typing but without FetA antigen typing.

The ferric enterochelin receptor FetA (formerly FrpB) is animmunodominant protein present in outer membrane vesicle vaccines(14, 17). During the preparation of the manuscript, Marsh etal. electronically published ahead of print the identificationof three fetA-negative isolates among 768 invasive isolatescollected in the United States between 1990 and 2006 (10). FetAdeletion was mediated by duplicated repeat sequence 3 (dRS3)repetitive elements. Deletion via flanking repeats is reminiscentof what has been observed in strains lacking the porin geneporA (18). FetA deletion was also a trait of ST-192 strainsisolated from cases and healthy carriers in Burkina Faso (7).

In this paper, we report the frequency of fetA deletion in Germany,provide evidence by geographic mapping and multilocus ST (MLST)(9) that fetA deletion is a sporadic event, and report a detailedgenetic analysis of deletion events.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Meningococcal strains. Strains were collected at the German National Reference Laboratoryfor Meningococci as part of the nationwide laboratory surveillanceof meningococcal disease. The majority of strains have beendescribed recently (5). For genetic studies, we include fourfetA-negative strains recently identified among noninvasiveisolates, which are sent to the reference laboratory on an unsystematicbasis or as part of a carriage study (11). Furthermore, therecently published Bavarian carrier strain collection was screenedfor fetA deletions (4). Finally, a representative ST-192 strainwas analyzed for the genetic mechanism of its fetA deletion,described recently (7). This strain was isolated in BurkinaFaso among a series of clonally related capsule null locus strains.

FetA antigen ST and MLST. FetA typing and MLST were performed as described previously(9, 16). Allele and ST assignments were achieved by comparisonwith the databases at www.neisseria.org. The fetA gene was routinelyamplified using the primers S13-2 (5'-CAT ACC CAA ATC ACC ACTCG-3') and S15 (5'-TTG CAG CGC GTC RTA CAG GCG-3'). In casesof negative results, the primers S1 (5'-CGG CGC AAG CGT ATTCGG-3') and S8 (5'-CGC GCC CAA TTC GTA ACC GTG-3') were tested.If no amplification of fetA was achieved, the presence of fetAwas determined by Southern blot hybridization using a fetA probegenerated from a fetA-positive strain (MC58) by PCR with theprimers S1 and S8. The carrier isolates from the Bavarian carriagestudy were analyzed by dot blot hybridization according to recentlypublished protocols (3), using the S1/S8 probe. Negative resultswere confirmed by S1/S8 PCR. In cases of fetA-negative strains,the remnants of the fetA chromosomal locus were investigatedeither by two PCR assays employing flanking primers (CM21 [5'-AACGCA TCG AAA TCC ACA GC-3'] and CM24 [5'-CTT TGA GGT TGG CGGTAT CG-3'] or CM28 [5'-ATG CCC GCA ATC TCA AAT CC-3'] and CM26[5'-GGT CGG ACA AAC CGG AAC G-3'], respectively) and subsequentsequencing of the PCR products or by cloning of the locus. Briefly,chromosomal DNA of fetA-negative meningococcal strains was restrictedwith MluI, which cuts in the genes adjacent to fetA, and analyzedby Southern blot hybridization using probes flanking fetA (i.e.,PCR product CM21/CM22 [5'-GGT CGT CCG CCG TAA ACT C-3'] andPCR product CM23 [5'-TTT GAC TGC TTT AGC CGT ATG-3']/CM24).MluI fragments hybridizing with both probes were cloned intothe EcoRI site of pBluescript SK (Stratagene, The Netherlands)and subsequently sequenced by primer walking.

Geographic maps. Latitude and longitude coordinates (map date, WSG 84) of zipcodes of the year 2003 were obtained from GfK Macon (Waghäusel,Germany). The Regiograph 8 (GfK Macon) and Fireworks MX Macromedia(Adobe Systems Inc., San Jose, CA) programs were used to generateand edit maps.

Nucleotide sequence accession numbers. The sequences of the intergenic regions between NMB1987 andNMB1989 or NMB1990, respectively, identified in the fetA-negativestrains have been deposited in the EMBL nucleotide sequencedatabase under accession numbers AM748025 to AM748034 and AM748728to AM748731.

RESULTS AND DISCUSSION

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RESULTS AND DISCUSSION

We report the frequency, geographic distribution, and lineagesof fetA-negative isolates in Germany. Furthermore, we providea detailed dissection of the genetic mechanisms giving riseto loss of the gene.

Among strains from 2,201 patients suffering from invasive meningococcaldisease in Germany between 2001 and 2007, we identified 11 fetA-negativestrains (0.5%). The strains were almost randomly distributedthroughout Germany (Fig. 1). This finding suggests sporadicemergence of invasive fetA-negative strains. All invasive strainsbelonged to different STs, with the exception of two isolatesfrom an outbreak of ST-11 disease among schoolchildren and twoST-35 cases which were not epidemiologically linked (Table 1).The genetic diversity, together with the geographic distribution,indicates that, in Germany, there are no pathogenic geneticlineages regularly lacking fetA. The above-mentioned outbreakdue to ST-11 strains comprised fetA-negative and fetA-positivestrains, which otherwise proved to be identical, as confirmedby MLST, fumC single-nucleotide polymorphism typing, and IS1301restriction fragment length polymorphism typing (6). Interestingly,van der Ende et al. reported an outbreak of seven cases dueto ST-11 strains, of which five were caused by strains witha stable point mutation in the porA gene resulting in the lossof expression of PorA (19). The authors hypothesized that thestrains were transmitted between cases and were unlikely tohave emerged during infection.

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FIG. 1. Geographic distribution of cases of invasive meningococcal disease in Germany caused by strains lacking the fetA gene. Time period: December 2001 through February 2007.

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TABLE 1. Characteristics of Neisseria meningitidis strains lacking the fetA gene^a

We determined precisely the mechanisms of fetA loss by DNA sequenceanalysis not only in the 11 invasive strains from the nationallaboratory surveillance and 12 carrier strains from the Bavariancarrier strain collection but also in five additional fetA-negativeisolates from other sources. The results are depicted in Fig.2. Marsh et al. elucidated the responsibility of dRS3 repeatsfor fetA loss in the three strains analyzed (10). We now demonstratea variety of mechanisms of intragenomic recombination or rearrangementleading to loss of fetA and, in some instances, also of thedownstream gene fetB. This includes (i) RS13-mediated recombination,in one case in conjunction with the uptake of the insertionsequence IS1655; (ii) recombination mediated by Correia elements;and (iii) recombination via dRS3 repeats.

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FIG. 2. Genetic analysis of strains lacking the fetA gene with emphasis on repeat elements. Strains Z2491 and MC58 were included as reference strains (13, 15). Strains with prefix "NM" were deduced from a recent publication describing three fetA-negative strains from the United States (10). Strains with the prefix "DE" are from the National Reference Center for Meningococci and mostly represent invasive isolates. Strains with the prefix " ${alpha}$ " were isolated in the course of the Bavarian carriage study (4). Strain ${gamma}$ 30 was isolated during a swabbing campaign whose results were published recently (11). Strain 10115V3B is an ST-192 capsule null locus isolate from Burkina Faso (7).

Strain 10115V3B was a representative of 17 fetA-negative ST-192strains isolated in Burkina Faso between 2003 and 2004 (7).The intergenic region was not sequenced in all strains; however,PCR analysis revealed its identical length, suggesting a singlemechanism of fetA deletion in all ST-192 isolates. Accordingto our knowledge, the ST-192 clone is the first clone to beuniformly associated with a fetA deletion.

FetA is a variable gene whose antigenic variability might beat least partially driven by flanking noncoding repeat elements,as postulated recently by Bentley et al. (1). These repeat arrays,however, apparently also support gene loss. FetA, formerly FrpB,is an iron-repressed TonB-dependent receptor which binds ferricenterobactin. In gonococci, FetA expression can be down-regulatedby phase variation (2). Inspection of the corresponding homopolymerictracts within the meningococcal fetA promoter sequence of strainMC58 (15) suggests that phase variation is possible in thisspecies also (data not shown). Mutation of FetA is nonlethal,iron uptake is not affected, and serum sensitivity is only mildlyenhanced (14). FetA is expressed during invasive infection,but a clear role in the bloodstream needs to be defined. Thefact that fetA-negative strains emerge now and then and causeinvasive disease is noteworthy with regard to the use of outermembrane vesicle vaccines harboring FetA. It will be interestingto see whether the use of such vaccines in New Zealand (12)will enhance the frequency of such strains.

We next focused on the proportion of fetA deletion in carriageisolates. We have recently demonstrated the high rate of capsulesynthesis gene-silencing events in carried meningococci, which,however, are not fixed in the population (20). We have now analyzedthe same strain collection for the frequency of fetA deletionby using dot blot hybridization and, in the case of negativeresults, by PCR. A total of 821 strains were included, and 12independent strains were fetA negative, in comparison to 11independent strains of 2,201 for the invasive disease cases.Herein, we defined dependence of strains as the spatiotemporallinkage of identical STs. In a chi-square test, the differencebetween the collections with regard to the number of fetA-negativestrains was significant (P = 0.01). However, one has to considerthat the Bavarian carrier strain collection was establishedin one federal state of Germany in winter 1999/2000, thus, beforethe collection of the invasive isolates. It was noteworthy that6 of 12 strains belonged to the ST-35 complex. Thus, the ST-35complex might tolerate fetA deletions similarly to the ST-192complex (7). This finding is supported by the fact that Marshet al. also found ST-35 in 2 of 3 strains with fetA deletion(10). Nevertheless, in the Bavarian carrier strain collection,only 6 of 45 ST-35 clonal complex strains were fetA negative.

It cannot be excluded for certain that fetA deletions mighthave occurred during laboratory passage in some cases. We estimatea number of four passages on artificial media prior to typingand storage. However, the observation of two epidemiologicallylinked fetA-negative strains within the ST-11 outbreak describedabove, as well as of the uniformly fetA-negative genotype ofST-192 strains from Burkina Faso (7), supports the view of awithin-host evolution of fetA deletion variants.

In conclusion, fetA typing is not hampered by rare and sporadicgene deletion events. Deletion is supported by flanking, noncodingrepeat arrays. The reasons for selection against loss of fetAare unclear. Due to the geographic and temporal independenceof the strain collections, our study does not have the powerto clearly demonstrate that the carrier state is more permissivefor fetA deletion than invasive disease, but we could demonstratea trend for more fetA deletions in carriage than in invasivedisease. This phenomenon could be explained either by the importanceof the ferric enterochelin receptor for systemic spread duringinfection or by the differences of clonal lineages and theirbiology which have recently been epidemiologically demonstrated(21).

ACKNOWLEDGMENTS

The National Reference Center for Meningococci is supportedby the German Ministry of Health via the Robert Koch-Institute.This study was part of the center's activities.

FOOTNOTES

* Corresponding author. Mailing address: Institute for Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Str. 2, E1, 97080 Würzburg, Germany. Phone: 49 931 201 46036. Fax: 49 931 201 46445. E-mail: hclaus{at}hygiene.uni-wuerzburg.de

Published ahead of print on 11 July 2007.

REFERENCES

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