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Infection and Immunity, April 2002, p. 2245-2248, Vol. 70, No. 4
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.4.2245-2248.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Role of Neisseria meningitidis luxS in Cell-to-Cell Signaling and Bacteremic Infection

Klaus Winzer,1 Yao-hui Sun,2 Andrew Green,1 Marie Delory,2 David Blackley,1 Kim R. Hardie,1 Thomas J. Baldwin,1 and Christoph M. Tang3*

Institute of Infections and Immunity, Queen's Medical Centre, Nottingham NG9 2EX,1 Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College of Technology, Science, and Medicine, London SW7 2AZ,3 Department of Paediatrics, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom2

Received 17 September 2001/ Returned for modification 5 November 2001/ Accepted 15 January 2002


   ABSTRACT
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Numerous pathogenic bacteria contain luxS, which is required for autoinducer-2 production. Here, we demonstrate that Neisseria meningitidis contains a functional copy of luxS that is necessary for full meningococcal virulence; strains with a luxS deletion are defective for bacteremia, a prerequisite of meningococcal pathogenesis.


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Bacteria have evolved mechanisms to coordinate gene expression in response to population density. Known as quorum sensing, these mechanisms involve the production and detection of signaling molecules (called autoinducers or pheromones), which modulate critical functions including virulence factor production, plasmid conjugal transfer, activation of secretion systems, swarming, swimming and twitching motility, biofilm differentiation, and bioluminescence (for reviews, see references 2, 13, 15, 28, and 30). Thus, bacteria in populations display properties denied individual cells.

Gram-positive and gram-negative bacteria are known to utilize distinct signal molecules for quorum sensing. Gram-positive bacteria export and detect small peptides, while gram-negative bacteria produce membrane-permeative N-acyl homoserine lactones (13, 15). Recently, however, potential quorum sensing systems based on a signal molecule called autoinducer-2 (AI-2) were shown to be present in both gram-positive and gram-negative species (2, 23, 24). The luxS gene product is required for AI-2 production by Salmonella enterica serovar Typhimurium (24), Escherichia coli (19, 23), Shigella flexneri (6), Helicobacter pylori (9, 11), Porphyromonas gingivalis (4, 5), Streptococcus pyogenes (12), and Actinobacillus actinomycetemcomitans (8). The chemical structure of AI-2 appears to be conserved, possibly allowing interspecies communication (2, 3, 18).

Neisseria meningitidis colonizes the human nasopharynx and can cause meningitis and/or septicemia (16). Here, we demonstrate that N. meningitidis possesses a functional luxS necessary for AI-2 production and full meningococcal virulence.

Serogroup B N. meningitidis possesses a functional luxS gene. Investigation of the MC58 genome sequence (25) using LuxS from Vibrio harveyi (LuxSVh) led to the identification of NMB1981 (N. meningitidis serogroup B annotated sequence), a 504-bp open reading frame (ORF) with 80% identity to LuxSVh which is annotated as being of unknown function. To establish whether NMB1981 encodes a functional product, the gene was amplified with rTth DNA polymerase XL (Perkin-Elmer, Warrington, United Kingdom), ligated in both orientations into pGEM T-Easy (Promega, Madison, Wis.), and then transferred into Escherichia coli DH5{alpha}, which cannot produce AI-2 due to a mutation in luxS (24). Strains were grown at 37°C in Luria-Bertani medium, and AI-2 production was assayed using V. harveyi BB170 as the reporter (3). Exogenous AI-2 induces premature bioluminescence by this reporter strain. Only clones containing NMB1981 expressed through the vector-encoded promoter (pGEMluxS-F) produced culture supernatants with AI-2 activity (Fig. 1A); no activity was detected if the insert was in the opposite orientation (pGEMluxS-R). Identical results were obtained with the NMB1981 homologue from strain SD (data not shown). Thus, NMB1981 complements the defective luxS in DH5{alpha} and was designated luxSNm.



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  		FIG. 1. (A) Complementation of E. coli DH5{alpha} with luxSNm. The growth profiles and extracellular AI-2 activity of E. coli DH5{alpha} carrying either pGEM T-Easy or the same vector containing luxSNm in the forward or reverse orientation were measured. Growth profiles: empty vector, closed squares; luxSNm in forward orientation, closed circles; luxSNm in reverse orientation, open circles. AI-2 activity: V. harveyi BB120 (wild type, after 24 h), black bar; luxSNm in forward orientation, grey bars; luxSNm in reverse orientation, white bars. Activities are presented as the fold increase of bioluminescence in relation to E. coli DH5{alpha} carrying an empty vector at the respective time point. (B) Growth profiles and AI-2 activity of N. meningitidis. N. meningitidis strains MC58 (closed circles) and MC58{Delta}luxS (open circles) were grown, and the optical densities at 600 nm of cultures were measured. Culture supernatants of MC58 (grey bars), MC58{Delta}luxS (white bars), and V. harveyi BB120 (black bar) were assayed for AI-2 activity. Activities are presented as the fold increase of bioluminescence in relation to the control (sterile medium) at the respective time points. The results for MC58{Delta}luxSEct were identical to those for the wild type and are not shown. For panels A and B, single representative experiments are shown, although similar results were obtained on three occasions for both assays.

 
Expression of N. meningitidis AI-2 activity is dependent on luxSNm. Next, we investigated whether N. meningitidis initiates luxSNm-dependent AI-2 production. A total of 432 bp of luxSNm (nucleotides 4 to 436 of the ORF) was deleted by inverse PCR and replaced with the kanamycin resistance gene from Tn903, yielding pGEM{Delta}luxS-kan. The insert of pGEM{Delta}luxS-kan was then cloned into pGIT5.3, a vector containing the Neisseria uptake sequence, and the resulting plasmid (pGIT5.3{Delta}luxS-kan) was introduced into N. meningitidis by transformation; Southern hybridization confirmed the integration of the deleted allele in MC58{Delta}luxS and B16/B6{Delta}luxS. A complemented strain, MC58{Delta}luxSEct, was constructed to enable the attribution of functions to luxSNm. luxSNm, under the control of an opa promoter, was introduced in the intergenic region between NMB102 and NMB103 (genes oriented in a tail-to-tail fashion), with ermC downstream of luxS. The construct (pYH204) was introduced into MC58{Delta}luxS by transformation, generating MC58{Delta}luxSEct; Southern analysis confirmed that integration into the NMB102/NMB103 intergenic region had occurred. N. meningitidis bacteria were grown at 37°C on brain heart infusion plates with Levinthal's supplement or in Mueller-Hinton medium for 24 h with shaking for AI-2 production. A list of the bacterial strains and plasmids used in this study is presented in Table 1.


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  		TABLE 1. Bacterial strains and plasmids used in this study

 
AI-2 activity was detected in MC58 culture supernatants from early logarithmic growth onward (Fig. 1B). While no AI-2 activity was observed in supernatants of MC58{Delta}luxS, the single ectopic luxS allele in MC58{Delta}luxSEct restored AI-2 activity to wild-type levels. Identical results were obtained with strain B16/B6 and its corresponding mutants (not shown).

N. meningitidis luxS mutants are attenuated for bacteremic infection. To investigate the influence of luxSNm on meningococcal pathogenesis, the ability of MC58{Delta}luxS to cause disseminated disease was compared directly against that of MC58. Bacteria were grown overnight on brain heart infusion agar and suspended in phosphate-buffered saline, and the number of CFU was determined. Bacteria (100 µl) in phosphate-buffered saline were injected intraperitoneally into five-day-old infant rats (Wistar; Harlan, Bicester, United Kingdom). The virulence of strains was assessed using groups of at least three animals in each of three experiments by determining their competitive index (CI). The CI is the proportion of mutant to wild-type bacteria recovered from the bloodstream following inoculation with a 1:1 ratio of the strains (26); results were compared by using a one-tailed Student's t test. MC58{Delta}luxS showed decreased survival in infected animals when compared with MC58 (P < 0.01; Fig. 2). Similar results were obtained with B16/B6 (not shown). Providing luxSNm in trans (MC58{Delta}luxSEct) restored the virulence to wild-type levels, demonstrating that the loss of luxS, not any polar effect, is responsible for attenuation.



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  		FIG. 2. MC58{Delta}luxS is defective for bacteremic infection compared with the parental strain, MC58. This defect is restored by supplying a single, intact copy of luxS in trans to MC58{Delta}luxS (MC58{Delta}luxSEct); the difference in the virulences of MC58{Delta}luxS and MC58{Delta}luxSEct is highly significant (P < 0.001). The CI is the proportion of the strains in the bloodstream 21 to 24 h following infection with a 1:1 ratio of the strains.

 
Complete genome sequences of bacterial pathogens provide an important resource for microbial research (21), though the annotations may miss biologically important homologies. While a luxS homologue was identified in the annotated Z2491 serogroup A strain sequence (14), the annotated MC58 genome (25) does not contain a luxS homologue. However, investigation of the MC58 database revealed an ORF closely related to luxSVh. We demonstrate that luxSNm complements DH5{alpha} and that N. meningitidis expresses AI-2 in a luxS-dependent fashion with maximal production during late exponential growth, consistent with a role in quorum sensing.

Studies with the infant rat model show that luxSNm contributes to the ability of the bacterium to cause bacteremia, a prerequisite for meningococcal pathogenicity. This model has been used to demonstrate the influence of other well-characterized virulence determinants on disseminated infection (20, 27). Although CIs provide a sensitive measure of a strain's virulence by eliminating host-to-host variation (22, 26), our findings may underestimate the impact of luxSNm on pathogenesis. AI-2 produced by the wild type could partially cross-feed a luxSNm mutant in mixed-inoculum experiments. However, the results are also consistent with a metabolic function of the LuxS protein, which is distinct from quorum sensing (18, 29).

Little is known about the regulatory networks governing the expression of N. meningitidis virulence factors apart from that of a LysR homologue, required for entry into host cells (7). Identification of the response elements and the genes controlled by AI-2-dependent signaling may help elucidate this aspect of pathogenesis and identify novel virulence determinants for this important human pathogen. Furthermore, the demonstration that luxSNm is required for full virulence suggests that interrupting this pathway may provide a means for preventing meningococcal disease (10).


   ACKNOWLEDGMENTS
 
We are grateful to Sharmila Bakshi, David Holden, Harry Smith, Nick West, and Paul Williams for valuable comments during preparation of the manuscript.

C.M.T. is an MRC Clinician Scientist, and A.G. was supported by a studentship from the University of Nottingham. Work in C.M.T.'s laboratory is supported by the Meningitis Research Fund. K.W. is supported by the MRC, and K.H. is a British Society for Antimicrobial Chemotherapy Fellow.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious Diseases, Center for Molecular Microbiology and Infection, Flowers Building, Imperial College of Technology, Science, and Medicine, Armstrong Rd., London SW7 2AZ, United Kingdom. Phone: (44) 207-594-3072. Fax: (44) 207-594-3076. E-mail: c.tang{at}ic.ac.uk. Back

Editor: V. J. DiRita


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Infection and Immunity, April 2002, p. 2245-2248, Vol. 70, No. 4
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.4.2245-2248.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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