INFECTION AND IMMUNITY, Dec. 1990, p. 4145-4148 Vol. 58, No. 120019-9567/90/124145-04$02.00/0Copyright C) 1990, American Society for Microbiology

Conservation of Neisseria gonorrhoeae Pilus Expression RegulatoryGenes pilA and pilB in the Genus Neisseria

MUHAMED K. TAHA AND CHRISTIAN MARCHAL*Unite des Antigenes Bacteriens, Institut Pasteur, 75724 Paris Cedex 15, France

Received 21 June 1990/Accepted 10 September 1990

The pili of Neisseria gonorrhoeae mediate bacterial adhesion to the host-susceptible tissues. We havepreviously reported the identification of two genes, pilA and pilB, which act in trans to regulate pilus expression.Besides this regulatory function, pilA participates in an essential function for bacterial viability. Here we showthat pilA and pUB homologs are also present in a variety of other members of the Neisseriaceae family ofbacteria in contrast to the gonococcal pilin gene which hybridizes only to the pathogenic Neisseria species.

Pathogenic neisseria comprise two principal species. Neis-seria gonorrhoeae is a causative agent of sexually transmit-ted disease worldwide. Clinical manifestations of gonorrhearange from a simple mucosal infection to salpingitis andarthritis. Sequelae of gonorrhea infections include ectopicpregnancy and sterility. Neisseria meningitidis causes indi-vidual epidemics of meningitis and septicemia in developedcountries, and major outbreaks still regularly occur in theSahel region of Africa and in China. The genus Neisseriaalso consists of commensal species that are not implicated ininfection under normal conditions. Besides this genus, thegenera Branhamella, Moraxella, and Acinetobacter are clas-sically classified as members of the family Neisseriaceae.The overall DNA homology is important among Neisseriaspecies, and the two pathogenic Neisseria species in partic-ular share an extremely high degree of homology (2, 4).The pili of N. gonorrhoeae are a major virulence factor.

Piliated (P+) gonococci attach more successfully to variouscells of susceptible tissues than nonpiliated (P-) variants (5,6, 10). Furthermore, in contrast to certain P- variants, P+strains are able to establish infection in human volunteers (J.Boslego, J. Ciak, P. Hitchcock, J. Swanson, E. Tramont, J.Sadoff, and M. J. Koomey, 1988. Abstr. Sixth Int. Patho-genic Neisseria Conf. 1988, p. 107). Piliation and virulencecan change with culture conditions (3), and this variationmay be important for the spread of bacteria from host to hostor to different anatomical sites.

Pilin is the major subunit of the pilus, and its expression iscontrolled at the chromosomal locus pilE. Strain MS11 hastwo expression sites, pilEl and pilE2, although most strainscontain only one. We have identified two closely linkedgenes, pilA and pilB, which act in trans to regulate pilintranscription. The two genes are divergently transcribed,with overlapping promoters. They map downstream of thepilEl and opaEl loci in the chromosome of MS11 (Fig. 1). InEscherichia coli, PilA alone activates the pilin gene pro-moter and represses it in conjunction with PilB. GonococcalpilB mutants are hyperpiliated, and one particular pilAmutant was shown to exhibit a P- phenotype (11). PilA andPilB have amino acid sequence similarities with members ofthe two component sensor-regulator family of proteins. PilBhas homology with histidine kinase sensors and has proper-ties of an integral membrane protein. PilA has homologywith response regulators, but it also participates in an

* Corresponding author.

essential function in the bacterium, since an intact copy ofpilA seems to be required for cell viability (M. K. Taha, B.Dupuy, W. Saurin, M. So, and C. Marchal, Mol. Microbiol.,in press). Besides pilin gene, pilA and pilB appear to regulateother genes whose expression is also affected in the pilA andpilB mutants (unpublished data).The importance of pilA and pilB in gene regulation in N.

gonorrhoeae led us to examine the presence of these twogenes in related bacteria. Intragenic pilA and pilB probes(Fig. 1) were used for Southern blot analysis of chromosomalDNA from a variety of bacteria (Fig. 2 and Table 1). Cultureswere passaged every 18 to 22 h on G medium plates with Gsupplements (Diagnostics Pasteur, Marnes la Coquette,France). DNA isolation and Southern blots were done aspreviously reported (11). Under stringent conditions, bothprobes hybridized to a single DNA fragment in all of theNeisseria species tested except Neisseria animalis and Neis-seria denitrificans. Moreover, in all cases but one (Neisseriaflava NRL34410; Fig. 2, section I, lane 13), both probeshybridized to the same restriction fragment. This indicatesthat pilA and pilB homologs are genetically linked in most ofthe Neisseria species tested. Whether the exception ob-served for N. flava reflects a restriction site polymorphism isunclear at present. The pilA and pilB probes did not hybrid-ize to DNA from Branhamella catarrhalis, Moraxella spe-cies, or Acinetobacter species but did hybridize to DNAfrom a strain of the M6 bacterial group (related to the genusMoraxella). No DNA hybridization was observed with ei-ther probe outside the Neisseriaceae family of bacteria.However, the pilin gene probe pNG1100 (7) hybridized onlyto DNA from the pathogenic Neisseria species (Fig. 2C), asalso observed by others with dot blot analysis (1). This probeidentifies a single pilin locus in N. meningitidis strains, incontrast to several expression and silent loci present in theN. gonorrhoeae genome (9).

Neisseria species, Moraxella species, Pseudomonasaeruginosa, and Vibrio cholerae express pilins that shareextensive amino acid homology in their N-terminal ends, butthis homology is weaker at the DNA level and would not bedetected under high-stringency conditions of hybridization.These data may reinforce the notion of an extreme geneticconservation of pilA and pilB homologs in the genus Neis-seria.

In N. gonorrhoeae MS11, pilA is transcribed at a lowerlevel than the pilin gene but at a higher level than pilB(unpublished data). To determine whether the pilA homologsin the cross-hybridizing bacterial species correspond to

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4146 NOTES INFECT. IMMUN.

pilEl opa El pA pIB pil E21.tl,111 > S/S/P///////D r~~~~~~~~~,o

c,oco ~ xcogE cu Co 097°X > E sE c

- - I' 1 kbFIG. 1. Schematic representation of piIA-piIB region in strain MS11A. Black bars represent restriction fragments of pilA and pilB cloned

in pUC18 (11) and a fragment of pilEl cloned in pBR322 (7) which were used as hybridization probes.

expressed genes, we monitored pilA transcription by RNAdot blot analysis. Total RNA was isolated as previouslydescribed (11). Serial dilutions of RNA (1 volume) weresuspended in 4 volumes of 20 mM HEPES (N-2-hydroxyeth-ylpiperazine-N'-2-ethanesulfonic)-5 mM sodium acetate-1

mM EDTA-50% formamide-7% formaldehyde, incubated at65°C for 10 min, put on ice, and spotted on N-hybond filter(Amersham). The filter was then dried, treated with 2xSSPE, dried again, exposed to UV (254 nm) light for 4 min,and hybridized as done previously (11) with T4 DNA poly-

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FIG. 2. Detection of pilA and pilB in Neisseria strains by Southern hybridization. Chromosomal DNA from Neisseria strains weredigested with Clal and probed with nick-translated pilA (panels A), pilB (panels B), and pilin (panels C) gene probes. The pilEl probe(pNG1100) and the internal pilA and pilB probes are denoted by dark bars in Fig. 1. Section I lanes: 1, Neisseria lactamica NRL35046; 2, N.lactamica NRL30011; 3, Neisseria cinerea NRL33683; 4, N. cinerea NRL33720; 5, N. meningitidis NRL30012b; 6, N. meningitidisNRL30012a; 7, N. meningitidis NRL35063; 8, B. catarrhalis NRL34355; 9, B. catarrhalis NRL33723; 10, Neisseria flavescens NRL30009;11, Neisseria mucosa NRL34415; 12, Neisseria subflava NRL6284; 13, N. flava NRL34410; 14, Neisseria perflava NRL34803; a, N.gonorrhoeae MS11A. This group of strains was obtained from J. Knapp. Section II lanes: 1, Neisseria polysaccharea N462; 2, Neisseria siccaN3265; 3, N. subflava N3260; 4, N. cinerea N415; 5, N. lactamica N411; 6, N. meningitidis N6922; 7, N. meningitidis N6930; 8, N.meningitidis N6933; 9, N. meningitidis N404; 10, N. gonorrhoeae N6903; 11, N. gonorrhoeae N6911; 12, N. gonorrhoeae N6910; 13, N.gonorrhoeae N6934; 14, N. gonorrhoeae N403; a, N. gonorrhoeae MS11A. This group of strains was obtained from J. Y. Riou.

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TABLE 1. Southern hybridization of DNA from bacterial species to specific gene probesa

DNA hybridization to: DNA hybridization to:Strain Strain

pilA pilB pilE pilA pilB pilE

Neisseria gonorrhoeae N. perflavaMS11 + + + N407 + NDb _N403 + + + NRL34803 + + -N6934 + + + N. flavescensN6910 + + + N414 + ND -N6911 + + + NRL30009 + + -

N6903 + + + N. sicca N3265 + + -

N812 (F62) + ND + N. polysaccharea N462 + + -

N. mucosa N405 and NRL 34415 + + -

N. meningitidis N. animalis N413 - ND -

N404 A + + + N. denitrificans N412 - ND -

N6933 B + + + Branhamella catarrhalisN6930 C + + + N417 - ND -N6922 Y + + + NRL33723 - - -NRL35063 + + + NRL34355 - - -NRL30012a + + + Moraxella nonliquefaciens 2288 - ND -

NRL30012b + + + M. nonliquefaciens 6188 - ND -

N. lactamica M6 (related to Moraxella) 18688 + ND -

N411 + + - M. osloensis - ND -

NRL30011 + + - Acinetobacter hemolytica 10188 - ND -

NRL35046 + + - A. lwoffii 5488 - ND -

N. cinerea A. calcoaceticus var. glucidolytica 26987N415 + + - - ND -

NRL33720 + + - Pseudomonas aeruginosa 16488 - ND -

NRL33683 + + - P. fluorescens 51387 - ND -

N. flava P. cepecia 27687 - ND -

N3264 + + - Vibrio cholerae - ND -

NRL34410 + + - Bordetella pertussis - ND -

N. subflava Yersinia enterocolitica - ND -

N3260 + + - Escherichia coli K-12 -

NRL6284 + + - E. coli J96 - ND -

a Hybridization with pilA or pilB intragenic probes or pilE (pilin gene probe pNG1100) derived from N. gonorrhoeae MS11 (Fig. 1) is indicated by a plus whenone or more bands were observed and by a minus when no signal was detected.

b ND, Not determined.

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FIG. 3. Detection of pilA transcripts from Neisseria strains by dot blot analysis. (A) Serial dilutions of RNA were hybridized withintragenic pilA probe. (B) Control RNA samples were treated with 0.4 N NaOH at 65°C for 45 min before proceeding as described for panelA. Lanes a, N. gonorrhoeae MS11A; 1, N. gonorrhoeae N812; 2, N. gonorrhoeae N6903; 3, N. meningitidis N404; 4, N. meningitidis N6933;5, Neisseriaflavescens N414; 6, Neisseria cinerea N415; 7, Neisseria subflava N3260; 8, B. catarrhalis N417. The numbers on the left indicatemicrograms of total RNA spotted onto the filter.

A

B

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4148 NOTES

merase-labeled pilA probe (Fig. 1). A pilA transcript wasobserved in all N. gonorrhoeae and all nonpathogenic Neis-seria strains tested. A smaller amount ofpilA transcript wasdetected in N. meningitidis (Fig. 3). This difference inmessage level may reflect a lower degree of homologybetween N. meningitidis RNA and the pilA probe, since thesignal obtained in the Southern blot was also weaker (Fig. 2).Our data so far indicate that pilA and pilB homologs are

present in many members of the Neisseriaceae family ofbacteria and particularly in the genus Neisseria. Hybridiza-tion of the gonococcal pilA probe with strain M6 genome andthe absence of hybridization with DNA from N. animalisand N. denitrificans may suggest that the first species isgenetically linked to the genus Neisseria and that the othertwo do not belong to it. Neisseria species of animal originother than human were previously shown to be more dis-tantly related to the human isolates by comparing their G+Ccontent and their DNA hybridization levels (2).As in the case of N. gonorrhoeae, pilA and pilB homologs

are closely linked on the chromosome and pilA homologs aretranscribed. It is then likely that this two-component regu-lation is conserved and functional in the whole genus Neis-seria and that it is specific to this genus. Other traits such aspili are associated with pathogenic Neisseria species andrepresent virulence factors. These results are reminiscent ofthe situation in V. cholerae in which toxR regulatory genehomologs are found in all toxinogenic and nontoxinogenicstrains, while only toxinogenic strains carry the ctxAB toxingenes (8). In the nonpathogenic Neisseria species, the pilA-pilB system may also regulate the expression of a differenttype of pilin.

We thank M. Kiredjian, J. Knapp, D. Mazigh, A. Rambach, J. Y.Riou, and A. Ullman for DNA and bacterial strains, and M. So forthe pilin gene probe.

This work was supported by INSERM (CRE 893009), CNRS (UA040557), NATO (0207/88), and CCAR (27790).

LITERATURE CITED

1. Aho, E. L., G. L. Murphy, and J. G. Cannon. 1987. Distributionof specific DNA sequences among pathogenic and commensalNeisseria species. Infect. Immun. 55:1009-1013.

2. Hoke, C., and N. A. Vedros. 1982. Taxonomy of the Neisseriae:deoxyribonucleic acid base composition, interspecific transfor-mation, and deoxyribonucleic acid hybridization. Int. J. Syst.Bacteriol. 32:57-66.

3. Keevil, C. W., N. C. Major, D. B. Davies, and A. Robinson.1986. Physiology and virulence determinants of Neisseria gon-orrhoeae grown in glucose-, oxygen- or cystine-limited contin-uous culture. J. Gen. Microbiol. 132:3289-3302.

4. Kingsbury, D. T. 1967. Deoxyribonucleic acid homologiesamong species of the genus Neisseria. J. Bacteriol. 94:870-874.

5. Mardh, P.-A., and L. Westrom. 1976. Adherence of bacteria tovaginal epithelial cells. Infect. Immun. 13:661-666.

6. McGee, Z. A., A. P. Johnson, and D. Taylor-Robinson. 1981.Pathogenic mechanisms of Neisseria gonorrhoeae: observationon damage to human fallopian tubes in organ culture by gono-cocci of colony type 1 or type 4. J. Infect. Dis. 143:413-422.

7. Meyer, T. F., N. Miawer, and M. So. 1982. Pilus expression inNeisseria gonorrhoeae involves chromosomal rearrangement.Cell 30:45-52.

8. Miller, V. L., and J. J. Mekalanos. 1984. Synthesis of choleratoxin is positively regulated at the transcriptional level by toxR.Proc. Natl. Acad. Sci. USA 81:3471-3475.

9. Segal, E., P. Hagblom, H. S. Seifert, and M. So. 1986. Antigenicvariation of gonococcal pilus involves assembly of separatedsilent gene segments. Proc. Natl. Acad. Sci. USA 83:2177-2181.

10. Swanson, J. 1973. Studies on gonococcus infection. IV. Pili:their role in attachment of gonococci to tissue culture cells. J.Exp. Med. 127:571-589.

11. Taha, M. K., M. So, H. S. Seifert, E. Billyard, and C. Marchal.1988. Pilin expression in Neisseria gonorrhoeae is under bothpositive and negative transcriptional control. EMBO J. 7:4367-4378.

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