Type-4 pili of Kingella denitrificans1

Gene 192 (1997) 171–176

Type-4 pili of Kingella denitrificans 1 Susan Weir a, Li-Wei Lee b, Carl F. Marrs b,* a Department of Molecular Biology and Microbiology, Tufts School of Medicine, 136 Hanison, Boston, MA 02111, USA b Department of Epidemiology, University of Michigan, 109 Observatory Street, Ann Arbor, MI 48105, USA Received 20 February 1996; accepted 22 March 1996; Received by P.A. Manning

Abstract Kingella denitrificans possess type-4 pili, and the type strain, ATCC 33394, contains at least four complete copies of type-4 pilinencoding genes. Previously reported hybridization patterns of K. denitrificans chromosomal DNA seen using a Neisseria gonorrhoeae pilin gene region probe, had been interpreted as representing possible partial, silent gene loci. This now appears to be due to cross-reaction to multiple copies of 18-bp inverted repeat structures. Data are presented on a variety of colony variants which have changed from a spreading-corroding (SC ) phenotype to a nonspreading-noncorroding (N ) phenotype. Interestingly, while the SC to N transition is most often associated with loss of piliation in other bacteria containing type-4 pili, many of the K. denitrificans N variants still produce pilin, and some still produce pili. © 1997 Elsevier Science B.V. Keywords: Gene organization; DNA repeats; Colony morphology; Hybridization; Virulence; Colonization; Twitching motility; Phase variation

1. Introduction The genus Kingella is most closely related to the genera Neisseria and Eikenella (Dewhirst et al., 1989; Rossau et al., 1989). Kingella kingae, originally classified as Moraxella kingii (Henriksen and Bøvre, 1968), was transferred to the new genus Kingella within the family Neisseriaceae in 1976 (Henriksen and Bøvre, 1976). The species Kingella denitrificans and Kingella indologenes were added to the genus (Snell and Lapage, 1976). In 1990 K. indologenes was transferred back out of Kingella * Corresponding author. Tel. +1 313 7472407; Fax +1 313 7643192; e-mail: [email protected] 1 Presented at the Workshop on ‘Type 4 pili – biogenesis, adhesins, protein export, and DNA import’, Schloss Ringberg, Germany, 26–29 November 1995. Abbreviations: aa, amino acid(s); bp, base pair(s); EM, electron microscopy; IR, inverted repeat(s); K., Kingella; kdpA-D, genes encoding pilin of K. denitrificans; KpdD, pilin encoded by kdpD gene; N., Neisseria; N, nonspreading-noncorroding colony-morphology variants; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; pilE1, pilin gene of N. gonorrhoeae; pilT-U, Pseudomonas aeruginosa genes required for pilus twitching; SC, spreading-corroding colony-morphology variants; SET, 25% sucrose/1 mM EDTA/50 mM Tris · Cl, pH 8.0; SSC, 0.15 M NaCl/0.015 M Na · citrate, pH 7.6; TE, Tris · Cl/1 mM EDTA, pH 8.0; tfpA, gene 3 encoding pilin of Moraxella nonliquefaciens. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 8 28 - 1

and renamed Suttonella indologenes (Dewhirst et al., 1990). In 1993 Kingella orale was added as a new species of the genus Kingella (Dewhirst et al., 1993). K. denitrificans and K. kingae are fastidious Gram− rods and common inhabitants of the human nasopharynx (Hollis et al., 1972; Yagupsky et al., 1995). Ordinarily, these organisms are not pathogenic, but do occasionally cause serious opportunistic infections. K. kingae has been reported in over 30 citations to cause a wide variety of diseases including bacteremia, empyema, endocarditis, epiglottitis, intervertebral disk infection, meningitis, osteomyelitis and septic arthritis. K. denitrificans is even less commonly found to cause disease, but has been implicated in cases of endocarditis (Brown et al., 1987; Geraci and Wilson, 1982; Goldman et al., 1980; Hassan and Hayek, 1993; Khan et al., 1986; Swann and Holmes, 1984), empyema (Molina et al., 1988), chorioamnionitis (Maccato et al., 1991) and granulomatous disease secondary to AIDS (Minamoto and Sordillo, 1992). Nothing is known of the factors that contribute to the virulence of this organism. In earlier work, we showed that K. denitrificans possess type-4 pili and phenotypes associated with this trait ( Weir and Marrs, 1992). In Neisseria gonorrhoeae these structures are essential for this organism to colonize mucosal surfaces ( Kellogg et al., 1968). Members of Neisseriaceae with type-4 pili are

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described as having a type of movement called twitching motility ( Henrichsen, 1975). This can be detected on an agar plate as a spreading of growth away from the main colony. These SC colonies are also capable of corroding agar and producing a pellicle when grown in static broth (Bøvre and Frøholm, 1972a; Pedersen et al., 1972; Sandhu et al., 1974). They are naturally competent, being capable of taking up DNA from the environment (Bøvre and Frøholm, 1972b). Noncorroding (N ) variants no longer pit agar or spread and are generally incompetent. Phase variation, a switch between piliated and nonpiliated variants, can be detected by changes in colony types. Electron micrographs, colony morphology observations, DNA transformation patterns and immunoblots with antiserum against Moraxella bovis Q pili, suggested that K. denitrificans and K. kingae have type-4 pili ( Weir and Marrs, 1992). This was confirmed when N-terminal aa sequencing of purified pilin protein of K. denitrificans showed a match to the conserved N-terminal aa found on type-4 pilin proteins ( Weir and Marrs, 1992). We used a 120-bp PCR fragment containing the conserved N-terminal region of a tfpA pilin gene of Moraxella nonliquefaciens ( Tønjum et al., 1991) and a 1400-bp DNA restriction fragment containing pilE1 plus some flanking DNA from N. gonorrhoeae strain MS11 ( Koomey et al., 1991) as probes in Southern hybridization analysis and found that both K. denitrificans and K. kingae had multiple bands of hybridization ( Weir and Marrs, 1994). However, while the tfpA PCR probe only hybridized with up to three bands per strain, the pilE1 DNA probe hybridized to a large number of bands in each strain. The type-4 pilin system of N. gonorrhoeae is composed of one, and sometimes two, expression copies that are transcribed into pilin proteins. There are multiple silent copies that lack portions of the N terminus and are not capable of being transcribed (Meyer et al., 1984). It is these silent copies that contribute to sequence variation by recombining with the expression copy to produce antigenically distinct pilins. We hypothesized that K. denitrificans and K. kingae might be similar to N. gonorrhoeae in having both expression genes and multiple silent copies of type-4 pilin-encoding genes, and that it was these silent copies to which the N. gonorrhoeae pilE1 DNA probe hybridized ( Weir and Marrs, 1994).

2. Results and discussion 2.1. Organization of pilus genes and presence of repeat sequences In experiments which will be presented in detail elsewhere, we cloned and sequenced four complete type-4 pilin-encoding genes from K. denitrificans ATCC

33394. Fig. 1 is a diagrammatic representation of the relative positions of these genes and some interesting structural features associated with them. We have named these genes kdpA, kdpB, kdpC and kdpD (Kingella denitrificans pilin). The genes kdpA, kdpB and kdpC are located at separate chromosomal loci and encode for identical pilin proteins. In contrast, kdpD is immediately 3∞ of kdpB and has only 53% identity in the deduced aa sequence. The gene locus containing kdpB and kdpD in tandem is similar to the genetic organization found for type-4 pilin-encoding genes in Eikenella corrodens ( Rao and Progulske-Fox, 1993; Tønjum et al., 1993). K. denitrificans is unique in having two additional complete pilin-encoding genes at separate loci. No other bacterial species has been reported to have more than two complete type-4 pilin-encoding genes within the same bacterial genome. Another important feature shown in Fig. 1 is the presence of sequences flanking kdpA and kdpC, which are composed of paired 18-bp inverted repeats ( IR) separated by 7–9 bp. The kdpB and kdpD region has a single 18-bp sequence 5∞ of kdpB. A fragment of the K. denitrificans genome which hybridized to the N. gonorrhoeae pilE1 DNA probe, but not to the M. nonliquefaciens tfpA PCR probe, was cloned. We expected it to contain one of the silent gene loci hypothesized by us previously. However, we discovered that it contained two sets of the 18-bp IR sequences, and no nt sequence related to type-4 pilin genes (Fig. 1). It thus appears that our hypothesis about the similarity between K. denitrificans and N. gonorrhoeae pilin gene organization is untrue, and instead the large number of hybridizing bands seen in Southern hybridization of genomic K. denitrificans DNA probed with pilE1 is due to crossreactivity with flanking sequences which are multiply repeated in Kingella.

2.2. Studies on N variants of K. denitrificans Given the large number of complete type-4 pilin genes present in K. denitrificans ATCC 33394, it is interesting to study the N variants of this strain. One would predict that their relatively high frequency of occurrence should not be due to simple knock out mutations of all four pilin genes. Some representative N variants are shown in Fig. 2, demonstrating the loss of pitting and spreading on agar. Some phase variation events in N. gonorrhoeae MS11, which has two complete pilin genes, can be detected grossly by size changes in restriction fragments containing the expression loci (Segal et al., 1986). In N. gonorrhoeae strains with a single copy of pilE are nonreverting N types because none of the silent copies can supply promoter and N-terminal sequence to reconstitute the expression copy. Fig. 3 shows that no size change can be detected in EcoRI or MspI fragments

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Fig. 1. The genetic organization of four type-4 pilin-encoding genes ( labeled A, B and C ) in K. denitrificans ATCC 33394. Arrows under genes show direction of transcription. Angled lines labeled IR represent 18-bp IR sequences and 1/2 IR in pKd20 represents the single copy of the 18-bp IR sequence. Map D represents an unlinked, non-pilus gene (orfX ) which is also flanked by the 18-bp IR sequences. B=BamHI; C=ClaI; E=EcoRI; H=HindIII; M=MspI; N=NdeI; P=PstI; Pv=PvuII.

Fig. 2. Variants of K. denitrificans ATCC 33394 were grown on 5% human blood agar. These variants were photographed with reflected light after 48 h growth. A, SC; B, N1; C, N5; D, N6.

when 14 independently isolated K. denitrificans N variants were hybridized with the tfpA PCR probe. Thus, no gross changes appear to take place in any of the pilin gene loci in going from SC to N phenotypes. Immunoblots of these same 14 K. denitrificans N variants as detected by antisera raised against pilin from the SC variant demonstrated that most still produced pilin ( Fig. 4). N3, N4, N5 and N7 pilins reacted identically to the parent SC variant, while N6, N8 and N10

produced no immunoreactive pilin, and N1 produced a slower migrating pilin. N11 either overproduced pilin or manufactured a pilin with enhanced reactivity with the antisera, while N14 either underproduced pilin or produced one altered so that it demonstrated a decrease in reactivity with the antisera. N1, N2, N9, N11, N12 and N13 differ from the SC variant and the other N variants by possessing a smeared band smaller in size than the major pilin band that may correspond to the truncated and secreted S-pilin observed in some N variants of N. gonorrhoeae (Haas et al., 1987). EM examination of these N variants showed that most of them were still capable of making pili, although they were less abundant than those of the parent SC variant. Fig. 5 shows EM micrographs of representative N variants. Southern hybridization resulted in identical bands for all N variants and the parent SC variant (data not shown). Table 1 is a composite of results from the immunoblots and EM. Of the 14 N variants, only N10 was nonpiliated and produced no immunoreactive pilin. N1, N3, N4 and N12 were nonpiliated but still produced pilin comparable in size and quantity to the SC variant, except for N1 whose pilin was slightly larger. N6 and N8 were of particular interest because they did not produce any pilin detectable with this antiserum on the immunoblot, but were found to be piliated by EM. These may represent cases where the pili on the SC variant were composed of pilin from one of the three

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Fig. 4. Immunoblot analysis of SC and N variants of K. denitrificans ATCC 33394 using antisera directed against denatured pilin of this strain. Lanes 1 and 9: SC; lanes 2–8 contain N1–N7, respectively, while lanes 10–16 contain N8–N14, respectively. Proteins of whole cell extracts of bacteria were boiled in loading buffer, separated according to molecular weight by 0.1% SDS-15% PAGE, and transferred to a PVDF (Bio-Rad ) membrane ( Towbin et al., 1979). Rabbit antiserum was raised against denatured purified pilin of K. denitrificans and diluted 1:1000. Fig. 3. The tfpA PCR probe hybridized to chromosomal DNA digested with either EcoRI (panel A) or MspI (panel B) from SC and N variants of K. denitrificans ATCC 33394. Lanes 1–13 contain variants N1–N13, respectively; lane 15: SC. Crude DNA was isolated from K. denitrificans strains by harvesting growth from one agar plate and suspending in phosphate-buffered saline (0.01 M phosphate, 0.85% NaCl, pH 7.6). After centrifugation, the pellets were suspended in SET and lysozyme (10 mg/ml in SET ). This was placed on ice with occasional mixing for 15 min. 100 ml of proteinase K (20 mg/ml in water), 1 ml of 0.5 M EDTA, and 500 ml of 10% Sarkosyl were added and mixed well. This was incubated at 65°C for 1 h to lyse the bacteria. Extractions were performed with a 1:1 mix of phenol/chloroform. The DNA was ethanol precipitated, centrifuged, and resuspended in TE buffer. Chromosomal DNA (5 mg) was digested with EcoRI and MspI and fractionated by electrophoresis through a 1% agarose gel. Transfer of DNA from the gene to Hybond-N (Amersham) nylon membrane was performed by the method of Southern (1975). The 120-bp PCR product of the N terminus of the type-4 pilin-encoding gene of M. nonliquefaciens 7784 was labeled with [a-32P]dCTP (Dupont-New England Nuclear) using a Megaprime DNA labeling kit (Amersham). The nylon filters containing the transferred DNA were hybridized with the probe at 56°C in 6×SSC and washed with 2×SSC.

genes which code for identical aa, while N6 and N8 produce pili composed of KdpD pilins (or vice versa). The pili present on N6 and N8 also appeared morpho-

logically different from the SC variant, being fewer in number and aggregated into bundles (Fig. 5E). In some type-4 pilus systems, single aa substitutions within the main pilin subunit gene can affect autoagglutination and other phenotypic characteristics, while still retaining the presence of pili (Chiang et al., 1995). Alternatively, in Pseudomonas aeruginosa it has been shown that mutations in genes located distant from pilin, such as pilT and pilU, can result in piliated organisms which have lost twitching motility and have altered colony morphology ( Whitchurch et al., 1991; Whitchurch and Mattick, 1994). Further investigation will be required to determine what kinds of alterations exist in our K. denitrificans strains.

3. Conclusions K. denitrificans ATCC 33394 has more complete type-4 pilin-encoding genes than any previously reported bacterium. The hypothesis that K. denitrificans contained multiple

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Fig. 5. EM micrographs of SC and N variants of K. denitrificans ATCC 33394. A, SC; B, N1; C, N2; D, N5; E, N6; F, N11. Specimens were prepared for EM by touching Formvar-coated grids to 24-h-old colonies. The grid was then washed with a drop of 1% ammonium acetate and blotted dry. The specimens were stained with a drop of 1% uranyl acetate for 10 s, blotted dry and then air dried. The grids were examined in a Zeiss EM-10-CA electron microscope.

Table 1 Composite of phenotypes seen for SC and N colony variants of K. denitrificans ATCC 33394 Varian

SC N1 N2 N3 N4 N5 N6 N7 N8 N9 N10 N11 N12 N13 N14

Pilin immunoblots

EMa

Quantity

Size (kDa)

++ ++ ++ ++ ++ ++ − ++ − ++ − ++++ ++ ++ +

16 >16 16 16 16 16 16 16 16 16 16 16 16 16 16

numerous pili no pili occasional pili no pili no pili rare pili bundles rare pili bundles occasional pili no pili occasional pili no pili occasional pili occasional pili

−, no pilin detected by immunoblots; +, small amounts of pilin detected by immunoblots; ++, moderate amounts of pilin detected by immunoblots; ++++, large amounts of pilin detected by immunoblots. aNumber and morphology of pili.

partial, silent copies of type-4 pilin genes analogous to the gene organization of N. gonorrhoeae has not been confirmed, rather the multiple bands of hybridization

previously reported appear to be due to cross-reaction with multiple copies of 18-bp IR structures. N colony variants have multiple distinguishable phenotypes, and many still produce pilin and/or pili.

Acknowledgement We thank Tone Tønjum for providing Kingella strains, protocols, and advice, and Karen DeVries for technical assistance. This work was supported by Public Health Service grant R01-EY07125 from the National Eye Institute.

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