Molecular Microbiology (1993) 7(2), 285-288

Q pili enhance the attachment of Moraxella bovis tobovine corneas in vitro

W. W. Ruehl,̂ -^* C. Marrs,̂ ' M. K. Beard,*V. Shokookj,^ J. R. Hinojoza,^ S. Banks,^ D. Bieber^and J. S. Mattick^Departments of ^Pathology and ^Comparative f^edicine,Stanford University School of Medicine, Stanford,California USA.^Department of Epidemiology, School of Epidemioiogyand Public Health, University of Michigan, Ann Arbor,Michigan, USA.^Department ofAnimai Health, The University of Sydney,Camden, New South Wales, Australia.^Centre for Molecular Biology and Biotechnoiogy,University of Queensiand, St Lucia, Queensland,Australia.

Summary

Moraxella bovis, the causative agent of infectiousbovine keratoconjunctivttis, exhibits several virulencefactors, including pili, haemotysin, leukotoxin, andproteases. The pili are filamentous appendages whichmediate bacterial adherence. Prior studies haveshown that Q-piliated M. bovis Epp63 are more infec-tious and more pathogenic than l-pitiated and non-piliated isogenic variants, suggesting that Q pili perse,or traits associated with Q-pilin expression, promotethe earty association of Q-pillated bacteria with bovinecomeal tissue. In order to better evaluate the role of Qpili in M. bovis attachment, several M. bovis strainsand a recombinant P. aeruginosa strain which elabo-rates M. bovis Q pili but not P. aeruginosa PAK pili,were evaluated using an in vitro corneal attachmentassay. For each strain tested, piliated organismsattached better than non-piliated bacteria. M. bovisEpp63 Q-pJIiated bacteria adhered better than eitherthe l-piliated or non-piliated isogenic variants. Finally,recombinant P. aeruginosa organisms elaborating M.bovis Q pili adhered better than the parent P. aerugi-nosa strain which did not produce M. bovis pili. Theseresults indicate that the presence of pili, especially Qpili, enhances the attachment of bacteria to bovinecornea In vitro.

Received 18 November 1991; revised 16 July 1992; accepted 16 Septem-ber 1992. *For correspondence: Deprenyl Animal Health Inc, 8000 West110th Street, Suite 115, Overland Park, Kansas 66210, USA, Tel, (913) 3382120: Fax (913) 338 3804,

Introduction

Moraxeila bovis is the primary cause of infectious bovinekeratoconjunctivitis (IBK), a highly contagious oculardisease of cattle that can result in temporary (or occas-ionally permananent) blindness (Hughes and Pugh, 1970).Haemolysin and pili have been established as M. bovisvirulence factors (Pugh and Hughes, 1968; Pedersen etal.,1972). The pili are surface appendages of the bacteriumcomposed of repeating, homologous polypeptide sub-units termed pilin. The pili of M. bovis are classified as type4, and the pilin molecule contains a methylated phenyla-lanine (mPhe) residue at the amino terminus (Mattick etai.,1987). We have previously described, characterized, andsequenced two distinct pilin molecules, termed I and Q,from M. bovis strain Epp63 (Ruehl et ai, 1988) and havealso cloned and sequenced both pilin genes (Marrs etai.,1985;Fulkse?a/., 1990).

Type 4 pili are also found on Moraxeila nonliquefaciens(Froholm and Sletten, 1977), Neisseria gonorrhoeae (Her-modson et ai, 1978; Schoolnik et ai, 1984), Neisseriameningitidis (Hermodson et ai, 1978), Bacteriodes nodo-sus (McKern et ai, 1983; 1985), and Pseudomonasaeruginosa (Sastry et ai., 1983). The pilin subunits of thesespecies range in size from 145 to 160 amino acids and aretranslated as prepilin. Prepilin has a six or seven residueleader sequence which when cleaved leaves the modifiedamino acid, methylphenylalanine, as the first reside of themature protein (Bradley, 1980; Mattick etai., 1987; Ruehletai, 1988). The 32 residue amino-terminal regions of alltype 4 mPhe pilins exhibit at least 90% homology and arevery hydrophobic. The carboxy-terminal two-thirds ofthese subunits, however, is relatively hydrophilic and hasmany variable and hypervariable domains. As such, thisregion is the source of both structural and antigenicvariations between species (Mattick et ai, 1987).

It appears that the hydrophobic amino-terminal regionmay have a dual function. First, it has been proposed that itacts, perhaps in conjunction with the short leadersequence, as a signal sequence marking the peptide fortransport to the cell membrane (Elleman and Hoyne,1984). Second, it has been hypothesized that the region isinvolved in pili morphogenesis. There have been twomechanisms proposed for involvement of this region inmorphogenesis: the region could be responsible forsubunit-subunit interactions during pili assembly (Her-modson et ai, 1978; McKern et ai, 1983; Sastry ef ai.

286 W. W. RuehieXal

1 2 3 4 5

CL 1 0 -

Fig. 1. Attachment in vitro of labelled M. bovis and P, aeruginosa tobovine corneal epithelium. Various piliated or non-piliated M. bovis andP. aeruginosa strains were incubated with intact bovine corneas. Detailsof the experimentai methods and data analysis are in the text, 1,Q-ptliated M. bovis Epp63; 2, recombinant P, aeruginosa whichexpresses M. bovis Epp63 Q pilj; 3, l-piliated M. bovis Epp63; 4,non-piliated M. bovis Epp63; 5, PAK-piliated P. aeruginosa. The errorbars indicate ±2 standard deviations.

1983); and/or the amino-terminal region might interactwith other essential factors involved in the assemblysystem. The second hypothesis v*/as suggested on thebasis of the highly conserved sequences in this regionbetween the various bacterial species expressing type 4pili, and is supported by the fact that 6. nodosus pili andM. bovis pili can be assembled from cloned pilin genesexpressed by recombinant P. aeruginosa (Mattick et ai,1987; Beard e( a/., 1990),

Attachment of pathogenic M. bovis to the bovinecorneal epithelial surface is an early {probably the first)event leading to colonization, then to infection and clinicaldisease. This attachment process appears to be medi-ated, at least in part, by the pili v r̂hich are elaborated on thebacterial cell surface. It has been observed that piliated M.bovis attach better to bovine corneas in vitro than donon-piliated bacteria of the same strain {Jackman andRosenbusch, 1984). Furthermore, piliated M. bows causedisease in the experimentally inoculated eyes of calves,while non-piliated isogenic variants do not (Ruehl et al.,1988), Additionally, the Q-piliated variant of strain Epp63was found to be significantly more infectious for experi-mentally inoculated eyes than l-piliated or non-piliatedvariants of Epp63. These results indicated that Q pili perse, or traits associated with Q-pilin expression, promotethe early association of Q-piliated bacteria with bovinecorneal tissue.

We have constructed a recombinant P. aeruginosastrain which expresses M. bovis Epp63 Q pilin andelaborates Q-pili filaments to the exclusion of the normalindigenous Pseudomonas (PAK) pili (Beard ef al., 1990).We then compared this recombinant P. aeruginosa with

the parent, non-piliated P. aeruginosa strain and variousM. bovis strains for their ability to adhere to intact bovinecorneas in vitro.

Results and Discussion

Data regarding attachment of M. bovis, parent P. aerugi-nosa, and recombinant P. aeruginosa bacteria to bovinecorneas is presented in Fig. 1. The previous finding(Jackman and Rosenbusch, 1984) that piliated M. bovis118F attached to corneas significantly better than non-piliated bacteria was confirmed for two additional strains,Epp63 and Tifton-1, suggesting that this is a generalphenomenon for M. bovis.

It is apparent in Fig. 1 that M. bovis Q piliated Epp63bacteria attached significantly better than l-piliated ornon-piliated isogenic variants of the same strain. Thisgreater adherence capability correlates well with the priorobservation that Q-piliated organisms are significantlymore infectious than l-piliated or non-piliated variants ofthe same strain when inoculated into the eyes of calves(Ruehl et ai, 1988; Lepper and Power, 1988), and sug-gests an attachment role for Q pili per se, or for somefactor associated with Q-piliation.

Figure 1 also shows that a recombinant P. aeruginosabacteria which elaborates M. bovis Q pili, but which nolonger expresses PAK pili, attached significantly betterthan the PAK-pili-expressing P. aeruginosa parent strain.The enhancement of attachment provided by Q pili wasgreater for M. bovis than for P. aeruginosa bacteria. Themolecular basis for this is unknown but might be related tothe fact that the recombinant organisms apparentlyexpress fewer pili per bacterial cell than the Q-piliated M.bovis (Fig. 2). Alternatively, other pilus-associated orco-regulated adhesins may be present in Q-piliated M.bovis but absent from the Q-piliated recombinant P.aeruginosa that contains only the Q-pilin subunit genefrom M. bovis (Beard et ai., 1990), Yet another possibleexplanation is that the assembly or presentation of the Qpilus is slightly different in P. aeruginosa compared withthe native Q pilus, and that this could result in the reducedadherence of the recombinant.

There are now several examples of Escherichia coli piliin which the adhesive molecule is a minor pilus componentseparate from the major structural subunit (Lindberg et ai.1984; Klemm and Christiansen, 1987). In contrast, theevidence to date for type 4 piliated bacteria has indicatedthat the major structural subunit is also the adhesin. Irvin efai (1989) and Lee ef al. (1989) have reported fhat theadherence of P. aeruginosa PAK-piliated bacteria tohuman epithelial cells is directly attributable to the pilinsubunif molecule. There is also some evidence that the piliof N. gonorrhoeae is the adhesin (Schoolnik et ai., 1984;Rothbard et ai, 1985). In agreement with the above

Pill-enhanced attachment of Moraxella bovis to corneas in vitro 287

Fig, 2. Transmission electron micrographs of piliated and non-piliated bacteria. Organisms were grown 20-24h at 37°C on GC or L-agar, then transferredto capper grids, negatively stained with 1 % phosphotungstate and photographed using a transmission electron microscope- Strain: A, non-piliated M.bovis Epp63, XI2000; B, Q-piliated M. bows Epp63, x 18000; C, recombinant P. aeruginosa which expresses M. bovis Epp63Q pili. x 18000.

results, this paper provides evidence the M. bovis Q-pili-mediated adherence must be a direct result of thepresence of the Q-pilin molecule.

Experimental procedures

Microorganisms

The bacterial strains used in this study were: M. bovis Epp63(non-piliated, l-piliated, and Q-piliated isogenic variants), M.bovis Tifton-1 (piliated and non-piliated), P. aeruginosa recom-binant PAK/2Pfs (MXB/Mxb6) which express M. bovis Epp63 Qpilin, but not PAK pilin, and exhibits plasmid-mediated resistanceto carbenicillin (Beard etal.. 1990), and the parental P. aeruginosastrain PAK/2Pfs (ATCC 53308) (Bradley, 1974) which expressPAK pilin and fails to grow in the presence of carbenicillin.

Solid media for cultivating microorganisms and performingcolony counts consisted of blood agar (Remel), GC {gonococcal)agar base (Difco Laboratories) to which was added 2% (v/v)IsoVitaleX (BBL Microbiology Systems), and L-agar with orwithout 75 ̂ ig ml ' carbenicillin added. The expression of pilinand the presence of pili were confirmed by immunoblotting(Beard eta/., 1990) and electron microscope inspection (Fig. 2) ofphosphotungstate-stained organisms, respectively.

Preparation of radioiabelled bacteria

For each strain, bacterial growth from two plates was gentlysuspended in 750iil of ice-cold Hanks Balanced Salt Solution(HBSS) (Sigma) and 250ti.l of the following salt solution; 50mMTris-HCI, pH 8, 50mM Na acetate, 140mM NaCI, 5mM CaCIs,4 mM KCI, 2 mM MgCI^, 1 % bovine serum albumin (BSA), and 1 %glycerol. To determine the concentration of eaoh of thesebacterial suspensions, a 1 -̂1 aliquot of each suspension wasdiluted 1:10 ,̂ inoculated onto a blood agar plate, incubatedovernight at 37°C, and the colonies counted. Also, a single drop of

each bacterial suspension was transferred to a microscope slideand Gram-stained. Ten fields were examined using x1000magnification to determine the number of bacterial aggregatespresent. If more than five bacterial aggregates were counted, thesuspension was discarded.

The remainder of each bacterial suspension was labelled byincubation with 20|j.Ci ml ' of L-(4'5-^H]-proline (Amersham) for2 min at 22''C, followed by incubation with 20M.g ml ' unlabelledproline under the same conditions.

Preparation of corneas

Adult cattle eyes were obtained from a local slaughter house andwere transported in ice-cold HBSS to which 100M.g ml " ' genta-micin had been added. Only comeas without visible opacity wereused. Each entire cornea was placed on a flat surface with theepithelial cell layer facing upward. A second matching plasticsurface containing holes was placed on top thereby formingwells: the floor of eaoh well thus consisted exclusively of cornealepithelium. The wells were filled with HBSS until use in theadherence assay, at which time the HBSS was removed and thetissue was washed twice with phosphate-buffered saline (PBS).

Adherence assay

The adherence assay was a modification of one described byJackman and Rosenbusch (1984). One-hundred microlitres of theradiolabelled bacterial suspension were added to each well andthe apparatus was incubated 60 min at 37°C in a moist environ-ment. The reaction was terminated by removal of the unattachedbacteria and the wells washed with PBS. Using a biopsy skinpunch a 9 mm diameter piece of exposed cornea was harvestedfrom each well, transferred to 1 ml of 2% SDS and boiled for 10min. The tubes were centrifuged 2 min; IOOM-I of the supernatantfrom each tube (containing radiolabelled bacterial components)were added to 3 ml of scintillation fluid, and radioactivitydetermined as counts per minute (c.p.m.) using a Beckman 8000

288 W. W. Ruehl et al.

beta counter. In each assay, the background radioactivity (c.p.m.)was determined for one well which contained buffer only. Thepercentage of bacteria attached was calculated by the followingformula:

(cfu attached to cornea) - (cfu onbackground cornea) X (100)

(cfu added to experimental cornea)

The results were derived from three to five experiments with eachmicroorganism. The significance of the differnces in attachmentbetween strains was determined using Students Mest with pairedvalues and a two-tailed hypothesis.

Acknowledgements

This work was supported in part by NIH Eye Institute grant R01EY07125-01 (CM.) and USDA grant 87-CRCR-1 -2403 (W.W.R.).

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