Immunization Against Experimental Pseudomonas Oeruginosaand

Immunization Against Experimental Pseudomonas oeruginosa and Serratia marcescens Keratitis

Vaccination With Lipopo/ysaccharide Endotoxins and Proteases

Arnold S. Kreger,* David M. Lyerly,*f Linda D. Hozleff4 and Richard 5. Berk§

Rabbits vaccinated with lipopolysaccharide endotoxins or with purified protease preparations from Pseu- domonas aeruginosa and Serratia marcescens before corneal challenge with the viable bacteria exhibited significantly less corneal damage than rabbits not vaccinated with the bacterial products. However, the rabbits vaccinated with the lipopolysaccharide endotoxin preparations were significantly better protected than rabbits vaccinated with the bacterial proteases. Rabbits vaccinated with antisera raised against the proteases showed significantly less corneal damage than rabbits vaccinated with normal rabbit serum, and the passive protection was not significantly different than that elicited by active immunization against the bacterial proteases. The ability of the antiserum raised against the pseudomonas elastolytic protease to passively protect against severe corneal damage produced by experimentally induced pseudomonas keratitis was confirmed in mice. These findings support the idea that the bacterial endotoxins and proteases are virulence factors during the development of pseudomonas and serratia keratitis. Invest Ophthalmol Vis Sci 77:932-939, 1986

The opportunistic bacterial pathogens Pseudomonas disease in mice than proteolytic strains. Although their aeruginosa and Serratia marcescens produce fulmi- intracorneal administration does not produce rapid nating and highly destructive corneal infections in hu- liquefactive necrosis as does administration of the pro- mans and laboratory animals.1"4 Proteases of the bac- teases, the lipopolysaccharide endotoxins of the bacteria teria are thought to be important in the pathogenesis also have been implicated in the pathogenesis of corneal of pseudomonas and serratia corneal diseases because disease because they elicit extensive opacification and intracorneal administration of submicrogram amounts polymorphonuclear leukocyte infiltration of the cor- of the highly purified proteases rapidly elicits corneal nea,"12 which are characteristic features of pseudo- damage that closely resembles, both grossly and mi- monas and serratia keratitis. In this communication, croscopically, the damage produced during corneal in- we report that the severity of experimentally induced fections with the bacteria.4"7 The enzymes (1) cause corneal disease by P. aeruginosa and S. marcescens is liquefactive necrosis of the cornea,4"7 which is a char- significantly reduced by immunization against either acteristic feature of pseudomonas and serratia keratitis, the lipopolysaccharide endotoxins or the proteases of and (2) digest the proteoglycan ground substance,4'6'8'9 the bacteria. These findings further support the idea a major structural component of the cornea. Also, Ka- that the bacterial products are important in the patho- waharajo and Homma10 reported that nonproteolytic genesis of pseudomonas and serratia keratitis. strains of/*, aeruginosa cause much less severe corneal Materials and Methods From the Department of Microbiology and Immunology,* The Bowman Gray School of Medicine of Wake Forest University, Win- Bacteria ston-Salem, North Carolina, and the Departments of Anatomy}: and Immunology/Microbiology,§ Wayne State University School of P. aeruginosa strain 5-31 (Fisher immunotype 2; Medicine, Detroit, Michigan. Supported by grants EY-01104, EY-02986, EY-01935, and Core Habs immunotype 11), isolated from a human corneal vision grant EY-04068 from the National Eye Institute. infection, was originally studied and was kindly sup- t Present address: Department of Anaerobic Microbiology, Virginia plied by John Gerke.13 5". marcescens strain BG (bio- Polytechnic Institute and State University, Blacksburg, VA 24061. type Ala) was obtained from the culture collection of Submitted for publication: August 2, 1985. the Department of Microbiology and Immunology of Reprint requests: Arnold S. Kreger, PhD, Department of Micro- biology and Immunology, Bowman Gray School of Medicine, 300 the Bowman Gray School of Medicine. The bacteria 414 South Hawthorne Road, Winston-Salem, NC 27103. were cultivated as previously described.

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Table 1. Grossly observable corneal disease in nonimmunized and immunized rabbits challenged with P. aeruginosa 5-31 Number of corneas exhibiting corneal disease grade of* Average corneal Vaccine 0 I 2 3 4 5 6 damage ± SD*f PBS-incomplete FA (control) [10]:|: 0 0 0 0 5 4 1 4.6 ± 0.2 Pseudomonas elastase-incomplete FA [13] 0 2 4 3 2 2 0 2.9±0.4(<0.001) Heptavalent pseudomonas LPS endotoxin [20] 14 5 1 0 0 0 0 0.4 ±0.1 (<0.001) Normal rabbit serum (control) [10] 0 0 0 0 5 5 0 4.5 ± 0.2 Anti-pseudomonas elastase antiserum [12] 0 0 2 3 4 3 0 3.7±0.3(<0.01)

* Results shown are 9 days after challenge. Corneal disease was graded ac- the 95% significance level for the active immunization studies is P = 0.025. cording to the descriptions in the legend for Figure 1. The 95% significance level for the passive immunization study is P = 0.05. t P values are in parentheses. Because of multiple-comparison corrections, X Numbers in brackets indicate the number of corneas challenged.

Vaccines and Active Immunization Protocols complete FA. Rabbits were bled 7 to 10 days after the last vaccinations and precipitating antibody titers (op- All investigations involving animals reported in this timal proportions zones) of the sera from the control study conformed to the ARVO Resolution on the Use animals and from the animals vaccinated with the pro- of Animals in Research. The heptavalent pseudomonas teases were determined against the purified proteases lipopolysaccharide (LPS) vaccine (Pseudogen®, lot as described by Garvey et al.20 The rabbits were chal- #43152; Warner-Lambert/Parke, Davis & Co., Detroit, lenged 7 to 10 days after the last vaccinations with the MI) was kindly provided by George Cole and Robert proteases. Brackett. The vaccine has been previously described1516 and contains the Fisher immunotype 2 LPS, which Antisera and Passive Immunization Protocols corresponds to our P. aeruginosa challenge strain 5- 31. Twenty New Zealand white rabbits, each weighing Monospecific rabbit antisera were raised against the ca. 2.7 kg, were bled prior to vaccination (for preim- purified proteases by the same vaccination protocol munization sera) and were injected intramuscularly on used to actively immunize rabbits, and the antisera days 0, 4, 9, and 14 with 135 fig of LPS vaccine in 0.25 were lyophilized and stored at 4°C. The monospecif- ml of sterile 0.9% saline (50 ng LPS per kg). The animals icity of the antisera was confirmed by crossed immu- tolerated this regimen well, with no local abscesses, noelectrophoresis against crude protease preparations illness, or unexpected deaths occurring. A phenol- as previously described.21 In addition to estimating the killed, heat-treated serratia LPS vaccine was prepared precipitating antibody titers of the antisera (as men- as described by McMeel et al17 for a pseudomonas LPS tioned in the preceding paragraph), the ability of the vaccine, and six rabbits were injected intravenously IgG fractions of the control serum and the antisera to with the vaccine over a 3-wk period as described by inhibit the cornea-damaging activity of the pseudo- McMeel et al.17 Agglutinating antibody titers of the monas and serratia proteases was determined. The IgG preimmunization sera and of the sera obtained from fractions were obtained by chromatography on a col- rabbits 7 to 10 days after the last injections with the umn of DEAE Affi-Gel Blue (Bio-Rad Laboratories, LPS vaccines were determined by a tube agglutination Inc.; Richmond, CA) as recommended by the manu- method (against heat-killed bacteria) as described by facturer. Mixtures (1 ml) containing the purified pseu- Garvey et al.18 The rabbits were challenged 7 to 10 domonas or serratia protease (1 protease unit) and IgG days after the last injections with the LPS vaccines. (3 mg) from either control rabbit serum or monospe- The elastolytic protease of P. aeruginosa 5-31 and cific antiprotease antiserum were incubated in 0.05 M the nonelastolytic, neutral metalloprotease of S. mar- Tris-HCl buffer (pH 7.5) at 25°C for 60 min. After cescens BG were purified as previously described.6'19 incubation, samples (30 to 40 /il) of the mixtures were Groups of rabbits (13 rabbits per group) were vacci- injected intracorneally into rabbits (as described in the nated subcutaneously every 2 wk for a total of four Experimental keratitis and statistical analysis section vaccinations with 0.5 ml of water-in-oil emulsions of the Materials and Methods), and the corneas were containing 1 mg of purified protease in incomplete observed for 8 hr to note any reduction in grossly ob- Freund adjuvant (FA). Twenty-four control animals servable cornea-damaging activity of the proteases. were vaccinated with an emulsion composed of 0.02 Two series of experiments utilized passive immu- M phosphate-buffered saline (PBS, pH 7.4) and in- nization protocols. In the first series of experiments,

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Fig. 1. Corneal disease observed in nonimmunized and actively immunized rabbits challenged with P. aeruginosa 5-31 or S. marcescens BG. Results shown are 9 days after challenge with viable bacteria suspended in PBS. A, Normal cornea (grade 0) typically observed in rabbits fully protected by vaccination with the preparation of heptavalent pseudomonas lipopolysaccharide endotoxins. B, Mild conjunctivitis and faint opacity (grade 1) typically observed in rabbits partially protected by the vaccination protocols. C and D Dense opacity over part of pupillary area and dense opacity over entire pupillary area (grades 2 and 3, respectively) typically observed in rabbits partially protected by the vaccination protocols. E through H, Extensive opacity and central liquefactive necrosis (grade 4), and extensive liquefactive necrosis and corneal protrusion (descemetocele formation) (grade 5) typically observed in nonvaccinated rabbits and in rabbits not protected by the vaccination protocols. The lesions shown in E and G (grades 4 and 5, respectively) were produced by P. aeruginosa 5-31, and the lesions shown in F and H (grades 4 and 5, respectively) were produced by S. marcescens BG. Grade 6 corneal damage includes corneal perforation.

three different groups of rabbits (10 to 14 rabbits per according to the criteria described in the legend for group) were vaccinated intramuscularly, 24 hr prior to Figure 1. and 48 hr after corneal challenge, with 5 ml of 2X The mice vaccinated with normal rabbit serum and concentrated preparations (150 mg/ml) of (1) control with anti-pseudomonas elastase antiserum were chal- rabbit serum, (2) monospecific anti-pseudomonas lenged with P. aeruginosa 5-31 by the previously de- elastase antiserum, and (3) monospecific anti-serratia scribed procedure of Hazlett et al.22 Briefly, mice were protease antiserum. In the second series of experiments, anesthetized with ether, placed beneath a stereoscopic 6-wk-old, female C57BL/6J mice (Jackson Laborato- microscope, and the left eye of each animal was ries; Bar Harbor, ME) were injected intraperitoneally, wounded by gently scratching the corneal surface with 1 to 3 hr prior to and 72 hr after corneal challenge, a sterile 26-gauge needle. Five microliters of bacterial with 0.2 ml of 2X concentrated control rabbit serum cell suspension (ca. 1 X 108 viable organisms) were or monospecific anti-pseudomonas elastase antiserum. delivered onto incised corneas with a micropipet having a sterile disposable tip, and the subsequent course of Experimental Keratitis and Statistical Analysis the infection was examined visually at 3-day intervals The control rabbits and the rabbits that were vac- for 4 wk. The corneas also were examined by light mi- cinated with the LPS and protease preparations and croscopy. The grades of grossly observable corneal with the anti-protease antisera were anesthetized by damage used for the mice were similar to those used intramuscular injections of ketamine hydrochloride (35 for the rabbits, except grades 2 and 3 were combined mg/kg) and xylazine hydrochloride (3 mg/kg) and by into grade 2, and grades 4, 5, and 6 were designated 3, topical application of 0.5% tetracaine hydrochloride. 4, and 5, respectively. The central portion of the left cornea of each rabbit The average corneal damage indicated in Tables 2, challenged with P. aeruginosa 5-31 was abraded with 3, and 4 is a weighted mean estimated after determining a sterile 18-gauge needle in a crosshatching manner the number of corneas exhibiting the various grades of and the challenge dose (ca. 109 viable organisms in 50 corneal disease, and is expressed as n\ of 0.9% saline) was immediately dropped onto the Average corneal damage traumatized cornea. The central portion of the left cor- S(corneal damage grade nea of each rabbit challenged with S. marcescens BG _ X # of corneas in the grade) was injected (30-gauge, 0.5-in needle) with the challenge dose (ca. 6 X 104 viable organisms in 40 ix\ of total # of corneas challenged 0.9% saline). The subsequent course of the keratitis The average corneal damage values were analyzed was examined visually each day for 9 days, and the for statistical significance by generalized categorical severity of the grossly observable lesions was scored data analysis using weighted least squares.2324 A mul-

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Table 2. Grossly observable corneal disease is nonimmunized and immunized rabbits challenged with S. marcescens BG

Number of corneas exhibiting corneal disease grade of* Average corneal Vaccine 0 1 2 3 4 5 6 damage ±SD*-f

PBS-incomplete FA (control) [14] % 0 0 0 0 5 8 1 4.7 ±0.2 Serratia protease-incomplete FA [13] 0 1 5 5 1 1 0 2.7±0.3(<0.001) Phenol-killed 5". marcescens BG[6] 0 2 4 0 0 0 0 1.7 ± 0.2 (<0.001) Normal rabbit serum (control) [13] 0 0 0 0 5 7 1 4.7 ± 0.2 Anti-serratia protease antiserum [14] 0 0 2 8 3 1 0 3.2±0.2(<0.001) * Results shown are 9 days after challenge. Corneal disease was graded ac- the 95% significance level for the active immunization studies is P = 0.025. cording to the descriptions in the legend for Figure 1. The 95% significance level for the passive immunization study is P = 0.05. t P values are in parentheses. Because of multiple-comparison corrections, X Numbers in brackets indicate the number of corneas challenged.

tiple-comparison correction was used, when necessary, challenge with the viable bacteria exhibited significantly to correct the P values (<0.05) considered significant. less corneal damage than rabbits not vaccinated with the bacterial products (Fig. 1, Tables 1 and 2). However, Light Microscopy the rabbits vaccinated with the pseudomonas LPS Mice were killed by intraperitoneal injection of so- preparation were significantly better protected than dium pentobarbital. The eyes were enucleated, washed rabbits vaccinated with the pseudomonas elastolytic briefly with sodium phosphate buffer (0.2 M, pH 7.4), protease (P = <0.001), and rabbits vaccinated with the and fixed intact for 3 hr at 4°C in sodium phosphate serratia LPS preparation were significantly better pro- buffer containing 1% osmium tetroxide and 2.5% glu- tected than rabbits vaccinated with the serratia protease taraldehyde. After fixation, specimens were rinsed in (P - 0.002). The main difference between the results sodium phosphate buffer (0.1 M, pH 7.4) and dehy- obtained with the LPS and protease preparations was drated with ethanol and propylene oxide. Specimens that vaccination with the proteases protected against were infiltrated with a mixture of Epon-araldite and corneal melting (liquefactive necrosis) but not against propylene oxide (1:1 for 24 hr, followed by 3:1 for 24 corneal opacification, but vaccination with the LPS hr) and were infiltrated with Epon-araldite for 1 wk preparations protected against both corneal melting before embedding. Thick sections (\.5-fim) were and opacification. The sera of the LPS-vaccinated rab- stained with a modified Richardson stain25 and were bits had agglutination titers of 300 to 600; the titers of examined and photographed with a Zeiss (Oberkochen, the preimmunization (control) sera were <5. West Germany) automatic photomicroscope with The two groups of rabbits vaccinated with the an- bright-field optics. tiprotease antisera exhibited significantly less corneal damage than rabbits vaccinated with normal rabbit Results serum (Tables 1 and 2), and the protection was not significantly different than that elicited by active im- Active and Passive Immunization of Rabbits Against munization against the bacterial proteases. The ability Pseudomonas and Serratia Keratitis of the antisera to passively immunize against corneal The four groups of rabbits vaccinated with the LPS melting was associated with high titers of precipitating preparations and protease preparations before corneal antibodies against the proteases (optimal proportions

Table 3. Passive immunization of mice against corneal disease produced by P. aeruginosa 5-31

Number of corneas exhibiting corneal response*

Corneal perforation Clear Slight opacity and eye shrinkage Average corneal Vaccine (Grade 0) (Grade 1) (Grade 5) damage ± SD*i[

Normal rabbit serum 0 0 51 Anti-pseudomonas elastase antiserum [105] 20 17 68 3.4±0.2(<0.001)

* Results shown are 4 wk after challenge. % Numbers in brackets indicate the number of corneas challenged. t P value is in parentheses. The 95% significance level is P = 0.05.

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Fig. 2. Light microscopy of uninfected mouse corneas and of mouse corneas 4 wk after corneal challenge of unimmunized and passively immunized mice with P, aetuginosa 5-31. A, Prior to corneal scarification and topical application of PBS, mice received 0.2 ml of PBS or 2X normal rabbit serum intraperitoneally (uninfected controls). The corneal epithelium (E) and stroma (ST) appear normal (X570). B and C, Corneas protected (grade 0) by vaccination with the antielastase antiserum exhibited either a normal ap- pearing epithelium (E; Fig. 2B) or an epithelium (E) that contained goblet cells (arrow) and widened intercellular junctions (Fig. 2C). In both cases, stromal collagen is more normally arranged than collagen in corneas from unvaccinated, infected mice. Scattered blood vessels (BV) are also apparent. Descemet's membrane and the endothe- lium appear slightly thick- ened (X400 Fig. 2B; X320 Fig. 2C). D, Corneas partially protected (grade 1) by the antielastase antiserum exhibited an epithelium containing numerous conjunc- tiva! goblet cells (arrow) that resurfaced the corneal epithelium. Although the super- ficial stroma (S) is disorganized, the collagen in the deep stroma (D) is more reg- ularly arranged than collagen in unimmunized corneas (X400). E, Corneas not protected (grade 5) by the antielastase antiserum and corneas from unvaccinated, infected mice exhibited epithelial basal cells with markedly widened intercellular junctions (arrow). In addition, the stroma is disorganized, Descemet's membrane (DM) is tortuous, and both anterior and posterior synechia are seen. Cataractous changes in the lens (L) are also apparent (X120).

zones of 0.05 to 0.1% antigen) and with the ability of IgG from the antisera did not cause corneal melting the IgG fractions of the antisera to inhibit the cornea- or opacification by 8 hr postinjection. damaging activity of the proteases. Intracorneal injections of protease preparations preincubated with IgG Passive Immunization of Mice Against from normal rabbit serum elicited extensive corneal Pseudomonas Keratitis melting and opacification by 3 hr postinjection, but The ability of the anti-pseudomonas elastase anti- injections of protease preparations preincubated with serum to passively protect against severe corneal dam-

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age produced by experimentally induced pseudomonas than anti-pseudomonas elastase IgG.35 It has also been keratitis was confirmed in mice (Table 3 and Fig. 2). reported that mice immunized against burn wound sepsis by vaccination with pseudomonas LPS clear the Discussion bacteria from their tissues faster than nonimmunized mice,34 and that protective anti-LPS antibodies act as Various investigators have observed that rabbits opsonins to increase the efficiency of phagocytosis and vaccinated with phenol- and heat-killed P. aerugi- killing of P. aeruginosa by host phagocytes.36'37 We nosa,l7>26 with sterile culture filtrates and slime extracts have found during our studies that antibodies against of P. aeruginosa,26'27 and with gamma globulin ob- the pseudomonas and serratia proteases inhibit the tained from rabbits vaccinated with the killed bacteria28 cornea-damaging activity of the enzymes; however, are protected against severe corneal disease by the ho- there is no published data to indicate that the antibodies mologous bacterial strains. Also, mice have been ac- inhibit in vivo growth or enhance clearance of the bac- tively immunized against pseudomonas keratitis by teria from the cornea. Thus, the pseudomonas and ser- oral or intraperitoneal administration of phenol-killed ratia LPS preparations may be more effective immu- suspensions of the homologous challenge strain.29 nogens than the bacterial proteases in our rabbit ker- Studies with purified antigens and their antisera have atitis model because anti-LPS antibodies enhance shown that vaccination with a multicomponent vaccine clearance of the bacteria from infected corneal tissue, containing the common protective antigen (OEP) of whereas anti-protease antibodies only inhibit a tissue- P. aeruginosa and the elastolytic protease and alkaline damaging bacterial virulence factor without aiding protease of the bacterium partially protects rabbits and ' elimination of the bacteria. 30 31 horses against pseudomonas keratitis ' and acts syn- An important question that should be examined in ergistically with the antibiotic dibekacin to protect mice future studies is whether the pseudomonas elastolytic against severe corneal damage by a heterologous strain protease and the serratia neutral metalloprotease used 32 of P. aeruginosa. Also, rabbit antiserum containing in our studies are produced in vivo during the devel- antibodies against the pseudomonas OEP and proteases opment of corneal infections. Kawaharajo and (detected by a passive hemagglutination assay) has been Homma10 found that P. aeruginosa strain PA-103, a reported to act synergistically with dibekacin to pas- strain that does not produce detectable amounts of the sively protect mice against severe corneal damage by elastolytic protease in vitro but produces the alkaline 32 a heterologous strain of P. aeruginosa. In addition, protease in vitro,1038 causes less severe corneal disease 33 Spierer and Kessler recently observed that the com- in mice than elastolytic isolates of the bacterium. bined topical application of 2-mercaptoacetyl-L-phe- However, Ohman et al38 did not observe a significant nylalanine-L-leucine (an inhibitor of the P. aeruginosa difference between the severity of corneal damage pro- elastase) and suboptimal doses of gentamicin to rabbit duced in mice by strain PA-103 and by elastase-pro- corneas infected with P. aeruginosa inhibits corneal ducing strains of P. aeruginosa. Also, Ohman et al did melting during the early stages of the developing ker- not observe a significant difference in the mouse corneal atitis (first 28 hr) significantly better than treatment virulence of P. aeruginosa mutant strain PAO-E64, a with gentamicin alone. The results in our paper extend strain that produces an elastase with reduced enzymatic the findings in the previous publications by demon- activity,39 and the parental strain. Ohman et al sug- strating that the severity of experimentally induced gested that if proteolytic activity is required for corneal corneal disease by P. aeruginosa and S. marcescens is damage by P. aeruginosa strains PAO-E64 and PA- significantly reduced by immunization against either 103, then it is supplied by the alkaline protease of the the LPS endotoxins or the proteases of the bacteria. strains rather than the elastolytic protease. Intracorneal These findings further support the previously proposed injection of very small amounts of the pseudomonas 3 12 idea " that the bacterial products are virulence factors elastase (ie, 0.03 protease units; 30 /xl of a solution during the development of pseudomonas and serratia containing 1 unit per ml) produces rapid and extensive keratitis. liquefactive necrosis of the cornea.6 Therefore, an al- Our observation that vaccination against the pseu- ternative explanation for the observations of Ohman domonas and serratia LPS endotoxins protects rabbits et al38 is that strains PA-103 and PAO-E64 may pro- significantly better against experimental keratitis than duce an amount of elastolytic protease activity in vivo does vaccination against the bacterial proteases is con- that is sufficient to cause severe corneal damage, even sistent with previous observations made with a burn though strain PA-103 does not produce detectable wound sepsis model. Cryz et al found that P. aeruginosa amounts of elastase in vitro and strain PAO-E64 pro- LPS is a very effective immunogen against pseudo- duces a mutant enzyme with reduced activity. The monas burn wound sepsis in mice34 and that anti-LPS elastolytic protease has been detected, by indirect im- IgG protects mice against burn wound sepsis better munofluorescence tests, in lung tissue sections of rats

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having an experimentally induced chronic pseudo- 6. Kreger AS and Gray LD: Purification of Pseudomonas aeruginosa monas pneumonia caused by strain PA-103.40 Another proteases and microscopic characterization of pseudomonal protease-induced rabbit corneal damage. Infect Immun 19:630, possible explanation for the apparent lack of correlation 1978. between in vitro elastase production and mouse corneal 7. Kawaharajo K, Abe C, Homma JY, Kawano M, Gotoh E, Ta- virulence for strain PA-103 is based on the idea that if naka N, and Morihara K: Corneal ulcers caused by protease and a virulent strain does not produce a particular factor, elastase from Pseudomonas aeruginosa. Jpn J Exp Med 44:435, that does not mean the factor is not important for dis- 1974. ease production, only that it is not essential for the 8. Kessler E, Kennah HE, and Brown SI: Pseudomonas protease. Purification, partial characterization, and its effect on collagen, particular strain examined. Thus, strain PA-103 may proteoglycan, and rabbit corneas. Invest Ophthalmol Vis Sci 16: not require the elastase for disease production as long 488, 1977. as it can produce the alkaline protease. Howe and Ig- 9. Brown SI, Bloomfield SE, and Tam WI: The cornea-destroying lewski41 recently reported that (1) strain PA-103 mu- enzyme of Pseudomonas aeruginosa. Invest Ophthalmol 13:174, tants that are markedly deficient in alkaline protease 1974. 10. Kawaharajo K and Homma JY: Pathogenesis of the mouse ker- production in vitro are less virulent for the mouse cor- atitis produced with Pseudomonas aeruginosa. Jpn J Exp Med nea than is the parental strain, and (2) the addition of 45:515, 1975. subdamaging amounts of either alkaline protease or 11. Bernstein HN and Maddox YT: Cornea pathogenicity of Serratia elastolytic protease to corneas infected with the alkaline marcescens in the rabbit. Trans Am Acad Ophthalmol Otolar- protease-deficient mutants results in infections that are yngol 77:432, 1973. 12. Marzulli FN, Evans JR, and Yoder PD: Induced Pseudomonas indistinguishable from infections caused by the parental keratitis as related to cosmetics. J Soc Cosmet Chem 23:89, 1972. strain. Their data suggest the possibility that although 13. Gerke JR and Magliocco MV: Experimental Pseudomonas an extracellular protease is required for maximal mouse aeruginosa infection of the mouse cornea. Infect Immun 3:209, corneal virulence of strain PA-103, a single specific 1971. protease is not required (ie, either the elastolytic pro- 14. Gray LD and Kreger AS: Rabbit corneal damage produced by Pseudomonas aeruginosa infection. Infect Immun 12:419, 1975. tease or the alkaline protease will suffice). The obser- 38 15. Pennington JE: Lipopolysaccharide pseudomonas vaccine: effi- vation that toxin A-deficient mutants derived from cacy against pulmonary infection with Pseudomonas aeruginosa. either strain PA-103 or PAO are less virulent for the J Infect Dis 140:73, 1979. mouse cornea than are their toxigenic parental strains 16. Hanessian S, Regan W, Watson D, and Haskel TH: Isolation underscores the possible multifactorial requirements and characterization of antigenic components of a new heptavalent Pseudomonas vaccine. Nature 229:209, 1971. for corneal damage by P. aeruginosa. 17. McMeel JW, Wood RM, and Senterfit LB: Active immunization against pseudomonas infection of the cornea. Trans Am Acad Key words: immunization, keratitis, Pseudomonas aerugi- Ophthalmol Otolaryngol 64:490, 1960. nosa, Serratia marcescens, endotoxin, proteases 18. Garvey JS, Cremer NE, and Sussdorf DH: Methods in Immu- nology, 3rd edition. New York, W.A. Benjamin, 1977, pp. 372- Acknowledgments 374. 19. Lyerly D and Kreger A: Purification and characterization of a The authors thank George Cole and Robert Brackett Serratia marcescens metalloprotease. Infect Immun 24:411,1979. (Warner-Lambert/Parke; Davis & Co., Detroit, MI) for sup- 20. Garvey JS, Cremer NE, and Sussdorf DH: Methods in Immu- plying the heptavalent pseudomonas lipopolysaccharide vac- nology, 3rd edition. New York, W.A. Benjamin, 1977, pp. 280- cine and for immunotyping the P. aeruginosa strain used in 283. this study, and thank Patrick A. D. Grimont (Pasteur Insti- 21. Lyerly DM and Kreger AS: Importance of serratia protease in tute, Paris) for biotyping the S. marcescens strain used in the pathogenesis of experimental Serratia marcescens pneu- this study. monia. Infect Immun 40:113, 1983. 22. Hazlett LD, Rosen D, and Berk RS: Experimental eye infections References caused by Pseudomonas aeruginosa. Ophthalmic Res 8:311, 1976. 1. Kreger AS: Pathogenesis of Pseudomonas aeruginosa ocular dis- 23. Grizzle JE, Starmer CF, and Koch GG: Analysis of categorical eases. Rev Infect Dis 5:S931, 1983. data by linear models. Biometrics 25:489, 1969. 2. Laibson PR: Annual review. Cornea and sclera. Arch Ophthalmol 24. Forthofer RN and Lehnen RG: Public Program Analysis. A New 88:553, 1972. Categorical Data Approach. Belmont, CA, Lifetime Learning 3. Kamata R, Yamamoto T, Matsumoto K, and Maeda H: A ser- Publications, 1981, pp. 75-97. ratial protease causes vascular permeability reaction by activation 25. Richardson KC, Jarett L, and Finke EH: Embedding in epoxy of the Hageman factor-dependent pathway in guinea pigs. Infect resins for ultrathin sectioning in electron microscopy. Stain Immun 48:747, 1985. Technol 35:313, 1960. 4. Lyerly D, Gray L, and Kreger A: Characterization of rabbit cor- 26. Jackson E and Hartman FW: Experimental Bacilluspyocyaneus neal damage produced by serratia keratitis and by a serratia pro- keratitis. J Lab Clin Med 12:442, 1927. tease. 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