Pneumolysin in pneumococcal adherence and colonization

Microbial Pathogenesis 1998; 25: 337–342 Article No. mi980239

MICROBIAL PATHOGENESIS

SHORT COMMUNICATION

Pneumolysin in pneumococcal adherence and colonization Jeffrey B. Rubinsa∗, Amber Hanson Paddocka, Darlene Charboneaua, Anne M. Berryb, James C. Patonb & Edward N. Janoffc a

Pulmonary and cInfectious Diseases Divisions, Department of Medicine, Veterans Affairs Medical Center and University of Minnesota School of Medicine, Minneapolis, MN 55417, U.S.A., b Department of Microbiology, Women’s and Children’s Hospital, North Adelaide, S.A., 5006, Australia (Received May 12, 1998; accepted in revised form September 11, 1998)

The universal and highly conserved production of pneumolysin, the major pneumococcal cytolysin, among clinical isolates of Streptococcus pneumoniae and the previously reported association of pneumolysin production with increased pneumococcal adherence to respiratory epithelium in organ cultures suggest that this toxin might be important for nasopharyngeal colonization. We confirmed that pneumolysin-deficient mutant pneumococcal strains had decreased adherence to respiratory epithelial cells in vitro compared with their isogeneic wild-type strains. However, neither early nor sustained colonization by type 14 S. pneumoniae in an established murine model was dependent on bacterial production of pneumolysin. We conclude that pneumolysin production is not a major determinant of successful nasopharyngeal colonization by pneumococci.  1998 Academic Press Key words: Streptococcus pneumoniae, pneumolysin, colonization, adherence.

Introduction Streptococcus pneumoniae is a mucosal pathogen which is adapted to colonize and replicate in the human respiratory tract. The bacterial factors which facilitate pneumococcal colonization of the respiratory mucosa have not been completely defined. Because adherence to epithelial cells is

∗ Correspondence to: Pulmonary (111N), One Veterans Drive, Minneapolis, MN 55417, U.S.A. 0882–4010/98/120337+06 $30.00/0

considered the initial step in successful colonization, pneumococcal virulence factors which function as adhesins to isolated epithelial cells or epithelial tissue organ cultures have been sought. The polysaccharide capsule, which is the major virulence factor in invasive infection, appears not to augment adherence and may in some cases actually impede it [1]. In contrast, components associated with the pneumococcal cell wall appear to be major pneumococcal adherence factors [2]. Several lines of evidence suggest that pneumolysin, a major pneumococcal cytolytic toxin,  1998 Academic Press

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contributes to the pathogenesis of invasive disease, including early events in pneumococcal pneumonia [3, 4]. As it is a cytoplasmic protein released only after autolysis, pneumolysin is considered unlikely to participate directly in the interaction between the pneumococcal cell surface and the host epithelium. Nevertheless, it may contribute indirectly to the colonization process through its various inhibitory effects on specific and non-specific host defences, and by directly damaging epithelial surfaces. Consistent with this supposition, recent studies have indicated that toxin production is associated with increased pneumococcal adherence to intercellular spaces in respiratory epithelium in vitro [5, 6]. However, the role of pneumolysin in respiratory tract colonization has not been investigated. Thus, in order to define the contribution of pneumolysin to nasopharyngeal colonization by S. pneumoniae, we assessed the ability of pneumolysin-deficient mutant strains and their isogeneic wild-type strains to adhere to respiratory epithelial cells in vitro and to sustain nasopharyngeal colonization in vivo.

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Effect of pneumolysin on adherence to respiratory epithelial cells in vitro Because attachment to target cells is considered a prerequisite for successful colonization, adherence to respiratory epithelium is generally used as a surrogate measure of pneumococcal ability to colonize the respiratory mucosa. To test the effect of pneumolysin production on pneumococcal adherence to human respiratory epithelial cells, we compared the adherence of pneumolysin-deficient mutant pneumococcal strains of different serotypes with that of their wild-type congeners. Adherence studies of epithelial cells incubated with pneumolysin-sufficient and pneumolysin-deficient strains revealed that pneumolysin production was associated with morphological evidence of epithelial cell injury, including mild degrees of cytoplasmic blebs and cell rounding. Lack of pneumolysin production significantly reduced pneumococcal adherence to respiratory epithelial cells for serotypes 2 and 14 and the unencapsulated R36 mutant strains, and appreciably reduced adherence of pneumolysindeficient serotype 3 mutant strains, compared

Figure 1. Respiratory epithelial cell adherence of pneumolysin-sufficient and pneumolysin-deficient strains. Adherence of pneumolysin-deficient (PLY−, Ε) mutant strains to human respiratory epithelial cells was compared with that of isogenic pneumolysin-sufficient (PLY+, Φ) wild-type strains for unencapsulated R36 and types 2, 3 and 14 pneumococci. Data bars represent the geometric mean±SE for 6–8 replicates from 2–3 separate experiments. ∗ indicates P<0.05 for comparison by one-way analysis of variance and post-hoc Bonferroni test.

with their respective pneumolysin-sufficient congeners (Fig. 1). However, the relative difference in adherence between pneumolysin-sufficient and pneumolysin-deficient strains was small compared with the differences in adherence between serotypes. Thus, pneumolysin production appears to contribute in a small but significant degree to pneumococcal adherence to human respiratory epithelial cells in vitro.

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Figure 2. Nasopharyngeal colonization by pneumococcal serotypes in a murine model. Mice were inoculated intranasally with suspensions (107 cfu) of S. pneumoniae serotypes 14 and 23F, and were sacrificed at the times indicated. Numbers of pneumococci in nasal lavage were quantified as described in Materials and methods. Data points represent geometric mean±SE of 6–8 mice from two experiments.

Role of pneumolysin in nasal colonization Although epithelial cell adherence is required for bacterial colonization, successful nasopharyngeal colonization presumably entails an interplay of a number of bacterial factors and host defences. Thus, in order to ascertain whether pneumolysin contributes to upper respiratory tract colonization in vivo, we studied nasopharyngeal colonization by pneumolysinsufficient and pneumolysin-deficient strains in an established murine model [7]. Nasal instillation of 107 cfu of serotypes 14 and 23F S. pneumoniae, which do not cause lethal infection in mice, produced sustained nasal carriage over 2 weeks without evidence of clinical illness (Fig. 2). Numbers of pneumococci recovered from nasal lavage gradually diminished between 2 and 4 weeks after intranasal instillation, during which time concentrations of serum and nasal lavage antibodies reactive with components of bacterial lysates increased (not shown). Despite showing markedly greater adherence for epithelial cells in vitro, nasopharyngeal colonization by the unencapsulated R36 strain was no greater

and was actually sustained more poorly than that of type 14 pneumococci in this model (Fig. 2). These data suggest that other bacterial properties in addition to epithelial cell adherence were required for successful colonization. The contribution of pneumolysin production to nasopharyngeal colonization was studied using a type 14 S. pneumoniae mutant expressing modified pneumolysin lacking haemolytic and complement activities (H−C−), which contribute independently to invasive infection [8]. In addition, because other properties of pneumolysin besides its haemolytic and complement activities may affect the pathogenesis of pneumococcal infection [9], colonization with an isogeneic pneumolysin-deficient mutant strain with complete disruption of the pneumolysin gene was studied. Finally, as a control for any unanticipated effects from genetic manipulation, a control strain with deletion and then reconstitution of the pneumolysin gene was included. Despite lack of functional pneumolysin, both mutant strains showed nasopharyngeal colonization comparable to or exceeding that of the wild-type and reconstituted mutant type 14 pneumococcal strains (Fig. 3). Numbers of pneumolysin-deficient bacteria recovered from nasopharyngeal lavage up to 4 weeks after intranasal instillation were equal to or higher than those of pneumolysin-sufficient type 14 pneumococci. Thus, neither initiation nor persistence of colonization by type 14 S. pneumoniae in this murine model was dependent on bacterial production of pneumolysin.

Discussion The ubiquitous production of pneumolysin by all pneumococcal isolates [10] and the high degree of conservation of the pneumolysin gene between disparate strains [11] suggest that pneumolysin production is important for key phases of this respiratory mucosal pathogen’s life-cycle. Because S. pneumoniae is highly adapted to colonize the human respiratory mucosa, the ubiquitous and highly conserved production of pneumolysin might indicate a role for this toxin in colonization. However, our studies suggest that, whereas pneumolysin may facilitate bacterial adherence to epithelial cells, it is not a major determinant of successful nasopharyngeal colonization by pneumococci. Mutant type 14 S.

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Figure 3. Nasopharyngeal colonization by pneumolysin-sufficient and pneumolysin-deficient type 14 mutant pneumococci. Mice were inoculated intranasally with 5×107 cfu of S. pneumoniae and numbers of bacteria in nasal lavage quantified at the indicated times. Data points are as described for Fig. 2. Ε indicates wild-type serotype 14 strain; PLN-A (Β) represents mutant type 14 with complete deletion of the pneumolysin gene; H−C− (Φ) represents mutant type 14 pneumococci expressing pneumolysin lacking haemolytic and complement-activation properties; and H+C+ (Χ) represents mutant type 14 transformed with the native pneumolysin gene.

pneumoniae deficient in pneumolysin production colonized the murine nasopharynx as effectively as did pneumolysin-sufficient pneumococci both at early and late time points. In contrast, we did observe that pneumolysin production was associated with increased bacterial adherence to human respiratory epithelial cells in vitro. Pneumolysin-deficient mutant strains of types 2, 3, 14 and unencapsulated R36 pneumococci had appreciably reduced adherence, compared with their respective pneumolysin-sufficient strains. These findings are in agreement with observations of Rayner et al. [5], who reported that pneumolysin produced earlier and more severe damage to human respiratory mucosa in organ culture and was associated with increased pneumococcal adherence. However, our studies and those of Rayner et al. [5] both indicate that pneumolysin-deficient pneumococcal mutants

still have appreciable adherence to human respiratory epithelium. Thus, we consider pneumolysin’s effect on bacterial adherence to the respiratory epithelium to be relatively minor in vivo compared with other pneumococcal factors implicated in adherence, including peptide permeases and cell wall constituents [2, 12, 13]. This study and other investigations of pneumococcal colonization are constrained by important limitations of the available experimental methods and models. In our colonization studies, we used a previously described murine model of nasopharyngeal colonization [7]. Such colonization models clearly reflect the interplay of bacterial and host factors in addition to epithelial cell adherence. For example, we found the unencapsulated R36 pneumococcal strain consistently adhered in greater numbers to epithelial cells than did encapsulated strains in vitro. Yet, colonization with R36 pneumococci in vivo was of significantly shorter duration than that of types 14 or 23F in our studies. Similarly, previous reports suggest that factors affecting phase variation in colony morphology, such as autolysin, which strongly affect pneumococcal adherence to epithelial cells, do not appear to be important determinants of colonization in murine models [2]. Although these in vivo models of colonization may be superior to simple adherence assays for studying pneumococcal pathogenesis, they also have important limitations. Because of the extreme susceptibility of mice to many pneumococcal serotypes, such models are limited to the study of pneumococcal serotypes which are relatively less virulent for mice [7]. In addition, clearance of colonization in these models appears to coincide with the appearance of anti-pneumococcal antibodies in serum and nasal wash. Therefore, the results of such studies must be interpreted in the context of the known differences in systemic and mucosal immunity between different murine strains, as well as those of mice and humans.

Materials and methods Bacteria strains Types 14 and 23F S. pneumoniae was purchased from American Type Culture Collection (ATCC, Rockville, MD). The clinical type 14 S. pneumoniae strain was isolated from blood of a bacteraemic patient at the Minneapolis VA.

Pneumolysin in colonization

Pneumolysin-deficient type 3 and unencapsulated R36 S. pneumoniae produced by chemical mutagenesis and their respective wildtype strains were graciously provided by Dr Mary Johnson, Tulane University, New Orleans, LA [14]. A pneumolysin-deficient mutant type 2 S. pneumoniae (PLN-A) was constructed previously from strain D39 by insertion-duplication mutagenesis as described [15]. Pneumolysin-deficient type 14 S. pneumoniae mutants were produced by direct transformation of type 14 pneumococci with donor DNA from either PLNA or D39 derivatives with point mutations in the pneumolysin gene that abolished both haemolytic and complement activation properties (Trp433→Phe+Cys428→Gly+Asp385→Asn, designated H−C−) [16]. C-terminal fragments of the pneumolysin gene carrying these point mutations were cloned into pVA891, and insertion-duplication mutagenesis was used to exchange these for the homologous portion of the chromosomal pneumolysin gene [16]. This procedure was also used to construct a H+C+ derivative (functionally homologous to the D39 strain) by insertion of pVA891 carrying the Cterminal portion of the native pneumolysin gene [16]. Pneumolysin-deficient mutant strains produced type 2 and type 14 capsules, respectively, as characterized by Quellung reaction using antisera obtained from Statens Seruminstitut (Copenhagen, Denmark). Mutant strains were selected using erythromycin plates as described [15]. Mutant strains were confirmed to lack haemolytic and complement activities as previously described [16]. Bacteria were stored frozen on glass beads at −70°C. All bacterial colonies selected for subculture appeared transparent or semi-transparent under oblique transmitted illumination [17].

Animals Specific pathogen-free white female National Institutes of Health Swiss outbred mice (20–30 g) were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). Animals were housed in a pathogen-free barrier facility fully accredited by the American Association for the Accreditation of Laboratory Animal Care. Animal studies were performed in accordance with the guidelines established in the NIH ‘Guide for the

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Care and Use of Laboratory Animals’ (Department of Health, Education and Welfare Publication No. [NIH] 85-23, Office of Science and Health Reports, Division of Research Resources, Bethesda, MD), and the research was approved by the Animal Study Subcommittee of the Veterans Affairs Medical Center (Minneapolis, MN).

Adherence assay Respiratory epithelial cells (A549, ATCC) were grown in LabTech 8-well chamber slide (Nunc, Naperville, IL) in HAMS F12+10% FBS medium until confluent. After removal of culture medium, epithelial cells were washed four times with phosphate-buffered saline (PBS) before suspensions of bacteria at 107 cfu in 100 ll PBS were added to duplicate wells. Slides were centrifuged at 1500 g for 10 min at room temperature to appose bacteria to cell monolayers, and then incubated for 30 min at 36°C. After removing wells and gaskets from slides, monolayers were washed by dipping slides four times into a beaker of sterile PBS, and then stained using Diff-Quik (Baxter, McGaw Park, IL). Numbers of adherent diplococci per 40 cells in 3–5 fields were visually counted at 100× magnification, and the average number of bacteria per cell in duplicate wells calculated, as previously described [18]. The coefficient of variation for this adherence assay using pneumococci and A549 cells was 20%.

Nasal colonization model Nasopharyngeal colonization of mice was established by slight modification of a previously described model [7]. Briefly, groups of 3–5 mice were anaesthetized with i.p. injection of 50 mg/ kg sodium pentobarbital, and 50 ll of bacterial suspension was injected intranasally (i.n.). Inoculum size was confirmed by quantitative culture on blood agar plates. At selected times after i.n. infection, mice were killed by i.p. injection of 50–100 ll Beuthanasia (Schering-Plough Animal Health, Kenilworth, NJ). The trachea was surgically exposed and cannulated with a catheter (PE 20 tubing, Clay Adams, Sparks, MD) directed cephalad. After positioning the animal with its nose dependent, 2 ml of sterile PBS was infused slowly through the tracheal catheter and nasal lavage was collected from the nares. The

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number of viable bacteria in nasal lavage was determined by quantitative culture. Bacteria were identified as pneumococci by colony morphology of a-haemolytic organisms on blood agar plates and by optochin sensitivity.

Statistics Where indicated, geometric means and standard errors (SE) of geometric means were calculated on log-transformed data. Statistical significance was calculated by comparison of multiple means by one-way analysis of variance (ANOVA) and Bonferroni’s multiple comparison tests, and by comparison of means of two groups by twotailed t-test.

Acknowledgements This work was supported by the Veterans Affairs Research Service and by the National Institutes of Health (AI-042240 to JBR, AI-31373, AI-39445 and HL-57880 to ENJ).

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