Sortase A (SrtA) is required to anchor neuraminidase, β-galactosidase, and possibly other LPXTG motif proteins to the pneumococcal cell surface. We examined the role of SrtA in Streptococcus pneumoniae nasopharyngeal (NP) colonization in the chinchilla model. The srtA mutant colonized the nasopharynx at a significantly lower level than the D39 parent strain during the second and third week of the carriage, and was eliminated from nasopharynx one week earlier than the D39 pneumococci. Our data indicate that SrtA contributes to pneumococcal NP colonization in this animal model.
Otitis media (OM) (middle ear inflammation/infection) in its various clinical forms, is recognized as one of the most common childhood diseases. The prevalence, medical cost and hearing related morbidity of OM are significant. Streptococcus pneumoniae remains one of the major pathogens of OM and accounts for 30% of the cases of acute OM, and 5% of the cases of chronic OM with effusion . The natural course of OM is thought to proceed from nasopharyngeal (NP) colonization, to retrograde ascension of the pathogen via the eustachian tube, and invasion of middle ear with the subsequent induction of disease. The asymptomatic carrier state in children is believed to be the reservoir of the pathogen . The regulation of colonization and interrelationship between S. pneumoniae virulence factors that promote carrier state remains an important research question.
Sortases are bacterial enzymes that mediate the covalent attachment of specific surface proteins of gram-positive bacteria to the cell wall. These enzymes typically cleave a LPXTG motif (leucine, proline, X, threonine, and glycine, where X is any amino acid) between the threonine and glycine residues and covalently attaches the threonine to the amino group of the pentaglycine cell wall cross bridge resulting in cell wall attached protein [3,4]. S. pneumoniae strains vary in their content of sortase genes but all S. pneumoniae strains examined contain sortase A (srtA) [5,6]. A previous study demonstrated that SrtA is required to anchor neuraminidase, β-galactosidase and presumably other LPXTG motif proteins to the pneumococcal cell surface . A S. pneumoniae srtA mutant has been developed which exhibits a reduced concentration of neuraminidase A (Nan A) associated with the cell surface. It demonstrates decreased adherence to human pharyngeal cells in vitro . Another recent study indicates that a S. pneumoniae srtA mutant was attenuated in murine models of pneumonia, bacteremia and NP colonization . The purpose of this study was to compare the ability of a srtA mutant with that of the parent strain in the chinchilla NP colonization model in order to further define the role of LPXTG-mediated cell anchoring of proteins in colonization by S.pneumoniae.
Materials and methods
Bacterial strains, preparation of inocula
S. pneumoniae serotype 2, strain D39 (ATCC 7466) and an isogenic srtA derivative of D39 deficient in Srt A have been described in detail previously . Briefly, srtA was deleted in D39 by allelic replacement and confirmed by PCR as described. In order to maintain selection pressure, erythromycin (1 μg/ml) was added to the blood agar when D39srtA was cultured. Following growth on plates, colonies were transferred to Todd-Hewitt broth (Difco Laboratory, Detroit, MI) containing erythromycin (5 μg/ml). Prior to inoculation of the chinchillas, both strains were passed once in mice as previously described . Approximately four colonies from the mouse-passed culture were grown on blood agar and transferred to Todd-Hewitt broth. After an overnight incubation at 37 °C, the cultures were centrifuged at 3500g for 20 min, washed twice with and resuspended in Dulbecco's phosphate-buffered saline (D-PBS) and used for intranasal (i.n.) inoculation described below. The S. pneumoniae concentration in the inoculum was confirmed by standard colony plate count.
Assessment of NP colonization and invasion of the middle ear
Forty chinchillas (Chinchilla lanigera) (400–600 g), free of middle ear diseases as determined by otoscopy and tympanometry, were randomly assigned by sex and weight to two cohorts. Twenty chinchillas from each group were inoculated intranasally with 0.5 ml of a suspension containing approximately 107 CFU of S. pneumoniae D39 or D39 srtA/ml, respectively. Four chinchillas from each cohort, pre-selected and randomized, were evaluated by tympanocentesis and NP lavage on days 1, 7, 14, 21 and 28 post inoculation with D39 or srtA as previously described . Tympanocentesis was performed on both ears of the chinchillas by aspiration with a tuberculin syringe fitted with a 25-gauge needle. If no middle ear fluid (MEF) was present, the bullas were lavaged with 0.5 ml sterile saline. Subsequent to tympanocentesis, NP lavage was performed on each chinchilla as described previously . Chinchillas were not subjected to repeat tympanocentesis or nasal lavage. Tympanocentesis and bulla lavage were always performed before NP lavage to prevent contamination of the middle ear. The MEF or lavage, and nasal lavage were cultured on blood agar, incubated overnight at 37 °C in a humidified atmosphere with 5% CO2 and the number of CFU per ml were determined by standard dilution and plate count.
Organisms recovered from NP lavage and MEF or middle ear lavage of chinchillas were tested for erythromycin resistance by standard macrodilution broth method. For neuraminidase activity assays, representative colonies were inoculated into 10 ml of BHI with or without erythromycin (5 μg/ml), and incubated in a humidified atmosphere with 5% CO2 overnight at 37 °C. The cultures were centrifuged and neuraminidase activity was measured in both the bacterial cell pellets and cell free culture supernatants as described by Winter et al. . Assays were performed in duplicate on two separate occasions.
Data are expressed as means ± standard error from at least duplicate samples. Bacterial culture results below the detection limit of the viable count assays (10 CFU/ml NP lavage fluid) were ascribed values just below the detection limit (9 CFU/ml). Differences of S. pneumoniae concentration in NP lavage fluid between the D39 and the srtA mutant cohorts were analyzed by the Mann Whitney Rank Sum Test. A P value of <0.05 was accepted as the minimal level of significance.
Effect of srtA gene disruption on NP colonization
The relative ability of the parent and the srtA-deficient mutant to colonize and persist in the nasopharynx for up to 28 days post i.n. challenge is shown in Fig. 1. There was no significant difference in the concentration of the parent or mutant in the lavage samples obtained on days 1 and 7 post inoculation. However, a statistically significant reduction in the concentration of the srtA mutant, compared with the D39 parent, was evident by day 14. The srtA S. pneumoniae were eliminated from 3 of the 4 chinchillas sampled at this time point. By day 21 no srtA S. pneumoniae were detected in the lavage fluid, whereas the D39 parent persisted in the nasopharynx of all four animals. By 28 days post inoculation both the D39 parent and the srtA mutant were eliminated from the nasopharynx. Two chinchillas from the D39 cohort and none from the srtA cohort developed OM with culture positive MEFs.
Phenotypes of D39 and its srtA-deficient mutant
Bacteria recovered from the NP lavage samples of animals challenged with the srtA mutant were erythromycin resistant, MIC > 32 μg/ml, while those from the animals challenged with D39 were erythromycin sensitive, MIC < 1 μg/ml. Cell associated neuraminidase activity for the srtA mutant was below the lower limit of detection of the assay (0.5 mU/μg of cell protein), while D39 expressed 10.5 mU/μg of cell protein.
S. pneumoniae express a number of virulence factors on the cell surface, which are involved in the pathogenesis of pneumococcal diseases. Several important virulence factors such as choline binding proteins are attached to the cell surface by choline residues of the teichoic acids through non-covalent binding. However, like other gram-positive bacteria, S. pneumoniae also use covalent attachment of some of the surface proteins. A genome analysis of S. pneumoniae R6 revealed 13 surface proteins with C-terminal LPTGX motifs that are processed by sortase . A srtA mutant has been used for evaluation of its virulence in different models both in vitro and in vivo [6,7].
A prior investigation into the role of SrtA in the mouse model of intraperitoneal infection conducted by Kharat and Tomasz indicated no clear role of SrtA in the virulence of strain R36A expressing a type III capsule . However, a recent report by Paterson and Mitchell  demonstrated that a srtA mutant of D39 was attenuated during competitive infections in the mouse model. In order to obtain a complete assessment of bacterial clearance kinetics data from nasopharynx, we used a chinchilla NP colonization model to evaluate the role of SrtA in the persistence of bacteria in nasopharynx. The chinchillas were monitored for up to 28 days, when S. pneumoniae were no longer present in the lavage fluids. Our results demonstrate that the extent and duration of NP colonization is altered significantly by disruption of the srtA gene during the second and third weeks post i.n. challenge. The mechanisms responsible for the differences between the parent and the srtrA mutant are not clear.
The possibility of localization defects in the 13 proteins that are the substrates of sortase could contribute to the lower level of NP colonization of the srtA mutant compared to that of D39. Two surface proteins β-galactosidase and NanA have shown drastic changes in the localization from the cell wall to the culture medium in a S. pneumoniae srtA mutant . β-Galactosidase has been reported to alter pneumococcal adherence and invasion of human pharyngeal cells, which could play a possible role in this animal model of NP colonization .
Finally, changes in the localization of NanA in the srtA mutant might also be involved in its defect of NP colonization. We have previously demonstrated that disruption of nanA diminishes the ability of S. pneumoniae to colonize and immunization with native or recombinant neuraminidase affords a degree of protection in the chinchilla OM model . Consistent with the results of Kharat and Tomasz , we could not detect any neuraminidase activity from srtA NP or middle ear isolate cell pellets in vitro. However, neuraminidase activity was detectable in the culture supernatant (data not shown), which indicates that neuraminidase is secreted by S. pneumoniae srtA. It is assumed that the extracellular neuraminidase would be available in the nasopharynx in vivo and possibly plays a significant role in the colonization of S. pneumoniae in the nasopharynx during the first week of carriage. Whereas differences in NP colonization between the srtA mutant and parent were not apparent for 7 days post i.n. inoculation, the altered colonization by the srtA-deficient mutant was evident by significantly lower bacterial counts on days 14 and 21. In contrast, a comparison of the bacterial clearance kinetics data of srtA with the nanA mutant from our previous report, which showed a rapid clearance starting from day1 post i.n. inoculation of a nanA mutant of D39, is of interest . The lesser effect of the srtA mutation in comparison to the nanA deletion demonstrates the subtle role played by cell anchoring of LPXTG-proteins in this model and reinforces previous findings in the mouse model . This is in marked contrast to the role of SrtA in other Gram positive organisms such as Staphylococcus aureus and Listeria monocytogenes, where SrtA plays a much larger role in virulence . This may relate to the fact that most LPXTG anchored proteins in the pneumococcus are enzymes and they are still functionally active when released from the cell surface. This is in contrast to the anchorage of adhesins and invasins in Staphylococcus aureus and Listeria monocytogenes which presumably need to be anchored to the cell surface to perform their function. In conclusion, the data from the present study indicate that disruption of srtA diminishes the ability of S. pneumoniae to colonize and persist in the chinchilla nasopharynx.
This study was supported, in part, by a grant from the National Institute on Deafness and Other Communication Disorders, National Institute of Health (RO1 DC3105-09) to TFD. Work in Glasgow was supported by the Wellcome Trust.