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Infection and Immunity, January 2005, p. 599-608, Vol. 73, No. 1
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.1.599-608.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Mutation in the sap Operon Attenuates Survival of Nontypeable Haemophilus influenzae in a Chinchilla Model of Otitis Media

Kevin M. Mason, Robert S. Munson Jr., and Lauren O. Bakaletz*

Department of Pediatrics, Columbus Children's Research Institute, The Ohio State University College of Medicine and Public Health, Columbus, Ohio

Received 23 June 2004/ Returned for modification 30 July 2004/ Accepted 20 September 2004


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ABSTRACT
 
Bacteria have evolved strategies to resist killing by antimicrobial peptides (APs), important effectors of innate immunity. The sap (sensitivity to antimicrobial peptides) operon confers resistance to AP-mediated killing of Salmonella. We have recently shown that sapA gene expression is upregulated in the middle ear in a chinchilla model of nontypeable Haemophilus influenzae (NTHI)-induced otitis media. Based on these findings, we constructed an NTHI strain containing a Lux reporter plasmid driven by the sapA promoter and demonstrated early yet transient expression of the sap operon within sites of the chinchilla upper airway upon infection. We hypothesized that the sap operon products mediate NTHI resistance to APs. In order to test this hypothesis, we constructed a nonpolar mutation in the sapA gene of NTHI strain 86-028NP, a low-passage-number clinical isolate. The sapA mutant was approximately eightfold more sensitive than the parent strain to killing by recombinant chinchilla ß-defensin 1. We then assessed the ability of this mutant to both colonize and cause otitis media in chinchillas. The sapA mutant was significantly attenuated compared to the parent strain in its ability to survive in both the nasopharynx and the middle ear of the chinchilla. In addition, the mutant was impaired in its ability to compete with the parent strain in a dual-strain challenge model of infection. Our results indicate that the products of the sap operon are important for resisting the activity of APs and may regulate, in part, the balance between normal carriage and disease caused by NTHI.


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INTRODUCTION
 
Antimicrobial peptides (APs), which are widely distributed in animals and plants, are key components of the primary innate host defense (28). Most APs are cationic, amphipathic molecules of 12 to 50 amino acids that interact with the bacterial cytoplasmic membrane, which is comprised of negatively charged phospholipids. The peptides insert into the cytoplasmic membrane and form channels that result in the leakage of cytoplasmic contents and subsequent cell death (23, 49, 64). First isolated from the skin of Xenopus laevis, similar molecules were later found in invertebrates (e.g., the horseshoe crab) and in neutrophil granules of several mammalian species, indicating a common and ancestral host defense mechanism (66). These positively charged peptides exhibit broad antimicrobial activity against gram-negative and gram-positive bacteria, fungi, and enveloped viruses, in addition to demonstrating properties similar to those of chemotactic agents, cytotoxins, and opsonins (16).

Humans produce various types of APs, such as lysozyme, lactoferrin, collectins, cathelicidins, thrombocidins, and defensins (10, 31, 35, 47, 51). Members of an important class, the defensins, contain six invariant cysteine residues that form three intramolecular disulfide bonds folded into a beta-sheet structure (16, 49). One group of these peptides, the ß-defensins, are expressed in a human middle ear epithelial cell line and in middle ear mucosae from patients with chronic otitis media (OM) with effusion (32-34, 44). Recently, it was demonstrated that lysozyme and ß-defensins can inhibit the growth of clinical isolates of OM pathogens via bacterial membrane damage (32). These data suggest an important role for APs in maintaining the health of the uppermost airway and perhaps the prevention of OM.

In 1992, Groisman and colleagues screened a transposon mutant library to identify genes that are required for resistance to APs (19). Three mutants mapped to the sap (sensitivity to antimicrobial peptides) operon (sapABCDF) in Salmonella (46). Further work revealed the presence of a sap operon in the plant pathogen Erwinia chrysanthemi that conferred sensitivity to plant-specific APs (35). Although the mechanism of AP resistance remains unclear, the sap genes have been implicated in playing a role in quorum sensing and the regulation of bioluminescence and in mediating AP sensitivity via a lipid A modification in Vibrio fischeri and Proteus mirabilis, respectively (13, 42). Groisman and colleagues proposed that the sap operon of Salmonella functions to detoxify protamine by transport into the cytoplasm followed by protease degradation (18, 46). In Escherichia coli, the SapD protein confers ATP dependence to the potassium uptake Trk system that mediates potassium homeostasis, contributing to extracytoplasmic protease activity by OmpT and the subsequent degradation of toxic peptides (25, 60, 61).

The sapD and sapF genes of nontypeable Haemophilus influenzae (NTHI) contain an ATP-binding domain and may share translocation ATPase activity. Sequence analysis of the NTHI sap locus revealed that SapD and SapF have homology to proteins in the ATP-binding-cassette family of transporters, including bacterial homologs in the Opp and SpoOK systems, which are proteins involved in oligopeptide uptake in Salmonella enterica serovar Typhimurium and Bacillus subtilis, respectively (27, 46, 48). SapA was predicted to localize to the periplasm due to the presence of a signal sequence and homology to the periplasmic solute binding proteins DppA from E. coli (1) and Salmonella OppA (46), which are involved in peptide transport. The SapB and SapC proteins have sequence homology with the membrane components of the Opp and SpoOK transport systems and are thus predicted to be integral membrane proteins.

Gram-positive and gram-negative bacteria limit the effectiveness of APs by various mechanisms, including reducing the net negative charge of the cell envelope via the incorporation of D-alanine into teichoic acid polymers or D-lysine into phosphatidylglycerol or via a modification of lipid A. A reduction in negative charge contributes to the repulsion of cationic APs. Alternative strategies for resisting the toxicity of APs include the efflux of APs via an energy-dependent pump, alterations of the cytoplasmic membrane fluidity, or cleavage with surface-associated proteases (9, 20, 21, 42, 49, 50, 55). The variety of mechanisms of resistance that bacteria have evolved suggests an important role for APs in host defense against microorganisms.

We have recently shown an increase in NTHI sapA gene expression when this microbe is recovered from the middle ear in a chinchilla model of OM compared to that when the organism is grown in vitro (40). In the present study, we extend this work and demonstrate that the NTHI sap gene locus is transcribed as a single message containing six open reading frames, sapABCDFZ. In order to localize the expression of the sap operon in NTHI during infection, we constructed an NTHI reporter strain that has a bioluminescent reporter, the lux operon, driven by the sap promoter. Via biophotonic imaging, we showed that the sap operon is transiently expressed in the middle ear, the eustachian tube, the nasopharynx, and the oropharynx. We then showed that a nonpolar mutation in sapA renders NTHI strain 86-028NP more sensitive to killing by recombinant chinchilla ß-defensin 1 [(r)cBD-1] (26). This mutant was also significantly impaired in its ability to survive in the middle ear and to establish long-term colonization of the nasopharynx, and the loss of SapA rendered the microorganism less fit for survival in the middle ear when in competition with the parent strain. Collectively, these data suggest an important role for the sap gene products in NTHI colonization as well as survival and in the maintenance of infection of the middle ear.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. NTHI strain 86-028NP is a minimally passaged clinical isolate obtained from a pediatric patient who underwent tympanostomy and tube insertion for chronic OM with effusion at Columbus Children's Hospital, and this strain has been extensively characterized in chinchilla models of OM (5, 30, 62). The genome of strain 86-028NP is currently being sequenced (www.microbial-pathogenesis.org/) (45). This strain has been maintained frozen in skim milk containing 20% glycerol (vol/vol). NTHI was grown on chocolate agar (Becton Dickinson and Co., Sparks, Md.) or in brain heart infusion broth (BHI broth; Becton Dickinson and Co.) supplemented with 2 µg of hemin chloride/ml (dissolved in 20 mM NaOH; Sigma-Aldrich, St. Louis, Mo.) and 2 µg of NAD/ml (Sigma-Aldrich) (sBHI broth) at 37°C in 5% CO2. The NTHI sapA::Kan mutant was grown on chocolate agar or in sBHI broth containing 20 µg of kanamycin/ml (Invitrogen Corp., Carlsbad, Calif.). NTHI strain 1885MEE is also a low-passage-number clinical isolate obtained from a child with chronic OM and has been used in a chinchilla model of OM (30, 58).

Animal infection model. Healthy adult chinchillas (Chinchilla lanigera) with no evidence of middle ear infection by either video otoscopy or tympanometry were used to monitor sapA expression in vivo and to assess the biological consequences that might have resulted from a mutation in the sapA gene. This chinchilla model of NTHI-induced OM has been well characterized (3-6, 30, 62).

RT-PCR. RNAs were isolated from strain 86-028NP and assessed for purity and integrity as previously described (40). Gene expression was assessed by reverse transcriptase PCR (RT-PCR) by use of a two-step RT-PCR kit (Qiagen Inc.) according to the manufacturer's protocol. mRNAs were reverse transcribed and amplified by RT-PCRs with primers generated to amplify the intergenic regions of the sapABCDFZ genes. Controls were analyzed in parallel to verify the absence of DNA in the RNA preparations (–RT control) as well as the absence of primer dimers in control samples lacking template RNA. RT-PCR products were analyzed by gel electrophoresis, and in all cases, single products were observed at the appropriate sizes.

Southern hybridization. Genomic DNAs were purified from 15 H. influenzae isolates, including strain Rd, 13 low-passage-number clinical isolates of NTHI that were recovered from patients undergoing tympanostomy and tube insertion for chronic OM, and a clinical isolate recovered from a patient with cystic fibrosis. The last 14 isolates were designated by the following strain numbers: 86-028NP, 1728, 1729, 1714, 214, 1236, 165, 1060, 1128, 10548, 3224A, 3185A, 1885MEE, and 27W11679INI. Two micrograms of genomic DNA was digested with MfeI, and the fragments were resolved in a 0.8% agarose gel and then blotted onto a Nytran SuPerCharge membrane by use of a Turbo Blotter kit (Schleicher and Schuell BioScience Inc., Keene, N.H.). Membranes were hybridized to a probe generated by PCR amplification of the coding sequence of the 86-028NP sapA gene with the primers 5'-AGGTGCTGGATTATAGACTTGTTC-3' and 5'-TCACGAACAAGCAAGAAAAGT-3'. The amplicon was purified by use of a QIAquick PCR purification kit (Qiagen Inc.) and was labeled with horseradish peroxidase by use of an ECL direct nucleic acid labeling and detection system (Amersham Biosciences, Piscataway, N.J.). All procedures were performed according to the manufacturers' directions, and developed blots were exposed to Fuji Super Rx X-ray film (Fuji Photo Film Co. Ltd., Tokyo, Japan). Likewise, genomic DNAs were purified from the parent strain 86-028NP and from six candidate sapA mutants by use of a Puregene DNA isolation kit (Gentra Systems, Minneapolis, Minn.). Genomic DNAs were digested with HaeIII, and the fragments were resolved, blotted, and probed as described above.

Generation of an NTHI strain expressing luxCDABE under control of the sapA promoter. The promoterless luxCDABE operon of Photorhabdus luminescens was obtained as a SnaBI-to-PstI fragment from plasmid pSB417 (65) (kindly provided by B. Ahmer). The 5.8-kb fragment was gel purified by use of a Perfectprep gel cleanup kit (Brinkmann), and the ends were blunted by a 3' fill-in reaction using Pfu Turbo Hotstart polymerase (Stratagene) and a 10 mM deoxynucleoside triphosphate mix (Invitrogen Corp.). For vector preparation, pGZRS-39A, a derivative of pGZRS-1 (63) (kindly provided by S. West), was isolated from an overnight culture by use of a QIAprep Spin miniprep kit (Qiagen Inc.). The plasmid DNA was linearized by KpnI digestion and purified by gel extraction, and 3' overhangs were filled in as described above. In order to minimize ligation of the vector alone, we removed 5' phosphate groups by treating the vector with calf intestinal alkaline phosphatase (Invitrogen Corp.). This prepared vector was ligated overnight with the luxCDABE-containing fragment by the use of T4 DNA ligase (Invitrogen Corp.). The ligation products were used to transform high-efficiency ElectroMAX DH10B cells (Invitrogen Corp.) by electroporation with a Gene Pulser II (Bio-Rad) according to the manufacturer's protocol. Kanamycin-resistant clones were passaged to ensure purity, and plasmid DNAs were purified from kanamycin-resistant clones by use of a plasmid mini kit (Qiagen Inc.) according to the manufacturer's protocol. A plasmid with the correct restriction map was saved and named pKMLN-0.

The sapA gene promoter region was PCR amplified with the primers 5'-GCGCGGATCCGTAATTCTACACTTTG-3' and 5'-GCGCGTCGACTGATGAATGTTGATTA-3' (restriction sites are underlined). The amplicon was purified by use of a QIAquick PCR purification kit (Qiagen Inc.), restriction digested, and cloned as a SalI-to-BamHI fragment into the SalI- and BamHI-digested pKMLN-0 vector. One microgram of plasmid DNA was electroporated into NTHI strain 1885MEE according to a modified protocol (40) and was then plated for overnight growth on chocolate agar containing 20 µg of kanamycin/ml. Plasmid DNAs were purified from kanamycin-resistant, luminescent clones, and a plasmid with the correct restriction map was saved and named pKMLN-01.

Luminescent imaging of NTHI strain 1885 containing pKMLN-01. NTHI 1885/pKMLN-01 was grown overnight on chocolate agar and monitored for luminescence with an IVIS imaging system and LivingImage software (Xenogen Corp., Alameda, Calif.). Live real-time noninvasive imaging of luminescence in a chinchilla model of OM was performed in our laboratory as described elsewhere (45a). For this study, we expanded upon these observations to specifically monitor sap promoter activity during acute OM development. NTHI strain 1885/pKMLN-01 was used to inoculate the nasopharynx (1.4 x 107 CFU) and middle ears (600 CFU) of a chinchilla. The animal was monitored in prone and lateral positions for luminescence at 2, 4, and 6 days postinoculation by use of the IVIS imaging system with a 3-min exposure time. Additionally, at 3, 5, and 7 days postinoculation, middle ear fluids obtained by epitympanic tap and nasal lavage fluids were collected, assayed for luminescence on a microplate reader (Genios; Tecan), and plated for a semiquantitative estimation of CFU/ml.

sapA mutant construction. The sapA gene in strain 86-028NP, as well as an additional 1-kb sequence upstream and downstream, was amplified from 86-028NP genomic DNA by a PCR using Pfu Turbo Hotstart DNA polymerase (Stratagene Corp.), the forward primer 5'-ACGAGTAATATGATCCGCCTTTGT-3', and the reverse primer 5'-AAGTGCGATGGTATTTTGACGAA-3'. After purification (Qiagen, Inc.) and the introduction of an A tail, the PCR product was TA cloned into pGEM-T Easy (Promega, Madison, Wis.) and transformed into ElectroMax DH10B cells (Invitrogen Corp.). A plasmid with the correct restriction fragments was saved as pSAP1, and the DNA was sequenced for verification. For insertional inactivation of sapA, pSAP1 was linearized by PmeI digestion and then gel purified by use of a Perfectprep gel cleanup kit (Brinkmann, Westbury, N.Y.). A kanamycin resistance gene was obtained from SmaI-digested pUC18K2 plasmid, a derivative of pUC18K (43) that contained an extra nucleotide for in-frame cloning (kindly provided by J. Kaper). The gel-purified kanamycin resistance gene was ligated overnight with the linearized pSAP1 plasmid generated as described above by the use of T4 DNA ligase (Invitrogen Corp.). The ligation products were used to transform high-efficiency ElectroMAX DH10B cells by electroporation as described above. Plasmid DNAs were purified from kanamycin-resistant clones, and a plasmid with the correct restriction map was saved and named pSAP2.

One microgram of pSAP2 was linearized with ApaI and used to transform NTHI strain 86-028NP according to a modified MIV transformation protocol which included an additional treatment with 1 mM 3',5'-cyclic AMP (Sigma-Aldrich) for 30 min at 37°C (8, 39). Transformants were selected on chocolate agar supplemented with 20 µg of kanamycin/ml, and the correct allelic exchange was determined by Southern analysis.

Bactericidal assays. Bactericidal assays were performed as previously described (2, 24), with the following modifications. Briefly, bacteria were grown overnight on chocolate agar plates, inoculated into 5 ml of prewarmed sBHI broth, grown for 3 h at 37°C in 5% CO2 until mid-log phase, and then diluted with sBHI to an optical density at 490 nm of 0.65. The bacteria were diluted further in 10 mM sodium phosphate (pH 7.4) to a concentration of 107 CFU/ml and were maintained on ice until used in the assay. In a 96-well polypropylene plate (Falcon), 90 µl of 10 mM sodium phosphate containing 1% sBHI was added to each well. The antimicrobial peptide (r)cBD-1 or human BD-3 (hBD-3) (PeproTech, Rocky Hill, N.J.) was serially diluted in the wells so that each well retained 90 µl of the appropriate concentration of peptide. Ten microliters of the bacterial solution was added to each well, and the plate was incubated for 1 h at 37°C in 5% CO2. Bacterial concentrations were determined by growth on chocolate agar, and data were expressed as percentages of killing relative to the bacterial concentration at the start of the assay.

Biological evaluation of the sapA mutant. We used a chinchilla model of OM to assay the relative ability of the sapA mutant to colonize the nasopharynx and to multiply and survive in the middle ear compared to the parental isolate. We also assayed the ability of the sapA mutant to compete with the parent in the middle ear microenvironment. Briefly, NTHI strain 86-028NP and the sapA mutant strain were grown overnight on chocolate agar and chocolate agar supplemented with 20 µg of kanamycin/ml, respectively, and then were adjusted to an optical density at 490 nm of 0.65 in 0.9% (wt/vol) sodium chloride in water. For assessment of the virulence of the mutant strain alone, two chinchillas were inoculated both intranasally and transbullarly with approximately 108 and 2,500 CFU of the sapA mutant, respectively. The actual inocula received were confirmed by plate counts. For competition experiments, parent and mutant bacteria were diluted to a concentration of 2,500 CFU/ml, and equal volumes were combined. A 300-µl volume of the mixture was used to inoculate the left and right transbullar cavities of two chinchillas. Animals were evaluated by video otoscopy and tympanometry daily or every 2 days from the time of bacterial inoculation until clearance.

Nasopharyngeal lavage was performed every 2 days by passive inhalation of 500 µl of pyrogen-free sterile saline with recovery of lavage fluid. Epitympanic taps were attempted when an effusion was considered to be of sufficient volume to be retrieved. Nasopharyngeal lavage and epitympanic tap fluids were maintained on ice until they were used for serial dilution. Dilutions of nasopharyngeal lavage fluids were then cultured on chocolate agar plates containing ampicillin (15 µg/ml) to limit the growth of other normal nasopharyngeal flora and on chocolate agar containing ampicillin and 20 µg of kanamycin/ml in order to select for the sapA mutant. Similarly, dilutions of epitympanic tap fluids were cultured on chocolate agar plates (Becton Dickinson and Co.) and on chocolate agar plates containing 20 µg of kanamycin/ml.

Statistical methods. The biometrics laboratory of The Ohio State University's College of Medicine and Public Health conducted all statistical analyses. A stratified log-rank test was used to compare cohorts for relative times to bacterial clearance of the nasopharynx and middle ear, as determined by a culture-negative status, and data were illustrated with Kaplan-Meier survival analysis curves. An alpha level of 0.05 was accepted as significant. Competitive fitness data were compared as median CFU per milliliter by group and day and were analyzed by an exact Mann-Whitney U test.


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RESULTS
 
The sap gene cluster is cotranscribed as a single message in strain 86-028NP. Using gene-specific primers designed to amplify the nucleotide sequence spacing the junction region between each pair of adjacent genes, we demonstrated that the NTHI sap genes are cotranscribed as a single message. RNAs were isolated from strain 86-028NP and reverse transcribed, and PCR products were detected in all cases (Fig. 1). No product was detected in control samples lacking RT (–RT).



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  		FIG. 1. sap genes are cotranscribed as a single operon. The sap gene cluster contains six genes (sapA to -Z) and is 6.8 kb long. A nonpolar kanamycin cassette (kan) was used to insertionally inactivate the sapA gene where indicated. The RNA was prepared from mid-log-phase broth cultures, and the mRNA was reverse transcribed. Primers spanning the six open reading frames were used to amplify cDNAs in the presence (+) or absence (–) of RT. PCR products were separated by gel electrophoresis.

The sapA gene is present in other H. influenzae strains. A sapA gene fragment was used to probe the genomic DNAs of multiple NTHI clinical OM isolates, H. influenzae Rd, and an NTHI clinical isolate obtained from a patient with cystic fibrosis. We demonstrated by Southern blotting that a sapA gene is present in all NTHI clinical isolates examined (Fig. 2).



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  		FIG. 2. The sapA gene is conserved in NTHI. A sapA gene fragment was PCR amplified and used to probe genomic DNAs isolated from numerous NTHI clinical isolates, as listed in Materials and Methods.

Biophotonic imaging of sap promoter activity in vivo. Previously, we demonstrated by the use of differential fluorescence induction and RT-PCR analysis that sapA gene expression was up-regulated in middle ear fluids obtained 48 h after NTHI 86-028NP inoculation in a chinchilla model of OM (40). For this study, we constructed a Lux reporter NTHI strain (Fig. 3A) and monitored bioluminescence to specifically track sap operon transcription in a noninvasive manner in the chinchilla (Fig. 4). NTHI strain 1885MEE/pKMLN-01 was used to inoculate the nasopharynx (NP) and middle ear cavities of a chinchilla. The sap operon was expressed in vitro, as detected by bioluminescence imaging on chocolate agar (Fig. 3B). Since the inoculum was prepared from an overnight growth on chocolate agar, we readily detected bioluminescent bacteria in both nares after passive inhalation of 1.4 x 107 CFU of NTHI 1885MEE/pKMLN-01 (data not shown). No bioluminescence was detected in either middle ear, suggesting that the inoculum of 600 CFU was below the limit of detection via biophotonic imaging.



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  		FIG. 3. Construction of a sap promoter-driven Lux reporter plasmid. (A) The promoterless luxCDABE operon was cloned into the unique KpnI site of pGZRS-39A, generating the promoterless Lux reporter plasmid pKMLN-0. The sapA promoter region (Psap) was PCR amplified and cloned into SalI- and BamHI-digested pKMLN-0, generating the sap promoter-driven Lux reporter plasmid pKMLN-01. (B) NTHI strain 1885MEE was transformed with plasmid pKMLN-01 by electroporation, and a kanamycin-resistant clone was analyzed for bioluminescence by use of a Xenogen imaging system.



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  		FIG. 4. Analysis of sap promoter activity and localization of expression in a chinchilla model of OM. NTHI 1885MEE/pKMLN-01 was used to inoculate the nasopharynx and middle ear of a chinchilla, which was monitored for bioluminescence 2, 4, and 6 days after the bacterial challenge in prone (A, C, and E, respectively) and left lateral (B, D, and F, respectively) positions for 3 min each by use of an IVIS imaging system. Note the foci of infection present in both tympanic bullae on days 2 and 4 (A and C), with clearance on day 6 (E). Similarly, note the presence of bioluminescent NTHI from the pharynx to the middle ear via the eustachian tube in panel B. The loss of a pharyngeal focus of infection can be seen in panel D (at 4 days postchallenge), with full loss of the biophotonic signal by day 6 (F). The scale of intensity was set based on the bioluminescence signal at 2 days postchallenge. (G) Enumeration of bacteria in middle ear ({blacktriangleup}) and nasal lavage ({blacksquare}) fluids. At 3, 5, and 7 days postchallenge, middle ear and nasal lavage fluids were collected and plated on chocolate agar to determine the numbers of bacteria (CFU per milliliter). Middle ear data are presented as means ± standard deviations for the left and right ears of one chinchilla. (H) Bioluminescence within the nasal cavity. Bioluminescence was assessed at 7 days postchallenge after sacrifice and sagittal bisectioning of the skull. Bioluminescent bacteria were detected in the nares and nasoturbinates and as punctate signals at the opening of the nasopharynx (near the septal window) as well as at the distal end of the nasopharynx.

At 2 days postchallenge, however, we detected a strong bioluminescent signal emanating from both middle ear cavities (prone) and extending down into the eustachian tubes and to the pharynx (prone and lateral), indicating that the sap operon is expressed in multiple niches of the uppermost respiratory tract (Fig. 4A and B). We observed a marked decrease in bioluminescent signal at 4 days (Fig. 4C and D) and 6 days (Fig. 4E and F) postchallenge. In order to determine whether this decrease paralleled clearance of the microorganisms from the middle ear, we collected effusions and plated them on chocolate agar for a semiquantitative determination of the bacterial loads in fluids recovered from both ears at 3, 5, and 7 days postchallenge (Fig. 4G). In addition, middle ear fluids were analyzed for bioluminescence at these same time points. Collectively, these data allowed us to quantitate the amount of bioluminescence per bacterial cell (data not shown). We observed a threefold decrease in the amount of bacteria recovered from middle ear fluids from days 3 to 5 postchallenge and a further twofold decrease from days 5 to 7. The levels of bioluminescence per bacterial cell did not remain equal over the 7-day period. We observed a moderate 2.6-fold decrease followed by a complete loss of detectable bioluminescence per bacterial cell from 3 to 5 and from 5 to 7 days postchallenge, respectively (data not shown).

We were unable to detect a bioluminescent signal from the nasopharynx of the live animal over the 6-day time course. Colonization at this site was nevertheless verified by a semiquantitative determination of the numbers of CFU per milliliter of nasal lavage fluid, also recovered at 3, 5, and 7 days postchallenge (Fig. 4G). As expected, the nasopharyngeal lavage fluids contained 4 log less bacteria than were present in middle ear fluids. Since this bacterial load should still be detectable with the Xenogen imaging system, we theorized that the strong bioluminescence signal from the middle ear interfered with our ability to detect a relatively weaker signal in the nasopharynx. We thus monitored for bioluminescence within the nasal cavity after sacrifice and sagittal bisectioning of the skull. We detected bioluminescent bacteria in both the proximal (near the septal window) and distal ends of the nasopharynx, indicating that the sap operon is expressed at these sites of colonization (Fig. 4H).

Generation of a nonpolar mutation in the NTHI sap operon. Although we demonstrated expression of the sap operon in the chinchilla upper respiratory tract, we next wanted to determine whether this expression was essential for the virulence of NTHI, mediating survival of the bacterium in the nasopharynx and middle ear microenvironments. SapA is predicted to localize to the periplasm due to its signal sequence and its homology to other periplasmic solute binding proteins involved in peptide transport (46). We thus reasoned that a mutation in the sapA gene would disrupt the function of the sap operon, thereby allowing us to ascertain the involvement of SapA in NTHI survival in a chinchilla model of OM. To that end, we inserted a promoterless kanamycin resistance cassette into the sapA gene to construct a nonpolar mutant, using the strategy employed by Menard et al. (43) (Fig. 1). This construction resulted in a disruption of the sapA gene but did not affect downstream transcription of the remainder of the sap operon, as verified by RT-PCR analysis (data not shown). We additionally verified the mutant construction by Southern blot analysis (data not shown). The sapA mutation did not affect the growth of the bacteria on chocolate agar or in BHI broth supplemented with 2 µg of hemin/ml. The mutant growth rate and growth pattern were similar to those of the parent strain, indicating that the disruption of sapA had no deleterious affects on bacterial growth.

Mutation in sapA confers sensitivity to (r)cBD-1. Members of our laboratory previously described the cloning of a cDNA encoding a chinchilla ß-defensin and demonstrated both expression of the transcript in the uppermost airway and antimicrobial activity in vitro (26). We wanted to determine whether the mutation in sapA altered the sensitivity to killing by (r)cBD-1. Using a bactericidal assay, we assessed the ability of (r)cBD-1 to kill NTHI strain 86-028NP compared to its ability to kill the sapA mutant strain. The mutation in the sapA gene rendered the bacterium more sensitive than the parent strain to the antimicrobial activity of (r)cBD-1 over a concentration range of 2.5 to 20 µg of (r)cBD-1/ml (Fig. 5) in all cases. Specifically, when we incubated cells with 5 µg of (r)cBD-1/ml, we observed an eightfold increase in killing of the mutant strain compared to killing of the parent strain. Likewise, due to the homology between cBD-1 and human ß-defensin 3, we tested the susceptibility of the sapA mutant to killing by recombinant human ß-defensin 3 [(r)hBD-3]. We observed a >8-fold increase in killing of the mutant strain compared to killing of the parent strain at a concentration of 0.4 µg of (r)hBD-3/ml (data not shown). These concentrations of (r)cBD-1 and (r)hBD-3 are within the physiological range for antimicrobial peptides in the airway and nasal secretions (14, 41, 57).



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  		FIG. 5. The sapA mutant is more sensitive to killing by recombinant chinchilla ß-defensin. The sapA mutant (gray bars) and the parent strain (black bars) were incubated with increasing concentrations of (r)cBD-1 for 1 h and then plated on chocolate agar for determination of the numbers of bacteria. Data represent percentages of killing relative to the bacterial concentration at the start of the assay. The data shown are representative of three separate experiments.

The sapA mutant is attenuated for survival in vivo. Having demonstrated that the sap operon plays a role in resistance to antimicrobial peptides, we next assessed whether the sapA mutant was impaired in its ability to colonize the nasopharynx or to multiply in the chinchilla middle ear compared to the parent strain (Fig. 6). Similar to the parent strain, the sapA mutant colonized the nasopharynx at a level of approximately 104 CFU/ml and increased 10-fold by day 4 after the challenge. Subsequent sampling, however, revealed that the sapA mutant was unable to sustain this level of colonization, and its concentration decreased steadily until it was completely cleared by day 12 postchallenge, a minimum of 22 days earlier than the parent strain. A comparison of the mean times until the bacterial concentration first dropped below 100 CFU/ml for the two groups (Fig. 6A) showed that there was a statistically significant difference between the groups in time to clearance (P = 0.039). A similar trend in clearance was observed when the sapA mutant was inoculated into the middle ear of a chinchilla in a transbullar model of frank disease induction (Fig. 6B). Early in the disease course, the sapA mutant increased from the inoculum of 2,500 CFU to approximately 108 CFU/ml during the first 48 h and maintained this level for 8 days. However, subsequent quantitation of bacterial cell numbers revealed that the mutant strain was unable to maintain this bacterial load, steadily decreasing until it was cleared a minimum of 16 days earlier than the parent strain. This difference in time to clearance was statistically significant (P = 0.043). Furthermore, we observed that products of the sap operon also mediated suppression of an early inflammatory immune response, as evidenced by increased erythema, vessel dilatation, and darkening behind the tympanic membrane in animals that were challenged with the sapA mutant (data not shown).



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  		FIG. 6. A mutation in sapA attenuates the survival of NTHI in vivo. The chinchilla middle ear and nasopharynx were inoculated with the sapA mutant ({blacktriangleup}, dotted line) or the parent strain ({blacksquare}, solid line) and then monitored for OM development and viable bacterial counts. (A) Relative abilities of the strains to colonize the nasopharynx. The sapA mutant was not recovered from nasal lavage fluids beginning 12 days following inoculation. The data are presented as means ± standard deviations for two animals (n = 2). (B) Relative abilities of the strains to survive in the middle ear. The sapA mutant was cleared from the middle ear beginning 8 days after inoculation. The data are presented as means ± standard deviations for the left and right ears of two animals (n = 4).

In order to further evaluate the loss in relative virulence displayed by the sapA mutant, we coinoculated both the parent and mutant strains into the middle ear cavity and monitored them for competitive fitness (Fig. 7). Four days after middle ear coinfections of approximately 1,250 CFU of each strain, we observed an approximately 3-log difference in relative bacterial counts between the parent and the mutant in middle ear fluids collected from chinchillas, with the sapA mutant being present at a lower concentration. During the subsequent 48 h, the mutant was clearly attenuated in its ability to compete for survival in the middle ear, resulting in a significant reduction in the concentration of the mutant on days 6 and 8 postchallenge (P = 0.029 for both days).



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  		FIG. 7. The sapA mutant ({blacktriangleup}, dotted line) was unable to compete with the parent strain ({blacksquare}, solid line) for survival in the middle ear environment. The parent and sapA mutant strain were coinoculated at a 1:1 ratio and monitored for the ability to induce OM and viable bacterial counts over time. The sapA mutant was attenuated for survival in the middle ear, resulting in a 7-log decrease in the amount of the sap A mutant strain recovered from middle ear fluids relative to the parent strain at 8 days postchallenge. The data are presented as means ± standard deviations for the left and right ears from two animals (n = 4).


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DISCUSSION
 
It was shown previously that the sap gene products mediate AP resistance in S. enterica serovar Typhimurium, V. fischeri, P. mirabilis, and E. chrysanthemi (13, 37, 42, 46). The sap gene products from NTHI share homology with SapABCDF proteins of S. enterica serotype Typhimurium which belong to the ATP-binding-cassette (ABC) family of transporters classified as the peptide/opine/nickel uptake transporter (PepT) subfamily (11, 12, 54). Members of this subfamily, however, demonstrate diversity in substrate specificity by transporting oligopeptides, aminoglycoside antibiotics, dipeptides, nickel, ß-galactoside, ß-glucoside, and maltose (12, 54).

The sapZ gene, which produces an as yet uncharacterized hypothetical protein, is cotranscribed with sapABCDF in NTHI strain 86-028NP. SapZ is predicted to be a transmembrane protein with homologs in the sap operon-containing bacteria Pasteurella multocida, S. enterica serovar Typhimurium, S. enterica, and E. coli O157:H7 as well as in Neisseria meningitidis and Pseudomonas aeruginosa, which do not contain a sap operon. In those bacteria that contain the sap gene cluster, however, the sapZ homologue is not located in the sap operon.

It is currently unclear whether SapA mediates direct binding and subsequent uptake of APs into the bacterial cytoplasm, thereby targeting them for degradation, or whether the sap gene products mediate other mechanisms of bacterial resistance to APs. Moreover, few studies have addressed the biological significance that a mutation in the sap operon renders upon the microorganism in vivo, and none have determined the biological consequences of such a mutation in a bacterium-induced disease model. Our work begins to define the importance of the sap operon biologically, specifically as it relates to the virulence of NTHI. Our data suggest that, similar to SapABCDF in Salmonella, the Sap gene products of NTHI function in part to mediate resistance to APs in vivo.

In an ongoing investigation of the role of innate immunity in the pathogenesis of OM, our laboratory recently identified and cloned cBD-1, a homolog of human ß-defensin 3 (26). The cBD-1 gene is highly expressed in the chinchilla NP, skin, and tongue mucosa compared to sites that are typically not colonized with normal flora. This increased expression on mucosal surfaces may reflect a primary defense against infection by pathogenic microbes at these portals of entry. Other investigators have demonstrated the expression of lysozyme, lactoferrin, hBD-1, and hBD-2 by human tubotympanal epithelial cells in culture (36). Further work revealed that hBD-2 was expressed at a higher level in a small number of diseased human middle ear mucosa samples than in nondiseased control tissues, and this expression was upregulated by interleukin-1{alpha}. In vitro, hBD-2 inhibits the growth of Moraxella catarrhalis, Streptococcus pneumoniae, and H. influenzae, which are all common OM pathogens (32, 33, 36, 56, 59). NTHI survival following invasion of the middle ear cavity may depend upon its ability to resist APs that play a role in maintaining sterility at this site. Our ability to produce recombinant chinchilla ß-defensin allowed us to more fully analyze the important role of the host innate immune response to NTHI, a commensal opportunist that can cause OM.

We demonstrated that the sap genes are expressed in the middle ear, the eustachian tubes, and both the anterior and posterior portions of the nasopharynx. This ubiquitous expression in the uppermost airway suggests an important role for this operon in adaptation to these microenvironments. The decrease in bioluminescence (Fig. 4, compare day 4 to day 6) cannot be attributed to bacterial clearance from the middle ear, since we measured only a twofold decrease in bacterial cell numbers in middle ear fluids recovered from chinchillas at 5 to 7 days postinfection, to a level well above the limit of detection of bioluminescence in our assay. Furthermore, the loss of bioluminescence was not the result of plasmid instability since the number of kanamycin-resistant clones recovered from the middle ear fluid at 7 days postinfection equaled that of clones that grew in the absence of antibiotic selection and since all colonies were bioluminescent on chocolate agar, as expected (data not shown). Collectively, these data suggest that transcription from the sap promoter resulting in lux expression may occur early in the course of infection as the bacterium adapts to its environment, yet this expression appears to be transient. A limitation to monitoring Lux activity via biophotonic imaging, however, is the amount of available oxygen, as oxygen is required for the luminescence reaction (17). It has been determined that gas exchange can occur both into and out of the middle ear cavity via the eustachian tube and also by passive diffusion across the middle ear mucosa (22, 53). Furthermore, the chinchilla eustachian tube appears to be continually semipatulous where a constant airflow may be maintained by the passage of air into the middle ear cleft (15). The presence of an air-fluid interface in the chinchilla bulla 7 days after infection still allows gas exchange through the middle ear mucosa (29). Importantly, we demonstrated that bacteria recovered from middle ear fluids 7 days after infection do not bioluminesce when exposed to atmospheric oxygen, arguing against the possibility that the Lux substrate was produced in vivo but was not utilized due to oxygen limitation.

A mutation in the first gene of the operon, sapA, conferred sensitivity to (r)cBD-1, a human ß-defensin 3 homologue (26), at concentrations within the physiological range for APs (14, 41, 57). These data demonstrate in vitro that the products of the sap operon in NTHI mediate resistance to a ß-defensin that is expressed in the upper airway of the chinchilla, perhaps suggesting an important role for the Sap proteins in NTHI virulence, particularly in the establishment of colonization at these sites.

We hypothesized that a mutation in the sapA gene would attenuate the survival of this organism in a relevant animal model of NTHI-induced OM. Indeed, we showed that the sapA mutant attenuated NTHI survival in a chinchilla model of OM when this mutant was introduced either alone or in competition with the parent strain into this host. We observed a significant difference in the time to clearance of the sapA mutant when it was coinoculated into the middle ear with the parent strain versus the time to clearance for inoculation of the mutant alone. The inoculation of high levels of bacteria directly into the middle ear may initially overwhelm the threshold of a protective innate immune response, thereby allowing the bacteria to grow and to initiate early infection. An infection leading to enhanced inflammation, however, would augment earlier clearance (relative to the parent strain) of those microorganisms that were unable to mediate resistance to APs. In contrast, the sapA mutant was immediately impaired in its ability to compete with the parent strain in establishing infection, as demonstrated by its inability to attain the concentration of bacteria observed for the parent strain in the middle ear fluid. This smaller population would be highly sensitive to attack by APs, thereby being eradicated from the mixed population early. It is clear from these data that the sap gene products are required for colonization and virulence in a chinchilla model of bacterium-induced infection.

We maintain that there exists a low constitutive level of AP production by mucosal cells in both the nasopharynx and sterile sites such as the middle ear. It has been reported that bacterial exposure to sublethal concentrations of APs may modulate an AP-resistant phenotype in bacteria (52). AP expression at sites that are colonized with normal flora would help to maintain the delicate balance between benign colonization and infection by the microorganism as well as protect against invading pathogens at that site. Similarly, mucosal cells lining a normally sterile niche would protect against invading pathogens or opportunistic commensals by increasing the production of inflammatory mediators, resulting in the rapid up-regulation of APs as a first line of defense at this site. In this manner, the middle ear mucosa may up-regulate the expression of APs to maintain the sterility of the middle ear environment. In the pediatric nasopharynx, an environment constantly exposed to mucosal fluids, there may be pressure to select for microbes possessing mechanisms of AP resistance. Indeed, for NTHI, cell wall modifications that mediate AP resistance have been reported (38). We reasoned that bacteria that had colonized the nasopharynx or infected the middle ear cavity would possess mechanisms to resist the antimicrobial weapons of the host immune response.

In agreement with those of other investigators, our data indicate that the sap operon gene products mediate antimicrobial peptide resistance in vitro and, furthermore, that SapA is required for the survival of NTHI, during both colonization of the chinchilla nasopharynx and infection of the middle ear cavity. We observed increased erythema, vessel dilatation, and darkening behind the tympanic membrane in animals that were challenged with the sapA mutant (data not shown), suggesting that the products of the sap operon may mediate the suppression of an early inflammatory immune response. It is tempting to speculate that the early production of inflammatory mediators, resulting in an up-regulation of cBD-1 production by the chinchilla host, combined with the loss of the ability to import and degrade this AP thereby rendered the sapA mutant more sensitive to membrane attack and AP killing, thus mediating the attenuated phenotype we observed in vivo. In addition, others have demonstrated that multiple APs can act synergistically, resulting in enhanced killing of bacteria (7, 57). We do expect that although (r)cBD-1 alone is efficient at killing NTHI in vitro, the in vivo environment may potentially contain a variety of APs that are up-regulated and released as a means to control infection, specifically at normally sterile sites.

The early, transient expression of the sap genes in vivo does not contradict the results that showed reduced survival of the sapA mutant later in infection. Early expression of the sap system precludes resistance to low-level antimicrobial peptides present at a normally sterile anatomical niche. The bacterial load may signal an up-regulation of defensin expression by the host, thereby mediating effective clearance of the microorganism, albeit later during infection. Hence, the sapA mutant would be impaired in its ability to resist these higher levels of antimicrobial peptides. It is important, however, that resistance to antimicrobial peptides may be only one function of the sap system in vivo.

Collectively, these data argue that the ability to resist antimicrobial peptides and perhaps other mechanisms of attack by the host are necessary for NTHI survival and implicate the product of the sapA gene as a virulence factor in NTHI infection. Preliminary data from our laboratory suggest that the Sap proteins may play a role in iron acquisition via either SapA-mediated binding and transport or the regulation of iron acquisition pathways of the microorganism (K. M. Mason, R. S. Munson, Jr., and L. O. Bakaletz, Abstr. 104th Gen. Meet. Am. Soc. Microbiol., p. 116, 2004). Products of the sap operon may function to allow bacteria to sense their microenvironment and to respond in part by mediating resistance to APs.


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ACKNOWLEDGMENTS
 
We thank Jennifer Freeman and Molly Bruggeman for excellent technical assistance, Yingjie Zhang for initial RT-PCR analysis, and Joe Jurcisek for assistance with data preparation. We thank SmithKline Beecham for the NTHI strains. We thank Jennifer Neelans for manuscript preparation.

This work was supported by grant R01-DC03915 to L.O.B. from NIDCD/NIH and a National Research Service Award to K.M.M. from NIDCD/NIH.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pediatrics, Center for Microbial Pathogenesis, Columbus Children's Research Institute, The Ohio State University College of Medicine and Public Health, 700 Children's Dr., Rm. W591, Columbus, OH 43205-2696. Phone: (614) 722-2915. Fax: (614) 722-2818. E-mail: BakaletL{at}pediatrics.ohio-state.edu. Back

Editor: D. L. Burns


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Infection and Immunity, January 2005, p. 599-608, Vol. 73, No. 1
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.1.599-608.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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