A functional tonB gene is required for both virulence and competitive fitness in a chinchilla model of Haemophilus influenzae otitis media
© Morton et al.; licensee BioMed Central Ltd. 2012
Received: 18 November 2011
Accepted: 25 June 2012
Published: 25 June 2012
Haemophilus influenzae requires heme for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient.
An insertional mutation in tonB was constructed and the impact of the mutation on virulence and fitness in a chinchilla model of otitis media was determined. The tonB insertion mutant strain was significantly impacted in both virulence and fitness as compared to the wildtype strain in this model.
The tonB gene of H. influenzae is required for the establishment and maintenance of middle ear infection in this chinchilla model of bacterial disease.
Haemophilus influenzae is a fastidious facultatively anaerobic Gram-negative bacterium that causes a range of human infections including otitis media, meningitis, epiglottitis and pneumonia [1, 2]. H. influenzae lacks all enzymes in the biosynthetic pathway for the porphyrin ring, and as a result is unable to synthesize protoporphyrin IX (PPIX), the immediate precursor of heme. Since H. influenzae cannot synthesize PPIX the organism has an absolute growth requirement for an exogenous source of either heme or PPIX in the presence of iron [3, 4]. As a result of this growth requirement, H. influenzae has evolved a complex multifunctional array of uptake mechanisms to ensure that it is able to utilize available porphyrin or iron in vivo. These mechanisms include numerous outer membrane proteins (OMPs) that bind one or more host heme- and iron- containing proteins including hemoglobin, hemoglobin-haptoglobin, heme-hemopexin and heme-human serum albumin complexes, and ferritransferrin [5–9]. All of these H. influenzae OMPs that bind host heme- or iron- containing proteins are TonB-dependent transporters (TBDT) . The outer membrane of Gram-negative bacteria hinders the uptake of essential nutrients, and, while small molecules passively diffuse through porins, substrates that are too large to pass through the porins and/or are present at very low concentrations require energized transport [10, 11]. A cytoplasmic transmembrane protein complex composed of three proteins, TonB, ExbB and ExbD, spans the periplasm and interacts with specific TBDTs. This TonB complex transduces the proton motive force of the cytoplasmic membrane to energize transport of substrates through a specific TBDT . Mutations of many of the TBDTs of H. influenzae have been shown to have significant impacts on virulence in animal models of infection, for example complete deletion of the complement of hemoglobin-haptoglobin binding proteins (Hgps) in a nontypeable strain significantly reduces the severity of middle ear infection in the chinchilla model of otitis media . Deletion of the complement of hgps in a type b strain had a significant impact on virulence in a weanling rat model of bacteremia but not in a 5-day old infant rat model of bacteremia . Similarly, mutation of the heme-hemopexin acquisition protein HxuC impacted virulence in the weanling but not in the 5-day old infant rat . However, a mutant strain lacking both the Hgps and HxuC was unable to sustain bacteremia in 5-day old rats . Mutation of an additional TBDT designated Hup (heme utilization protein) had no impact on virulence in rat models of disease . In addition to the TBDTs many other proteins have been shown to be involved in heme acquisition and in virulence in rat models of invasive disease, including the periplasmic heme-binding protein HbpA, lipoprotein e(P4) and the tellurite resistance protein TehB [14–16]. These data highlight the complexity of the heme acquisition systems of H. influenzae and their potential roles in virulence.
The tonB gene of H. influenzae has previously been mutated and shown to be essential for the utilization of transferrin bound iron, hemoglobin, hemoglobin-haptoglobin, heme-hemopexin and low levels of heme [17, 18]. In addition a tonB mutant of the type b H.influenzae strain Eagan was avirulent in an infant rat model of bacteremia .
The goal of the present study was to determine if the TonB system is essential for the establishment and maintenance of otitis media in the chinchilla, a widely used model of human otitis media.
Bacterial strains and growth conditions
Nontypeable H. influenzae strain 86-028NP is a minimally passaged nasopharyngeal isolate from a pediatric patient who underwent tympanostomy and tube insertion for chronic otitis media at Columbus Children’s Hospital. Strain 86-028NP has been extensively characterized in chinchilla models of otitis media [19–21]. H. influenzae was maintained long-term in 10% skim milk at −80°C. H. influenzae was routinely grown on chocolate agar with bacitracin (BBL, Becton-Dickinson, Sparks, MD, USA) at 37°C. When necessary, H. influenzae was grown on brain heart infusion (BHI) agar (Difco, Becton-Dickinson, Sparks, MD, USA) supplemented with 10 μg ml-1 heme and 10 μg ml-1 β-NAD (supplemented BHI; sBHI) and the appropriate antibiotic(s). Heme-deplete growth was performed in BHI broth supplemented with 10 μg ml-1 β-NAD alone (heme-deplete BHI; hdBHI).
Human serum albumin (HSA), and heme (as hemin) were purchased from Sigma. Stock heme solutions (1 mg ml-1 heme in 4% v/v triethanolamine) were prepared as previously described . Heme-albumin complexes were prepared as previously described .
Construction of a tonB insertional mutant
The genome of strain 86-028NP has been fully sequenced , and the exbB exbD and tonB genes in this strain are designated with the locus numbers NTHI0358, NTHI0359 and NTHI0360 respectively. An insertional mutation in tonB (NTHI0360) was constructed as follows. A pair of primers was designed for use in the PCR, based on the available H. influenzae genomic sequence, to amplify an ~1930-bp region encompassing the entire tonB gene. Primers were designated TONB-1 and TONB-2 and had the respective sequences 5’-AATGGCAAGATCAAAACGG-3’ and 5’-CCTTATGTTGGATTACTTGG-3’. PCRs were performed in a 50 μl volume using 100 ng of H. influenzae chromosomal DNA as template, and the reactions contained 2 mM MgCl2, 200 μM each dNTP and 2 U of FastStart Taq DNA Polymerase (Roche, Indianapolis, IN, USA). The PCR was carried out for 30 cycles with each cycle consisting of denaturation at 95°C for 1 min, annealing for 1 min at 52°C and primer extension at 72°C for 2 min with one final extension for 30 min. A PCR product of the expected size was obtained and successfully cloned into the TA cloning vector pCR2.1-TOPO (Invitrogen); a plasmid harbouring the correct insert was confirmed by automated sequencing and designated pDJM15. pDJM15 was linearized at a unique Ssp I restriction site within the coding sequence of tonB. The zeocin resistance marker was excised from pEM7/Zeo (Invitrogen) by digestion with Eco RV and Pvu II and the ~600-bp fragment containing the zeocin marker was ligated to linearized pDJM15 to yield pDJM19. H. influenzae strain 86-028NP was transformed to zeocin resistance using pDJM19 by the static aerobic method as previously described with selection on zeocin at 25 μg ml -1[7, 12]. A zeocin resistant 86-028NP transformant was confirmed as correct by sizing and sequencing of a PCR product and was designated HI2280.
Chinchilla models of otitis media
A total of 15 adult chinchillas (Chinchilla lanigera) with no evidence of middle ear infection by either otoscopy or tympanometry at the beginning of the study were used. Animals were rested for at least 7 days upon arrival to acclimate them to the vivarium. After acclimation, chinchillas were challenged with H. influenzae in two separate experiments.
In the first experiment groups of five chinchillas were challenged in both ears transbullarly with approximately 2000 c.f.u. of either NTHi strain 86-028NP or its tonB mutant derivative HI2280 in order to compare virulence of the two strains. Transbullar inocula were delivered in 300 μl 0.1% gelatin in PBS by direct injection of bacterial suspensions into the superior bullae. Actual challenge dosages received were confirmed by plate count. On days 4, 7, 11, 14 and 17 days post challenge middle ear effusions (MEE) were collected by epitympanic tap, i.e. withdrawl of fluids from the middle ear cavity using a 1.5 inch 25-gauge hypodermic needle . On epitympanic tap the minimum amount of fluid required to perform a dilution series and plating to determine bacterial titers was withdrawn. Ears were scored as “dry” (i.e. no detectable MEE) when an ear was successfully tapped and no evidence of effusion was seen when the plunger of a 1 ml syringe was pulled back maximally. In some cases ears were scored as “ISVP” (insufficient volume to plate) when there was any evidence of effusion, which in some cases manifested as bubbles in the hub of the syringe, but the volume was insufficient to perform a dilution series. Although bacterial titers could not be determined for ISVP ears such ears were considered positive for the presence of MEE.
Bacterial titers were determined using a modification of the track-dilution method of Jett et al as previously described [7, 27]. Serial dilutions (0 to 10-5) of freshly recovered MEE were made in 0.1% gelatin in PBS and 10 μl aliquots from each dilution were plated in triplicate on sBHI and all plates were incubated at 37°C for 48 hours to quantify c.f.u. NTHi ml -1. Since heme is not required for anaerobic growth plates were incubated both aerobically and anaerobically to remove any potential impact of the tonB mutation on detection of bacteria.
In the second experiment a group of 5 chinchillas was challenged transbullarly with an inoculum containing equal numbers of NTHi strain 86-028NP and its tonB mutant derivative HI2280 (total of approximately 2500 c.f.u.) to quantify differential fitness of the two strains. All ears were tapped for collection of MEE on days 4, 7, 11, 15 and 18 days post infection. Each recovered MEE was plated on sBHI and on sBHI containing 7.5 μg ml -1 zeocin to determine total bacterial titer and the titer of the mutant strain respectively (7.5 μg ml -1 zeocin was used in these experiments since it is the lowest concentration we have found to be adequate for differentiation of zeocin-sensitive and zeocin-resistant strains). The competitive index (CI) was calculated for all ears at all time-points in experiment 2 and is defined as the ratio of the output mutant/wildtype ration to the input mutant/wildtype ratio.
Animal procedures have been described in detail elsewhere [19, 26, 28]. The protocol for use of animals in this study was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center.
Statistical comparisons of growth between strains under the same growth conditions in vitro were made using the Mann–Whitney test. Percentages of infected ears yielding a detectable effusion and percentages of effusions with detectable wildtype or mutant bacteria were compared using Fisher’s Exact test.
Analyses were performed using Analyse-It for Microsoft Excel v2.22 (Analyze-It Software Inc., Leeds, England). A P value < 0.05 was taken as statistically significant.
Results and discussion
In addition to the impact of tonB mutation on heme source utilization, we and others have previously shown that utilization of many of these heme sources is abrogated by mutations in specific TBDTs [6–8, 30]. For example utilization of heme-HSA is dependent on a functional HxuC protein , and the utilization of hemoglobin-haptoglobin complexes requires the presence of a functional hemoglobin-haptoglobin binding protein (HgpA, HgpB, or HgpC) [6, 25].
Having established that the tonB mutant strain exhibited the expected in vitro phenotype, we compared the mutant and wildtype strains for their respective abilities to establish and maintain infection of the middle ear in chinchilla models of otitis media.
The TonB protein has previously been shown to be essential for virulence of H. influenzae type b strains in the infant rat model of bacteremia . However, there have been no reports of the impact of tonB mutations on virulence in other clinically relevant models of H. influenzae disease, including models of otitis media. Two separate experiments were performed, one to assess the impact of the tonB mutation on virulence by comparison of two groups of chinchillas infected with individual strains, and the second to assess the impact of the mutation on competitive fitness by infection of a cohort of animals with equal numbers of both strains.
These data demonstrate that expression of TonB is essential for both virulence and competitive fitness of a nontypeable H. influenzae strain in a chinchilla model of otitis media. It has previously been shown that a tonB mutant of a type b strain is avirulent in the infant-rat model of invasive disease. Thus TonB is essential for establishment of H. influenzae disease in multiple clinically relevant animal models of H. influenzae disease. Since TonB is required for the function of TBDTs and all of the H. influenzae TBDTs appear to be involved in acquisition of heme these data indicate that heme acquisition is an essential process during infection caused by H. influenzae. This observation is further supported by the reports of the impact of mutations in specific H. influenzae TBDTs, including the hemoglobin-haptoglobin binding proteins (Hgps) and the heme-hemopexin acquisition protein (HxuC), on virulence in animal models [12, 13].
In conclusion expression of TonB is required for the establishment and maintenance of infection in an animal model of H. influenzae otitis media.
This work was supported in part by Public Health Service Grant AI29611 from the National Institute of Allergy and Infectious Disease to TLS. The authors gratefully acknowledge the support of the Children’s Hospital Foundation.
- Turk DC: The pathogenicity ofHaemophilus influenzae. J Med Microbiol. 1984, 18: 1-16. 10.1099/00222615-18-1-1.PubMedView ArticleGoogle Scholar
- Murphy TF, Faden H, Bakaletz LO, Kyd JM, Forsgren A, Campos J, Virji M, Pelton SI: NontypeableHaemophilus influenzaeas a pathogen in children. Pediatr Infect Dis J. 2009, 28: 43-48. 10.1097/INF.0b013e318184dba2.PubMedView ArticleGoogle Scholar
- Panek H, O'Brian MR: A whole genome view of prokaryotic haem biosynthesis. Microbiology. 2002, 148: 2273-2282.PubMedView ArticleGoogle Scholar
- White DC, Granick S: Hemin biosynthesis inHaemophilus. J Bacteriol. 1963, 85: 842-850.PubMedPubMed CentralGoogle Scholar
- Morton DJ, Stull TL: Haemophilus. Iron Transport in Bacteria. Edited by: Crosa JH, Mey AR, Payne SM. 2004, American Society for Microbiology, Washington, DC, 273-292.View ArticleGoogle Scholar
- Morton DJ, Whitby PW, Jin H, Ren Z, Stull TL: Effect of multiple mutations in the hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA, HgpB, and HgpC ofHaemophilus influenzaetype b. Infect Immun. 1999, 67: 2729-2739.PubMedPubMed CentralGoogle Scholar
- Morton DJ, Smith A, Ren Z, Madore LL, VanWagoner TM, Seale TW, Whitby PW, Stull TL: Identification of a haem-utilization protein (Hup) inHaemophilus influenzae. Microbiology. 2004, 150: 3923-3933. 10.1099/mic.0.27238-0.PubMedView ArticleGoogle Scholar
- Morton DJ, Seale TW, Madore LL, VanWagoner TM, Whitby PW, Stull TL: The haem-haemopexin utilization gene cluster (hxuCBA) as a virulence factor ofHaemophilus influenzae. Microbiology. 2007, 153: 215-224. 10.1099/mic.0.2006/000190-0.PubMedView ArticleGoogle Scholar
- Morton DJ, Williams P: Siderophore-independent acquisition of transferrin-bound iron byHaemophilus influenzaetype b. J Gen Microbiol. 1990, 136: 927-933. 10.1099/00221287-136-5-927.PubMedView ArticleGoogle Scholar
- Schauer K, Rodionov DA, de RH: New substrates for TonB-dependent transport: do we only see the 'tip of the iceberg'?. Trends Biochem Sci. 2008, 33: 330-338. 10.1016/j.tibs.2008.04.012.PubMedView ArticleGoogle Scholar
- Nikaido H: Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev. 2003, 67: 593-656. 10.1128/MMBR.67.4.593-656.2003.PubMedPubMed CentralView ArticleGoogle Scholar
- Morton DJ, Bakaletz LO, Jurcisek JA, VanWagoner TM, Seale TW, Whitby PW, Stull TL: Reduced severity of middle ear infection caused by nontypeableHaemophilus influenzaelacking the hemoglobin/hemoglobin-haptoglobin binding proteins (Hgp) in a chinchilla model of otitis media. Microb Pathog. 2004, 36: 25-33. 10.1016/j.micpath.2003.08.007.PubMedView ArticleGoogle Scholar
- Seale TW, Morton DJ, Whitby PW, Wolf R, Kosanke SD, VanWagoner TM, Stull TL: Complex role of hemoglobin and hemoglobin-haptoglobin binding proteins inHaemophilus influenzaevirulence in the infant rat model of invasive infection. Infect Immun. 2006, 74: 6213-6225. 10.1128/IAI.00744-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Morton DJ, Smith A, VanWagoner TM, Seale TW, Whitby PW, Stull TL: Lipoproteine(P4) ofHaemophilus influenzae: Role in heme utilization and pathogenesis. Microbes Infect. 2007, 9: 932-939. 10.1016/j.micinf.2007.03.013.PubMedPubMed CentralView ArticleGoogle Scholar
- Morton DJ, Seale TW, Bakaletz LO, Jurcisek JA, Smith A, VanWagoner TM, Whitby PW, Stull TL: The heme-binding protein (HbpA) ofHaemophilus influenzaeas a virulence determinant. Int J Med Microbiol. 2009, 299: 479-488. 10.1016/j.ijmm.2009.03.004.PubMedPubMed CentralView ArticleGoogle Scholar
- Whitby PW, Seale TW, Morton DJ, VanWagoner TM, Stull TL: Characterization of theHaemophilus influenzae tehBgene and its role in virulence. Microbiology. 2010, 156: 1188-1200. 10.1099/mic.0.036400-0.PubMedPubMed CentralView ArticleGoogle Scholar
- Jarosik GP, Sanders JD, Cope LD, Muller-Eberhard U, Hansen EJ: A functionaltonBgene is required for both utilization of heme and virulence expression byHaemophilus influenzaetype b. Infect Immun. 1994, 62: 2470-2477.PubMedPubMed CentralGoogle Scholar
- Jarosik GP, Maciver I, Hansen EJ: Utilization of transferrin-bound iron byHaemophilus influenzaerequires an intacttonBgene. Infect Immun. 1995, 63: 710-713.PubMedPubMed CentralGoogle Scholar
- Bakaletz LO, Kennedy B-J, Novotnoy LA, Duquesne G, Cohen J, Lobet Y: Protection against development of otitis media induced by nontypeableHaemophilus influenzaeby both active and passive immunization in a chinchilla model of virus-bacterium superinfection. Infect Immun. 1999, 67: 2746-2762.PubMedPubMed CentralGoogle Scholar
- Kennedy BJ, Novotnoy LA, Jurcisek JA, Lobet Y, Bakaletz LO: Passive transfer of antiserum specific for immunogens derived from a nontypeableHaemophilus influenzaeadhesin and lipoprotein D prevents otitis media after heterologous challenge. Infect Immun. 2000, 68: 2756-2765. 10.1128/IAI.68.5.2756-2765.2000.PubMedPubMed CentralView ArticleGoogle Scholar
- Suzuki K, Bakaletz LO: Synergistic effect of adenovirus type 1 and nontypeableHaemophilus influenzaein a chinchilla model of experimental otitis media. Infect Immun. 1994, 62: 1710-1718.PubMedPubMed CentralGoogle Scholar
- Poje G, Redfield RJ: General methods for culturingHaemophilus influenzae. Methods Mol Med. 2003, 71: 51-56.PubMedGoogle Scholar
- Harrison A, Dyer DW, Gillaspy A, Ray WC, Mungur R, Carson MB, Zhong H, Gipson J, Gipson M, Johnson LS, Lewis L, Bakaletz LO, Munson RS: Genomic sequence of an otitis media isolate of nontypeableHaemophilus influenzae: comparative study withH. influenzaeserotype d, strain KW20. J Bacteriol. 2005, 187: 4627-4636. 10.1128/JB.187.13.4627-4636.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Morton DJ, Madore LL, Smith A, VanWagoner TM, Seale TW, Whitby PW, Stull TL: The heme-binding lipoprotein (HbpA) ofHaemophilus influenzae: role in heme utilization. FEMS Microbiol Lett. 2005, 253: 193-199. 10.1016/j.femsle.2005.09.016.PubMedView ArticleGoogle Scholar
- Morton DJ, VanWagoner TM, Seale TW, Whitby PW, Stull TL: Differential utilization byHaemophilus influenzaeof hemoglobin complexed to the three human haptoglobin phenotypes. FEMS Immunol Med Microbiol. 2006, 46: 426-432. 10.1111/j.1574-695X.2006.00052.x.PubMedView ArticleGoogle Scholar
- Gitiban N, Jurcisek JA, Harris RH, Mertz SE, Durbin RK, Bakaletz LO, Durbin JE: Chinchilla and murine models of upper respiratory tract infections with respiratory syncytial virus. J Virol. 2005, 79: 6035-6042. 10.1128/JVI.79.10.6035-6042.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Jett BD, Hatter KL, Huycke MM, Gilmore MS: Simplified agar plate method for quantifying viable bacteria. Biotechniques. 1997, 23: 648-650.PubMedGoogle Scholar
- Bakaletz LO, Leake ER, Billy JM, Kaumaya PTP: Relative immunogenicity and efficacy of two synthetic chimeric peptides of fimbrin as vaccinogens against nasopharyngeal colonization by nontypeableHaemophilus influenzaein the chinchilla. Vaccine. 1997, 15: 955-961. 10.1016/S0264-410X(96)00298-8.PubMedView ArticleGoogle Scholar
- Saeed-Kothe A, Yang W, Mills SD: Use of the riboflavin synthase gene (ribC) as a model for development of an essential gene disruption and complementation system forHaemophilus influenzae. Appl Environ Microbiol. 2004, 70: 4136-4143. 10.1128/AEM.70.7.4136-4143.2004.PubMedPubMed CentralView ArticleGoogle Scholar
- Cope LD, Yogev R, Muller-Eberhard U, Hansen EJ: A gene cluster involved in the utilization of both free heme and heme:hemopexin byHaemophilus influenzaetype b. J Bacteriol. 1995, 177: 2644-2653.PubMedPubMed CentralGoogle Scholar
- Hong W, Mason K, Jurcisek J, Novotny L, Bakaletz LO, Swords WE: Phosphorylcholine decreases early inflammation and promotes the establishment of stable biofilm communities of nontypeableHaemophilus influenzaestrain 86-028NP in a chinchilla model of otitis media. Infect Immun. 2007, 75: 958-965. 10.1128/IAI.01691-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Hong W, Juneau RA, Pang B, Swords WE: Survival of bacterial biofilms within neutrophil extracellular traps promotes nontypeableHaemophilus influenzaepersistence in the chinchilla model for otitis media. J Innate Immun. 2009, 1: 215-224. 10.1159/000205937.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.