1 Department of Microbiology and Immunology, State University of New York at Buffalo, Buffalo, NY 14214, USA
2 Witebsky Center for Microbial Pathogenesis and Immunology, State University of New York at Buffalo, Buffalo, NY 14214, USA
3 Division of Infectious Diseases, Department of Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA
Correspondence
Anthony A. Campagnari
aac{at}acsu.buffalo.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the M. catarrhalis 7169 mhuA nucleic acid sequence and MhuA amino acid sequence reported in this paper is AY574198.
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INTRODUCTION |
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M. catarrhalis is a strict human pathogen that has been reported to colonize a significant number of young, healthy children (Faden, 2001). It also appears that this organism can maintain a presence in the human lung, primarily in individuals with COPD (Bakri et al., 2002
). Despite these data, there is very little information regarding the basic biological mechanisms that M. catarrhalis uses to survive in the human host. One major hurdle that all successful pathogens must overcome is the lack of free iron in vivo. It is well-established that, like most other organisms, M. catarrhalis has a strict requirement for iron (Campagnari et al., 1994
, 1996
; Furano & Campagnari, 2003
; Luke & Campagnari, 1999
; Luke et al., 1999
). M. catarrhalis can obtain iron for growth and metabolism through the binding of either transferrin or lactoferrin, two of the human iron-binding proteins (Campagnari et al., 1994
; Luke & Campagnari, 1999
; Luke et al., 1999
; Schryvers et al., 1998
; Stojiljkovic et al., 1996
). These two specific receptor systems are similar in composition, with an integral outer-membrane protein (OMP) component and an outer-membrane lipoprotein component that work together to bind the iron-carrier protein and internalize the iron moiety. These well-known transferrin- and lactoferrin-binding systems have been described for many other pathogens, including pathogenic species of the genera Neisseria and Haemophilus (Cornelissen & Sparling, 1994
; Gray-Owen & Schryvers, 1996
; Schryvers, 1989
). Investigators have provided evidence that these surface receptors have vaccine potential, although significant heterogeneity has been reported (Ala'Aldeen & Borriello, 1996
; Ala'Aldeen et al., 1994
; Johnson et al., 1999
; Masri & Cornelissen, 2002
; Rokbi et al., 2000
; West et al., 2001
).
We now present data demonstrating that M. catarrhalis can utilize haemoglobin (Hb) for in vitro growth. We have identified and characterized an OMP, MhuA, that we hypothesize to be an integral component of a newly identified haemoprotein-utilization system for this bacterium. A mAb specific to MhuA, 3F5-5E5, demonstrates that expression of this surface-exposed protein is conserved among a wide panel of M. catarrhalis clinical isolates and that the epitope recognized by this mAb appears to be species-specific, as we have not detected this determinant on any other bacterium evaluated. These data provide new information regarding another mechanism utilized by M. catarrhalis for survival in the human host and also provide new insight into the biological systems available to this important mucosal pathogen.
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METHODS |
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General DNA manipulations.
M. catarrhalis chromosomal DNA was prepared by using a previously described method (Russo et al., 1993). All standard molecular-biology reagents, including T4 ligase and restriction endonucleases, were purchased from either Promega or New England Biolabs and were utilized according to standard protocols. PCR-amplification analyses were performed by using genomic M. catarrhalis 7169 DNA with Platinum Taq High Fidelity Polymerase (Invitrogen Life Technologies). All PCR products and plasmid constructs were purified by using MinElute kits or QIAprep spin kits, respectively (Qiagen). DNA sequencing was performed by the RPCI Biopolymer Facility, Roswell Park Cancer Institute, Buffalo, NY, USA, and analysed with MacVector software (version 7.2; Genetics Computer Group).
Cloning and mutagenesis of M. catarrhalis 7169 mhuA.
mhuA was identified through BLAST searches based on homology to other TonB-dependent proteins in GenBank. PCR primers were designed for cloning mhuA based on the nucleotide sequence submitted under Incyte Genomics sequence 6, patent WO0078968, GenBank accession no. AX067431. PCR amplification between 75 bp upstream of the predicted 5' transcription start site and 197 bp upstream of the 3' predicted stop site was performed by using primers 605 (5'-TGATTGGTGATAAAAGTAGG-3') (sense) and 606 (5'-TGTTGGCATCTAAGGGGTC-3') (antisense). Primers 605 and 606 resulted in a 2782 bp product that was ligated into pGEM-T Easy (Promega), resulting in pTB6-AH. E. coli XL-1 Blue cells were transformed with pTB6-AH by using electroporation. PCR analysis, restriction digestion and sequence analysis were performed on this plasmid to confirm the nucleotide organization of the 7169 mhuA.
A deletioninsertion isogenic mutant of 7169 mhuA was constructed by inverse PCR, using pTB6-AH as a template. Primers 653 (5'-ATCTCGAGCAAGGCTATGGGCTAAGCAAAGTG-3') (sense) and 654 (5'-CGCAGATCTAATACATTTTCTCGCACGCTGG-3') (antisense), with engineered XhoI and BglII sites, respectively (underlined), were designed to create a 2081 bp deletion internal to the ORF of mhuA. Amplification of aphA-3, the non-polar kanamycin-resistance cassette from pUC18K, was performed by using primers 417 (5'-TATAAGATCTGGGTGACTAACTAGGAGGAATAAATGGCTA-3') (sense) and 491 (5'-TATACTCGAGGTCGACTCTAGAGGATCCCCGGGTCATTA-3') (antisense) with complementing restriction sites for directional cloning (Ménard et al., 1993). Restriction digestion of the PCR products and ligation resulted in pTB6-AHkan (mhuA with an internal deletion and containing aphA-3). E. coli XL-1 Blue cells were transformed with pTB6-AHkan by using electroporation. Sequence analysis confirmed proper insertion of the kanamycin-resistance cassette.
Primers 605 and 606 were used to amplify a 1551 bp product from pTB6-AHkan that was used to naturally transform M. catarrhalis 7169 as described previously (Furano & Campagnari, 2003). All resulting kanamycin-resistant clones were analysed by using PCR and sequence analysis to ensure proper allelic exchange; one clone, 7169 : : mhuA, was chosen for further study. Sequence analysis of 7169 : : mhuA chromosomal DNA was performed to confirm that the inactivated mhuA gene had recombined properly into the chromosome.
Hbagarose binding.
Hbagarose binding was performed essentially as described by Dashper et al. (2000) with the following modifications. Briefly, 200 µl Hbagarose (Sigma) was washed with 100 mM NaCl, 25 mM Tris/HCl (pH 7·4). Washes were performed three times by resuspending the agarose in 500 µl buffer and centrifuging (7000 r.p.m. for 5 min). Zwittergent-extracted OMP preparations (50 µl) from either chemically defined medium (CDM)/Hb-grown 7169 or 7169 : : mhuA were incubated with the Hbagarose for 3 h at 20 °C with mixing. The samples were centrifuged and the supernatants were removed. The Hbagarose was washed three times as before and wash fractions were concentrated by using Centricon filters (Millipore). Bound proteins were eluted for 2 min by using either 3 M NaCl or 2 M guanidine/HCl (50 µl). Concentrated wash samples and Hbagarose-bound and eluted proteins were subjected to SDS-PAGE and Western blot (mAb 3F5-5E5) analyses.
Growth analyses.
Chelex-treated CDM was used to culture M. catarrhalis in the presence of 10 µM desferal with or without 5 µM human Hb (CDM/Hb; Sigma), 8 µM bovine haemin (CDM/Hm; Sigma) or 100 µM Fe(NO3)3 (CDM 100; Sigma) (Campagnari et al., 1994). Hb was solubilized by using 10 mM HEPES (pH 7·4), Hm was solubilized with 0·1 M NaOH and Fe(NO3)3 was solubilized in H2O. Hb, Hm and Fe(NO3)3 were sterilized before addition to cultures by using a 0·2 µm pore-size filter (Pall). CDM 0, CDM 100 and CDM/Hb cultures (10 ml) containing 10 µM desferal were inoculated to a starting OD600 (BioPhotometer; Eppendorf) of 0·1 from overnight GC agar plate-grown M. catarrhalis 7169 or 7169 : : mhuA. For studies with Hm, CDM 0 and CDM/Hm cultures (10 ml) were also inoculated to a starting OD600 of 0·1 from overnight GC agar plate-grown M. catarrhalis 7169 or 7169 : : mhuA, but these strains were cultured at 37 °C with rotary shaking at 225 r.p.m. overnight (16 h) in either CDM 0 or CDM/Hm, centrifuged at 3000 r.p.m. for 10 min and fresh 10 ml cultures (CDM 0 or CDM/Hm plus desferal) were inoculated to a starting OD600 of 0·06 for these studies. All strains were cultured at 37 °C with rotary shaking at 225 r.p.m. over the time course indicated. Representative curves are shown for CDM 100 and CDM/Hm growth analyses that have been repeated at least three times, and the CDM/Hb curve shown is the mean±SD of three independent experiments.
RT-PCR analysis of mhuA.
Transcriptional analysis of mhuA was performed by using wild-type M. catarrhalis 7169. RNA was isolated from this strain after culture in CDM 0, CDM 100, CDM/Hm or CDM/Hb (as described above) by using an RNeasy Mini kit (Qiagen). The resulting concentration of RNA was standardized for all four conditions to 15 ng µl1 for use in RT-PCR analysis with primers internal to mhuA: primer 543, 5'-TCACTTACAATGAAGCCAGCCG-3' (sense) and primer 544, 5'-TGATAACCCACTACTTTTAGCCCC-3' (antisense), resulting in a product of 1217 bp.
OMP preparation, SDS-PAGE and Western blot.
M. catarrhalis strains 7169 and 7169 : : mhuA were cultured in 250 ml CDM broth containing 10 µM desferal plus 5 µM Hb, 8 µM Hm, 100 µM Fe(NO3)3 or no exogenous iron (conditions described above). After 16 h, cultures were harvested and OMPs were isolated by Zwittergent extraction (Campagnari et al., 1994, 1996
). OMPs were analysed by SDS-PAGE (7 % gel). Western blot, colony-lift assays and flow-cytometry analyses with mAb 3F5-5E5 were performed by using our standard methods (Campagnari et al., 1994
, 1996
; Luke et al., 1999
).
mAb 3F5-5E5.
mAb 3F5-5E5 (anti-MhuA) was developed against iron-stressed M. catarrhalis 25240 by injecting BALB/c mice using a previously described protocol (Campagnari et al., 1996).
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RESULTS |
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In order to determine whether MhuA was involved in the ability of M. catarrhalis 7169 to utilize haemoproteins for growth, an isogenic mhuA mutant was constructed and termed 7169 : : mhuA, as described above.
MhuA binding of Hbagarose
To determine the ability of MhuA to bind Hb directly, OMPs from M. catarrhalis 7169 were tested for their ability to bind Hbagarose. The eluted protein fractions were analysed by SDS-PAGE, revealing the presence of multiple bands (Fig. 1a). However, a Western blot (Fig. 1b
) probed with mAb 3F5-5E5 demonstrated that MhuA was present both in the Hbagarose-bound fraction (Fig. 1b
, lane 3) and in the 2 M guanidine/HCl-eluted fraction (Fig. 1b
, lane 6). Furthermore, it is important to note that MhuA was not detected in the concentrated wash samples (Fig. 1b
, lane 4), nor was there any evidence of this protein in the 3 M NaCl-elution fraction (Fig. 1b
, lane 5). These studies confirm that MhuA can bind directly to human Hb and these data also suggest that there is a strong interaction between these two components.
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In order to determine the level of conservation exhibited by MhuA, whole bacterial samples were prepared from a series of M. catarrhalis clinical isolates from various geographical locations. Fig. 5 is a representative Western blot analysis probed with mAb 3F5-5E5, demonstrating that all of these isolates expressed MhuA. In addition, all of these strains were reactive by colony-lift analyses and flow cytometry (data not shown). These studies confirm that the Hb-utilization protein MhuA is surface-exposed and conserved among strains associated with human disease. These analyses were also performed on the Gram-negative organisms Moraxella bovis, Moraxella lacunata, Haemophilus ducreyi, Haemophilus influenzae, Neisseria gonorrhoeae, Neisseria cinerea, Klebsiella pneumoniae and Pseudomonas aeruginosa. MhuA expression was not detected in any of these other species, suggesting that the epitope recognized by mAb 3F5-5E5 may be specific to M. catarrhalis (data not shown).
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DISCUSSION |
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Whilst it has previously been shown that this M. catarrhalis strain expresses transferrin- and lactoferrin-receptor systems and can utilize these iron-carrier proteins for growth, the actual mechanism as to how these systems function has not been clearly defined (Campagnari et al., 1994; Luke & Campagnari, 1999
; Luke et al., 1999
; Schryvers et al., 1998
). It was initially thought that these proteins probably function in the same manner as the other, well-studied transferrin and lactoferrin receptors. However, in the case of the transferrin receptors in Moraxella, these genes are arranged in the opposite orientation and there is a third, undefined ORF present; thus, there may be functional differences. Until now, the lactoferrin and transferrin receptors are the only known iron-acquisition systems that have been reported for M. catarrhalis. In addition, this bacterium does not secrete siderophores and no haemolysins have been identified (Campagnari et al., 1994
).
Haem is the most abundant source of iron in the human body (Otto et al., 1992). Previous studies have demonstrated that numerous bacteria can utilize haem, Hb and other haemoprotein complexes as both haem and iron sources (Chen et al., 1996
; Genco & Dixon, 2001
; Lewis et al., 1997
, 1998
, 1999
; Stojiljkovic et al., 1996
; Wandersman & Stojiljkovic, 2000
). Multiple different haemoprotein-utilization systems, some of which are redundant, have been characterized for pathogenic species of the genera Neisseria, Haemophilus, Escherichia, Shigella, Yersinia, Pseudomonas, Vibrio and Serratia (Morton et al., 1999
; Ochsner et al., 2000
; Wandersman & Stojiljkovic, 2000
). Many of these systems are related by their reliance on the energy-transducing TonB/ExbB/ExbD protein complex that is present in the inner membrane and periplasmic space. It is thought that, during inflammation, both Hb and free haem are present on mucosal surfaces, making them viable options as iron sources for these pathogens (Schryvers & Stojiljkovic, 1999
).
As M. catarrhalis is a mucosal pathogen, the availability of haem and Hb would provide an obvious advantage, particularly in the middle ear during AOM. To date, there has not been any such system described for M. catarrhalis. In fact, there are conflicting reports involving this bacterium and Hb utilization. One study showed that an undefined M. catarrhalis strain bound both haem and Hb in a solid-phase binding assay (Stojiljkovic et al., 1996). However, a separate study used a disc-diffusion method to demonstrate that M. catarrhalis could not use Hb as a sole iron source for growth (Aebi et al., 1996
). Our data demonstrate clearly that M. catarrhalis can use human Hb as a sole iron source for in vitro growth. The most likely explanation for the discrepancies of the previous reports and our data is the fact that our studies utilized a broth-culture method that may be more sensitive and specific than the previously reported techniques that were utilized.
Another important observation presented in our studies is the identification of the MhuA protein, which appears to be a highly conserved OMP that binds Hb and is probably involved in the subsequent utilization of this iron source. Whilst our studies have not as yet defined the specific role of MhuA, the significant growth difference between the mutant and the wild-type, as we show in the growth studies, relates directly to the disruption of a single gene. These data provide further support for the involvement of MhuA in Hb utilization. The binding of other unidentified proteins to the Hbagarose column is consistent with previous data obtained by other investigators studying the Hb-binding activity of proteins from various organisms (Archambault et al., 2003; Bracken et al., 1999
; Dashper et al., 2000
; Lee & Levesque, 1997
; Sengupta et al., 1999
). Due to the varied haem and iron sources that are present in the human host and the varied degree of receptor specificities for haemoproteins, it is not surprising that multiple receptor systems exist for haem uptake in many bacterial species (Cope et al., 1995
, 1998
; Genco & Dixon, 2001
; Lewis et al., 1999
; Wandersman & Stojiljkovic, 2000
). Like these related organisms, other Hb-binding and -utilization proteins are likely to exist for M. catarrhalis, suggesting the possibility of redundant systems.
This study is the first to demonstrate the ability of M. catarrhalis to grow with Hb as the sole iron source and to define a putative Hb receptor. Future studies will be designed to determine the actual function of MhuA in this Hb system and also to determine whether in vivo expression elicits human antibodies. However, far more studies are needed to begin to define the steps involved in colonization and survival on human mucosal surfaces. It will be important to continue to characterize the mechanism(s) of iron uptake via haemoproteins by M. catarrhalis, in order to increase our knowledge of the biological systems utilized by this insufficiently studied bacterium and the role that these systems play in colonization and pathogenesis.
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ACKNOWLEDGEMENTS |
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Received 8 December 2004;
revised 6 January 2005;
accepted 7 January 2005.
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