Identification of biofilm proteins in non-typeable Haemophilus Influenzae
© Gallaher et al; licensee BioMed Central Ltd. 2006
Received: 16 March 2006
Accepted: 19 July 2006
Published: 19 July 2006
The Erratum to this article has been published in BMC Microbiology 2013 13:261
Non-typeable Haemophilus influenzae biofilm formation is implicated in a number of chronic infections including otitis media, sinusitis and bronchitis. Biofilm structure includes cells and secreted extracellular matrix that is "slimy" and believed to contribute to the antibiotic resistant properties of biofilm bacteria. Components of biofilm extracellular matrix are largely unknown. In order to identify such biofilm proteins an ex-vivo biofilm of a non-typeable Haemophilus influenzae isolate, originally from an otitis media patent, was produced by on-filter growth. Extracellular matrix fraction was subjected to proteomic analysis via LC-MS/MS to identify proteins.
265 proteins were identified in the extracellular matrix sample. The identified proteins were analyzed for COG grouping and predicted cellular location via the TMHMM and SignalP predictive algorithms. The most over-represented COG groups identified compared to their frequency in the Haemophilus influenzae genome were cell motility and secretion (group N) followed by ribosomal proteins of group J. A number of hypothetical or un-characterized proteins were observed, as well as proteins previously implicated in biofilm function.
This study represents an initial approach to identifying and cataloguing numerous proteins associated with biofilm structure. The approach can be applied to biofilms of other bacteria to look for commonalities of expression and obtained information on biofilm protein expression can be used in multidisciplinary approaches to further understand biofilm structure and function.
Bacteria exist in both planktonic and biofilm states [1, 2]. Recent findings indicate chronic infections are associated with the formation of in vivo biofilm which renders the bacteria resistant to antibiotic treatment . This resistance has been believed to be due to the structural properties of the biofilm which have been described as "matrix encased microbrial communities" . More recently, studies of Pseudomonas aeruginosa biofilm indicated that simple lack of anti-biotic penetration is not the cause of resistance  and "anoxic regions where bacteria are poorly killed due to very low metabolic rates" in has been hypothesized . Formation of biofilm includes adherence events wherein the bacteria become sessile and secrete extracellular matrix. The end result is a highly structured multicellular complex with cavities and channels . Historically, molecular and biochemical studies of bacteria have examined the planktonic state rather than biofilm state. Understanding the molecular nature of the biofilm structure is of interest in developing strategies to combat chronic biofilm infections.
Results and discussion
292 total protein identifications arising from analysis of all 20 gel slices were made with 27 being redundant (25 seen in two slices and one seen in three) for a total of 265 unique proteins identified in the extracellular matrix sample. Proteins corresponding to four of the five strains of NTHi proteins in the search data file were observed with the exception being the aegyptius strain. The analyte NTHi strain had been isolated from an OM patient  and is genetically uncharacterized. The majority of the proteins, 158, could not be assigned to a specific strain in that protein-identifying peptide sequences (or sequence tag) were shared by each of the four strains. Other identifying peptide sequences indicated that 32 strain-specific proteins were present with four proteins specific only for KW20, 10 specific for R2846, five specific for R2866 and 13 specific for 86-028NP. The other 77 identified proteins could be assigned to a combination of two or three strains. Determinants for strain specificity were usually based upon one amino acid difference in one of the protein-identifying peptides. The gene ompA, which codes for outer membrane protein P5, serves as an example. Strain KW20's OmpA contains glutamic acid at position 118 whereas each of the other strains has the conservative substitution aspartic acid at the corresponding position. The difference in mass of seven Da between the glutamic acid and aspartic acid-containing peptides in the doubly charged parent ion is easily resolved by MS and the corresponding CID fragmentation pattern subsequently obtained allows the specific identification. Figure 3 presents a distribution of the strain specificities observed for each protein. Tables containing all identified proteins are available as additional files in rtf format or tab-delimited text [stab2_rtf.rtf and stab2_txt.txt, respectively.]
A broader search using the full NCBI non-redundant database found only one non-HI peptide, a 14 amino acid peptide corresponding to a thioredoxin protein found in two Neisseria bacteria that differ from the corresponding HI thioredoxin peptide at three positions. This peptide and the mix of peptides corresponding to various annotated HI strain proteins most likely are representative of wild HI bacteria strain heterogeneity involved in chronic infection. Our observations of proteins corresponding to various HI strains, and in one case another species homolog, is supportive of and consistent with the idea of a supragenome "distributed throughout naturally occurring infectious populations" of HI, as hypothesized by Shen et al after a thorough sequence-based genetic analysis of 10 different clinical isolates of HI .
Identified ECM biofilm proteins annotated as hypothetical or uncharacterized.
Putative N-acetylmannosamine-6-phosphate epimerase
RimM protein, required for 16S rRNA processing
1 3 4
No related COG
Putative translation initiation inhibitor
Fe-S cluster protector protein
1 3 4
Predicted RNA-binding protein containing KH domain, possibly ribosomal protein
Starvation-inducible DNA-binding protein
No related COG
1 2 4
Pyridoxine biosynthesis enzyme
Oligoribonuclease (3'->5' exoribonuclease)
Identified proteins of the most over-represented COG category.
COG1862: Preprotein translocase subunit YajC [Haemophilus influenzae R2846]
Preprotein translocase subunit YajC
COG2854: ABC-type transport system involved in resistance to organic solvents, auxiliary component [Haemophilus influenzae R2846]
ABC-type exporter of toluene and related compounds, periplasmic component ABC-type exporter of toluene and related compounds, periplasmic component
protein-export protein (secB) [Haemophilus influenzae Rd KW20]
Preprotein translocase subunit SecB
COG0541: Signal recognition particle GTPase [Haemophilus influenzae R2866]
Signal recognition particle GTPase
Outer membrane protein P4, NADP phosphatase [Haemophilus influenzae 86-028NP]
Predicted secreted acid phosphatase
COG0740: Protease subunit of ATP-dependent Clp proteases [Haemophilus influenzae R2866]
Protease subunit of ATP-dependent Clp proteases
COG0823: Periplasmic component of the Tol biopolymer transport system [Haemophilus influenzae R2866]
Periplasmic component of the Tol biopolymer transport system
GTP-binding protein [Haemophilus influenzae Rd KW20]
Predicted membrane GTPase involved in stress response
All identifications were also screened for the presence of signal sequence secretory signal  and transmembrane domains . 21 proteins were positive for signal sequence and 20 were positive for at least one TMH domain with six of these proteins containing both predicted signal sequence and TMD, often overlapping. These putative secreted or membrane bound proteins were in 12 different COG categories and also included the COG unclassifiable protein mentioned earlier. These proteins are included in supplemental table 1 [see additional file 3].
Given that the sample analyzed was preparatively isolated to be that which corresponds to extracellular matrix, we would have expected to have seen a larger proportion of secreted proteins, or possibly membrane proteins, in the analysis. We, though, identified large numbers of ribosomal proteins, metabolic enzymes or other proteins normally associated with intracellular localization and function. Electron microscopy of sample (presented in supplemental information; supplemental figures 1 and 2 [see additional file 3]) demonstrates that whole cell contamination of the sample has not occurred. The presence of lysed cell components cannot be ruled out. It is not known if such proteins act as components of the biofilm structure. The idea that these types of proteins may contribute to biofilm structure is possible in that dead cells and cell death have been reported to be part of biofilm structure and function [30, 31] and an earlier proteomic approach in another bacteria, Shewanella oneidensis MR-1, identified ribosomal proteins as well in their analysis . Further, proteins normally associated with intracellular function are observed outside the cell which has generated interest in a non-classical secretion pathway . Among such normally intracellular proteins that have been demonstrated to also be found outside gram positive bacterial cells  are a number of proteins we have observed in this biofilm EMC sample, including ribosomal proteins, enolase, superoxide dismutase, elongation factor Tu and chaperonins DnaK and GroL (GroL is the COG gene name for GroEL proteins).
Overall 43 proteins (~16% of all identified proteins) are either annotated as periplasmic, membrane or membrane associated or were identified by signal peptide or TMH analysis (indicative of either periplasmic or membrane location). Nine annotated ABC transporters were identified in the sample which corresponds to 3.4% of the identified biofilm proteins. Eight additional proteins annotated transporters or periplasmic were also observed, including multi-membrane spanning transport proteins. Of note is that members of the ABC transporter protein class have been shown to be essential for biofilm formation including a membrane bound component of the ABC transporter in Bacillus subtilis , the lapEBC cluster of Pseudomonas fluorescens  and the adc operon of Streptococcus gordonii . These 43 proteins are presented in supplemental table 1 [see additional file 3].
Recently, a chaperonin gene, groEL1, has been shown to be essential for biofilm formation in the gram-positive actinobacterium Mycobacterium smegmatis . As mentioned, we see a groEl protein in our sample, and as noted above, GroEL is known to be localized outside bacterial cells as well as intracellularly. Our identification of groEL in biofilm sample ECM would seem to suggest a pan-bacterial role for groEl in biofilm formation, but there is a caveat. The groEL in HI is most similar to groEL2 of the mycobacterium, sharing a methionine-glycine rich carboxyl region whereas biofilm formation in the mycobacterium was attributed to the groEL1 gene, a homologous gene that has a histidine rich carboxyl terminus. Does the GroEL in HI or other bacteria which express only one GroEL form use this gene in biofilm formation? Of interest is that in this same study , GroEL2 is reported to physically associate with the protein KasA with the protein-complex levels being enhanced during biofilm formation. KasA is 3-oxoacyl-(acyl-carrier-protein) synthase, a FabB gene with a COG number 0304. In our sample the HI homolog is also present and is the earlier mentioned acyl carrier protein which migrated in SDS-PAGE at an anomalous molecular weight. A second protein reported to associate with KasA/FabB, referred to as SMEG4308 (COG0492), which has an HI homolog, was not seen in our analysis.
Also in our biofilm ECM sample is the universal stress protein UspA. A protein of the uspA family in E. coli has been shown to interact with and act as a substrate for GroEl-mediated phosphorylation . Of further note is that, recently, UspA of the periodontopathic Porphyromonas gingivalis bacterium was reported to be necessary for biofilm formation .
Our analysis also identified two ompA outer membrane component proteins of NTHi, the P5 and P6 (also known as peptidoglycan-associated outer membrane lipoprotein). This class of outer membrane proteins can serve as adhesins and have been implicated in biofilm formation . Of note in terms of pathogenicity and potential clinical issues is that P6 has been shown to induce human macrophage mediated immunogenicity associated with inflammatory events . Recently ompA of E. coli has been demonstrated to regulate biofilm formation . In this same study, two DNA binding transcriptional regulatory proteins, Hha and YbaJ of E. coli were also implicated in the biofilm formation. Hha positively regulates ompA expression. We did not see HHa or YbaJ in our analysis but did see two ompA proteins which is consistent with the ECM nature of our sample.
Another protein recently indicated to be found in NTHi biofilm cell envelope is a peroxiredoxin-glutaredoxin  which corresponds to HI0572 with COG number 678 and COG gene designation AHP1. The protein is observed to be present in greater abundance in biofilm and bacterial strains with expression deficient mutations showing a 25 – 50% decrease in biofilm formation. In our analysis this peroxiredoxin, AHP1, was identified, as was another peroxiredoxin, AHPC.
This study has provided the results of an initial inquiry in to the protein structural components of biofilm. An ex vivo biofilm of NTHi bacteria (strain 9274), which was originally isolated from an otitis media patient, was grown on nitrocellulose membrane. Extracellular matrix proteins were isolated from the biofilm by sonication and washing of the filter and differential centrifugation and these proteins were resolved by SDS-PAGE and analyzed by LC-MS/MS ("proteomics"). In this manner 265 NTHi proteins were identified. Proteins identified indicated this isolate is a genetically unique strain (or non-clonal mix of strains) based upon sequences of identified peptides, sharing properties of four different well characterized (i.e. genomically sequenced) HI strains. All identifications are provided in supplemental information [see additional file 1 or 2].
Identified proteins were analyzed in terms of their COG group and functional categorization of COG, and ostensible cellular localization, e.g. presence of signal peptide or transmembrane helices or annotation indicating cellular localization. Hypothetical or uncharacterized proteins were characterized. Of these one was not able to be placed in a COG. Three were novel identifications for HI via the proteomic approach.
Importantly, a number of HI proteins homologous to proteins specifically implicated in biofilm formation in other bacteria were observed in our sample, including GroEL and a GroEl-associated acyl carrier, KasA/FabB, OmpA, UspA and peroxyredoxin.
This inquiry provides a starting point to further address questions of bacterial biofilm structure where information provided here can be applied in genetic, biochemical, biophysical or other types of studies. The method and information obtained also indicates how biofilms from other bacteria can also be evaluated and cross-correlated to answer broader questions of common biofilm structural components.
Biofilm and ECM protein isolation
NTHi biofilm growth and preparation has been characterized previously (13). Extracellular matrix proteins were isolated by briefly washing Millipore filter grown biofilm (fed on chocolate agar) in phosphate buffered saline (PBS) with sonication. This wash eluent was centrifuged to remove whole bacterial cells and supernatant was subjected to SDS-PAGE using Invitrogen NuPAGE 4 – 12%. Proteins were visualized by coomassie blue stain.
LC-MS/MS: (a) In-gel tryptic digest
Protein bands from SDS-PAGE were excised from the gels and destained with 50% acetonitrile in 50 mM ammonium carbonate. In-gel tryptic digest was carried out using reductively methylated trypsin (Promega, Madison, WI). Prior to digestion, samples were reduced with DTT (10 mM in 50 mM ammonium carbonate for 60 minutes at 56°C) and subsequently alkylated with iodoacetamide (55 mM in 50 mM ammonium carbonate for 45 minutes in the dark at room temperature). The digestion reaction was carried out overnight at 37°C. Digestion products were extracted from the gel with a 5% formic acid/50% acetonitrile solution (2X) and one acetonitrile extraction followed by evaporation using an APD SpeedVac (ThermoSavant). The dried tryptic digest samples were cleaned with ZipTip (Millipore CB18B).
(b) Analysis of tryptic peptides by tandem mass spectrometry for protein identifcation
The sample was resuspended in 10 μL of 60% acetic acid, injected via autosample (Surveyor, ThermoFinnigan) and subjected to reverse phase liquid chromatography using ThermoFinnigan Surveyor MS-Pump in conjunction with a BioBasic-18 100 × 0.18 mm reverse-phase capillary column (ThermoFinnigan, San Jose, CA). Mass analysis was done using a ThermoFinnigan LCQ Deca XP Plus ion trap mass spectrometer equipped with a nanospray ion source (ThermoFinnigan) employing a 4.5-cm long metal needle (Hamilton, 950-00954) in a data-dependent acquisition mode. Electrical contact and voltage application to the probe tip took place via the nanoprobe assembly. Spray voltage of the mass spectrometer was set to 2.9 kV and heated capillary temperature at 190 C. The column equilibrated for 5 min at 1.5 μL/min with 95% solution A and 5% solution B (A, 0.1% formic acid in water; B, 0.1% formic acid in acetonitrile) prior to sample injection. A linear gradient was initiated 5 min after sample injection ramping to 35% A and 65% B after 50 min and 20% A and 80% B after 60 min. Mass spectra were acquired in the m/z 400–1800 range.
(c) Protein identification
Protein identification was carried out with the MS/MS search software Mascot 1.9 (Matrix Science) with confirmatory or complementary analyses with TurboSequest as implemented in the Bioworks Browser 3.2, build 41 (ThermoFinnegan).
- Abbreviations are NTHi:
Non-typeable Haemophilus influenzae
Cluster of orthologous groups
liquid chromatography tandem mass spectrometry
transmembrane helix or domain.
This work was funded in part by a grant from the Deafness Research Foundation (to P.W.). The authors wish to acknowledge the support of their respective Institutions. We wish to thank, and express our appreciation to, Dr. Julian Whitelegge for reading the manuscript.
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