Proteomic and Electron Microscopy Study of Bacteriophages From Bartonella Henselae And Bartonella Grahamii

Key words

Rochalimaea, Cat Scratch Disease, Zoonotic Disease, Evolution of Pathogenic Bacteria, Horizontal Gene Transfer

Introduction

Bartonella (known also as Rochalimaea) is a genus of Gram-negative bacteria representing facultative intracellular parasites. These bacteria can on occasion infect healthy people,but are considered as opportunistic pathogens causing strong infections in immunecompromised patients such as those with AIDS.

Bartonella-s are transmitted by blood sucking arthropods, e.g. ticks, fleas, sand flies and mosquitoes. They use zoonotic reservoir for their transfer to human. After their invasion of primary niche - endothelial cells of reservoir animal, they are released in period intervals to bloodstream, where they invade erythrocytes. Bacterial infection might be transferred to human through occasional scratch by infected animal.

Bartonella henselae is a zoonotic pathogen using cats as a reservoir and causing cat scratch disease (CSD) [1]. The disease has serious symptoms characterized by lymphadenopathy and persistent fever. It might have even more severe symptoms in AIDS patients, where it can cause bacillary angiomatosis, bacillary peliosis hepatis, endocarditis and bacteremia associated with relapsed fever [2]. B. henselae has been identified as a one of eyes infecting bacterial species occurring with increasing incidence [3,4]. Another species involved in intraocular inflammatory disease is Bartonella grahamii, which uses rodents as a reservoir [5]. Infection in this case triggers even behavioural changes in patients [6].

Bacteriophages are one of the key elements playing an important role in acquisition of new genetic information by various bacterial species leading towards evolution of bacteria [7]. Bacterial survival and adaptation to new environmental conditions and host species are likewise mediated by bacteriophages. Furthermore, they might enhance bacterial evasion or inactivation of host defense mechanisms. In addition, bacteriophage genomes often contain a variety of genes horizontally transferred to various bacteria by phage infection. Importance of our knowledge concerning bacteriophages increases, if they are carrier of genes encoding virulence factors. Phage infection raises virulence of the bacteria and sometimes converts a non-pathogenic strain to a dangerous pathogen [8]. Thus, questions regarding evolution of bacteriophages, acquiring of new genes, their diversification and horizontal transfer between bacterial species are one of the issues with biological and medical relevance.

Several authors have demonstrated the occurrence of bacteriophagelike particles amongst Bartonella-s [9-12]. The Particles conserved among different Bartonella species are capable to package host DNA and export it outside of host cells. It suggests possible role of these bacteriophage particles in genetic exchange [13].

The present study confirmed the occurrence of before reported bacteriophage particles in both B. henselae and B. grahamii with diameter approximately 40 nm and with similar morphology and relative molecular weights of three main bacteriophage proteins. Furthermore, large enveloped and non-enveloped tailed bacteriophages were found in both cultures as well. To characterize structural bacteriophage proteins, we have performed sensitive proteomic analysis and identified bacteriophage-associated proteins.

Materials and Methods

Growth of Bacterial Strains

B. henselae Houston-1 and B. grahamii were routinely grown for 5-10 days on blood agar containing 5 % (v/v) horse blood (CSB agar; Statens veterinärmedicinska anstalt (SVA), Uppsala, Sweden) in a humidified atmosphere with 5 % (v/v) CO2  at 35°C.

Isolation of Bacteriophages

The isolation procedure was adopted from Anderson et al. (1994) [10] with a few additional modifications. Bacteria were scraped from agar plates and immediately transferred into sterile Dulbecco´s phosphate buffered saline (DPBS; pH 7,2). Bacterial biomass obtained by growth on each plate was collected by addition of 0,5 ml of DPBS. Bacteria were pelleted by twice repeated centrifugation for 1 min at 10000 × g. The supernatant after second centrifugation was collected and bacteriophages were precipitated by addition of the solution consisted of 20 % (w/v) PEG 8000 and 2,5 M NaCl. The mixture was incubated overnight at 4°C. Bacteriophage pellet was collected by centrifugation at 10000 × g for 10 min. Then particles were suspended in sterile SM buffer [14] and left for a few hours on ice before complete resuspension. The bacteriophage suspension was further stored at 4°C.

SDS-PAGE

Bacteriophage suspension was solubilized in NuPAGE® LDS 4×LDS sample buffer (Invitrogen AB, Sweden), cleaned-up using ProteoExtract™ Protein Precipitation Kit (Calbiochem, Merck-4Biosciences, Sweden) and solubilized again in NuPAGE® LDS 4×LDS sample buffer. Sample was run at reducing conditions on 4-12 % NuPAGE Bis-Tris Gel using MOPS running buffer. The gels were stained by Bio-Safe Coomassie Stain (Biorad, Sweden),individual lanes cut off and used for proteomic analysis

Proteomic Analysis

Excised polyacrylamide slices have been further reduced, alkylated and in-gel digested by trypsin [15]. Nanoflow LC-MS/MS analysis was performed on a 7-tesla hybrid linear iontrap (LTQ) FT mass spectrometer (Thermo Electron, Bremen, Germany) modified with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark) according to the protocol of Nielsen et al (2005) [16]. Protein and peptides were identified by searching of tandem mass spectra using Mascot search engine against the non-redundant NCBI protein database. Analysis was performed twice and only statistically significant Mascot matches with individual ion scores indicating identity or extensive homology above cut off score for 95 % of correctly assigned peptides (p≤0.05) and at the same time existing in both analyses were selected. Only proteins identified by minimum two peptides were included in the study and the proteins assigned through single peptide matches were excluded from further analysis.

Transmission Electron Microscopy

Sample preparation was performed according to the protocol of Barbian and Minnick (2000) [11]. Bacteriophage suspension was deposited on Silicon Monoxide Type-A support grids (300 mesh copper) and stained by 1 % (w/v) sodium phosphotungstate. The grids were observed under Zeiss Supra 35VP electron microscope.

Results

To investigate occurrence of bacteriophages in the cultures of B.henselae (BH) Houston-1 and B. grahamii (BG), bacteriophages have been isolated from in vitro cultured bacteria. These preparations have been characterized by both morphologic and proteomic analysis.

Size and Morphology of Bacteriophage-like Particles in B. Henselae and B. Grahamii

In the current study, the electron microscopy images showed the presence of icosahedral particles in two ranges of sizes. In both BH and BG bacteriophage preparations, icosahedral particles having average diameter between opposite vertices equal to 42 nm were present (Fig. 1 A). The average value of the diameters between opposite faces was 36 nm (Fig. 1 A). Furthermore, in both bacterial cultures there were also observed larger enveloped bacteriophages with icosahedral to round shaped core (Fig. 1 B, C, D) and tailed bacteriophages (data not shown). Both detected enveloped and non-enveloped phages were larger in their diameters than before described 40 nm particles. The diameter of enveloped particles varied between 110-160 nm, while cores of enveloped particles had diameters in range between 88-110 nm. The tailed phages consisted of a capsid with average diameter 87 nm; tail and fibers were about 175 nm and 83 nm long.

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Figure1:

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Figure1. Bacteriophages isolated from cultures of facultative intracellular parasites Bartonella grahamii and henselae.

Protein Analysis of B. Henselae and B. Grahamii Bacteriophages

SDS-PAGE analysis of BH bacteriophages showed several protein bands with following relative molecular weights: 21; 27; 30;32; 42; 46 and 63 kDa, where major bands had Mr equal to 32; 46 and 63 kDa. Following values relative molecular weights of protein bands were detected by analysis of BG bacteriophage preparations: 14; 19; 25; 27; 30; 32; 33; 36; 37; 42; 45; 51; 60 and 63 kDa.

Further proteomic analysis using LC-MS/MS assisted in identification of several of these proteins and also included other low-abundant proteins phage proteins. Results of this analysis including information concerning their potential functions are summarized in both Table 1 and Table 2.

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Table1:

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Table 1. List of proteins identified by LC-MS/MS analysis of bacteriophage preparations from B. henselae. Proteins were identified by minimum two peptide matches and using statistically significant Mascot scores above threshold indicating 95% correctly assigned peptides (p≤0.05). Minimum Mascot score value of matches used in this study was 63.

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Table2:

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Table 2.The List of proteins identified by LC-MS/MS analysis of bacteriophage preparations from B. grahamii. The proteins identified by minimum two peptide matches and with statistically significant Mascot scores above threshold value indicating 95% correctly assigned peptides (p≤0.05) were included. Minimum Mascot score value of matches used in this study was 68.

Discussion

In our present study, bacteriophages of different types were detected by electron microscopy in the bacteriophage suspensions prepared from cultures of both BH and BG.By morphologic analysis we have identified before described bacteriophage-like particles, non-enveloped tailed and novel enveloped bacteriophages. The Proteomic analysis of structural phage proteins has assisted in description of their functions and in the classification of identified bacteriophages.

The size and morphology of the phages with smaller diameter corresponds to bacteriophage-like particles occurring in Bartonella culture supernatants [9,10]. Nevertheless, the existence of bacteriophages with diameter > 40 nm was reported by Barbian and Minnick (2000) [11], who detected round to icosahedral phageswith diameter 80 nm in B. baciliformis culture. But according to their transmission electron micrographs, particle sizeswere rather heterogeneous in their sizes with diameters 40-80 nm. Likewise, the presence of bacteriophages with diameter >40 nm was confirmed in BG [17] withhead size in the range 50-70 nm.

Tailed phages are the most efficient gene-transfer particles developed in the process of evolution. Previous observations performed using Bartonella cultures suggested the presence of tailed bacteriophages. Umemori et al. (1992) [9] reported the presence of tailed bacteriophages with diameter 40 nm and short tail with length 16 nm. Furthermore, Berglund et al. (2009) [17] observed the presence of sheathed tails approximately 100 nm long in BG. Our proteomic analysis confirmed the presence of tail proteins with homology to viruses of family Myoviridae in both bacterial cultures. Major tail sheath protein FI identified in BH and BG bacteriophage preparations is occurring in T4-like viruses and bacteriophages P2 (Table 1 and 2). Likewise, structural protein GP20 with homology to T4 bacteriophage capsid [18] and major structural unit of contractile P2 bacteriophages phage tail tube protein FII were identified in BH culture (Table 1). Among other tail proteins confirming presence of P2-like phages were P2-like phage tail protein, P2 GPu protein functioning in tail assembly of λ-like phages and phage tail measure protein participating on phage tail assembly and determining length of phage tail. Another protein identified in BG bacteriophage preparation is putative phage portal protein (Access. No. YP_002972503) with partial similarity to protein gp1, which is forming bacteriophage portal vertex structure of bacteriophage P22. It has a function during membrane binding and penetration of host membrane as well as a plug holding phage DNA inside of capsid [19]. Single-strand binding proteins functioning in protection of ssDNA intermediates during DNA metabolism, were identified in both BG and BH bacteriophage preparations. Further large subunit of DNA packaging enzyme (gp15), several anti-repressor proteins and phage-related proteins have been found as well. Finally, another component of phage tail (gp27), baseplate (gp26, gp25), tail sheath (gp18), portal protein (gp17) and neck protein (gp13) were identified.

Bacteriophages with similar morphology to the bacteriophage-like particles and the large enveloped phages described in the current study were found in proteobacterium Pseudomonas sp. This phytopathogenic bacterium is the host of two different bacteriophages, a dsDNA bacteriophage Psp231a belonging to the family Podoviridae (P.phaseicola, [20]) and an enveloped dsRNA bacteriophage Φ6-Φ12 [21,22]. The Bacteriophage-like particles (diameter 42 nm) are comparable to icosahedral phages Psp231a with diameter size 55 nm and with easily separable tails. The Structure of another Cystoviridae bacteriophage Φ12 described by Wei et al (2009) which is surrounded by a lipid-containing envelope required for infectivity reminds the enveloped phage particles in our Bartonella preparations, though Bartonella bacteriophage sizes are smaller than in family Cystoviridae. This phage is able to infect not only Pseudomonas sp., however also other Gram-negative bacteria..

One of the most interesting proteins found in BH bacteriophage preparations is phage protein CAF27122. The protein displays significant similarity to the phage protein gp79 having inhibitory effect on host transcription [23] or having cytotoxic effect on bacterial host [24]. These characters might be useful for development of phage therapy or study of potential drug targets against bacterial pathogens.

Functions of hemin-binding proteins and Pap31, a protein with high homology to hemin-binding protein in BH (99 % identity) has not been fully elucidated yet. However, common feature of these cell surface proteins is that they are conserved in several plant and animal pathogens within α-proteobacteria [25]. Pap31 was originally considered as a phage-associated membrane protein in BH [10,26]. But later it was suggested, that this protein is a bacteriophage receptor on the outer surface of the bacterium serving for bacterial iron acquisition [27]. Heme acquisition by Pap31 makes this protein potentially important virulence factor in the pathogenesis of BH [28]. Membranes of enveloped phagessuch as Φ12, fuse during the first stage of infection with bacterial outer membrane. Therefore during the process called targeted protein-dependent fusion, phage phospholipids and membrane proteins might co-purify with the bacterial outer membrane of infected cells [29].

Conclusions

Present study has shown the occurrence of bacteriophages from different families in human-pathogenic bacteria B. henselae and B.grahamii. Most of the results of our observations suggests, that B. henselae is a host of tailed dsDNA bacteriophages belonging to order Caudovirales and family Myoviridae (similar to bacteriophage P2), and enveloped bacteriophages similar to dsRNA viruses from family Cystoviridae. The Small-size bacteriophage-like particles could correspond to defective or satellite phages. Similar conclusions could be drawn for B. grahamii, though less experimental evidences are available.

Competing Interests

The authors declare that they have no competing interests.

Author's Contributions

JN performed experimental work including bacterial culture, bacteriophage isolation, SDS-PAGE and transmission electron microscopy, experimental planning and manuscript writing. MLN conducted proteomic analysis using LC-MS/MS and contributed to the manuscript writing by method description. All authors read and approved manuscript.

Acknowledgement

We would like to acknowledge Dr. Alex Mira, who has shared his experiences with Bartonella culture and the bacteriophage isolation. We also thank to Prof. Roman Zubarev and Prof. Siv Andersson, who provided their laboratories and funding to perform this work.