joe the tick

November 6, 2009

Bacterial antibiotic resistance genes discovered

Filed under: Lyme Disease, Lyme Disease News, Uncategorized — Tags: , , , , , , , , , — joethetick @ 6:37 pm

Antibacterial soap, hand sanitizer and antibiotics are all substances that we use in an attempt to kill bacteria that might make us sick.Whether we are concerned about getting strep throat, bacterial meningitis or something else, these prevention methods can offer protection.

However, some bacteria, such as those that cause Staph and MRSA infections, are becoming increasingly resistant to antibiotics. Since the 1930s, researchers have been aware that bacteria may be able to resist treatment because they can morph into the L-form, or bacteria lacking cell walls.

Until the 1980s, not much else could be known about the L-form, but now, researchers at the Bloomberg School of Public Health have used a wide variety of modern molecular tools to learn more about the origin and biological functions of the L-form bacteria.

Ying Zhang, a professor of molecular microbiology and immunology at Bloomberg, is the senior author of the study, which was published in PLoS ONE last month.

Not all bacteria can transform into the L-form, but those that can include Bacillus anthracis (anthrax), Treponema pallidum (syphilis), Mycobacterium tuberculosis (tuberculosis), Heliobacter pylori (stomach ulcers and cancer), Borrelia burgdorferi (Lyme disease) and Escherichia coli (food poisoning). Zhang’s team used E. coli to create a culture of L-form bacteria.

Although it had been difficult to culture L-form bacteria before, Zhang and his team created a new method that more closely simulated the in vivo conditions in which these bacteria form.

“The presence of antibiotic stress is cell wall inhibiting, like penicillin,” Zhang said. To prevent the cells from bursting because of this increased stress, Zhang’s team added sucrose to the cell media.
This culture represented the mechanism that occurs in the body. “L forms are formed in response to stress,” Zhang said. “They have a different mode of survival and replication from classical bacteria.” The cell wall-deficient bacteria cluster together in the shape of a fried egg rather than the smooth, homogeneous appearance of wild-type bacteria cultures.

Not only are L-form bacteria difficult to culture and therefore study, but this “fried egg” cluster is part of what makes the L-form bacteria resistant to antibiotics, in addition to the fact that they do not have cell walls for commonly used antibiotics to disintegrate.

Once Zhang and his team were able to successfully culture L-form E. coli, they screened for and identified mutants that fail to grow at the L-form. From these mutants, they were able to discover a series of genes that were linked with the inability to grow in the L-form.

“These fall into four to five different categories involving extracellular matrix synthesis, membrane proteins, membrane biogenesis, DNA repair as well as iron metabolism and energy metabolism,” Zhang said.

Their identification of these genes and their effect on L-form bacterial expression is a resounding discovery because it was impossible to do before, what with the difficulty of culturing the L-forms of various bacteria. Zhang noted, however, that although his team managed to create and study a culture of L-form bacteria, their study cannot be universal.

“What we can culture is only a small percentage – probably less than 1 percent – of all bacteria on earth,” Zhang said.
“They exist in nature and grow easily, but we’re limited to what we can grow and the form of bacteria that can grow. Bacteria can grow a variety of different forms even for the same species, and can change forms under different conditions. L-forms are one example of changing under antibiotic stress.”

These L-forms of various bacteria may be the underlying reason for chronic resistant and recurring diseases, such as sarcoidosis, various forms of inflammatory bowel diseases and rheumatoid arthritis. Zhang is confident that there will be many practical applications of this discovery.

“It is possible, with our discovery of the L-form genes to develop new antibiotics and more effective ones that can be used with current ones as well as new vaccines to . . . allow these forms to be eliminated by the immune system,” he said.

jhunewsletter.com
By Aleena Lakhanpal

October 29, 2009

Distribution of Antibodies Reactive to Borrelia lonestari and Borrelia burgdorferi in White-Tailed Deer (Odocoileus virginianus) Populations in the Eastern United States

Filed under: Lyme Disease — Tags: , , , , , , , , , , , , — joethetick @ 9:40 am

Southern tick-associated rash illness is a Lyme-like syndrome that occurs in the southern states.

Borrelia lonestari, which has been suggested as a possible causative agent of southern tick-associated rash illness, naturally infects white-tailed deer (WTD; Odocoileus virginianus) and is transmitted by the lone star tick (Amblyomma americanum). To better understand the prevalence and distribution of Borrelia exposure among WTD, we tested WTD from 21 eastern states for antibodies reactive to B. lonestari using an indirect immunofluorescent antibody assay and Borrelia burgdorferi using the IDEXX SNAP® 4Dx® test. A total of 107/714 (15%) had antibodies reactive to B. lonestari, and prevalence of antibodies was higher in deer from southern states (17.5%) than in deer from northern states (9.2%). Using the SNAP 4DX test, we found that 73/723 (10%) were positive for B. burgdorferi, and significantly more northern deer (23.9%) were positive compared with southern deer (3.8%). Our data demonstrate that WTD are exposed to both Borrelia species, but antibody prevalence for exposure to the two species differs regionally and distributions correlate with the presence of Ixodes scapularis and A. americanum ticks.

Jessica H. Murdock, Michael J. Yabsley, Susan E. Little, Ramaswamy Chandrashekar, Thomas P. O’Connor, Joe N. Caudell, Jane E. Huffman, Julia A. Langenberg, Simon Hollamby.

October 2, 2009

Borrelia Able To Shroud Their Colonies With Protective Biofilms

Filed under: Lyme Disease, Lyme Disease Documentary, Lyme Disease Research, Under Our Skin — Tags: , , , , , , , , , , , , , — joethetick @ 9:18 pm

At the end of UNDER OUR SKIN, Dr. Alan MacDonald presents a revolutionary new hypothesis that Borrelia are able to shroud their colonies with protective biofilms and this may explain why these pathogens can be so difficult to eradicate with short courses of antibiotics.

His collaborator, Eva Sapi, Ph.D., Associate Professor of Biology and Environmental Science at the University of New Haven, is exploring why and how these biofilms form, in hopes of developing more effective treatments for chronic Lyme sufferers.

The green fragments on the screen are DNA from Borrelia burgdorferi strain B31, fluorescently tagged to glow under the special illumination of a dark field microscope.

Doctors Finally Believe That Lyme Disease Is In North Carolina

Filed under: Lyme Disease, Lyme Disease Awareness — Tags: , , , , , , , , , , , , — joethetick @ 8:38 pm

Sarah Avery from newsobserver.com reported on Oct 1st that doctors have finally realized that the Lyme disease epidemic is in North Carolina.

After years of educating patients that it’s unlikely to get Lyme disease in North Carolina, state health leaders are now advising that the tick-borne illness can, in fact, be acquired here.

In at least four cases this year, Lyme was confirmed among patients who never left their home counties, ruling out the prospect that they picked up the Borrelia bacterial infection while traveling.

Based on the new evidence, Dr. Megan Davies, state epidemiologist, said the state is now working to bring Lyme Awareness to doctors throughout North Carolina, who for years were reluctant to even test patients for Lyme disease because it wasn’t considered much of a possibility.

September 8, 2009

Actress Parker Posey Diagnosed with Lyme Disease

Filed under: Lyme Celebraties, Lyme Disease — Tags: , , , , , , , , , , , , , , , — joethetick @ 8:21 pm

Parker Posey

Parker Posey

40-year-old Actress Parker Posey pulled out of an off-Broadway play, called “This,” to focus on battling her tick-borne illness.

Lyme Disease is easily treatable with a short course of antibiotics if diagnosed during it’s early-stage of infection, as it has been for Posey. If left untreated, the Borrelia bacteria can lead to severe heart, neurological and mental problems.

Producers of “This” plan to go ahead with the play in November, but have yet to name Posey’s replacement.

Parker Posey, had been planning to play a single-mom poet dealing with life and love in Melissa James Gibson’s This at the Mainstage Theater. Previews start Nov. 6 and, according to the production’s artistic director, they will kick off as scheduled with a replacement to be announced later.

July 15, 2009

Complement factor H binding by different Lyme disease and relapsing fever Borrelia in animals and human

Filed under: Lyme Disease, Lyme Disease News, Lyme Disease Research — Tags: , , , , , , , , , , , — joethetick @ 9:36 pm
Borreliae employ multiple immune evasive strategies such as binding to complement regulatory proteins [factor H (fH) and factor H like-1 (FHL1)], differential regulation of surface membrane proteins, antigenic variation, and binding of plasminogen/plasmin and matrix metalloproteinases. As a complement regulatory subunit, fH serves as a cofactor for the factor I-mediated cleavage of C3b. fH binding by Borrelia has been correlated with pathogenesis as well as with host diversity. Here we show the differential binding of borrelial proteins to fH from human and animal sera.
Findings
Affinity ligand binding experiments, 2-D electrophoresis, and protein identification and peptide de novo sequencing based on mass spectrometry, revealed novel fH putative binding proteins of Lyme- and relapsing fever Borrelia. An OspA serotype-associated differential human and animal fH binding by B. garinii was also observed, which could be related with the ability of some strains from serotypes 4 and 7 to invade non-nervous system tissues. Also, the variable affinity of binding proteins expressed by different Borrelia to animal fH correlated with their host selectivity.
Conclusion
The novel animal and human putative fH binding proteins (FHBPs) in this study underscore the importance of evasion of complement in the pathogenesis of Borrelia infections.
Findings
Binding of fH on the borrelial cell surface is critical for resistance to complement-mediated killing by inhibiting the formation of the terminal complement complex [1,2]. Human fH binding has been reported and its association with the pathogenic nature of Borrelia species was predicted earlier [1,3,4]. Complement resistant strains (e.g. B. afzelii and B. hermsii) survive successfully in body compartments where complement concentration is high, whereas it is proposed that B. garinii strains do not bind fH on their surface and thus are prone to complement-mediated killing; therefore, they would be able to invade the nervous system where complement concentration is low [5]. However, it has been reported that some OspA serotypes of B. garinii can infect and disseminate through the skin [6], and resist human complement mediated killing [7].
The nature of human and animal fH binding ability to Borrelia is complex. To date, the majority of studies have focused on human fHBPs of B. burgdorferi s.s., B. afzelii and B. hermsii, using purified fH or recombinant proteins [3,8-10]. In contrast, here we have analyzed a wider panel of Borrelia species, as well as human and different animal sera as source of native fH. We also present how the reservoir competence for Borrelia parallels their fH binding ability in different animal species, identifying known as well as not yet described putative fHBPs.
Materials and methods
First, the reactivity of sheep anti-human fH polyclonal antibody to fH from human and different animal species was assessed (Figure (Figure1).1Figure 1). Human and animal (mouse, rat, guinea pig, cattle, horse, dog and cat) serum samples, free of antibodies against B. burgdorferi, were purchased (Sigma-Aldrich), albumin depleted [11]., fractionated by non-reducing SDS-PAGE (10 μg/well/animal species) and transferred to nitrocellulose membranes. Purified human fH served as a positive control. Membranes were blocked overnight at 4°C in SuperBlock buffer (Pierce, Rockford, IL, USA) and then incubated for 2 hours (37°C with shaking) with sheep anti-human fH polyclonal antibody (Abcam, Cambridge, UK) diluted 1:1,500 in TTBS buffer [10 mM Tris/HCl (pH 8.3), 0.05% Tween-20 and 150 mM NaCl] with 1% skim milk. Membranes were washed 3 times with TTBS, incubated with rabbit anti-sheep HRPO antibody (Abcam) diluted to 1:400,000 in TTBS with 1% skim milk for 1 hour (37°C with shaking) and then washed 3 times as above. The reaction was developed by chemiluminescence with SuperSignal West Dura substrate (Pierce).

Figure 1

Figure 1
Figure 1

Binding ability of anti-factor H antibody to human and animal fH. Non-reducing one-dimensional immunoblot of albumin depleted human and animal sera against sheep anti-human fH polyclonal antibody. Purified human fH was used as a positive control.
Subsequently, affinity ligand binding immunoblot (ALBI) assays were performed to detect fHBPs of Lyme disease and relapsing fever borreliae (Table 1). Borrelial strains were grown in BSK-II medium at 33°C, harvested, washed 5 times with PBS supplemented with 5 mM MgCL2 and then resuspended in ultra pure water containing 1% trifluoroacetic acid (Sigma-Aldrich), 1% of nuclease mix and 1% of a protease inhibitor cocktail (GE Healthcare). Cells were sonicated and total protein concentration was measured (Bradford). Proteins were fractionated by non reducing SDS-PAGE, immunoblotted, and the membranes were cut in 3 mm strips, which were incubated 2 hours either with 1 ml of purified human fH (1,500 μg/ml) as a positive control or human and animal sera (1:4 dilution). fH bound to borrelial proteins was detected with sheep anti-human fH antibody and rabbit anti-sheep HRPO conjugate, as indicated above. Binding was detected by chemiluminescence.

Table 1
Table 1

fH binding proteins, binding strength, calculated molecular mass and isoelectric points estimated in 2-DE.
To isolate and identify the borrelial proteins showing human/animal fH binding ability, 2-D electrophoresis (2-DE) coupled with MALDI-TOF-TOF was employed. For that, borrelial proteins were cleaned (Bio-Rad Laboratories S.A., Barcelona, Spain) and solubilized in Destreak solution (GE Healthcare, Madrid, Spain). Protein solutions (100 μg) were loaded by rehydration on 7 cm IPG strips (pH 3-11NL or pH 4-7; GE Healthcare), and were focused for 9,142 Vhr using the IPGphor system (GE Healthcare). Strips were equilibrated and subjected to SDS-PAGE on duplicated 15% polyacrylamide gels. One gel was stained with the Silver Stain Plus Kit (Bio-Rad), and the second was subjected to ALBI assay as described above, to ascertain fHBPs. The protein spots of interest were excised from 2-DE gels and digested [12] (Proteineer, Bruker-Daltonics). Digested aliquots were mixed with α-cyano-4-hydroxycinnamic acid in 33% aqueous acetonitrile and 0.25% trifluoroacetic acid. This mixture was deposited onto a 600 μm AnchorChip prestructured MALDI probe (Bruker-Daltonics) and allowed to dry. MALDI-MS data were obtained in an automated analysis loop (Ultraflex, Bruker-Daltonics) equipped with a LIFT-MS/MS device. Spectra were acquired in the positive-ion mode at 50 Hz laser frequency, and 100 to 1000 individual spectra were averaged. Automated analysis of mass data was performed (FlexAnalysis software; Bruker-Daltonics). MALDI-MS and MALDI-MS/MS data were combined (BioTools, Bruker-Daltonics) to search a non-redundant protein database (NCBInr) using Mascot software (Matrix Science). When Mascot search failed to assign a peptide match with Borrelia proteins, manual de novo sequencing [13] was attempted based on MALDI-MS/MS spectra.
As coiled-coil elements were demonstrated to be involved in the presentation of the fH binding sites [14,15]., putative coiled-coil formation analysis for novel sequences was performed by using PepCoil software [16]http://bioweb.pasteur.fr/seqanal/interfaces/pepcoil.html. Lipoprotein signal peptide analysis was done following the description of Setubal et al. [17].
Results
A wide repertoire of human fHBP was detected in Borrelia species
Both B. afzelii and B. burgdorferi s.s. ~26 kDa proteins bound human fH (Figure (Figure2,2Figure 2, panels A and B, lanes 1 and 2, respectively). A ~19 kDa protein of B. garinii serotype 4 and a ~17 kDa protein of B. garinii serotype 7 showed human fH binding ability (Figure (Figure2,2Figure 2, panels A and B, lanes 4 and 7, respectively; Figure Figure3),3Figure 3), while other B. garinii serotypes (3, 6, and 8) did not express any human fHBP (Figure (Figure2,2Figure 2, panels A and B, lanes 3, 6 and 8, respectively). Also, a ~26 kDa fHBPs was observed in B. garinii serotype 5 when using purified human fH (Figure (Figure2,2Figure 2, Panel A, lane 5), although the corresponding band when using human serum (Figure (Figure2,2Figure 2, Panel B, lane 5) was not observed, probably due to the lesser amount of fH present in human serum compared to the amount of purified fH used in the experiment. B. valaisiana, B. andersonii, B. bissettii and B. japonica also expressed human fHBPs (Figure (Figure2,2Figure 2, panels A and B, lanes 9, 10, 12 and 13, respectively), although B. bissettii binding was weaker when using human serum than purified human factor H, again probably due to the lesser amount of fH present in human serum. Conversely, B. lusitaniae did not show any human fHBP in any of the assays (Figure (Figure2,2Figure 2, panels A and B, lane 11). B. parkeri expressed a ~23 kDa human fHBP in our study (Table 1; Figure Figure2,2Figure 2, panels A and B, lane 15). A ~20 kDa human fH putative ligand of B. hermsii was also observed in our study (Table 1; Figure Figure2,2Figure 2, panels A and B, lane 14). Consistent with earlier report [3], in the case of B. anserina, the causative agent of avian borreliosis, and B. coriaceae, the causative agent of epizootic bovine abortion, no human fHBPs were observed.

Figure 2

Figure 2
Figure 2

Affinity ligand binding (ALBI) assays. Non-reducing one-dimensional immunoblot of whole cell sonicates of different Borrelia species against purified human fH (Panel A), human serum (Panel B), and mouse serum (Panel C).

Figure 3

Figure 3
Figure 3

Example of 2D ALBI assay. Two-dimensional (pH 3-11NL) immunoblot of a whole cell sonicate of B. garinii serotype 4 (strain PBi) against human serum, showing a reactive protein of ~19 kDa.
Animal fHBPs were also observed in different Borrelia species
Some of the murine fH putative ligands that showed no affinity to human fH in the study were ~15 kDa protein of B. afzelii, ~28 kDa protein of B. garinii serotype 4, ~28 and ~40 kDa proteins of B. bissettii, ~15 and ~26 kDa proteins of B. japonica, and ~40 and ~58 kDa proteins of B. coriaceae (Table 1; Figure Figure2,2Figure 2, panel C, lanes 1, 4, 12, 13 and 17, respectively).
Amongst Lyme disease Borreliae, only B. japonica ~15 kDa, ~19 kDa and ~24 kDa proteins showed rat fH binding (Table 1), while no affinity for guinea pig fH was observed. However, B. hermsii ~20 kDa protein showed affinity for both rat and guinea pig fH (Table 1). As with mouse fH, B. coriaceae ~58 kDa protein showed affinity for rat but not for guinea pig fH (Table 1). As expected, none of the B. anserina proteins showed affinity for rodent fH.
Four Lyme disease- (B. afzelii, B. valaisiana, B. andersonii and B. japonica) and none of the relapsing fever Borrelia expressed canine fH binding proteins (Table 1). The feline fH binding pattern in the array of Borrelia studied herein was similar to that of canine fH, except in VS116 (B. valaisiana) and 21123 (B. andersonii) strains, which were negative.
Finally, none of the Borrelia species tested, except B. coriaceae (~40 and ~58 kDa proteins), bound bovine fH (Table 1). Likewise, none of the Borrelia species tested possessed fHBPs that bind horse fH.
Identification of human and animal fHBPs.
From the ALBI assays, we were able to identify some human fHBPs, not yet described as part of the complement evasion system of Borrelia. Although Mascot software failed to find any significant protein hit for the ~19 kDa protein of B. garinii serotype 4 (Table 1,
Figure Figure3),3Figure 3), manual de novo sequencing generated the peptide sequence SNEKLEEDEENEAQQVNSLQNR
(Figure (Figure4).4Figure 4). The short input BLAST search showed a complete sequence homology with a hypothetical protein of B. garinii PBi (Genbank AAU07257). Unfortunately, neither information regarding the function and topology of this hypothetical protein was available in the protein databases nor proofs of binding were provided to exclude the probability of a contamination, although in silico analysis indicated that there was a high probability of two coiled-coil formations near the C-terminus (120 to 147 and 118 to 152 residues with a probability of 1.00). Although coiled-coil motifs are not specific of fHBPs, their presence has been described to be required for the formation of the fH binding site [14], therefore supporting the role of this novel protein in human fH binding [14,15].

Figure 4

Figure 4
Figure 4

MALDI-TOF based identification and de-novo sequencing. (Top) MALDI-MS spectrum from the ~19 kDa protein of B. garinii ST4 (strain PBi). Relevant mass signals employed for database searching have been labeled and known trypsin and keratin peptide signals (more …)
B. japonica ~22 kDa human fHBP (Table 1) was identified as OspE-related lipoprotein (GenBank-accession AAC62921), while ~26 kDa human fH binding proteins of B. burgdorferi s.s. and B. afzelii (Table 1) were identifed as CspA (cspA gene, BBA68) and BaCRASP-1 (ortholog of CspA; mmsa71 gene) respectively.
The ~15 kDa murine fH binding B. afzelii protein (Table 1) was identified as an outer membrane protein [GenBank-accession YP_853823], whilst rat fH binding ~19 kDa protein of B. japonica (Table 1) was identified as OspE-related lipoprotein (GenBank-accession AAC62921). Both proteins showed acidic pIs (4.0 – 5.2) in 2-DE in agreement with their theoretical pI (data not shown). The Mascot search for mouse fH-binding ~28 kDa protein of B. garinii serotype 4 showed a significant match and pI in agreement with the recently described BgCRASP-1 (likely to be orthologous of CspA; GenBank-accession CAH10086).
We identified a ~58 kDa B. coriaceae protein (Table 1) as a member of the bacterial extracellular solute-binding protein (BESBP) family. In fact, this is a probable lipoprotein according to the known features for lipoprotein signal peptides in spirochaetes [17]. For the ~40 kDa bovine fHBP of this species (Table 1) no confident protein match was found. Manual de novo sequencing for ~20 kDa B. hermsii protein (Table 1) using the corresponding MS/MS fragmentation spectra yielded two putative sequences: TLDNLLK (815.9 Da) and YLLVIFLLLSLASCDLFLK (2185.2 Da), which revealed high homology with the earlier characterized FhbA [18] of B-hermsii. (GenBank-accession AAY42861).
Discussion
Among the putative human fHBPs, we have found a ~19 kDa B. garinii serotype 4 protein different (only 12.3% of amino acid sequence similarity) from the earlier reported BgCRASP-1 described in this genospecies [19], as well as from the earlier cited B. burgdorferi s.s. or B. afzelii fHBPs. It is proposed that the fHBPs of Lyme disease borreliae possess linear sequence elements involved in fH binding [20]. However, a sequence similarity of 15 to 18% between CspA and the rest of Erp related fHBPs (ErpA, ErpC and ErpP) indicates that this may not always be the case. A further detailed study has provided evidence that fH binding is rather dependant on protein conformation [5], and formation of coiled-coil motifs [10,14]. The high probability of coiled-coil formation observed in in silico analysis for this putative ~19 kDa human fHBP and its amino acid divergence from other fHBPs strengthens these findings.
B. garinii is the most heterogeneous species in terms of plasmid content and OspA serotype [21,22], and these differences could account for different fHBP expression and complement susceptibility. Therefore, it is not at all surprising that some of the B. garinii serotypes that bound human fH in this study (OspA serotypes 4 and 7) also resisted complement mediated killing in a previous experiment [7]. Human fH binding by B. valaisiana could correspond to its proposed ability to produce erythema migrans [23], and the absence of human fH binding by B. anserina and B. coriaceae observed in our experiments correlates with the fact that these species do not infect humans.
The host selectivity of different Borrelia species correlates with their complement sensitivity, and thus also with their fH binding profile [7,24,25]. We show novel murine fH putative ligands expressed by Lyme disease Borrelia and B. coriaceae. The murine fH binding ability of B. bissettii and B. japonica strengthens their survival in rodents [26,27]. Moreover, the putative murine fHBP identified herein in B. garinii OspA serotype 4 correlates with the described ability of these strains to survive in mice [28].
We and others have reported that cattle and horses are not suitable hosts for Lyme disease related Borreliae [7,24,29], which strongly correlates with the inability of borrelial proteins to bind bovine and equine fH. However, binding of bovine fH only by B. coriaceae (~40 kDa & ~58 kDa proteins) suggests that cattle are a primary host for this Borrelia species. We have found a ~58 kDa protein that appears to have a bovine fH binding ability, to be a BESBP that may be an immune evasion tool of B. coriaceae in bovine. Like in other fHBPs, the ~58 kDa protein is a putative lipoprotein, as per the requirements described by Setubal et al. [17]., which suggest that it is located in the membrane.
The role of carnivores as hosts for Borrelia is probably limited. In this study we have noticed canine fHBPs in B. afzelii and B. valaisiana and, although with weak signals, also in B. andersoni and B. japonica, that could account for the ability of these genospecies to infect dogs [30]. Feline fHBPs has been detected, as well, in B. afzelii and B. japonica, although neither good data regarding reservoir competence nor pathogenicity of Borrelia in these hosts is available.
In summary, the fH binding strategy employed by Borrelia species in different hosts is complex. Novel, putative human and animal fHBPs have been found in this study, which highlights the multiplicity of the immune evading arsenal that Borrelia possesses.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MRB carried out the affinity ligand binding assays, the 2-DE electrophoresis and drafted the manuscript. RE participated in the design of the study and carried out the preparation of protein lysates of the Borrelia species tested. EC performed the identification of protein spots. HG and IJ also participated in the design of the study and helped in the 2-DE electrophoresis. PA conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements
Dr. Mangesh Bhide was supported by a grant from the Spanish Ministry of Education and Science (SB2004-0188). Financial support was also from Instituto de Salud Carlos III (grant EM03/06) and RETIC EBATRAG (G03/057).
We thank Frank M. Hodgkins for reviewing the English version of this manuscript.
References
  • Kraiczy P, Skerka C, Kirschfink M, Zipfel PF, Brade V. Mechanism of complement resistance of pathogenic Borrelia burgdorferi isolates. Int Immunopharmacol. 2001;1:393–401. doi: 10.1016/S1567-5769(00)00041-2. [PubMed]
  • Alitalo A, Meri T, Rämö L, Jokiranta TS, Heikkilä T, Seppälä IJ, Oksi J, Viljanen M, Meri S. Complement evasion by Borrelia burgdorferi: serum-resistant strains promote C3b inactivation. Infect Immun. 2001;69:3685–3691. doi: 10.1128/IAI.69.6.3685-3691.2001. [PubMed]
  • McDowell JV, Tran E, Hamilton D, Wolfgang J, Miller K, Marconi RT. Analysis of the ability of spirochete species associated with relapsing fever, avian borreliosis, and epizootic bovine abortion to bind factor H and cleave c3b. J Clin Microbiol. 2003;41:3905–3910. doi: 10.1128/JCM.41.8.3905-3910.2003. [PubMed]
  • Rodríguez de Córdoba S, Esparza-Gordillo J, Goicoechea de Jorge E, Lopez-Trascasa M, Sánchez-Corral P. The human complement factor H: functional roles, genetic variations and disease associations. Mol Immunol. 2004;41:355–367. doi: 10.1016/j.molimm.2004.02.005. [PubMed]
  • Metts MS, McDowell JV, Theisen M, Hansen PR, Marconi RT. Analysis of the OspE determinants involved in binding of factor H and OspE-targeting antibodies elicited during Borrelia burgdorferi infection in mice. Infect Immun. 2003;71:3587–3596. doi: 10.1128/IAI.71.6.3587-3596.2003. [PubMed]
  • Oteo JA, Backenson PB, del Mar Vitutia M, García Moncó JC, Rodríguez I, Escudero R, Anda P. Use of the C3H/He Lyme disease mouse model for the recovery of a Spanish isolate of Borrelia garinii from erythema migrans lesions. Res Microbiol. 1998;149:39–46. doi: 10.1016/S0923-2508(97)83622-4. [PubMed]
  • Bhide MR, Travnicek M, Levkutova M, Curlik J, Revajova V, Levkut M. Sensitivity of Borrelia genospecies to serum complement from different animals and human: a host-pathogen relationship. FEMS Immunol Med Microbiol. 2005;43:165–172. doi: 10.1016/j.femsim.2004.07.012. [PubMed]
  • McDowell JV, Wolfgang J, Tran E, Metts MS, Hamilton D, Marconi RT. Comprehensive analysis of the factor H binding capabilities of Borrelia species associated with Lyme disease: delineation of two distinct classes of factor H binding proteins. Infect Immun. 2003;71:3597–3602. doi: 10.1128/IAI.71.6.3597-3602.2003. [PubMed]
  • Stevenson B, El-Hage N, Hines MA, Miller JC, Babb K. Differential binding of host complement inhibitor factor H by Borrelia burgdorferi Erp surface proteins: a possible mechanism underlying the expansive host range of Lyme disease spirochetes. Infect Immun. 2002;70:491–497. doi: 10.1128/IAI.70.2.491-497.2002. [PubMed]
  • Hovis KM, Jones JP, Sadlon T, Raval G, Gordon DL, Marconi RT. Molecular analyses of the interaction of Borrelia hermsii FhbA with the complement regulatory proteins factor H and factor H-like protein 1. Infect Immun. 2006;74:2007–2014. doi: 10.1128/IAI.74.4.2007-2014.2006. [PubMed]
  • Colantonio DA, Dunkinson C, Bovenkamp DE, Van Eyk JE. Effective removal of albumin from serum. Proteomics. 2005;5:3831–3835. doi: 10.1002/pmic.200401235. [PubMed]
  • Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc. 2006;1:2856–2860. doi: 10.1038/nprot.2006.468. [PubMed]
  • Tannu NS, Hemby SE. De novo protein sequence analysis of Macaca mulatta. BMC Genomics. 2007;8:270. doi: 10.1186/1471-2164-8-270. [PubMed]
  • McDowell JV, Harlin ME, Rogers EA, Marconi RT. Putative coiled-coil structural elements of the BBA68 protein of Lyme disease spirochetes are required for formation of its factor H binding site. J Bacteriol. 2005;187:1317–1323. doi: 10.1128/JB.187.4.1317-1323.2005. [PubMed]
  • McDowell JV, Wolfgang J, Senty L, Sundy CM, Noto MJ, Marconi RT. Demonstration of the involvement of outer surface protein E coiled coil structural domains and higher order structural elements in the binding of infection-induced antibody and the complement-regulatory protein, factor H. J Immunol. 2004;173:7471–7480. [PubMed]
  • Lupas A, Van Dyke M, Stock J. Predicting coiled coils from protein sequences. Science. 1991;252:1162–1164. doi: 10.1126/science.252.5009.1162.
  • Setubal JC, Reis M, Matsunaga J, Haake DA. Lipoprotein computational prediction in spirochaetal genomes. Microbiology. 2006;152:113–121. doi: 10.1099/mic.0.28317-0. [PubMed]
  • Hovis KM, McDowell JV, Griffin L, Marconi RT. Identification and characterization of a linear-plasmid-encoded factor H-binding protein (FhbA) of the relapsing fever spirochete Borrelia hermsii. J Bacteriol. 2004;186:2612–2618. doi: 10.1128/JB.186.9.2612-2618.2004. [PubMed]
  • Wallich R, Pattathu J, Kitiratschky V, Brenner C, Zipfel PF, Brade V, Simon MM, Kraiczy P. Identification and functional characterization of complement regulator-acquiring surface protein 1 of the Lyme disease spirochetes Borrelia afzelii and Borrelia garinii. Infect Immun. 2005;73:2351–2359. doi: 10.1128/IAI.73.4.2351-2359.2005. [PubMed]
  • Alitalo A, Meri T, Lankinen H, Seppälä I, Lahdenne P, Hefty PS, Akins D, Meri S. Complement inhibitor factor H binding to Lyme disease spirochetes is mediated by inducible expression of multiple plasmid-encoded outer surface protein E paralogs. J Immunol. 2002;169:3847–3853. [PubMed]
  • Escudero R, Barral M, Pérez A, Vitutia MM, García-Pérez AL, Jiménez S, Sellek RE, Anda P. Molecular and pathogenic characterization of Borrelia burgdorferi sensu lato isolates from Spain. J Clin Microbiol. 2000;38:4026–4033. [PubMed]
  • Wilske B, Preac-Mursic V, Gobel UB, Graf B, Jauris S, Soutschek E, Schwab E, Zumstein G. An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis. J Clin Microbiol. 1993;31:340–350. [PubMed]
  • Rijpkema SG, Tazelaar DJ, Molkenboer MJ, Noordhoek GT, Plantinga G, Schouls LM, Schellekens JF. Detection of Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii and group VS116 by PCR in skin biopsies of patients with erythema migrans and acrodermatitis chronica atrophicans. Clin Microbiol Infect. 1997;3:109–116. doi: 10.1111/j.1469-0691.1997.tb00259.x. [PubMed]
  • Kurtenbach K, Sewell HS, Ogden NH, Randolph SE, Nuttall PA. Serum complement sensitivity as a key factor in Lyme disease ecology. Infect Immun. 1998;66:1248–1251. [PubMed]
  • Brade V, Kleber I, Acker G. Differences of two Borrelia burgdorferi strains in complement activation and serum resistance. Immunobiology. 1992;185:453–465. [PubMed]
  • Ishii N, Isogai E, Isogai H, Kimura K, Nishikawa T, Fujii N, Nakajima H. T cell response to Borrelia garinii, Borrelia afzelii, and Borrelia japonica in various congenic mouse strains. Microbiol Immunol. 1995;39:929–935. [PubMed]
  • Ishiguro F, Takada N, Yano Y, Harada M, Masuzawa T, Iida H. Prevalence of antibodies to Borrelia spirochetes among wild small rodents in central and western Japan. Microbiol Immunol. 1995;39:419–424. [PubMed]
  • Huegli D, Hu CM, Humair PF, Wilske B, Gern L. Apodemus species mice are reservoir hosts of Borrelia garinii OspA serotype 4 in Switzerland. J Clin Microbiol. 2002;40:4735–4737. doi: 10.1128/JCM.40.12.4735-4737.2002. [PubMed]
  • Gern L, Estrada-Peña A, Frandsen F, Gray JS, Jaenson TG, Jongejan F, Kahl O, Korenberg E, Mehl R, Nuttall PA. European reservoir hosts of Borrelia burgdorferi sensu lato. Zentralbl Bakteriol. 1998;287:196–204. [PubMed]
  • Bhide M, Travnicek M, Curlik J, Stefancikova A. The importance of dogs in eco-epidemiology of Lyme borreliosis: a review. Vet Med Praha. 2004;49:135–142.

Powered by WordPress