Study of the Protein Complex, Pore Diameter, and Pore-forming Activity of the Borrelia burgdorferi P13 Porin

Bárcena-Uribarri, Iván; Thein, Marcus; Barbot, Mariam; Sans-Serramitjana, Eulàlia; Bonde, Mari; Mentele, Reinhard; Lottspeich, Friedrich; Bergström, Sven; Benz, Roland

doi:10.1074/jbc.m113.539528

Cited by 15 publications

(11 citation statements)

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“…The conductance of the toxin pores was investigated as a function of the hydrodynamic radii of different nonelectrolytes dissolved in the salt solution. These molecules have been used previously to determine the effective diameters of several channels reconstituted into planar lipid bilayers [40, 41, 69–71]. Inspection of the results from nonelectrolytes suggested that the RtxA pore has a diameter of around 1.9 nm.…”

Section: Discussionmentioning

confidence: 99%

Pore forming activity of the potent RTX-toxin produced by pediatric pathogen Kingella kingae: Characterization and comparison to other RTX-family members

Bárcena-Uribarri

Benz

Winterhalter

et al. 2015

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Pediatric septic arthritis in patients under age of four is frequently caused by the oral Gram-negative bacterium Kingella kingae. This organism may be responsible for a severe form of infective endocarditis in otherwise healthy children and adults. A major virulence factor of K. kingae is RtxA, a toxin that belongs to the RTX (Repeats-in-ToXin) group of secreted pore forming toxins. To understand the RtxA effects on host cell membranes, the toxin activity was studied using planar lipid bilayers. K. kingae strain PYKK081 and its isogenic RtxA-deficient strain, KKNB100, were tested for their ability to form pores in artificial membranes of asolectin/n-decane. RtxA, purified from PYKK081, was able to rapidly form pores with an apparent diameter of 1.9 nm as measured by the partition of nonelectrolytes in the pores. The RtxA channels are cation-selective and showed strong voltage-dependent gating. In contrast to supernatants of PYKK081, those of KKNB100 did not show any pore forming activity. We concluded that RtxA toxin is the only secreted protein from K. kingae forming large channels in host cell membranes where it induces cation flux leading to programmed cell death. Furthermore, our findings suggested that the planar lipid bilayer technique can effectively be used to test possible inhibitors of RTX toxin activity and to investigate the mechanism of the toxin binding to the membrane.

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Section: Discussionmentioning

confidence: 99%

Pore forming activity of the potent RTX-toxin produced by pediatric pathogen Kingella kingae: Characterization and comparison to other RTX-family members

Bárcena-Uribarri

Benz

Winterhalter

et al. 2015

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…At least four genes upregulated by c-di-GMP encode known or putative porins: DipA (encoded by bb0418) (92) and the three P13 paralogs (encoded by bbh41, bbi31, and bbq06) (93). P13 paralogs are thought to function as general diffusion porins, while DipA has a high affinity for dicarboxylates, including glutamate and pyruvate (92), which may help protect B. burgdorferi against oxidative damage within the fed midgut milieu.…”

Section: Resultsmentioning

confidence: 99%

Cyclic di-GMP Modulates Gene Expression in Lyme Disease Spirochetes at the Tick-Mammal Interface To Promote Spirochete Survival during the Blood Meal and Tick-to-Mammal Transmission

et al. 2015

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e Borrelia burgdorferi, the Lyme disease spirochete, couples environmental sensing and gene regulation primarily via the Hk1/ Rrp1 two-component system (TCS) and Rrp2/RpoN/RpoS pathways. Beginning with acquisition, we reevaluated the contribution of these pathways to spirochete survival and gene regulation throughout the enzootic cycle. Live imaging of B. burgdorferi caught in the act of being acquired revealed that the absence of RpoS and the consequent derepression of tick-phase genes impart a Stay signal required for midgut colonization. In addition to the behavioral changes brought on by the RpoS-off state, acquisition requires activation of cyclic di-GMP (c-di-GMP) synthesis by the Hk1/Rrp1 TCS; B. burgdorferi lacking either component is destroyed during the blood meal. Prior studies attributed this dramatic phenotype to a metabolic lesion stemming from reduced glycerol uptake and utilization. In a head-to-head comparison, however, the B. burgdorferi ⌬glp mutant had a markedly greater capacity to survive tick feeding than B. burgdorferi ⌬hk1 or ⌬rrp1 mutants, establishing unequivocally that glycerol metabolism is only one component of the protection afforded by c-di-GMP. Data presented herein suggest that the protective response mediated by c-di-GMP is multifactorial, involving chemotactic responses, utilization of alternate substrates for energy generation and intermediary metabolism, and remodeling of the cell envelope as a means of defending spirochetes against threats engendered during the blood meal. Expression profiling of c-di-GMP-regulated genes through the enzootic cycle supports our contention that the Hk1/Rrp1 TCS functions primarily, if not exclusively, in ticks. These data also raise the possibility that c-di-GMP enhances the expression of a subset of RpoS-dependent genes during nymphal transmission. Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature within an enzootic cycle that involves an arthropod vector and vertebrate reservoir hosts, typically, small rodents and birds (1). In the northeastern United States, the primary vector for B. burgdorferi is the black-legged deer tick, Ixodes scapularis (2, 3). Because B. burgdorferi cannot be passaged transovarially, naive larvae acquire spirochetes by feeding on infected reservoir hosts. Successful colonization of the vector requires B. burgdorferi to establish an intimate association with rapidly differentiating, highly endocytic midgut epithelial cells (4-6). In order to accomplish this feat, spirochetes must resist deleterious substances within the midgut lumen, such as host-and tick-derived innate immune effector molecules, reactive oxygen species (ROS), and salivary enzymes imbibed from the feeding site (4, 7-11). At the same time, B. burgdorferi also must alter its metabolic machinery to exploit the availability of alternative carbon sources (e.g., glycerol, N-acetylglucosamine [GlcNAc], chitobiose) as the supply of ingested glucose diminishes (12-15). During nymphal transmission, spirochetes are almost certai...

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“…Unlike the majority of the surface-exposed lipoproteins, all of the borrelial OMPs identified thus far are encoded on the ~900 kB linear chromosome [ 11 , 33 , 39 , 41 , 43 – 46 ]. While it is known that the borrelial OM contains membrane spanning OMPs with β-barrel structure and/or pore-forming capabilities that are important in overall physiology and host interactions, fewer than 10 borrelial proteins have been identified as potential OMPs and only half of those have thus far been characterized [ 22 , 33 , 39 , 41 , 45 – 52 ]. Of the known borrelial OMPs, B. burgdorferi proteins BamA (BB0795), BesC (BB0142), DipA (BB0418), P66 (BB0603), and P13 (BB0034) have been shown to form a β-barrel, to form pores in the borrelial OM, or to be functional orthologs to known OMPs.…”

Section: Introductionmentioning

confidence: 99%

Consensus computational network analysis for identifying candidate outer membrane proteins from Borrelia spirochetes

et al. 2016

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BackgroundSimilar to Gram-negative organisms, Borrelia spirochetes are dual-membrane organisms with both an inner and outer membrane. Although the outer membrane contains integral membrane proteins, few of the borrelial outer membrane proteins (OMPs) have been identified and characterized to date. Therefore, we utilized a consensus computational network analysis to identify novel borrelial OMPs.ResultsUsing a series of computer-based algorithms, we selected all protein-encoding sequences predicted to be OM-localized and/or to form β-barrels in the borrelial OM. Using this system, we identified 41 potential OMPs from B. burgdorferi and characterized three (BB0838, BB0405, and BB0406) to confirm that our computer-based methodology did, in fact, identify borrelial OMPs. Triton X-114 phase partitioning revealed that BB0838 is found in the detergent phase, which would be expected of a membrane protein. Proteolysis assays indicate that BB0838 is partially sensitive to both proteinase K and trypsin, further indicating that BB0838 is surface-exposed. Consistent with a prior study, we also confirmed that BB0405 is surface-exposed and associates with the borrelial OM. Furthermore, we have shown that BB0406, the product of a co-transcribed downstream gene, also encodes a novel, previously uncharacterized borrelial OMP. Interestingly, while BB0406 has several physicochemical properties consistent with it being an OMP, it was found to be resistant to surface proteolysis. Consistent with BB0405 and BB0406 being OMPs, both were found to be capable of incorporating into liposomes and exhibit pore-forming activity, suggesting that both proteins are porins. Lastly, we expanded our computational analysis to identify OMPs from other borrelial organisms, including both Lyme disease and relapsing fever spirochetes.ConclusionsUsing a consensus computer algorithm, we generated a list of candidate OMPs for both Lyme disease and relapsing fever spirochetes and determined that three of the predicted B. burgdorferi proteins identified were indeed novel borrelial OMPs. The combined studies have identified putative spirochetal OMPs that can now be examined for their roles in virulence, physiology, and disease pathogenesis. Importantly, the studies described in this report provide a framework by which OMPs from any human pathogen with a diderm ultrastructure could be cataloged to identify novel virulence factors and vaccine candidates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0762-z) contains supplementary material, which is available to authorized users.

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Study of the Protein Complex, Pore Diameter, and Pore-forming Activity of the Borrelia burgdorferi P13 Porin

Cited by 15 publications

References 60 publications

Pore forming activity of the potent RTX-toxin produced by pediatric pathogen Kingella kingae: Characterization and comparison to other RTX-family members

Pore forming activity of the potent RTX-toxin produced by pediatric pathogen Kingella kingae: Characterization and comparison to other RTX-family members

Cyclic di-GMP Modulates Gene Expression in Lyme Disease Spirochetes at the Tick-Mammal Interface To Promote Spirochete Survival during the Blood Meal and Tick-to-Mammal Transmission

Consensus computational network analysis for identifying candidate outer membrane proteins from Borrelia spirochetes

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