The Cyanobacterial Neurotoxin L- -methylamino-n-alanine (BMAA) As An Emerging Public Health Concern In The Columbia River Watershed

Transcription

1 The Cyanobacterial Neurotoxin L- -methylamino-n-alanine (BMAA) As An Emerging Public Health Concern In The Columbia River Watershed Stuart W. Dyer 1,2, Tawnya D. Peterson 2, Joseph A. Needoba 2 1 OHSU School of Medicine 2 OHSU-PSU School of Public Health April, 2018

2 FlowCam images, Franz Lake Slough, WA (2016): Dolichospermum sp. bloom

3 Cyanobacteria are an emerging global health threat to ecosystems and humans Lake Erie, USA (2011) Toledo

4 Cyanobacteria are an emerging local health threat to ecosystems and humans Ross Island Lagoon (2015) Photo: S. Dyer Ross Island Lagoon (2015) Photo: S. Dyer

5 Cyanotoxins are the main pubic health concern in the management of HABs Microcystin Saxitoxin Anatoxin Hepatotoxic 0.3 g/l (drinking) 20 g/l (no contact) Nephero/neurotoxic 0.2 g/l (drinking) 3 g/l (no contact) neurotoxic 0.7 g/l (drinking) 3 g/l (no contact)

6 The unique and ubiquitous cyanotoxin: β-n-methylamino-l-alanine (BMAA) BMAA detected in representative species of all cyanobacterial morphotypes present for duration of bloom forming season Innate biochemical and ecological function are unknown

7 The developing science of BMAA: BMAA isolated from cycad seeds Controversy over dose needed to trigger neuropathology (1963), 268: Chamorro native with advanced Guam-PD (Rep Village ca., 1960)

8 ca First evidence supporting a cyanobacterial origin for BMAA First plausible hypothesis for Guam-PD Pteropus vampyrus (fruit bat) soup wordpress

9 ca First evidence indicating that BMAA is a fundamental cyanobacterial metabolite BMAA detected in 95% of axenic cyanobacterial cultures analyzed (n=30)

10 ca ca. 2016

11 BMAA disrupts the tightly coupled carbon/nitrogen metabolism of cyanobacteria GS/GOGAT

12 Amotile cyanobacteria use buoyancy regulation as the physiochemical like between access to energy and nutrient pools Carbohydrate Ballast Promotes downward movement ENERGY NUTRIENTS Adapted from Walsby (1994) Proteinaceous Gas Vesicles Promote upward movement

13 warm temperatures promote cyanobacterial dominance Ross Island Lagoon Willamette River, OR (2016) mm Hr rate of DVM increases with colony size m Hr

14 Large initial nutrient inputs promote colonization of the air/water interface ~72 Hrs rapid doubling time for most planktonic cyanobacteria (< 24 hours), coupled with favorable CO 2 uptake kinetics

15 increased temperature & nutrient loads promote surface trapping ~10 3 cells L -1 ~10 7 cells L -1 Photo: S. Dyer

16 Surface trapping in HABs drives food-web perturbations and cyanobacterial stress responses Dolichospermum sp., dominant: Franz Lake Slough (2016) Foodweb Oxygen depletion Shading Toxins Microcystis sp., dominant: Ross Island Lagoon (2016) HAB Organisms Nutrient limitation Increased PAR Increased potential for oxidative damage

17 Cyanotoxins provide a selective advantage Microcystin Saxitoxin Anatoxin Increased fitness under highlight/oxidative conditions covalent binding to large subunit of RuBisCo Increased fitness under periods of osmotic stress Bioflim formation decreases H 2 O loss and salt stress Increased fitness due to reduced grazing (?) Very fast death factor...

18 Well then what s the deal with BMAA?

19 The Guam-PD hypothesis remains contentious: Chernoff et al., (2017) Before public health actions are taken based on the [Guam-PD] hypothesis, it is imperative that fundamental issues and inconsistencies concerning the central assumptions are discussed and resolved, Including: Issues with detection and quantification

20 LC-MS/MS quantification of BMAA within complex environmental samples

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22 Disambiguation of BMAA from its isomers DAB and AEG based on elution time BMAA DAB AEG Dyer unpublished results

23 Quantification based on the area ratio of L-BMAA: 15 N-BMAA LOD: 2.4 ng/g-cells LLOQ: 23 ng/g-cells Dyer unpublished results

24 whole water grab biofilm scraping

25 samples collected at the start of cyanobacterial bloom progression

26 ca BMAA is a public health concern BMAA is present in the Lower Columbia River watershed

27 Thank you Committee Members Dr. Joseph Needoba Dr. Tawnya Peterson Dr. Bradley Tebo Dr. John Perona Lab Members Lyle Cook Nikolai Danilchik Taylor Dodril Interns Desire Povijua IEH faculty Dr. Richard Johnson Dr. Mickiko Nakano OHSU Bioanalytical Shared Resource/ Pharmacokinetics Core Dr. Dennis Koop Lisa Bleyle OHSU Medicinal Chemistry Core Dr. Aaron Nilsen

28 Questions?

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30 HOW CAN WE MONITOR NONMONOTONIC CHANGES IN HAB DYNAMICS?

31 Nephel-o-metr-ic relating to the measurement of light scattering Turbid-i-meter an instrument for measuring the concentration of suspended solids in a liquid medium An instrument that measures the scattering of light as a proxy for the concentration of suspended solids in a liquid medium

32 Intact gas vesicles (GVs) provide buoyancy and strongly scatter light Adapted from Walsby, 1994.

33 Pressure Nephelometer An instrument that is able to modulate pressure within a nephelometric cell, measure the resulting scattering of light, and determine pressure/turbidity relationships

34 Critical collapse pressure is species-specific Cyanobacteria Pc (psig) Pt (psig) Reference Anabaena flos-aquae CCAP1403/13f Walsby, 1980 Aphanizomenon flos-aquae CCAP1401/ Konopka et al., 1978 Chroococcales sp Walsby & Bleything, 1988; Microcystis sp. BC84/ Walsby et al., 1983 Lyngbya/Oscillatoria agardhii PCC7801 (red) Walsby & Bleything, 1988 Other Bacteria Amoebobacter roseus Walsby, 1971 Aebobacter purpureus Overmann & Pfennig, 1992 Ancylobacter aquaticus Koch & Pinette, 1987 Pelodictyon phaeochlathratiforme Overmann et al., 1991 Prosthecomicrobium pneumaticum Walsby, 1976 Halobacteria Halobacterium salinarium Walsby, 1971

35 Generating a high-resolution pressure nephelometry measurement with an environmental sample from the 2017 Ross Island Lagoon HAB psig stepwise integration of nephelometric spectrum recorded at each pressure step ( P=0.75 psig) yields Helobacter Merismopedia Chroococcales Anabaena Microcystis Lyngbya DETECTOR Dyer unpublished results

36 Generating a traditional pressure nephelometry measurement with an environmental sample from the 2017 Ross Island Lagoon HAB Nephelometric turbidity (counts) psig nephelometric spectrum ( nm) recorded at each pressure step stepwise integration of nephelometric spectrum recorded at each pressure step ( P=2.5 psig) yields... DETECTOR Dyer unpublished results

37 Critical collapse pressure is determined by gas vesicle structure GV width is inversely proportional to strength Strength is proportional to protein investment Changes in GV critical collapse pressure may indicate cellular stress or changes in nutrient status Dyer unpublished results

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39 Growth Profiling Via Tubidity Nonlinear Regression Equation: Sigmoidal f = y0+a/(1+exp(-(x-x0)/b)) R Rsqr Adj Rsqr Standard Error of Estimate Statistical Tests: Normality Test (Shapiro-Wilk) Passed (P = ) W Statistic= Significance Level = Constant Variance Test Failed (P = )

40 Turbidimetric growth profiling of M. aeruginosa from Carolina Biological Supply showing small Pcrit 0.5 during exponential growth

41 psig Field of View (FOV) ~10mm P TOT = P 2 -P 1 P(t) = 0.25psig/s t TOT = P tot /(0.25psig/s) Rate of migration (ROM) = FOV/ t TOT ROM peak1 =0.33 m/hr

42 Ross Island Lagoon Thermocline Progression (2017) Dyer unpublished results

43 Thermocline progression leads to increased physical separation between access to nutrient and energy pools ENERGY NUTRIENTS Dyer unpublished results

44 LIGHT MATTERS! Both quality and quantity of light have important effects on cyanobacterial metabolism Complementary chromatic adaptation Chlorosis MMA production Toxin production Buoyancy Regulation Etc., Etc.,