Brian Stevenson Ph.D. spirochete lab

@bstevensonlab.bsky.social

Mostly about bacterial gene/protein regulation & function, esp. with Borrelia burgdorferi and Lyme disease. Professor at U Kentucky, views are my own. He/him Lab= http://microscopist.net/Stevenson_Lab.html UK= https://medicine.uky.edu/users/bstev0

Screenshot of 7/17 UK Now, the daily propaganda email from the University of Kentucky. Shows an attractive, bearded scientist who is holding up a tick.

Two people kissing, within a large frame

Onion satire: picture of puzzled man with cell phone and computer, with caption:
“Study: 97% of average American’s day spent retrieving 6-digit codes”

Graphical Abstract

The Lyme disease agent Borrelia burgdorferi has limited metabolic capabilities and depends on salvage of riboflavin via the BB0316-BB0319 transport system for generation of critical flavin cofactors FMN and FAD. Riboflavin is important for NAD+ regeneration to fuel glycolysis. FAD-dependent coenzyme A disulfide reductase (CoADR) is the critical link between riboflavin availability and glycolytic redox balance driving glycolytic success in B. burgdorferi.

Video of feeding Dermacentor tick, from Uni of Nomi's Lyme disease page. Dermacentor ticks cannot vector Lyme disease

Screen cap of U Nomi's opening page on Lyme disease. Shows a Dermacentor spp. tick, which cannot vector Lyme disease

Screenshot of intro to "Matters Microbial"

Figure 5 from the article:
CCB disruption of actin-based mobility. (A) Fluorescence micrographs of R. conorii and host actin after 48 hours of infection with noted compounds added for the last 24 hours of infection. R. conorii (green) containing actin tails (red) are readily observed in mock (DMSO) treated cells, but the frequency of actin polymerization is reduced in CCB and Cytochalasin D treated cells. (B) Quantification of R. parkeri with observable actin tails after 48 hours of infection of EA.hy926 cells with noted compounds. Treatment with all three CCBs and the actin polymerization inhibitor Cytochalasin D all significantly reduced rickettsial actin polymerization. (C) Similar analysis of actin polymerization in R. conorii-infected EA.hy926 cells. Scale bar = 20 µm. *P < 0.05 by one-way ANOVA with Dunnett’s multiple comparisons test.

Fig 1. Overview of machine learning workflow.

From the paper:

Figure 1. (A) Structure of the E. coli BAM complex (PDB ID: 9HG9) showing BamA (grey), BamB (orange), BamD (brown), BamC (green) and BamE (purple). (B) AlphaFold-predicted structure of B. burgdorferi BamA (residues 25-821; average predicted local-distance difference test (pLDDT) value of 86.1) superimposed with BamA from E. coli (Uniprot: P0A940; gold; average pLDDT 92.5; Cα root-mean-square deviation (RMSD) of 3.5 Å). Both proteins are illustrated without the N-terminal signal sequence, and the 16-stranded β-barrel of B. burgdorferi BamA is colored grey, while the periplasmic POTRA domains are colored as follows: POTRA1 (residues 28-102), red; POTRA2 (residues 103-180), green; POTRA3 (residues 181-275), blue; POTRA4 (residues 276-353), purple; and POTRA5 (residues 354-430), orange. (C) Superimposed B. burgdorferi POTRA domains 1-5 (RMSD 1.63 to 3.05 Å).

From the publication:
Fig. 2. Experimental design of the study. At 7 weeks of age, 32 female mice were randomly assigned to one of four groups (n = 8 mice per group): (1) uninfected (white), (2) infected with B. burgdorferi strain A (red), (3) infected with B. burgdorferi strain I (blue), and (4) co-infected with both strains (purple). Female mice were infected via nymphal tick bite; uninfected females were infested with uninfected nymphs. At 10 weeks of age and at 3 weeks post-infection, female mice were mated with uninfected male mice. At 13-14 weeks of age and at 6-7 weeks post-infection, female mice produced 139 offspring. At weeks 1 and 2 of age, mouse pups were euthanized (n = 47), exsanguinated, and milk was obtained from their stomachs. A total of 88 offspring were weaned and were assigned to group 1 (G1; n = 45 offspring) and group 2 (G2; n = 43 offspring). The G1 pups and G2 pups were challenged with both B. burgdorferi strains via nymphal tick bite at 6 weeks of age and at 11 weeks of age, respectively.

Graphical Abstract

Motility cessation in bacteria is a key regulatory strategy that provides multiple survival advantages including enhanced community cooperation, niche adaptation and evasion of host immune responses. This process is controlled by associated mechanisms such as post-translational modifications and second messenger signalling that stabilise non-motile bacterial attachment. This review aims to provide a comprehensive overview of the molecular mechanisms in motility suppression and the functional and environmental conditions that trigger the transition from motile to non-motile states.

Two sciency-guys standing next to a multimillion dollar washing machine.
(Actually, a high throughput DNA sequencer)

Model from the paper (current data suggest that this is probably not completely accurate)
Figure 8
BadR represses rpoS to regulate host-specific adaptation in B. burgdorferi. The rpoS gene is upregulated in a badR-deficient B. burgdorferi strain. Log2 fold change of rpoS derived from two microarrays from (pH 7.6/32°C) grown cultures. P-values from pooled (overall) and individual arrays (Rep.1, Rep.2) are indicated using n = 4 for both A3 (WT) and badR− (A). Quantitative RT-PCR fold changes of rpoS are indicated with P ≤ 0.01. Error bars indicate s.e.m. (B). Predicted BadR regulatory network. Many activators of rpoS have been identified (e.g. DsrABb, BosR, RpoN, RpoD, CsrABb). Our study is the first to demonstrate BadR as a repressor of rpoS and thus may facilitate the spirochaete's transition back into ticks. A model for BadR regulation entails the following: 1.) BadR binds upstream of rpoS to repress rpoS in unfed ticks; 2.) Since ROK regulators are nutrient-responsive, BadR regulation may be modulated directly or indirectly by nutrient inducers; 3.) Nutrient surges from the blood meal may provide the inducer to allow BadR's derepression of rpoS; and 4.) BadR regulates chitobiose utilization genes and may activate genes to enhance the spirochaetes maintenance in ticks (lp28-4 genes). Square = activation, oval = repression by BadR respectively (C).

From the Preprint:
Fig. 2. Neutral lipids in B. burgdorferi. (A, B) Sankey diagrams illustrating
the relative abundance of neutral lipids identified via tandem mass
spectrometry. The width of each flow is proportional to the integrated peak area under the curve of the daughter ion, with the distribution of
cholesteryl esters (ChE, A) and triglycerides (TG, B). (C, D) Widefield
epifluorescence microscopy images of fixed B. burgdorferi cells show
lipid staining with either the neutral lipid-specific LipidTOXTM (C) or the
non-specific dye Nile Red (D). Scale bar = 5 μm

Fig 1. Maximum likelihood tree of the 86 genomes based on cgMLST and plasmid presence/absence matrix. Figure includes information about species, reservoir host, tick vector, pathogenicity and genome elements. Species are listed and colored in different colors. Reservoir hosts are shown as icons; unknown hosts are indicated by “?”; for insectivores and carnivores a hedgehog icon is used. Tick vector species are listed. Pathogenicity is shown in different colors (green: nonpathogenic, red: pathogenic, red and “?”: potentially pathogenic, white and “?”: unknown). Genome elements (chromosome and plasmids): presence is indicated in green (complete genome element) and red (incomplete genome element), absence is indicated in grey; “X”: fusion plasmid, “-”: linear plasmid that carry a PFam32 that is typically found on a circular plasmid, “O”: circular plasmid that carry a PFam32 that is typically found on a linear plasmid. “$”: cp32-28-4 (circular plasmid with syntenic gene contents to cp32 plasmid family but encodes a lp28−4 type PFam32 protein), “^”: inverted repeat plasmid, “~”: dimer fusion, “+”: sequence was additionally added, “2”: two cp9 plasmids with different PFam57 proteins. “(X)”: the same cp32−11 type PFam32 was found on a non-fused plasmid (cp32−11) and on a fusion plasmid (cp32−9 + 11); “!”: plasmid name used in this study differs to the published plasmid name (S3 Table). S2 Fig adds information about the continent, vls/vlsE located plasmid, fusion plasmids, and shows reservoir host and vector not as icons, but in separate color-coded columns. S1 Table presents this information in tabular form with additional information on plasmid length.

Fig. 8. Predicted 3rd dimensional structure of B. persica LMU-C01 Vmp proteins. One representative from each Vlp subfamily (A, alpha; B, beta; C, gamma; D, delta) and the Vsp protein (E) are presented. Protein structures were modeled using AlphaFold (v1.5.2) and visualized using ChimeraX (v1.9). Membrane orientation and surface rendering were modeled using PPM database. Different colors correspond to the pLDDT score (dark blue, very high pLDDT>90; light blue, high 70<pLDDT<90; yellow, moderate 70>pLDDT>50; orange, low pLDDT<50). The membrane attachment properties of the Vsp (F) and a Vlp representative (G) are shown. The two-colored plates represent the double layered bacterial membrane where the blue plate corresponds to the outer membrane. Hydrophobic properties of the Vmp proteins represented by Vlp alpha protein are presented on the surface structure of the protein (H). Light blue represents hydrophilic residues, whereas yellow color represents hydrophobic residues.

Stevenson lab personnel through the years, photographed in various exotic locations

FIG 9 Model of murine host immune responses to Bb, tick factors, and tick-transmitted Bb relative to the control. Graphical summary of the study showing the infection conditions—subcutaneous needle inoculation of multi-strain Bb (BbSC), uninfected tick feeding (Tick–Bb), and multi-strain Bb-infected tick feeding (Tick+Bb)—in C3H/HeN mice, with sequential analyses, including flow-cytometric profiling of immune cell populations at the bite/inoculation site and in spleen, and serum proteome profiling to quantify 19 cytokines and 20 chemokines.

Figure 3 from the article:
Scenic stops and extended stay: immune interference across the host landscape shapes tissue colonization, persistence, and clinical manifestation during B. burgdorferi infection. This schematic integrates pathogen-intrinsic strategies and host immune disruptions across spatial, temporal, and immunological scales. It frames the journey from early tissue colonization to prolonged immune stalemate. The central panel depicts the host landscape, emphasizing the translational continuum between experimental host models and human disease. Surrounding concentric rings organize immune interference by function rather than by individual molecules. The inner pink ring delineates four major modes of host-pathogen interaction that shape immune interference and persistence. The outer ring depicts representative mechanisms corresponding to each immune-interference category, illustrated as discrete modules.

Illuminated stereoview of the Moon, showing both Moon images

The Ixodes ricinus complex life cycle comprises four life stages (egg, larva, nymph and adult), where each post-egg stage needs a blood meal to develop into the next stage or reproduce. Larvae and nymphs feed on many vertebrate species, including mammals, songbirds and lizards, whereas adults feed on a narrower range of hosts, most commonly deer. Lyme borreliae are not passed from infected female ticks to their offspring; thus, the bacteria are maintained in an enzootic cycle between immature ticks (larvae and nymphs) and reservoir hosts, typically small mammals and songbirds.
For illustrative purposes, this figure typifies the life cycle of Ixodes scapularis in the northeastern USA. Eggs are laid in the late spring; larvae will hatch in mid-summer and then seek their first host in late summer. Engorged larvae will detach, drop off the host and moult into nymphs. Unfed nymphs emerge and quest for their second host the following late spring through summer, which is therefore the epidemiologically most risky time of the year. Engorged nymphs detach, drop off the host and moult into adults in the leaf litter. Beginning in the fall, adult ticks quest for a third host, most commonly deer. Engorged females detach from the host and drop into the leaf litter, where they overwinter. Adult ticks that are unable to locate hosts in the fall will also overwinter and resume questing in early spring. Fed I. scapularis females will oviposit an egg mass (up to 1,500–2,000 eggs), and then die. Although both nymphs and adult females may be infected, because of their small size (about the size of a poppy seed), their large numbers, and a spring/summer activity period that coincides with increased outdoor human activity, nymphs are responsible for most human infections. Because they are larger (about the size of a sesame seed) and have red coloration, adult female ticks are easier to notice and remove before the bacteria can be transmitted

Your death will come on an ordinary day, in the middle of unfinished plans, and the world will continue on without you.
So live a little

Darkened theater, with "The First Omen" displayed on the screen

Scientist dude holding a vial that contains engorged ticks

Graphic abstract of the article:
"Spirochete motility is driven by periplasmic flagella, yet how sheath architecture is organized and regulated has remained unclear. This study reveals that coordinated asymmetric localization of sheath proteins underlies flagellar architecture and motility in Leptospira.

The periplasmic flagellum of Leptospira consists of a FlaB1-based core filament that is asymmetrically surrounded by the sheath proteins FcpA and FcpB.

FcpA functions as a major flagellar coiling protein required for filament curvature.

FcpB acts as a structural wedge that reinforces flagellar rigidity.

FlaA2 controls the asymmetric localization of FcpA and FcpB, thereby coordinating flagellar architecture and motility."

Fig 1 from the paper. Data showing that bbd18 and its upstream sRNA SR0736 (ittA) impact expression of specific spirochete gene products and murine infectivity.

Figure from this publication:
Human foreskin-based cultivation system supports the growth of various
T. pallidum strains (n=8), from the Nichols-like as well as the SS14-like cluster. Note that human foreskin fibroblast cells were prepared as an equal mixture of three tested cell lines. Each T. pallidum strain was cultivated in a single in vitro well, representing a sole experimental replicate used for data acquisition.

Figure 1 from paper:
Characteristic features of lyme arthritis in different mouse strains after Borrelia burgdorferi infection. (A) X-ray examination of the hind limb joints on day 14 after infection to assess soft tissue edema in the joints. The upper row is the infection group, and the lower row is the PBS control group. (B) H&E-stained joint tissue on day 14 post-infection demonstrates inflammatory cell infiltration and tissue edema. Orange arrows mark pronounced synovial cell hyperplasia with structural disorganization. Green arrows indicate extensive connective tissue proliferation replacing native architecture. Purple arrows designate scattered necrotic cellular debris. Yellow arrows identify focal bone erosions. Blue arrows show dense inflammatory cell infiltration throughout joint tissue. Red arrows highlight isolated inflammatory cells within joint space. The left panels are at 4× magnification, while the right panels are magnified at 20×, showcasing detailed enlargements of specific regions within the 4× images. Scale bars: 200 μm at 4× magnification, and 50 μm at 20× magnification. (C) Dynamic changes in the diameter of the rear ankle joints of mice (C3H and C57BL/6) within 56 days after infection. The diameter of the joint of the hind limb was measured weekly using a digital caliper, and the results are expressed as mean ± standard error (n = 6/group, *P < 0.05 and **P < 0.01 compared to C57BL/6 control group, #P < 0.05 and ##P < 0.01 compared to C3H control group). (D) Pathological severity scores in H&E-stained joint tissue from C3H and C57BL/6 mice (*P < 0.05 and **P < 0.01). Regarding the rectangular areas detected by the forensic tools, this is attributable to our utilization of whole slide imaging in pathology, which constructs digital imaging by stitching together square fields of view and supports free magnification from 0× to 400×. Blank areas within the scanning range (where there is no tissue on the slide) are identified as a white background.

Empty champagne bottles from celebrations of funded grants

Screenshot from Bladerunner, showing incept date for "Pris"

Embedded tick with heart shaped scutum, and words “I’m stuck on you!”

Happy Valentines Day to all who read this!

Figure 2. qPCR data of B. mayonii (blue dots) and B. burgdorferi (red dots) with mean spirochetemia as a bar in SCID mice at 7, 14, 21, and 28 dpi (n=4 per infection group). At 7 dpi, three B. mayonii-infected SCID had undetectable levels of spirochetes in the blood, thus only one dot is present with no mean bar. X-axis is days post-inoculation. Y-axis is copies of flaB per mL of blood. Unpaired t test with Welch’s correction was used to compare means at each timepoint. 7 dpi p=0.0333. 14 dpi p=0.0468. 21 dpi p=0.2782. 28 dpi p=0.3606. * p<0.05, ns, not significant.

Screen shot of Rocky Mountain Laboratories in Hamilton, Montana, from the 1937 film "Green Light"

$Neighbor-joining tree of the ParA-like proteins encoded on cp32 elements. The tree was constructed from all the proteins encoded by the parA-like genes of the cp32s of the sequenced B. burgdorferi sensu lato genomes and those known from strains CA15, Sh-2-82, and Far04. Numbers on the tree branches are bootstrap support values from 1,000 trials. Blue and yellow boxes highlight the different sequence types (tree branches) for the cp32 ParA proteins. These types correlate with episomes named in large red type. The species which harbored each cp32 are indicated by the color of the strain name at the right branch tips (see color key in upper left of figure). The DN127 protein labeled DN127frag in the cp32-12 branch is a ParA fragment from the DN127 cp32-Quad element. All the replicons in the figure carry highly related complements of non-partition cluster genes, except for seven “lp32” plasmids, noted by a pink background in the figure, which encode cp32-like ParA proteins but do not carry erp sequences (these will be described elsewhere [S. Casjens, unpublished data]). Replicon cp26 is an unrelated, universally present circular “plasmid” and is shown only for reference. Bar, 0.05 fractional change.$