A few days ago I wrote a blog summarizing what TRP120 does (very politely: “host manipulation”; less politely: “cellular chaos with a smile”), and I compared WT vs. ΔTRP120 mutant… (👀click here). But then your inner scientist (and your inner anxious gremlin) might have asked the obvious question:
If the gene is disrupted… how do you know the mutant isn’t still making TRP120 protein in some sneaky, loophole-y way?
So this post is the story of how I made anti-TRP120 serum to confirm the disruption at the protein level, and then discovered a plot twist: TRP120 is mostly outside the cells… like it pays rent in the culture supernatant. ✨
Why protein-level confirmation matters (a.k.a. DNA is honest, but proteins are the receipts)
Genotypes are great, PCR is comforting, and fluorescent reporters can be very persuasive… but in microbiology, proteins are where the actual drama happens. Even with a clean disruption, the practical question is: Is full-length TRP120 absent?
In my workflow, the ΔTRP120 clone was specifically checked for TRP120 protein expression using western blot with mouse antiserum against rTRP120.
Because if TRP120 is still produced (even partially), it can still mess with host cells and muddy everything downstream.
Step 1… Make recombinant TRP120 (a protein-shaped mugshot) 🧬➡️🧫
To generate a specific antibody, I first needed an antigen that screams “TRP120” loudly and clearly.
The gene encoding full-length Eja TRP120 (EHF_0993) was codon-optimized, synthesized, and cloned… then moved into pET33b(+) to express 6×His-tagged TRP120 (rTRP120) in E. coli BL21 (DE3), followed by affinity purification using cobalt resin.
In other words… I taught bacteria to make the bacterial protein… so I could make mammals produce antibodies… to catch the bacteria doing bacterial things. Science is a circle of life. 🦁🔬
Step 2… Turn rTRP120 into anti-TRP120 serum (immune system, please clock in) 💉🐭
To raise antibodies, mouse antiserum against TRP120 was generated in ten C57BL/6 male mice, using rTRP120 bands cut from SDS-PAGE, homogenized with Quil A adjuvant. The mice were immunized intraperitoneally three times at two-week intervals… and serum was collected 10 days after the last immunization.
And because serum is precious (and because I am emotionally attached to my reagents😂), the anti-rTRP120 serum was stored at −80 °C with 50% glycerol.
This serum became my TRP120 truth detector.
Step 3… “Is TRP120 actually gone?” (Western + IF, no vibes-based conclusions) ✅❌
Once I had anti-TRP120 serum, I used it to check WT vs. ΔTRP120 at the protein level.
On the microscopy side, immunofluorescence with anti-rTRP120 serum showed TRP120 signal in WT-infected cells… but TRP120 was not detected in ΔTRP120-infected DH82 cells, supporting the idea that the mutant truly wasn’t producing detectable TRP120.
Translation… the mutant didn’t just “change its relationship status.” It fully ghosted TRP120’s appearance at the party. 😌
Plot twist… TRP120 is an extrovert protein and lives outside the cells 🌊🧼
Here’s the part that made me sit up like a raccoon hearing a trash can open at 3 a.m.:
When I looked at where TRP120 ends up, most of it was in the culture supernatant.
Using dot-immunoblotting (non-denatured protein) and western blotting (denatured protein), TRP120 was detected in WT supernatant but not ΔTRP120, and quantitation showed roughly ~1.6 ng/µL secreted versus ~0.12 ng/µL intracellular… meaning >90% of TRP120 was released into the culture medium.
So TRP120 isn’t just an intracellular effector doing quiet sabotage… it’s more like a bacterial influencer dropping content into the extracellular space. 📣
“But is that secretion… or just cellular chaos?” (reasonable suspicion is healthy) 🤨
The cautious part of my brain immediately asked: Is TRP120 outside because it’s secreted, or because cells are lysing and spilling everything?
To address that, the supernatant prep included a high-speed centrifugation step (15,000 × g, 10 min, 4°C) to remove cellular debris or released bacteria before analysis.
Also, uninfected DH82 supernatant served as a negative control in the dot blot setup.
So the workflow was designed to focus on extracellular TRP120… not “oops, everything exploded.”
Why would Eja shove TRP120 outside? My evidence-based SPECULATIONS 😈🧠
TRP120’s “outside-the-cell” lifestyle actually makes a lot of sense when you zoom out and look at what TRP120 is known to do in Ehrlichia biology more broadly.
A major theme in the literature is that TRP120 is a moonlighting effector… it can behave like a ligand mimic, a nucleomodulin, and a ubiquitin-pathway manipulator, all in one protein🙂. If you’re a bacterium trying to reprogram a host cell without being invited, having an effector that can touch multiple control panels is basically cheating… lovingly… scientifically… aggressively.
One strong idea is surface and receptor-facing signaling. TRP120 has been described as engaging host signaling by acting as a ligand mimetic, including triggering pathways like Wnt via interaction with host receptors (Frizzled family) in E. chaffeensis work. If you want to poke receptors, being extracellular (or at least host-accessible beyond your little bacterial bubble) is a tactical advantage… you can influence signaling early, broadly, and maybe even in neighboring cells.
Another angle is neighborhood control… not just the infected cell, but also the nearby immune ecosystem. Secreted or externally exposed effectors can reshape how immune cells behave in the local area, smoothing the path for infection and persistence. In my own dataset context, culture supernatant differences (WT vs. ΔTRP120) correlated with changes in monocyte marker readouts, consistent with TRP120 contributing to extracellular immune modulation.
Then there’s the whole “TRP120 does not know how to be normal” category. 😂TRP120 has been reported to inter ost proteins and influence chromatin and transcriptional programs in infected cells. It also has work supporting a nucleomodulin-like role, including DNA binding and nuclear effects. Those functions don’t require secretion per se… but they do fit the bigger pattern: TRP120 seems built to travel, interface, and meddle.
And finally… 😝the ubiquitin storyline. TRP120 has been described as “moonlighting” with HECT E3 ubiquitin ligase–related activity or interactions, with studies showing TRP120 ubiquitination and links to host HECT E3 ligases like Nedd4L, and broader effects on infection biology when these interactions are perturbed. A protein that can tap into ubiquitin networks is basically holding a master key to host regulation… and if Eja can deploy that influence efficiently, secretion or external enrichment could help ensure TRP120 is available where it needs to be, when it needs to be.
So my personal synthesis is this… TRP120 outside the cell is NOT random. It looks like strategy: broadcast influence, touch receptors, shape immune behavior, keep the host environment permissive, and buy time for the bacterium’s lifecycle… all while making the scientist running the experiment whisper, “why are you like this?” into the incubator. 😭
The goodbye-for-now TRP120 epilogue 🔬
Okay… real talk… I should probably cite a bunch of papers here as references… but sorry, I’m not doing that right now hahahah 😅 (If you’re a TRP120 expert reading this, please don’t throw pipette tips at your screen.)
And also… this is probably my last TRP120 update from my side for a while. I’m packing up my brain and moving on to my next research journey… a totally different topic, a totally new rabbit hole… but I already know I’ll be back. I’ll visit TRP120 again someday… like checking on a chaotic ex who somehow still has your Netflix password. 🙃
To all the TRP120 researchers out there… I just want to say: even if we’ve never met, I weirdly feel like we’re connected. We’ve all been orbiting the same stubborn little molecular troublemaker, watching it slip out of cells like it’s late for an appointment, arguing with blots at midnight, and trying to translate “what is it doing???” into something that looks like science😅.
And even if I didn’t cite you here (or maybe even in my paper) the way I should have… please know this… I always felt your presence in the background of my work. I read your papers, borrowed your mental frameworks, learned from your controls, and let your findings shape what questions I dared to ask. I might not have written your names on this page today, but your ideas still traveled with me at the bench… quietly, consistently, and honestly.
So thank you… truly. For pushing the TRP120 story forward, for leaving breadcrumbs, for making this molecule feel a little less mysterious and a little more human-understandable. If TRP120 is a shared language, then we’ve all been having the same conversation… just in different labs, different years, different time zones.
I’m stepping away now… but I’m not gone. I’ll come back. And when I do… I’ll be the same person, still cheering for every clean band, every elegant mechanism, every “WAIT THAT’S ACTUALLY SO COOL” moment that this little bacterium and its molecules keep forcing out of us. 🧡🧪
