🧬 biology

Uropathogenic Escherichia coli remodels LPS to evade Caspase-11-dependent pyroptosis to establish intracellular bacterial communities

Uropathogenic *Escherichia coli* (UPEC) upregulates the phosphotransferase LpxT to modify its lipopolysaccharide (LPS) lipid A, thereby evading Caspase-11 detection and pyroptosis to successfully establish intracellular bacterial communities and cause recurrent urinary tract infections.

Original authors: Yu Pang, Xueping Li, Si Zhang, Lingyan Jiang, Qian Wang, Hongmin Sun, Chen Ji, Jianwei Mu, Qiyue Zhang, Xiao Zhang, Peng Ding, Le Liu, Jiarui Zhou, Jianmin Qian, Yangyang Li, Jingliang Qin, Bin Liu, S

Published 2026-07-31

📖 4 min read☕ Coffee break read

CC BY 4.0

Original authors: Yu Pang, Xueping Li, Si Zhang, Lingyan Jiang, Qian Wang, Hongmin Sun, Chen Ji, Jianwei Mu, Qiyue Zhang, Xiao Zhang, Peng Ding, Le Liu, Jiarui Zhou, Jianmin Qian, Yangyang Li, Jingliang Qin, Bin Liu, Simon Swift, Lu Feng, Lei Wang

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

Imagine your body is a high-tech fortress, and your bladder is the main courtyard. Usually, this courtyard is a harsh, sterile place where intruders are quickly spotted and thrown out. But some sneaky bacteria, specifically a type called Uropathogenic Escherichia coli (UPEC), have learned to break in and hide. Once inside, they don't just sit there; they throw a massive, rapid party, multiplying from a single invader into a crowd of 100,000 neighbors in just a few hours. They build these "bacterial neighborhoods" right inside the cells lining your bladder, creating a fortress within a fortress.

To stop this, your immune system has a special security guard called Caspase-11. This guard patrols the inside of your cells, looking for a specific "uniform" worn by bad bacteria: a molecule called Lipopolysaccharide (LPS). When Caspase-11 spots this uniform, it sounds the alarm, blowing a hole in the infected cell to kill the bacteria and flush them out. This process is called pyroptosis. But here's the mystery: if Caspase-11 is so good at spotting LPS, how does UPEC manage to throw its massive party without getting caught? Scientists have long wondered how these bacteria hide their "uniform" from the immune system's most sensitive sensors.

In this study, researchers from Nankai University and Tianjin University decided to play detective. They wanted to find out how UPEC tricks the Caspase-11 guard. By looking at the bacteria's genetic instructions while they were partying inside mouse bladders, they discovered a clever disguise. The bacteria were turning up the volume on a gene called lpxT. Think of lpxT as a chemical painter. Its job is to add an extra phosphate group—a tiny, negatively charged tag—to the LPS uniform.

The researchers found that when UPEC adds this extra tag, it changes the "texture" of the bacterial surface. It's like the bacteria are wearing a suit that is now covered in static electricity. The immune system's Caspase-11 guard, which usually grabs onto the LPS uniform to trigger an attack, finds it slippery and hard to hold onto. Because the guard can't get a good grip, it doesn't sound the alarm, and the bacteria are left alone to multiply.

The team tested this idea by creating a version of UPEC that couldn't use the lpxT painter. Without this gene, the bacteria couldn't add the extra phosphate tag. When these "naked" bacteria tried to invade mouse bladders, the Caspase-11 guard spotted them immediately. The mice's immune systems kicked into high gear, destroying the infected cells and clearing out the bacteria. The mutant bacteria failed to form their massive neighborhoods and were much less likely to cause recurring infections.

Conversely, when the researchers made the bacteria wear even more of these phosphate tags, the bacteria became even better at hiding. They formed larger, more stable communities and were harder for the immune system to clear. The study confirmed that this phosphate modification directly stops Caspase-11 from activating, which in turn stops the cell from blowing itself up to save the host.

This discovery is a big deal because it explains how UPEC pulls off one of its most dangerous tricks: massive, rapid replication inside human cells. It also suggests that the ability to cause recurring urinary tract infections (UTIs) depends on this specific chemical disguise. The researchers showed that without this lpxT modification, the bacteria struggle to cause the initial infection and are even worse at causing the stubborn, recurring infections that plague millions of people. By understanding exactly how the bacteria hide, scientists might one day be able to design treatments that strip away this disguise, leaving the bacteria exposed for the immune system to destroy.

Technical Summary: Uropathogenic Escherichia coli Remodels LPS to Evade Caspase-11-Dependent Pyroptosis

Problem Statement
Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs). A critical aspect of UPEC pathogenesis is its ability to invade superficial bladder epithelial cells (BECs), escape into the host cytosol, and rapidly replicate clonally to form biofilm-like intracellular bacterial communities (IBCs) containing approximately $10^5$ bacteria. This extensive replication occurs despite the presence of the noncanonical inflammasome, a cytosolic immune surveillance system mediated by Caspase-4 (in humans) and Caspase-11 (in mice). This system detects lipopolysaccharide (LPS) from Gram-negative bacteria, triggering Caspase-4/11 activation, Gasdermin-D (GSDMD) cleavage, and pyroptosis (inflammatory cell death), which eliminates infected cells. While other pathogens like Francisella novicida and Yersinia pestis evade Caspase-11 via lipid A deacylation, they achieve only modest replication or basic survival. The mechanism by which UPEC evades Caspase-11 recognition to support massive cytosolic replication and establish IBCs remains unknown.

Methodology
The study employed a multi-faceted approach combining in vivo mouse models, in vitro cell culture, and molecular biology techniques:

Transcriptional Profiling: RNA sequencing (RNA-seq) was performed on UPEC CFT073 scraped from the bladders of C3H/HeN mice at 6 hours post-infection (p.i.), the peak of initial IBC formation.
Strain Construction: Isogenic strains were generated, including a lpxT deletion mutant ( $\Delta$ lpxT), a complemented strain ( $\Delta$ lpxT+), and an overexpression strain (lpxT+).
In Vivo Infection Models: Mice were infected transurethrally to assess acute infection (bacterial titers at 2, 6, and 24 h p.i., IBC enumeration via confocal microscopy) and chronic/recurrent infection (28-day model). Caspase-11 knockout (Caspase-11 $^{-/-}$ ) mice were used to validate the specific role of this enzyme.
Lipid A Analysis: Thin-layer chromatography (TLC) with $^{32}$ P labeling was used to quantify lipid A modifications (specifically 1-pyrophosphate lipid A) in bacterial strains grown in vitro and in infected bladders.
Cellular Assays: Human BEC line 5637 cells were infected to measure cell death (LDH release, trypan blue staining), pyroptosis markers (GSDMD cleavage via Western blot), and cytokine release (IL-1 $\alpha$ , IL-1 $\beta$ , IL-6 via ELISA).
Interaction Studies: Immunofluorescence and colocalization analysis were used to examine the interaction between LPS and Caspase-4. RNA interference (siRNA) and chemical inhibitors were used to silence or inhibit Caspase-4 and Guanylate-binding protein 1 (GBP1).

Key Contributions and Results

Identification of lpxT Upregulation: Transcriptomic analysis revealed that lpxT, encoding a phosphotransferase that adds a phosphate group to the 1-position of lipid A, is significantly upregulated (13.06-fold) during IBC formation. This upregulation was confirmed via qRT-PCR and GFP reporter assays at 6, 16, and 24 hours p.i.
Role in Virulence and IBC Formation: Deletion of lpxT ( $\Delta$ lpxT) did not affect bacterial invasion into BECs but significantly reduced bacterial colonization in mouse bladders at 24 hours p.i. and drastically reduced the number of IBCs formed at 6 hours p.i. (a 77.3% reduction). The lpxT+ overexpression strain showed increased IBC formation. Furthermore, the $\Delta$ lpxT strain failed to establish chronic cystitis or recurrent UTIs in the 28-day model, indicating lpxT is essential for both initial and subsequent rounds of IBC formation.
Lipid A Modification: TLC analysis confirmed that LpxT is responsible for generating 1-pyrophosphate lipid A. In wild-type (WT) UPEC, approximately 35.83% of lipid A carried this modification, which increased to 57.98% in the lpxT+ strain. In infected mouse bladders, the expression of lpxT was induced, correlating with increased 1-pyrophosphate lipid A levels.
Evasion of Caspase-4/11: The study demonstrated that the lack of lpxT triggers Caspase-4-dependent pyroptosis. In 5637 cells, $\Delta$ $Δ$ lpxT infection caused higher LDH release and cell death compared to WT, which was reversed by Caspase-4 inhibition or silencing. Conversely, lpxT+ infection resulted in lower cell death.
- Mechanism of Evasion: Colocalization studies showed that LpxT-mediated phosphorylation reduces the direct interaction between bacterial LPS and Caspase-4. The colocalization percentage dropped from 21.8% (WT) to 10.7% (lpxT+). This reduction in interaction led to decreased cleavage of GSDMD and reduced release of inflammatory cytokines IL-1 $\alpha$ and IL-1 $\beta$ .
- Specificity: The effect was specific to the noncanonical inflammasome; LpxT did not significantly affect the TLR4 pathway (IL-6 release) or GBP1-mediated LPS release.
Rescue in Knockout Models: In Caspase-11 $^{-/-}$ mice, the virulence defect of the $\Delta$ lpxT strain was completely restored. The bacterial titers and IBC numbers in $\Delta$ lpxT-infected knockout mice were comparable to those in WT-infected mice, confirming that LpxT functions specifically to dampen Caspase-11 activation to facilitate UPEC replication.

Significance
The paper claims that UPEC utilizes a novel mechanism of immune evasion distinct from the known deacylation of lipid A. By upregulating lpxT during infection, UPEC phosphorylates the 1-position of lipid A, creating 1-pyrophosphate lipid A. This modification increases the negative charge of the LPS, thereby hindering its interaction with the CARD domain of Caspase-4/11. This evasion allows UPEC to avoid Caspase-11-dependent pyroptosis, enabling the massive cytosolic replication required to form IBCs and establish recurrent UTIs. The authors identify the lpxT-encoded modification as a critical virulence factor and a potential target for therapeutic intervention against recurrent UPEC infections.

Technical Summary: Uropathogenic Escherichia coli Remodels LPS to Evade Caspase-11-Dependent Pyroptosis

More like this