Rui Bao

The Pseudomonas aeruginosa type III secretion system effector ExoU is a potent phospholipase A2 that causes rapid host-cell injury, but how it engages intracellular host networks during infection remains incompletely understood. Here, using an endogenously Flag-tagged infection model in male-derived A549 cells, we identified MYH6 and DDX3Y as ExoU-associated host factors. Knockdown of either protein reduced PA14-induced cytotoxicity, indicating that both contribute to the cellular response to ExoU-positive P. aeruginosa. An in vitro pull-down assay using purified proteins further supported a direct but relatively weak association between ExoU-S142A and DDX3Y. Quantitative phosphoproteomic analysis revealed extensive host phosphorylation changes associated with ExoU catalytic activity and identified multiple phosphorylation sites on DDX3Y. Phosphorylation at S506 was detected following PA14 infection but not following exoU-S142A infection. Phospho-deficient DDX3Y mutants at S101, S141, and S506 showed increased association with ExoU, whereas the combined S101A/S141A/S506A mutant further enhanced this association and reduced infection-induced cytotoxicity. Conversely, the phosphomimetic S506E mutant showed reduced association with ExoU and restored cytotoxicity relative to S506A. RNA sequencing and DDX3Y RIP analyses further showed that ExoU catalytic activity was associated with a broader inflammatory transcriptional response and with changes in the DDX3Y-bound transcriptome, including altered association with IL6ST and NFKBIZ transcripts. Together, these findings identify MYH6 and DDX3Y as distinct ExoU-associated host factors and support a model in which ExoU catalytic activity is linked to multisite regulation of DDX3Y, altered ExoU-DDX3Y association, and changes in DDX3Y-associated RNAs. These results extend the current understanding of ExoU-mediated cytotoxicity beyond direct membrane damage.

During infection, Klebsiella pneumoniae must withstand host-derived oxidative stress not only by detoxifying reactive oxygen species (ROS) but also by repairing oxidative damage to cellular physiology. How these detoxification and stress-adaptive responses are coordinated remains unclear. Here, we identify a plasmid-encoded tacAT locus as an OxyR-responsive regulatory module that supports peroxide tolerance and in vivo fitness in K. pneumoniae . Guided by the conservation of the OxyR DNA-binding domain and its consensus binding motif, we surveyed promoter-proximal regions of the K. pneumoniae CRK3022 chromosome and plasmids for candidate OxyR-responsive loci. This analysis identified tacAT , a putative type II toxin–antitoxin (TA) module, as the only plasmid-associated candidate. OxyR directly bound the tacAT promoter and contributed to tacAT induction during hydrogen peroxide (H _o O ₂ ) stress. The TacAT complex exhibited DNA-binding and transcriptional regulatory activity, promoting the expression of genes linked to protein quality control and membrane/envelope homeostasis, including clpB, htpG, cadC, bhsA , and marA . Deletion of tacAT compromised survival under lethal peroxide challenge and attenuated bacterial dissemination, tissue pathology, and inflammatory responses in a murine bacteremia model. These findings define an OxyR–TacAT regulatory branch that connects peroxide sensing with stress-adaptive gene expression, revealing how a TA-associated module can be integrated into stress-adaptive programs during host-associated oxidative stress. Highlight: Promoter motif screening identifies tacAT as a candidate OxyR-responsive TA-associated module OxyR directly binds the tacAT promoter and contributes to peroxide-induced tacAT expression TacAT promotes stress-response genes linked to protein quality control and envelope homeostasis TacAT supports oxidative stress survival and in vivo fitness of Klebsiella pneumoniae Graphical Proposed model of the OxyR-TacAT regulatory branch in K. pneumoniae . During infection, K. pneumoniae encounters host-derived oxidative stress. Oxidized OxyR activates classical antioxidant responses and contributes to tacAT induction by binding an OxyR-like motif in the tacAT promoter region. The induced TacAT complex binds OP1 within its own promoter and promotes the expression of stress-response genes, including clpB and htpG , which are associated with protein quality control, and cadC , bhsA , and marA , which are associated with membrane/envelope homeostasis. This regulatory branch supports bacterial survival under severe oxidative stress and contributes to in vivo fitness during systemic infection. Dashed arrows indicate proposed or indirect regulatory effects.

Two-component systems (TCSs) in Pseudomonas aeruginosa are essential for sensing and responding to diverse environmental cues, including those associated with ion homeostasis and virulence within the host. However, how specific TCSs discriminate between chemically similar metal signals to fine-tune adaptation remains unclear. Here, we identify the TCS ColRS, conserved across the Pseudomonas genus, as a critical sensor that distinguishes zinc (Zn ²⁺ ) and copper (Cu ²⁺ ) to drive divergent survival strategies. First, we establish that the response regulator ColR directly binds the promoters of lipid A modification genes ( arnB , eptA ), the metal responsive regulator czcR , and the alginate and motility regulator Z ( amrZ ), genetically linking metal sensing to resistance and virulence circuitry. Phenotypically, ColRS translates these inputs into context-dependent states: enhancing polymyxin tolerance and suppressing motility under copper stress, while sustaining type III secretion system (T3SS) virulence and motility under zinc stress. Mechanistically, this discrimination relies on the sensor kinase ColS, which utilizes a non-canonical E96-H105 interface as a specificity filter to recognize Cu ²⁺ (Kd ≈ 1.11 μM) and distinguish it from Zn ²⁺ (Kd ≈ 19.8 μM). Global proteomics confirms this bifurcation, revealing that Zn ²⁺ triggers focused metabolic tuning, whereas Cu ²⁺ induces broad envelope and motility rewiring. Collectively, our findings reveal ColRS as a pivotal molecular hub that decodes the duality of host metal immunity to optimize pathoadaptation.

Two-component systems convert environmental signals into transcriptional responses, but how one response-regulator phosphorylation state produces different outputs at different promoters remains unclear. Here we show that promoter architecture determines how the ParR response regulator in Pseudomonas aeruginosa interprets phosphorylation. Phenotypic, transcriptomic, biochemical and promoter-engineering analyses showed that phosphorylation of the conserved ParR receiver residue Asp57 lowered DNA-occupancy thresholds across target promoters. Individual promoters, however, converted this shared increase in binding into activation, repression or phosphorylation-dependent sign switching. Chromosomally D57 ParR variants reproduced these behaviours at endogenous loci and altered antibiotic susceptibility, biofilm formation and virulence-associated phenotypes. Mapping and engineering of representative promoters identified a two-tier cis-regulatory code: half-site sequence compatibility determines productive ParR engagement, whereas spacer length determines regulatory sign and magnitude. Spacer swaps were sufficient to reprogram promoter logic between regulatory modes. These findings separate regulator state from promoter decoding and show how bacterial promoter architecture can diversify the outputs of a shared phosphorylation signal. Importance: Pseudomonas aeruginosa is an opportunistic pathogen that survives antimicrobial treatment and adapts to diverse host environments by rapidly changing gene expression. How a single sensory pathway generates different, and sometimes opposing, responses across many genes has remained unclear. We show that the regulatory protein ParR does not impose one fixed transcriptional program. Instead, the DNA architecture of each target promoter determines whether the same phosphorylation signal activates, represses, or reverses gene expression. The sequence and spacing of ParR recognition sites form a compact regulatory code, and changing spacer length alone can reprogram promoter behavior. Different ParR states were also associated with changes in antibiotic susceptibility, biofilm formation, and virulence-associated phenotypes. Our findings explain how one bacterial signaling pathway can generate diverse physiological outcomes without additional regulators and provide a framework for understanding regulatory flexibility in Pseudomonas aeruginosa and for engineering genetic circuits with multiple outputs. Graphical

Two-component systems convert environmental signals into transcriptional responses, but how one response-regulator phosphorylation state produces different outputs at different promoters remains unclear. Here we show that promoter architecture determines how the ParR response regulator in Pseudomonas aeruginosa interprets phosphorylation. Phenotypic, transcriptomic, biochemical and promoter-engineering analyses showed that phosphorylation of the conserved ParR receiver residue Asp57 lowered DNA-occupancy thresholds across target promoters. Individual promoters, however, converted this shared increase in binding into activation, repression or phosphorylation-dependent sign switching. Chromosomally D57 ParR variants reproduced these behaviours at endogenous loci and altered antibiotic susceptibility, biofilm formation and virulence-associated phenotypes. Mapping and engineering of representative promoters identified a two-tier cis-regulatory code: half-site sequence compatibility determines productive ParR engagement, whereas spacer length determines regulatory sign and magnitude. Spacer swaps were sufficient to reprogram promoter logic between regulatory modes. These findings separate regulator state from promoter decoding and show how bacterial promoter architecture can diversify the outputs of a shared phosphorylation signal. Graphical Highlights: ParR coordinates antibiotic resistance, biofilm formation and virulence-linked phenotypes in P. aeruginosa . Promoters convert ParR phosphorylation into activation, repression or sign switching. Half-site compatibility and spacer length form a cis-regulatory code for ParR output.

In Pseudomonas aeruginosa , the sigma factor AlgU functions as a critical stress-responsive regulator governing essential physiological processes. This study assessed algU -dependent phenotypes through targeted gene knockout, including effects on alginate secretion and host cell invasion. Furthermore, an innovative fusion protein strategy addressed complex preparation challenges, enabling the determination of a high-resolution (2.101 Å) structure of the AlgU-MucA ^cyto complex and revealing an exceptionally low backbone deviation of merely 0.01 Å relative to the reported 6IN7 complex. Structural analysis revealed an extensive mutation-resistant interaction network at the binding interface, stabilized primarily by salt bridges, hydrogen bonds, and hydrophobic interactions. Subsequent mutational validation confirmed that triple or sextuple alanine substitutions at critical interaction sites maintained complex binding integrity while significantly compromising thermal stability, indicating that although dispensable for binding, these residues are indispensable for complex stability. Crucially, pull-down assays identified SspB as the essential adaptor bridging the AlgU-MucA ^cyto complex to the ClpX/ClpP protease; AlphaFold3 modeling and quantitative BLI measurements reveal that SspB engages AlgU via its N-terminal domain and binds ClpX through its disordered C-terminus, enabling substrate delivery and proteolysis-coupled AlgU release. Collectively, these findings elucidate a dual-safeguard regulatory mechanism: inherent mutation resistance at the binding interface prevents aberrant AlgU activation by common clinical mucA mutations, while SspB-mediated proteolysis ensures stimulus-responsive AlgU liberation. This evolutionary adaptation confers resistance against detrimental AlgU overactivation in P. aeruginosa and provides novel therapeutic targets. IMPORTANCE: Deletion of the algU gene affected the production of pyoverdine, pyocyanin, and alginate in Pseudomonas aeruginosa , as well as its cell invasiveness. By fusing AlgU and MucA ^cyto , we were able to obtain an AlgU-MucA ^cyto protein complex with both high yield and purity, and successfully obtained a crystal structure of the AlgU-MucA ^cyto complex. Structural analysis revealed an extensive and robust network of salt bridges, hydrogen bonds, and charge interactions between the two proteins. Multi-site alanine substitutions (up to sextuple mutations) in interfacial charged clusters of the AlgU-MucA ^cyto complex fail to disrupt binding but severely impair thermal stability, revealing that these residues are binding-redundant yet stability-essential. Structural modeling and BLI measurements identify SspB as a dual-interface adaptor: its N-terminal domain recognizes the preassembled AlgU-MucA ^cyto complex (via the AlgU hairpin surface), whereas its flexible C-terminal tail engages ClpX, thereby bridging AlgU-MucA ^cyto to ClpXP and positioning MucA ^cyto for proteolysis-coupled AlgU release.

Ubiquitous in biological systems, caseinolytic protease P (ClpP) represents a premier target for antimicrobial and antitumor therapies due to its central role in proteostasis. In Pseudomonas aeruginosa, PaClpP1 and PaClpP2 display distinct oligomeric states and enzymatic properties, yet the molecular basis underlying this divergence remains elusive. Here, by combining biochemical characterization, structural analysis, and molecular dynamics simulations, we identify Arg140 in PaClpP2 as the critical structural determinant governing its autoinhibited state. Mutation of this residue to alanine (R140A) relieves steric constraints, promoting the extension of the handle domain and conferring a robust proteolytic activity. Enzymatic assays further reveal that bortezomib and Z-Ile-Leu (ZIL) exert opposing, conformation-dependent effects on PaClpP. Molecular docking suggests that steric hindrance by R140 prevents canonical binding and induces a conformational switch, thereby activating the enzyme. Collectively, our findings establish the handle domain as a pivotal conformational switch, offering a structural foundation for the design of isoform-specific modulators.

Helicobacter pylori is a significant pathogen associated with a range of diseases, most notably gastritis and gastric cancer, making it a critical target for medical intervention. β-lactam antibiotics, known for their potent bactericidal properties, play a central role in clinical regimens aimed at eradicating H pylori. However, the increasing resistance of H pylori to β-lactams, compounded by the rise of multidrug-resistant strains, presents a major challenge to effective treatment. The molecular mechanisms underlying β-lactam resistance in H pylori are multifactorial, including (1) mutations in penicillin-binding proteins, (2) production of β-lactamases, (3) alterations in outer membrane permeability, and (4) activation of efflux pumps. These mechanisms not only reduce the efficacy of β-lactam antibiotics but also contribute to the emergence of multidrug-resistant H pylori strains. Understanding these molecular pathways is essential for the development of novel therapeutic strategies to overcome resistance and enhance the effectiveness of treatment for H pylori infections.