Carbon source competition within the wound microenvironment can significantly influence infection progression.

It is becoming increasingly apparent that commensal skin bacteria have an important role in wound healing and infection progression. However, the precise mechanisms underpinning many of these probiotic interactions remain to be fully uncovered. In this work, we demonstrate that the common skin commensal Cutibacterium acnes can limit the pathogenicity of the prevalent wound pathogen Pseudomonas aeruginosa in vivo. We show that this impact on pathogenicity is independent of any effect on growth, but occurs through a significant downregulation of the Type Three Secretion System (T3SS), the primary toxin secretion system utilised by P. aeruginosa in eukaryotic infection. We also show a downregulation in glucose acquisition systems, a known regulator of the T3SS, suggesting that glucose availability in a wound can influence infection progression. C. acnes is well known as a glucose fermenting organism, and we demonstrate that topically supplementing a wound with glucose reverses the probiotic effects of C. acnes. This suggests that introducing carbon source competition within the wound microenvironment may be an effective way to prevent or limit wound infection.

Pseudomonas aeruginosa breaches respiratory epithelia through goblet cell invasion in a microtissue model.

Pseudomonas aeruginosa, a leading cause of severe hospital-acquired pneumonia, causes infections with up to 50% mortality rates in mechanically ventilated patients. Despite some knowledge of virulence factors involved, it remains unclear how P. aeruginosa disseminates on mucosal surfaces and invades the tissue barrier. Using infection of human respiratory epithelium organoids, here we observed that P. aeruginosa colonization of apical surfaces is promoted by cyclic di-GMP-dependent asymmetric division. Infection with mutant strains revealed that Type 6 Secretion System activities promote preferential invasion of goblet cells. Type 3 Secretion System activity by intracellular bacteria induced goblet cell death and expulsion, leading to epithelial rupture which increased bacterial translocation and dissemination to the basolateral epithelium. These findings show that under physiological conditions, P. aeruginosa uses coordinated activity of a specific combination of virulence factors and behaviours to invade goblet cells and breach the epithelial barrier from within, revealing mechanistic insight into lung infection dynamics.

Quantifying Yersinia pseudotuberculosis Type III Secretion System Activity Following Iron Starvation and Anaerobic Growth.

A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or immunomodulatory effector proteins into host cells. The T3SS is a target for antimicrobial discovery campaigns since it is accessible extracellularly and largely absent from non-pathogenic bacteria. Recent studies demonstrated that the T3SS of Yersinia and Salmonella are regulated by factors responsive to iron and oxygen, which are important niche-specific signals encountered during mammalian infection. Described here is a method for iron starvation of Yersinia pseudotuberculosis, with subsequent optional supplementation of inorganic iron. To assess the impact of oxygen availability, this iron starvation process is demonstrated under both aerobic and anaerobic conditions. Finally, incubating the cultures at the mammalian host temperature of 37 °C induces T3SS expression and allows quantification of Yersinia T3SS activity by visualizing effector proteins released into the supernatant. The steps detailed here offer an advantage over the use of iron chelators in the absence of iron starvation, which is insufficient for inducing robust iron starvation, presumably due to efficient Yersinia iron uptake and scavenging systems. Likewise, acid-washing laboratory glassware is detailed to ensure the removal of residual iron, which is essential for inducing robust iron starvation. Additionally, using a chelating agent is described to remove residual iron from media, and culturing the bacteria for several generations in the absence of iron to deplete bacterial iron stores. By incorporating standard protocols of trichloroacetic acid-induced protein precipitation, SDS-PAGE, and silver staining, this procedure demonstrates accessible ways to measure T3SS activity. While this procedure is optimized for Y. pseudotuberculosis, it offers a framework for studies in pathogens with similar robust iron uptake systems. In the age of antibiotic resistance, these methods can be expanded to assess the efficacy of antimicrobial compounds targeting the T3SS under host-relevant conditions.

Secretion of functional interferon by the type 3 secretion system of enteropathogenic Escherichia coli.

BACKGROUND: Type I interferons (IFN-I)-a group of cytokines with immunomodulatory, antiproliferative, and antiviral properties-are widely used as therapeutics for various cancers and viral diseases. Since IFNs are proteins, they are highly susceptible to degradation by proteases and by hydrolysis in the strong acid environment of the stomach, and they are therefore administered parenterally. In this study, we examined whether the intestinal bacterium, enteropathogenic Escherichia coli (EPEC), can be exploited for oral delivery of IFN-Is. EPEC survives the harsh conditions of the stomach and, upon reaching the small intestine, expresses a type III secretion system (T3SS) that is used to translocate effector proteins across the bacterial envelope into the eukaryotic host cells. RESULTS: In this study, we developed an attenuated EPEC strain that cannot colonize the host but can secrete functional human IFNα2 variant through the T3SS. We found that this bacteria-secreted IFN exhibited antiproliferative and antiviral activities similar to commercially available IFN. CONCLUSION: These findings present a potential novel approach for the oral delivery of IFN via secreting bacteria.

Burkholderia pseudomallei BipD modulates host mitophagy to evade killing.

Mitophagy is critical for mitochondrial quality control and function to clear damaged mitochondria. Here, we found that Burkholderia pseudomallei maneuvered host mitophagy for its intracellular survival through the type III secretion system needle tip protein BipD. We identified BipD, interacting with BTB-containing proteins KLHL9 and KLHL13 by binding to the Back and Kelch domains, recruited NEDD8 family RING E3 ligase CUL3 in response to B. pseudomallei infection. Although evidently not involved in regulation of infectious diseases, KLHL9/KLHL13/CUL3 E3 ligase complex was essential for BipD-dependent ubiquitination of mitochondria in mouse macrophages. Mechanistically, we discovered the inner mitochondrial membrane IMMT via host ubiquitome profiling as a substrate of KLHL9/KLHL13/CUL3 complex. Notably, K63-linked ubiquitination of IMMT K211 was required for initiating host mitophagy, thereby reducing mitochondrial ROS production. Here, we show a unique mechanism used by bacterial pathogens that hijacks host mitophagy for their survival.

Long-chain unsaturated fatty acids sensor controlling the type III/VI secretion system is essential for Edwardsiella piscicida infection.

Edwardsiella piscicida is an acute marine pathogen that causes severe damage to the aquaculture industry worldwide. The pathogenesis of E. piscicida is dependent mainly on the type III secretion system (T3SS) and type VI secretion system (T6SS), both of which are critically regulated by EsrB and EsrC. In this study, we revealed that fatty acids influence T3SS expression. Unsaturated fatty acids (UFAs), but not saturated fatty acids (SFAs), directly interact with EsrC, which abolishes the function of EsrC and results in the turn-off of T3/T6SS. Moreover, during the in vivo colonization of E. piscicida, host fatty acids were observed to be transported into E. piscicida through FadL and to modulate the expression of T3/T6SS. Furthermore, the esrCR38G mutant blocked the interaction between EsrC and UFAs, leading to dramatic growth defects in DMEM and impaired colonization in HeLa cells and zebrafish. In conclusion, this study revealed that the interaction between UFAs and EsrC to turn off T3/T6SS expression is essential for E. piscicida infection.

Metabolism of L-arabinose converges with virulence regulation to promote enteric pathogen fitness.

Virulence and metabolism are often interlinked to control the expression of essential colonisation factors in response to host-associated signals. Here, we identified an uncharacterised transporter of the dietary monosaccharide ʟ-arabinose that is widely encoded by the zoonotic pathogen enterohaemorrhagic Escherichia coli (EHEC), required for full competitive fitness in the mouse gut and highly expressed during human infection. Discovery of this transporter suggested that EHEC strains have an enhanced ability to scavenge ʟ-arabinose and therefore prompted us to investigate the impact of this nutrient on pathogenesis. Accordingly, we discovered that ʟ-arabinose enhances expression of the EHEC type 3 secretion system, increasing its ability to colonise host cells, and that the underlying mechanism is dependent on products of its catabolism rather than the sensing of ʟ-arabinose as a signal. Furthermore, using the murine pathogen Citrobacter rodentium, we show that ʟ-arabinose metabolism provides a fitness benefit during infection via virulence factor regulation, as opposed to supporting pathogen growth. Finally, we show that this mechanism is not restricted to ʟ-arabinose and extends to other pentose sugars with a similar metabolic fate. This work highlights the importance integrating central metabolism with virulence regulation in order to maximise competitive fitness of enteric pathogens within the host-niche.

Fur-mediated regulation of hydrogen sulfide synthesis, stress response, and virulence in Edwardsiella piscicida.

The production of endogenous hydrogen sulfide (H2S) is an important phenotype of bacteria. H2S plays an important role in bacterial resistance to ROS and antibiotics, which significantly contributes to bacterial pathogenicity. Edwardsiella piscicida, the Gram-negative pathogen causing fish edwardsiellosis, has been documented to produce hydrogen sulfide. In the study, we revealed that Ferric uptake regulator (Fur) controlled H2S synthesis by activating the expression of phsABC operon. Besides, Fur participated in the bacterial defense against ROS and cationic antimicrobial peptides and modulated T3SS expression. Furthermore, the disruption of fur exhibited a significant in vivo colonization defect. Collectively, our study demonstrated the regulation of Fur in H2S synthesis, stress response, and virulence, providing a new perspective for better understanding the pathogenesis of Edwardsiella.

MolPhase, an advanced prediction algorithm for protein phase separation.

We introduce MolPhase, an advanced algorithm for predicting protein phase separation (PS) behavior that improves accuracy and reliability by utilizing diverse physicochemical features and extensive experimental datasets. MolPhase applies a user-friendly interface to compare distinct biophysical features side-by-side along protein sequences. By additional comparison with structural predictions, MolPhase enables efficient predictions of new phase-separating proteins and guides hypothesis generation and experimental design. Key contributing factors underlying MolPhase include electrostatic pi-interactions, disorder, and prion-like domains. As an example, MolPhase finds that phytobacterial type III effectors (T3Es) are highly prone to homotypic PS, which was experimentally validated in vitro biochemically and in vivo in plants, mimicking their injection and accumulation in the host during microbial infection. The physicochemical characteristics of T3Es dictate their patterns of association for multivalent interactions, influencing the material properties of phase-separating droplets based on the surrounding microenvironment in vivo or in vitro. Robust integration of MolPhase's effective prediction and experimental validation exhibit the potential to evaluate and explore how biomolecule PS functions in biological systems.

Direct Salmonella injection into enteroid cells allows the study of host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.

PurA is the main target of aurodox, a type III secretion system inhibitor.

Anti-microbial resistance (AMR) is one of the greatest threats to global health. The continual battle between the emergence of AMR and the development of drugs will be extremely difficult to stop as long as traditional anti-biotic approaches are taken. In order to overcome this impasse, we here focused on the type III secretion system (T3SS), which is highly conserved in many Gram-negative pathogenic bacteria. The T3SS is known to be indispensable in establishing disease processes but not essential for pathogen survival. Therefore, T3SS inhibitors may be innovative anti-infective agents that could dramatically reduce the evolutionary selective pressure on strains resistant to treatment. Based on this concept, we previously identified a polyketide natural product, aurodox (AD), as a specific T3SS inhibitor using our original screening system. However, despite its promise as a unique anti-infective drug of AD, the molecular target of AD has remained unclear. In this paper, using an innovative chemistry and genetic biology-based approach, we show that AD binds to adenylosuccinate synthase (PurA), which suppresses the production of the secreted proteins from T3SS, resulting in the expression of bacterial virulence both in vitro and in vivo experiments. Our findings illuminate the potential of PurA as a target of anti-infective drugs and vaccination and could open a avenue for application of PurA in the regulation of T3SS.

The read-through transcription-mediated autoactivation circuit for virulence regulator expression drives robust type III secretion system 2 expression in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis in humans worldwide. The major virulence factor responsible for the enteropathogenicity of this pathogen is type III secretion system 2 (T3SS2), which is encoded on the 80-kb V. parahaemolyticus pathogenicity island (Vp-PAI), the gene expression of which is governed by the OmpR-family transcriptional regulator VtrB. Here, we found a positive autoregulatory feature of vtrB transcription, which is often observed with transcriptional regulators of bacteria, but the regulation was not canonically dependent on its own promoter. Instead, this autoactivation was induced by heterogeneous transcripts derived from the VtrB-regulated operon upstream of vtrB. VtrB-activated transcription overcame the intrinsic terminator downstream of the operon, resulting in transcription read-through with read-in transcription of the vtrB gene and thus completing the autoregulatory loop for vtrB gene expression. The dampening of read-through transcription with an exogenous strong terminator reduced vtrB gene expression. Furthermore, a V. parahaemolyticus mutant with defects in the vtrB autoregulatory loop also showed compromises in T3SS2 expression and T3SS2-dependent cytotoxicity in vitro and enterotoxicity in vivo, indicating that this autoregulatory loop is essential for sustained vtrB activation and the consequent robust expression of T3SS2 genes for pathogenicity. Taken together, these findings demonstrate that the regulatory loop for vtrB gene expression based on read-through transcription from the upstream operon is a crucial pathway in T3SS2 gene regulatory network to ensure T3SS2-mediated virulence of V. parahaemolyticus.

Calcium Induces the Cleavage of NopA and Regulates the Expression of Nodulation Genes and Secretion of T3SS Effectors in Sinorhizobium fredii NGR234.

The type III secretion system (T3SS) is a key factor for the symbiosis between rhizobia and legumes. In this study, we investigated the effect of calcium on the expression and secretion of T3SS effectors (T3Es) in Sinorhizobium fredii NGR234, a broad host range rhizobial strain. We performed RNA-Seq analysis of NGR234 grown in the presence of apigenin, calcium, and apigenin plus calcium and compared it with NGR234 grown in the absence of calcium and apigenin. Calcium treatment resulted in a differential expression of 65 genes, most of which are involved in the transport or metabolism of amino acids and carbohydrates. Calcium had a pronounced effect on the transcription of a gene (NGR_b22780) that encodes a putative transmembrane protein, exhibiting a 17-fold change when compared to NGR234 cells grown in the absence of calcium. Calcium upregulated the expression of several sugar transporters, permeases, aminotransferases, and oxidoreductases. Interestingly, calcium downregulated the expression of nodABC, genes that are required for the synthesis of nod factors. A gene encoding a putative outer membrane protein (OmpW) implicated in antibiotic resistance and membrane integrity was also repressed by calcium. We also observed that calcium reduced the production of nodulation outer proteins (T3Es), especially NopA, the main subunit of the T3SS pilus. Additionally, calcium mediated the cleavage of NopA into two smaller isoforms, which might affect the secretion of other T3Es and the symbiotic establishment. Our findings suggest that calcium regulates the T3SS at a post-transcriptional level and provides new insights into the role of calcium in rhizobia-legume interactions.

Type III secretion system effector YfiD inhibits the activation of host poly(ADP-ribose) polymerase-1 to promote bacterial infection.

Modulation of cell death is a powerful strategy employed by pathogenic bacteria to evade host immune clearance and occupy profitable replication niches during infection. Intracellular pathogens employ the type III secretion system (T3SS) to deliver effectors, which interfere with regulated cell death pathways to evade immune defenses. Here, we reveal that poly(ADP-ribose) polymerase-1 (PARP1)-dependent cell death restrains Edwardsiella piscicida's proliferation in mouse monocyte macrophages J774A.1, of which PARP1 activation results in the accumulation of poly(ADP-ribose) (PAR) and enhanced inflammatory response. Moreover, E. piscicida, an important intracellular pathogen, leverages a T3SS effector YfiD to impair PARP1's activity and inhibit PAR accumulation. Once translocated into the host nucleus, YfiD binds to the ADP-ribosyl transferase (ART) domain of PARP1 to suppress its PARylation ability as the pharmacological inhibitor of PARP1 behaves. Furthermore, the interaction between YfiD and ART mainly relies on the complete unfolding of the helical domain, which releases the inhibitory effect on ART. In addition, YfiD impairs the inflammatory response and cell death in macrophages and promotes in vivo colonization and virulence of E. piscicida. Collectively, our results establish the functional mechanism of YfiD as a potential PARP1 inhibitor and provide more insights into host defense against bacterial infection.

Identification of a new export signal that targets early subunits to the flagellar type III secretion export machinery.

Type III secretion systems (T3SSs) are essential for motility and virulence in many bacterial pathogens. Proteins destined for the flagellar T3SS contain at least two export signals in their N-terminal D0 domain. Here, we describe a third carboxy (C)-terminal signal in early flagellar subunits that facilitates subunit targeting to the export machinery. Mutational analysis identified critical residues within the flagellar hook subunit C-terminal export signal. The flagellar ATPase and cytoplasmic ring components were not required for this targeting, indicating that core export machinery components facilitate substrate targeting via the C-terminal export signal. More broadly, these results demonstrate that multiple distinct export signals within type III secretion substrates facilitate distinct export events at the T3SS export machinery. Our data establish key events in the export mechanism of type III secretion systems: targeting of subunits to and their sequential interactions with key components of the export machinery. IMPORTANCE: Many bacterial pathogens utilize T3SS to inject virulence proteins (effectors) into host cells or to assemble flagella on the bacterial cell surface. Bacterial flagella present a paradigm for how cells build and operate complex cell-surface "nanomachines." Efficient subunit targeting from the bacterial cytosol to type III secretion systems is essential for rapid assembly and secretion by T3SSs. Subunits are thought to dock at the export machinery before being unfolded and translocated into the export channel. However, little is known about how subunits dock at the export machinery and the events that occur post docking. Here, we identified a new export signal within the C-termini of subunits that is essential for targeting of subunits to the type III export machinery. We show that this new export signal and previously identified export signals are recognized separately and sequentially, revealing a pathway for subunit transit through the type III export machinery in which sequential recognition events carry out different roles at major steps in the export pathway.

Structural insights into peptidoglycan glycosidase EtgA binding to the inner rod protein EscI of the type III secretion system via a designed EscI-EtgA fusion protein.

Bacteria express lytic enzymes such as glycosidases, which have potentially self-destructive peptidoglycan (PG)-degrading activity and, therefore, require careful regulation in bacteria. The PG glycosidase EtgA is regulated by localization to the assembling type III secretion system (T3SS), generating a hole in the PG layer for the T3SS to reach the outer membrane. The EtgA localization was found to be mediated via EtgA interacting with the T3SS inner rod protein EscI. To gain structural insights into the EtgA recognition of EscI, we determined the 2.01 Å resolution structure of an EscI (51-87)-linker-EtgA fusion protein designed based on AlphaFold2 predictions. The structure revealed EscI residues 72-87 forming an α-helix interacting with the backside of EtgA, distant from the active site. EscI residues 56-71 also were found to interact with EtgA, with these residues stretching across the EtgA surface. The ability of the EscI to interact with EtgA was also probed using an EscI peptide. The EscI peptide comprising residues 66-87, slightly larger than the observed EscI α-helix, was shown to bind to EtgA using microscale thermophoresis and thermal shift differential scanning fluorimetry. The EscI peptide also had a two-fold activity-enhancing effect on EtgA, whereas the EscI-EtgA fusion protein enhanced activity over four-fold compared to EtgA. Our studies suggest that EtgA regulation by EscI could be trifold involving protein localization, protein activation, and protein stabilization components. Analysis of the sequence conservation of the EscI EtgA interface residues suggested a possible conservation of such regulation for related proteins from different bacteria.

Cytosolic sorting platform complexes shuttle type III secretion system effectors to the injectisome in Yersinia enterocolitica.

Bacteria use type III secretion injectisomes to inject effector proteins into eukaryotic target cells. Recruitment of effectors to the machinery and the resulting export hierarchy involve the sorting platform. These conserved proteins form pod structures at the cytosolic interface of the injectisome but are also mobile in the cytosol. Photoactivated localization microscopy in Yersinia enterocolitica revealed a direct interaction of the sorting platform proteins SctQ and SctL with effectors in the cytosol of live bacteria. These proteins form larger cytosolic protein complexes involving the ATPase SctN and the membrane connector SctK. The mobility and composition of these mobile pod structures are modulated in the presence of effectors and their chaperones, and upon initiation of secretion, which also increases the number of injectisomes from ~5 to ~18 per bacterium. Our quantitative data support an effector shuttling mechanism, in which sorting platform proteins bind to effectors in the cytosol and deliver the cargo to the export gate at the membrane-bound injectisome.

Sensing for survival: specialised regulatory mechanisms of Type III secretion systems in Gram-negative pathogens.

For centuries, Gram-negative pathogens have infected the human population and been responsible for numerous diseases in animals and plants. Despite advancements in therapeutics, Gram-negative pathogens continue to evolve, with some having developed multi-drug resistant phenotypes. For the successful control of infections caused by these bacteria, we need to widen our understanding of the mechanisms of host-pathogen interactions. Gram-negative pathogens utilise an array of effector proteins to hijack the host system to survive within the host environment. These proteins are secreted into the host system via various secretion systems, including the integral Type III secretion system (T3SS). The T3SS spans two bacterial membranes and one host membrane to deliver effector proteins (virulence factors) into the host cell. This multifaceted process has multiple layers of regulation and various checkpoints. In this review, we highlight the multiple strategies adopted by these pathogens to regulate or maintain virulence via the T3SS, encompassing the regulation of small molecules to sense and communicate with the host system, as well as master regulators, gatekeepers, chaperones, and other effectors that recognise successful host contact. Further, we discuss the regulatory links between the T3SS and other systems, like flagella and metabolic pathways including the tricarboxylic acid (TCA) cycle, anaerobic metabolism, and stringent cell response.

Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system.

In animal pathogens, assembly of the type III secretion system injectisome requires the presence of so-called pilotins, small lipoproteins that assist the formation of the secretin ring in the outer membrane. Using a combination of functional assays, interaction studies, proteomics, and live-cell microscopy, we determined the contribution of the pilotin to the assembly, function, and substrate selectivity of the T3SS and identified potential new downstream roles of pilotin proteins. In absence of its pilotin SctG, Yersinia enterocolitica forms few, largely polar injectisome sorting platforms and needles. Accordingly, most export apparatus subcomplexes are mobile in these strains, suggesting the absence of fully assembled injectisomes. Remarkably, while absence of the pilotin all but prevents export of early T3SS substrates, such as the needle subunits, it has little effect on secretion of late T3SS substrates, including the virulence effectors. We found that although pilotins interact with other injectisome components such as the secretin in the outer membrane, they mostly localize in transient mobile clusters in the bacterial membrane. Together, these findings provide a new view on the role of pilotins in the assembly and function of type III secretion injectisomes.

Pseudomonas aeruginosa two-component system CprRS regulates HigBA expression and bacterial cytotoxicity in response to LL-37 stress.

Pseudomonas aeruginosa is a highly pathogenic bacterium known for its ability to sense and coordinate the production of virulence factors in response to host immune responses. However, the regulatory mechanisms underlying this process have remained largely elusive. In this study, we investigate the two-component system CprRS in P. aeruginosa and unveil the crucial role of the sensor protein CprS in sensing the human host defense peptide LL-37, thereby modulating bacterial virulence. We demonstrate that CprS acts as a phosphatase in the presence of LL-37, leading to the phosphorylation and activation of the response regulator CprR. The results prove that CprR directly recognizes a specific sequence within the promoter region of the HigBA toxin-antitoxin system, resulting in enhanced expression of the toxin HigB. Importantly, LL-37-induced HigB expression promotes the production of type III secretion system effectors, leading to reduced expression of proinflammatory cytokines and increased cytotoxicity towards macrophages. Moreover, mutations in cprS or cprR significantly impair bacterial survival in both macrophage and insect infection models. This study uncovers the regulatory mechanism of the CprRS system, enabling P. aeruginosa to detect and respond to human innate immune responses while maintaining a balanced virulence gene expression profile. Additionally, this study provides new evidence and insights into the complex regulatory system of T3SS in P. aeruginosa within the host environment, contributing to a better understanding of host-microbe communication and the development of novel strategies to combat bacterial infections.