ABSTRACT Pseudomonas aeruginosa produces multiple toxins and exoenzymes that contribute to its survival and ability to cause disease. In the current study, we examined whether the type II-secreted cytotoxin Exotoxin A (ToxA) is required for P. aeruginosa growth and disease severity in infected murine corneas. Using Δ toxA mutants and complemented strains on a PAO1 background, we report that although ToxA is produced during corneal infection, ToxA deletion did not significantly affect bacterial replication, neutrophil recruitment, or disease severity in infected corneas. These findings contrast with an earlier study identifying a role for ToxA in P. aeruginosa keratitis.

Pseudomonas aeruginosa is a globally recognized pathogen causing pulmonary, skin, and severe corneal infections (keratitis), with the potential to induce irreversible blindness if untreated. Spatial transcriptomic analysis of P. aeruginosa infected corneas identified elevated expression of the outer membrane proteins OprF and OprL and PA1414, which encodes the small RNA SicX in the corneal stroma compared with corneal epithelium. Comparative spatial transcriptomics analysis of corneas infected with an oprF transposon (TN) mutant showed reduced expression of the type III effector protein ExoT, which was absent in an oprF deficient mutant (Δ oprF ) and in contrast to PA14, did not inhibit reactive oxygen species (ROS) production by neutrophils. Corneal infection with the Δ oprF mutant resulted in reduced corneal virulence and lower CFU compared to the parental PA14 strain. Collectively, our findings demonstrate a coordinated virulence program connecting OprF functionality with the release of ExoT and its ability to block ROS production and survive in infected corneas.

AbstractMultilayered epithelia lining our tissue surfaces normally resist traversal by opportunistic bacteria. Previously, we developed a strategy to experimentally perturbate this resistancein situin the corneas of mouse eyes and used it to show that traversal of a multilayered epithelium byPseudomonas aeruginosarequires ExsA, the transcriptional activator of its type 3 secretion system (T3SS). Here, we developed a novel strategy for quantitively localizing individual traversing bacteria within thein situmultilayered corneal epithelium and explored contributions of T3SS components. The results showed that T3SS translocon and T3SS effector mutants had reduced epithelial traversal efficiency. Surprisingly, a ΔpscCmutant unable to assemble the T3SS needle traversed as efficiently as wild-typeP. aeruginosa, while a ΔexsDmutant ‘constitutively on’ for T3SS expression was traversal defective. Dispensability of the T3SS needle for effector-mediated traversal was confirmed using a mutant lacking the T3SS operon except the effector genes (ΔpscU-Lmutant). That mutant reacquired the ability to traverse if complemented with rhamnose-inducibleexsA, but not if the effector genes were also deleted (ΔpscU-LΔexoSTY). Western immunoblot confirmed ExoS in culture supernatants of rhamnose-inducedexsA-complemented ΔpscU-Lmutants lacking all T3SS needle protein genes. Together, these results show that epithelial traversal byP. aeruginosacan involve T3SS effectors and translocon proteins independently of the T3SS needle previously thought essential for T3SS function. This advances our understanding ofP. aeruginosapathogenesis and has relevance to development of therapeutics targeting the T3SS system.ImportanceWhile the capacity to cross an epithelial barrier can be a critical step in bacterial pathogenesis, our understanding of mechanisms involved is derived largely from cell culture experimentation. The latter is due to practical limitations ofin vivo/in situmodels and challenge of visualizing individual bacteria in the context of host tissue. Here, factors used byP. aeruginosato traverse an epithelial multilayerin situwere studied by: 1) leveraging the transparent properties and superficial location of the cornea, 2) using our established method for enabling bacterial traversal susceptibility, and 3) developing a novel strategy for accurate and quantitative localization of individual traversing bacteriain situ. Outcomes showed that T3SS translocon and T3SS effector proteins synergistically contribute to epithelial traversal efficiency independently of the T3SS needle. These findings challenge the assumption that the T3SS needle is essential for T3SS effectors or translocon proteins to contribute to bacterial pathogenesis.

Bacteria in nature can exist in multicellular communities called biofilms. Biofilms also form in the course of many infections. Pseudomonas aeruginosa infections frequently involve biofilms, which contribute materially to the difficulty to treat these infections with antibiotic therapy. Many biofilm-related characteristics are controlled by the second messenger, cyclic-di-GMP, which is upregulated on surface contact. Among these factors is the exopolysaccharide Psl, which is a critically important component of the biofilm matrix. Here we describe the discovery of a P. aeruginosa bacteriophage, which we have called Clew-1, that directly binds to and uses Psl as a receptor. While this phage does not efficiently infect planktonically growing bacteria, it can disrupt P. aeruginosa biofilms and replicate in biofilm bacteria. We further demonstrate that the Clew-1 can reduce the bacterial burden in a mouse model of P. aeruginosa keratitis, which is characterized by the formation of a biofilm on the cornea. Due to its reliance on Psl for infection, Clew-1 does not actually form plaques on wild-type bacteria under standard in vitro conditions. This argues that our standard isolation procedures likely exclude bacteriophage that are adapted to using biofilm markers for infection. Importantly, the manner in which we isolated Clew-1 can be easily extended to other strains of P. aeruginosa and indeed other bacterial species, which will fuel the discovery of other biofilm-tropic bacteriophage and expand their therapeutic use.

Multilayered epithelia lining our tissue surfaces normally resist traversal by opportunistic bacteria. Previously, we developed a strategy to experimentally perturb this resistance in situ in the corneas of mouse eyes and used it to show that traversal of a multilayered epithelium by Pseudomonas aeruginosa requires ExsA, the transcriptional activator of its type 3 secretion system (T3SS). Here, we developed a novel strategy for quantitatively localizing individual traversing bacteria within the in situ multilayered corneal epithelium and explored the contributions of T3SS components. The results showed that T3SS translocon and T3SS effector mutants had reduced epithelial traversal efficiency. Surprisingly, a ΔpscC mutant unable to assemble the T3SS needle traversed as efficiently as wild-type P. aeruginosa, while a ΔexsD mutant "constitutively on" for T3SS expression was traversal defective. The dispensability of the T3SS needle for effector-mediated traversal was confirmed using a mutant lacking the T3SS operon except for the effector genes (ΔpscU-L mutant). That mutant reacquired the ability to traverse if complemented with rhamnose-inducible exsA, but not if the effector genes were also deleted (ΔpscU-LΔexoSTY). Western immunoblot confirmed ExoS in culture supernatants of rhamnose-induced exsA-complemented ΔpscU-L mutants lacking all T3SS needle protein genes. Together, these results show that epithelial traversal by P. aeruginosa can involve T3SS effectors and translocon proteins independently of the T3SS needle previously thought essential for T3SS function. This advances our understanding of P. aeruginosa pathogenesis and has relevance to the development of therapeutics targeting the T3SS system.IMPORTANCEWhile the capacity to cross an epithelial barrier can be a critical step in bacterial pathogenesis, our understanding of the mechanisms involved is derived largely from cell culture experimentation. The latter is due to the practical limitations of in vivo/in situ models and the challenge of visualizing individual bacteria in the context of host tissue. Here, factors used by P. aeruginosa to traverse an epithelial multilayer in situ were studied by (i) leveraging the transparent properties and superficial location of the cornea, (ii) using our established method for enabling bacterial traversal susceptibility, and (iii) developing a novel strategy for accurate and quantitative localization of individual traversing bacteria in situ. Outcomes showed that T3SS translocon and T3SS effector proteins synergistically contribute to epithelial traversal efficiency independently of the T3SS needle. These findings challenge the assumption that the T3SS needle is essential for T3SS effectors or translocon proteins to contribute to bacterial pathogenesis.

Abstract We have established improved methods to access the mouse airways and lungs for pulmonary research. We have applied a non-surgical, bronchoscopic system with a camera to bacterial infection studies and found it reduced mortality and weight change in infected mice compared to our previously used procedure, a tracheostomy involving surgery. The two procedures were compared with and without bacteria to identify the inflammatory responses to the surgery and to the bacteria involved. We found the mouse tolerated the bronchoscope much better than the tracheostomy which allowed delineation of the effects of the bacteria from those of the surgical wound. The bronchoscope appeared to reduce stress to the mice, compared to the tracheostomy, and provided a significant benefit of reducing morbidity and mortality, thus reducing the number of mice needed for experiments. We found that mice receiving sterile beads instilled in the lung by tracheostomy lost more weight compared to mice undergoing bronchoscopy. Adding bacteria gave rise to even greater weight loss displaying significantly higher in surgery compared to bronchoscopy groups. Furthermore for surgery, immune cells (especially neutrophils) were increased in broncho-alveolar lavage fluid, as were pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. The effects from the surgical wound are significant and should be recognized and incorporated into experimental designs to best reflect the condition the mouse is intended to model.

Macrophages infected with Gram-negative bacteria expressing Type III secretion system (T3SS) activate the NLRC4 inflammasome, resulting in Gasdermin D (GSDMD)-mediated IL-1β secretion and pyroptosis. Here we examined inflammasome signaling in neutrophils infected with Pseudomonas aeruginosa strain PAO1 that expresses the T3SS effectors ExoS and ExoT. IL-1β secretion by neutrophils required the T3SS needle and translocon proteins and GSDMD. In macrophages, PAO1 and mutants lacking ExoS and ExoT ( ΔexoST ) stimulated NLRC4 for IL-1β secretion. While IL-1β release from ΔexoST infected neutrophils was also NLRC4-dependent, this was redirected to NLRP3-dependence by PAO1 infection via the ADP ribosyl transferase activity of ExoS. Genetic and pharmacologic approaches revealed that NLRP3, but not NLRC4, was essential for bacterial killing and limiting disease severity in a murine model of P. aeruginosa corneal infection. This reveals a novel role for ExoS ADPRT in regulating inflammasome subtype usage by neutrophils versus macrophages and an unexpected role for NLRP3 in P. aeruginosa keratitis.### Competing Interest StatementThe authors have declared no competing interest.

Macrophages infected with Gram-negative bacteria expressing Type III secretion system (T3SS) activate the NLRC4 inflammasome, resulting in Gasdermin D (GSDMD)-dependent, but GSDME independent IL-1β secretion and pyroptosis. Here we examine inflammasome signaling in neutrophils infected with Pseudomonas aeruginosa strain PAO1 that expresses the T3SS effectors ExoS and ExoT. IL-1β secretion by neutrophils requires the T3SS needle and translocon proteins and GSDMD. In macrophages, PAO1 and mutants lacking ExoS and ExoT ( ΔexoST ) require NLRC4 for IL-1β secretion. While IL-1β release from ΔexoST infected neutrophils is also NLRC4-dependent, infection with PAO1 is instead NLRP3-dependent and driven by the ADP ribosyl transferase activity of ExoS. Genetic and pharmacologic approaches using MCC950 reveal that NLRP3 is also essential for bacterial killing and disease severity in a murine model of P. aeruginosa corneal infection (keratitis). Overall, these findings reveal a function for ExoS ADPRT in regulating inflammasome subtype usage in neutrophils versus macrophages and an unexpected role for NLRP3 in P. aeruginosa keratitis.

The laboratory mouse is used extensively for human disease modeling and preclinical therapeutic testing for efficacy, biodistribution, and toxicity. The variety of murine models available, and the ability to create new ones, eclipses all other species, but the size of mice and their organs create challenges for many in vivo studies. For pulmonary research, improved methods to access murine airways and lungs, and track substances administered to them, would be desirable. A nonsurgical endoscopic system with a camera, effectively a bronchoscope, coupled with a cryoimaging fluorescence microscopy technique to view the lungs in 3D, is described here that allows visualization of the procedure, including the anatomical location at which substances are instilled and fluorescence detection of those substances. We have applied it to bacterial infection studies to characterize better and optimize a chronic lung infection murine model in which we instill bacteria-laden agarose beads into the airways and lungs to extend the duration of the infection and inflammation. The use of the endoscope as guidance for placing a catheter into the airways is simple and quick, requiring only momentary sedation, and reduces post-procedural mortality compared with our previous instillation method that includes a trans-tracheal surgery. The endoscopic method improves speed and precision of delivery while reducing the stress on animals and the number of animals generated and used for experiments.

Type III secretion systems are integral to the pathogenesis of many Gram-negative bacterial pathogens. A hallmark of these secretion systems is that they deliver effector proteins vectorially into the targeted host cell via a translocation pore.

Macrophages infected with Gram-negative bacteria expressing flagellin or Type III secretion system (T3SS) structural proteins are known to activate the NLRC4 inflammasome, resulting in caspase-1 and Gasdermin D (GSDMD) cleavage, IL-1β secretion and pyroptotic cell death. We examined the role of these mediators in IL-1β secretion by neutrophils infected with Pseudomonas aeruginosa strain PAO1 that expresses the Type III secretion system (T3SS) effectors ExoS and ExoT. We found that IL-1β secretion by neutrophils was dependent on expression of the T3SS needle and translocon proteins. Although pro-GSDMD and pro-GSDME were processed in PAO1 infected neutrophils, only GSDMD was required for IL-1β secretion. PAO1 – induced IL-1β secretion by macrophages was NLRC4 dependent, IL-1β secretion by neutrophils utilized NLRC4 only in the absence of P. aeruginosa exoenzymes. Instead, PAO1 – induced IL-1β secretion required NLRP3, which mediated by ExoS ADP ribosyl transferase activity. Overall, these findings reveal fundamental differences between neutrophils and macrophages in IL-1β secretion in response to pathogenic bacteria.

ABSTRACT Macrophages infected with Gram-negative bacteria expressing Type III secretion system (T3SS) activate the NLRC4 inflammasome, resulting in Gasdermin D (GSDMD)-mediated IL-1β secretion and pyroptosis. Here we examined inflammasome signaling in neutrophils infected with Pseudomonas aeruginosa strain PAO1 that expresses the T3SS effectors ExoS and ExoT. IL-1β secretion by neutrophils required the T3SS needle and translocon proteins and GSDMD. In macrophages, PAO1 and mutants lacking ExoS and ExoT ( ΔexoST ) stimulated NLRC4 for IL-1β secretion. While IL-1β release from ΔexoST infected neutrophils was also NLRC4-dependent, this was redirected to NLRP3-dependence by PAO1 infection via the ADP ribosyl transferase activity of ExoS. Genetic and pharmacologic approaches revealed that NLRP3, but not NLRC4, was essential for bacterial killing and limiting disease severity in a murine model of P. aeruginosa corneal infection. This reveals a novel role for ExoS ADPRT in regulating inflammasome subtype usage by neutrophils versus macrophages and an unexpected role for NLRP3 in P. aeruginosa keratitis.

Many Gram-negative pathogens use a type III secretion system (T3SS) to promote disease by injecting effector proteins into host cells. Common to many T3SSs is that injection of effector proteins is feedback inhibited. The mechanism of feedback inhibition and its role in pathogenesis are unclear. In the case of P. aeruginosa, the effector protein ExoS is central to limiting effector injection. ExoS is bifunctional, with an amino-terminal RhoGAP and a carboxy-terminal ADP-ribosyltransferase domain. We demonstrate that both domains are required to fully feedback inhibit effector injection. The RhoGAP-, but not the ADP-ribosyltransferase domain of the related effector protein ExoT also participates. Feedback inhibition does not involve translocator insertion nor pore-formation. Instead, feedback inhibition is due, in part, to a loss of the activating trigger for effector injection, and likely also decreased translocon stability. Surprisingly, feedback inhibition is abrogated in phagocytic cells. The lack of feedback inhibition in these cells requires phagocytic uptake of the bacteria, but cannot be explained through acidification of the phagosome or calcium limitation. Given that phagocytes are crucial for controlling P. aeruginosa infections, our data suggest that feedback inhibition allows P. aeruginosa to direct its effector arsenal against the cell types most damaging to its survival.

Many Gram-negative bacterial pathogens interact with mammalian cells by using type iii secretion systems (T3SS) to inject virulence proteins into host cells.A subset of these injected protein 'effectors' are enzymes that inhibit the function of host proteins by catalyzing the addition of unusual posttranslational modifications.The E. coli and Citrobacter rodentium NleB effectors, as well as the Salmonella enterica SseK effectors are glycosyltransferases that modify host protein substrates with N-acetyl glucosamine (GlcNAc) on arginine residues.This post-translational modification disrupts the normal functioning of host immune response proteins.T3SS effectors are thought to be inactive within the bacterium and fold into their active conformations after they are injected, due to the activity of chaperones that keep the effectors in a structural state permissive for secretion.While performing mass spectrometry experiments to identify glycosylation substrates of nleB orthologs, we unexpectedly observed that the bacterial glutathione synthetase (GshB) is glycosylated by NleB on arginine residue R256.NleB-mediated glycosylation of GshB resulted in enhanced GshB activity, leading to an increase in glutathione production, and promoted C. rodentium survival in oxidative stress conditions.these data represent, to our knowledge, the first intra-bacterial activity for a T3SS effector and show that arginine-GlcnAcylation, once thought to be restricted to host cell compartments, also plays an important role in regulating bacterial physiology.

Pseudomonas aeruginosa is an opportunistic bacterium of which the main virulence factor is the Type III Secretion System. The ATPase of this machinery, PscN (SctN), is thought to be localized at the base of the secretion apparatus and to participate in the recognition, chaperone dissociation and unfolding of exported T3SS proteins. In this work, a protein-protein interaction ELISA revealed the interaction of PscN with a wide range of exported T3SS proteins including the needle, translocator, gate-keeper and effector. These interactions were further confirmed by Microscale Thermophoresis that also indicated a preferential interaction of PscN with secreted proteins or protein-chaperone complex rather than with chaperones alone, in line with the release of the chaperones in the bacterial cytoplasm after the dissociation from their exported proteins. Moreover, we suggest a new role of the gate-keeper complex and the ATPase in the regulation of early substrates recognition by the T3SS. This finding sheds a new light on the mechanism of secretion switching from early to middle substrates in P. aeruginosa. Highlights T3SS substrates are secreted sequentially but information on the switches are missing Interaction of the T3SS ATPase with secreted proteins were investigated by different approaches Microscale Thermophoresis revealed a lower affinity for chaperones alone compared to complexes The Gate-keeper complex binds to the ATPase and increases its affinity for the needle complex A new role of the Gate-keeper complex is proposed, directly acting on the T3SS ATPase

Type III protein secretion systems (T3SS) deliver effector proteins from the Gram-negative bacterial cytoplasm into a eukaryotic host cell through a syringe-like, multi-protein nanomachine. Cytosolic components of T3SS include a portion of the export apparatus, which traverses the inner membrane and features the opening of the secretion channel, and the sorting complex for substrate recognition and for providing the energetics required for protein secretion. Two components critical for efficient effector export are the export gate protein and the ATPase, which are proposed to be linked by the central stalk protein of the ATPase. We present the structure of the soluble export gate homo-nonamer, CdsV, in complex with the central stalk protein, CdsO, of its cognate ATPase, both derived from Chlamydia pneumoniae. This structure defines the interface between these essential T3S proteins and reveals that CdsO engages the periphery of the export gate that may allow the ATPase to catalyze an opening between export gate subunits to allow cargo to enter the export apparatus. We also demonstrate through structure-based mutagenesis of the homologous export gate in Pseudomonas aeruginosa that mutation of this interface disrupts effector secretion. These results provide novel insights into the molecular mechanisms governing active substrate recognition and translocation through a T3SS.

Pseudomonas aeruginosa is an important opportunistic pathogen that employs a type III secretion system (T3SS) to inject effector proteins into host cells. Using a protein depletion system, we show that the endoribonuclease RNase E positively regulates expression of the T3SS genes. We also present evidence that RNase E antagonizes the expression of genes of the type VI secretion system and limits biofilm production in P. aeruginosa. Thus, RNase E, which is thought to be the principal endoribonuclease involved in the initiation of RNA degradation in P. aeruginosa, plays a key role in controlling the production of factors involved in both acute and chronic stages of infection. Although the posttranscriptional regulator RsmA is also known to positively regulate expression of the T3SS genes, we find that RNase E does not appreciably influence the abundance of RsmA in P. aeruginosa. Moreover, we show that RNase E still exerts its effects on T3SS gene expression in cells lacking all four of the key small regulatory RNAs that function by sequestering RsmA. IMPORTANCE The type III secretion system (T3SS) is a protein complex produced by many Gram-negative pathogens. It is capable of injecting effector proteins into host cells that can manipulate cell metabolism and have toxic effects. Understanding how the T3SS is regulated is important in understanding the pathogenesis of bacteria with such systems. Here, we show that RNase E, which is typically thought of as a global regulator of RNA stability, plays a role in regulating the T3SS in Pseudomonas aeruginosa. Depleting RNase E results in the loss of T3SS gene expression as well as a concomitant increase in biofilm formation. These observations are reminiscent of the phenotypes associated with the loss of activity of the posttranscriptional regulator RsmA. However, RNase E-mediated regulation of these systems does not involve changes in the abundance of RsmA and is independent of the known small regulatory RNAs that modulate RsmA activity.