Bart W. Bardoel

Pseudomonas evades immune recognition of flagellin in both mammals and plants.

The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants.

Pseudomonas aeruginosa Alkaline Protease Blocks Complement Activation via the Classical and Lectin Pathways

The complement system rapidly detects and kills Gram-negative bacteria and supports bacterial killing by phagocytes. However, bacterial pathogens exploit several strategies to evade detection by the complement system. The alkaline protease (AprA) of Pseudomonas aeruginosa has been associated with bacterial virulence and is known to interfere with complement-mediated lysis of erythrocytes, but its exact role in bacterial complement escape is unknown. In this study, we analyzed how AprA interferes with complement activation and whether it could block complement-dependent neutrophil functions. We found that AprA potently blocked phagocytosis and killing of Pseudomonas by human neutrophils. Furthermore, AprA inhibited opsonization of bacteria with C3b and the formation of the chemotactic agent C5a. AprA specifically blocked C3b deposition via the classical and lectin pathways, whereas the alternative pathway was not affected. Serum degradation assays revealed that AprA degrades both human C1s and C2. However, repletion assays demonstrated that the mechanism of action for complement inhibition is cleavage of C2. In summary, we showed that P. aeruginosa AprA interferes with classical and lectin pathway-mediated complement activation via cleavage of C2.

Staphylococcal Complement Inhibitor: Structure and Active Sites

The pathogenic bacterium Staphylococcus aureus counteracts the host immune defense by excretion of the 85 residue staphylococcal complement inhibitor (SCIN). SCIN inhibits the central complement convertases; thereby, it reduces phagocytosis following opsonization and efficiently blocks all downstream effector functions. In this study, we present the crystal structure of SCIN at 1.8 Å resolution and the identification of its active site. Functional characterization of structure based chimeric proteins, consisting of SCIN and the structurally but nonfunctional homologue open reading frame-D, indicate an 18-residue segment (Leu-31—Gly-48) crucial for SCIN activity. In all complement activation pathways, chimeras lacking these SCIN residues completely fail to inhibit production of the potent mediator of inflammation C5a. Inhibition of alternative pathway-mediated opsonization (C3b deposition) and formation of the lytic membrane attack complex (C5b-9 deposition) are strongly reduced for these chimeras as well. For inhibition of the classical/lectin pathway-mediated C3b and C5b-9 deposition, the same residues are critical although additional sites are involved. These chimeras also display reduced capacity to stabilize the C3 convertases of both the alternative and the classical/lectin pathway indicating the stabilizing effect is pivotal for the complement inhibitory activity of SCIN. Because SCIN specifically and efficiently inhibits complement, it has a high potential in anti-inflammatory therapy. Our data are a first step toward the development of a second generation molecule suitable for such therapeutic complement intervention.

Exposure of intestinal epithelial cells to UV-killed Lactobacillus GG but not Bifidobacterium breve enhances the effector immune response in vitro.

Background: Intestinal bacteria and intestinal epithelial cells (IEC) may modulate the mucosal immune response. In this study, immune modulation by Lactobacillus GG (LGG) and Bifidobacterium breve (Bb1, Bb2) in the presence or absence of IEC was addressed in an in vitro transwell co-culture model. Methods: UV-killed LGG,Bb1, Bb2 or Toll-like receptor (TLR) 2 or nucleotide oligomerization domain (NOD) 2 ligands were added directly to unstimulated or anti-CD3/CD28-stimulated PBMC, or applied apically to human IEC (HT-29) co-cultured with PBMC. A mixture of live bacteria was used as reference. The effect on T helper 1 (IFN-γ, IL-12), T helper 2 (IL-13), inflammatory (TNF-α) and regulatory (IL-10) cytokine secretion was determined. Results: Both UV-killed LGG and Bb enhanced IL-12, IFN-γ, TNF-α and IL-10, and reduced IL-13 secretion when added directly to stimulated PBMC, similar to live bacteria. IEC reduced IL-13, IFN-γ and IL-10 secretion by stimulated PBMC. Apically added LGG, TLR2 and NOD2 ligands,but not Bb, enhanced IFN-γ, IL-12 and/or TNF-α secretion. Bacteria did not induce cytokine secretion when added to HT-29/unstimulated PBMC co-cultures, whereas direct incubations with PBMC did. Conclusion: UV-killed LGG as well as Bb supported a T helper 1 and/or regulatory phenotype when added directly to activated PBMC, similar to live bacteria. In contrast, LGG, TLR2 or NOD2 ligands – but not Bb – enhanced T helper 1 type cytokine secretion when added to IEC, while IL-10 secretion remained suppressed. Co-cultures combining IEC and PBMC may reveal differences between bacterial strains relevant for the in vivo situation.

Pseudomonas syringae Evades Host Immunity by Degrading Flagellin Monomers with Alkaline Protease AprA

Bacterial flagellin molecules are strong inducers of innate immune responses in both mammals and plants. The opportunistic pathogen Pseudomonas aeruginosa secretes an alkaline protease called AprA that degrades flagellin monomers. Here, we show that AprA is widespread among a wide variety of bacterial species. In addition, we investigated the role of AprA in virulence of the bacterial plant pathogen P. syringae pv. tomato DC3000. The AprA-deficient DC3000 ΔaprA knockout mutant was significantly less virulent on both tomato and Arabidopsis thaliana. Moreover, infiltration of A. thaliana Col-0 leaves with DC3000 ΔaprA evoked a significantly higher level of expression of the defense-related genes FRK1 and PR-1 than did wild-type DC3000. In the flagellin receptor mutant fls2, pathogen virulence and defense-related gene activation did not differ between DC3000 and DC3000 ΔaprA. Together, these results suggest that AprA of DC3000 is important for evasion of recognition by the FLS2 receptor, allowing wild-type DC3000 to be more virulent on its host plant than AprA-deficient DC3000 ΔaprA. To provide further evidence for the role of DC3000 AprA in host immune evasion, we overexpressed the AprA inhibitory peptide AprI of DC3000 in A. thaliana to counteract the immune evasive capacity of DC3000 AprA. Ectopic expression of aprI in A. thaliana resulted in an enhanced level of resistance against wild-type DC3000, while the already elevated level of resistance against DC3000 ΔaprA remained unchanged. Together, these results indicate that evasion of host immunity by the alkaline protease AprA is important for full virulence of strain DC3000 and likely acts by preventing flagellin monomers from being recognized by its cognate immune receptor.

Molecular battle between host and bacterium: recognition in innate immunity

During infection, our innate immune system is the first line of defense and has evolved to clear invading bacteria immediately. To do so, recognition is the key element. However, how does the innate immune system distinguish self from nonself, and how does it recognize all bacteria (estimated to be far over a million species)? The answer lies in the recognition of evolutionary conserved structures. In this review, we approach this phenomenon from the bacterial perspective. What are the evolutionary conserved structures in bacteria, and what strategies are there in the human innate immune system to sense these structures? We illustrate most examples both at the functional as well as at the molecular level. Furthermore, we highlight how pathogenic bacteria can evade this recognition to survive better in the human host which in turn can result in life‐threatening diseases. Copyright

Inhibition of Pseudomonas aeruginosa Virulence: Characterization of the AprA–AprI Interface and Species Selectivity

Pseudomonas aeruginosa secretes the virulence factor alkaline protease (AprA) to enhance its survival. AprA cleaves one of the key microbial recognition molecules, monomeric flagellin, and thereby diminishes Toll-like receptor 5 activation. In addition, AprA degrades host proteins such as complement proteins and cytokines. P. aeruginosa encodes a highly potent inhibitor of alkaline protease (AprI) that is solely located in the periplasm where it is presumed to protect periplasmic proteins against secreted AprA. We set out to study the enzyme-inhibitor interactions in more detail in order to provide a basis for future drug development. Structural and mutational studies reveal that the conserved N-terminal residues of AprI occupy the protease active site and are essential for inhibitory activity. We constructed peptides mimicking the N-terminus of AprI; however, these were incapable of inhibiting AprA-mediated flagellin cleavage. Furthermore, we expressed and purified AprI of P. aeruginosa and the homologous (37% sequence identity) AprI of Pseudomonas syringae, which remarkably show species specificity for their cognate protease. Exchange of the first five N-terminal residues between AprI of P. syringae and P. aeruginosa did not affect the observed specificity, whereas exchange of only six residues located at the AprI surface that contacts the protease did abolish specificity. These findings are elementary steps toward the design of molecules derived from the natural inhibitor of the virulence factor AprA and their use in therapeutic applications in Pseudomonas and other Gram-negative infections.

Identification of an immunomodulating metalloprotease of Pseudomonas aeruginosa (IMPa)

Phagocytosis by neutrophils is the essential step in fighting Pseudomonas infections. The first step in neutrophil recruitment to the site infection is the interaction of P‐selectin (on endothelial cells) with P‐selectin glycoprotein ligand‐1 (PSGL‐1) on neutrophils. Pseudomonas aeruginosa secretes various proteases that degrade proteins that are essential for host defence, such as elastase and alkaline protease. Here we identify PA0572 of P. aeruginosa as an inhibitor of PSGL‐1 and named this secreted hypothetical protease immunomodulating metalloprotease of P. aeruginosa or IMPa. Proteolytic activity was confirmed by cleavage of recombinant and cell‐surface expressed PSGL‐1. Functional inhibition was demonstrated by impaired PSGL‐1‐mediated rolling of IMPa‐treated neutrophils under flow conditions. Next to PSGL‐1, IMPa targets CD43 and CD44 that are also involved in leucocyte homing. These data indicate that IMPa prevents neutrophil extravasation and thereby protects P. aeruginosa from neutrophil attack.

Complement resistance mechanisms of Klebsiella pneumoniae.

The current emergence of antibiotic-resistant bacteria causes major problems in hospitals worldwide. To survive within the host, bacterial pathogens exploit several escape mechanisms to prevent detection and killing by the immune system. As a major player in immune defense, the complement system recognizes and destroys bacteria via different effector mechanisms. The complement system can label bacteria for phagocytosis or directly kill Gram-negative bacteria via insertion of a pore-forming complex in the bacterial membrane. The multi-drug resistant pathogen Klebsiella pneumoniae exploits several mechanisms to resist complement. In this review, we present an overview of strategies used by K. pneumoniae to prevent recognition and killing by the complement system. Understanding these complement evasion strategies is crucial for the development of innovative strategies to combat K. pneumoniae.

Human CD45 is an F-component-specific receptor for the staphylococcal toxin Panton–Valentine leukocidin

The staphylococcal bi-component leukocidins Panton–Valentine leukocidin (PVL) and γ-haemolysin CB (HlgCB) target human phagocytes. Binding of the toxins’ S-components to human complement C5a receptor 1 (C5aR1) contributes to cellular tropism and human specificity of PVL and HlgCB. To investigate the role of both leukocidins during infection, we developed a human C5aR1 knock-in (hC5aR1KI) mouse model. HlgCB, but unexpectedly not PVL, contributed to increased bacterial loads in tissues of hC5aR1KI mice. Compared to humans, murine hC5aR1KI neutrophils showed a reduced sensitivity to PVL, which was mediated by the toxin’s F-component LukF-PV. By performing a genome-wide CRISPR–Cas9 screen, we identified CD45 as a receptor for LukF-PV. The human-specific interaction between LukF-PV and CD45 provides a molecular explanation for resistance of hC5aR1KI mouse neutrophils to PVL and probably contributes to the lack of a PVL-mediated phenotype during infection in these mice. This study demonstrates an unsuspected role of the F-component in driving the sensitivity of human phagocytes to PVL.Human CD45 serves as a receptor for the Staphylococcus aureus Panton–Valentine leukocidin (PVL) F-component LukF-PV and contributes to the species specificity behind PVL intoxication of human immune cells.