Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus α-hemolysin-mediated toxicity

Staphylococcus aureus causes a wide variety of infections and antibiotic resistant strains are a major problem in hospitals. One of the best studied virulence factors of S. aureus is the pore-forming toxin alpha hemolysin (αHL) whose mechanism of action is incompletely understood. We performed a genome- wide loss-of-function screen using CRISPR/Cas9 technology to identify host targets required for αHL susceptibility in human myeloid cells. We found gRNAs for ten genes enriched after intoxication with αHL and focused on the top five hits. Besides a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), the host receptor for αHL, we identified three proteins, Sys1 golgi trafficking protein (SYS1), ADP-ribosylation factor 1 (ARFRP1), and tetraspanin-14 (TSPAN14) which regulate the presentation of ADAM10 on the plasma membrane post-translationally. Interestingly, we also showed that cells lacking sphingomyelin synthase 1 (SGMS1) resist αHL intoxication, but have only a slightly reduced ADAM10 surface expression. SGMS1 regulates lipid raft formation, suggesting that αHL requires these membrane microdomains for attachment and cytotoxicity

Human Complement Inhibits Myophages against Pseudomonas aeruginosa

Therapeutic bacteriophages (phages) are primarily chosen based on their in vitro bacteriolytic activity. Although anti-phage antibodies are known to inhibit phage infection, the influence of other immune system components is less well known. An important anti-bacterial and anti-viral innate immune system that may interact with phages is the complement system, a cascade of proteases that recognizes and targets invading microorganisms. In this research, we aimed to study the effects of serum components such as complement on the infectivity of different phages targeting Pseudomonas aeruginosa. We used a fluorescence-based assay to monitor the killing of P. aeruginosa by phages of different morphotypes in the presence of human serum. Our results reveal that several myophages are inhibited by serum in a concentration-dependent way, while the activity of four podophages and one siphophage tested in this study is not affected by serum. By using specific nanobodies blocking different components of the complement cascade, we showed that activation of the classical complement pathway is a driver of phage inhibition. To determine the mechanism of inhibition, we produced bioorthogonally labeled fluorescent phages to study their binding by means of microscopy and flow cytometry. We show that phage adsorption is hampered in the presence of active complement. Our results indicate that interactions with complement may affect the in vivo activity of therapeutically administered phages. A better understanding of this phenomenon is essential to optimize the design and application of therapeutic phage cocktails

Colistin resistance mutations in phoQ can sensitize Klebsiella pneumoniae to IgM-mediated complement killing

International audienceAbstract Due to multi-drug resistance, physicians increasingly use the last-resort antibiotic colistin to treat infections with the Gram-negative bacterium Klebsiella pneumoniae. Unfortunately, K. pneumoniae can also develop colistin resistance. Interestingly, colistin resistance has dual effects on bacterial clearance by the immune system. While it increases resistance to antimicrobial peptides, colistin resistance has been reported to sensitize certain bacteria for killing by human serum. Here we investigate the mechanisms underlying this increased serum sensitivity, focusing on human complement which kills Gram-negatives via membrane attack complex (MAC) pores. Using in vitro evolved colistin resistant strains and a fluorescent MAC-mediated permeabilization assay, we showed that two of the three tested colistin resistant strains, Kp209_CSTR and Kp257_CSTR, were sensitized to MAC. Transcriptomic and mechanistic analyses focusing on Kp209_CSTR revealed that a mutation in the phoQ gene locked PhoQ in an active state, making Kp209_CSTR colistin resistant and MAC sensitive. Detailed immunological assays showed that complement activation on Kp209_CSTR in human serum required specific IgM antibodies that bound Kp209_CSTR but did not recognize the wild-type strain. Together, our results show that developing colistin resistance affected recognition of Kp209_CSTR and its killing by the immune system

Soluble MAC is primarily released from MAC-resistant bacteria that potently convert complement component C5

The membrane attack complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC formation is initiated when complement enzymes on the bacterial surface convert complement component C5 into C5b. Although the MAC is a membrane-inserted complex, soluble forms of MAC (sMAC), or terminal complement complex (TCC), are often detected in sera of patients suffering from infections. Consequently, sMAC has been proposed as a biomarker, but it remains unclear when and how it is formed during infections. Here, we studied mechanisms of MAC formation on different Gram-negative and Gram-positive bacteria and found that sMAC is primarily formed in human serum by bacteria resistant to MAC-dependent killing. Surprisingly, C5 was converted into C5b more potently by MAC-resistant compared to MAC-sensitive Escherichia coli strains. In addition, we found that MAC precursors are released from the surface of MAC-resistant bacteria during MAC assembly. Although release of MAC precursors from bacteria induced lysis of bystander human erythrocytes, serum regulators vitronectin (Vn) and clusterin (Clu) can prevent this. Combining size exclusion chromatography with mass spectrom-etry profiling, we show that sMAC released from bacteria in serum is a heterogeneous mixture of complexes composed of C5b-8, up to three copies of C9 and multiple copies of Vn and Clu. Alto-gether, our data provide molecular insight into how sMAC is generated during bacterial infections. This fundamental knowledge could form the basis for exploring the use of sMAC as biomarker

Selective Exoenzymatic Labeling of Lipooligosaccharides of Neisseria gonorrhoeae with α2,6-Sialoside Analogues

The interactions between bacteria and their host often rely on recognition processes that involve host or bacterial glycans. Glycoengineering techniques make it possible to modify and study the glycans on the host's eukaryotic cells, but only a few are available for the study of bacterial glycans. Here, we have adapted selective exoenzymatic labeling (SEEL), a chemical reporter strategy, to label the lipooligosaccharides of the bacterial pathogen Neisseria gonorrhoeae, using the recombinant glycosyltransferase ST6Gal1, and three synthetic CMP-sialic acid derivatives. We show that SEEL treatment does not affect cell viability and can introduce an α2,6-linked sialic acid with a reporter group on the lipooligosaccharides by Western blot, flow cytometry and fluorescent microscopy. This new bacterial glycoengineering technique allows for the precise modification, here with α2,6-sialoside derivatives, and direct detection of specific surface glycans on live bacteria, which will aid in further unravelling the precise biological functions of bacterial glycans

Colistin resistance mutations in phoQ can sensitize Klebsiella pneumoniae to IgM-mediated complement killing

Due to multi-drug resistance, physicians increasingly use the last-resort antibiotic colistin to treat infections with the Gram-negative bacterium Klebsiella pneumoniae. Unfortunately, K. pneumoniae can also develop colistin resistance. Interestingly, colistin resistance has dual effects on bacterial clearance by the immune system. While it increases resistance to antimicrobial peptides, colistin resistance has been reported to sensitize certain bacteria for killing by human serum. Here we investigate the mechanisms underlying this increased serum sensitivity, focusing on human complement which kills Gram-negatives via membrane attack complex (MAC) pores. Using in vitro evolved colistin resistant strains and a fluorescent MAC-mediated permeabilization assay, we showed that two of the three tested colistin resistant strains, Kp209_CSTR and Kp257_CSTR, were sensitized to MAC. Transcriptomic and mechanistic analyses focusing on Kp209_CSTR revealed that a mutation in the phoQ gene locked PhoQ in an active state, making Kp209_CSTR colistin resistant and MAC sensitive. Detailed immunological assays showed that complement activation on Kp209_CSTR in human serum required specific IgM antibodies that bound Kp209_CSTR but did not recognize the wild-type strain. Together, our results show that developing colistin resistance affected recognition of Kp209_CSTR and its killing by the immune system

Novel insights in antimicrobial and immunomodulatory mechanisms of action of PepBiotics CR-163 and CR-172

Objectives: Our group recently developed a new group of antimicrobial peptides termed PepBiotics, of which peptides CR-163 and CR-172 showed optimized antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus without inducing antimicrobial resistance. In this study, the antibacterial mechanism of action and the immunomodulatory activity of these two PepBiotics was explored. Methods: RAW264.7 cells were used to determine the ability of PepBiotics to neutralize Lipopolysaccharide (LPS)-and Lipoteichoic acid (LTA)-induced activation of macrophages. Isothermal titration calorimetry and competition assays with dansyl-labeled polymyxin B determined binding characteristics to LPS and LTA. Combined bacterial killing with subsequent macrophage activation assays was performed to determine so-called ‘silent killing’. Finally, flow cytometry of peptide-treated genetically engineered Escherichia coli expressing Green Fluorescent Protein (GFP) and mCherry in the cytoplasm and periplasm, respectively, further established the antimicrobial mechanism of PepBiotics. Results: Both CR-163 and CR-172 were shown to have broad-spectrum activity against ESKAPE pathogens and E. coli using a membranolytic mechanism of action. PepBiotics could exothermically bind LPS/LTA and were able to replace polymyxin B. Finally, it was demonstrated that bacteria killed by PepBiotics were less prone to stimulate immune cells, contrary to gentamicin and heat-killed bacteria that still elicited a strong immune response. Conclusions: These studies highlight the multifunctional nature of the two peptide antibiotics as both broad-spectrum antimicrobial and immunomodulator. Their ability to kill bacteria and reduce unwanted subsequent immune activation is a major advantage and highlights their potential for future therapeutic use

Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies

The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector functions result from C5 cleavage into C5a and C5b, development of C5 inhibitors, such as clinically approved monoclonal antibody eculizumab, are of great interest. Here, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance, we determined a binding affinity of 119.9 pM for UNbC5-1 and 7.7 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 (3.6 Å resolution) revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5 that is not recognized by eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5

Host–receptor post-translational modifications refine staphylococcal leukocidin cytotoxicity

Staphylococcal bi-component pore-forming toxins, also known as leukocidins, target and lyse human phagocytes in a receptor-dependent manner. S-components of the leukocidins Panton-Valentine leukocidin (PVL), γ-haemolysin AB (HlgAB) and CB (HlgCB), and leukocidin ED (LukED) specifically employ receptors that belong to the class of G-protein coupled receptors (GPCRs). Although these receptors share a common structural architecture, little is known about the conserved characteristics of the interaction between leukocidins and GPCRs. In this study, we investigated host cellular pathways contributing to susceptibility towards S. aureus leukocidin cytotoxicity. We performed a genome-wide CRISPR/Cas9 library screen for toxin-resistance in U937 cells sensitized to leukocidins by ectopic expression of different GPCRs. Our screen identifies posttranslational modification (PTM) pathways involved in the sulfation and sialylation of the leukocidin-receptors. Subsequent validation experiments show differences in the impact of PTM moieties on leukocidin toxicity, highlighting an additional layer of refinement and divergence in the staphylococcal host-pathogen interface. Leukocidin receptors may serve as targets for anti-staphylococcal interventions and understanding toxin-receptor interactions will facilitate the development of innovative therapeutics. Variations in the genes encoding PTM pathways could provide insight into observed differences in susceptibility of humans to infections with S. aureus