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Type II Secretion System: Shooting Toxins Next to Cells
Another toxin of interest is ToxA, the most toxic protein secreted by P. aeruginosa with a Lethal Dose 50 (LD50) of 0.2 mg in mice [38]. While being secreted in the extracellular milieu by the type II secretion system (T2SS), ToxA has an intracellular target. Indeed, this toxin with ADPRT activity mediates its entry into target host cells through its cell-binding domain, then ADP-ribosylates host elongation factor 2 to block protein synthesis [39, 40]. The T2SS is also known to secrete diverse proteases involve in tissue invasion including LasA, LasB, and Prpl. Both LasA and LasB are metalloproteases. LasA has low elastolytic and high staphylolytic activities [41], it also highly increases the elastolytic activity of LasB [42]. LasB has a strong elastolytic activity and allows for tissue invasion by increasing membrane permeability of epithelial cells. LasB also inactivates immunoglobulin A (IgA), IgG and complement components [43-46]. The loss of either LasB or LasA significantly decreases the ability of P. aeruginosa to invade epithelial cells in vitro and the loss of both proteases leads to an even lower invasion phenotype [47]. Prpl is another T2SS secreted protease (a lysine endoproteinase) that degrades proteins such as complement components, immunoglobulins, elastin, lactoferrin, and transferrin [48].
Next Target: Lipids
A different virulence strategy of P. aeruginosa relies on its ability to degrade host cell phospholipids. In addition to the previously mentioned phospholipases (PldA, PldB and Prpl), P. aeruginosa uses the hemolytic phospholipase C PlcH (secreted by the T2SS). PlcH can degrade phospholipids (phosphatidylcholine and sphingomyelin) found in eukaryotic membranes and in lung surfactant [49] and suppresses bacterium-induced neutrophil respiratory burst [50]. Furthermore, to increase the efficacy of its phospholipases, P. aeruginosa produces extracellular glycolipids called rhamnolipids. Indeed, these rhamnolipids act as detergents on the phospholipids of the lung surfactant [51], disrupt mucociliary transport and ciliary beating [52], and inhibit phagocytosis [53].
Iron Is All I Need
Once host cells have been destroyed, P. aeruginosa has the ability to retrieve important nutriments, molecules and cofactors necessary to its growth, including iron. Iron is an essential cofactor of many proteins (iron–sulfur cluster‐containing proteins, enzymes in mitochondrial respiration…) and is critical to P. aeruginosa physiology [54]. To capture this essential metal, P. aeruginosa uses iron chelating molecules named siderophores: pyoverdin and pyochelin. Both siderophores have been shown to be essential virulence factors in diverse mice infection models [55-57]. P. aeruginosa can also get iron via the uptake of siderophores produced by other bacteria (xenosiderophores), or via the uptake of heme molecules from the host hemoproteins, or by using phenazine compounds. Phenazine-1-carboxylic acid (PCA) is the precursor of pyocyanin, the blue-green compound typical of P. aeruginosa (this bacterium used to be nicknamed “pyocyanic bacilli”). PCA is able to reduce Fe3+ bound to host proteins into Fe2+, allowing the uptake of iron via the Feo system [58]. If pyocyanin is less potent than PCA for iron-uptake, it nevertheless plays other roles during the infection. Indeed, in vitro studies have shown that pyocyanin inhibits cell respiration, ciliary function, epidermal cell growth, and also induces neutrophils apoptosis [59-61]. Moreover, it was shown to be a major virulence factor in vivo [62].
LPS: Smooth or Rough
Overall, P. aeruginosa infections are characterized by severe tissue damages and a strong inflammatory response. The immune system recognizes different components of the bacterial envelope via Toll like Receptors (TLRs). More specifically, the lipopolysaccharide (LPS), peptidoglycan and flagellin are recognized by TLR4, TLR2 and TLR5, respectively, driving the induction of pro‐inflammatory cytokines and type I interferon (IFN) responses [63]. The LPS is composed of 3 parts: the lipid A, which can overstimulate the immune system and lead to a fatal “septic shock” [64]; the oligosaccharide core; and the O-antigen side chain, a variable part that can be present (smooth LPS) or absent (rough LPS). A P. aeruginosa strain with smooth LPS was shown to be significantly more virulent in mice than its rough LPS mutant [65]. This lower virulence might be related to the involvement of LPS in P. aeruginosa motility. Indeed, both “swimming” and “swarming” motilities were impaired in mutants that lacked smooth LPS [14, 66]. Furthermore, P. aeruginosa strains with rough LPS are sensitive to human serum whereas strains with smooth LPS are not [67].
Hacking the Immune System
Infections by P. aeruginosa are characterized by a highly inflammatory immune response. Unfortunately, P. aeruginosa can efficiently disrupt this immune response. Indeed, numerous virulence factors previously mentioned (Table 2) degrade proteins of the immune system (e.g. immunoglobulins, complement components…), inhibit antibacterial cell functions (e.g. oxidative burst, phagocytosis…) but also disturb cytokine signaling. For example, the alkaline protease ArpA (secreted by the Type I Secretion System) and the elastase LasB, can inactivate human interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and degrade two key pro-inflammatory cytokines, IL-6 and IL-8 as well [68-70]. These two proteases also inhibit neutrophils function by interfering with their chemotaxis [71], reduce phagocytic activities of leukocytes [72], inhibit natural killers [73] and disrupt lymphocytes via degradation of interleukin 2 [74].
Antibiotics Against P. aeruginosa: Choose Carefully
P. aeruginosa is intrinsically resistant to oxazolidinones, macrolides, lincosamides, streptogramins, daptomycin, glycopeptides, rifampin, trimethoprim-sulfamethoxazole, tetracycline, some β-lactams (aminopenicillins with or without β-lactamase-inhibitors, as well as 1st and 2nd generation cephalosporins) [12]. This impressive drug-resistance is due to a particularly low membrane permeability (12 to 100 fold lower than Escherichia coli permeability) associated with several efficient efflux systems and antibiotic inactivating enzymes [75]. Luckily, some antibiotics can still affect most P. aeruginosa isolates: fluoroquinolones, aminoglycosides, polymyxins, and some β-lactams (3rd and 4th generation cephalosporins, monobactams, carbapenems, and novel β-lactam/β-lactamase inhibitor combinations) [12]. Unfortunately, the overuse of these drugs can lead to the selection of mutations increasing P. aeruginosa antibiotic resistance. Moreover, this pathogen can acquire additional resistance mechanisms through acquisition of mobile genetic elements like plasmids.
All the Roads Lead to Resistance
The membrane of P. aeruginosa is the first line of defense against antibiotics. As the efficient diffusion/uptake of antibiotics in P. aeruginosa require specific porin or transporters, resistance can by acquired via mutations in these components: e.g. β-lactam antibiotics and fluoroquinolones enter bacterial cells through porin channels, and the loss of the OprD porin leads to carbapenem resistance [76, 77]. Likewise, aminoglycosides uptake involves oxygen- or nitrogen-dependent electron transport chains, and it was shown that absence of oxygen (e.g. inside biofilms) or functional deficiency of ATPases contribute to resistance to these antibiotics [78, 79]; Finally, colistin (polymyxin) promotes its own uptake by interacting with the LPS, and modification of the lipid A part contributes to polymyxin resistance [80, 81].
Even when a drug successfully reaches the cytoplasm of P. aeruginosa cells, it can be rapidly expelled by efflux systems. P. aeruginosa possesses multiple efflux systems that undeniably play a critical role in its intrinsic resistance to different antibiotics including β-lactams, tetracycline, chloramphenicol, and fluoroquinolones [82, 83]. Overexpression of some of these efflux systems (by mutations in their regulators) further contribute to the multi-drug-resistance (MDR) phenotype of P. aeruginosa: e.g. overexpression of MexAB-OprM leads to increased resistance to β-lactams and quinolones [84, 85].
Cystic Fibrosis Lungs: a Bacterial Paradise
CF is the most common life-shortening autosomal recessive disorder for Caucasian (affecting 1 in 2500 newborns), and is caused by mutations in the CF-transmembrane conductance regulator (CFTR) [104]. These mutations (over 300 have been documented by the CF Genetic Analysis Consortium) lead to a defective chloride (Cl−) channel, which disrupts electrolyte secretion, thus increasing the viscosity of the mucus secreted at the surface of epithelial cells and affecting osmolarity [104]. The main lung defenses against bacterial colonization (mucociliary clearance, polymorphonuclear neutrophil phagocytosis, and local production of antibacterial cationic peptides) are not performing efficiently in the CF environment, resulting in chronic bacterial infections [105, 106]. Nonetheless, in this specific environment, bacteria must survive the limitations in growth factors, dehydration, the host’s immune defenses, and the yearlong antibiotic therapies. Most CF patients become permanently colonized by P. aeruginosa, thus the same bacterial lineage can persist in the lungs for years or even decades [107]. With the advent of cheaper genome sequencing technologies, evolution of P. aeruginosa in the lungs of CF patients has been extensively studied and genetic basis of several phenotypes have now been identified.
Mucoidy Origins
The most characterized phenotype of P. aeruginosa isolates from CF patients is mucoidy. In the 1960’s Doggett et al reported “An atypical Pseudomonas aeruginosa associated with cystic fibrosis” [108, 109]. This “atypical” P. aeruginosa was defined by an uncommon aspect on agar plates and liquid cultures [108]. This mucoid phenotype corresponds to an overproduction of alginate (an exopolysaccharide). It was then reported that this mucoid phenotype was absent from recently P. aeruginosa-infected CF patients, whereas it was present in patients with chronic infection [110]. Later, the mechanism of mucoidy conversion was identified by Martin et al [111]. They discovered that a mutation in the mucA gene leads to a loss of function of this inhibitor of the sigma factor AlgU, the latter regulating the transcription of genes involved in the alginate biosynthetic pathway. The couple MucA/AlgU, localized at the cytoplasmic membrane, controls the expression of more than 200 genes related to membrane homeostasis (membrane proteins, LPS, peptidoglycan synthesis…). Its activation upon membrane stress, starts with the proteolysis of MucA catalyzed by AlgW. Once free from its anti-sigma factor MucA, AlgU binds to the RNA polymerase and direct the transcription of its regulon [112]. Recently a portion of mucA was shown to be essential in P. aeruginosa [113]. Indeed, overexpression of algU in the absence of MucA prevents cell growth, and this phenotype can be rescued by the overproduction of RpoD (the housekeeping sigma factor). In CF strains, mutations of mucA are commonly found, which implies an important role for alginate overproduction in P. aeruginosa’s adaptation to the CF lung environment. This role might be to protect P. aeruginosa from the constant inflammation in the lungs as it increases resistance to antibody-independent opsonic killing [114], confers resistance to phagocyte-generated hypochlorite [115], decreases phagocytosis of planktonic mucoid bacteria by neutrophils and macrophages [116], and reduces polymorphonuclear chemotaxis while inhibiting activation of the complement system [117]. Additionally, alginate plays a role in antibiotic resistance since biofilms formed by an alginate-overproducing strain were shown to be more resistant to tobramycin than biofilms formed by an isogenic non-mucoid strain [118]. Conversely, the use of alginate lyase proved to increase antibiotic efficacy against mucoid P. aeruginosa biofilms [119].
Evolution of P. aeruginosa in CF: Less Virulence, More Resistance
Longitudinal studies of P. aeruginosa’s genome throughout years of lung infection in CF patients, shined a light on its evolution in this environment. Such a study of the phenotypic and genotypic changes in P. aeruginosa isolates, was performed in a cohort of 40 CF children during the first 3 years of life [120]. A high degree of genotypic variability was found as each patient had unique genotypes. Surprisingly, the early isolates were generally non-mucoid and antibiotic susceptible, which is unusual for CF isolates. These results support the hypothesis of an initial infection by “classical” non-mucoid P. aeruginosa strains which then evolve during the chronic infection to become the mucoid “CF” strain.
Table of contents :
Contents
Acknowledgments/Remerciements
Contents
Introduction
Chapter I: Pseudomonas aeruginosa infections, a growing health problem.
1- A Versatile Pathogen
2- The Main Weapons of P. aeruginosa
A) First Contacts
B) Type III Secretion System: Shooting Toxins in Cells
C) Virulence Without a T3SS
D) Type II Secretion System: Shooting Toxins Next to Cells
E) Next Target: Lipids
F) Iron Is All I Need
G) LPS: Smooth or Rough
H) Hacking the Immune System
3- Current antibiotic treatments and associated resistance mechanisms
A) Antibiotics Against P. aeruginosa: Choose Carefully
B) All the Roads Lead to Resistance
C) Biofilms: United We Stand
4- P. aeruginosa in Cystic Fibrosis: From Acute to Chronic Infections.
A) Cystic Fibrosis Lungs: a Bacterial Paradise
B) Mucoidy Origins
C) Evolution of P. aeruginosa in CF: Less Virulence, More Resistance
Chapter II: Bacteriophages, the most abundant bacterial predators.
1- The Three Faces of Phage Research.
A) An Ubiquitous Predator.
B) Phage Therapy Part 1: Chaotic Beginnings.
C) Phages and the Birth of Molecular Biology.
2- Introduction to phage biology.
A) Phage Diversity and their Classification.
B) Phage Life Cycles.
C) Adsorption: First Step Toward Infection.
D) Preventing Phage Adsorption: First Line of Defense
E) Genome Injection or Ejection?
F) Superinfection Exclusion: A Prophage Protects Against Phages
G) Restriction-Modification Systems: Cutting Genome of Phages
H) CRISPR-Cas: Adaptive Immunity against Phages
I) From Bacterial Cell to Virocell
J) Viral Genome Replication
K) DNA Packaging and Phage Assembly
L) Host Cell Lysis: Three Steps and Two Choices to Release Phages
M) Abortive Infection: Bacterial Altruistic Suicide
3- Phage Therapy Part II: A New Hope
A) Clinical Experience of Phage Therapy: Does It Work?
B) Compassionate Phage Therapy around the World: Sporadic Successes
C) The Importance of Dosage and Timing
D) Choosing the Right Phage
E) Phage Product Formulation
F) Phage Cocktail Design Strategies
G) Interactions Between Phage Therapy and the Immune System
Results
1- A bacteriophage protein favors bacterial host takeover by impairing stress response.
2- The combination of bacteriophages prevents resistant outgrowth in vitro but does not increase pulmonary phage therapy efficacy in immunocompromised animals.
Perspectives
1- A bacteriophage protein favors bacterial host takeover by impairing stress response.
A) Hypotheses and Complementary Work
B) From a Protein of Unknown Function to a Potent New Drug
2- The combination of bacteriophages prevents resistant outgrowth in vitro but does not increase pulmonary phage therapy efficacy in immunocompromised animals.
A) Hypotheses and Complementary Work
B) Phage Receptor Cocktail Design
C) Exploiting Phage Resistance to Steer Bacterial Evolution
D) Phages/Antibiotics Combinations: Everything Can Happen
References .
