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Chapter 41Pseudomonas aeruginosa Weihui Wu1, Yongxin Jin1, Fang Bai1 and Shouguang Jin1,2 1 Nankai University, Tianjin, China, 2University of Florida, Gainesville, FL, USA INTRODUCTION Because of the large genome, P. aeruginosa encodes a large number of enzymes for various metabolic pathways, confer- Pseudomonas aeruginosa is a Gram-negative, rod-shaped, ring high nutritional versatility. In addition, about 8% of the asporogenous, and monoflagellated bacterium. It has a pearl- genome encodes regulatory genes, which enables the bacte- escent appearance and grape-like or tortilla-like odour. rium to adapt to complex growth environments. P. aeruginosa grows well at 25
C to 37
C, and its ability to grow at 42
C helps distinguish it from many other Pseudomonas species. P. aeruginosa is a ubiquitous microor- PATHOGENESIS ganism which has the ability to survive under a variety of P. aeruginosa encodes numerous virulence factors that environmental conditions. It not only causes disease in plants enable it to establish various human infections. It has and animals, but also in humans, causing serious infections evolved a complicated regulatory network to control tem- in immunocompromised patients with cancer and patients poral and spatial expression of selected virulence factors suffering from severe burns and cystic fibrosis (CF). for maximum benefit for bacterial survival. Most strains of P. aeruginosa produce one or more pigments, including pyocyanin (blue-green), pyoverdine (yellow-green and fluorescent), and pyorubin (red-brown). Adhesins Previous investigations have suggested that pyocyanin not Adhesion of P. aeruginosa to the host tissues is a crucial only contributes to the persistence of P. aeruginosa in the early step in infection and pathogenesis. Regardless of lungs of CF patients, but also interferes with many mam- biotic or abiotic surfaces, P. aeruginosa exploit this prop- malian cell functions, including cell respiration, ciliary erty to colonize, replicate, overcome environmental shear beating, epidermal cell growth, calcium homeostasis and forces and obtain nutrients. Generally, the surfaces that prostacyclin release from lung endothelial cells [1]. pathogens attach to are specific. P. aeruginosa uses cell- However, the precise molecular mechanism mediated by surface components or appendages to promote the attach- pyocyanin pathology is unknown. ment to cells or to inanimate surfaces. There are at least P. aeruginosa strains produce two distinct types of three different adherence factors or adhesins: type IV pili, O antigen (O-Ag): a common polysaccharide antigen mediating adhesion to epithelial host cells; flagella, (A-band) composed of a homopolymer of D-rhamnose, and binding to mucin on epithelial cells; and the core an O-specific antigen (B-band) composed of a heteropoly- oligosaccharide of LPS, mediating adhesion to the cystic mer of three to five distinct sugars in its repeat units. So far, fibrosis transmembrane conductance regulator (CFTR) of P. aeruginosa isolates have been classified into 20 epithelial cells. serotypes by the International Antigenic Typing Scheme (IATS) [2]. The lipopolysaccharide (LPS) of P. aeruginosa is less toxic than that of other Gram-negative rods, facilitat- Type IV Pili-Mediated Adhesion ing its establishment of chronic infections by eliciting a low P. aeruginosa surfaces have type IV pili (T4P) that are inflammatory response [3]. retractable and flexible filaments. Pili filaments are homo- The genome of P. aeruginosa consists of a single circu- polymers of pilin protein and have an average diameter of lar chromosome. P. aeruginosa has a relatively large 5.2 nm and length of 2.5 μm. T4P of P. aeruginosa have genome (5.57 Mb) and high G 1 C content (6567%). multiple functions, such as surface motility, biofilm and Molecular Medical Microbiology. DOI: http://dx.doi.org/10.1016/B978-0-12-397169-2.00041-X © 2015 Elsevier Ltd. All rights reserved. 753 aeruginosa [4]. Among the seven secretion systems in the Gram-negative P. aeruginosa displays reduced cassette) transporter. aeru. P. aeruginosa are required for multiple The type I secretion system (T1SS) is composed of functions. T4bP include a in in vivo and in vitro conditions [9]. immune evasion. Flagella in P. CFTR trans. the vir- this channel plays an important role in maintaining the ulence factors are injected directly into the cytosol of host . which favours the sur- Additionally. moderate mucus of epithelial cell membranes. There are two types of T2SS in P. depending on the cell surfaces. CFTR mutant has been shown to enhance eukaryotic cells. aeruginosa. trans. aeruginosa infections. Several studies have demonstrated LasA. cells of the host. such as adhesion. sequence 108117. of heparan sulphate proteoglycans (HSPGs) on polarized The type II secretion system (T2SS) uses a two-step epithelial host cells. which results in the activation of the process to deliver extracellular proteins. There are two subfamilies of T4P. which binds haem from that activate the host innate immune system [8]. have a C-terminal secretion signal. P. type infection and pathology when infected with P. although there are a large tains five secretion systems: type I. The P. Flagellin can zation of iron and requires has genes. motility and biofilm forma- an outer-membrane protein and an ABC (ATP-binding tion. many other proteins for subsequent internalization of the ginosa lacking T4P results in a loss of adherence to P. The proteins secreted by the T2SS include or ten monosaccharides. lar domain of CFTR. aeruginosa has evolved a number of complex secretion systems.754 PART | 6 Disseminating Bacterial Infections microcolony formation. which lubricate and in the extracellular secretion of the alkaline protease protect tissues from pathogens. Conversely. into the periplasm and the second step involves T2SS complex-mediated further secretion into the extracellular Core Oligosaccharide (OS) of LPS-Mediated space. T4aP interact with the glycosphingoli. type II.or Tat-dependent delivery from the cytosol the PI3K/Akt signalling pathway and bacterial invasion [6]. aeruginosa and is a vaccine target. Each of the CFTR. A CFTR formation of DNA. aeruginosa infection [10]. aeruginosa has a single polar flagellum which is com- bacteria that have been found so far. and subsequently involves Sec. Recent haemoglobin. Both types of P. aeruginosa: the Binding of CFTR Xcp (extracellular protein) and the Hxc (homologous to Core OS is a branched oligosaccharide that contains nine Xcp) systems. D-region structure. PrpL. LasB. type III. that the core OS is the ligand for the CFTR receptor and The type III secretion system (T3SS) can inject toxic mediates the internalization by epithelial cells. which is only exposed at the tip of T4aP [5]. Upon ports chloride ions across epithelial cell membranes and cellcell contact between bacterial and host cells. Mucins can be divided into AprA. aeruginosa con- posed of a flagellin named FliC. ponent for P. The other T1SS involves utili- mucin have been reported to bind flagella. So the function of the core OS-specific binding to CFTR is pids (GSLs). the subtype called the tight adherence pili (Tad). which is located in the first extracellu- T4P play an important role in adherence to host epi. HasAp is considered to be a crucial com- studies have demonstrated that at the basolateral surface. T4P can trigger the host immune defences vival and propagation of P. to induce subsequent host Secreted Toxins and Exoenzymes PI3K/Akt signalling pathway and bacterial invasion [6]. About 90% of the adhesion capability of mation of membrane lipid rafts that contain CFTR and P. The proteins secreted by the T1SS invasion of epithelial host cells and pathogenicity [7]. as receptors on the apical surface of polarized epithelial host cells. which is a virulence factor involved in various secreted mucins and cell-associated mucins. The interaction can activate the for- thelial cells.and lipid raft-mediated internalization of bacterial three classes of T4P has a distinct assembly system. adhesion. defect results in mucus overload. exotoxin A and phospolipase C. cell signalling and phage attachment. and the receptor-binding site is buried in the infection sites. proteins directly into the cytosol of eukaryotic cells. type number of genes involved in flagella assembly and func- V and type VI. The first step epidermal growth factor receptor (EGFR). The secreted pro- also bind to asialo GM1 and Toll-like receptor 5 (TLR5) tein HasAp is a haemophore. aeruginosa to host cells is dependent on T4P. Recent studies have discovered T4P can also bind to N-glycans. The Apr system is involved flagella-mediated adhesion are mucins. tion. aeruginosa. aeruginosa. aeruginosa IVa pili (T4aP) and type IVb pili (T4bP). which deliver virulence factors either into the Flagella-Mediated Mucin Binding extracellular environment or into the host cell cytosol. There are two types Several studies have shown that the receptors for of T1SS in P. Disruption of T4P results in LPS outer core specifically recognizes CFTR amino acids reduced virulence of P. P. aeruginosa survival in the early stages of flagella mediate adhesion to heparan sulphate (HS) chains infection [11]. Flagella-defective P. asialo GM1 and asialo GM2 on epithelial both advantageous and disadvantageous. T4aP cells in corneal epithelial cells has been demonstrated to are the dominant adhesins in adherence to the epithelial develop keratitis caused by P. 41. aeruginosa can secrete two different types of PLC. PE. aeruginosa and have a wide translocase function of the C-terminal portion of the protein range of substrates. alkaline protease and member of the repeats in the toxin (RTX) family of pro- phospholipase C. The effectors for the 40 kDa. result. protruding from the cytosol to pre-proenzyme (53. AP enzymes termed mono-ADP-ribosyltransferases. oncogenesis and (Fig. One of the substrates of elastases is (auto-transporters) or another helper protein (two-partner elastin. the synthesis of diacylglycerol (DAG) from the hydrolysis domain II (amino acids 253364) helps PE to cross of phosphatidylinositol or phosphatidylcholine (PC). eukaryotic cells. (ENaC) in CF [18]. including apoptosis. The pro. Various toxins and exoenzymes are secreted through which is also necessary for its secretion [17]. Recent ing in the inhibition of protein synthesis and cell death. The N-terminal domain Ia (amino Phospholipase C (PLC) in eukaryotic cells catalyses acids 1252) is responsible for the receptor binding. face of the protein. otic cell by binding to a special receptor on the surface of AP consists of two domains. It can also degrade human gamma Exotoxin A (PE) is one of the most important virulence interferon (IFN-γ). and the other is the otic protease furin is necessary for its toxic effect on non-haemolytic PLC (PlcN). P. including exo. structural domains. like the T2SS. A 22-kDa HSI-I T6SS are Tse1. dependent secretion into the periplasm. one is the haemolytic PLC (PlcH). ADP-ribosyltransferase activity of this protein domain (PD) with a metal-binding motif. . HSI-II and HSI-III. Both LasA and LasB have elastolytic activity and are The type VI secretion system (T6SS) is also a needle. also neces- two-step process secretion system. LasB is synthesized as a phage tail-like structure. Following Sec. and domain III (amino acids process of DAG metabolism is very important for various 405613) has ADP-ribosyltransferase activity [15] signalling processes. shown to enhance the elastolytic activity of Pseudomonas tion in the environment [12].1 Schematic representation of the struc- tural and functional domains of exotoxin A. aeruginosa. presumably for the purpose of competi. The HSI-II T6SS was shown to peptide (2. an N-terminal proteolytic the cell. the pro-peptide (18 kDa). a an endoplasmic reticulum retention sequence. The cellular membranes. which target other LasA fragment is also found in P. aeruginosa. the above-mentioned secretion systems. aeruginosa and was bacterial species. Tse2 and Tse3. is an important virulence factor in P. AP can activate the epithelial sodium channel virulence. all of which contribute to bacterial teins. Recent research has also elastase and other proteases [16]. the proteins are Elastases are the most abundant among the many pro- secreted through the outer membrane utilizing the teases that are secreted by P. high stability against most proteases. The protein uses an while the function of the HSI-III T6SS is not known. aeruginosa. C-terminal domain with a Ca21-binding motif. It is a member of a class of stimulated neutrophil oxygen consumption. The precursor of like complex. mature. (amino acids 609613) at the C-terminus of PE which is The type V secretion system (T5SS) is.6 kDa). secreted protease (33 kDa).6 kDa) with three domains: the signal the bacterial surface [13]. and inhibit opsonized zymosan- factors in P. Alkaline protease (AP) is a Zn21-metalloprotease. and the enhance bacterial internalization in epithelial cells [14]. Therefore. elastases. The cleavage of the motif by eukary. secretion of AP depends on the T1SS in P. LasB protease is a found that the HSI-I T6SS needle is a dynamic contractile neutral zinc metalloprotease. rhaminolipid. sary for the toxicity of the protein. One is located in domain II and exposed at the sur. The 40-kDa LasA can degrade elastin. Proteins secreted by the T5SS include EstA.1). The tein is secreted through the T2SS and enters into the eukary. secreted by the T2SS of P. There are two important amino acid motifs in inflammation. aerugi. The second important motif is REDLK causing damage to the host cells. the extracellular LasA protein has a molecular weight of nosa: HSI-I. research has found that Ca21 binding can induce AP fold- The structure of PE has been elucidated and has three ing and secretion [19]. Chapter | 41 Pseudomonas aeruginosa 755 cells through needle-like bacterial surface structures. and a modifies and inactivates elongation factor 2 (eEF-2). Three T6SSs have been found in P. a toxin A. aeruginosa. Recent research has FIGURE 41. autoproteolytic mechanism to remove the pro-peptide. which is the element of the connective tissue with secretion). LepB and LepA. Without a functional type III The T3SS of P. apoptosis of infected host cells twitching motility which is also due to increased rhamno. and causing nosyl-L-rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate mortality. whose capable of inducing apoptosis at high frequency in epithe- expression is regulated by a quorum sensing (QS) system. They are glycosides and have three specifically interact with the C-terminus of the ExoS. L-rham. The nee. rial persistence in the lung. ExoT. the toxic effect of ExoS is depen- (dirhamnolipid) and 3-(3-hydroxyalkanoyloxy)alkanoic dent on its ADP-ribosylating activity. How low calcium or host cell contact leads to the model and a rat lung infection model. The ExoS preferentially ADP- lung surfactant [20]. It can activity and a GTPase-activating protein (GAP) activity. ribosylating activity requires host 14-3-3 proteins which ious chain lengths. Iron-limiting conditions promote Chlamydia species. PlcH is also responsible for the destruction of the adenylate cyclase ExoY. and further analysis showed still not very clear. ExoT. such as low calcium [23]. rlhB and rhlC. In humans. freeing ExsA to acti- effector molecules. infection activation of type III secretion is not yet understood. lial and fibroblast cell lines. for lipid production [22]. is the two systems has also been demonstrated [24]. favours clearance of bacterial pathogens. secreted from the cell through the type III secretion appa- dle is presumed to be inserted into the host cell to inject the ratus. Components of the type III (ExsD) which inhibits its activator function. plants. called PlcB. normally bound to ExsC. Most importantly. such as P. The T3SS is composed of more than 30 proteins that and direct contact with host cells. Mutants of the exoS gene The function of the rhamnolipids in P. Generally. In a mouse model three rhamnolipids species are L-rhamnosyl-3-hydroxy. aeruginosa. aeruginosa [27. also to modulate swarming motility and affect biofilm Apoptosis can either be advantageous or detrimental architecture in P. and also mucociliary transport and clearly observed in human and animal tissues following ciliary beating [21]. There are four known effector molecules: ExoS and pathway. aeruginosa. ExsA is bound by a repressor evading host immune attack. aeruginosa harbours multiple regulators to tightly effector proteins.inducing condition. The parts: a glycon part and an aglycon part linked to each other ADPRT activity of ExoS causes programmed cell death in via an O-glycosidic linkage. PlcH and PlcN are with a type III-secreting isolate correlated with severe dis- secreted by the T2SS and PlcB is secreted via the Sec ease. vate T3SS operons [29]. Apoptotic cells were of macrophages. degrade PC and sphingomyelin in eukaryotic cell mem. . However. aeruginosa biofilms. however. an acute cytotoxin ExoU with lipase activity. Excess ExsC now binds ExsD. aeruginosa is failed to induce apoptosis. ExoU and ExoY. an acute mouse pneumonia ExsE. A successful control of respiratory Toxins Directly Injected into the Host Cells infection requires that the host maintain the right balance P. extracellular pathogens.28]. aeruginosa. ExoS plays a significant role in bacte- decanoyl-3-hydroxydecanoate (monorhamnolipid). ultimately regulators. Numerous studies indicate that PlcH is a viru. Expression of the T3SS assemble into a ‘needle-like’ complex on the bacterial cell genes is under the control of a transcriptional activator. of acute pneumonia. rhamnolipids are required in the detachment from in infections involving intracellular pathogens. There is evidence that the to the host throughout the course of infection. encodes a T3SS involved in the secretion of at least four P. including ExoS. expression of the whole T3SS regu- important virulence mechanism. acid (HAA) in P. aeruginosa strains harbouring the exoS gene are carried out by gene products of rlhA. the rhamnolipids were found infection by P. and an branes. all of which contribute to cytotoxicity at different levels responding to environmental stimuli. control the expression of the large T3SS gene cluster. aeruginosa also a chronic disabling disease. a small intracellu- homology with that of flagella and a structural similarity of lar repressor protein (ExsE). Its ADP mono. as in the case of P. The most frequently seen various types of cultured cells [25. Normally. cell proliferation and differentiation.or dirhamnose linked to 3-hydroxy fatty acids of var.756 PART | 6 Disseminating Bacterial Infections identified a new PLC. Rhamnolipids are composed of transport. Interestingly. Rhamnolipid biosynthesis is P. yeast and insects. affecting the bacterial lon is repressed due to the accumulation of intracellular infectivity in a burn model. as failure infections due in part to the numerous virulence factors to do so may convert an acute self-limiting infection into mentioned above. aeruginosa has been shown to be an secretion apparatus. both having an ADP-ribosyltransferase (ADPRT) lence factor for mammals. Under a secretion machinery share significant amino acid sequence type III. surface. Later.26]. dissemination. belonging to the AraC family of transcriptional the cytoplasmic compartments of host cells. P. ribosylates several of the Ras family of GTP-binding Rhamnolipids are biosurfactants and are surface-active proteins required for the regulation of intracellular vesicle amphipathic molecules. designed to deliver effector molecules directly into ExsA. apoptosis of host inflammatory cells may be advantageous to the pathogens. aeruginosa is capable of causing various human tissue between apoptotic and anti-apoptotic pathways. Rhamnolipids were initially identified that the ADP-ribosylating activity of ExoS is essential for as a heat-stable haemolysin which can affect the function inducing host cell apoptosis [26]. (iii) periplasmic transfer and modification. Alginate seems to conversion in vivo to highly mucoid colony morphology protect P. AlgR. aeruginosa is a leading cause of morbidity and mortal. Regulation of alginate synthesis is complex. aeruginosa: (i) precursor synthesis. Twelve genes. allows P. resulting in the activation of σ22 and alginate overproduc- tion. the degradation of MucA (Fig. such as T3SS expression and to persist in the lungs of CF patients. Several important proteases and mechanisms relate to FIGURE 41. due to alginate overproduction. complement activation and decreasing phagocytosis by neu- ity in CF patients. 41. 41. involving many regulatory genes responding to various environmental cues. 41.3 Regulation of alginate biosynthesis in P. It is considered to be flagellum. MucA is an inner-membrane protein with one transmem- brane domain and acts as an anti-sigma factor by binding to σ22. Alginate protects P.2 Alginate biosynthesis pathway and relevant genes. MucA is degraded. and (iv) export through the outer membrane (Fig. alginate overproduction influences the expres- D-mannuronic and L-guluronic acid. encode the core alginate biosynthesis machinery. In response to environmental stress signals. AlgB and AmrZ [31] (Fig. (ii) polymerization and cytoplasmic membrane transfer. it has Overproduction) notable effects on the biofilm architecture. These gene products are responsible for the four steps of alginate biosynthesis in P. The MucA encoding gene is in the same operon as algU where the algU gene is followed by four downstream genes called ‘mucAmucBmucCmucD’.2). Recent research suggests a high level of coordi- the main cause of a poor prognosis and high mortality. . aeruginosa from the host immune response by inhibiting P. Chapter | 41 Pseudomonas aeruginosa 757 Mucoid Conversion (Alginate Although alginate is not essential for all biofilms. aeruginosa from various environmental stresses. nated regulation with respect to virulence. aeruginosa. a polymer of Meanwhile. as well as by sequestering the free strains infecting the CF pulmonary tract is the frequent radicals that are released from these cells. Alginate. aeruginosa sion of other virulence factors. designated the algD operon.3). with AlgT/U serving a major central role [30]. AlgU/T or σ22 is essential for alginate production. The algD promoter is regulated by the σ22 factor and three tran- scription factors. MucA22 is a mutant FIGURE 41.3). The most striking characteristic about trophils and macrophages. the Las sys- AlgW and alginate overproduction. AlgW has a PDZ tem is found to play an important role in the formation of domain. but FimS and AlgR alginate-overproducing P. aeruginosa. quorum sensing. undergo autolysis. P. fimS-algR immune systems. Biofilms formed by production in mucoid P. coli DegS. The last gene in the operon AlgR. biofilm. Recent research shows that it may be indepen- patients. aeruginosa. Among three known QS systems. aeruginosa strains show are both required for type IV pilus. QS plays an important role in P. Interaction with Other Virulence Factors Mucoid conversion is always accompanied by lower tox- icity. rhamnolipid production and motility [32]. exotoxin A. The psl locus encodes the cognate environmental sensor for AlgB. The algBkinB mannose-rich polysaccharide that plays an essential role genes form an operon that is also under σ22 control. FimS is a sensory component that Biofilms show high tolerance to antibiotics and the host interacts with AlgR. AlgR is also a global regulator that not only immune systems. aeruginosa iron regulation. but negatively regulates flagellum that the DNA in biofilms is similar to P. DNA is also a major matrix component of P. encoded upstream of algR. AmrZ positively regulates type IV pilin expression ate the dispersal of mature biofilms. whereas high quinolone signals result in It is the functional homologue of E. AlgR also controls highly structured architecture which is thought to result hydrogen cyanide production. aeruginosa geno- biosynthesis and self-expression. biofilms formed in seems to form one operon which is under the positive the CF lung result in antibiotic resistance and are believe control of σ22 factor. a peri. aeruginosa and even initi- sis. including the non-mucoid P. but can form biofilm normally. and KinB is a negative film formation [35]. Pellicle is a regulation is triggered by degradation of MucA by activated glucose-rich polysaccharide and the pel locus contains intramembrane proteases AlgW and/or MucP [32]. protecting it from proteolysis. aeruginosa biofilm plasm. Researchers found that DNase treatment can AmrZ (AlgZ) is also essential for alginate biosynthe. MucB is a negative regulator of σ22 factor. and an mature biofilm in P. aeruginosa biofilm matrix. T4P involved in the proteolysis of abnormal proteins that is and protease IV. called pellicle. For P. This lysis is regulated by P. and the QS system is also linked to swarming and DNA release. elastase. Downstream of algB is kinB which encodes encoded by psl and pel loci. those involved in carbohydrate metabolism. Such at a liquidair surface. aeruginosa. Clearly. which is required for proteolysis of MucA. These two domains pel operon decreases in the QS system defective strain cooperate to regulate the proteolytic activity of AlgW [34]. thus a model suggests membrane stress activates formation. which is . aeruginosa tiple virulence factors of P. aeruginosa in CF patients through approximately 58 genes and represses the Rhl QS system. CVS regulates the expression of many invasive viru- nate overproduction. quinolone QS signals. which encodes another negative regulator of algi. suggesting that this DNA may be the result of as both a transcriptional activator and a repressor of mul. as mutations in quinolone QS do not AlgW is a positive regulator of alginate overproduction. whole-cell lysis. aeruginosa [33]. inhibit biofilm formation in P. MucD is a periplasmic protease lence factors. activates the expression of in persistence of P. which high autolysis. which can bind to three different sites upstream of Biofilm Formation the algD promoter. Biofilm formation and QS gene expression are maximal at low iron concentrations. dent of alginate production. The plasmic protein that binds the periplasmic domain of cAMP/Vfr-dependent signalling (CVS) pathway is MucA. The function of defective in mucA mutants. Infections by P. such as by the T3SS. aeruginosa are very difficult to eradicate tissue-damaging proteases LasA and LasB. regulates alginate synthesis but also other genes including Some P. aeruginosa strains cannot produce alginate virulence factors. Many invasive virulence factors are down- Antibiotic Resistance regulated in mucoid isolates. aeruginosa. aeruginosa can also form biofilm regulator of alginate production in wild-type strains. The biofilms formed by AlgB is needed for algD expression in mucoid these strains are composed of a polysaccharide matrix P. which depend on AlgU and MucC has not been elucidated. and flagellar due to their high resistance to antibiotics. FimS is not required for alginate to be the major cause of mortality.758 PART | 6 Disseminating Bacterial Infections variant commonly associated with mucoid isolates from CF motility. is mucD. AlgB in cellsurface and cellcell interactions as well as bio- regulates alginate gene expression. The transcription of the LA loop that prevents MucA binding. including T3SS. required for resistance to oxidative and heat stresses. AlgR is a protein of the two-component regulator family. responds to misfolded or accumulated proteins in the peri. promotion of resistance to antimicrobials and host Therefore. but do not depend on alginate overproduction. KinB genes for the synthesis of the glucose-rich component of and AlgB also control a large number of genes. but dependent on AlgU. Further studies show and twitching motility. AmrZ functions mic DNA. Two mechanisms contribute to the high expression level play important roles in the resistance of aminoglycoside of ampC. homologues of Resistance Genes AmpD (AmpDh2 and AmpDh3). AmpE.4 Activation of ampC gene expression under various growth conditions. aerugi- tion. Although not as common as resistance mechanisms. attributable to both intrinsic resistance and acquired resis. leading except the carbapenems [36]. In the case Efflux-Mediated Resistance of derepression.38]. mutations in the ampD structural gene are The reduction of drug accumulation in the cytoplasm is the cause. Chapter | 41 Pseudomonas aeruginosa 759 FIGURE 41. these regulatory mechanisms are not well understood and All four classes of β-lactamases have been identified in need further study. Aminoglycoside modifications. The three most generally studied ampD-related mutations. The full derepression of AmpC in P.4). which are common determinants in P. while the other Aminoglycosides is a class D oxacillinase encoded by poxB. which is Genes Mediating Resistance to encoded by ampC. as well as rRNA methylases. to antibiotic inactivation. belongs to class C. Two classes of β-lactamases are typically harboured on the chromosome: cephalosporinase. P. When the (AMEs). There . induction and derepression. 41. efflux pumps and membrane impermeability. uncommon [39]. the mutated AmpR link to dere- resistance mechanisms in P. increased and its binding to AmpR converts AmpR into transcriptional activator for the ampC promoter. aeruginosa encounters specific β-lactams or nosa that are resistant to aminoglycoside are found to be β-lactamase inhibitors that can bind to penicillin-binding carrying more than one modifying enzyme and exhibit proteins (PBPs). The latter mainly refers to acquisition of resistance concentration of muropeptides in the cytoplasm perma- genes on mobile genetic elements. resulting in an increase in ampC expression (Fig. aeruginosa has been described. culminating in a constitutive elevation of resistance primarily refers to chromosomally encoded ampC expression (Fig. commonly observed in clinical isolates. while the intrinsic nently increases.4). Some clinical strains of P. In these also an important mechanism to resist antibiotics. and PBP4 also are ampC-Mediated Resistance to β-Lactams involved in the regulation of ampC expression [37. In the case of induc- among clinical isolates. when P. so the tance. the AmpD amidase is modified or decreased. aerugino- expressional level of AmpC is significantly increased. strains. the concentration of muropeptides is broad-spectrum aminoglycoside resistance [40]. 41. aeruginosa is resistant to almost all classes of β-lactams. The expression Resistance to aminoglycoside is due to acquired or chro- of AmpC can be induced when encountering benzyl peni- mosomally encoded aminoglycoside-modifying enzymes cillin and narrow-spectrum cephalosporins. sa except in CF isolates where these mechanisms are P. genes. aeruginosa is compli- cated and not always a single-step process compared with the case in Enterobacteriaceae. aeruginosa are the resistance pression of AmpC in P. aeruginosa. In some accumulation: one is through membrane impermeability. novobiocin. OpmG. NalD and NalC. The MexCD-OprJ efflux pump shares a high degree of Membrane Impermeability similarity to MexAB-OprM. indi- of mexAB-oprM is regulated directly or indirectly by three cating that this pump is involved in intrinsic resistance. repressors. is capable of in clinical isolates [45]. The homology with MexAB-OprM and MexCD-OprJ. aeruginosa. chloramphenicol and tetracycline. dysfunction of these specific porin channels expression of MexCD-OprJ. Deletion of strains. fluoroqui. aeruginosa and is most romycin. and is involved in intrinsic resistance. Certain hydrophilic antibiotics. a transcriptional acti. which are protein for the β-lactams compared to MexAB-OprM. Mutations in mexZ or the mexZ- OprM [41]. aeruginosa genome has Transcription of mexCD-oprJ can be observed in wild-type identified 163 known or predicted outer-membrane cells. MexXY is the frequently linked to β-lactam resistance in clinical iso. such as tor. The MexEF-OprN efflux pump also shares amino acid The major general porin in P. tetracyclines. as well as its own expression. MexR binds as a The expression of mexXY is controlled by a single regula- stable homodimer to the mexA promoter and represses tor. expression of mexXY. The loss of another porin. but a mutational event within lates and associated with hyperexpression of mexXY [43]. Expression mexXY increases susceptibility in wild-type strains. trimethoprim. which is located upstream of MexCD-OprJ but β-lactams. Efflux stimulation of MexT requires mutations within a gene. and a toxic metabolites [42]. chloramphenicol. membrane protein. There are several different clinical isolates. leading to the hyperexpression of MexCD. trimethoprim and novobiocin. fluoroquinolones. inactivating mutations are not present in mexT. The substrates of MexCD. aminoglycosides. which locates upstream of mexXY but tran- transcription of the mexAB-oprM operon. hydrophilic molecules from pass- col. can lead to decreased susceptibility of P. porin channels that contribute to the inherent resistance OprJ. channels that span the outer membrane and are water- entially exports the fourth-generation cephalosporins. strains. iates aminoglycoside resistance in P. On the contrary. an outer-membrane factor (OMF). macrolides and β-lactams. aeruginosa to Mutations in nfxB have been described in laboratory and certain antibacterial agents. cefepime). to the nfxB-mexC intergenic region negatively regulates the Conversely. but its loss of OprF has not been found to be a major cause of regulation is unique. The mechanisms involving OprD- positively regulating its expression. All Gram-negative bacteria have an outer membrane that OprJ include tetracycline. macrolides.760 PART | 6 Disseminating Bacterial Infections are two ways to achieve the purpose of reducing drug can convert inactive MexT into an active form. Sequence analysis of the P. eryth- drug efflux pump discovered in P. MexT. pumps belonging to the resistance-nodulation-division mexS. . In order to get inside the cell. belonging to the LysR family. Mutations in mexR. has negative regulator. filled. aeruginosa genome. mexZ. but it prefer. aminoglycosides. of porins [44]. aeruginosa is OprF. which is located upstream of mexT and encodes a (RND) family are the most significant contributors to putative oxidoreductase/dehydrogenase homologue. OprD. MexR. ing through.g. nalC and nalD have been mexX intergenic region have been described in clinical iso- reported in clinical isolates. Binding of MexZ to to the mexA promoter region but downstream of the mexR the mexZ-mexX intergenic region negatively regulates the binding sites. aeruginosa PAO1. Unusually. MexAB-OprM is also able to export many other types of The mexXY system lacks a gene coding for an outer- antibiotics. with a total of believed that inactivation of MexS causes a build-up of 12 RND systems encoded on the P. OpmH and MexAB-OprM is constitutively expressed in wild-type OpmI) to form a functional efflux pump. NalC is an indirect repressor of MexAB. these mole- MexCD-OprJ does not have an extensive substrate profile cules must pass through porins. including tetracyclines. been commonly linked with the resistance to carbapenems vator. wild-type mediated resistance include decreased transcription of strains carry inactivating mutations in the mexT gene. (e. RND pumps consist of a periplasmic membrane fusion which up-regulates mexEF-oprN expression to remove the protein (MFP). Besides β-lactams and β-lactamase inhibitors. some fluoro- transcribes divergently from the operon. cytoplasmic membrane (RND) transporter. Binding of NfxB quinolones. and the other is by membrane-associated pumps. It use OprM and possibly other nolones. aeruginosa to antimicrobial agents. naturally prevents large. metabolites that serve as effector molecules for MexT. NfxB. fluoroquinolones. only pump of the 12 identified RND systems that med- lates. MexEF-OprN is not suppressed by a antibiotic resistance. oprD and mutations that disrupt the translational produc- Additional cis-acting mutations or deletions within mexT tion of a functional porin for the outer membrane. NalD also binds scribes divergently from the operon. but the levels of protein cannot be detected. The MexXY efflux pump extrudes specific β-lactams The MexAB-OprM efflux pump was the first multi. of P. and quinolones. one of them may not be the sole mechanism to increase MexAB-OprM transcription. The proteins. outer-membrane proteins (OpmB. with 64 of these grouped into three families expression of MexCD-OprJ is controlled by a single regula. It is antimicrobial resistance in P. chlorampheni. can diffuse through porins. while pqs autoinduces PQS syn- susceptible host. and motility pheno- acute and chronic infections in a variety of hosts. thesis and further activates rhl system expression. In addition. produce AI molecules of 3OC12HSL. aeruginosa harbours three QS systems: two LuxI/LuxR- type QS circuits (las and rhl) that are based on the intercel- Regulation of Virulence Genes lular signals N-acyl homoserine lactones (AHLs). P. respond. and activate the expression of indicated target genes. the rhl system negatively formation and reduced virulence. aeruginosa QS systems are arranged hierar- infections are associated with motility and cytotoxicity chically with the las system positively regulating both the via the T3SS. aeruginosa is one of the the non-LuxI/LuxR-type 2-heptyl-3-hydroxy-4-quinolone- most complex systems known to date. During colonization of a regulates the pqs system. Chapter | 41 Pseudomonas aeruginosa 761 FIGURE 41. Quorum Sensing Regulation P. Additionally. cated and extensive array of regulatory networks to Moreover. LasI. and The signalling network of P. causing both factor production. aeruginosa control the switch between planktonic and biofilm life- membrane permeability can be regulated. respectively. P.5).5 P. RhlR and PqsR. Three autoinducer (AI) synthases. three QS systems are through the regulation of virulence and adapt to fluctuating environmental cues. while chronic infections relate to biofilm rhl and pqs systems. C4HSL and PQS. RhlI and PqsABCDH. biofilm maturation. Many traits controlled by these relies on numerous signalling pathways to sense. each of these systems is further modulated by a . and is the best based system called the Pseudomonas quinolone signalling studied among all microorganism systems. aeruginosa quorum sensing circuits. respectively. A novel styles and consequently influence whether they cause two-component regulatory system (PprA and PprB) was acute or chronic infections. identified and shown to regulate membrane permeability. The P. but the downstream genes responsible for such phenotype have not been identified yet [46]. Acute types. aeruginosa (PQS) system (Fig. 41. it has been demonstrated that P. The AIs are detected by the cytoplasmic transcription factors LasR. aeruginosa has evolved a sophisti. concentrations are achieved. rhlR gene expression. Binding responses before the cells reach ‘a quorum’. 41.6). genome sequencing has in any microorganism analysed thus far [48]. aerugi- nosa QS circuit. LysR-type receptor PqsR (also called MvfR) (Fig. LadS and RetS. in both cases activating PqsR-dependent altering the expression of multiple downstream virulence gene expression and virulence to levels similar to those genes. aeruginosa infec- been designated as orphan LuxR homologues [47]. controlled by LasR3OC12HSL. the Pseudomonas quinolone signal (PQS). encodes 55 HKs. including those responsible for the production of rhamno- lipids. which is gues lacking a LuxI-type cognate partner. has been GacS. which is also synthases (LasI and RhlI) to produce autoinducers N-3- capable of potentiating PqsR binding to the pqsABCD oxo-dodecanoyl homoserine lactone (3OC12HSL) and promoter. while RetS . GacA. Several additional accessory regulators modulate the Interestingly. the PQS circuit is intimately translational and post-translational level. which have central to the control of the course of P. rendering them inactive. QS molecule. to control viru. and alkaline protease.5). LadS (lost autoinduces PQS synthesis and further activates rhlI and adherence sensor) phosphorylates GacS.5). In a mouse acute pneumonia model. tiple genes involved in T3SS. meanwhile repressing mul- The third P. The PQS is favours chronic infection and results in increased inflam- then sensed and bound with high affinity by its cognate mation in the lungs of infected mice [49]. exotoxin A. thereby directly to PqsR. activation of the LasIR system promotes the later over 60 two-component systems. QscR likely controls a specific regulon phate group to its cognate response regulator (RR). designated iron homeostasis. respectively. the molecules recognize and where it either is converted into PQS by PqsH or binds activate their cognate receptors. Further.and which in turn promotes the expression of two small RhlR-dependent regulons and prevents aberrant QS untranslated regulatory RNAs. Both the Las and Rhl systems con. ulence phenotype via two-component regulatory systems.and PQS-dependent QS systems. AHL-Dependent QS Systems PQS-dependent signalling is critical in the P. aeruginosa possesses several LuxR homolo- critical two-component systems is GacS/GacA. 41. HHQ can be released into the extracellular N-butyryl-HSL (C4HSL). P. a pqsH scription of target genes involved in acute infection and mutant. loss of RsmA results in reduced colonization during the lence gene expression. 89 RRs and 14 HK-RR hybrids. Two sensor kinases. 41. forming Thus. When sufficient medium and subsequently taken up by neighbouring cells. Thus. Thus. and represses those responsible for assembly and function of the T3SS. a hierarchical relationship exists between the Las which are basically formed by two proteins: a histidine and Rhl systems: binding of the LasR3OC12HSL com- kinase (HK) and a response regulator (RR). tied to the LasI/LasR and RhlI /RhlR QS systems and therefore influences virulence factor production. including those encoding the LasA and does not display a defect in PqsR-mediated gene expres- LasB elastases. The LasR3OC12HSL complex activates tran- observed in response to PQS itself. expression of pqsH and pqsR is positively GacS/A system. PQS is produced by gene products initial infection stages of acute infection but ultimately encoded by the pqsABCD operon and pqsH. The pqsABCD gene products can direct The las and rhl systems use dedicated autoinducer synthesis of the precursor molecule HHQ. LasR and RhlR. RsmZ and RsmY. sion or virulence. T6SS) disease and thus the switch orphan LuxR homologue called QscR (quorum sensing between acute and chronic infections. QS and T6SS.762 PART | 6 Disseminating Bacterial Infections plethora of regulators that function at the transcriptional. host cell damage. aeruginosa plex directly up-regulates transcription of rhlR and rhlI. T4P) and chronic respond to endogenously synthesized AHL(s). GacS transfers a phos- reported. indicating that HHQ is also a functional RhlRC4HSL induces the expression of several genes. The GacS/GacA control repressor) that can form mixed dimers with system consists of a transmembrane hybrid sensor HK. aeruginosa QS system utilizes 2-heptyl-3. and possesses one of the activation of the RhlIR system (Fig. An (exopolysaccharides. PqsRPQS GacS/GacA/RsmYZ pathway (Fig. largest pools of two-component system proteins identified Besides RhlR and LasR. tion by inversely regulating the expression of virulence These highly conserved LuxR-type orphans may factors associated with acute (T3SS. tors (LasR and RhlR) induce transcription of their cognate P. Upon autophosphorylation. The Gac/Rsm Regulator Pathway tain an autoinducing feed-forward loop where their regula- In addition to the AHL. LasR and RhlR. type IV pili formation and hydroxy-4-quinolone as a signalling molecule. whereas the rhl system have been found to modulate gene expression via the negatively regulates pqsABCD and pqsR. Furthermore. that overlaps with the already overlapping LasR. aeruginosa controls its environmental lifestyle and vir- synthase genes that allow rapid signal amplification. which produces HHQ but completely lacks PQS. One of the revealed P. of RsmZ and RsmY to the small RNA-binding protein RsmA activates the production of genes involved in bio- 4-Quinolone-Dependent QS System film formation. Several pro- P. The sensor kinase LadS works in parallel to GacS. which bind to the vation conditions. secreted toxins. including: T4P. In a mouse model of acute pneumo. phorylation of GacA and leading to a reduction in RsmYZ expression. and phos- CyaB. cyclic di- GMP (c-di-GMP). the intracellular adenylate cyclases CyaA and domains responsible for c-di-GMP synthesis.52]. cyaA and cyaB mutants reveal an essential role for CyaB and Vfr during infection. is responsible for the production of these metabolites. plays an important role as a secondary messenger molecule in many bacterial species. forming a adaptation. The intra- Nucleotide-Based Signalling Molecules cellular concentration of c-di-GMP within a cell is cAMP/Vfr Signalling fine-tuned by the opposing actions of two types of The P. which is a clear consequence of the inability of directly interacts with GacS and prevents GacS auto. FimL and FimV increase and PilH. Infection studies using a mouse pneumonia model with vfr. Under amino acid star- the production of the small RNAs RsmZ and RsmY. aeruginosa. AlgQ up-regulates the pro- proteinprotein complex with GacS that blocks RsmY and RsmZ duction of the nucleoside diphosphate kinase Ndk. Furthermore. Another nucleotide-based signalling molecule. PilJ. GacA phosphorylation via GacS stimulates comprises the cellular alarmones.6 GAC regulatory network in P. Vfr negatively regulates the expression of flagellar genes by repressing expression of fleQ. Modulation of cAMP homeostasis occurs via the Chp (chemotaxis-like che- mosensory system) gene cluster in P. Small regu- The second group of nucleotide-based signalling mole- latory protein RsmA binds to the promoters of multiple genes. ChpC. Deletion of algQ leads to cell death in the late logarithmic (regulator of exopolysaccharide and type III secretion) phase. ExoY. Synthesis of cAMP is phodiesterases (PDE) containing EAL or HD-GYP driven primarily by CyaB and to a lesser extent by CyaA. establishing a link between Chp and T4P. this mutant to adapt from logarithmic growth to survival phosphorylation. In P. suggesting that these . these molecules rapidly accumulate in RsmA protein and inactivate it. where PilG. PilI. In domains involved in c-di-GMP degradation. leading to the hypothesis regulator). thereby contributing to cytotoxicity. EAL domains and thus may be capable of both synthesiz- homologous transcription factor Vfr (virulence factor ing and degrading c-di-GMP. Chapter | 41 Pseudomonas aeruginosa 763 infections. c-di-GMP Signalling nia. ChpA. aeruginosa. PilK and ChpB reduce cAMP levels. aerugino- sa and secreted via the T3SS directly into the cytoplasm of target host cells. mode. aeruginosa. while the sensor kinase cells. and positively regulates the various bacterial species have been identified that contain production of virulence factors important for acute degenerate GGDEF domains. where it modulates cAMP activity. interfering with the subsequent phos. aeruginosa. and the las and rhl QS systems. In contrast. which production. activating RsmZ and RsmY production. which encodes the master regulator of flagellar biogenesis. enhancing cules 50 -diphosphate 30 -diphosphate guanosine (ppGpp) several acute virulence factors while repressing virulence factors associ. a series of proteins in P. Additionally. ppGpp and pppGpp Signals FIGURE 41. mutations in mucA and consequent activation of AlgU and the response regula- tor AlgR have been reported to inhibit cAMPVfr signal- ling. and 50 -triphosphate 30 -diphosphate guanosine (pppGpp) ated with chronic infections. degradative enzymes. aeruginosa PAO1 genome encodes three adenylate enzymes: diguanylate cyclases (DGC) containing GGDEF cyclases. aeruginosa genes [51. and a secreted effector. the retS mutant is unable to establish infection [50]. ExoY is produced by P. T3SS. demonstrating that cAMPVfr signalling constitutes a complex signalling cascade with multiple regulatory inputs. cAMP influences gene regulation through its teins have been identified that contain both GGDEF and activity as an allosteric activator of the CRP. T2SS. Microarray analysis indicates that the that they act by balancing the internal cellular concentra- cAMPVfr complex controls the expression of nearly 200 tion of this molecule. triggering a switch from cell growth to survival RetS acts in an opposite manner to the LadS and GacS. synthesis of secondary metabolites. has been evaluated in a small-scale clinical tion with PopB protected mice against P. One QS identified to elicit Th17 responses. difficile colitis. Intranasal immuniza- inhibitor. sion and secretion of T3SS effectors [58]. PopB was recently models and interfered with biofilm formation. slow. affecting the growth of P.60]. OprF and OprI gave promising results that deserve further study. which is jeopardizing our fight against this bacte. For example. an effective vaccine is not yet available on the resistance is lower compared to traditional antibiotics.55]. Since the inhibition of viru. Natural and synthetic com- pounds have been identified to prevent the formation of ANTI-PSEUDOMONAS APPROACHES biofilm. which might be the primary Some compounds conferred protection in various infection mechanism for the protection [61]. such as EDTA and alginate lyase. Tests of vaccines based on affect normal flora as much as traditional antibiotics. PopD. surfaces of environmental stress adaptation. aeruginosa attach- is associated with control of biofilm formation and other ment blocking compounds decreased the colonization of group behaviours. cation of T3SS effectors. Together. Immunization against LPS or flagella resulted in Due to the specificity. trial for the treatment of lung infections in cystic fibrosis tion as allosteric sites or as c-di-GMP receptors. prevention approaches. Antibodies against PcrV blocked the translo- repress the production of QS-regulated virulence genes. [61] and antibodies against flagella inhibit the swimming tions. identified chemicals that inhibit the expres- tion are underway. aeruginosa terial dispersal. and clinical trials on vaccines against P. aeruginosa infec- Aiello et al. synergised with antibiotics in eradicating the biofilm rium. Chemicals that inhibit the ADP-ribosyltransferase activity of ExoS and the phospholipase activity of ExoU have also been identi- Targeting Virulence fied [59. and film and disrupt mature biofilm. Efforts have been made to prevent the formation of bio- cytotoxicity. Most of these compounds have been shown to model [63]. aeruginosa virulence factors. Based on the structures of the auto. several new treatment strategies are in the pipeline. motility. Inhibition of virulence factors disarms the pathogens. which leads to the rise of resis. some the development of new effective antibiotics has been biofilm inhibitors. these enzymes influence a wide range of phenotypes in diverse bacterial species including biosynthesis of Targeting Biofilm adhesins and exopolysaccharides. Vaccines and Immunotherapy which leaves them vulnerable to the host immune system and eventually being cleared. targeting virulence factors will not varying degrees of failure. PopB and PcrV. secretion. antibody tant strains. QS inhi- imposes a great threat to human health. Psl. alginate. patients and showed promising effects [53]. bacteria. such as Candida albicans infections or Clostridium motility of the bacteria [62]. antibodies can also pressure on the bacteria. there is an urgent need for new treatment and [56. mature biofilm can be disrupted. cation of T3SS effectors. Targeting Quorum Sensing The T3SS contains a needle tip complex. Research From large-scale screening of small-molecule libraries. Modulation of c-di-GMP medical instruments coated with P. inhibit bacterial virulence factors. composed of Numerous natural or synthetic compounds have been iden. such as bacteriophages and Targeting Type III Secretion System agents targeting P. garlic. the chance for the development of antibiotic However. Through degrading the matrix. exotoxin A tox- oid. Thus. When tested. lian cells from T3SS-mediated cytotoxicity without ria through inhibiting essential bacterial functions. In addition. which is required for the translo- tified as QS inhibitors. compounds have tion with PcrV significantly improved the survival of also been synthesized to compete with the autoinducers for P. In addition to natu- Application of these antibiotics imposes a selection ral and synthesized compounds. aeruginosa. Active and passive immuniza- inducers and corresponding receptors. At the same time. Presently. bitors also reduce biofilm formation. These inhibitors protected cultured mamma- Traditional antibiotics inhibit the growth of or kill bacte. great efforts have been made to lence factors usually does not affect the growth of the develop effective vaccines or therapeutic antibodies. elastase. aeruginosa-infected mice in a pneumonia or burn receptors. long-term use of antibiotics tends against PcrV blocks the translocation of effector proteins to destroy beneficial normal flora and induce superinfec. For example. the bacteria and reduced the chances of ventilator- associated pneumonia [54. and inducing bac- The increasing antibiotic resistance of P.764 PART | 6 Disseminating Bacterial Infections domains no longer generate c-di-GMP but instead func. market. In the past decades. alkaline proteases. aeruginosa .57]. Tremblay J. [11] Delepelaire P. Hachani A. one important aspect of research is to better structure. target cells. Vollmer W. J Mol Biol 2001. 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Henderson GP.and SR-type lipopolysaccharides of reference [19] Zhang L. [21] Johnson MK. [22] Glick R. especially its conversion internalization in epithelial cells. Garvis S. and secretion. O-units in the R. J Bacteriol 2010.287:3255665. Ho TC. et al. aeruginosa [13] Basler M. Golan DE. of Pseudomonas aeruginosa: a review. Understanding the mechanisms of antibiotic resistance and [12] Russell AB.50:80924. 247388. Jensen GJ. LeRoux M. terial resistance against most commonly used antibiotics. Mol Microbiol 2003.46:8599. [5] Craig L. Cystic underlying conditions. Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity. Type I secretion in gram-negative bacteria. S. et al. Zhang L. The other aspect relates 2012. PLoS One 2009. . Nature 2012. Zaidi T. Gilmour C.8:e1002616. PLoS Pathog shown to be essential for the protection [64]. Ohman DE. 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