These bacteria was multidrug resistance (MDR) from opportunistic pathogen in which infections with its mostly acquired in the hospitals causing acute or chronic infection in immunocompromised patients(ICPs) with chronic obstructive pulmonary disease (COPD), cystic fibrosis, cancer, traumas, burns, sepsis and ventilator related pneumonia(VAP) including those caused by COVID-19 [1-3]. This pathogen form sometimesbiofilms which help it's for survive in a hypoxic atmosphere and other abnormal conditions [4,5]. In addition to that treatments of infections with P. aeruginosa was extremely difficult due to its fast mutations occurred by this bacteria and adaptation to acquired antibiotics resistance [6]. In additions to that these bacteria also one of the top-listed pathogens found in surface and also have ability to grow and multiply in disinfection solutions [7].

Epidemiological studies appeared nearly 700,000 patients was died as a result from antibiotic resistance in European countries infection with these bacteria reach to 12.9% in each as a result from nosocomial infections (NCIs) complicated with antibiotics resistance in which become as first problem health [8,9]. In Spain country and in 2016 the researches showed that both Escherichia coli and P. aeruginosa were most common cause NCIs [10]. These study appeared that the ability of this bacteria maybe related to enzymes secretion, virulence factors, genes expression and antibiotics resistance [11] as shown in Figure 1.

Figure 1: Pathogenesis of P. aeruginosa in Human Internal organs

Virulence factors 

Pseudomonus aeruginosa can adapted to environment and suddenly external conditions by secreted a variety of enzymes and by help of other virulence factors, it had ability to invasive host and causes huge kinds of infections [12]. Lipopolysaccaride was important surface structural component to protect the external leaflet and posion host cells and the endotoxicity of the lipid A in LPS able to tissue damage, attachment and recognition by host receptors [13]. Endotoxin of this bacteria may be related to antibiotics tolerance and biofilm formation [14]. Second, Out Membrane Proteins (OMPs) contributes to nutrient exchange, adhesion and antibiotic resistance [15]. In addition, drug resistance caused by the formation of biofilms is associated with the flagellum, pili and other adhesions [16]. Fourth, there are six types of secretion systems, including flagella (T6SS-associated), pili (T4SS) and multi-toxin components type 3 secretion system (T3SS), which function at colonisation of the host, adhesion, swimming and swarming responding chemotactic signaling. Finally, exopolysaccharides, such as alginate, Psl and Pel, may help facilitate the formation of biofilms, while impairing bacterial clearance [17]. 

Pyocyanin is also toxic to hosts to cause disease severity, damage host tissue and impair organs’ function [18]. In addition, LasA and LasB elastases, alkaline protease (AprA) LipC lipases, phospholipase C and esterase A enzymes comprise a large group of lytic enzymes that modulate other virulence factors [19]. Moreover, rhamnolipids-mediated lung surfactant degrading and tight junction destroying can directly injure trachea or lung epithelial cells [20]. Furthermore, siderophores (pyoverdine and pyochelin) as iron uptake systems help in bacterial survival in iron-depleted environment to augment virulence strength [21]. Finally, antioxidant enzymes, such as catalases (KatA, KatB and KatE), alkyl hydroperoxide reductases and superoxide dismutases, neutralize activity of Reactive Oxygen Species (ROS) in phagocyte environments to avoid clearance [22]. Virulence factors related to membranes lipopolysaccharide as a virulence factor widely interacting with hosts and also target for vaccines. LPS, an important classical structural component of the Outer Membrane (OM) of most Gram-negative bacteria, is a known potent agonist that elicits robust innate inflammatory immunity, its distal end may be capped with O antigen, a long polysaccharide that can range from a few to hundreds of sugars in length, which is critical for bacterial physiology and pathogenesis [23]. At the early stage, scientists were interested in developing vaccines to prevent infection by focusing on LPS, which were later proven highly difficult due to the various serotypes and inefficacious outcomes [24-26]. 

Pathogen-Associated Molecular Patterns (PAMPs), as small molecular motifs conserved in a class of microorganisms, can be sensed by Toll-Like Receptors (TLRs) and other Pattern Recognition Receptors (PRRs) to activate innate immune responses, which effectively protect the host from infection [27]. LPS, as the prototypical PAMP, can be recognized by multiple host receptors, including TLRs, PRRs and nucleotide-binding oligomerization domain-like (NOD-like) receptors [28]. The LPS-PRRs/NOD-like molecules activate inflammasome to produce proinflammatory cytokines [29]. Activating TNF-α and IL-1β, two of the most eminent inflammatory cytokines [30]. Furthermore, LPS amongst five major Gram-negative bacteria have the ability to induce the production of NF-κB and proinflammatory IL-8 in a TLR4-dependent manner, suggesting that the pathogenesis of bacterial enhancement of chronic inflammatory diseases may be related to its serotype- specific LPS response [31]. 

Apparently, LPS exhibits a crucial role in regulating the interaction of bacteria with their host, is the main cause of tissue degeneration and chronic damage. LPS induces respiratory tract infections by regulating epithelial-mesenchymal transition (EMT)-mediated airway remodeling [32]. The mutations of LPS can result in attenuated virulence [33,34]. Caffeine alleviates the excessive inflammatory response caused by P. aeruginosa infection by inhibiting the activation of LPS-mediated TLR4/MyD88/NF-κB/miR-301b signaling pathway and improves lung tissue injury [35]. Notably, LPS mutations confer bacteria gain tolerance to phage infection, taken together, in addition to the direct interaction with the host PRRs receptors, LPS may use its unique molecular features to adjust bacterial pathogenesis and damage host immune defense, ultimately benefiting the fitness and invasive strength [36].

Bacterial Virulence Factors Associated withOuter Membrane Vesicles

The bacterial components that can be released spontaneously to the environment during growth by many Gram-negative bacteria. Bacterial-derived OMVs have been characterized as a novel secretion mechanism that can deliver a variety of bacterial proteins and lipids into host cells without direct contact with host cells [37,38]. These vesicles can package and enrich a wide variety of proteins and nucleic acids, including lipoproteins, periplasmic proteins (E. coli cytolysin A, enterotoxigenic E. coli heat-labile enterotoxin and Actinobacillus   actinomycetemcomitans leukotoxin), plasmid containing chromosomal DNA fragments, phage DNA, virulence factors (LPS, alkaline phosphatase, phospholipase C, β-lactamase, and, P. aeruginosa secretion of OMVs have been implicated in many virulence-associated behaviors, including the acquisition of drug resistance, the regulation of bacterial density and host immune escape [39,40]. Mechanistically, P. aeruginosa secretes OMVs to deliver virulence factors and sRNAs into lung epithelial cells through the diffusion the mucus layer, some studies also illustrate that OMVs could lead to an increased hydrophobicity of cell surface, resulting in enhanced ability to form biofilms [41]. These vesicales was controlled by quorum sensing systems, which enable bacteria to colonize and immune escape [42,43]. Interestingly, OMVs are naturally immunogenic and self-adjuvation, making them have potential to be developed as antibacterial vaccine, such as OMV vaccine for Neisseria meningitides, therefore, OMVs are not only an important functional constitute but also a potential biotechnological engineering carrier for vaccination or drug delivery. More details about virulence factors and their associated treatment strategies in P. aeruginosa are listed in Table 1 [44,45].

Mechanism of P. aeruginsoa Secreatory System

Gram-negative pathogens cause various types of nosocomial infections and secreted virulence factors often mediate their interactions with the host [46]. Bacteria can modulate the host immune response through the secretion system for secreting virulence factors into host cells, which facilitates immune escape and enable bacterial colonization, currently, 6 types of secretion systems (T1SS to T6SS) have been identified in P. aeruginosa. Based on the secretion routes of transport proteins, the secretion systems are divided into two major classes, one-step secretion system (T1SS, T3SS, T4SS and T6SS) and two-step secretion system (T2SS and T5SS). The one-step secretion system directly secretes proteins from bacterial cytosol to the surface, while the two-step secretion system requires a brief periplasmic stay of the secreted proteins on the export way and then releases the proteins to outside environments of the bacterium (Figure 2) [47].

Figure 2: Mechanisms of Antimicrobial Resistance in P. Aeruginosa

First step in Secretion System

Two different T1SS types in P. aeruginosa elucidated, Apr secretion system and Has secretion system, the Apr secretion system consists of three major components: AprD (ATP-binding cassette transporter, ABC transporter), AprE (adaptor), AprF (outer membrane factor, OMF) and secretes two proteins: AprA (alkaline protease) and AprX (protein with unknown function) [48]. T1SS is found in a variety of bacteria (P. aeruginosa Salmonella enterica, Neisseria meningitidis and E. coli) [49]. T1SS major transport proteins (such as proteases and lipases). The substrate protein containing a C-terminal uncleaved secretion signal were recognized by the ABC transporter, were directly transferred across bacterial inner and outer membranes in a one-step process [50]. The Has secretion system is composed of  HasD (ABC transporter), HasE (adaptor), HasF and OMF [51]. Has secretion system participates in iron regulation by secreting an extracellular haem-binding protein (hasAp) [52]. Thus far, data relating to T1SS is very limited and its function in pathogenesis and significance for bacterium physiology and fitness are largely unknown, requiring further elucidation in order to know whether it has potential important functions.

The T3SS of P. aeruginosa, playing a key role in virulence like quorum sensing, was first discovered in 1996 [53] and is one of the most extensively-studied secreted toxins with increasing evidence for its important virulent effects [54]. The T3SS regulon comprises five distinct operons, including the pscN to pscU, the exsD-pscB to pscL operons, the popN-pcr1-pcr2-pcr3-pcr4-pcrD-pcrR operon, the pcrGVH-popBD operon and finally the exsCEBA operon [55,56].

Table 1: Most of P. aeruginosa Pathogenic Factors 

Second Step Secretion Systems

Different from the One-Step secretion, Two-step secretion requires a brief periplasmic phase of the secreted proteins on the export route before being exported to the outside of the cell through general export pathways, which plays an important role in the transport of periplasmic and outer membrane proteins.

T2SS

The function of T2SS is one of the less characterized secretion systems and is thought to secrete folded proteins from the periplasm [57]. Two different pathways exist in T2SS: the general secretory (Sec) and twin-arginine translocation (Tat) [58]. The secreted proteins are first transited through the inner membrane, stays briefly in the periplasm and then secreted into the extracellular environment [59,60]. The Sec pathway consists of a protein targeting component, a motor protein and a membrane integrated conducting channel called SecYEG translocase, the secreted proteins with a SecB-specific signal sequence might be guided to the periplasm or the extracellular environment [61]. The twin-arginine translocation (Tat) pathway of Gram-negative consists of TatA and TatB, which can decide whether the secreted is retained in the periplasm or translocated to the extracellular space with a twin-arginine motif [62]. Functionally, T2SS participates in the secretion of guanylate cyclase ExoA, proteases lasA/B and multiple other factors and many of which have emerged as potential therapeutic targets [63,64]. 

The T5SS of P. aeruginosa is composed of five subtypes (type Va to Ve) and exports the proteins across the inner membrane via the Sec pathway [65]. The proteins of the V-type secretion system are often referred to as autotransporters (ATs). Typically, the T5SS consists of only one polypeptide chain with a β-barrel translocator domain in the membrane and an extracellular passenger or effector region [66]. Under the regulation of the Bam complex (β-barrel assembly mechanism) and TAM complex (translocation and assembly module), outer membrane proteins fold to form a β-barrel conformation and insert into the outer membrane [67].

Virulence Regulatory System

The regulation of all these virulence factors is cell density- dependent through release of autoinducers of critical Quorum Sensing (QS) (e.g., Las, Rhl, Pqs and Iqs), a mass communication system [68]. Quorum sensing may help large population fitness by a hierarchical signal pattern to survive in fierce host environments and thrive, leading to persistent infection in individuals with cystic fibrosis, which cannot be completely cured even with tremendous progress in drug development, drastically improved medcare systems and living conditions [69]. Hence, QS systems along with some other critical virulence factors, such as six types of secretion systems (of toxic molecules), Two-Component Systems (TCSs), have become an intense interest in mechanistic understanding of this bacterium [69].

Antibiotics Resistance

The acquisition of drug resistance by P. aeruginosa depends primarily on multiple intrinsic and acquired antibiotic resistance mechanisms, including the biofilm-mediated formation of resistant and multi-drug-resistant persistent cells [70]. Therefore, P. aeruginosa can quickly develop resistance to various antibiotics, including aminoglycosides, quinolones and β-lactams [71]. 

In the course of long-term evolution, bacteria have developed a variety of ancient genetic resistance mechanisms that have significant genetic plasticity against harmful antibiotic molecules, enabling them to respond to various environmental threats, including possible harm (e.g., antibiotics, chemical compounds and antimicrobial peptides) to their survival. The acquisition of antibiotic resistance with P. aeruginosa is quite diverse and some primary mechanisms including outer membrane permeability, efflux systems and antibiotic-inactivating enzymes will be addressed below [72] (Figure 2).

Mechanisms of antimicrobial resistance in P. aeruginosa can be divided into intrinsic antibiotic resistance: (1: Outer membrane permeability, 2: Efflux systems and 4: Antibiotic-modifying enzymes or 5: Antibiotic-inactivating enzymes), acquired antibiotic resistance (6: Resistance by mutations and acquisition of resistance genes) and adaptive antibiotic resistance (3: Biofilm-mediated resistance). Alteration of outer membrane protein porins decreases the penetration of drugs into cells by reducing membrane permeability. The efflux system directly pumps out drugs. Drug-hydrolyzing and modification enzymes render them inactive. Similarly, some enzymes cause target alterations so that the drug cannot bind its target, resulting in drug inactivity. Antibiotic resistance genes carried on plasmids can be acquired via horizontal gene transfer from the same or different bacterial species [73]. Quorum-sensing signaling molecules activate the formation of biofilms, which act as physical barriers and prevent antibiotics penetrating the cell.

Outer Membrane Permeability

To treat P. aeruginosa infections, most antibiotics need to penetrate the cell membrane to reach the intracellular compartment to function [75]. The outer membrane of P. aeruginosa can act as a specific hurdle inhibiting antibiotic penetration. The outer membrane of P. aeruginosa is chiefly composed of bilayer phospholipid molecules, LPS and porins embedded in phospho-lipids. The outer membrane is responsible for the specific and non-specific uptake of extracellular substances relying on different porin functions, including non-specific porins (OprF), specific porins (OprB, OprD, OprE, OprO and OprP), gated porins (OprC and OprH) and efflux porins (OprM, OprN and OprJ) [76,77]. 

P. aeruginosa manipulates different porins to limit antibiotic penetration and increase antibiotic resistance. OprF promotes the formation and attachment function of P. aeruginosa biofilm to protect the bacterium against antibiotics [78]. Mutations of specific porins OprD involving conformational features cause carbapenem resistance, a serious challenge for medical treatment practices [79]. Outer membrane protein H (OprH) of P. aeruginosa enhances the stability of the outer membrane through direct interaction with LPS to regulate antibiotic resistance [80]. Efflux porins (OprM, OprN and OprJ) contribute to the active efflux of several antibiotics, including norfloxacin, tetracycline and β-lactame drugs [81]. These results demonstrate that the elegant effects and diverse mechanisms by which porins determine antibiotic resistance.

Summary

As opportunistic pathogen, P. aeruginosa has a complex regulatory system that is closely connected and mutually regulated to cope with the harsh external and internal environment, which causes substantial morbidity, debilitating diseases, shortened life span and high mortality in humans In this comprehensive review and other articles, scientists have discussed the virulence factors of P. aeruginosa, such as LPS, adherence factors, elastase, secretion systems and OMVs [82]. We also introduced the recent progress in new antibiotic formulations and compounds, phage therapy strategy, vaccine approaches, nanoparticle fabrication as well as gene editing and nucleic acid-based antibiotics. Furthermore, we have included a large set of immune responses from hosts, including cell types, innate and adaptive immunity and emerging advances in immunological research.

Our paper is a rarely extensive review that covers both bacterial pathogenesis and host defense in a great depth, serving as an irreplaceable reference for both students, doctors and scientists who want to better understand P. aeruginosa. This comprehensive and analytic summary of current literature may enrich our knowledge in the balance between P. aeruginosa invasion and host responses. Despite the vast progress made over the years, a number of questions ranging from basic to clinical and applied aspects remain to be answered and further increased research efforts are still needed to study P. aeruginosa, which will improve our design to more effectively combat the infection caused by emerging drug-resistance strains.

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