What Is New in the Anti-Pseudomonas aeruginosa Clinical Development Pipeline Since the 2017 WHO Alert?

The spread of antibiotic-resistant bacteria poses a substantial threat to morbidity and mortality worldwide. Carbapenem-resistant Pseudomonas aeruginosa (CRPA) are considered "critical-priority" bacteria by the World Health Organization (WHO) since 2017 taking into account criteria such as patient mortality, global burden disease, and worldwide trend of multi-drug resistance (MDR). Indeed P. aeruginosa can be particularly difficult to eliminate from patients due to its combinatory antibiotic resistance, multifactorial virulence, and ability to over-adapt in a dynamic way. Research is active, but the course to a validated efficacy of a new treatment is still long and uncertain. What is new in the anti-P. aeruginosa clinical development pipeline since the 2017 WHO alert? This review focuses on new solutions for P. aeruginosa infections that are in active clinical development, i.e., currently being tested in humans and may be approved for patients in the coming years. Among 18 drugs of interest in December 2021 anti-P. aeruginosa development pipeline described here, only one new combination of ß-lactam/ß-lactamase inhibitor is in phase III trial. Derivatives of existing antibiotics considered as "traditional agents" are over-represented. Diverse "non-traditional agents" including bacteriophages, iron mimetic/chelator, and anti-virulence factors are significantly represented but unfortunately still in early clinical stages. Despite decade of efforts, there is no vaccine currently in clinical development to prevent P. aeruginosa infections. Studying pipeline anti-P. aeruginosa since 2017 up to now shows how to provide a new treatment for patients can be a difficult task. Given the process duration, the clinical pipeline remains unsatisfactory leading best case to the approval of new antibacterial drugs that treat CRPA in several years. Beyond investment needed to build a robust pipeline, the Community needs to reinvent medicine with new strategies of development to avoid the disaster. Among "non-traditional agents", anti-virulence strategy may have the potential through novel and non-killing modes of action to reduce the selective pressure responsible of MDR.

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through lack of the Rieske cytochrome bc1 sub-unit.

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of PQS-dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat substrate(s) responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the primary signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. This defect was also phenocopied by deletion of cytB or cytC1. Thus, either lack of the Rieske sub-unit or mutation of cytochrome bc1 genes results in the perturbation of PQS-dependent autoinduction resulting in eDNA deficient biofilms, reduced antibiotic tolerance and compromised virulence factor production.

Bacterial injection machines: Evolutionary diverse but functionally convergent.

Many human pathogens use Type III, Type IV, and Type VI secretion systems to deliver effectors into their target cells. The contribution of these secretion systems to microbial virulence was the main focus of a workshop organised by the International University of Andalusia in Spain. The meeting addressed structure-function, substrate recruitment, and translocation processes, which differ widely on the different secretion machineries, as well as the nature of the translocated effectors and their roles in subverting the host cell. An excellent panel of worldwide speakers presented the state of the art of the field, highlighting the involvement of bacterial secretion in human disease and discussing mechanistic aspects of bacterial pathogenicity, which can provide the bases for the development of novel antivirulence strategies.

A Type VI Secretion System Trans-Kingdom Effector Is Required for the Delivery of a Novel Antibacterial Toxin in Pseudomonas aeruginosa.

Pseudomonas aeruginosa has evolved multiple strategies to disarm and take advantage of its host. For this purpose, this opportunist pathogen has particularly developed protein secretion in the surrounding medium or injection into host cells. Among this, the type VI secretion system (T6SS) is utilized to deliver effectors into eukaryotic host as well as target bacteria. It assembles into a contractile bacteriophage tail-like structure that functions like a crossbow, injecting an arrow loaded with effectors into the target cell. The repertoire of T6SS antibacterial effectors of P. aeruginosa is remarkably broad to promote environmental adaptation and survival in various bacterial communities, and presumably in the eukaryotic host too. Here, we report the discovery of a novel pair of antibacterial effector and immunity of P. aeruginosa, Tle3 and Tli3. Tli3 neutralizes the toxicity of Tle3 in the periplasm to protect from fratricide intoxication. The characterization of the secretion mechanism of Tle3 indicates that it requires a cytoplasmic adaptor, Tla3, to be targeted and loaded onto the VgrG2b spike and thus delivered by the H2-T6SS machinery. Tla3 is different from the other adaptors discovered so far and defines a novel family among T6SS with a DUF2875. Interestingly, this led us to discover that VgrG2b that we previously characterized as an anti-eukaryotic effector possesses an antibacterial activity as well, as it is toxic towards Escherichia coli. Excitingly Tli3 can counteract VgrG2b toxicity. VgrG2b is thus a novel trans-kingdom effector targeting both bacteria and eukaryotes. VgrG2b represents an interesting target for fighting against P. aeruginosa in the environment and in the context of host infection.

Killing from the inside: Intracellular role of T3SS in the fate of Pseudomonas aeruginosa within macrophages revealed by mgtC and oprF mutants.

While considered solely an extracellular pathogen, increasing evidence indicates that Pseudomonas aeruginosa encounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-type P. aeruginosa PAO1 strain by live and electron microscopy. P. aeruginosa first resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis, both in a macrophage cell line and primary human macrophages. Two bacterial factors, MgtC and OprF, recently identified to be important for survival of P. aeruginosa in macrophages, were found to be involved in bacterial escape from the phagosome as well as in cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes of P. aeruginosa were down-regulated within macrophages in both mgtC and oprF mutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern of mgtC and oprF mutants, a T3SS mutant (ΔpscN) exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellular P. aeruginosa towards macrophages. Moreover, this macrophage damage caused by intracellular P. aeruginosa was found to be dependent on GTPase Activating Protein (GAP) domain of ExoS. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life of P. aeruginosa which allow internalized bacteria to lyse macrophages.

Genome wide identification and experimental validation of Pseudomonas aeruginosa Tat substrates.

In bacteria, the twin-arginine translocation (Tat) pathway allows the export of folded proteins through the inner membrane. Proteins targeted to this system are synthesized with N-terminal signal peptides bearing a conserved twin-arginine motif. The Tat pathway is critical for many bacterial processes including pathogenesis and virulence. However, the full set of Tat substrates is unknown in many bacteria, and the reliability of in silico prediction methods largely uncertain. In this work, we performed a combination of in silico analysis and experimental validation to identify a core set of Tat substrates in the opportunistic pathogen Pseudomonas aeruginosa. In silico analysis predicted 44 putative Tat signal peptides in the P. aeruginosa PA14 proteome. We developed an improved amidase-based Tat reporter assay to show that 33 of these are real Tat signal peptides. In addition, in silico analysis of the full translated genome revealed a Tat candidate with a missassigned start codon. We showed that it is a new periplasmic protein in P. aeruginosa. Altogether we discovered and validated 34 Tat substrates. These show little overlap with Escherichia coli Tat substrates, and functional analysis points to a general role for the P. aeruginosa Tat system in the colonization of environmental niches and pathogenicity.

Pseudomonas aeruginosa zinc uptake in chelating environment is primarily mediated by the metallophore pseudopaline.

Metal uptake is vital for all living organisms. In metal scarce conditions a common bacterial strategy consists in the biosynthesis of metallophores, their export in the extracellular medium and the recovery of a metal-metallophore complex through dedicated membrane transporters. Staphylopine is a recently described metallophore distantly related to plant nicotianamine that contributes to the broad-spectrum metal uptake capabilities of Staphylococcus aureus. Here we characterize a four-gene operon (PA4837-PA4834) in Pseudomonas aeruginosa involved in the biosynthesis and trafficking of a staphylopine-like metallophore named pseudopaline. Pseudopaline differs from staphylopine with regard to the stereochemistry of its histidine moiety associated with an alpha ketoglutarate moiety instead of pyruvate. In vivo, the pseudopaline operon is regulated by zinc through the Zur repressor. The pseudopaline system is involved in nickel uptake in poor media, and, most importantly, in zinc uptake in metal scarce conditions mimicking a chelating environment, thus reconciling the regulation of the cnt operon by zinc with its function as the main zinc importer under these metal scarce conditions.

Game of Trans-Kingdom Effectors.

TplE, a type VI secreted (phospho)lipase, has been identified as the third trans-kingdom effector of Pseudomonas aeruginosa, targeting both prokaryotic and eukaryotic hosts. Indeed, TplE triggers the killing of bacterial competitors and promotes autophagy in epithelial cells once localized to the endoplasmic reticulum.

The T6SSs of Pseudomonas aeruginosa Strain PAO1 and Their Effectors: Beyond Bacterial-Cell Targeting.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many diseases such as chronic lung colonization in cystic fibrosis patients and acute infections in hospitals. The capacity of P. aeruginosa to be pathogenic toward several hosts is notably due to different secretion systems. Amongst them, P. aeruginosa encodes three Type Six Secretion Systems (T6SS), named H1- to H3-T6SS, that act against either prokaryotes and/or eukaryotic cells. They are independent from each other and inject diverse toxins that interact with different components in the host cell. Here we summarize the roles of these T6SSs in the PAO1 strain, as well as the toxins injected and their targets. While H1-T6SS is only involved in antiprokaryotic activity through at least seven different toxins, H2-T6SS and H3-T6SS are also able to target prokaryotic as well as eukaryotic cells. Moreover, recent studies proposed that H2- and H3-T6SS have a role in epithelial cells invasion by injecting at least three different toxins. The diversity of T6SS effectors is astounding and other effectors still remain to be discovered. In this review, we present a table with other putative P. aeruginosa strain PAO1 T6SS-dependent effectors. Altogether, the T6SSs of P. aeruginosa are important systems that help fight other bacteria for their ecological niche, and are important in the pathogenicity process.

Structural Basis of Lipid Targeting and Destruction by the Type V Secretion System of Pseudomonas aeruginosa.

The type V secretion system is a macromolecular machine employed by a number of bacteria to secrete virulence factors into the environment. The human pathogen Pseudomonas aeruginosa employs the newly described type Vd secretion system to secrete a soluble variant of PlpD, a lipase of the patatin-like family synthesized as a single macromolecule that also carries a polypeptide transport-associated domain and a 16-stranded ß-barrel. Here we report the crystal structure of the secreted form of PlpD in its biologically active state. PlpD displays a classical lipase α/ß hydrolase fold with a catalytic site located within a highly hydrophobic channel that entraps a lipidic molecule. The active site is covered by a flexible lid, as in other lipases, indicating that this region in PlpD must modify its conformation in order for catalysis at the water-lipid interface to occur. PlpD displays phospholipase A1 activity and is able to recognize a number of phosphatidylinositols and other phosphatidyl analogs. PlpD is the first example of an active phospholipase secreted through the type V secretion system, for which there are more than 200 homologs, revealing details of the lipid destruction arsenal expressed by P. aeruginosa in order to establish infection.

Genome-wide Screen of Pseudomonas aeruginosa in Saccharomyces cerevisiae Identifies New Virulence Factors.

Pseudomonas aeruginosa is a human opportunistic pathogen that causes mortality in cystic fibrosis and immunocompromised patients. While many virulence factors of this pathogen have already been identified, several remain to be discovered. In this respect we set an unprecedented genome-wide screen of a P. aeruginosa expression library based on a yeast growth phenotype. Fifty-one candidates were selected in athree-round screening process. The robustness of the screen was validated by the selection of three well known secreted proteins including one demonstrated virulence factor, the protease LepA. Further in silico sorting of the 51 candidates highlighted three potential new Pseudomonas effector candidates (Pec). By testing the cytotoxicity of wild type P. aeruginosa vs. pec mutants toward macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa. Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions.

A Macrophage Subversion Factor Is Shared by Intracellular and Extracellular Pathogens.

Pathogenic bacteria have developed strategies to adapt to host environment and resist host immune response. Several intracellular bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis, share the horizontally-acquired MgtC virulence factor that is important for multiplication inside macrophages. MgtC is also found in pathogenic Pseudomonas species. Here we investigate for the first time the role of MgtC in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in the systemic infection model of zebrafish embryos, and strikingly, the attenuated phenotype is dependent on the presence of macrophages. In ex vivo experiments, the P. aeruginosa mgtC mutant is more sensitive to macrophage killing than the wild-type strain. However, wild-type and mutant strains behave similarly toward macrophage killing when macrophages are treated with an inhibitor of the vacuolar proton ATPase. Importantly, P. aeruginosa mgtC gene expression is strongly induced within macrophages and phagosome acidification contributes to an optimal expression of the gene. Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC shares a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to host immune response in relation to the different bacterial lifestyles. In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus highlighting MgtC as a promising new target for anti-virulence strategies.

Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network.

UNLABELLED: Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction. IMPORTANCE: Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, including attack, this opportunistic bacterial pathogen is able to avoid host clearance by triggering its own internalization in nonphagocytic cells. We previously showed that a protein secretion/injection machinery, called the H2 type VI secretion system (H2-T6SS), promotes P. aeruginosa uptake by epithelial cells. Here we investigate which H2-T6SS effector enables P. aeruginosa to enter nonphagocytic cells. We show that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubules and, more particularly, with the γ-tubulin ring complex (γTuRC) known as the microtubule-nucleating center. This interaction precedes a microtubule- and actin-dependent internalization of P. aeruginosa. We thus discovered an unprecedented target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first example of a bacterial protein interacting with the γTuRC.

Intracellular phase for an extracellular bacterial pathogen: MgtC shows the way.

Pseudomonas aeruginosa is an extracellular pathogen known to impair host phagocytic functions. However, our recent results identify MgtC as a novel actor in P. aeruginosa virulence, which plays a role in an intramacrophage phase of this pathogen. In agreement with its intracellular function, P. aeruginosamgtC gene expression is strongly induced when the bacteria reside within macrophages. MgtC was previously known as a horizontally-acquired virulence factor important for multiplication inside macrophages in several intracellular bacterial pathogens. MgtC thus provides a singular example of a virulence determinant that subverts macrophages both in intracellular and extracellular pathogens. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC has a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to the host in relation to the different bacterial lifestyles. MgtC thus appears as an attractive target for antivirulence strategies and our work provides a natural peptide as MgtC antagonist, which paves the way for the development of MgtC inhibitors.

Gene transfer: conjugation.

Conjugation is a gene transfer process in which a recipient bacterium receives DNA from a donor bacterium by cell-to-cell contact through conjugative pili. Conjugation is mediated by certain plasmids or transposons. Here, we describe plasmid conjugation.

The target cell genus does not matter.

Two type VI secreted phospholipases D of Pseudomonas aeruginosa were identified as trans-kingdom virulence effectors, targeting both prokaryotic and eukaryotic host cells. Each of them triggers killing bacterial competitors and internalization into non-phagocytic cells. These type VI lipolytic enzymes are widely distributed among pathogens and may constitute a conserved strategy.

HoPaCI-DB: host-Pseudomonas and Coxiella interaction database.

Bacterial infectious diseases are the result of multifactorial processes affected by the interplay between virulence factors and host targets. The host-Pseudomonas and Coxiella interaction database (HoPaCI-DB) is a publicly available manually curated integrative database (http://mips.helmholtz-muenchen.de/HoPaCI/) of host-pathogen interaction data from Pseudomonas aeruginosa and Coxiella burnetii. The resource provides structured information on 3585 experimentally validated interactions between molecules, bioprocesses and cellular structures extracted from the scientific literature. Systematic annotation and interactive graphical representation of disease networks make HoPaCI-DB a versatile knowledge base for biologists and network biology approaches.