Antivirulence and antipathogenic activity of Mayan herbal remedies against Pseudomonas aeruginosa.

ETHNOPHARMACOLOGICAL RELEVANCE: The Yucatan Peninsula has a privileged wealth of vascular plants with which various Mayan herbal formulations have been developed. However, studies on their antipathogenic and antivirulence properties are scarce. AIM OF THE STUDY: Identify antivirulence properties in Mayan herbal remedies and determine their antipathogenic capacity in burn wounds infected with Pseudomonas aeruginosa. MATERIALS AND METHODS: An ethnobotanical study was conducted in Mayan communities in central and southern Quintana Roo, Mexico. Furthermore, the antipathogenic capacity of three Mayan herbal remedies was analyzed using an animal model of thermal damage and P. aeruginosa infection. Antivirulence properties were determined by inhibiting phenotypes regulated by quorum sensing (pyocyanin, biofilm, and swarming) and by the secretion of the ExoU toxin. The chemical composition of the most active herbal remedy was analyzed using molecular network analysis. RESULTS: It was found that topical administration of the remedy called "herbal soap" (HS) for eleven days maintained 100% survival of the animals, reduced establishment of the bacteria in the burn and prevented its systemic dispersion. Although no curative effect was recorded on tissue damaged by HS treatment, its herbal composition strongly reduced swarming and ExoU secretion. Through analysis of Molecular Networks, it was possible to carry out a global study of its chemical components, and identify the family of oxindole monoterpenoid alkaloids and carboline and tetrahydropyrididole alkaloids. In addition, flavonols, flavan-3-ols, and quinic acid derivatives were detected. CONCLUSIONS: The antipathogenic and antivirulence capacity of ancient Mayan remedies makes them a potential resource for developing new antibacterial therapies to treat burns infected by P. aeruginosa.

To cheat or not to cheat: cheatable and non-cheatable virulence factors in Pseudomonas aeruginosa.

Important bacterial pathogens such as Pseudomonas aeruginosa produce several exoproducts such as siderophores, degradative enzymes, biosurfactants, and exopolysaccharides that are used extracellularly, benefiting all members of the population, hence being public goods. Since the production of public goods is a cooperative trait, it is in principle susceptible to cheating by individuals in the population who do not invest in their production, but use their benefits, hence increasing their fitness at the expense of the cooperators' fitness. Among the most studied virulence factors susceptible to cheating are siderophores and exoproteases, with several studies in vitro and some in animal infection models. In addition to these two well-known examples, cheating with other virulence factors such as exopolysaccharides, biosurfactants, eDNA production, secretion systems, and biofilm formation has also been studied. In this review, we discuss the evidence of the susceptibility of each of those virulence factors to cheating, as well as the mechanisms that counteract this behavior and the possible consequences for bacterial virulence.

Resistance against two lytic phage variants attenuates virulence and antibiotic resistance in Pseudomonas aeruginosa.

Background: Bacteriophage therapy is becoming part of mainstream Western medicine since antibiotics of clinical use tend to fail. It involves applying lytic bacteriophages that self-replicate and induce cell lysis, thus killing their hosts. Nevertheless, bacterial killing promotes the selection of resistant clones which sometimes may exhibit a decrease in bacterial virulence or antibiotic resistance. Methods: In this work, we studied the Pseudomonas aeruginosa lytic phage φDCL-PA6 and its variant φDCL-PA6α. Additionally, we characterized and evaluated the production of virulence factors and the virulence in a Galleria mellonella model of resistant mutants against each phage for PA14 and two clinical strains. Results: Phage φDCL-PA6α differs from the original by only two amino acids: one in the baseplate wedge subunit and another in the tail fiber protein. According to genomic data and cross-resistance experiments, these changes may promote the change of the phage receptor from the O-antigen to the core lipopolysaccharide. Interestingly, the host range of the two phages differs as determined against the Pseudomonas aeruginosa reference strains PA14 and PAO1 and against nine multidrug-resistant isolates from ventilator associated pneumonia. Conclusions: We show as well that phage resistance impacts virulence factor production. Specifically, phage resistance led to decreased biofilm formation, swarming, and type III secretion; therefore, the virulence towards Galleria mellonella was dramatically attenuated. Furthermore, antibiotic resistance decreased for one clinical strain. Our study highlights important potential advantages of phage therapy's evolutionary impact that may be exploited to generate robust therapy schemes.

A Brominated Furanone Inhibits Pseudomonas aeruginosa Quorum Sensing and Type III Secretion, Attenuating Its Virulence in a Murine Cutaneous Abscess Model.

Quorum sensing (QS) and type III secretion systems (T3SSs) are among the most attractive anti-virulence targets for combating multidrug-resistant pathogenic bacteria. Some halogenated furanones reduce QS-associated virulence, but their role in T3SS inhibition remains unclear. This study aimed to assess the inhibition of these two systems on Pseudomonas aeruginosa virulence. The halogenated furanones (Z)-4-bromo-5-(bromomethylene)-2(5H) (C-30) and 5-(dibromomethylene)-2(5H) (named hereafter GBr) were synthesized, and their ability to inhibit the secretion of type III exoenzymes and QS-controlled virulence factors was analyzed in P. aeruginosa PA14 and two clinical isolates. Furthermore, their ability to prevent bacterial establishment was determined in a murine cutaneous abscess model. The GBr furanone reduced pyocyanin production, biofilm formation, and swarming motility in the same manner or more effectively than C-30. Moreover, both furanones inhibited the secretion of ExoS, ExoT, or ExoU effectors in all tested strains. The administration of GBr (25 and 50 µM) to CD1 mice infected with the PA14 strain significantly decreased necrosis formation in the inoculation zone and the systemic spread of bacteria more efficiently than C-30 (50 µM). Molecular docking analysis suggested that the gem position of bromine in GBr increases its affinity for the active site of the QS LasR regulator. Overall, our findings showed that the GBr furanone displayed efficient multi-target properties that may favor the development of more effective anti-virulence therapies.

Anti-Virulence Properties of Plant Species: Correlation between In Vitro Activity and Efficacy in a Murine Model of Bacterial Infection.

Several plant extracts exhibit anti-virulence properties due to the interruption of bacterial quorum sensing (QS). However, studies on their effects at the preclinical level are scarce. Here, we used a murine model of abscess/necrosis induced by Pseudomonas aeruginosa to evaluate the anti-pathogenic efficacy of 24 plant extracts at a sub-inhibitory concentration. We analyzed their ability to inhibit QS-regulated virulence factors such as swarming, pyocyanin production, and secretion of the ExoU toxin via the type III secretion system (T3SS). Five of the seven extracts with the best anti-pathogenic activity reduced ExoU secretion, and the extracts of Diphysa americana and Hibiscus sabdariffa were identified as the most active. Therefore, the abscess/necrosis model allows identification of plant extracts that have the capacity to reduce pathogenicity of P. aeruginosa. Furthermore, we evaluated the activity of the plant extracts on Chromobacterium violaceum. T3SS (ΔescU) and QS (ΔcviI) mutant strains were assessed in both the abscess/necrosis and sepsis models. Only the ΔescU strain had lower pathogenicity in the animal models, although no activity of plant extracts was observed. These results demonstrate differences between the anti-virulence activity recorded in vitro and pathogenicity in vivo and between the roles of QS and T3S systems as virulence determinants.

Anti-Pathogenic Properties of the Combination of a T3SS Inhibitory Halogenated Pyrrolidone with C-30 Furanone.

Antimicrobial resistance is one of the current public health challenges to be solved. The World Health Organization (WHO) has urgently called for the development of strategies to expand the increasingly limited antimicrobial arsenal. The development of anti-virulence therapies is a viable option to counteract bacterial infections with the possibility of reducing the generation of resistance. Here we report on the chemical structures of pyrrolidones DEXT 1-4 (previously identified as furan derivatives) and their anti-virulence activity on Pseudomonas aeruginosa strains. DEXT 1-4 were shown to inhibit biofilm formation, swarming motility, and secretion of ExoU and ExoT effector proteins. Also, the anti-pathogenic property of DEXT-3 alone or in combination with furanone C-30 (quorum sensing inhibitor) or MBX-1641 (type III secretion system inhibitor) was analyzed in a model of necrosis induced by P. aeruginosa PA14. All treatments reduced necrosis; however, only the combination of C-30 50 µM with DEXT-3 100 µM showed significant inhibition of bacterial growth in the inoculation area and systemic dispersion. In conclusion, pyrrolidones DEXT 1-4 are chemical structures capable of reducing the pathogenicity of P. aeruginosa and with the potential for the development of anti-virulence combination therapies.

CesL Regulates Type III Secretion Substrate Specificity of the Enteropathogenic E. coli Injectisome.

The type III secretion system (T3SS) is a complex molecular device used by several pathogenic bacteria to translocate effector proteins directly into eukaryotic host cells. One remarkable feature of the T3SS is its ability to secrete different categories of proteins in a hierarchical manner, to ensure proper assembly and timely delivery of effectors into target cells. In enteropathogenic Escherichia coli, the substrate specificity switch from translocator to effector secretion is regulated by a gatekeeper complex composed of SepL, SepD, and CesL proteins. Here, we report a characterization of the CesL protein using biochemical and genetic approaches. We investigated discrepancies in the phenotype among different cesL deletion mutants and showed that CesL is indeed essential for translocator secretion and to prevent premature effector secretion. We also demonstrated that CesL engages in pairwise interactions with both SepL and SepD. Furthermore, while association of SepL to the membrane does not depended on CesL, the absence of any of the proteins forming the heterotrimeric complex compromised the intracellular stability of each component. In addition, we found that CesL interacts with the cytoplasmic domains of the export gate components EscU and EscV. We propose a mechanism for substrate secretion regulation governed by the SepL/SepD/CesL complex.

Antivirulence Activity of a Dietary Phytochemical: Hibiscus Acid Isolated from Hibiscus sabdariffa L. Reduces the Virulence of Pseudomonas aeruginosa in a Mouse Infection Model.

Hibiscus sabdariffa L. (Hs) calyxes, rich in organic acids, are included in diets in different countries. In recent years, some phytochemicals have been shown to reduce bacterial virulence at sublethal concentrations by interfering with quorum sensing (QS) systems. Therefore, in this study the antivirulence properties of Hs calyxes and two γ-lactones (hibiscus acid [HA] and its methyl ester) in Pseudomonas aeruginosa were analyzed. Acetone and methanol extracts of Hs showed anti-QS activity by inhibiting violacein production (60% to 80% with 250 µg/mL). In molecular docking analysis, the γ-lactones registered a good binding score, which suggests strong interaction with the active site of LasR protein. To verify their effect in vitro, they were isolated from Hs and evaluated in six QS-regulated phenotypes, as well as in ExoU toxin that is released by the type III secretion system (T3SS). At 500 µg/mL they reduced alkaline protease (29-52%) and elastase (15-37%) activity, biofilm formation (∼75%), and swarming (50%), but there was no effect on pyocyanin production, hemolytic activity, or type III secretion. In a mouse abscess/necrosis model, HA at sublethal concentrations (15 and 31.2 µg/mL) affected infection establishment and prevented damage and systemic spread. In conclusion, HA is the first molecule identified with antivirulence properties in Hs with the potential to prevent infections caused by P. aeruginosa.

Tetradecanoic Acids With Anti-Virulence Properties Increase the Pathogenicity of Pseudomonas aeruginosa in a Murine Cutaneous Infection Model.

Blocking virulence is a promising alternative to counteract Pseudomonas aeruginosa infections. In this regard, the phenomenon of cell-cell communication by quorum sensing (QS) is an important anti-virulence target. In this field, fatty acids (FA) have gained notoriety for their role as autoinducers, as well as anti-virulence molecules in vitro, like some saturated FA (SAFA). In this study, we analyzed the anti-virulence activity of SAFA with 12 to18 carbon atoms and compared their effect with the putative autoinducer cis-2-decenoic acid (CDA). The effect of SAFA on six QS-regulated virulence factors and on the secretion of the exoenzyme ExoU was evaluated. In addition, a murine cutaneous infection model was used to determine their influence on the establishment and damage caused by P. aeruginosa PA14. Dodecanoic (lauric, C12:0) and tetradecanoic (myristic, C14:0) acids (SAFA C12-14) reduced the production of pyocyanin by 35-58% at 40 and 1,000 µM, while CDA inhibited it 62% at a 3.1 µM concentration. Moreover, the SAFA C12-14 reduced swarming by 90% without affecting biofilm formation. In contrast, CDA reduced the biofilm by 57% at 3 µM but did not affect swarming. Furthermore, lauric and myristic acids abolished ExoU secretion at 100 and 50 µM respectively, while CDA reduced it by ≈ 92% at 100 µM. Remarkably, the coadministration of myristic acid (200 and 1,000 µM) with P. aeruginosa PA14 induced greater damage and reduced survival of the animals up to 50%, whereas CDA to 500 µM reduced the damage without affecting the viability of the PA14 strain. Hence, our results show that SAFA C12-14 and CDA have a role in regulation of P. aeruginosa virulence, although their inhibition/activation molecular mechanisms are different in complex environments such as in vivo systems.

AiiM Lactonase Strongly Reduces Quorum Sensing Controlled Virulence Factors in Clinical Strains of Pseudomonas aeruginosa Isolated From Burned Patients.

Pseudomonas aeruginosa is an opportunistic bacterium associated with healthcare infections in intensive care units (ICUs), ventilator-associated pneumonia (VAP), surgical site infections, and burns. This bacterium causes 75% of death in burned patients, since it can develop a persistent biofilm associated with infections, express several virulence factors, and antibiotic-resistance mechanisms. Some of these virulence factors are proteases such as elastase and alkaline protease, or toxic metabolites such as pyocyanin and is one of the few microorganisms able to produce cyanide, which inhibits the cytochrome oxidase of host cells. These virulence factors are controlled by quorum sensing (QS). In this work, 30 P. aeruginosa clinical strains isolated from burned patients from a tertiary hospital in Mexico City were studied. Antibiotic susceptibility tests were done, and virulence factors (elastase, alkaline protease, HCN, and pyocyanin) were determined in presence of an N-acylhomoserine lactonase, AiiM able to hydrolyze a wide range of acyl homoserine lactones. The treatment reduced significantly the activities of elastase and alkaline protease, and the production of pyocyanin and HCN in all producer strains but not the secretion of toxins through the type III secretion system. Our work suggests that AiiM treatment may be an effective therapy to combat P. aeruginosa infection in burn patients.

Two Pseudomonas aeruginosa clonal groups belonging to the PA14 clade are indigenous to the Churince system in Cuatro Ciénegas Coahuila, México.

Pseudomonas aeruginosa is a widely distributed environmental bacterium but is also an opportunistic pathogen that represents an important health hazard due to its high intrinsic antibiotic resistance and its production of virulence factors. The genetic structure of P. aeruginosa populations using whole genome sequences shows the existence of three clades, one of which (PA7 clade) has a higher genetic diversity. These three clades include clinical and environmental isolates that are very diverse in terms of geographical origins and isolation date. Here, we report the characterization of two distinct clonal P. aeruginosa groups that form a part of the PA14 clade (clade 2) sampled from the Churince system in Cuatro Ciénegas Basin (CCB). One of the clonal groups that we report here was isolated in 2011 (group 2A) and was displaced by the other clonal group (2B) in 2015. Both Churince groups are unable to produce pyoverdine but can produce other virulence-associated traits. The existence of these unique P. aeruginosa clonal groups in the Churince system is of ecological and evolutionary significance since the microbiota of this site is generally very distinct from other lineages, and this is the first time that a population of P. aeruginosa has been found in CCB.

Inactivation of the quorum-sensing transcriptional regulators LasR or RhlR does not suppress the expression of virulence factors and the virulence of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an environmental bacterium but is also an opportunistic pathogen. The aim of this work is to evaluate the contribution of P. aeruginosa LasR and RhlR transcriptional regulators of the quorum-sensing response (QSR) to the production of virulence factors, and to its virulence in a mouse abscess model. The QSR is a complex regulatory network that modulates the expression of several virulence factors, including elastase, pyocyanin and rhamnolipids. LasR, when complexed with the auto-inducer 3-oxo-dodecanoyl lactone (3O-C12-HSL), produced by LasI, is at the top of the QSR regulatory cascade since it activates transcription of some genes encoding virulence factors (such as the gene coding for elastase, lasB) and also transcription of both rhlR and rhlI, encoding the synthase of the auto-inducer butanoyl-homoserine lactone (C4-HSL). In turn RhlR, coupled with C4-HSL, activates the transcription of genes encoding for the enzymes involved in pyocyanin and rhamnolipid production. Several efforts have been made to obtain inhibitors of LasR activity that would suppress the QSR. However, these attempts have used chemical compounds that might not be specific for LasR inactivation. In this work we show that individual inactivation of either lasR or rhlR did not block the QSR, nor did it impair P. aeruginosa virulence, and that even a lasR rhlR double mutant still presented residual virulence, even lacking the production of virulence factors. These results show that the inhibition of either lasR or rhlR is not a straightforward approach to blocking P. aeruginosa virulence, due to the great complexity of the QSR.

Functional Characterization of EscK (Orf4), a Sorting Platform Component of the Enteropathogenic Escherichia coli Injectisome.

The type III secretion system (T3SS) is a supramolecular machine used by many bacterial pathogens to translocate effector proteins directly into the eukaryotic host cell cytoplasm. Enteropathogenic Escherichia coli (EPEC) is an important cause of infantile diarrheal disease in underdeveloped countries. EPEC virulence relies on a T3SS encoded within a chromosomal pathogenicity island known as the locus of enterocyte effacement (LEE). In this work, we pursued the functional characterization of the LEE-encoded protein EscK (previously known as Orf4). We provide evidence indicating that EscK is crucial for efficient T3S and belongs to the SctK (OrgA/YscK/MxiK) protein family, whose members have been implicated in the formation of a sorting platform for secretion of T3S substrates. Bacterial fractionation studies showed that EscK localizes to the inner membrane independently of the presence of any other T3SS component. Combining yeast two-hybrid screening and pulldown assays, we identified an interaction between EscK and the C-ring/sorting platform component EscQ. Site-directed mutagenesis of conserved residues revealed amino acids that are critical for EscK function and for its interaction with EscQ. In addition, we found that T3S substrate overproduction is capable of compensating for the absence of EscK. Overall, our data suggest that EscK is a structural component of the EPEC T3SS sorting platform, playing a central role in the recruitment of T3S substrates for boosting the efficiency of the protein translocation process. IMPORTANCE: The type III secretion system (T3SS) is an essential virulence determinant for enteropathogenic Escherichia coli (EPEC) colonization of intestinal epithelial cells. Multiple EPEC effector proteins are injected via the T3SS into enterocyte cells, leading to diarrheal disease. The T3SS is encoded within a genomic pathogenicity island termed the locus of enterocyte effacement (LEE). Here we unravel the function of EscK, a previously uncharacterized LEE-encoded protein. We show that EscK is central for T3SS biogenesis and function. EscK forms a protein complex with EscQ, the main component of the cytoplasmic sorting platform, serving as a docking site for T3S substrates. Our results provide a comprehensive functional analysis of an understudied component of T3SSs.

EscO, a functional and structural analog of the flagellar FliJ protein, is a positive regulator of EscN ATPase activity of the enteropathogenic Escherichia coli injectisome.

Type III secretion systems (T3SSs) are multiprotein molecular devices used by many Gram-negative bacterial pathogens to translocate effector proteins into eukaryotic cells. A T3SS is also used for protein export in flagellar assembly, which promotes bacterial motility. The two systems are evolutionarily related, possessing highly conserved components in their export apparatuses. Enteropathogenic Escherichia coli (EPEC) employs a T3SS, encoded by genes in the locus of enterocyte effacement (LEE) pathogenicity island, to colonize the human intestine and cause diarrheal disease. In the present work, we investigated the role of the LEE-encoded EscO protein (previously Orf15 or EscA) in T3SS biogenesis. We show that EscO shares similar properties with the flagellar FliJ and the Yersinia YscO protein families. Our findings demonstrate that EscO is essential for secretion of all categories of T3SS substrates. Consistent with its central role in protein secretion, it was found to interact with the ATPase EscN and its negative regulator, EscL, of the export apparatus. Moreover, we show that EscO stimulates EscN enzymatic activity; however, it is unable to upregulate ATP hydrolysis in the presence of EscL. Remarkably, EscO partially restored the swimming defect of a Salmonella flagellar fliJ mutant and was able to stimulate the ATPase activity of FliI. Overall, our data indicate that EscO is the virulence counterpart of the flagellar FliJ protein.