rhlA Genes Research Articles

Metabolomics responses and tolerance of Pseudomonas aeruginosa under acoustic vibration stress.

Sound has been shown to impact microbial behaviors. However, our understanding of the chemical and molecular mechanisms underlying these microbial responses to acoustic vibration is limited. In this study, we used untargeted metabolomics analysis to investigate the effects of 100-Hz acoustic vibration on the intra- and extracellular hydrophobic metabolites of P. aeruginosa PAO1. Our findings revealed increased levels of fatty acids and their derivatives, quinolones, and N-acylethanolamines upon sound exposure, while rhamnolipids (RLs) showed decreased levels. Further quantitative real-time polymerase chain reaction experiments showed slight downregulation of the rhlA gene (1.3-fold) and upregulation of fabY (1.5-fold), fadE (1.7-fold), and pqsA (1.4-fold) genes, which are associated with RL, fatty acid, and quinolone biosynthesis. However, no alterations in the genes related to the rpoS regulators or quorum-sensing networks were observed. Supplementing sodium oleate to P. aeruginosa cultures to simulate the effects of sound resulted in increased tolerance of P. aeruginosa in the presence of sound at 48 h, suggesting a potential novel response-tolerance correlation. In contrast, adding RL, which went against the response direction, did not affect its growth. Overall, these findings provide potential implications for the control and manipulation of virulence and bacterial characteristics for medical and industrial applications.

Assessing the antibiofilm activity of flavonol esters against Pseudomonas aeruginosa PAO1 biofilm: an in vitro, molecular docking, and molecular dynamics study

Pseudomonas aeruginosa is one of the opportunistic pathogens that may cause serious health problems and can produce several virulence factors, which are responsible for various infections, particularly in immunocompromised patients. They are responsible for producing infections on indwelling medical devices by attaching on to them and forming a biofilm. Antibiofilm, antivirulence, and gene expression studies of P. aeruginosa biofilm treated with esters of flavonols were evaluated. Pyocyanin, cell surface hydrophobicity, LasA protease estimation, rhamnolipid estimation, and pyoverdine estimation were performed to evaluate the antivirulence activities of the test compounds against P. aeruginosa. Previous studies on the antivirulence activity of flavonoids against P. aeruginosa demonstrate that even if they can inhibit bacterial growth, relatively high concentrations of the compound are generally required for the inhibition of virulence factors. The esters showed more than 40% inhibition in all the tested virulence factors at their sub minimum inhibitory concentration. The gene expression studies of selected esters toward lasB and rhlA genes show downregulation of rhlA which suggests the inhibition in biofilm formation through rhamnolipid inhibition, quorum sensing inhibition, or biofilm formation inhibition. Communicated by Ramaswamy H. Sarma

New Record: Molecular Depiction of Rhamnolipids (rhlA) Gene in Locally Isolated Strains of Pseudomonas aeruginosa

Soil samples were collected from oil-contaminated sites which were located in west Qurna, Basrah, Iraq. Pseudomonas species were initially isolated on mineral salts and Pseudomonas agar media and identified using morphological and biochemical characterizations. Then, specific primers for the rhlA gene belonging to Pseudomonas aeruginosa were designed based on the primer design conditions, and PCR was performed to amplify the 888 bp size fragment of the rhlA gene; additionally, the primary PCR products were purified and sent for sequencing. The band of about 888bp was determined on the gel, the amplified rhlA gene sequencing findings were revised, only 366 bp were ready to analyze using the (BLAST) software, and the final result was identified as a partial sequence of chromosomal rhlA gene related to Pseudomonas aeruginosa with percent identity of 99.45%. The query gene’s incomplete matching with another partial rhlA record on NCBI was caused by variations in two base pair sequences (T in sequence 348 and C in sequence 353, respectively), and despite the small difference, this results in variation in the amino acids produced; so that a new record number, ON637169, was assigned when the sequence was deposited in GenBank. The relation among the new record of partial rhlA gene with the same number of the other rhlA gene sequences (60 records) was demonstrated by creating a phylogenetic tree.

Phytochemical screening, antibacterial, anti-biofilm and quorum sensing inhibiting activity of Alpinia nigra leaf extract against infectious pathogen Pseudomonas aeruginosa PAO1

The recurrent cycles of several bacterial diseases are an endorsement of the formation of biofilms that are intractable to antibiotic therapy. Biofilm-associated diseases have affected millions of people worldwide and are unassailable due to frequent changes of surface antigens and gene expression. Therefore, there is a need to find out an alternative approach to cure biofilm infections. Plant-derived bioactive compounds and their derivative have shown a significant role in combatting bacterial infection. In this scenario, we performed phytochemical screening of ethyl acetate leaf extract (L-EtAc) of Alpina nigra and antibacterial, anti-biofilm activity towards Pseudomonas aeruginosa PAO1. Antibacterial and growth curve analysis of bacterial cells confirmed the significant bacterial cell death in L-EtAc treated sample. Relatively less biofilm formation was observed in the treated sample as revealed through fluorescence microscopy. Further, quorum-sensing inhibition was observed by applying a similar concentration of L-EtAc on Chromobacterium violaceum bacteria. The interaction between the biofilm-forming protein and phytochemical constituent was validated through in-silico analysis and further confirmed with qRT-PCR. The qRT-PCR data revealed reduced lasI, lasR, lasB, rhlI, rhlR and rhlA gene expression, which may be directly associated with biofilm and extracellular matrix formation. This study might be helpful for the development of new natural product for curing biofilm-associated disease.

The Xanthophyll Carotenoid Lutein Reduces the Invasive Potential of Pseudomonas aeruginosa and Increases Its Susceptibility to Tobramycin.

Recently, the xanthophyll carotenoid lutein has been qualified as a potential quorum sensing (QS) and biofilm inhibitor against Pseudomonas aeruginosa. To address the potential of this xanthophyll compound as a relevant antivirulence agent, we investigated in depth its impact on the invasion capabilities and aggressiveness of P. aeruginosa PAO1, which rely on the bacterial ability to build and maintain protective barriers, use different types of motilities and release myriad virulence factors, leading to host cell and tissue damages. Our data, obtained on the PAO1 strain, indicate that all-trans lutein (Lut; 22 µM) disrupts biofilm formation and disorganizes established biofilm structure without affecting bacterial viability, while improving the bactericidal activity of tobramycin against biofilm-encapsulated PAO1 cells. Furthermore, this xanthophyll affects PAO1 twitching and swarming motilities while reducing the production of the extracellular virulence factors pyocyanin, elastase and rhamnolipids as well as the expression of the QS-regulated lasB and rhlA genes without inhibiting the QS-independent aceA gene. Interestingly, the expression of the QS regulators rhlR/I and lasR/I is significantly reduced as well as that of the global virulence factor regulator vfr, which is suggested to be a major target of Lut. Finally, an oxidative metabolite of Lut, 3′-dehydrolutein, induces a similar inhibition phenotype. Taken together, lutein-type compounds represent potential agents to control the invasive ability and antibiotic resistance of P. aeruginosa.

The correlation between biofilm formation capability and antibiotic resistance pattern in Pseudomonas aeruginosa

The infections caused by Pseudomonas aeruginosa are life-threatening, due to high intrinsic antimicrobial resistance of this microorganism. The integrons and biofilm formation of P. aeruginosa have a significant role in antibiotic resistance. Therefore, this study aimed to evaluate antibiotic resistance pattern in Pseudomonas aeruginosa isolates with biofilm formation ability. This cross-sectional study from January 2017 to December 2017 was conducted on 78 isolates (58 clinical and 20 environmental) of P. aeruginosa recovered from the 547 samples (439 of clinical and 108 of environmental samples). The isolates were identified by phenotypic and genotypic tests. Kirby-Bauer disk diffusion method was used for susceptibility testing. The prevalence of class 1, 2 and 3 Integrons, rhlA, and lasB genes were determined using Polymerase Chain Reaction (PCR). Biofilm formation was determined using the microtiter plate method. Data analyzed using Stata 14 software and Chi-Square test.The most prevalent resistance was observed against Ticarcillin/Clavulanic Acid (55%). Generally, 56.4% of isolates were producers of strong biofilm in both environmental and clinical isolates. The prevalence of strong biofilm producers in clinical isolates was more than environmental. A significant correlation was observed between Int1, Int2, and rhlA genes with biofilm formation capability (P = 0.02).Regarding >50% of both environmental and clinical isolates were producers of strong biofilm and because the source of clinical isolates may be from the environment, the necessary hygiene measurements should be taken. No significant correlation was observed between lasB gene with biofilm formation capability.

AgNP combined with quorum sensing inhibitor increased the antibiofilm effect on Pseudomonas aeruginosa.

Pseudomonas aeruginosa biofilm lifestyle exhibits multidrug resistance in chronic bacterial infections. Alternative antimicrobial compounds or combination drug therapies must be urgently developed. In this work, the antibiofilm effect of Ag nanoparticle (AgNP) combined with the quorum sensing inhibitor (QSI) 4-nitropyridine N-oxide (4NPO) on P. aeruginosa biofilms was investigated. The biofilm biomass of P. aeruginosa was considerably reduced by 1.56-50mg/L AgNP. However, 4NPO enhanced the ability of AgNP to inhibit P. aeruginosa biofilm formation (P < 0.05). The combination of AgNP with 4NPO could continuously inhibit biofilm development after 12h, and 50mg/L AgNP combined with 6.25mg/L 4NPO thoroughly suppressed biofilm growth. The expression levels of QS genes and exopolysaccharide genes of biofilm treated with the combination of AgNP with 4NPO (AgNP-4NPO combination) were lower than those treated with AgNP alone (P < 0.05). Additional extracellular proteins and polysaccharides were determined in the samples treated with AgNP-4NPO combination. Based on proteomic analysis, this result was attributed to cell rupture caused by antimicrobial agents and intracellular materials released. The combination of the two antimicrobial agents could weaken the swimming ability of bacterial cells by damaging bacterial flagella and blocking rhlA gene expression. Thus, AgNP combined with QSI showed stronger antibiofilm ability than AgNP alone. These results may contribute to the development of antimicrobial agents.

Inhibitive Effect of Eugenol and Its Nanoemulsion on Quorum Sensing–Mediated Virulence Factors and Biofilm Formation by Pseudomonas aeruginosa

The aim of the present study was to evaluate the quorum sensing (QS) inhibition potential of eugenol and eugenol nanoemulsion against QS-dependent virulence factor production and gene expression, as well as biofilm formation in Pseudomonas aeruginosa. In the current study, eugenol nanoemulsion at a sub-MIC of 0.2 mg/mL specifically inhibited about 50% of the QS-mediated violacein production in Chromobacterium violaceum, as well as the production of N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) and C4-HSL N-acyl homoserine lactone signal molecules, pyocyanin, and swarming motility in P. aeruginosa. The inhibitive effect of eugenol and its nanoemulsion on the expression of the QS synthase genes was concentration dependent, displaying 65 and 52% expression level for lasI, respectively, and 61 and 45% expression level for rhlI, respectively, at a concentration of 0.2 mg/mL. In addition, the inhibitive effect of eugenol and its nanoemulsion on the expression of the rhlA gene responsible for the production of rhamnolipid was also concentration dependent, displaying 65 and 51% expression level for the rhlA gene, respectively, at a concentration of 0.2 mg/mL. Eugenol and its nanoemulsion also displayed 36 and 63% respective inhibition of biofilm formation by P. aeruginosa at the 0.2 mg/mL concentration. Therefore, the nanoemulsion could be used as a novel QS-based antibacterial and antibiofilm agent for the control of harmful bacteria.

Effects of an autoinducer analogue on antibiotic tolerance in Pseudomonas aeruginosa.

Antibiotic tolerance causes chronic, refractory and persistent infections. In order to advance the development of a new type of drug for the treatment of infectious diseases, we herein investigated the effects of a newly synthesized analogue of the Pseudomonas aeruginosa quorum-sensing autoinducer named AIA-1 ( a uto i nducer a nalogue) on antibiotic tolerance in P. aeruginosa . A P. aeruginosa luminescent strain derived from PAO1 was injected into neutropenic ICR mice and bioluminescence images were acquired for a period of time after treatments with antibiotics and AIA-1. In vitro susceptibility testing and killing assays for the planktonic and biofilm cells of PAO1 were performed using antibiotics and AIA-1. The expression of quorum-sensing-related genes was examined using real-time PCR. In vivo and in vitro assays showed that AIA-1 alone did not exert any bactericidal effects and also did not affect the MICs of antibiotics. However, the combined use of AIA-1 and antibiotics exerted markedly stronger therapeutic effects against experimental infection than antibiotics alone. The presence of AIA-1 also enhanced the killing effects of antibiotics in planktonic and biofilm cells. Although AIA-1 did not inhibit the expression of lasB and rhlA genes, which are directly regulated by quorum sensing, it clearly suppressed expression of the rpoS gene. The new compound, AIA-1, did not alter the antibiotic susceptibility of P. aeruginosa by itself; however, its addition enhanced the antibacterial activity of antibiotics. AIA-1 did not inhibit quorum sensing, but reduced the antibiotic tolerance of P. aeruginosa by suppressing rpoS gene expression.

Obtaining transgenic alfalfa plants for improved phytoremediation of petroleum-contaminated soils

The possibility of using transgenic plants and their complexes with microorganisms to clean up soil from pollution is a topical research area. In our work the transgenic alfalfa plants with the rhlA gene, responsible for the biosynthesis of biosurfactant–rhamnolipids, were obtained. Rhamnolipids help reduce the surface tension of the hydrocarbon oil and its desorption from soil particles, thereby facilitating its recycling by microorganism. Cultivation of the control and the transgenic alfalfa plants in soil polluted with 4% oil showed the advantage of plants emitting rhamnolipids: oil utilization was 20% higher than with the control plants. When used together, the transgenic plants and Candida maltosa microorganism increased the degree of degradation of the oil to 86%. The results suggest bright prospects for the application of transgenic plants and the complex of transgenic plants and microorganisms to increase the efficiency of bioremediation.

Molecular Basis of Tobacco Induced Bacterial Biofilms

Objective: Tobacco smoke exposure has been correlated to recalcitrant chronic rhinosinustis. Prior work has demonstrated tobacco smoke mediated sinonasal biofilm formation. Thus our objectives were: 1) Construct a series of reporter constructs for pseudomonas biofilm forming genes. 2) Determine which biofilm forming genes are regulated by tobacco smoke. Method: Promoter regions of 6 genes involved in pseudomonas biofilm formation (LasI, LasR, RhlA, PilF, FlgK, and AlgC) were subcloned into the luciferase reporter plasmid pMini-CTX-Lux. Subcloning was confirmed with PCR with subsequent conjugation into Pseudomonas (PAO1). Each subclone was then challenged with serial tobacco smoke exposure and luciferase activity quantified. Results: Elevated levels of luminescence, indicating specific promoter activation was identified after daily tobacco-smoke exposure in the LasI, PilF, FlgK, and AlgC genes. The promoter activation of these genes was significantly elevated ( P &lt; .05) after 2 consecutive exposures with no additional activation following a third exposure (Kruskall-Wallis non-parametric test). The level of promoter activation of the LasB gene was not affected by tobacco smoke exposure, while the RhlA gene decreased progressively by daily exposure. Conclusion: Utilizing the luciferase reporter construct mLuxI we demonstrate activation of several key biofilm forming genes by exposure to tobacco smoke. This confirms our prior finding that tobacco smoke induces biofilms in sinonasal microbes. Interestingly, not all biofilm pathways interrogated demonstrated activation, suggesting unique tobacco mediated biofilm forming pathways in pseudomonas.

A rhodanese-like protein is highly overrepresented in the mutant S. clavuligerus oppA2::aph: effect on holomycin and other secondary metabolites production.

SummaryA protein highly overrepresented in the proteome of Streptomyces clavuligerus oppA2::aph was characterized by MS/MS as a rhodanese‐like enzyme. The rhlA gene, encoding this protein, was deleted from strains S. clavuligerus ATCC 27064 and S. clavuligerus oppA2::aph to characterized the RhlA enzyme activity, growth on different sulfur sources and antibiotic production by the mutants. Whereas total thiosulfate sulfurtransferase activity in cell extracts was not affected by the rhlA deletion, growth, cephamycin C and clavulanic acid production were impaired in the rhlA mutants. Holomycin production was drastically reduced (66–90%) in the rhlA mutants even when using S. clavuligerusΔrhlA pregrown cells, suggesting that this enzyme might be involved in the formation of the cysteine precursor for this sulfur‐containing antibiotic. While growth on thiosulfate as the sole sulfur source was particularly low the volumetric and specific antibiotic production of the three antibiotics increased in all the strains in the presence of thiosulfate. This stimulatory effect of thiosulfate on antibiotic production was confirmed by addition of thiosulfate to pre‐grown cells and appears to be a general effect of thiosulfate on oxidative stress as was also evident in the production of staurosporin by S. clavuligerus.

Inactivation of the rhlA gene in Pseudomonas aeruginosa prevents rhamnolipid production, disabling the protection against polymorphonuclear leukocytes

Many of the virulence factors produced by the opportunistic human pathogen Pseudomonas aeruginosa are quorum-sensing (QS) regulated. Among these are rhamnolipids, which have been shown to cause lysis of several cellular components of the human immune system, e.g. monocyte-derived macrophages and polymorphonuclear leukocytes (PMNs). We have previously shown that rhamnolipids produced by P. aeruginosa cause necrotic death of PMNs in vitro. This raises the possibility that rhamnolipids may function as a 'biofilm shield'in vivo, which contributes significantly to the increased tolerance of P. aeruginosa biofilms to PMNs. In the present study, we demonstrate the importance of the production of rhamnolipids in the establishment and persistence of P. aeruginosa infections, using an in vitro biofilm system, an intraperitoneal foreign-body model and a pulmonary model of P. aeruginosa infections in mice. Our experimental data showed that a P. aeruginosa strain, unable to produce any detectable rhamnolipids due to an inactivating mutation in the single QS-controlled rhlA gene, did not induce necrosis of PMNs in vitro and exhibited increased clearance compared with its wild-type counterpart in vivo. Conclusively, the results support our model that rhamnolipids are key protective agents of P. aeruginosa against PMNs.

A New Metabolic Link between Fatty Acid de NovoSynthesis and Polyhydroxyalkanoic Acid Synthesis: THE PHAG GENE FROM PSEUDOMONAS PUTIDAKT2440 ENCODES A 3-HYDROXYACYL-ACYL CARRIER PROTEIN-COENZYME A TRANSFERASE

To investigate the metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid (PHA) synthesis, we isolated mutants of Pseudomonas putida KT2440 deficient in this metabolic route. The gene phaG was cloned by phenotypic complementation of these mutants; it encoded a protein of 295 amino acids with a molecular mass of 33,876 Da, and the amino acid sequence exhibited 44% amino acid identity to the primary structure of the rhlA gene product, which is involved in the rhamnolipid biosynthesis in Pseudomonas aeruginosa PG201. S1 nuclease protection assay identified the transcriptional start site 239 base pairs upstream of the putative translational start codon. Transcriptional induction of phaG was observed when gluconate was provided, and PHA synthesis occurred from this carbon source. No complementation of the rhlA mutant P. aeruginosa UO299-harboring plasmid pBHR81, expressing phaG gene under lac promoter control, was obtained. Heterologous expression of phaG in Pseudomonas oleovorans, which is not capable of PHA synthesis from gluconate, enabled PHA synthesis on gluconate as the carbon source. Native recombinant PhaG was purified by native polyacrylamide gel electrophoresis from P. oleovorans-harboring plasmid pBHR81. It catalyzes the transfer of the acyl moiety from in vitro synthesized 3-hydroxydecanoyl-CoA to acyl carrier protein, indicating that PhaG exhibits a 3-hydroxyacyl-CoA-acyl carrier protein transferase activity.

Mapping of the immune response genes in the major histocompatibility complex of the Rhesus monkey.

Interest in the Ir genes of rheus monkeys stems from their phylogenetic relationship to man and the extensive data already available on the major histocompatibility complex of the monkey. At least two independent dominant H-linked Ir genes have been identified in the rhesus. These genes control the ability of monkeys to respond to the random linear copolymer of glutamyl alanine (GA), or the dinitrophenyl conjugate of glutamyl lysine (DNP-GL). These synthetic polymers can elicit weak delayed-type skin reactions and strong humoral responses in some monkeys. In a series of unrelated monkeys phenotyped for the serologically defined RhL-A specificities of both segregant series, there were no correlations between any RhL-A specificity and responder status to the GA or DNP-GL polymers. However, segregation analysis of 21 rhesus families sired by 3 fathers indicated the capacity of the offspring to form antibodies was associated with genes coded for in the RhL-A complex. In three monkeys, verified recombination within the RhL-A complex between the genes coding for the serologically defined determinants (SD loci) and the gene(s) controlling the lymphocyte-activating determinants (Lad loci) responsible for mixed lymphocyte reactivity was established. In two of these monkeys the immune response genes controlling the DNP-GL response segregated with the Lad genes, while in the third case the Ir-GL gene segregated with the SD loci, tentatively localizing the Ir-GL gene between the SD and Lad loci. In addition, we have shown that genetically distinct genes control responsiveness to DNP-GL and GA. These genes were separated by recombination, thus one monkey inherited the Lad, Ir-GL, and SD loci from one paternal haplotype and by crossing over inherited the gene controlling GA responsiveness from the other paternal haplotype. The fine structure mapping of the RhL-A gene complex is compared with the H-2 and HL-A gene complexes. Several striking similarities were noted.