Ravi Kumar Gupta

Detection and quantification of quinolone signalling molecule: A third quorum sensing molecule of Pseudomonas aeruginosa by high performance-thin layer chromatography

Sophisticated network of quorum sensing involves the production of chemical signals which regulate the combined expression of virulence genes and biofilm formation in Pseudomonas aeruginosa. Two well-characterized acyl homoserine lactone based las and rhl systems together with alkyl quinolone based Pseudomonas quinolone signalling (PQS) are fundamental components of this network. Third signalling molecule, 2-heptyl-3-hydroxy-4-quinolone (PQS) is of paramount importance because of its interconnecting role in quorum sensing hierarchy in P. aeruginosa. Accurate detection of PQS molecule is very important to understand the involvement of this system in infection process of P. aeruginosa. In this study, high performance-thin layer chromatography (HP-TLC) method was developed for detection as well as quantification of PQS signal molecules in P. aeruginosa, which combines conventional method like TLC with sophisticated instrumentation. This method was validated using parameters like linearity, accuracy, precision, reproducibility and sensitivity. Intra- and inter-day accuracy and precision values were determined which were found to be within acceptable level and hence showed reproducibility. Measurement of PQS in the range of 0.01nmol indicated excellent sensitivity of this approach for quantifying PQS molecule. Automated sampling, rapid and simultaneous analysis of large number of samples and minimal errors make this method more suitable for analysis of PQS signalling molecules. Production of PQS was found to be strain dependent since variation in amount of PQS was observed among different P. aeruginosa isolates. Further, PQS production was also dependent on growth phase of P. aeruginosa with maximum production in late stationary phase.

Quorum sensing signal molecules cause renal tissue inflammation through local cytokine responses in experimental UTI caused by Pseudomonas aeruginosa

Quorum sensing (QS) plays a pivotal role in the virulence of Pseudomonas aeruginosa. Urinary tract infections (UTIs) caused by P. aeruginosa leads to tissue destruction and inflammation. Interaction of host immune cells and pathogen at the epithelial surface are regulated by cytokines. Role of virulence factors has been implicated in the induction of renal cytokine responses. However, information regarding the role of QS, an important virulence factor of P. aeruginosa, is lacking. This study examined role of QS signal molecules for their ability to induce pro and anti-inflammatory renal cytokines and inflammation in experimental UTI. For this, both standard parent P. aeruginosa strain (PAO1) capable of producing QS signal molecules and its isogenic single and double mutant strains lacking this ability were used. Mice infected with QS producer strain PAO1, evoked significant renal tissue inflammation and cytokine response with high neutrophil migration and AHL production in vivo. Mild inflammation, low level of renal cytokines and other factors were observed in mice infected with QS single and double mutant strains. These results provide first evidence regarding the role of QS signal molecules and point towards the importance of AHLs in induction of renal cytokines and tissue inflammatory responses.

Multiple virulence factors regulated by quorum sensing may help in establishment and colonisation of urinary tract by Pseudomonas aeruginosa during experimental urinary tract infection

PURPOSE Damage caused by an organism during infection is attributed to production of virulence factors. Different virulence factors produced by the organism contribute to its pathogenicity, individually. During infectious conditions, role of virulence factors produced by the pathogen is different, depending upon the site of involvement. Pseudomonas aeruginosa is an opportunistic nosocomial pathogen known to cause infections of the respiratory tract, burn wound, urinary tract and eye. Importance of virulence factors produced by P. Aeruginosa during infections such as keratitis, burn wound and respiratory tract is known. The present study was designed to understand the importance of different virulence factors of P. aeruginosa in urinary tract infection in vivo. MATERIALS AND METHODS An ascending urinary tract infection model was established in mice using standard parent strain PAO1 and its isogenic mutant, JP2. Mice were sacrificed at different time intervals and renal tissue homogenates were used for estimation of renal bacterial load and virulence factors. RESULTS Both parent and mutant strains were able to reach the renal tissue. PAO 1 PAO1 was isolated from renal tissue till day 5 post-infection. However, the mutant strain was unable to colonise the renal tissue. Failure of mutant strain to colonise was attributed to its inability to produce protease, elastase and rhamnolipid. CONCLUSION This study suggests that protease, elastase and rhamnolipid contribute to pathogenesis and survival of P. aeruginosa during urinary tract infection.

Acyl homoserine lactones from culture supernatants of Pseudomonas aeruginosa accelerate host immunomodulation.

The virulence of Pseudomonas aeruginosa is multifactorial and under the control of quorum sensing signals, such as acyl homoserine lactones (AHLs). The importance of these molecules in the establishment of infection has been previously reported. These molecules either improve the virulence potential of P. aeruginosa or modulate the host immune response. To establish the immune modulating potential of quorum sensing signal molecules, previous studies have only used synthetic AHLs. However, there can be differences in the biological properties of synthetic and natural AHLs. The use of naturally extracted AHLs from the culture supernatant of P. aeruginosa is likely to simulate natural conditions more than the use of synthetic AHLs. Therefore, in the present study, the immune modulating potential of synthetic and naturally extracted AHLs was compared using a thymidine uptake assay, immunophenotyping and sandwich ELISA in order to assess mouse T-cell proliferation and production of Th1 and Th2 cytokines. Natural AHLs were able to suppress T-cell proliferation, even at low concentrations, compared to synthetic AHLs. The majority of cells undergoing proliferation were CD4+, as revealed by immunophenotyping. The inhibition of T-cells was stronger with natural AHLs compared to synthetic AHLs. Moreover, the natural AHLs were also able to shift immune responses away from host protective Th1 responses to pathogen protective Th2 responses.

Leaf extract of Azadirachta indica (neem): a potential antibiofilm agent for Pseudomonas aeruginosa

Pseudomonas aeruginosa is well known for its ability to form biofilm on indwelling medical devices. These biofilms are difficult to remove because of their high tolerance to conventional antibiotics. Therefore, there is a need to look for alternative agents such as medicinal plants, which can eradicate or inhibit biofilm effectively. This study evaluated the role of neem in inhibiting biofilm formation by P aeruginosa Factors contributing to adherence and biofilm formation were also studied. Results demonstrated that neem leaves extract was quite effective in disrupting formation and structure of biofilms. Moreover, the level of exopolysaccharide, alginate, hydrophobic interactions and uroepithelial cell attachment, which contributes to biofilm formation, was also affected significantly. Results confirm the effectiveness of neem extract in inhibiting biofilm formation. Such studies can lead to the discovery of safe antimicrobial drugs from natural sources without the risk of resistance.

Rhl quorum sensing affects the virulence potential of Pseudomonas aeruginosa in an experimental urinary tract infection

Pseudomonas aeruginosa quorum sensing systems (QSS) las, rhl and PQS play a significant role in pathogenicity. Although multiple studies have explored the role of the las QSS in various types of infections, including urinary tract infections (UTIs), the role of the rhl QSS is still poorly understood. In this study, an attempt was made to explore the role of the rhl QSS in the pathogenesis of UTI. The wild-type strain P. aeruginosa PAO1 and its isogenic rhl QSS mutant strains, PDO-100 and PDO-111, were used. Strains were studied for the expression of extra-cellular virulence factors and the ability to induce experimental UTI in a mouse model. Evaluation was based on in vitro estimation of virulence factors (elastase, protease, rhamnolipid) and in vivo bacteriological and histopathological analysis of renal and bladder tissue. The cytokine levels were determined in the renal tissue of infected mice. Wild-type PAO1 showed the highest level of virulence traits. The tissue bacteriology and histopathology were indicative of establishment of UTI. The level of inflammatory cytokines was consistent with the bacteriology and pathology results. The rhl QSS mutant strains showed significantly reduced pathogenicity. On supplementation with C4-homoserine lactone, strain PDO-100 showed a significant increase in pathogenicity. The results indicate that the rhl QSS affects the virulence potential of P. aeruginosa and that, despite the presence of the las QSS, mutant strains exhibited reduced pathology. Therefore, the rhl QSS may be exploited in more detail for future drug intervention tactics.

Quorum Sensing Signal Molecules Produced by Pseudomonas aeruginosa Cause Inflammation and Escape Host Factors in Murine Model of Urinary Tract Infection

Quorum sensing (QS) is well established for its role in pathogenesis of various infections of Pseudomonas aeruginosa. However, its role in local tissue damage during urinary tract infection (UTI) is not yet fully established. To have insight in this, the present study was planned. UTI was established in mice using standard strain PAO1 and its isogenic QS mutant JP2. One group was challenged only with QS signals. Damage was assessed in terms of histopathology and pathology markers, malondialdehyde (MDA) and reactive nitrogen intermediates (RNI). Effect on pathogen motility, uroepithelial adhesion, and host serum sensitivity was also ascertained. PAO1-infected mice showed severe inflammation and tissue destruction, while mice infected with JP2 showed no significant destruction. JP2 was also unable to mount any tissue pathology markers, MDA and RNI, whereas PAO1 showed significantly higher levels of these two. Presence of only QS signals also showed considerable renal pathology. Strain JP2 also showed less motility, reduced uroepithelial cell adhesion, and increased serum sensitivity. Result highlights that QS signals induce local tissue pathology along with interference of host protective mechanisms during UTI.