Quorum sensing is necessary for the virulence of Pseudomonas aeruginosa during urinary tract infection
2009; Elsevier BV; Volume: 76; Issue: 3 Linguagem: Inglês
10.1038/ki.2009.183
ISSN1523-1755
AutoresRavi Kumar, Sanjay Chhibber, Kusum Harjai,
Tópico(s)Vibrio bacteria research studies
ResumoQuorum sensing is a signaling pathway used by bacteria to monitor their population density by detecting small diffusible molecules. To understand the role of quorum sensing in pathogenesis of urinary tract infections, wild type Pseudomonas aeruginosa, having both functional las and rhl quorum sensing systems, and its isogenic single and double mutants were used in a mouse model of ascending urinary tract infection. Mice were evaluated on the basis of bacteriological and histopathological examinations, detection of acyl homoserine lactone, and the pathologic index factors of renal and bladder tissue. Single as well as double mutant strains were able to reach renal tissue, but they were not able to multiply and cause tissue damage. Reduced rates of infection, mild histopathologic lesions, and decreased production of pathologic index factors like myeloperoxidase, malondialdehyde, and reactive nitrogen intermediates were found in these groups of mice as compared to mice infected with a standard strain (PAO1) possessing both functional sensing systems. Loss of any one of the quorum-sensing components significantly reduced the in vivo virulence of P. aeruginosa in this mouse model of ascending urinary tract infection. Our results suggests that quorum-sensing signals may act as virulence factors and are essential for P. aeruginosa multiplication and virulence during the course of urinary tract infection. Quorum sensing is a signaling pathway used by bacteria to monitor their population density by detecting small diffusible molecules. To understand the role of quorum sensing in pathogenesis of urinary tract infections, wild type Pseudomonas aeruginosa, having both functional las and rhl quorum sensing systems, and its isogenic single and double mutants were used in a mouse model of ascending urinary tract infection. Mice were evaluated on the basis of bacteriological and histopathological examinations, detection of acyl homoserine lactone, and the pathologic index factors of renal and bladder tissue. Single as well as double mutant strains were able to reach renal tissue, but they were not able to multiply and cause tissue damage. Reduced rates of infection, mild histopathologic lesions, and decreased production of pathologic index factors like myeloperoxidase, malondialdehyde, and reactive nitrogen intermediates were found in these groups of mice as compared to mice infected with a standard strain (PAO1) possessing both functional sensing systems. Loss of any one of the quorum-sensing components significantly reduced the in vivo virulence of P. aeruginosa in this mouse model of ascending urinary tract infection. Our results suggests that quorum-sensing signals may act as virulence factors and are essential for P. aeruginosa multiplication and virulence during the course of urinary tract infection. Urinary tract infections are the second most common nosocomial infections. They often occur in patients with an indwelling urinary catheter. They may involve urosepsis with significant morbidity and mortality. Incidence of urosepsis, which is defined as an inflammation of the upper urinary tract causing sepsis and bacteremia, occurs in approximately 16 percent of intensive care unit patients.1.Leone M. Albanese J. Garniser F. et al.Risk factors of nosocomial catheter associated urinary tract infection in a polyvalent intensive care unit.Intens Care Med. 2003; : 1-10Google ScholarPseudomonas aeruginosa is the third most common organism responsible for nosocomial infections and causes 35 percent of nosocomial urinary tract infections.2.Jarvis W.R. Martone W.J. Predominant pathogens in hospital infections.J Antimicrob Chemother. 1992; 29: 19-24Crossref PubMed Scopus (496) Google Scholar This organism is particularly notorious because it forms biofilms on the catheter surface that contribute to the infection process and make this organism difficult to eradicate using antibiotics. Such nosocomial infections are a major cause of concern because of recurrence and chronicity among several patient populations. The pathogenicity of Pseudomonas aeruginosa is attributed to the production of both cell-associated as well as extracellular virulence factors such as type IV pilli, exotoxin A, exoenzyme S, elastase, alkaline protease, hemolysins, phospholipase C, rhamnolipid, and siderophores. It has been shown that expression of most of the extracellular virulence factors as well as biofilm formation is controlled by quorum-sensing signals, which are also used by Pseudomonas aeruginosa to monitor its population density.3.Pesci E.C. Pearson J.P. Seed P.C. Iglewski B.H. Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa.J Bacteriol. 1997; 179: 3127-3132Crossref PubMed Scopus (665) Google Scholar, 4.Pesci E.C. Iglewski B.H. The chain of command in Pseudomonas quorum sensing.Trends Microbiol. 1997; 5: 132-134Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 5.VanDalden C. Iglewski B.H. Cell to cell signaling and Pseudomonas aeruginosa infections.Emerg Infect Dis. 1998; 4: 551-560Crossref PubMed Scopus (608) Google Scholar Quorum sensing exerts its action through small diffusible signal molecules, which, in Gram-negative bacteria, are often called N-acyl homoserine lactones.6.Fuqua C. Winans S.C. Greenberg E.P. Census and consensus in bacterial ecosystems: the LuxR–LuxI family of quorum-sensing transcriptional regulators.Annu Rev Microbiol. 1996; 50: 591-624Crossref PubMed Scopus (924) Google Scholar,7.Fuqua C. Greenberg P. Self perception in bacteria: quorum sensing with acylated homoserine lactones.Currt Opin Microbiol. 1998; 1: 183-189Crossref PubMed Scopus (247) Google Scholar In Pseudomonas aeruginosa, two types of quorum-sensing systems, Las and Rhl, have been recognized.8.Gambello M.J. Iglewski B.H. Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression.J Bacteriol. 1991; 173: 3000-3009Crossref PubMed Scopus (519) Google Scholar The Las system consists of LasI and LasR (encoding an acyl homoserine lactone synthase and a transcriptional activator, respectively) and the acyl homoserine lactone signal N-3-oxo–dodecanoyl homoserine lactone.9.Passador L. Cook J.M. Gambello M.J. et al.Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication.Sci. 1993; 260: 1127-1130Crossref PubMed Scopus (689) Google Scholar,10.Pearson J.P. Gray K.M. Passador L. et al.Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes.Proc Natl Acad Sci USA. 1994; 91: 197-201Crossref PubMed Scopus (800) Google Scholar The Rhl system consists of RhlI and RhlR (encoding an acyl homoserine lactone synthase and a transcriptional activator, respectively) and the acyl homoserine lactone signal N-butanoyl homoserine lactone.11.Ochsner U.A. Reiser J. Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.Proc Natl Acad Sci USA. 1995; 92: 6424-6428Crossref PubMed Scopus (422) Google Scholar,12.Winson M.K. Camara M. Latifi A. et al.Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa.Proc Natl Acad Sci USA. 1995; 92: 9427-9431Crossref PubMed Scopus (423) Google Scholar These two quorum-sensing systems work in a hierarchical manner and are being discussed as candidates of virulence markers by some workers.13.Passador L. Tucker K.D. Guertin K.R. et al.Functional analysis of the Pseudomonas aeruginosa autoinducer PAI.J Bacteriol. 1996; 178: 5995-6000PubMed Google Scholar,14.Hamood A.N. Griswold J.A. Duhan C.M. Production of extracellular virulence factors by Pseudomonas aeruginosa isolates obtained from trachea, urinary tract and wound infections.J Surg Res. 1996; 61: 425-432Abstract Full Text PDF PubMed Scopus (66) Google Scholar The role of quorum sensing in Pseudomonas aeruginosa virulence has only begun to be studied. Some of the clinical studies have suggested that quorum-sensing systems are fully functional in sputum samples from cystic fibrosis patients colonized with Pseudomonas aeruginosa15.Erickson D.L. Endersby R. Kirkham A. et al.Pseudomonas aeruginosa quorum-sensing systems may control virulence factor expression in the lungs of patients with cystic fibrosis.Infect Immun. 2002; 70: 1783-1790Crossref PubMed Scopus (212) Google Scholar as well as from lung tissue of mice infected with Pseudomonas aeruginosa.16.Wu H. Song Z. Hentzer M. et al.Detection of N-acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa.Microbiol. 2001; 146: 2481-2493Crossref Scopus (135) Google Scholar Besides these, a few experimental studies have also shown the contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in certain animal models. These studies have used strains of Pseudomonas aeruginosa having deletions of one or more quorum-sensing-related genes in addition to their parent strains and have observed reduced tissue damage and a low mortality rate caused by mutants in a model of acute pulmonary infection,17.Pearson J.P. Feldman M. Iglewski B.H. et al.Pseudomonas aeruginosa cell-to-cell signaling is required for virulence in a model of acute pulmonary infection.Infect Immun. 2000; 68: 4331-4334Crossref PubMed Scopus (260) Google Scholar burn wound model,18.Rumbaugh K.P. Griswold J.A. Iglewski B.H. Hamood A.N. Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in burn wound infections.Infect Immun. 1999; 67: 5854-5862Crossref PubMed Google Scholar keratitis model,19.Zhu H. Bandara R. Conibear T.C. et al.Pseudomonas aeruginosa with lasI quorum-sensing deficiency during corneal infection.Invest Ophthalmol Vis Sci. 2004; 45: 1897-1903Crossref PubMed Scopus (110) Google Scholar and model of chronic lung infection.16.Wu H. Song Z. Hentzer M. et al.Detection of N-acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa.Microbiol. 2001; 146: 2481-2493Crossref Scopus (135) Google Scholar,20.Willcox M.D.P. Zhu H. Conibear T.C.R. Hume E.B.H. et al.Role of quorum sensing by Pseudomonas aeruginosa in microbial keratitis and cystic fibrosis.Microbiology. 2008; 154: 2184-2194Crossref PubMed Scopus (68) Google Scholar However, little documentation exists with regard to urinary tract infections. A current understanding of the involvement of the acyl homoserine lactone quorum-sensing system in urinary tract infection is based only on the in vitro and in situ findings.21.Stickler D.J. Morris N.S. McLean R.J. Fuqua C. Biofilms on indwelling uretheral catheters produce quorum sensing signal molecules in situ and in vitro.Appl Environ Microbiol. 1998; 64: 3486-3490PubMed Google Scholar,22.Goldsworthy M.J.H. Gene expression of Pseudomonas aeruginosa and MRSA within a catheter associated urinary tract biofilm model.Biosci Horz. 2008; 1: 28-37Crossref Scopus (26) Google Scholar These workers reported production of quorum-sensing signals by uroisolates of Pseudomonas aeruginosa-forming biofilms on indwelling urethral catheters and positive reactions for acyl homoserine lactones on sections of colonized catheters freshly removed from patients. The aim of this study was to elucidate the contribution of quorum sensing in the pathogenesis of urinary tract infection. This was evaluated in a mouse model of ascending urinary tract infection using a standard Pseudomonas aeruginosa strain possessing both functional quorum-sensing systems and its isogenic quorum-sensing single and double mutant strains. Before the establishment of experimental urinary tract infection, the growth rate of wild-type PAO1 and its mutants was checked in vitro in a Luria Broth and M9 medium. No major differences in the growth rate of PAO1 and quorum-sensing mutants were found (data not shown). Pyelonephritic potential was assessed on the basis of quantitation of bacterial load in kidney and bladder tissue homogenates. The extent of pathology was ascertained by the histopathological examination of kidney and bladder tissue as well as by determining pathology index factors such as myeloperoxidase, malondialdehyde, and reactive nitrogen intermediates in kidney and bladder tissues. The results of these findings were further correlated with the production of quorum-sensing molecules in vivo by detecting acyl homoserine lactones in urine and renal tissue of experimental animals by a qualitative method. Kidney and bladder homogenates were plated on MacConkey agar plates for estimation of bacterial counts. Counts per gram of tissue were calculated and expressed as log CFU. Mice infected with standard PA01 strain showed significantly more renal and bladder bacterial count on post-infection days 1, 3, and 5 as compared with the mice infected with isogenic single and double mutant strains (P≤0.01). Log bacterial count of 4.63 CFU per gram of tissue was recovered from the kidneys of mice infected with standard strain PAO1 on post-infection day 1, which increased to log CFU of 6.95 on post-infection day 3 (P≤0.01) and showed a slight decline to log CFU of 6.17 on subsequent post-infection day 5 of killing (Figure 1). Mutant strains JP1, R1, JP2, and JP3 were able to reach the mouse kidney tissue as bacterial lodgment was observed on post-infection day 1, but these strains were cleared off by post-infection day 3 except for the mutant strain JP2, which showed a 2 log cycle decrease on post-infection day 3 and was completely cleared off on post-infection day 5 (Figure 1). Infection rate was calculated on the basis of recovery of Pseudomonas aeruginosa from renal tissue by determining the number of infected mice over a period of 5 days and was expressed as the percentage of mice infected. Standard strain PAO1 showed an infection rate of (80%), which was significantly higher than that of those infected with single- and double-mutant strains. The infection rate in mice infected with mutant strains was significantly lower and varied from 12 to 33%. However, maximum reduction of the infection rate (12%) was observed in double-mutant strain JP3 (Table 1).Table 1Infection rate in mice challenged with standard wild-type Pseudomonas aeruginosa and its isogenic mutant strains defective in quorum-sensing signal productionStrainsGenotypeMice infected (%)Occurrence of infection (n)PAO1Wild type8019/24JP1LasI338/24R1LasR133/24JP2LasIRhlI215/24JP3LasRRhlR123/24 Open table in a new tab Bladder lodgment of standard strain PAO1 was maximum on post-infection day 1 followed by a slight decrease on post-infection days 3 and 5 (P≤0.04). Mutant strain JP1 showed bladder counts on all 3 post-infection days, although the counts showed a slight decrease after post-infection day 1 (P≤0.13), suggesting its sustenance in the bladder. Mutant strain JP2 showed counts till post-infection day 3, whereas mutant strains R1 and JP3 were cleared off from the bladder after post-infection day 1 (Figure 2). Production of quorum-sensing molecules (acyl homoserine lactones) was detected in pooled urine and kidney homogenates of infected groups of mice. The production of acyl homoserine lactone molecules was detected by employing a cross-feeding assay using E. coli MG4 as a reporter strain. Mice infected with the standard strain PAO1 showed production of acyl homoserine lactones in pooled urine as well as kidney homogenates on all 3 post-infection days, whereas none of the mice infected with mutant strains showed production of acyl homoserine lactones on any of the post-infection days (Table 2).Table 2Detection of acyl homoserine lactone in urine and kidney homogenates of mice infected with standard strain PAO1 and its isogenic mutant strains using the cross-feeding assayPseudomonas aeruginosaPooled urineKidney homogenatesDay 1Day 3Day 5Day 1Day 3Day 5PAO1++++++JP1−−−−−−R1−−−−−−JP2−−−−−−JP3−−−−−− Open table in a new tab On histopathological analysis, the medulla, Cortex, Calyx, and subcalyx of each kidney were examined on post-infection day 3, the day of peak infection. In mice infected with the standard strain, kidney tissue sections showed severe inflammation in the interstitium of the medullary and cortical regions, along with infiltration of neutrophils in the glomeruli and the subepithelium of the renal pelvis. Destruction of tubules was also observed. By contrast, mice infected with single- and double-mutant strains showed mild inflammation only (Figures 3 and 4; Supplementary Figure 1, Supplementary Figure 2, Supplementary Figure 3).Figure 4Photomicrograph showing mild inflammation in the interstitium of mice infected with mutant strain JP1 (H&E × 40).View Large Image Figure ViewerDownload (PPT) Download .jpg (.11 MB) Help with files Supplementary Figure 1 Download .jpg (.13 MB) Help with files Supplementary Figure 2 Download .jpg (.11 MB) Help with files Supplementary Figure 3 On histopathological analysis of bladder tissue of mice infected with standard strain PAO1, severe inflammation was observed. The tissue showed infiltration of neutrophils and destruction of mucosa lining along with shedding of cells. Although mice infected with single- and double-mutant strains showed normal bladder tissue morphology without any significant changes, only mild inflammation and a very low level of mucosa destruction was observed in case of mice infected with mutant strain JP1 (Figures 5 and 6).Figure 6Photomicrograph showing no significant changes in the bladder tissue of mice infected with mutant strain JP1 (H&E × 40).View Large Image Figure ViewerDownload (PPT) Infiltration of neutrophils in the renal and bladder tissues of infected mice was also assessed by estimating myeloperoxidase, an enzyme secreted by neutrophils. Myeloperoxidase is known to produce some allied components that oxidize lipoproteins in cells. Myeloperoxidase has also been implicated in the production of tissue injury owing to the production of H2O2. The myeloperoxidase level was significantly high in case of mice infected with standard strain PAO1 on all post-infection days as compared with that in mice infected with mutant strains. In the former group, the myeloperoxidase level was maximum on post-infection day 3 and a slight decline in its level was observed on infection day 5, whereas in the latter groups significantly low levels (P≤0.001) of myeloperoxidase production were observed on all post-infection days (Table 3).Table 3Levels of pathology index factors myeloperoxidase, malondialdehyde, and reactive nitrogen intermediates in renal and bladder tissue homogenates of mice infected with standard strain PAO1 and its mutant strainsStrainPost-infection dayKidneyBladderMPOMDARNIMPOMDARNINormal uninfected mice0.001±0.0010.55±0.11.3±0.10.0001±0.0010.01±0.10.2±0.1PAO110.19±0.00854.2±13.9174.6±34.80.09±0.143.2±0.618±2.230.29±0.0171.2±11.7227.7±29.30.12±0.025.3±0.325.5±4.250.22±0.00882±15.0279±44.00.06±0.0056.3±0.313.8±2.5JP110.12±0.00531.1±5.1125.3±9.70.06±0.0062.2±0.310.5±2.130.05±0.00625.7±7.7198.7±23.20.03±0.0031.5±0.2613.0±0.850.02±0.00620.4±3.0131.9±27.50.01±0.0020.75±0.076.0±0.8R110.06±0.00718.9±4.464.1±18.80.01±0.0032.4±0.378.6±0.7530.01±0.00316.9±3.232.9±5.20.006±0.0021.8±0.153.7±0.2350.005±0.00212.7±3.519.5±1.60.0006±0.0031.48±0.121.94±0.41JP210.04±0.027.8±2.117.1± 3.40.02±0.0021.0±0.343.7±0.430.007±0.0013.4±1.49.7±3.10.001±0.0010.6±0.11.6±0.150.003±0.0012.9±1.13.3±1.40.0001±0.0010.5±0.011.1±0.1JP310.04±0.036.1±2.311.3±2.70.02±0.010.8±0.11.9±0.230.007±0.0014.9±2.18.1±2.00.002±0.0010.4±0.010.8±0.150.006±0.0011.1±1.52.7±1.10.0002±0.0010.2±0.010.2±0.1Significance level (P-values): (1, 3 and 5 days post-infection).MPO, MDA and RNI: PAOI vs JP1 (P≤0.001). PAOI vs R1 (P≤0.001). PAOI vs JP2 (P≤0.0001). PAOI vs JP3 (P≤0.0001). Open table in a new tab Significance level (P-values): (1, 3 and 5 days post-infection). MPO, MDA and RNI: PAOI vs JP1 (P≤0.001). PAOI vs R1 (P≤0.001). PAOI vs JP2 (P≤0.0001). PAOI vs JP3 (P≤0.0001). Malondialdehyde is used as an indicator of lipid peroxidation. Lipid peroxidation mediated by free radicals might yield a large number of reactive aldehydes, such as malondialdehyde, which are casually involved in pathophysiological changes associated with oxidative stress in cells and tissues. Therefore, we estimated that the level of cellular injury occurred because of the release of reactive oxygen species as a result of lipid peroxidation in terms of malondialdehyde levels in kidney and bladder tissue. Acute infection of kidneys of mice with standard strain PAO1 showed significantly increased malondialdehyde production. By contrast, infection with mutant strains significantly decreased malondialdehyde production and prevented lipid peroxidation-mediated tissue damage (P≤0.001) (Table 3). Reactive Nitrogen Intermediates are an index of the level of nitrite produced by neutrophils and macrophages with the help of NO synthase by the formation of peroxynitrite in response to inflammatory responses to microbial products. It involves the conversion of L-arginine to L-citrulline catalyzed by NO in active cells. Estimation levels of reactive nitrogen intermediates in renal and bladder tissues of mice infected with standard strain PAO1 showed increased production from 1 to 5 days of infection. Mice infected with mutant strains showed a decrease in the level of units of reactive nitrogen intermediates in renal and bladder tissues after post-infection day 1, which was found to be minimal on post-infection day 5 except in mice infected with strain JP1 where an increase in the renal reactive nitrogen intermediates level was observed on post-infection day 3, which later decreased on post-infection day 5 (P≤0.001) (Table 3). All strains were evaluated for their ability to induce urinary tract infection in mice. Initial standardization of the animal model was carried out by using Pseudomonas aeruginosa standard strain PAO1. Mice infected with the standard strain PAO1 were killed at different time intervals from 2 h to 7 h post-infection day (data not shown). On the basis of the recovery of Pseudomonas aeruginosa from the renal tissue, post-infection days 1, 3, and 5 were selected as days of killing for studying different parameters in the course of infection. Mutant strain JP1 (ΔLasI) and R1 (ΔLasR) were able to reach the kidneys and were recovered only on post-infection day 1. However, these strains were unable to establish in the renal tissue because no multiplication and increase in bacterial count was observed on subsequent post-infection days, whereas both the strains were cleared from the renal tissue after post- infection day 3. For establishment of infection and tissue damage, the pathogen should be able to express its virulence factors (which are quorum sensing regulated in case of Pseudomonas aeruginosa). Lack of production of quorum-sensing signals with no expression of Las-regulated virulence factors did not result in successful infection. JP1 was able to reach the kidney in lesser number as compared with the wild-type PAO1 strain. The innate immune response generated in terms of neutrophil infiltration was sufficient to clear these bacteria from the kidney tissue on post-infection day 3. Histopathological analysis showed mild inflammation in the interstitium and mild glomerular infiltration in the renal tissue. Reduced rate of infection, lodgment in renal tissue, pathological findings, and other pathology index factors such as myeloperoxidase, malondialdehyde, and reactive nitrogen intermediates were indicative of avirulence of quorum-sensing mutant strains in the urinary tract infection model. Observations made with respect to bladder tissue indicated bladder lodgment of mutant strain JP1 on all three post-infection days, indicating that this strain was able to lodge in the bladder tissue and was also able to sustain itself in the tissue for 3 days, but the pathological findings of bladder tissue showed only mild inflammation and very low levels of mucosa destruction. A possibly active Rhl system in this mutant regulating the production of biosurfactant rhamnolipid could have helped in its retention in high lipid-containing bladder tissue. Mutant strain R1 was cleared off from bladder tissue after post-infection day 1 and normal bladder tissue morphology with no significant changes was observed. As activation of the Rhl system requires involvement of LasR and mutant R1 is defective in LasR, it is possible that no Rhl activation would have taken place in this case. These findings were also supported by reduced levels of myeloperoxidase, malondialdehyde, and reactive nitrogen intermediates in the bladder tissues of both groups of animals, indicating that despite the presence of mutant strains in the bladder tissue, these strains were not able to cause any pathology and hence were avirulent. This reduced establishment of LasI and LasR mutant strains, along with reduced pathological markers in renal and bladder tissues, suggests that quorum-sensing signals (Las) may act as the probable virulence factor in the pathogenesis of urinary tract infection. The hierarchical arrangement shows that the Las system has an essential role in the functioning of quorum sensing.23.Wagner V.E. Li L.L. Isabella V.M. Iglewski B.H. Analysis of the hierarchy of quorum sensing regulation in Pseudomonas aeruginosa.Anal Bioanal Chem. 2007; 387: 469-479Crossref PubMed Scopus (59) Google Scholar The regulation of pathogenic traits by quorum-sensing circuitry operating in Pseudomonas aeruginosa is a highly effective strategy to establish an infection.24.Charu G. Srivastava S. Quorum sensing: the phenomenon of microbial communication.Current Sci. 2006; 90: 666-677Google Scholar Some of the quorum-sensing regulated virulence genes are under the control of both quorum-sensing systems and some others are regulated specifically by either the Las or Rhl system. Swimming, swarming, and twitching motilities, which have an important role at the initial stages of infection25.O'Toole G.A. Kolter R. Flagellar and twitching motilities are necessary for Pseudomonas aeruginosa biofilm development.Mol Microbiol. 1998; 30: 295-304Crossref PubMed Scopus (2047) Google Scholar and are required for adherence and colonization of mucosal surfaces such as urinary tract, are controlled by both the Las and Rhl systems.26.Juhas M. Eberl L. Tummler B. Quorum sensing: the power of cooperation in the world of Pseudomonas.Environ Microbiol. 2005; 7: 459-471Crossref PubMed Scopus (294) Google Scholar Ascending of the two mutants in the renal tissue in the present model of ascending urinary tract infection can be attributed to the three types of motilities regulated by both the Las and Rhl systems. It is possible that mutation in the Las quorum-sensing system may have been compensated by the Rhl quorum-sensing system with respect to motilities. For causing infection, the success of a pathogen depends not only on its ability to reach the renal tissue but also to multiply and increase their number and form successful communities or to develop biofilms (as in the case of Pseudomonas) in the renal tissue. The mere presence of the pathogen in any mucosal tissue may not necessarily correlate with the state of infection. The inability of mutants to multiply in the renal tissue, resulting in decreased pathological lesions, could be explained on the basis of regulation of virulence factors by quorum-sensing systems. Factors such as biofilm formation, elastase, alkaline protease and Exo A have a very important role in mucosal infections13.Passador L. Tucker K.D. Guertin K.R. et al.Functional analysis of the Pseudomonas aeruginosa autoinducer PAI.J Bacteriol. 1996; 178: 5995-6000PubMed Google Scholar,27.Shaber J.A. Carty N.L. Mcdonald N.A. et al.Analysis of quorum sensing deficient clinical isolates of Pseudomonas aeruginosa.J Med Microbio. 2004; 53: 841-853Crossref PubMed Scopus (172) Google Scholar and are shown to be regulated by the Las system of Pseudomonas aeruginosa. A deficiency in quorum sensing could potentially result in the inability of the mutants to form biofilms or elaborate important extracellular virulence factors in the renal tissue. This may have lead to early clearance and may not help the mutants to establish. Shih and Huang28.Shih P.C. Huang C.T. Effects of quorum sensing deficiency on Pseudomonas aeruginosa biofilm formation and antibiotic resistance.JAC. 2002; 49: 309-314Google Scholar also showed a defect in the biofilm accumulation rate in the quorum-sensing deficient mutants. Wu et al.16.Wu H. Song Z. Hentzer M. et al.Detection of N-acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa.Microbiol. 2001; 146: 2481-2493Crossref Scopus (135) Google Scholar correlated the clearance of mutant strains with a quicker, faster, and stronger immune response against these mutants in the early phase of bacterial infections. These workers reported a role of pronounced humoral immune response in terms of levels of cytokines, IFN-γ, and IL-4 in quicker bacterial clearance of mutant strains from lungs in a chronic lung infection model. In the second part of the study, double mutant strains JP2 (Δ LasI RhlI) and JP3 (Δ LasR RhlR) were used in the urinary tract infection model. Reduced infection rates (21 and 12%) were observed in mice infected with JP2 and JP3. Renal lodgment of JP2 and JP3 mutant strains was significantly less than that of standard strain PAO1. Although mutant strain JP2 was recoverable from the renal as well as the bladder tissue of infected mice on post-infection day 3, its log count continued to remain very low. Mutant strain JP3 was cleared on post-infection day 1 from the renal and bladder tissues. The histopathology and pathology index factors were also low in case of mice infected with these mutant strains. Less virulence of the double mutant strain JP2 as compared with the single mutant strain has also been observed by Rumbaugh et al.18.Rumbaugh K.P. Griswold J.A. Iglewski B.H. Hamood A.N. Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in burn wound infections.Infect Immun. 1999; 67: 5854-5862Crossref PubMed Google Scholar in the burn wound model. The effect of mutations on Pseudomonas aeruginosa virulence has also been shown to be consistent with the effect on production of virulence factors such as elastase and rhamnolipids by Pseudomonas aeruginosa.10.Pearson J.P. Gray K.M. Passador L. et al.Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes.Proc Natl Acad Sci USA. 1994; 91: 197-201Crossref PubMed Scopus (800) Google Scholar Elastase and rhamnolipid expression is shown to be important in the invasion of pathogen in the blood stream17.Pearson J.P. Feldman M. Iglewski B.H. et al.Pseudomonas aeruginosa cell-to-cell signaling is required for virulence in a model of acute pulmonary infection.Infect Immun. 2000; 68: 4331-4334Crossref PubMed Scopus (260) Google Scholar and in the burn wound infection model.18.Rumbaugh K.P. Griswold J.A. Iglewski B.H. Hamood A.N. Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in burn wound infections.Infect Immun. 1999; 67: 5854-5862Crossref PubMed Google Scholar Similarly, regulation of motilities has been shown to have a role in cystic fibrosis.16.Wu H. Song Z. Hentzer M. et al.Detection of N-acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa.Microbiol. 2001; 146: 2481-2493Crossref Scopus (135) Google Scholar The failure of mutants to disseminate in the skin tissue in the burn wound mouse model,18.Rumbaugh K.P. Griswold J.A. Iglewski B.H. Hamood A.N. Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in burn wound infections.Infect Immun. 1999; 67: 5854-5862Crossref PubMed Google Scholar as well as to produce less pathological lesions in the pneumonia model17.Pearson J.P. Feldman M. Iglewski B.H. et al.Pseudomonas aeruginosa cell-to-cell signaling is required for virulence in a model of acute pulmonary infection.Infect Immun. 2000; 68: 4331-4334Crossref PubMed Scopus (260) Google Scholar and in the chronic lung infection model,16.Wu H. Song Z. Hentzer M. et al.Detection of N-acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa.Microbiol. 2001; 146: 2481-2493Crossref Scopus (135) Google Scholar has indicated the role of a quorum-sensing mechanism in these infections. Pearson et al.17.Pearson J.P. Feldman M. Iglewski B.H. et al.Pseudomonas aeruginosa cell-to-cell signaling is required for virulence in a model of acute pulmonary infection.Infect Immun. 2000; 68: 4331-4334Crossref PubMed Scopus (260) Google Scholar have also shown association of avirulence of the mutant strains with lack of functioning Las and Rhl genes because the complementation of wild-type genes was able to restore the virulence of the mutant strains to nearly that of wild type. On the basis of the establishment of parent strain PAO1 having both quorum-sensing functional systems and avirulence of mutant strains in the urinary tract infection model, our results indicate that in vivo virulence of Pseudomonas aeruginosa in urinary tract infection is significantly reduced on the loss of any of the quorum-sensing system components, suggesting that functional quorum-sensing systems are essential for the virulence of Pseudomonas aeruginosa in the course of urinary tract infections. Further work regarding the elaboration of virulence factors by mutant strains and the correlation of these factors with mutations will throw more light on these observations. Pseudomonas aeruginosa standard strain PA01 and its isogenic single mutants JP1 (ΔLasI::Tcr), R1 (Δ LasR::Tcr) and isogenic double mutant strains JP2 (ΔLasI::Tcr Hgr ΔRhlI::Tn 501) and JP3 (ΔLasR::Tcr Hgr ΔRhlR::Tn 501) were very kindly provided by Dr. Barbara H. Iglewski, University of Rochester, New York, USA. E. coli MG4 (pKDT17) was used as a reporter strain for detection of exogenous acyl homoserine lactones. Plasmid pKDT17 contains LasR under control of a lac promoter and a LasB::LacZ translational fusion. Antibiotics Tetracycline and Mercuric chloride were added to the media (50 μg/ml and 7.5 μg/ml, respectively) for the growth and maintenance of double mutant strains (JP2 and JP3). Tetracycline (50 μg/ml) was used for single mutant strains (JP1 and R1). Ampicillin was added (100 μg/ml) for the growth of E. coli MG4 to maintain the plasmid. The method of Mittal et al.29.Mittal R. Chhibber S. Sharma S. Harjai K. Macrophage inflammatory protein-2, neutrophil recruitment and bacterial persistence in an experimental mouse model of urinary tract infection.Microbes infec. 2004; 6: 1326-1332Crossref PubMed Scopus (35) Google Scholar was used for the induction of urinary tract infection. For the use of animals, ethics approval was granted by the Panjab University Ethics Committee. Female LACA (Swiss Webster) mice, 6–8 weeks old, free of bacteriuria were used for the experiment. 50 μl of 108 CFU/ml of adjusted inoculum was introduced into the bladder of mice using a soft non-radio-opaque polyethylene tubing (outer diameter 0.61 mm, Clay Adams, A division of BD company, Parsipanny, NJ, USA). The catheter was withdrawn slowly without any further manipulation. Animals were killed on post-infection days 1, 3, and 5. Kidneys and bladders were aseptically removed and weighed. Kidney and bladder tissues were homogenized in 1 ml sterile saline solution. A quantitative bacterial count was made after plating appropriate dilutions of tissue homogenates on MacConkey agar plates. Log bacterial counts were calculated per gram of tissue as reported by Harjai et al.30.Harjai K. Mittal R. Chhibber S. Sharma S. Contribution of Tamm-Horsfall protein to the virulence of Pseudomonas aeruginosa in relation to urinary tract infections.Microbes infec. 2005; 7: 132-137Crossref PubMed Scopus (21) Google Scholar Kidney and bladder tissues were fixed in 10% buffered normal saline and dehydrated in 30–100% gradient ethanol. Paraffin wax blocks were prepared and 5 μ thin sections were stained with hematoxylin and eosin. The medulla, cortex, calyx, and subcalyx of each kidney tissue were evaluated. Induction of pathology was evaluated on the basis of malondialdehyde, the index of lipid peroxidation. The method of Anjaneyulu and Chopra,31.Anjaneyulu M. Chopra K. Effect of irbesartan on the antioxidant defence system and nitric oxide release in diabetic rat kidney.Am J Nephr. 2004; 24: 488-496Crossref PubMed Scopus (57) Google Scholar was used. Briefly, 0.5 ml of tissue homogenate was added to 0.5 ml of Tris HCl (0.1 M, pH7.4) and incubated at 37 °C for 2 h followed by the addition of 1 ml of ice-cold Trichloroacetic acid (10% w/v). Samples were centrifuged at 1000 r.p.m. for 10 min. 1 ml of supernatant was then taken and mixed with 1 ml of thiobarbituric acid (0.67% w/v). Tubes were covered and kept in a boiling water bath for 10 min. After cooling, absorbance was read at 532 nm. Tissue neutrophils were quantitated by using the myeloperoxidase assay by the method of Hang et al.32.Hang L. Haraoka M. Agace W.W. et al.Macrophage inflammatory protein-2 is required for neutrophil passage across the epithelial barrier of the infected urinary tract.J Immunol. 1999; 162: 3037-3044PubMed Google Scholar Briefly tissues were homogenized in 2 ml of 50 mM potassium phosphate pH 6.0 with 0.5% hexadecyl trimethyl ammonium bromide and 5 mM EDTA. The homogenate was sonicated and centrifuged at 12,000 g for 15 min. Supernatant was then mixed with 1:15 assay buffer (O-diansidine 0.167 mg/ml in 50 mM phosphate buffer with 5 μl of H2O2) and absorbance was read at 490 nm. Nitrite was estimated in the renal and bladder tissues of infected mice following the method of Rockett et al.33.Rockett K.A. Awburn M.M. Rockett E.J. et al.Possible role of nitric oxide in malarial immunosuppression.Parasite Immunol. 1994; 16: 243-249Crossref PubMed Scopus (160) Google Scholar Briefly, 100 μl of the sample was mixed with 200 μl of Griess reagent (Sigma Aldrich Chemicals Ltd., St Louis, MO, USA) followed by the addition of 100 μl of 10% trichloroacetic acid and incubated for 20 min at room temperature. After centrifugation, the optical density of supernatants was read at 540 nm. The amount of nitrite was determined using standard curves of sodium nitrite. Luria Bertani agar plates covered with 40 μl of X-gal (5-bromo-4-chloro-3-indoyl–β-D-galactopyranoside, 20 mg/ml stock solution in dimethyl formamide) were used for the cross-feeding assay. Kidney tissue homogenates and urine were extracted for acyl homoserine lactone with acidified ethyl acetate as described by Shaw et al.34.Shaw P.D. Ping G. Daly S.L. Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin layer chromatography.Proc Natl Acad Sci USA. 1997; 94: 6036-6041Crossref PubMed Scopus (679) Google ScholarE. coli MG4 was streaked on LB plate and ethyl acetate-extracted acyl homoserine lactones were spotted on LB plates approximately 1 cm apart. Appearance of blue color in the reporter strain was indicative of the production of acyl homoserine lactone. All the experiments were repeated three times to validate the reproducibility of the experiments. Results were analyzed statistically by a Student's t-test using Graphpad software to calculate P-values. Contingency grant for this work was kindly provided by the Department of BioTechnology, New Delhi, India. We gratefully acknowledge Barbara H. Iglewski for providing standard and mutant strains of Pseudomonas aeruginosa. We are also thankful to BN Dutta (retired) and Kusum Joshi (Dept of Histopathology, PGIMER) for their generous help in the histological examination. Figure S1. Photomicrograph showing glomerular infiltration, vascular permeability and protein casts in tubules along with destruction of tubules in the renal tissue of mice infected with standard strain PAO1 (H and E X40). Figure S2. Photomicrograph showing the medullary region of kidney with protein casts in tubules. Lining epithelial cells are swollen and vacuolated associated with marked inflammation in the renal tissue of mice infected with standard strain PAO1 (H and E X100). Figure S3. Photomicrograph showing glomerular infiltration, vascular permeability and protein casts in tubules, along with destruction of tubules in the renal tissue of mice infected with standard strain PAO1. Upper cellular glomerular surrounding tubules also shows casts in lumen and swollen and vacuolated lining epithelial cells (H and E X400). Supplementary material is linked to the online version of the paper at http://www.nature.com/ki Download .doc (.03 MB) Help with doc files Supplementary Figures Legends
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