rpos contributes to variations in the survival …...contribute to decreased susceptibility of...

Journal of Experimentall Microbiology and Immunology (JEMI) Vol. 14: 21-27 Copyright © April 2010, M&I UBC

RpoS Contributes to Variations in the Survival Pattern of Pseudomonas aeruginosa in Response to Ciprofloxacin

Marlo Firme, Hasan Kular, Crystal Lee, and Diana Song

Department of Microbiology and Immunology, University of British Columbia

Pseudomonas aeruginosa is of increasing concern in hospital settings because of frequently emerging antibiotic resistant strains implicated in nosocomial infections. There exists a complex network of gene expression regulatory mechanisms that contribute to decreased susceptibility of P.aeruginosa to difference classes of antibiotics. One protein recently shown to be involved in antibiotic resistance to ciprofloxacin is Lon, a protease that degrades misfolded proteins in addition to upregulating a number of stress response genes. In this study, RpoS and DnaK, two downstream targets of Lon, were assessed to determine their contribution to antibiotic susceptibility. RpoS is a transcriptional regulator involved in surviving various environmental conditions. DnaK is a chaperone that binds to nascent polypeptides to prevent protein misfolding, a function similar to that of Lon. We investigated the kinetics through which RpoS and DnaK affect susceptibility to ciprofloxacin by way of minimal inhibitory concentration (MIC) and time-dependent killing assays. Results indicate no significant difference in MIC but enhanced sensitivity to ciprofloxacin in dnaK strains contrasted by delayed susceptibility in rpoS mutants by a survival difference of 15%. Taken together, these results suggest that DnaK is involved in transient ciprofloxacin tolerance but global resistance to antibiotics is mediated by complex gene regulatory networks that may not be entirely dependent on RpoS.

Pseudomonas aeruginosa is a gram negative opportunistic pathogen and a major cause of nosocomial infections in immunocompromised individuals and patients with severe burns and cystic fibrosis (9). Infections with P. aeruginosa are especially difficult to treat due to the organism’s high intrinsic antibiotic resistance pertaining to efflux pumps (5) in addition to an outer membrane of low permeability (30). The success of this organism in causing a significant proportion of hospital-acquired diseases is in part related to complex regulatory networks that allow efficient adaptation to changing environmental conditions. One gene that contributes to pathogenesis and antibiotic resistance is lon, a gene that encodes for the ATP-dependent Lon protease involved in the degradation of abnormal proteins (e.g. during heat shock) and in protein quality control (22). Brazas et al. have shown that lon mutants are significantly more sensitive to the DNA gyrase inhibitor ciprofloxacin and that resistance to this antibiotic in wild type strains is mediated by a combination of mechanisms pertaining to the induction of stress responses. The exact mechanism of lon-mediated antibiotic resistance has yet to be elucidated, but it is known that Lon regulates a number of stress-responses

including response to DNA damaging agents such as the DNA gyrase inhibitor ciprofloxacin (3).

Two specific proteins downstream of Lon are RpoS (R.E.W. Hancock, personal communication) and DnaK (8), both of which are important for survival in stressful conditions. In particular, RpoS (σs), a sigma factor, is a general stress response regulator that affects the transcription of genes which confer increased tolerance to stresses such as nutrient limitation, heat and osmotic stress (23). RpoS is able to activate genes which have its corresponding promoter by increasing RNA polymerase recruitment (25). Genes controlled by RpoS have many functions including maintenance of growth in stationary phase, virulence, metabolism, and changes in cell envelope and morphology (26). Kayama et al. (14) have determined that RpoS regulates more than 50 genes in Pseudomonas aeruginosa and that it participates in ofloxacin tolerance. In addition, RpoS regulated genes also allow the cell to survive new stresses that it has not yet encountered, a term call cross-protection (23). Based on these observations, we investigated the role of RpoS in antibiotic resistance and analyzed the killing kinetics in P.aeruginosa in response to ciprofloxacin. In addition, we also examined the role of DnaK, a heat shock protein regulated by RpoS that assists in the folding of nascent

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peptides and binds misfolded peptides to reduce the accumulation of abnormal proteins, a role similar to that of the Lon protease. E.coli mutants in dnaK are more susceptible to the bactericidal effects of fluroquinolone antibiotics (29). Many more RpoS regulated genes also exist, which, like dnaK, may result in the mutant rpoS being more susceptible to antibiotics than wild type.

In this study, we investigated what we believed to be one possible mechanism by which Lon confers ciprofloxacin resistance in P.aeruginosa, which is through RpoS and its downstream protein DnaK. Transposon mutants in rpoS and dnaK were tested for altered susceptibilities to the three classes of antibiotics currently used in treating P.aeruginosa infections – fluoroquinolone (ciprofloxacin), β-lactam (piperacillin) and polymyxin B. Minimal inhibitory concentration (MIC) assays showed no significant difference between the wild type and mutants although killing assays with ciprofloxacin revealed variations in killing kinetics for the two mutants, suggesting that RpoS and DnaK are potentially involved in transient antibiotic tolerance.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The P. aeruginosa strains used in this study included the wild type PA14 as well as 2 mutants from the P.aeruginosa transposon mutant library from Harvard University (15). Transposon mutant identification numbers for rpoS and dnaK are PA3622 ID 32095 and PA4761 ID46422, respectively. Strains were routinely grown at 37oC on Luria-Bertani (LB) broth [1.0% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl; Difco Laboratories, Detroit, MI]. In all experiments, overnight cultures were grown with agitation (200 rpm) in LB broth. When required for transposon maintenance, LB agar plates were supplemented with gentamicin to a final concentration of 15 μg/ml.

Growth curves. P. aeruginosa strains were grown overnight in liquid BM2-swarming. Five-microliter portions of these cultures were added to 195 µl of fresh BM2-swarming medium in 96-well microtiter plates (Costar, USA). The growth of these cultures at 37°C under shaking conditions was monitored with a TECAN Spectrofluor Plus by determining the absorbance at 600 nm every 17 min for 20 h.

Gram stain. Cells were inoculated onto glass slides using heat-sterilized metal loop from wildtype, rpoS-, dnaK- cultures and heat fixed with a Bunsen burner. Slides were Gram stained and visualized under light microscope (Unilux-12 light microscope) with 100X objective oil immersion lens. Pictures of three field of views were taken of each of the antibiotic treated and untreated samples using NOKIA E71 cellular phone. Quantification of cell size was performed by comparing width to length ratios of individual cells with computer software Olympus Fluoview Ver.1.4a.

Antibiotics. Ciprofloxacin (Sigma, UK) Polymyxin B (Sigma, UK) Gentamicin and Piperacillin (Bayer, USA) were used for the MIC screening. Antibiotics were dissolved in dH2O at 32μg/ml, filter sterilized (0.2 μm pore size), and kept at -20oC until use.

Minimal inhibitory concentration assay. Wild-type and mutant PA14 strains were streaked onto LB agar plates supplemented with 15 μg/ml gentamycin and incubated for 24 hours at 37oC. Cultures were prepared from isolated colonies in fresh LB broth and incubated overnight at 37oC. Overnight cultures were diluted 1/30 in LB broth (a final volume of 900 µl) and turbidity of samples was measured at OD600nm. Cultures were then diluted in fresh LB broth using the following equation adapted from the protocol from MacFarlane et al.(16): 4.16/OD600 = μl to add to 1 ml LB media (16). From this 1ml

of diluted culture, 50 μl was added to each well of a 96-well plate pre-filled with 50 μl/well of antibiotics at indicated concentrations and incubated at 37oC for 18 h. Minimum inhibitory concentrations were defined as the lowest concentration of antibiotic at which no growth (no turbidity) was observed. Treatments were applied in triplicate and the mean absolute deviation was measured for cases where MICs were not consistent.

Time-dependent killing assay. Wild-type and mutant PA14 strains were streaked onto LB agar plates supplemented with 15 μg/ml gentamicin and incubated for 24 h at 37oC. Cultures were prepared from isolated colonies in 8 ml fresh LB broth and incubated overnight at 37oC at 200 rpm. The following day, overnight cultures were diluted 1/100 in fresh LB broth ( a final volume of 2 ml) and incubated at 37oC at 200 rpm in New Brunswick Scientific Excella E24 Incubator Shaker Series for approximately 3 hours until samples reach mid-log phase at 0.5 OD600. At this time, 1 ml was removed from each sample, centrifuged at 3000 xg in Eppendorf Centrifuge 5415D for 10 minutes to pellet cells, and resuspended in 10 ml fresh LB broth. Turbidity readings at OD600 were taken at time zero with Pharmacia Biotech Utrospec 3000 spectrophotometer prior to addition of ciprofloxacin. Serial dilutions of each culture were performed in 0.9% (w/v) sterile saline at time zero to plate samples on LB agar plates at final dilutions of 10-5, 10-6, 10-7. One hundred microliters of stock ciprofloxacin (32 μg/ml) was then added to each of the 10 ml wild-type and mutant cultures to obtain a final concentration of 0.4 μg/ml, four times the MIC level determined from MIC assays. Control samples include untreated wild-type and mutant cultures. Samples were incubated at room temperature shaking at 150rpm on Orbit Shaker Labline. At each indicated time point, turbidity reading was obtained at OD600 and cultures were plated at 10-5, 10-6, and 10-7 dilutions on LB agar plates after serial dilution in sterile saline. Plates were incubated at 37oC aerobically for 20 h and counted the following day to obtain cfu/ml of each sample. Turbidity and plate counts at each time point were normalized to measurements at time zero to obtain a normalized growth curve of percentage survival.

RESULTS

P. aeruginosa PA14 wild type and mutant strains

did not exhibit significant differences in antibiotic MICs MIC assays with the antibiotics polymixin B, ciprofloxacin and piperacillin were performed on the wild type and dnaK and rpoS mutant strains to determine any differences in antibiotic resistance over 18 hours. For the antibiotic polymyxin B, dnaK mutants were observed to have a lower minimal inhibitory concentration than either wild type or rpoS (Table 1)\. Contrarily, the wild type was shown to have a MIC lower than both rpoS and dnaK mutants for piperacillin (Table 1). No difference in MICs between the wild type and mutant strains was observed for ciprofloxacin. Despite different MIC values for polymyxin B and piperacillin between mutant and wild

TABLE 1. Minimum inhibitory concentrations (MICs) of P. aeruginosa PA14 wild type and mutant strains to various

antibiotics. Polymyxin B

(µg/mL) Ciprofloxacin (µg/mL)

Piperacillin (µg/mL)

PA14 (Wild Type)

2.0 0.1 8.0

rpoS 2.0 0.1 13.3 ± 5.2 dnaK 1.3 ± 0.4 0.1 16.0

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type strains, all variations differ by less than two folds, a range generally considered to be within the error of standard assessment protocols previously established by Breidenstein et al. (2).

Turbidity of the wild type and mutant strains increased in spite of ciprofloxacin treatment. Since no significant differences were observed from MIC assays, turbidity measurements were performed during killing assays to examine differences in the survival pattern of wild type and mutant strains over a shorter period of time. Specifically, wild type, rpoS and dnaK strains were treated with 4 times the MIC of ciprofloxacin and the turbidity of the cultures was measured to obtain an indication of growth or killing

over 70 minutes. All 3 strains, whether treated or untreated, exhibited a trend of increase in turbidity over 70 min (Fig. 1A, B, C). However, significant difference between treated and untreated samples of the same strain was only observed for dnaK mutants (Fig.1C). In the control samples without antibiotic treatment, dnaK mutants demonstrated the fastest rate of increase in turbidity, followed by rpoS mutants and subsequently by the wild type strain (Fig. 1D). With the addition of ciprofloxacin at time zero, rpoS mutants exhibitied the greatest rate of increase in turbidity, followed by the wild type. The dnaK mutantstrain showed only minimal changes in growth (Fig. 1E).

FIG. 1. Normalized growth curves of wild type and mutant P.aeruginosa strains in response to ciprofloxacin treatment determined by turbidity measurements at OD660nm. Turbidity readings at each time point were normalized to the OD600 at time zero to compare growth between treated and untreated samples of wild type (A), rpoS (B), dnaK (C) as well as growth between wild type and mutant strains -ciprofloxacin (D) and +ciprofloxacin (E).

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Viability of wild type and mutant strains decreased in the presence of ciprofloxacin. In order to better quantify the viability of mutant and wild type strains after antibiotic treatment, plate counts were performed on the cultures during the killing assays to determine the number of cells that were able to grow in response to ciprofloxacin. Survival, as determined from colony forming units (cfus), of both wild type and mutant strains decreased in response to treatment with 4 times the MIC of ciprofloxacin over 60 min (Fig. 2A). Both the rpoS mutant and wild type experienced an initial increase in cfusupon addition of ciprofloxacin at time zero followed by a rapid decrease in the wild type at 10 minutes and a gradual decline for the rpoS mutant at 25 minutes (Fig. 2A). dnaK mutants, contrarily, did not experience the initial increase and instead began to decrease in cfus starting immediately(Fig. 2A). At 60 minutes, survival of rpoS mutants was 15% higher than that of either wild type or dnaK mutant strains (Fig. 2A). Without ciprofloxacin treatment, all strains increased in cfus with the exception of dnaK mutants, which exhibited only minimal growth (Fig. 2B). Wild type strains entered a plateau in cell growth while rpoS mutants increased exponentially starting from 30 minutes (Fig. 2B). The killing assay was performed for a total of three times and the most representative data set is shown.

rpoS and dnaK mutants increased in cell size in response to ciprofloxacin. Since discrepancies between turbidity and colony forming units were observed, we performed gram stains on wild type and mutant strains to determine any differences in cell morphology or spatial distribution. Increase in cell size, determined from length to width ratio, was observed for both dnaK and rpoS mutants in response to four times the inhibitory concentration of ciprofloxacin (Table 2). Over 60 minutes, rpoS and dnaK mutants demonstrated a size increase of 22% and 17% respectively (Table 2). Differences seen in wild type samples upon treatment were insignificant (P > 0.05). It was also observed that dnaK mutants formed clusters that were dispersed upon antibiotic treatment (Fig. 3). This observation was seen in all three replicates of gram stain performed.

TABLE 2. Changes in cell size in response to 60 minutes of ciprofloxacin treatment

Length to width ratio Untreated Treated

% increase

P-value

Wild Type

1.88 2.01 6.74 0.22

ΔrpoS 2.19 2.81 22.06 0.0006 ΔdnaK 1.68 2.04 17.65 7.21x10-5

FIG. 2. Viability of wild type and mutant strains in response to ciprofloxacin treatment determined from plate counts. Survival of wild type and mutants with the addition of ciprofloxacin (A) and without (B). Plate counts for killing assays were performed twice the more representative data is shown.

DISCUSSION

P. aeruginosa is one of the leading causes of

nosocomial infections in immunocompromised individuals. Such infections are especially difficult to treat because of emerging antibiotic resistant strains, a trend that could be attributed to selective pressures from antibiotic treatment as well as the organism’s intrinsic ability to adapt to drug-stressed environments. Examples of mechanisms by which P. aeruginosa facilitate resistance to antibiotics include efflux pumps, presence of an outer membrane of low permeability, and expression of certain stress response genes such as lon. Specifically, two proteins downstream of Lon are RpoS and DnaK, the former of which is a global regulator of stress response genes pertaining to stationary phase growth and antibiotic tolerance. dnaK is one gene regulated by RpoS. It is a heat shock protein involved in peptide folding and is therefore a potential contributor to antibiotic resistance. Here, we have examined the role of RpoS and DnaK in mediating tolerance to different classes of antibiotics currently used in P. aeruginosa infections and have demonstrated that dnaK and rpoS mutants both elicit different survival patterns upon ciprofloxacin treatment during killing assays despite insignificant differences in minimal inhibitory concentrations determined from MIC assays.

Results from the MIC assays were not as expected since the absence of RpoS in mutant strains should

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render rpoS mutant cells incapable of expressing RpoS-dependent genes. Dysregulation of these genes should have led to increased antibiotic-susceptibility. This was not observed in the MIC assays results. Similarly, DnaK was also expected to contribute to antibiotic resistance since its assistance in the folding of nascent polypeptides is involved in surviving antibiotic stress. However, mechanistic effects of RpoS and DnaK on antibiotic resistance may not have been detectable in the MIC assays performed. Specifically, the turbidity observed after 18 hours of incubation could have been a result of growth from a relatively small population of surviving cells that does not accurately represent characteristics of the entire population. MIC assays cannot determine the proportion of cells killed nor the rate of killing upon antibiotic exposure. Additionally, RpoS and DnaK may have contributed to antibiotic resistance over a short period of time but their effects may not have been observed over the 18-hour period. Because of these reasons, killing assays were performed to better analyze the kinetics of antibiotic killing over a shorter time frame of 60 minutes.

The delayed sensitivity of rpoS P. aeruginosa mutants to ciprofloxacin treatment over 60 minutes in comparison to wild type and dnaK mutant strains (Fig. 2A) could be rationalized in part by suggesting that components of the RpoS-dependent stress response may potentially be disadvantageous to cell viability in the environmental conditions used during the killing assay. Whiteley et al. demonstrated increased antibiotic resistance in rpoS mutants to tropomycin (25) through their ability to form significantly thicker biofilms than

their wild type counterpart. Therefore, it is also possible that the delay in sensitivity noticed in this killing assay experiment is due to expression of biofilm-related genes that confer clumping of cells in ΔrpoS mutants despite constant shaking of cultures. Gram stains of the rpoS mutants, however, displayed no clustering or biofilm formation in comparison to the wild type sample (Fig. 3).

FIG. 3. Gram stain of treated and untreated wild type and mutant strains at 60 minutes. rpoS and dnaK mutants demonstrate overall increase in cell size in response to ciprofloxacin treatment.

An alternate explanation for the delayed ciprofloxacin sensitivity of rpoS mutants is related to the large repertoire of downstream genes involved in the RpoS global stress response. Little is known about the role of rpoS in antibiotic resistance in P. aeruginosa (18). Yet, it is speculated that downstream genes normally suppressed by RpoS may have been relieved from repression in the rpoS mutant such that expression of these genes contributed to delayed ciprofloxacin susceptibility (10). Possible phenotypic adaptations in the rpoS mutant include changes in outer membrane permeability through modification of phospholipid or lipopolysaccharide content, increased expression of drug efflux pumps to prevent antibiotic accumulation, or altered susceptibility of target enzymes such as DNA gyrase and topoisomerase to ciprofloxacin (10, 11).

As already described, rpoS mutants may also have experienced delayed susceptibility due to an increase in a ciprofloxacin efflux pump, mexAB-oprM (10). Forty percent of genes regulated by RpoS are related to quorum sensing (18). In particular, rpoS mutants are shown to have elevated levels of Rh1R-Rh1I-regulated gene transcription involved in the regulation of quorum sensing. This system enhances production of the

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quorum-sensing autoinducer, N-butyrl-L-homoserine lactone (C4-HSL), which increases the expression of mexAB-oprM efflux pumps to prevent ciprofloxacin accumulation in the cell (10, 21, 26). The presence of an increased number of these efflux pumps may have contributed to the 15% higher cell survival rate of rpoS mutants compared to the wild type and dnaK mutant strains.

All strains tested experienced an overall decrease in cell viability upon ciprofloxacin treatment in the killing assay (Fig. 2). The initial incline in growth for wild type and rpoS mutant strains could be attributed to the time lag it takes for ciprofloxacin to exert bacteriocidal effects on cells. It is possible that the more gradual decrease seen in rpoS mutants than in wild type is a result of higher transient antibiotic resistance in the mutant strain since the absence of RpoS potentially generates a different network of genes to allow for delayed antibiotic susceptibility. The initial incline is not present in dnaK mutants which show immediate decline in cell viability from ciprofloxacin at time zero. This could be a result of higher sensitivity of dnaK mutants to ciprofloxacin since ciprofloxacin works on protein folding, a function in which DnaK is involved. Absence of DnaK in the mutant would therefore result in a greater number of misfolded peptides and render cells more susceptible to protein damages from ciprofloxacin treatment.

Lack of RpoS expression in wild-type strains at exponential phase may also have contributed to the decline in percentage survival observed. As previously described, induction of RpoS significantly increases at the onset of stationary phase to mediate cell survival in response to stress (10). Because killing assays were performed in cells from exponential phase (0.50 OD600), RpoS levels in wild type strains would have been present only in minimal amounts that are insufficient to induce any noticeable differences between wild type cells and mutant strains. This therefore plays a role in the more rapid decline in viability for wild type strains as opposed to rpoS mutants.

Despite a decrease in cell viability from plate counts, an increase in culture turbidity could be explained in part by antibiotic-induced damage to cells that still allows for growth in culture during the 60-minute killing assay but not on solid media overnight. In this case, decreased viability from plate counts is a result of cells that were committed to death rather than cells that were already dead in liquid culture during the killing assay. Increase in turbidity could also have resulted from changes in color and increase in cell size of wild type and mutant strains. P.aeruginosa secrete pyocyanin and pyoverdin, both of which are virulence factors that mediate killing of mammalian cells via generation of reactive oxygen species or chelation of iron compounds in the host respectively (17, 20).

Secretion of pyocyanin gives P. aeruginosa strains a blue-green hue, which when combined with the yellow pigment in pyoverdine, contributes to the green color observed in pseudomonas cultures (20). This color is more intense in wild type cells than in mutant strains. Since turbidity readings were taken at 600nm, the wavelength at which green-yellow colors are absorbed, coloring of cultures from pyocyanin and pyovedine production likely contributed to the increase in absorbance seen in both wild-type and mutant strains. Increase in cell size for dnaK and rpoS mutants in response to antibiotic treatment also played a role in increasing turbidity despite reduced plate counts (Table 2). In the case of dnaK mutants, clustering of cells in culture is potentially another contributing factor to the increased turbidity observed (Fig. 3.)

In conclusion, our results indicated no significant difference in MIC between wild type, rpoS, or dnaK mutants and therefore no significant antibiotic resistance to ciprofloxacin. The immediate decline in cell viability of dnaK mutants during the first 20 minutes of ciprofloxacin exposure supports the involvement of DnaK in ciprofloxacin tolerance. This result, combined with the 15% greater survival of rpoS mutants compared to wild type and dnaK mutant strains after 60 minutes, suggests that RpoS-regulated genes (i.e. dnaK) are involved in antibiotic tolerance but global resistance to antibiotics is mediated by complex gene regulatory networks that may not be entirely dependent on RpoS.

FUTURE DIRECTIONS To better examine the clustering of cells in response

to antibiotic treatment, cultures should be visualized on wet mount slides via phase contrast microscopy to minimize artifacts from Gram stain (i.e. drying of water droplets and heat fixing). Killing assays should also be performed in stationary phase to elucidate effects on cell viability pertaining to differential RpoS expression levels in exponential and stationary phase growth. The role of RpoS in delayed ciprofloxacin susceptibility can be further implicated through Western blot analyses. Another way by which this correlation could be examined is via RpoS over-expression. The rpoS gene can be inserted into a high copy plasmid (e.g. pUCP18), placed under the control of a constitutively active promoter, and transformed into P.aeruginosa with a wild type background. Antibiotic killing of the transformants can be determined through time dependent ciprofloxacin killing assays. In the case that RpoS contributes to delayed ciprofloxacin susceptibility, cells that over-express this protein should exhibit a more gradual decline in cell viability compared to wild type cells. Other proteins downstream of Lon can also be assessed to determine if they are

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involved in antibiotic resistance. It would also be advisable to use knockout mutants for the genes tested rather than mutants from a transposon library to ensure that differences in antibiotic susceptibility is not a result of partial gene expression.

ACKNOWLEDGEMENTS

We thank Dr. Ramey for his advice in experimental design and data interpretation. We also thank Hancock lab for providing P.aeruginosa strains and Gold lab for supplying experimental equipment and reagents. We thank Jessica Labonté for her help throughout the project.

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