Enhanced Extracellular Growth of Staphylococcus aureus

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1 65 Enhanced Extracellular Growth of Staphylococcus aureus in the Presence of Selected Linear Peptide Fragments of Human Interleukin (IL) 1b and IL-1 Receptor Antagonist Siva Kanangat, 1 Michael S. Bronze, 2,a G. Umberto Meduri, 1,a Arnold Postlethwaite, 3,5 Frankie Stentz, 1 Elizabeth Tolley, 4 and Dennis Schaberg 2 1 Memphis Lung Research Program, Department of Medicine, Divisions of Pulmonary and Critical Care Medicine, 2 Infectious Diseases, and 3 Rheumatology, and 4 Department of Preventive Medicine, University of Tennessee, and 5 Department of Veterans Affairs Medical Center, Memphis, Tennessee Replication of Staphylococcus aureus is significantly enhanced in the presence of recombinant interleukin (IL) 1b. In this study, specific binding of IL-1b to the surface of S. aureus significantly increased growth of S. aureus in the presence of IL-1b and IL-1ra in a concentration-dependent manner. Although IL-1ra enhanced the growth of S. aureus, there was a significant reduction in IL-1b mediated growth enhancement of S. aureus when 25-fold excess amounts of IL-1ra (in comparison with the IL-1b concentration) were present in the culture medium. Thus, IL-1b may influence the growth of S. aureus through a receptor-mediated event. By using 5 linear peptides spanning limited regions of IL-1b, the growth-promoting regions were localized to amino acid residues and These results build on the newly evolved concept of direct interactions between the soluble mediators of inflammation and infectious agents. Host defense against invading microbes is primarily mediated by the innate immune response that consists of the elaboration of proinflammatory cytokines (tumor necrosis factor [TNF] a, interleukin [IL] 1b, and IL-6) and subsequent recruitment and activation of neutrophils and macrophages. Recent studies showed integrated and direct interactions between the invading infectious agents and the soluble mediators of the host s innate immune system [1, 2]. For example, certain bacteria express specific cytokine receptors [3, 4] and use cytokines as growth factors [3, 5] or as altered virulence factors [6]. A clinical corollary of this ability of bacteria to use cytokines as growth factors is that persons presenting with intense and protracted inflammation are prone to bacterial infections. Acute respiratory distress syndrome (ARDS) is characterized by prolonged elevated levels of proinflammatory cytokines, such as IL-1b, TNF-a, and IL-6 [7]. Persons who die of ARDS have sustained high levels of inflammatory cytokines, and many develop nosocomial bacterial infections, predominantly ventilator-associated pneumonia [8, 9]. The most common bacterial agents isolated from patients admitted to intensive care units are Staphylococcus aureus, Pseudomonas aeruginosa, and Aci- Received 19 May 2000; revised 6 September 2000; electronically published 10 November Financial support: Assisi Foundation of Memphis, University of Tennessee Medical Group, and Baptist Memorial Health Care Foundation. a Both authors contributed equally to this manuscript. Reprints or correspondence: Dr. Siva Kanangat, 956 Court Ave., H316, Memphis, TN (skanangat@utmem.edu). The Journal of Infectious Diseases 2001;183: by the Infectious Diseases Society of America. All rights reserved /2001/ $02.00 netobacter species. Although we consider bacterial infection in the inflamed host as an epiphenomenon, we recognize that inflammatory mediators could serve as markers of severe illnesses and may directly or indirectly alter the host immune responses against bacterial infections. However, such inflammatory mediators may exert direct effects on the survival and replication of bacteria, thereby enhancing the likelihood of an inflamed host s developing bacterial infections. We recently showed that these bacterial pathogens use IL-1b, IL-6, and TNF-a as growth factors [5, 7]. We observed a biphasic extracellular and intracellular growth curve when these organisms were grown in the presence of increasing concentrations of the cytokines. Bacterial growth was inhibited at lower concentrations of cytokines but was significantly enhanced in the presence of higher levels of cytokines. The cytokine levels that enhanced bacterial growth parallel those found in patients with unresolving ARDS [5, 7]. The present investigation was designed to further elucidate the complex interactions between bacteria and cytokines. We conducted in vitro experiments using S. aureus, to [1] determine the presence of IL-1 receptors on the surface of S. aureus, [2] to localize the region(s) of IL-1b that enhances the extracellular growth of S. aureus, [3] to determine the effects of IL-1ra (which binds to the IL-1 receptor on eukaryotic cells and regulates the expression of IL-1b) on the extracellular growth of S. aureus, and [4] to determine the effects of IL-1b and IL-1ra on the extracellular growth of S. aureus. Materials and Methods Bacteria. Fresh clinical isolates of S. aureus recovered from the bronchoalveolar lavage fluid or blood of patients admitted to

2 66 Kanangat et al. JID 2001;183 (1 January) the University of Tennessee Bowld Hospital (Memphis) were used without serial passage in vitro. We inoculated 3 or 4 well isolated colonies from the primary bacterial plate into 5.0 ml of nutrient broth and incubated the plates at 37 C for 6 8 h. The culture was then centrifuged at 2000 g, and the resulting bacterial pellet was resuspended in PBS at a concentration of 10 6 bacteria/ml. Aliquots of these original cultures were stored at 80 C in nutrient broth containing 15% glycerol, so that the biologic properties could be maintained for repeated experiments. IL-1b, IL-1ra, and IL-1b peptides. Functionally active, 99% pure IL-1b expressed in Escherichia coli was obtained in lyophilized form from R&D Systems and was reconstituted in sterile PBS (ph 7.2), was aliquoted on ice, and immediately was stored at 80 C. Recombinant human (rh) IL-1ra (Synergen) was stored in aliquots at 80 C. The IL-1b peptide fragments were prepared by J. M. Seyer (Department of Biochemistry, University of Tennessee and VA Medical Center, Memphis). Five peptide fragments spanning amino acid (aa) residues , , , , and were used in the current study. The peptide has biologic activity on human glioblastoma cells and induces intercellular adhesion molecule 1 expression; this was blocked by IL- 1ra [10]. Preliminary studies with the remaining 4 peptides failed to demonstrate biologic effects on human cells and were chosen as biologic controls. Bovine serum albumin (BSA) that was used as a stabilizer for the peptide fragments was used as a control (depicted as 0 in the Methods and Results sections) in all experiments. The peptide fragments were also stored at 80 C. Demonstration of IL-1 binding sites on the S. aureus surface. We used a competitive radioligand-binding assay to detect IL-1 binding sites on S. aureus. In brief, cfu/ml of S. aureus grown in RPMI containing 25 mm HEPES buffer and 10 mm L- glutamine were incubated with 100,000 cpm of 125 I-labeled rhil- 1b. Nonradioactive IL-1b was added, at concentrations of mg/ml, for determination of specific binding. Bacteria were incubated for 3 h in a 15 C water bath. The tubes then were centrifuged, and the pellets were washed thrice in RPMI. The radioactivity in the pellet was counted for 10 min by a gamma counter. Specific binding was calculated by subtracting nonspecific binding from total binding [3]. Monitoring growth of S. aureus in the presence of IL-1b peptide fragments, rhil-1ra, and whole recombinant IL-1b. The growth responses of S. aureus to IL-1b peptides copying limited regions of IL-1b and IL-1ra were monitored in RPMI (without serum or serum components), a simple minimal-nutrient tissue culture medium (Life Technologies). This synthetic medium is made without any complex organic materials that are present in conventional bacteriologic medium and has no known interference with any cytokines or cytokine receptors. We added 10 ng of the peptide fragment or various concentrations of whole IL-1b molecules (0, 1, 5, 10, and 50 ng) to 1.0 ml of RPMI in sterile round-bottom culture tubes. Each tube then received 10.0 ml ofthes. aureus suspension (10 5 cfu). Another set of tubes received various concentrations of IL-1ra (0, 3.5, 6.25, 12.5, 25.0, 50.0, and ng/ ml RPMI). To determine the effect of coincubating S. aureus with IL-1b and IL-1ra, we determined the concentration of IL-1b that produced half-maximal growth of S. aureus. This concentration of IL-1b was mixed with 125, 62.5, 33, or 0 ng of IL-1ra in 1.0 ml of RPMI and 10 mlofs. aureus inoculum (10 5 cfu) in sterile roundbottom culture tubes. The controls (0 cytokines) were RPMI and BSA, which was used as a stabilizer in the peptide preparations in the present study. The cultures were incubated at 37 C inahumidified incubator with 5% CO 2 for 6 7 h, with intermittent shaking. Culture aliquots were taken at the end of the incubation period and diluted 10-fold in 100 ml of volume in sterile round-bottom tissue culture plates. From each dilution, we plated 20 ml onto LB agar (Difco) plates, which were incubated for h at 37 C. The resulting bacterial colonies were counted manually and expressed as colony-forming units per milliliter. Statistical analysis. The results were analyzed with 1-way analysis of variance (ANOVA). In the second experiment, growth of S. aureus (expressed as colony-forming units/ml) was considered as the dependent variable, and the concentrations of IL-1b as the independent variable. For the third and fourth experiments, the independent variables were the IL-1ra concentrations. For the fifth experiment, the independent variables were the linear peptide fragments and the control. All comparisons were preplanned and compared various treatment levels with the control or 0 levels. P p.05 was considered statistically significant. Results were expressed as mean SE. Results Demonstration of IL-1 receptors on the S. aureus surface. Previous studies showed the presence of IL-1b receptors on E. coli [3]. By using a competitive radioligand-binding assay, in 6 which 5 10 cfu of S. aureus was incubated with either 100,000 cpm of 125 I-labeled IL-1b or increasing concentrations of nonlabeled IL-1b (0 1.0 mg/ml), we demonstrated specific binding of IL-1b to the surface of S. aureus (figure 1). Extracellular growth of S. aureus in the presence of IL-1b. Figure 2 shows the results of 2 independent experiments in which increasing concentrations of pure recombinant IL-1b were incubated with S. aureus. As we reported elsewhere [7], after a 6 7-h incubation, enhanced bacterial growth was observed in the presence of increasing concentrations of IL-1b, compared with controls in which cultures were grown in BSA ( cfu at 5.0 ng/ml of IL-1b vs cfu; P p.0001). The concentration of IL-1b that produced halfmaximal growth of S. aureus was estimated to be 1 5 ng/ml of culture. Growth response of S. aureus in the presence of IL-1ra. Because IL-1ra modulates the effect of IL-1b in eukaryotic cells, studies were done to determine the effect of IL-1ra on the extracellular growth of S. aureus. The results obtained from 3 independent experiments in which pure IL-1ra was added to the culture are shown in figure 3. Results from all 3 experiments showed a concentration-dependent increase in the extracellular growth of S. aureus. Significant growth enhancement was observed when the concentration of IL-1ra reached 50 and 100 ng/ml of culture: cfu and cfu, respectively, in contrast to the BSA controls ( cfu; P p.0001). Growth response of S. aureus in the presence of half-maximal

3 JID 2001;183 (1 January) S. aureus Growth with IL-1b and IL-1ra 67 Figure 1. Interleukin (IL) 1 binding sites on Staphylococcus aureus surface. S. aureus ( cfu) were incubated with 10 6 cpm of 125 I-labeled IL-1b. Nonradioactive IL-1b was added to tubes (0 1.0 mg/ml), for determination of specific binding. Bacteria (in presence of cold or radioactive IL-1b) were incubated at 15 C for 3 h. Bacterial pellets were collected and washed by repeated centrifugation. Pellet counts were measured for 10 min. Specific binding was determined by subtracting nonspecific binding from total binding. concentrations of whole IL-1b and IL-1ra molecules. To determine the inhibitory effect of IL-1ra on IL-1b activity, S. aureus cultures were incubated simultaneously with half-maximal concentrations of IL-1b and increasing concentrations of IL-1ra. The half-maximal concentration of IL-1b that enhanced the growth of S. aureus was 5.0 ng, on the basis of 2 growthresponse experiments that used increasing levels of pure recombinant IL-1b ( cfu with 5.0 ng versus cfu with BSA control; P p.0002; figure 2). The results obtained from 2 independent experiments (shown in figure 4) demonstrate an 2-fold reduction in the extracellular growth of S. aureus ( P p.0008) when the organisms were grown in the presence of IL-1b and IL-1ra, in contrast to control organisms grown solely in the presence of IL-1b. These data suggest a competition between IL-1b and IL-1ra for receptor occupancy on the surface of these organisms. Extracellular growth response of S. aureus in the presence of IL-1b peptide fragments. The results obtained in 4 independent experiments that used 5 peptide fragments and BSA are shown in figure 5. About fold enhancement ( cfu vs cfu) in the extracellular growth, compared with the BSA control, was observed with the peptide fragment ( P p.0001). Similar, but much less ( 5-fold), enhancement of S. aureus growth ( cfu vs cfu; P p.0075) was seen when peptide fragment was added to the culture medium. As shown, none of the other 3 peptide fragments significantly affected the extracellular growth of S. aureus. Discussion Cytokines are expressed and secreted by eukaryotic cells and, until recently, were thought to act only on eukaryotic cells as part of the host-defense response. Bacteria, despite their limited genetic machinery, have survived the elaborate defense strategies elicited by higher organisms. In fact, the association between protracted inflammatory responses characterized by high levels of IL-1b, TNF-a, and IL-6 and bacterial infections has been recognized in patients diagnosed with sepsis-induced Figure 2. Enhanced Staphylococcus aureus growth in the presence of interleukin (IL) 1b in 2 independent experiments. IL-1b (0, 1.0, 5.0, 10.0, and 50.0 ng) was added to 1.0 ml of RPMI devoid of antibiotics and serum in sterile round-bottom culture tubes. Each tube then received 10 6 cfu of S. aureus. Cultures were incubated at 37 C inahumidified incubator with 5% CO 2 for 6 7 h, with intermittent shaking. Culture aliquots were diluted 10-fold, 20.0 ml of each dilution was spread on Luria broth agar plates, plates were incubated h at 37 C, and bacterial colonies were counted (no. of bacteria expressed as cfu/ml). P values are probabilities from 1-way analysis of variance for preplanned comparisons of control (no added IL-1b) and added IL-1b.

4 68 Kanangat et al. JID 2001;183 (1 January) Figure 3. Enhanced Staphylococcus aureus growth in the presence of interleukin (IL) 1ra in 2 independent experiments. IL-1ra concentrations (0, 3.5, 6.25, 12.5, 25.0, 50.0, and 100 ng) were added to 1.0 ml of RPMI devoid of antibiotics and serum in sterile round-bottom culture tubes. Each tube then received 10 6 cfu of S. aureus. Cultures were incubated at 37 C in a humidified incubator with 5% CO 2 for 6 7 h, with intermittent shaking. Culture aliquots were diluted 10-fold, and 20.0 ml of each dilution was spread on Luria broth agar plates and incubated h at 37 C. Bacterial colonies were counted and expressed as cfu/ml. P values are probabilities from 1-way analysis of variance for preplanned comparisons of control (i.e., 0) and added IL- 1ra. ARDS [11]. Earlier studies demonstrated the presence of IL-1 and TNF receptors on the surfaces of gram-negative bacteria, and bacteria that express these receptors have altered virulence [3, 6]. We recently reported enhanced in vitro extracellular growth of fresh isolates of S. aureus, P. aeruginosa, and Acinetobacter species in the presence of pure recombinant IL-1b, IL-6, or TNF-a [7]. These reports focused on the direct relationship between proximal mediators of inflammation, the cytokines and chemokines, and infectious agents rather than on the cellular components of inflammation. In-depth understanding of these concepts might result in newer strategies for the treatment and management of infectious diseases, especially for persons with exaggerated and protracted inflammatory response such as ARDS. This investigation was designed to further characterize the observed effects of IL-1b on the concentration-dependent extracellular growth of S. aureus [7]. We found a high-affinity binding of IL-1b to S. aureus. This may be a direct IL-1b receptor ligand binding reaction akin to that in eukaryotic cells. However, there are likely other not yet identified molecules on the surface of S. aureus that bind to IL-1b. We used 5 linear peptide fragments of human IL-1b and whole biologically active molecules of IL-1ra and IL-1b, to study their effects on extracellular S. aureus growth. Of the 5 peptide fragments chosen, the fragment had the most pronounced effect on the growth of S. aureus. Elsewhere, this fragment was shown to be pyrogenic and to enhance sleep in rabbits [12]. However, this fragment lacks mitogenic activity on T cells in vitro, indicating its probable inability to interact specifically with the IL-1 receptor (although the possibility of a receptor-ligand interaction cannot be completely ruled out). Peptide enhanced the growth of S. aureus 22-fold, compared with the BSA control. Another peptide spanning aa also significantly enhanced the extracellular growth of S. aureus, although much less efficiently than the fragment. No significant effects were exerted on S. aureus growth by the other 3 fragments. No biologic activities in any other systems have been reported for any of the peptides studied, other than for that spanning aa Because the IL-1b peptide spanning aa activates human glioblastoma cells (in a receptor-mediated event [10]), we believe that the enhanced extracellular growth of S. aureus in the presence of peptide is a receptor-mediated event, even though we do not know the signaling pathways within S. aureus. The several fold increase in extracellular multiplication of S. aureus in the presence of peptide , compared with the whole IL- 1b molecule, could be because the peptide fragment is a better receptor agonist than the whole IL-1b molecule. Several proteinases are released from S. aureus into the extracellular medium [13]; therefore, it is possible that such enzymes may cleave the IL-1b molecule into peptide fragments. Such short peptide fragments may then be transported across the bacterial cell membrane and act as direct growth factors or as transcription factors for the production of bacterial growth factors. This remains speculative, since there is no direct proof of such cleavage activities of S. aureus extracellular proteinases Figure 4. Significant reduction of Staphylococcus aureus growth in presence of submaximal concentrations of interleukin (IL) 1b and 25- fold higher concentrations of IL-1ra in 2 independent experiments. IL- 1b (5.0 ng: half-maximal concentration that significantly enhanced S. aureus growth) was mixed with IL-1ra concentrations (0, 33.0, 62.5, and ng) in 1.0 ml of RPMI without antibiotics and serum in sterile round-bottom culture tubes. Each tube then received 10 6 cfu of S. aureus. Cultures were incubated at 37 C in a humidified incubator with 5% CO 2 for 6 7 h, with intermittent shaking. Culture aliquots were diluted 10-fold, and 20.0 ml of each dilution was spread on Luria broth agar plates and incubated h at 37 C. Bacterial colonies were counted and expressed as cfu/ml. P values are probabilities from one-way analysis of variance for preplanned comparisons between the control and combinations of IL-1b and IL-1ra.

5 JID 2001;183 (1 January) S. aureus Growth with IL-1b and IL-1ra 69 IL-4) or cytokine receptor antagonists (e.g., IL-1ra) as a growth factor demonstrates the versatility of these organisms to react with the dynamic microenvironment as it is affected by various host defense response strategies. When the results of the current investigation are combined, novel mechanistic views on the enhanced growth of S. aureus in the presence of IL-1b seem to emerge. Figure 5. Enhanced Staphylococcus aureus growth in the presence of specific peptides derived from human interleukin (IL) 1b in 4 independent experiments, in which 10.0 ng of each linear synthetic peptide was added to 1.0 ml of RPMI medium devoid of antibiotics and serum in sterile round-bottom culture tubes. Each tube received 10 6 cfu of S. aureus. Cultures were incubated at 37 C in a humidified incubator with 5% CO 2 for 6 7 h, with intermittent shaking. Culture aliquots were diluted 10-fold, and 20.0 ml of each dilution was spread on Luria broth agar plates and incubated for h at 37 C. Bacterial colonies were counted and expressed as cfu/ml. SEs were obtained from each peptide fragment and control separately. P values are probabilities from 1-way analysis of variance for preplanned comparisons between the control and various peptide fragments. on IL-1b, although there are reports of proteolytic cleavages of IL-2, TNF-a, and/or IFN-g by bacterial products of P. aeruginosa and Legionella pneumophila [1]. Our finding that IL-1ra enhanced the extracellular growth of S. aureus was unexpected. When added alone, concentrations of 50 and 100 ng/ml significantly enhanced the growth of S. aureus. IL-1ra acts on eukaryotic cells through the IL-1 receptor and transduces signals that down-regulate the production of IL-1b. Because S. aureus is not known to produce IL-1b, itis possible that IL-1ra transmits a signal similar to that for IL- 1b, resulting in the generation of molecules that enhance the extracellular growth of this bacterium. Alternatively, we envision the possibilities of different pathways activated by IL-1b and IL-1ra in prokaryotic cells. There may be different signaling pathways in these 2 totally divergent systems, and we are currently investigating this possibility. Hultgren et al. [14] reported the ability of S. aureus to use anti-inflammatory cytokines as growth factors, because the organism grew poorly in the joint spaces of IL-4 deficient mice. However, exposure of macrophages to IL-4 reduced the intracellular killing of S. aureus without impairing phagocytosis. We believe that the ability to use anti-inflammatory cytokines (e.g., Acknowledgments We thank Vivian Gomez for figure preparation and Gail Spake (Memphis Lung Research Program, Dept. of Medicine, Div. of Pulmonary and Critical Care Medicine, University of Tennessee) for editorial assistance. The bacterial strains used in these studies were the gift of Vickie Baselski (Department of Pathology, University of Tennessee, Memphis). References 1. Wilson M, Seymour R, Henderson B. Bacterial perturbation of cytokine networks. Infect Immun 1998;66: Casadevell A, Pirofski LA. Host-pathogen interactions: redefining the basic concepts of virulence and pathogenicity. Infect Immun 1999;67: Porat R, Clark BD, Wolff SM, Dinarello CA. Enhancement of growth of virulent strains of Escherichia coli by interleukin-1. Science 1991;254: Zav1yalov VP, Chernovskaya TV, Navolotskaya EV, et al. Specific high affinity binding of human interleukin 1b by CaflA usher protein of Yersinia pestis. 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