Global gene expression in Staphylococcus aureus biofilms.
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Journal: 2004/August - Journal of Bacteriology
ISSN: 0021-9193
Abstract:
We previously demonstrated that mutation of the staphylococcal accessory regulator (sarA) in a clinical isolate of Staphylococcus aureus (UAMS-1) results in an impaired capacity to form a biofilm in vitro (K. E. Beenken, J. S. Blevins, and M. S. Smeltzer, Infect. Immun. 71:4206-4211, 2003). In this report, we used a murine model of catheter-based biofilm formation to demonstrate that a UAMS-1 sarA mutant also has a reduced capacity to form a biofilm in vivo. Surprisingly, mutation of the UAMS-1 ica locus had little impact on biofilm formation in vitro or in vivo. In an effort to identify additional loci that might be relevant to biofilm formation and/or the adaptive response required for persistence of S. aureus within a biofilm, we isolated total cellular RNA from UAMS-1 harvested from a biofilm grown in a flow cell and compared the transcriptional profile of this RNA to RNA isolated from both exponential- and stationary-phase planktonic cultures. Comparisons were done using a custom-made Affymetrix GeneChip representing the genomic complement of six strains of S. aureus (COL, N315, Mu50, NCTC 8325, EMRSA-16 [strain 252], and MSSA-476). The results confirm that the sessile lifestyle associated with persistence within a biofilm is distinct by comparison to the lifestyles of both the exponential and postexponential phases of planktonic culture. Indeed, we identified 48 genes in which expression was induced at least twofold in biofilms over expression under both planktonic conditions. Similarly, we identified 84 genes in which expression was repressed by a factor of at least 2 compared to expression under both planktonic conditions. A primary theme that emerged from the analysis of these genes is that persistence within a biofilm requires an adaptive response that limits the deleterious effects of the reduced pH associated with anaerobic growth conditions.
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J Bacteriol 186(14): 4665-4684
PMID: 15231800

Global Gene Expression in <em>Staphylococcus aureus</em> Biofilms

Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, Wyeth Research, Pearl River, New York 10965, Wyeth Protein Technologies, Cambridge, Massachusetts 02140, Department of Microbiology and Immunology, The University of Texas Southwestern Medical Center, Dallas, Texas 753904
Corresponding author. Mailing address: Department of Microbiology and Immunology, Mail Slot 511, University of Arkansas for Medical Sciences, 4301 W. Markham, Little Rock, AR 72205. Phone: (501) 686-7958. Fax: (501) 686-5359. E-mail: ude.smau@skramreztlems.
Received 2004 Jan 13; Accepted 2004 Apr 5.
Copyright © 2004, American Society for Microbiology

Abstract

We previously demonstrated that mutation of the staphylococcal accessory regulator (sarA) in a clinical isolate of Staphylococcus aureus (UAMS-1) results in an impaired capacity to form a biofilm in vitro (K. E. Beenken, J. S. Blevins, and M. S. Smeltzer, Infect. Immun. 71:4206-4211, 2003). In this report, we used a murine model of catheter-based biofilm formation to demonstrate that a UAMS-1 sarA mutant also has a reduced capacity to form a biofilm in vivo. Surprisingly, mutation of the UAMS-1 ica locus had little impact on biofilm formation in vitro or in vivo. In an effort to identify additional loci that might be relevant to biofilm formation and/or the adaptive response required for persistence of S. aureus within a biofilm, we isolated total cellular RNA from UAMS-1 harvested from a biofilm grown in a flow cell and compared the transcriptional profile of this RNA to RNA isolated from both exponential- and stationary-phase planktonic cultures. Comparisons were done using a custom-made Affymetrix GeneChip representing the genomic complement of six strains of S. aureus (COL, N315, Mu50, NCTC 8325, EMRSA-16 [strain 252], and MSSA-476). The results confirm that the sessile lifestyle associated with persistence within a biofilm is distinct by comparison to the lifestyles of both the exponential and postexponential phases of planktonic culture. Indeed, we identified 48 genes in which expression was induced at least twofold in biofilms over expression under both planktonic conditions. Similarly, we identified 84 genes in which expression was repressed by a factor of at least 2 compared to expression under both planktonic conditions. A primary theme that emerged from the analysis of these genes is that persistence within a biofilm requires an adaptive response that limits the deleterious effects of the reduced pH associated with anaerobic growth conditions.

Abstract

Staphylococcus aureus is a prominent human pathogen that causes a wide variety of infections. Of particular interest in our laboratory are musculoskeletal infections including those associated with orthopedic implants. The hallmark characteristic of these infections is formation of a biofilm, which consists of multiple layers of bacteria encased within an exopolysaccharide glycocalyx. The presence of this glycocalyx protects the enclosed bacteria from host defenses and impedes delivery of at least some antibiotics (64). Moreover, bacteria within biofilms adopt a phenotype that confers intrinsic resistance to many antibiotics. For example, the reduced growth rate of biofilm-associated bacteria limits the efficacy of antibiotics that target cell wall biosynthesis, while the reduced oxidative metabolism limits the uptake of aminoglycosides (33, 64, 65). Consequently, biofilm-associated infections are recalcitrant to antimicrobial therapy and often require surgical intervention to debride infected tissues and/or remove colonized implants.

The formation of three-dimensional biofilms is a complex process that can be subdivided into the relatively distinct phases of attachment, accumulation, maturation, and dispersal (10). With respect to staphylococcal biofilms, the primary emphasis so far has been placed on the attachment and accumulation phases, which appear to be mediated by different types of adhesins. More specifically, a group of surface-exposed proteins collectively referred to as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) (48) appear to be the primary determinants responsible for the initial attachment to both native tissues and biomaterials, while the accumulation phase appears to be dependent on polysaccharide adhesins that promote adhesive interactions between bacterial cells (26). Although a number of candidate polysaccharides have been described, there is an emerging consensus that the primary determinant of the accumulation phase of staphylococcal biofilm formation is the polysaccharide intercellular adhesin (PIA), production of which is dependent upon the genes within the icaADBC operon (28). Composition studies have demonstrated that PIA consists of polymeric N-acetylglucosamine, and for this reason it has also been referred to as PNAG (40).

The ica operon was first identified in Staphylococcus epidermidis (28) and has been studied most extensively in that species. However, it is also present and appears to serve the same function in S. aureus (14). Most S. aureus strains appear to contain the entire ica operon (14, 22, 53), although there are reports to the contrary (3), and it is clear that there are strain-dependent differences with respect to the overall capacity to form a biofilm in vitro (5, 14, 53). The ica operon is subject to phase variation in S. epidermidis (75), and a number of studies have indicated that expression of ica in both S. epidermidis and S. aureus is also subject to environmental regulation. Perhaps most importantly, McKenney et al. (42) demonstrated that PNAG production in S. aureus is enhanced during in vivo growth. Rachid et al. (52) subsequently demonstrated that expression of ica is at least partially controlled by the stress response transcription factor σ. In addition, anaerobic growth was found to induce expression of the ica operon and PIA production in both S. epidermidis and S. aureus (15).

Recently, Conlon et al. (12) reported that icaR, which is located immediately upstream of the ica operon, encodes a repressor that is important for the environmental regulation of ica expression in S. epidermidis. However, studies done with S. aureus have demonstrated that regulation of ica expression and the ability to form a biofilm also involve regulatory elements other than σ and IcaR (66). Included among these additional regulatory loci are the accessory gene regulator (agr) and the staphylococcal accessory regulator (sarA). The agr locus encodes a two-component quorum-sensing system that modulates production of a regulatory RNA molecule (RNAIII) in a density-dependent manner. Induction of RNAIII synthesis results in reduced production of surface proteins (e.g., MSCRAMMs) and a concomitant increase in production of exotoxins (4, 45). Production of δ-toxin, which is encoded within the RNAIII locus, has been negatively correlated with biofilm formation (69, 70). This suggests that strains expressing agr at high levels would have a reduced capacity to form a biofilm, which is consistent both with our results (5) and results from other laboratories (70).

The sarA locus encodes a 14.5-kDa DNA-binding protein (SarA) that is required at least under some growth conditions for maximum expression from the agr and RNAIII promoters (29). This would imply that mutation of sarA would limit production of RNAIII and thereby enhance the ability to form a biofilm. However, recent reports have confirmed that mutation of sarA results in a reduced capacity to form a biofilm (5, 66). SarA also regulates expression of other genes in an agr-independent manner (6, 19, 72, 74), and Valle et al. (66) recently demonstrated that mutation of sarA results in reduced transcription of the ica operon and a reduced capacity to produce PNAG. They also suggested that SarA may promote biofilm formation in an indirect manner by suppressing transcription of a repressor of PNAG synthesis or a protein involved in the turnover of PNAG.

The persistence of bacteria within a biofilm also requires an adaptive response appropriate for the sessile lifestyle. The availability of complete bacterial genome sequences has facilitated the use of microarray technologies to identify genes that are differentially expressed by biofilm-encased bacteria. Using an array representing 99% of the Bacillus subtilis genome, Stanley et al. (63) identified 519 genes that were differentially expressed in biofilms as opposed to planktonic cultures. Similarly, Schembri et al. (59) found that 5 to 10% of the genes in the Escherichia coli genome were differentially expressed in biofilms, depending on which planktonic growth condition was used as a reference. Included among these genes were 30 of the 65 genes previously reported to be under the regulatory control of the general stress response regulator rpoS (36). Schembri et al. (59) subsequently demonstrated that an E. coli rpoS mutant was incapable of forming a biofilm. However, Whiteley et al. (71) found that expression of rpoS was repressed in Pseudomonas aeruginosa biofilms and that a P. aeruginosa rpoS mutant formed a more extensive biofilm than the corresponding wild-type strain. While these results confirm that biofilms represent a unique growth state by comparison to planktonic cultures, they also suggest the existence of species-specific pathways that contribute to biofilm formation and maintenance of the sessile lifestyle. To date, no comprehensive transcriptional analysis of S. aureus biofilms has been reported. However, Prigent-Combaret et al. (51) demonstrated that biofilm-encased E. coli encounter high osmolarity, oxygen limitation, and higher cell density than cells grown under planktonic conditions, and all of these factors are known to influence gene expression in S. aureus (11, 45).

To further investigate these issues, we generated sarA and ica mutations in a clinical isolate of S. aureus (UAMS-1) and examined their relative capacity to form a biofilm both in vitro and in vivo. We also used a custom-made Affymetrix GeneChip representing the combined genomes of six strains of S. aureus (N315, Mu50, COL, NCTC 8325, EMRSA-16 [strain 252], and MSSA-476) to investigate differential gene expression in a mature S. aureus biofilm.

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