Requirement of the agr Locus for Colony Spreading of Staphylococcus aureus

Abstract

The important human pathogen Staphylococcus aureus is known to spread on soft agar plates. Here, we show that colony spreading of S. aureus involves the agr quorum-sensing system. This finding can be related to the agr-dependent expression of biosurfactants, such as phenol-soluble modulins, suggesting a connection between spreading motility and virulence.

The Gram-positive bacterium Staphylococcus aureus is frequently encountered among the human microbiota (41). For most individuals, S. aureus is an apparently harmless commensal. However, once S. aureus passes the primary barriers imposed by the human skin or mucosa, it becomes evident that this organism is in fact a dangerous pathogen. S. aureus is then capable of infecting almost every tissue and organ, causing a wide range of acute and chronic diseases (16, 31). This ability to cause infections depends on a diverse array of cell wall-associated and extracellular virulence factors (44, 49, 64). The expression of many S. aureus virulence genes is coordinated by the accessory gene regulator (agr) quorum-sensing system (33, 42, 46), which responds to cell density-dependent stimuli. At high cell densities or in confined compartments, this system upregulates the expression of secreted virulence factors and downregulates the expression of cell wall-associated virulence factors (38-40, 42). Consequently, cell wall proteins and surface adhesins are expressed during the early, colonizing stages of infection whereas secreted proteins, such as hemolysins, lipases, and proteases, are expressed at later, tissue-damaging stages (14). Notably, when S. aureus is cultured in vitro, all hemolysins are upregulated at the transition from the late exponential to the stationary phase (38-40, 42, 64).

Activity of the agr system involves two major transcripts named RNAII and RNAIII. RNAII covers the agrABCD operon. The membrane protein AgrB is involved in (i) processing of the AgrD product into the activating auto-inducing octa-peptide AIP, (ii) secretion of AIP, and (iii) modification of AIP. AgrC is a histidine kinase that binds the extracellular AIP and, in turn, modulates the activity of the response regulator AgrA, which determines the synthesis of RNAII and RNAIII (14, 38-40, 63). RNAIII is the effector molecule of the agr locus that upregulates the transcription and, in some cases, the translation of secreted proteins (2, 37). Conversely, RNAIII downregulates the transcription of cell wall-associated proteins (11). Furthermore, the 5′ end of RNAIII encodes the toxin δ-hemolysin (also known as phenol-soluble modulin γ [PSMγ]).

Bacterial pathogens often employ motility mechanisms for host colonization. Almost 40 years ago, Henrichsen made a distinction among six different categories of bacterial surface motility, which he referred to as swimming, swarming, gliding, twitching, sliding, and darting (18). Swimming and swarming are dependent on flagella, whereas twitching has been shown to require type IV pili, as do some forms of gliding. Sliding and darting are forms of passive bacterial movement (16). Sliding is correlated with the production of surfactants, such as lipopeptides, lipopolysaccharides (LPS), or glycolipids (16). It was proposed that expansion forces of dividing S. aureus cells cause the motility phenomenon that was named darting motility (18). More recently, a different form of S. aureus motility was defined as colony spreading (18, 26), which resembles sliding, as it is independent of flagella or pili. In 2008 the so-called fudoh gene was implicated in the inhibition of colony spreading (25). This gene is present on the type II and III staphylococcal chromosomal cassettes mec (SCCmec) of certain methicillin-resistant S. aureus (MRSA) strains (25) but not on the six other presently known types of SCCmec (i.e., types I and IV to VIII) (7-9, 23). The underlying mechanisms of colony spreading as well as the precise role of the fudoh gene in this process have thus far remained elusive. Therefore, the aim of the present study was to define key determinants for colony spreading of S. aureus.

Correlation between staphylococcal colony spreading and the quorum-sensing system agr.

To determine which genetic features are important for colony spreading, a collection of MRSA and methicillin-sensitive S. aureus (MSSA) strains with different characteristics (Table 1) were tested for the ability to spread on tryptic soy soft agar plates (0.24% agar). The spreading assay was performed as described by Kaito et al. (25, 26) with minor modifications. Each plate (capacity, 10 ml) was dried for approximately 10 min in a laminar flow cabinet. From an overnight culture in tryptic soy broth, an aliquot of 2 μl was spotted in the center of a plate, which was subsequently dried for 5 min under laminar flow. The plates were then incubated overnight at 37°C. Images were recorded with a G:Box (Syngene, Leusden, Netherlands). The results are summarized in Table 2, and some representative images are presented in Fig. 1. When the ability to spread was compared to other features of the tested strains, it became apparent that there was a clear correlation between spreading and the presence of an intact agr system for quorum sensing (Table 2). For example, it was shown previously that S. aureus NCTC8325, MRSA252, MSSA476, MW2, RF122, Newman, and USA300 express RNAIII (19, 50). These strains were clearly able to spread. S. aureus COL expresses low levels of RNAIII due to an agr defect (19, 50), and our results show that this strain has minimal spreading ability (Table 2). The heavily mutagenized S. aureus strain RN4220 has a mutation in agrA which results in an agr-deficient phenotype. Consistent with the notion that agr might have a role in colony spreading, our results show that S. aureus RN4220 does not spread (Fig. 1). Furthermore, we tested clinical S. aureus isolates from patients with different staphylococcal infections (65). Previous analyses had shown that not all of these strains express RNAIII (65). Our present results show that all agr⁺ clinical isolates do spread but that the agr-deficient isolates are unable to spread (Table 2).

TABLE 1.

Bacterial strains and plasmids

Strain or plasmid	Descriptiona	Reference(s)
Strains
NCTC8325	HA-MSSA strain; agr+rsbU	35
NCTC8325−	HA-MSSA strain; agrrsbU	This work
NCTC8325-4	Derivative of NCTC8325, cured of all prophages; carries 11-bp deletion in rsbU	36
NCTC8325-4 agr	NCTC8325-4 derivative; agr::tet	6
RN4220	Restriction-deficient derivative of NCTC8325, cured of all known prophages	28
SH1000	NCTC8325-4 derivative; rsbU+agr+	22
SH1000−	NCTC8325-4 derivative; rsbU+agr	This work
HG001	NCTC8325 derivative; rsbU+agr+	43
HG001−	NCTC8325 derivative; rsbU+agr	This work
RN6390	NCTC8325 derivative, cured of prophages	42
RN6911	RN6390 derivative; agr::tetMsarA+ Tcr	34
SA113	ATCC 35556 derivative; PIA-dependent biofilm producer	24
15981	Clinical isolate; positive for biofilm production in TSBg	53
Newman	ATCC 25904; exhibits high-level clumping factor production; σB+	12
Newman Δagr	Newman derivative; Δagr::tetM	59
N315	MRSA	29
COL	MRSA	15, 47
Isolate C	HA-MRSA	65
Isolate D	HA-MRSA	65
Isolate E	HA-MSSA	65
Isolate F	HA-MSSA	65
Isolate R	CA-MSSA	65
Isolate V	CA-MSSA	65
Isolate X	HA-MSSA	65
Isolate Y	HA-MRSA	65
UMCG-M2	CA-MSSA strain (formerly referred to as isolate A); resulted from in vivo MRSA-to-MSSA conversion of strain UMCG-M4	7, 65
UMCG-M4	CA-MRSA strain (also referred to as isolate B)	7, 65
UMCG-M4 I	MRSA strain; derivative of the UMCG-M4 strain that lost hemolysin activity upon in vitro cultivation at 41°C	7
USA300	CA-MRSA	10
LAC USA300	CA-MRSA	58
LAC USA300 ΔPSMα	LAC USA300 with a PSMα gene locus deletion	58
Mu50	HA-VISA	29
MRSA252	HA-MRSA	21
MSSA476	CA-MSSA	21
MW2	CA-MRSA	3
RF122	Bovine mastitis isolate	20
Plasmids
pRN6662	pSK267 with S. aureusagrA	1
pALC2073RNAIII	pALC2073 with RNAIII coding region	54

^aHA, hospital-acquired; PIA, polysaccharide intercellular adhesin; TSBg, tryptic soy broth with glucose; CA, community-acquired; VISA, vancomycin-intermediate S. aureus.

TABLE 2.

Properties of investigated S. aureus strains in relation to colony spreading and hemolytic activity

Strain	Description	Presence or absence of:
		Spreadinga	agrb	Hemolysis	rsbU	SCCmec or other SCC elementc	fudoh
NCTC8325	HA-MSSA	+	+	+	−	−	−
RN4220	MSSA	−	−	−	−	−	−
SH1000	MSSA	+	+	+	+	−	−
NCTC8325-4	MSSA	+	+	+	−	−	−
HG001	MSSA	+	+	+	+	−	−
RN6390	MSSA	+	+	+	−	−	−
RN6911 Δagr::tetM	MSSA	−	−	−	−	−	−
SA113	MSSA	−	−	−	−	−	−
15981	MSSA	−	−	−	+	−	−
Newman	MSSA	+	+	+	+	−	−
N315	HA-MRSA	−	−	−	Unknown	II	+
COL	HA-MRSA	+/−	+/−	−	+	I	−
UMCG-M2 (isolate A)	CA-MSSA	+	+	+	Unknown	−	−
UMCG-M4 (isolate B)	CA-MRSA	+	+	+	Unknown	V(5C2&5)	−
Isolate C	HA-MRSA	−	−	−	Unknown	Unknown	−
Isolate D	HA-MRSA	−	−	−	Unknown	Unknown	−
Isolate E	HA-MSSA	+	+	+	Unknown	−	−
Isolate F	HA-MSSA	+	+	+	Unknown	−	−
Isolate R	CA-MSSA	+	+	+	Unknown	−	−
Isolate V	CA-MSSA	+	+	+	Unknown	−	−
Isolate X	HA-MSSA	−	−	−	Unknown	−	−
Isolate Y	HA-MRSA	−	−	−	Unknown	Unknown	−
USA300	CA-MRSA	+	+	+	+	IV	−
LAC USA300	CA-MRSA	+	+	+	Unknown	IV	−
Mu50	HA-VISA	−	−	−	+	II	+
MRSA252	HA-MRSA	+	+	−	+	II	+
MSSA476	CA-MSSA	+	+	+	+	SCChsd	−
MW2	CA-MRSA	+	+	+	+	IV	−
RF122	Bovine mastitis isolate	+	+	+	+	−	−

^a+/−, strain showed minimal spreading ability.

^b+/−, agr is present but defective.

^cFor strains with SCC elements, the type or identity is given.

FIG. 1.

Colony spreading of different S. aureus strains on soft agar plates. From an overnight culture, an aliquot of 2 μl was spotted in the middle of a tryptic soy agar (TSA) plate, which was then incubated overnight at 37°C. The analyses include standard laboratory strains of S. aureus (i.e., RN4220, SH1000, and HG001), as well as community-acquired (i.e., USA300) and hospital-acquired (i.e., N315, Mu50, MRSA252, and Newman) strains.

The agr system controls the expression of staphylococcal hemolysins via RNAIII. Lack of hemolytic activity is typical for strains with agr defects and mutants with delayed agr activation (51). To confirm the correlation between colony spreading and agr activity, all strains used in this study were tested for hemolytic activity on 5% sheep blood agar plates (13) (Table 2). The α- and β-hemolysin activities were directly detectable on blood agar plates. The production of δ-hemolysin was detected by measuring its synergistic activity with the β-hemolysin produced by strain RN4220, which at the same time inhibits the α-hemolysin. Thus, the synergistic activity of β- and δ-hemolysins is reflected by bright arrow-like zones on blood agar plates when the respective strains are striped in close proximity to each other (1). As expected, all strains with a functional agr locus showed clear δ-hemolysin activity, which directly confirmed the production of RNAIII.

An intact agr system is required for colony spreading.

Somerville et al. reported that during growth under aerobic conditions, spontaneous mutations in agr occur, thereby creating mixed bacterial populations with cells that are either alpha-hemolytic or nonhemolytic (50). Notably, this population heterogeneity occurs not only in laboratory cultures, but also in vivo, where it may enhance the ability of S. aureus to withstand the stress and insults imposed by the human immune defenses (4, 32, 50, 51). By plating agr⁺ strains onto blood agar plates, we also observed this population heterogeneity, which we exploited to test whether the nonhemolytic cells had also lost the ability to spread. This analysis is exemplified for nonhemolytic variants of the laboratory strain SH1000 (designated SH1000⁻) and the clinical isolate UMCG-M4 (designated UMCG-M4 I) (Table 3 ). Spontaneous agr mutations are known to arise preferentially in the coding region of agrC or the intergenic region between agrC and agrA, thereby preventing the translation of agrA (1, 51). This pattern implies that agr defects can be complemented at least partially by ectopic expression of RNAIII and, in some cases, also by ectopic agrA expression (1, 54). Accordingly, we performed a complementation analysis to verify the requirement of an intact agr system for colony spreading by using the agrA mutant S. aureus strain RN4220 and the spontaneous agr mutant SH1000⁻. Both strains were complemented with the pALC2073RNAIII plasmid for RNAIII expression or pRN6662 for expression of an intact agrA gene (39). As shown in Fig. 2A, both complemented strains carrying pALC2073RNAIII or pRN6662 were able to spread. It should be noticed, however, that the spreading abilities of the strains RN4220 and SH1000⁻ complemented with agrA and/or RNAIII were less pronounced than those of known agr⁺ strains (Fig. 1). The hemolytic activities of the complemented strains were also partially restored (Fig. 2B). Taken together, these results show unambiguously that an intact agr system is required for colony spreading of S. aureus (Table 3).

TABLE 3.

S. aureus strains used to confirm the roles of agr in colony spreading and hemolytic activity

Strain	Methicillin resistance phenotype	Presence or absence of:
		Spreading	agr	Hemolysis	rsbU	SCCmeca	fudoh
NCTC8325−	MSSA	−	−	−	−	−	−
RN4220(pRN6662)	MSSA	+	Restored	+	−	−	−
RN4220(pALC2073RNAIII)	MSSA	+	Restored	+	−	−	−
SH1000−	MSSA	−	−	−	+	−	−
SH1000−(pRN6662)	MSSA	+	Restored	+	+	−	−
NCTC8325-4 agr	MSSA	−	−	−	−	−	−
HG001−	MSSA	−	−	−	+	−	−
Newman Δagr	MSSA	−	−	−	+	−	−
UMCG-M4 I	MRSA	−	−	−	Unknown	V(5C2&5)	−

^aFor the strain with SCCmec, the type is given.

FIG. 2.

Involvement of the S. aureus agr system and phenol-soluble modulins in colony spreading. To complement the agr mutations in S. aureus strains RN4220 and SH1000⁻, the respective cells were transformed either with plasmid pALC2073RNAIII for RNAIII production or with plasmid pRN6662 for AgrA production. (A) Colony spreading by complemented agr mutant strains of S. aureus. wt, wild type. (B) Hemolysis by the agr⁺ strain S. aureus SH1000, an agr-deficient variant of SH1000 (SH1000⁻), and a variant complemented with agr [SH1000⁻(pRN6662)]. Note that hemolysis was partially restored in the S. aureus agr-deficient strain upon introduction of pRN6662, carrying agrA.

In contrast to agr, rsbU mutations or the presence of SCCmec elements showed no obvious correlations with colony spreading (Fig. 1). The lack of detectable effects of rsbU mutations means that reduced levels of the accessory sigma factor B (σ^B) do not have a strong impact on spreading (22, 60). More noticeable was the observation that the S. aureus MRSA252 strain, which carries SCCmec type II with a fudoh gene, did spread on soft agar plates (Fig. 1). This was an unexpected finding, as Kaito et al. (26) have reported that the presence of the fudoh gene in SCCmec type II would suppress the colony-spreading phenotype of MRSA strains (25). In line with this idea, the tested strains N315 and Mu50, which carry the fudoh gene within the type II SCCmec, were indeed unable to spread (Fig. 1). However, the strains N315 and Mu50 are agr deficient, whereas MRSA252 carries agr⁺, which suggests that agr is more generally important for spreading than the fudoh gene. The latter view is consistent with our finding that agr is a key determinant for spreading in MSSA strains (Tables 2 and 3).

The agr-deficient phenotype of S. aureus has been connected with the enhancement of biofilm formation in vitro and in vivo, where agr mutants form thicker biofilms than agr⁺ strains (27, 55-57, 61, 62). Indeed, all tested agr-deficient strains were stronger biofilm formers than their agr⁺ counterparts, and this finding was true even for the RN4220 strain complemented with agrA or RNAIII (data not shown). Furthermore, it was reported previously that upregulation of RNAIII is associated with the escape of S. aureus from biofilms grown in flow cells (61). These findings suggest that the agr system plays a decisive role in the choice of S. aureus cells between a sessile and a nonsessile lifestyle. Specifically, Boles and Horswill showed that agr-deficient cells have the ability to attach to surfaces and form biofilm due to the low expression levels of detachment factors such as hydrolases, proteases, and surfactants (5). Surfactants are especially interesting in this respect, because surfactant molecules lower the surface tension of surface-air interfaces, thereby improving surface wettability, which allows liquids to spread on hydrophobic surfaces. This property of surfactants could be directly connected to colony spreading since the surface motility of bacteria is dependent on moist conditions (16). Notably, the δ-hemolysin (PSMγ) encoded by RNAIII has strong surfactant properties (17, 56). This would directly link RNAIII production to colony spreading. Additionally, all other PSMs of S. aureus have surfactant properties and these properties are tightly controlled by the agr system (11, 45). To test the possible involvement of such compounds, we tested colony spreading for a mutant of S. aureus strain LAC USA300 lacking the psm-α operon (kindly provided by M. Otto). Indeed, the spreading ability of this mutant was strongly reduced compared to that of the parental LAC USA300 strain, and spreading of the mutant strain was restored by addition of the four chemically synthesized PSMα peptides (Fig. 3). This result directly shows the involvement of the PSMα peptides in colony spreading. Importantly, these PSMs have been implicated in leukocyte killing, which suggests a potential connection between spreading motility and staphylococcal virulence (58).

FIG. 3.

Phenol-soluble modulins α promote colony spreading. The role of PSMs in colony spreading was tested with the S. aureus LAC USA300 ΔPSMα strain, which lacks all four PSMα peptides. As a control, the parental strain, S. aureus LAC USA300 (marked wt), was used. To verify that the spreading defect of S. aureus LAC USA300 ΔPSMα was due to the absence of the PSMα peptides, spreading was tested upon addition of a mixture of the four chemically synthesized PSMα peptides to the cells. PSMα1 to PSMα4, each with a C-terminal four-residue glycine spacer and an ɛ-amino biotinyl lysine, were synthesized by standard 9-fluorenylmethoxy carbonyl (Fmoc) solid-phase peptide synthesis. The crude peptides were purified by reversed-phase high-performance liquid chromatography, and the molecular masses of the peptides were confirmed by electrospray ionization mass spectrometry. The purified PSMα1 to PSMα4 peptides were then dissolved in a mixture of dimethyl sulfoxide (DMSO) and 10 mM dithiothreitol (DTT) to yield a final concentration of 15 mM (each). For the present experiment, the α1, α2, α3, and α4 peptides were mixed in a 4.4:1:1.5:2.5 ratio, which is in accordance with the ratios of these peptides encountered in the medium of S. aureus USA300 (30). Next, the mixture was diluted 10-fold in phosphate-buffered saline (PBS). Twenty microliters of an overnight culture of the ΔPSMα mutant strain was centrifuged and subsequently resuspended in 20 μl of the diluted peptide mixture (yielding the sample labeled ΔPSMα-complemented). As a control, ΔPSMα cells were resuspended in 20 μl of PBS with a 10-fold dilution of 10 mM DTT in DMSO without the PSMα1 to PSMα4 peptides. Aliquots of 2 μl of the resuspended cells were added to soft agar plates to test colony spreading. All experiments were performed in duplicate and repeated three times.

General conclusion and outlook.

Taken together, our present findings show that an intact agr system is required for the movement of S. aureus over surface-air interfaces by colony spreading. A key question that remains to be answered in future studies is whether agr-dependent colony spreading plays a role during S. aureus infections. Clearly, many examples of the coexistence of agr⁺ and agr-deficient variants of S. aureus in vivo have been reported previously (48, 52, 57). This coexistence may suggest a division of tasks within an infecting S. aureus population, where nonsessile agr⁺ cells would have relatively high potentials for spreading over surfaces and growing invasively while sessile agr-deficient cells would have a relatively high potential for colonization, possibly through the formation of biofilms.