- Email: [email protected]
Features of Bacillus cereus swarm cells
Research in Microbiology 161 (2010) 743e749 www.elsevier.com/locate/resmic
Features of Bacillus cereus swarm cells Sonia Senesi a,*, Sara Salvetti b, Francesco Celandroni b, Emilia Ghelardi b b
a Dipartimento di Biologia, Via San Zeno, 37-39, 56127 Pisa, Pisa University, Italy Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Via San Zeno, 37-39, 56127 Pisa, Pisa University, Italy
Received 27 May 2010; accepted 21 September 2010 Available online 28 October 2010
Abstract When propagated on solid surfaces, Bacillus cereus can produce differentiated swarm cells under a wide range of growth conditions. This behavioural versatility is ecologically relevant, since it allows this bacterium to adapt swarming to environmental changes. Swarming by B. cereus is medically important: swarm cells are more virulent and particularly prone to invade host tissues. Characterisation of swarmingdeficient mutants highlights that flagellar genes as well as genes governing different metabolic pathways are involved in swarm-cell differentiation. In this review, the environmental and genetic requirements for swarming and the role played by swarm cells in the virulence this pathogen exerts will be outlined. Ó 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Bacillus cereus; Swarm cell differentiation; Genetic control; Virulence; Flagellum-dependent HBL-export
1. Introduction Flagellated bacteria may experience two main types of flagellum-driven motility, swimming and swarming, depending on whether they are propagated in liquid or solid media. Swimming is brought about by individual cells independently adapting their movement in response to compounds acting either as attractants or repellents. Swarming motility has long been recognised as a social behaviour enabling flagellated bacteria to co-operatively move across moist surfaces (Hauser, 1885; Henrichsen, 1972). This specialised form of surface translocation is coupled with a complex process leading vegetative cells to differentiate into a swarm state. Swarm cells are longer, harbour a higher number of peritrichously placed flagella, do not divide, and possess the unique ability co-ordinately move across the surface in multicellular rafts (Harshey, 2003). * Corresponding author. Tel.: þ39 050 2213695; fax: þ39 050 2213711. E-mail addresses: [email protected] (S. Senesi), sara.salvetti@for. unipi.it (S. Salvetti), [email protected] (F. Celandroni), ghelardi@ biomed.unipi.it (E. Ghelardi).
The ability to swarm has been described in both Gramnegative and Gram-positive bacteria, including Aeromonas, Burkholderia, Proteus, Pseudomonas, Salmonella, Serratia, Vibrio, Yersinia, Bacillus, and Clostridium (Fraser and Hughes, 1999; Harshey, 2003; Kearns and Losick, 2003; Macfarlane et al., 2001; Senesi et al., 2002, 2004; Verstraeten et al., 2008). The wide diffusion of this type of flagellum-dependent motility suggests that swarming confers an advantage for colonisation of natural environments, where microbial activities are often associated with surfaces. However, bacteria living on surfaces may produce different forms of organised surfaceadherent communities, the development of which is mainly conditioned by nutrient availability and moisture conditions (Hsueh et al., 2007; Shrout et al., 2006; Verstraeten et al., 2008). Swarming was first observed in Proteus mirabilis (Hauser, 1885), in which swarm cell differentiation occurs in cyclic rounds alternated with reversion to the undifferentiated vegetative state. Active division of vegetative cells gives rise to consolidation phases within the expanding community and results in the appearance of characteristic terraced colonies (Harshey, 2003; McCarter, 2004; Rather, 2005). However, morphogenesis of swarming colonies substantially varies
0923-2508/$ - see front matter Ó 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2010.10.007
744
S. Senesi et al. / Research in Microbiology 161 (2010) 743e749
among swarming-proficient micro-organisms depending on the bacterial species and growth conditions under which swarming occurs. Most often, swarming colonies do not display regular alternation of swarming and consolidation phases, but are characterised by a unique migration front of swarm cells at the colony border (Verstraeten et al., 2008). The collective swarmcell migration, either occurring within or at the colony rim, allows colony expansion, the extent of which depends on nutrient availability and viscosity of the surface. The ongoing of swarming process is controlled by a plethora of factors arising and acting within the microbial community. A robust literature documents considerable variability in regulatory, metabolic, chemosensory, motility, and quorum-sensing related genes required for swarming in different bacteria (Daniels et al., 2004; Fraser and Hughes, 1999; Verstraeten et al., 2008). Findings of particular interest demonstrate that swarming is coupled with a decrease in antimicrobial susceptibility and an increase in the production of secretory toxins and enzymes (Butler et al., 2010; Fraser and Hughes, 1999; Ghelardi et al., 2007; Kim and Surette, 2003, 2004; Lai et al., 2009; Overhage et al., 2008; Pearson et al., 2010). These attributes, together with the substantial alteration in metabolic bias and gene expression, demonstrated for swarming strains of Salmonella typhimurium, Pseudomonas aeruginosa, and Proteus mirabilis (Kim and Surette, 2004; Overhage et al., 2008; Pearson et al., 2010; Wang et al., 2004), strongly suggest that swarming should be regarded as a complex adaptive response requiring multiple and integrated cellular functions, rather than only as a surface-induced motility phenotype. Bacillus cereus is a Gram-positive, motile, spore-forming rod frequently isolated from the soil, where spores ensure its persistence under adverse conditions. Despite in soil it displays a full saprophytic life cycle (Vilain et al., 2006), when allowed access to mammalian tissues, B. cereus behaves as an opportunistic pathogen causing severe local and systemic infections. Indeed, although long known to be responsible for two forms of food poisoning, characterised by either diarrhea and abdominal distress or nausea and vomiting, in recent years there has been an increasing concern over B. cereus potential to cause extra-intestinal infections in healthy individuals, but mostly in immunocompromised, critically ill, or otherwise debilitated patients. These infections include severe endophthalmitis, bacteremia, septicemia, endocarditis, pneumonia, meningitis, gastritis, and cutaneous infections (Bottone, 2010; Callegan et al., 1999; Drobniewski, 1993; Logan and Turnbull, 1999). Bacillus cereus is the model species of the “Bacillus cereus group”, also known as B. cereus sensu lato, comprising five other closely related species: Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis (Drobniewski, 1993; Lechner et al., 1998; Nakamura, 1998). These bacteria share a significant degree of genetic similarity, so that both DNAeDNA hybridization as well as 16S and 23S rRNA sequence analyses have failed to clearly separate these taxa that are consequently considered as variants of a single species (Drobniewski, 1993). B. thuringiensis is well known for its ability to produce parasporal crystalline protein inclusions (parasporal bodies or crystals) that are encoded by plasmids carrying cry genes (Schnepf
et al., 1998). Loss of these genes makes B. thuringiensis indistinguishable from B. cereus by other physiological or morphological traits. B. cereus can move over surfaces by swarming or sliding motility mainly depending on nutrient availability (Hsueh et al., 2007; Senesi et al., 2002). Swarming is a high-energyconsuming process that requires the synthesis of large numbers of flagella and therefore may not be a viable option for surface translocation when nutrient are scarce. In lownutrient environments, surface motility is mainly due to sliding; this type of motility, also referred to as spreading, is produced by the expansive forces in a growing colony in combination with cell-surface properties resulting in reduced friction between the cell monolayer and substrate (Henrichsen, 1972). Sliding does not require flagella and is facilitated by the production of biosurfactant compounds that reduce the surface tension (Harshey, 2003; Kinsinger et al., 2003, 2005). In lownutrient environments, B. cereus enhances production of a biosurfactant, not yet chemically defined, which lowers surface tension and facilitates flagellum-independent sliding motility with a minimal output of energy (Hsueh et al., 2007). This review focuses on the ability of B. cereus to undergo swarming differentiation under a wide variety of growth conditions, addresses the question of how swarming increases the virulence potential this opportunistic pathogen may exert, and provides a general view of the genetic control under which swarming may occur. 2. B. cereus swarming behaviour and features of swarm cells The ability to swarm by B. cereus was recognised following the isolation of a spontaneous non-swarming mutant from the laboratory strain NCIB 8122 (Senesi et al., 2002). The mutant was characterised as carrying a genomic deletion in the fliY gene. While the parental strain developed large and rough colonies exhibiting irregular edges around the colony rim, the mutant produced small, smooth and round colonies (Fig. 1). Microscopic observations of the NCIB 8122 cells picked up from the growing communities showed the presence of swarm cells localized at the colony rim. Swarm cells were elongated (up to 4-fold increase in cell length), harboured a higher number of flagella (up to 40efold increase in flagellin), and gave rise to organised groups of tightly-bound cells generating finger-like structures migrating outward the colony border (Fig. 1). Conversely, cells in the colony centre were never organised in groups and did not exhibit elongation or hyperflagellation (Fig. 1). Swarming communities of B. cereus NCIB 8122 did not generate macroscopic layered consolidation phases forming spaced concentric rings, as viewed in P. mirabilis. Rather, the communities were found to be characterised by a unique consolidation phase, constituting the colony centre, surrounded by a migration front of swarm cells, as described for many other swarming-proficient species (Verstraeten et al., 2008). This was the first evidence demonstrating that surface translocation by B. cereus could be due to swarming and not only to sliding motility, as previously believed (Henrichsen, 1972).
S. Senesi et al. / Research in Microbiology 161 (2010) 743e749
745
Fig. 1. Representative morphological traits of Bacillus cereus NCIB 8122 and of its fliY mutant MP01. Colony produced on TrA-NaCl (1.0% tryptone, 0.5% NaCl, 1.0% agar) plates by NICB 8122 (a) and MP01 (b) after 3 h incubation at 37 C. Elongated (c) and hyperflagellate (d) swarm cells picked up from the rim of NCIB 8122 colony. Short (e) and oligoflagellate (f) cells picked up from the rim of MP01 colony. Colony border of NCIB 8122 (g) and MP01 (h) after 12 h incubation. Modified from Senesi et al. (2002).
Analysis of the growth conditions allowing swarming of B. cereus reference strains (Salvetti et al., 2007; Senesi et al., 2002) as well as natural isolates (Ghelardi et al., 2007), showed that this micro-organism is able to swarm under a wide variety of growth conditions, although the migration extent can significantly differ among swarming-proficient strains (Ghelardi et al., 2007). The medium viscosity required for swarming is not particularly critical, as B. cereus can swarm under a wide range of agar concentrations, corresponding to a 0.4e1.5% agar for many strains and up to 2.0% for some others. For the majority of the analysed B. cereus strains, an optimal agar concentration was around 0.7e1.0%. This behaviour is quite different from that of other bacteria, such as Salmonella, Serratia, Pseudomonas, Escherichia coli, and Bacillus subtilis, which only swarm on media containing lower agar percentages (0.5e0.8%) or that of Vibrio parahemolyticus and P. mirabilis readily swarming on higher agar percentages (1.5e3.0%) (Harshey, 2003). Swarming by B. cereus is influenced by the kind and level of nutrients, the optimal media for swarming being the rich ones, such as Luria Bertani or Triptone-NaCl (1% tryptone, 0.5% NaCl). Swarming by B. cereus optimally occurs at temperatures ranging from 25 to 38 C. Under optimal growth conditions, some B. cereus strains develop colony patterns characterised by cycles of swarming and
consolidation, as reported in Fig. 2. Dendritic colony patterns by B. cereus have also been observed (Hsueh et al., 2007). Development of dendritic patterns mainly occurs at low-nutrient concentrations, is associated with biosurfactant production, does not require flagella, and resembles sliding translocation (Hsueh et al., 2007). The flexibility of B. cereus in eliciting a swarming response under a wide range of medium viscosity is a trait of relevant ecological importance. Indeed, swarming by B. cereus can occur over a great variety of environmental surfaces, thus potentially facilitating this ubiquitous bacterium to colonise by swarming migration new growth environments. 3. B. cereus swarming and virulence The high frequency of food contamination by B. cereus, the ability of spores to germinate and grow in food as well as in the intestinal tract, together with the active production of one or more enterotoxins by almost all strains (Schoeni and Wong, 2005), explain why this micro-organism has been long recognised as a major cause of food-poisoning related diseases. The ability of B. cereus to behave as opportunistic human pathogen responsible for severe local and systemic extra-
746
S. Senesi et al. / Research in Microbiology 161 (2010) 743e749
Fig. 2. Swarming colony of Bacillus cereus ATCC 14579 produced on TrANaCl (1.0% tryptone, 0.5% NaCl, 0.7% agar) plates after 24 h incubation at 37 C.
intestinal infections (Bottone, 2010) is due to the production of several membrane-damaging toxins that are active against a variety of mammalian cells and tissues. Among the toxins produced by B. cereus, the haemolytic enterotoxin haemolysin BL (HBL) has been most intensively studied (Senesi and Ghelardi, 2010). HBL is a membrane-lytic system composed of three antigenically distinct proteins designated B, L1, and L2 (Beecher and MacMillan, 1991). These proteins are secreted independently and all three are necessary for maximal biological activity. The toxic activities so far identified for HBL include hemolysis, vascular permeability and necrosis in rabbit skin (Beecher and MacMillan, 1991; Beecher and Wong, 1994), in vitro degradation of explanted rabbit retinal tissue, and in vivo ocular necrosis and inflammation in rabbits (Beecher et al., 2000). As regards HBL diarrheal potential, this toxin has been shown to cause rapid fluid accumulation in ligated rabbit ileal loops (Beecher et al., 1995), which is a decisive test to reveal the diarrheal activity of enterotoxins. Several studies carried out with natural isolates and reference strains of B. cereus and its closest relative B. thuringiensis have demonstrated that swarming differentiation in these bacteria is coupled with an increase in HBL secretion (Ghelardi et al., 2002, 2007; Salvetti et al., 2007; Senesi et al., 2002). This finding appears of some relevance considering that the ability to swarm is a relatively widespread behaviour in B. cereus isolates; in addition, the higher percentage of swarming-proficient strains among clinical (62.5%) than food isolates (31.6%) suggests that strains able to swarm are well adapted to the human host (Ghelardi et al., 2007). The first evidence suggesting that B. cereus swarm cells can secrete increased amounts of HBL compared to vegetative
swimmer cells came from the observation that the reference strain NCIB 8122 secretes appreciable amounts of the L2 component of HBL (the only one this strain produces) during swarming differentiation (Senesi et al., 2002). Indeed, secretion of L2 by NCIB 8122 was easily detectable only at the colony rim constituted by the migration front of swarm cells (Senesi et al., 2002). Association between HBL secretion and swarming differentiation was better defined in the Cry laboratory strain IP2 of B. thuringiensis (Gominet et al., 2001), which secretes appreciable amounts of HBL in both liquid and solid media. Secretion of active HBL proteins was never observed in a non-swarming mutant of IP2 obtained by mini-Tn10 insertion in the flhA gene (Ghelardi et al., 2002; Bouillaut et al., 2005), which encodes an essential component of the flagellar export apparatus (Minamino and Macnab, 1999). Complementation with a plasmid harbouring flhA regained the ability of the mutant to produce flagella, to swarm, and to secrete HBL (Ghelardi et al., 2002). To exclude the possibility that the defect in secretion of active HBL was peculiar to the flhA mutant of B. thuringiensis, a collection of B. cereus natural isolates was subsequently analysed. All strains lacking flagella but possessing all hbl genes were found to be unable to secrete the HBL protein components that were instead intracellularly detected (Ghelardi et al., 2007). Further data demonstrated that the amount of HBL secreted by B. cereus is substantially influenced by the degree of cell flagellation. Reduced HBL export (about 3-fold) occurs in a B. cereus null mutant in flhF that is characterised by a lower number of flagella (1-3 per cell instead of 10e12) compared to the wild-type strain (Salvetti et al., 2007). Conversely, hyperflagellated swarm cells of B. cereus secrete higher levels of HBL than the vegetative swimmer cells. In fact, while motile but non-swarming B. cereus strains secrete similar amounts of HBL in liquid and solid media, swarming-proficient strains are characterised by a significant increase (about 22-fold) in toxin secretion when propagated in culture conditions promoting swarming differentiation (Ghelardi et al., 2007). Interestingly, it was also proven that swarming motility itself contributes to the B. cereus virulence behaviour. This conclusion derives from a comparative study analysing the behaviour of strain NCIB 8122, its swimming but non-swarming mutant in fliY, and the fliY-complemented strain, showing identical array of secreted enzymes/toxins, in an experimental model of rabbit endophthamitis (Callegan et al., 2006). While all strains grew to a similar number in the vitreous fluid, the deficiency in swarming prevented the fliY mutant from migrating to the anterior chamber of the eye, leading to less severe anterior segment disease in comparison to the parental and complemented strains. Over all, these data indicate that B. cereus swarm cells display a more virulent phenotype. Indeed, the higher number of flagella harboured by the swarm cells enhances the ability to adhere to host cells (Ramarao and Lereclus, 2006), promotes a more rapid colonisation of host tissues (Callegan et al., 2006), and leads to increase in HBL secretion (Ghelardi et al., 2002). All these properties are consistent with the high prevalence of swarming observed for strains isolated from clinical samples (Ghelardi et al., 2007).
S. Senesi et al. / Research in Microbiology 161 (2010) 743e749
4. Genetic control of B. cereus swarming differentiation Characterization of non-swarming mutants of B. cereus/B. thuringiensis was the approach used to identify genes governing the swarming process. The first non-swarming mutants analysed were all defective in assembly/functionality of flagella. The fliY mutant of B. cereus NCIB 8122 mentioned above is characterised by a genomic deletion in fliY, which encodes an essential component of the flagellar motoreswitch complex (Bischoff and Ordal, 1992). This strain is flagellated and motile, though displaying a more tumbling phenotype and defective chemotaxis (Senesi et al., 2002). Lack of swarming in the B. cereus fliY mutant is consistent with the demonstration obtained with other species that mutants defective in anyone of the flagellar genes, including those encoding the motor-switch proteins, are impaired in swarming (Belas et al., 1995; Burkart et al., 1998). In fact, complete abolishment of swarming migration was also observed in the non-flagellated flhA mutant of B. thuringiensis (Ghelardi et al., 2002), as well as in the flhF mutant of B. cereus ATCC 14579 (Salvetti et al., 2007). Interestingly, this mutant is motile in liquid media although displaying defective flagellation in both number and placement of flagella (Salvetti et al., 2007). FlhF is a signal recognition particle (SRP)-like GTPase, which is involved in the regulation of the number and placement of flagella in polar flagellates (Green et al., 2009). The role of FlhF in peritrichous flagellates is not clear. In B. subtilis, disruption of flhF showed that this gene resulted both dispensable (Carpenter et al., 1992) and required (Zanen et al., 2004) for flagellation. Although the role played by FlhF in regulating flagellation of peritrichous flagellates remains to be defined, B. cereus FlhF was shown to regulate the number and placement of flagella at the cell surface. Demonstration that disruption of fliY, flhA, and flhF was the only factor responsible for the non-swarming phenotype was achieved by complementing each mutant with a wild copy of the disrupted gene: the complemented strains exhibited complete restoration of the wild-type swarming phenotype (Ghelardi et al., 2002; Salvetti et al., 2007; Senesi et al., 2002). In a study aimed at identifying genes involved in swarming, but dispensable for cell flagellation, motility, and chemotaxis, several non-swarming mutants were isolated from the swarming-proficient strain B. thuringiensis 407 Cry by insertional mutagenesis (Salvetti et al., 2009). Analysis of the mutants and complemented strains led to the identification of six genes that appeared to be only required for swarming differentiation. These encode the monomeric form of sarcosine oxidase, catalase-2, an amino acid permease, a phenazine (PZ) biosynthesis protein belonging to the PhzF family, dGTP triphosphohydrolase, and acetyltransferase. Lack in monomeric sarcosine oxidase, likely determining an abnormal accumulation of glycine betaine, can mimic the physiological state acquired by cells in conditions of high-osmolarity that exert an inhibitory effect on swarming (Young et al., 1999). In B. subtilis, catalase2 has been reported to be dispensable for active growth, but necessary for coping with adverse growth conditions such as nutrient depletion or high cell density (Engelmann et al., 1995). The hypothesis on the role of catalase-2 in B. thuringiensis swarming was that this enzyme is required when vegetative cells
747
reach the high cell density at which a cycle of swarming migration occurs. Since amino acids act as inducers of swarming in many micro-organisms (Ko¨hler et al., 2000), the cryptic amino acid permease can be involved in a sensory route for perceiving extracellular signals inducing swarming by B. thuringiensis. PhzF proteins are involved in the production of PZ compounds, increasingly reported to act as secondary metabolites able to regulate global gene expression patterns (PriceWhelan et al., 2006). The altered secretion of PZs detected in the non-swarming mutant of B. thuringiensis suggests that these molecules act as signals inducing the gene expression changes required for swarming (Salvetti et al., 2009). The finding that proteins involved in diverse physiological processes have a role in swarming motility underlines the complexity of the molecular mechanisms governing the swarming behaviour. Despite random mutagenesis was used to search genes specifically required for swarming (Salvetti et al., 2009), no mutant defective in regulators was identified. Thus, it remains unknown how swarming differentiation is regulated in B. cereus. Comparison with other swarming species, in particular B. subtilis, is difficult to explore, as genes coding for SigD, DegU/S, and SwrA have not been identified in B. cereus. In B. subtilis, these genes are evoked to play a role in swarming (Calvio et al., 2005; Calvio et al., 2008; Murray et al., 2009; Patrick and Kearns, 2009). Only for PlcR, a role has been proposed in regulating B. cereus swarming (Hsueh et al., 2007). However, although a DplcR mutant does not swarm, PlcR is a global regulator in B. cereus and the inability of mutant cells to undergo swarming differentiation in likely due to a reduced expression of flagellin, which in turn interferes with the production of hyperflagellated swarm cells. Finally, no gene governing surfactant biosynthesis was identified in B. cereus although production of a surfactant has been observed in this micro-organism (Hsueh et al., 2007). No role of this surfactant has been suggested in B. cereus swarming, and it is unlikely that secretion of this surfactant could have functions similar to those played by surfactin in B. subtilis. Indeed, while surfactin has been reported to play a role in swarming differentiation in B. subtilis (Patrick and Kearns, 2009), surfactant production by B. cereus appears to promote sliding rather than swarming motility (Hsueh et al., 2007). 5. Conclusions Described later than in other swarming-proficient bacteria, the ability to swarm by B. cereus is now recognised as a relatively widespread behaviour among environmental and clinical isolates. B. cereus produces hyperflagellated and elongated swarm cells under a wide range of growth conditions; this versatile behaviour is of great ecological relevance. Indeed, this ubiquitous bacterium may colonise by swarming migration different kinds of solid surfaces adapting swarming to environment changes. Swarming by B. cereus is also of considerable medical importance. Despite B. cereus may display a full saprophytic life cycle in soil, it behaves as successful opportunistic human pathogen that is increasingly recognised to be responsible for local and severe systemic infections. The pathogenicity B. cereus exerts largely depends on the secretion of a wide array of
748
S. Senesi et al. / Research in Microbiology 161 (2010) 743e749
tissue-destructive/reactive toxins and on the ability to swarm by the infectious strains. The colonisation of host tissues is significantly enhanced by the collective migration of swarm cells, which, in addition, secrete high levels of the pore-forming HBL at the site of colonisation/infection. The combined effect of tissue invasion by swarming and the rapid secretion of high levels of HBL may contribute to the severity and rapidity of disease progression. The genetics of the swarming process in B. cereus is less known than for Gram-negative bacteria. As expected, all mutants defective in the assembly/functionality of flagella resulted completely deficient in swarming. However, many of the non-swarming mutants analysed were characterised by a deletion in genes involved in several cellular activities and metabolic pathways, which were required for swarming but dispensable for vegetative growth. These data support the view that swarming requires a significant differential regulation of multiple integrated cellular functions. A comprehensive understanding of the physiology of swarm cells should help to identify genes that are required for inducing transition of vegetative swimmer cells into swarm cells and vice versa, and for maintaining the swarm-cell state during swarming migration. This basic knowledge will better define the molecular mechanisms whereby swarming differentiation contributes to enhance the virulence potential this opportunistic pathogen exerts. References Beecher, D.J., MacMillan, J.D., 1991. Characterization of the components of hemolysin BL from Bacillus cereus. Infect. Immun. 59, 1778e1784. Beecher, D.J., Wong, A.C., 1994. Improved purification and characterization of hemolysin BL, a hemolytic dermonecrotic vascular permeability factor from Bacillus cereus. Infect. Immun. 62, 980e986. Beecher, D.J., Schoeni, J.L., Wong, A.C., 1995. Enterotoxic activity of hemolysin BL from Bacillus cereus. Infect. Immun. 63, 4423e4428. Beecher, D.J., Olsen, T.W., Somers, E.B., Wong, A.C., 2000. Evidence for contribution of tripartite hemolysin BL, phosphatidylcholine-preferring phospholipase C, and collagenase to virulence of Bacillus cereus endophthalmitis. Infect. Immun. 68, 5269e5276. Belas, R., Goldman, M., Ashliman, K., 1995. Genetic analysis of Proteus mirabilis mutants defective in swarmer cell elongation. J. Bacteriol. 177, 823e828. Bischoff, D.S., Ordal, W.G., 1992. Identification and characterization of FliY, a novel component of the Bacillus subtilis flagellar switch complex. Mol. Microbiol. 18, 2715e2723. Bottone, E.J., 2010. Bacillus cereus: a volatile human pathogen. Clin. Microbiol. Rev. 23, 382e398. Bouillaut, L., Ramarao, N., Buisson, C., Gilois, N., Gohar, M., Lereclus, D., Nielsen-LeRoux, C., 2005. FlhA influences Bacillus thuringiensis PlcRregulated gene transcription, protein production, and virulence. Appl. Environ. Microbiol. 71, 8903e8910. Burkart, M., Toguchi, A., Harshey, R.M., 1998. The chemotaxis system, but not chemotaxis, is essential for swarming motility in Escherichia coli. Proc. Natl. Acad. Sci. USA 95, 2568e2573. Butler, M.T., Wang, Q., Harshey, R.M., 2010. Cell density and mobility protect swarming bacteria against antibiotics. Proc. Natl. Acad. Sci. USA 107, 3776e3781. Callegan, M.C., Booth, M.C., Jett, B.D., Gilmore, M.S., 1999. Pathogenesis of gram-positive bacterial endophthalmitis. Infect. Immun. 67, 3348e3356. Callegan, M.C., Novosad, B.D., Ramirez, R., Ghelardi, E., Senesi, S., 2006. Role of swarming migration in the pathogenesis of Bacillus endophthalmitis. Invest. Ophthalmol. Vis. Sci. 47, 4461e4467.
Calvio, C., Celandroni, F., Ghelardi, E., Amati, G., Salvetti, S., Ceciliani, F., Galizzi, A., Senesi, S., 2005. Swarming differentiation and swimming motility in Bacillus subtilis are controlled by swrA, a newly identified dicistronic operon. J. Bacteriol. 187, 5356e5366. Calvio, C., Osera, C., Amati, G., Galizzi, A., 2008. Autoregulation of swrAA and motility in Bacillus subtilis. J. Bacteriol. 190, 5720e5728. Carpenter, P.B., Hanlon, D.V., Ordal, G.W., 1992. flhF, a Bacillus subtilis flagellar gene that encodes a putative GTP-binding protein. Mol. Microbiol. 6, 2705e2713. Daniels, R., Vanderleyden, J., Michiels, J., 2004. Quorum sensing and swarming migration in bacteria. FEMS Microbiol. Rev. 28, 261e289. Drobniewski, F.A., 1993. Bacillus cereus and related species. Clin. Microbiol. Rev. 6, 324e338. Engelmann, S., Lindner, C., Hecker, M., 1995. Cloning, nucleotide sequence, and regulation of katE encoding a sigma B-dependent catalase in Bacillus subtilis. J. Bacteriol. 177, 5598e5605. Fraser, G.M., Hughes, C., 1999. Swarming motility. Curr. Opin. Microbiol. 2, 630e635. Ghelardi, E., Celandroni, F., Salvetti, S., Beecher, D.J., Gominet, M., Lereclus, D., Wong, A.C.L., Senesi, S., 2002. Requirement of flhA for swarming differentiation, flagellin export, and secretion of virulenceassociated proteins in Bacillus thuringiensis. J. Bacteriol. 184, 6424e6433. Ghelardi, E., Celandroni, F., Salvetti, S., Ceragioli, M., Beecher, D.J., Senesi, S., Wong, A.C.L., 2007. Swarming behavior of and hemolysin BL secretion by Bacillus cereus. Appl. Environ. Microbiol. 73, 4089e4093. Gominet, M., Slamti, L., Gilois, N., Rose, M., Lereclus, D., 2001. Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence. Mol. Microbiol. 40, 963e975. Green, J.C., Kahramanoglou, C., Rahman, A., Pender, A.M., Charbonnel, N., Fraser, G.M., 2009. Recruitment of the earliest component of the bacterial flagellum to the old cell division pole by a membrane-associated signal recognition particle family GTP-binding protein. J. Mol. Biol. 391, 679e690. Harshey, R.M., 2003. Bacterial motility on a surface: many ways to a common goal. Annu. Rev. Microbiol. 57, 249e273. } Hauser, G., 1885. Uber F. F. G. W. Vogel, Leipzig, Germany. Henrichsen, J., 1972. Bacterial surface translocation: a survey and a classification. Bacteriol. Rev. 36, 478e503. Hsueh, Y.H., Somers, E.B., Lereclus, D., Ghelardi, E., Wong, A.C.L., 2007. Biosurfactant production and surface translocation are regulated by PlcR in Bacillus cereus ATCC 14579 under low-nutrient conditions. Appl. Environ. Microbiol. 73, 7225e7231. Ko¨hler, T., Curty, L.K., Barja, F., van Delden, C., Peche`re, J.C., 2000. Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J. Bacteriol. 182, 5990e5996. Kearns, D.B., Losick, R., 2003. Swarming motility in undomesticated Bacillus subtilis. Mol. Microbiol. 49, 581e590. Kim, W., Surette, M.G., 2003. Swarming population of Salmonella represent a unique physiological state coupled to multiple mechanisms of antibiotic resistance. Biol. Proced. Online 5, 189e196. Kim, W., Surette, M.G., 2004. Metabolic differentiation in actively swarming Salmonella. Mol. Microbiol. 54, 702e714. Kinsinger, R.F., Shirk, M.C., Fall, R., 2003. Rapid surface motility in Bacillus subtilis is dependent on extracellular surfactin and potassium ion. J. Bacteriol. 185, 5627e5631. Kinsinger, R.F., Kearns, D.B., Hale, M., Fall, R., 2005. Genetic requirements for potassium ion-dependent colony spreading in Bacillus subtilis. J. Bacterial 187, 8462e8469. Lai, S., Tremblay, J., De´ziel, E., 2009. Swarming motility: a muticellular behaviour conferring antimicrobial resistance. Environ. Microbiol. 11, 126e136. Lechner, S., Mayr, R., Fransis, K.P., Pruss, B.M., Kaplan, T., WiebnerGunkel, E., Stewart, G.S., Scherer, A.B., 1998. Bacillus weihenstephanensis sp. nov. is a new psychrotolerant species of the Bacillus cereus group. Int. J. Syst. Bacteriol. 48, 1373e1382. Logan, N.A., Turnbull, P.C., 1999. In: Murray, P.R. (Ed.), Manual of Clinical Microbiology. American Society for Microbiology, Washington, DC, pp. 357e369.
S. Senesi et al. / Research in Microbiology 161 (2010) 743e749 Macfarlane, S., Hopkins, M.G., Macfarlane, G.T., 2001. Toxin synthesis and mucin breakdown are related to swarming phenomenon in Clostridium septicum. Infect. Immun. 69, 1120e1126. McCarter, L.L., 2004. Dual flagellar systems enable motility under different circumstances. J. Mol. Microbiol. Biotechnol. 7, 18e29. Minamino, T., Macnab, R.M., 1999. Components of the Salmonella flagellar export apparatus and classification of export substrates. J. Bacteriol. 181, 1388e1394. Murray, E.J., Kiley, T.B., Stanley-Wall, N.R., 2009. A pitoval role for the response regulator DegU in controlling multicellular behaviour. Microbiol 155, 1e8. Nakamura, L.K., 1998. Bacillus pseudomycoides sp. nov. Int. Syst. Bacteriol. 48, 1031e1034. Overhage, J., Bains, M., Brazas, M.D., Hancock, R.E., 2008. Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factors and antibiotic resistance. J. Bacteriol. 190, 2671e2679. Patrick, J.E., Kearns, D.B., 2009. Laboratory strains of Bacillus subtilis do not exhibit swarming motility. J. Bacteriol. 191, 7129e7133. Pearson, M.M., Rasko, D.A., Smith, S.N., Mobley, H.L., 2010. Transcriptome of swarming Proteus mirabilis. Infect. Immun. 78, 2834e2845. Price-Whelan, A., Dietrich, L.E.P., Newman, D.K., 2006. Rethinking “secondary” metabolism: physiological roles for phenazine antibiotics. Nat. Chem. Biol. 2, 71e78. Ramarao, N., Lereclus, D., 2006. Adhesion and cytotoxicity of Bacillus cereus and Bacillus thuringiensis to epithelial cells are FlhA and PlcR dependent, respectively. Microbes Infect. 8, 1483e1491. Rather, P.N., 2005. Swarmer cell differentiation in Proteus mirabilis. Environ. Microbiol. 7, 1065e1073. Salvetti, S., Ghelardi, E., Celandroni, F., Ceragioli, M., Giannessi, F., Senesi, S., 2007. FlhF, a signal recognition particle-like GTPase, is involved in the regulation of flagellar arrangement, motility behaviour and protein secretion in Bacillus cereus. Microbiology 153, 2541e2552. Salvetti, S., Celandroni, F., Ceragioli, M., Senesi, S., Ghelardi, E., 2009. Identification of non-flagellar genes involved in swarm cell differentiation using a Bacillus thuringiensis mini-Tn10 mutant library. Microbiology 155, 912e921.
749
Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. 62, 775e806. Schoeni, J.L., Wong, A.C.L., 2005. Bacillus cereus food poisoning and its toxins. J. Food Prot. 68, 636e648. Senesi, S., Ghelardi, E., 2010. Production, secretion and biological activity of Bacillus cereus enterotoxins. Toxins 2, 1690e1703. Senesi, S., Celandroni, F., Salvetti, S., Beecher, D.J., Wong, A.C.L., Ghelardi, E., 2002. Swarming motility in Bacillus cereus and characterization of a fliY mutant impaired in swarm cell differentiation. Microbiology 148, 1785e1794. Senesi, S., Ghelardi, E., Celandroni, F., Salvetti, S., Parisio, E., Galizzi, A., 2004. Surface-associated flagellum formation and swarming differentiation in Bacillus subtilis are controlled by ifm locus. J. Bacteriol. 186, 1158e1164. Shrout, J.D., Chopp, D.L., Just, C.L., Givskov, M., Parsek, M.R., 2006. The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional. Mol. Microbiol. 62, 1264e1277. Verstraeten, N., Braeken, K., Debkumari, B., Fauvart, M., Fransaer, J., Vermant, J., Michiels, J., 2008. Living on a surface: swarming and biofilm formation. Trends Microbiol. 16, 496e506. Vilain, S., Luo, Y., Hildreth, M.B., Bro¨zel, V.S., 2006. Analysis of the life cycle of the soil saprophyte Bacillus cereus in liquid soil extract and in soil. Appl. Environ. Microbiol. 72, 4970e4977. Wang, Q., Frye, J.G., McClelland, M., Harshey, R.M., 2004. Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes. Mol. Microbiol. 52, 169e187. Young, G.M., Smith, M.J., Minnich, S.A., Miller, V.L., 1999. The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility. J. Bacteriol. 181, 2823e2833. Zanen, G., Antelmann, H., Wester, H., Hecker, M., van Dijl, J.M., Quax, W.J., 2004. FlhF, the third signal recognition particle-GTPase of Bacillus subtilis, is dispensable for protein secretion. J. Bacteriol. 186, 5956e5960.
Features of Bacillus cereus swarm cells Sonia Senesi a,*, Sara Salvetti b, Francesco Celandroni b, Emilia Ghelardi b b
a Dipartimento di Biologia, Via San Zeno, 37-39, 56127 Pisa, Pisa University, Italy Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Via San Zeno, 37-39, 56127 Pisa, Pisa University, Italy
Received 27 May 2010; accepted 21 September 2010 Available online 28 October 2010
Abstract When propagated on solid surfaces, Bacillus cereus can produce differentiated swarm cells under a wide range of growth conditions. This behavioural versatility is ecologically relevant, since it allows this bacterium to adapt swarming to environmental changes. Swarming by B. cereus is medically important: swarm cells are more virulent and particularly prone to invade host tissues. Characterisation of swarmingdeficient mutants highlights that flagellar genes as well as genes governing different metabolic pathways are involved in swarm-cell differentiation. In this review, the environmental and genetic requirements for swarming and the role played by swarm cells in the virulence this pathogen exerts will be outlined. Ó 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Bacillus cereus; Swarm cell differentiation; Genetic control; Virulence; Flagellum-dependent HBL-export
1. Introduction Flagellated bacteria may experience two main types of flagellum-driven motility, swimming and swarming, depending on whether they are propagated in liquid or solid media. Swimming is brought about by individual cells independently adapting their movement in response to compounds acting either as attractants or repellents. Swarming motility has long been recognised as a social behaviour enabling flagellated bacteria to co-operatively move across moist surfaces (Hauser, 1885; Henrichsen, 1972). This specialised form of surface translocation is coupled with a complex process leading vegetative cells to differentiate into a swarm state. Swarm cells are longer, harbour a higher number of peritrichously placed flagella, do not divide, and possess the unique ability co-ordinately move across the surface in multicellular rafts (Harshey, 2003). * Corresponding author. Tel.: þ39 050 2213695; fax: þ39 050 2213711. E-mail addresses: [email protected] (S. Senesi), sara.salvetti@for. unipi.it (S. Salvetti), [email protected] (F. Celandroni), ghelardi@ biomed.unipi.it (E. Ghelardi).
The ability to swarm has been described in both Gramnegative and Gram-positive bacteria, including Aeromonas, Burkholderia, Proteus, Pseudomonas, Salmonella, Serratia, Vibrio, Yersinia, Bacillus, and Clostridium (Fraser and Hughes, 1999; Harshey, 2003; Kearns and Losick, 2003; Macfarlane et al., 2001; Senesi et al., 2002, 2004; Verstraeten et al., 2008). The wide diffusion of this type of flagellum-dependent motility suggests that swarming confers an advantage for colonisation of natural environments, where microbial activities are often associated with surfaces. However, bacteria living on surfaces may produce different forms of organised surfaceadherent communities, the development of which is mainly conditioned by nutrient availability and moisture conditions (Hsueh et al., 2007; Shrout et al., 2006; Verstraeten et al., 2008). Swarming was first observed in Proteus mirabilis (Hauser, 1885), in which swarm cell differentiation occurs in cyclic rounds alternated with reversion to the undifferentiated vegetative state. Active division of vegetative cells gives rise to consolidation phases within the expanding community and results in the appearance of characteristic terraced colonies (Harshey, 2003; McCarter, 2004; Rather, 2005). However, morphogenesis of swarming colonies substantially varies
0923-2508/$ - see front matter Ó 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2010.10.007
744
S. Senesi et al. / Research in Microbiology 161 (2010) 743e749
among swarming-proficient micro-organisms depending on the bacterial species and growth conditions under which swarming occurs. Most often, swarming colonies do not display regular alternation of swarming and consolidation phases, but are characterised by a unique migration front of swarm cells at the colony border (Verstraeten et al., 2008). The collective swarmcell migration, either occurring within or at the colony rim, allows colony expansion, the extent of which depends on nutrient availability and viscosity of the surface. The ongoing of swarming process is controlled by a plethora of factors arising and acting within the microbial community. A robust literature documents considerable variability in regulatory, metabolic, chemosensory, motility, and quorum-sensing related genes required for swarming in different bacteria (Daniels et al., 2004; Fraser and Hughes, 1999; Verstraeten et al., 2008). Findings of particular interest demonstrate that swarming is coupled with a decrease in antimicrobial susceptibility and an increase in the production of secretory toxins and enzymes (Butler et al., 2010; Fraser and Hughes, 1999; Ghelardi et al., 2007; Kim and Surette, 2003, 2004; Lai et al., 2009; Overhage et al., 2008; Pearson et al., 2010). These attributes, together with the substantial alteration in metabolic bias and gene expression, demonstrated for swarming strains of Salmonella typhimurium, Pseudomonas aeruginosa, and Proteus mirabilis (Kim and Surette, 2004; Overhage et al., 2008; Pearson et al., 2010; Wang et al., 2004), strongly suggest that swarming should be regarded as a complex adaptive response requiring multiple and integrated cellular functions, rather than only as a surface-induced motility phenotype. Bacillus cereus is a Gram-positive, motile, spore-forming rod frequently isolated from the soil, where spores ensure its persistence under adverse conditions. Despite in soil it displays a full saprophytic life cycle (Vilain et al., 2006), when allowed access to mammalian tissues, B. cereus behaves as an opportunistic pathogen causing severe local and systemic infections. Indeed, although long known to be responsible for two forms of food poisoning, characterised by either diarrhea and abdominal distress or nausea and vomiting, in recent years there has been an increasing concern over B. cereus potential to cause extra-intestinal infections in healthy individuals, but mostly in immunocompromised, critically ill, or otherwise debilitated patients. These infections include severe endophthalmitis, bacteremia, septicemia, endocarditis, pneumonia, meningitis, gastritis, and cutaneous infections (Bottone, 2010; Callegan et al., 1999; Drobniewski, 1993; Logan and Turnbull, 1999). Bacillus cereus is the model species of the “Bacillus cereus group”, also known as B. cereus sensu lato, comprising five other closely related species: Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis (Drobniewski, 1993; Lechner et al., 1998; Nakamura, 1998). These bacteria share a significant degree of genetic similarity, so that both DNAeDNA hybridization as well as 16S and 23S rRNA sequence analyses have failed to clearly separate these taxa that are consequently considered as variants of a single species (Drobniewski, 1993). B. thuringiensis is well known for its ability to produce parasporal crystalline protein inclusions (parasporal bodies or crystals) that are encoded by plasmids carrying cry genes (Schnepf
et al., 1998). Loss of these genes makes B. thuringiensis indistinguishable from B. cereus by other physiological or morphological traits. B. cereus can move over surfaces by swarming or sliding motility mainly depending on nutrient availability (Hsueh et al., 2007; Senesi et al., 2002). Swarming is a high-energyconsuming process that requires the synthesis of large numbers of flagella and therefore may not be a viable option for surface translocation when nutrient are scarce. In lownutrient environments, surface motility is mainly due to sliding; this type of motility, also referred to as spreading, is produced by the expansive forces in a growing colony in combination with cell-surface properties resulting in reduced friction between the cell monolayer and substrate (Henrichsen, 1972). Sliding does not require flagella and is facilitated by the production of biosurfactant compounds that reduce the surface tension (Harshey, 2003; Kinsinger et al., 2003, 2005). In lownutrient environments, B. cereus enhances production of a biosurfactant, not yet chemically defined, which lowers surface tension and facilitates flagellum-independent sliding motility with a minimal output of energy (Hsueh et al., 2007). This review focuses on the ability of B. cereus to undergo swarming differentiation under a wide variety of growth conditions, addresses the question of how swarming increases the virulence potential this opportunistic pathogen may exert, and provides a general view of the genetic control under which swarming may occur. 2. B. cereus swarming behaviour and features of swarm cells The ability to swarm by B. cereus was recognised following the isolation of a spontaneous non-swarming mutant from the laboratory strain NCIB 8122 (Senesi et al., 2002). The mutant was characterised as carrying a genomic deletion in the fliY gene. While the parental strain developed large and rough colonies exhibiting irregular edges around the colony rim, the mutant produced small, smooth and round colonies (Fig. 1). Microscopic observations of the NCIB 8122 cells picked up from the growing communities showed the presence of swarm cells localized at the colony rim. Swarm cells were elongated (up to 4-fold increase in cell length), harboured a higher number of flagella (up to 40efold increase in flagellin), and gave rise to organised groups of tightly-bound cells generating finger-like structures migrating outward the colony border (Fig. 1). Conversely, cells in the colony centre were never organised in groups and did not exhibit elongation or hyperflagellation (Fig. 1). Swarming communities of B. cereus NCIB 8122 did not generate macroscopic layered consolidation phases forming spaced concentric rings, as viewed in P. mirabilis. Rather, the communities were found to be characterised by a unique consolidation phase, constituting the colony centre, surrounded by a migration front of swarm cells, as described for many other swarming-proficient species (Verstraeten et al., 2008). This was the first evidence demonstrating that surface translocation by B. cereus could be due to swarming and not only to sliding motility, as previously believed (Henrichsen, 1972).
S. Senesi et al. / Research in Microbiology 161 (2010) 743e749
745
Fig. 1. Representative morphological traits of Bacillus cereus NCIB 8122 and of its fliY mutant MP01. Colony produced on TrA-NaCl (1.0% tryptone, 0.5% NaCl, 1.0% agar) plates by NICB 8122 (a) and MP01 (b) after 3 h incubation at 37 C. Elongated (c) and hyperflagellate (d) swarm cells picked up from the rim of NCIB 8122 colony. Short (e) and oligoflagellate (f) cells picked up from the rim of MP01 colony. Colony border of NCIB 8122 (g) and MP01 (h) after 12 h incubation. Modified from Senesi et al. (2002).
Analysis of the growth conditions allowing swarming of B. cereus reference strains (Salvetti et al., 2007; Senesi et al., 2002) as well as natural isolates (Ghelardi et al., 2007), showed that this micro-organism is able to swarm under a wide variety of growth conditions, although the migration extent can significantly differ among swarming-proficient strains (Ghelardi et al., 2007). The medium viscosity required for swarming is not particularly critical, as B. cereus can swarm under a wide range of agar concentrations, corresponding to a 0.4e1.5% agar for many strains and up to 2.0% for some others. For the majority of the analysed B. cereus strains, an optimal agar concentration was around 0.7e1.0%. This behaviour is quite different from that of other bacteria, such as Salmonella, Serratia, Pseudomonas, Escherichia coli, and Bacillus subtilis, which only swarm on media containing lower agar percentages (0.5e0.8%) or that of Vibrio parahemolyticus and P. mirabilis readily swarming on higher agar percentages (1.5e3.0%) (Harshey, 2003). Swarming by B. cereus is influenced by the kind and level of nutrients, the optimal media for swarming being the rich ones, such as Luria Bertani or Triptone-NaCl (1% tryptone, 0.5% NaCl). Swarming by B. cereus optimally occurs at temperatures ranging from 25 to 38 C. Under optimal growth conditions, some B. cereus strains develop colony patterns characterised by cycles of swarming and
consolidation, as reported in Fig. 2. Dendritic colony patterns by B. cereus have also been observed (Hsueh et al., 2007). Development of dendritic patterns mainly occurs at low-nutrient concentrations, is associated with biosurfactant production, does not require flagella, and resembles sliding translocation (Hsueh et al., 2007). The flexibility of B. cereus in eliciting a swarming response under a wide range of medium viscosity is a trait of relevant ecological importance. Indeed, swarming by B. cereus can occur over a great variety of environmental surfaces, thus potentially facilitating this ubiquitous bacterium to colonise by swarming migration new growth environments. 3. B. cereus swarming and virulence The high frequency of food contamination by B. cereus, the ability of spores to germinate and grow in food as well as in the intestinal tract, together with the active production of one or more enterotoxins by almost all strains (Schoeni and Wong, 2005), explain why this micro-organism has been long recognised as a major cause of food-poisoning related diseases. The ability of B. cereus to behave as opportunistic human pathogen responsible for severe local and systemic extra-
746
S. Senesi et al. / Research in Microbiology 161 (2010) 743e749
Fig. 2. Swarming colony of Bacillus cereus ATCC 14579 produced on TrANaCl (1.0% tryptone, 0.5% NaCl, 0.7% agar) plates after 24 h incubation at 37 C.
intestinal infections (Bottone, 2010) is due to the production of several membrane-damaging toxins that are active against a variety of mammalian cells and tissues. Among the toxins produced by B. cereus, the haemolytic enterotoxin haemolysin BL (HBL) has been most intensively studied (Senesi and Ghelardi, 2010). HBL is a membrane-lytic system composed of three antigenically distinct proteins designated B, L1, and L2 (Beecher and MacMillan, 1991). These proteins are secreted independently and all three are necessary for maximal biological activity. The toxic activities so far identified for HBL include hemolysis, vascular permeability and necrosis in rabbit skin (Beecher and MacMillan, 1991; Beecher and Wong, 1994), in vitro degradation of explanted rabbit retinal tissue, and in vivo ocular necrosis and inflammation in rabbits (Beecher et al., 2000). As regards HBL diarrheal potential, this toxin has been shown to cause rapid fluid accumulation in ligated rabbit ileal loops (Beecher et al., 1995), which is a decisive test to reveal the diarrheal activity of enterotoxins. Several studies carried out with natural isolates and reference strains of B. cereus and its closest relative B. thuringiensis have demonstrated that swarming differentiation in these bacteria is coupled with an increase in HBL secretion (Ghelardi et al., 2002, 2007; Salvetti et al., 2007; Senesi et al., 2002). This finding appears of some relevance considering that the ability to swarm is a relatively widespread behaviour in B. cereus isolates; in addition, the higher percentage of swarming-proficient strains among clinical (62.5%) than food isolates (31.6%) suggests that strains able to swarm are well adapted to the human host (Ghelardi et al., 2007). The first evidence suggesting that B. cereus swarm cells can secrete increased amounts of HBL compared to vegetative
swimmer cells came from the observation that the reference strain NCIB 8122 secretes appreciable amounts of the L2 component of HBL (the only one this strain produces) during swarming differentiation (Senesi et al., 2002). Indeed, secretion of L2 by NCIB 8122 was easily detectable only at the colony rim constituted by the migration front of swarm cells (Senesi et al., 2002). Association between HBL secretion and swarming differentiation was better defined in the Cry laboratory strain IP2 of B. thuringiensis (Gominet et al., 2001), which secretes appreciable amounts of HBL in both liquid and solid media. Secretion of active HBL proteins was never observed in a non-swarming mutant of IP2 obtained by mini-Tn10 insertion in the flhA gene (Ghelardi et al., 2002; Bouillaut et al., 2005), which encodes an essential component of the flagellar export apparatus (Minamino and Macnab, 1999). Complementation with a plasmid harbouring flhA regained the ability of the mutant to produce flagella, to swarm, and to secrete HBL (Ghelardi et al., 2002). To exclude the possibility that the defect in secretion of active HBL was peculiar to the flhA mutant of B. thuringiensis, a collection of B. cereus natural isolates was subsequently analysed. All strains lacking flagella but possessing all hbl genes were found to be unable to secrete the HBL protein components that were instead intracellularly detected (Ghelardi et al., 2007). Further data demonstrated that the amount of HBL secreted by B. cereus is substantially influenced by the degree of cell flagellation. Reduced HBL export (about 3-fold) occurs in a B. cereus null mutant in flhF that is characterised by a lower number of flagella (1-3 per cell instead of 10e12) compared to the wild-type strain (Salvetti et al., 2007). Conversely, hyperflagellated swarm cells of B. cereus secrete higher levels of HBL than the vegetative swimmer cells. In fact, while motile but non-swarming B. cereus strains secrete similar amounts of HBL in liquid and solid media, swarming-proficient strains are characterised by a significant increase (about 22-fold) in toxin secretion when propagated in culture conditions promoting swarming differentiation (Ghelardi et al., 2007). Interestingly, it was also proven that swarming motility itself contributes to the B. cereus virulence behaviour. This conclusion derives from a comparative study analysing the behaviour of strain NCIB 8122, its swimming but non-swarming mutant in fliY, and the fliY-complemented strain, showing identical array of secreted enzymes/toxins, in an experimental model of rabbit endophthamitis (Callegan et al., 2006). While all strains grew to a similar number in the vitreous fluid, the deficiency in swarming prevented the fliY mutant from migrating to the anterior chamber of the eye, leading to less severe anterior segment disease in comparison to the parental and complemented strains. Over all, these data indicate that B. cereus swarm cells display a more virulent phenotype. Indeed, the higher number of flagella harboured by the swarm cells enhances the ability to adhere to host cells (Ramarao and Lereclus, 2006), promotes a more rapid colonisation of host tissues (Callegan et al., 2006), and leads to increase in HBL secretion (Ghelardi et al., 2002). All these properties are consistent with the high prevalence of swarming observed for strains isolated from clinical samples (Ghelardi et al., 2007).
S. Senesi et al. / Research in Microbiology 161 (2010) 743e749
4. Genetic control of B. cereus swarming differentiation Characterization of non-swarming mutants of B. cereus/B. thuringiensis was the approach used to identify genes governing the swarming process. The first non-swarming mutants analysed were all defective in assembly/functionality of flagella. The fliY mutant of B. cereus NCIB 8122 mentioned above is characterised by a genomic deletion in fliY, which encodes an essential component of the flagellar motoreswitch complex (Bischoff and Ordal, 1992). This strain is flagellated and motile, though displaying a more tumbling phenotype and defective chemotaxis (Senesi et al., 2002). Lack of swarming in the B. cereus fliY mutant is consistent with the demonstration obtained with other species that mutants defective in anyone of the flagellar genes, including those encoding the motor-switch proteins, are impaired in swarming (Belas et al., 1995; Burkart et al., 1998). In fact, complete abolishment of swarming migration was also observed in the non-flagellated flhA mutant of B. thuringiensis (Ghelardi et al., 2002), as well as in the flhF mutant of B. cereus ATCC 14579 (Salvetti et al., 2007). Interestingly, this mutant is motile in liquid media although displaying defective flagellation in both number and placement of flagella (Salvetti et al., 2007). FlhF is a signal recognition particle (SRP)-like GTPase, which is involved in the regulation of the number and placement of flagella in polar flagellates (Green et al., 2009). The role of FlhF in peritrichous flagellates is not clear. In B. subtilis, disruption of flhF showed that this gene resulted both dispensable (Carpenter et al., 1992) and required (Zanen et al., 2004) for flagellation. Although the role played by FlhF in regulating flagellation of peritrichous flagellates remains to be defined, B. cereus FlhF was shown to regulate the number and placement of flagella at the cell surface. Demonstration that disruption of fliY, flhA, and flhF was the only factor responsible for the non-swarming phenotype was achieved by complementing each mutant with a wild copy of the disrupted gene: the complemented strains exhibited complete restoration of the wild-type swarming phenotype (Ghelardi et al., 2002; Salvetti et al., 2007; Senesi et al., 2002). In a study aimed at identifying genes involved in swarming, but dispensable for cell flagellation, motility, and chemotaxis, several non-swarming mutants were isolated from the swarming-proficient strain B. thuringiensis 407 Cry by insertional mutagenesis (Salvetti et al., 2009). Analysis of the mutants and complemented strains led to the identification of six genes that appeared to be only required for swarming differentiation. These encode the monomeric form of sarcosine oxidase, catalase-2, an amino acid permease, a phenazine (PZ) biosynthesis protein belonging to the PhzF family, dGTP triphosphohydrolase, and acetyltransferase. Lack in monomeric sarcosine oxidase, likely determining an abnormal accumulation of glycine betaine, can mimic the physiological state acquired by cells in conditions of high-osmolarity that exert an inhibitory effect on swarming (Young et al., 1999). In B. subtilis, catalase2 has been reported to be dispensable for active growth, but necessary for coping with adverse growth conditions such as nutrient depletion or high cell density (Engelmann et al., 1995). The hypothesis on the role of catalase-2 in B. thuringiensis swarming was that this enzyme is required when vegetative cells
747
reach the high cell density at which a cycle of swarming migration occurs. Since amino acids act as inducers of swarming in many micro-organisms (Ko¨hler et al., 2000), the cryptic amino acid permease can be involved in a sensory route for perceiving extracellular signals inducing swarming by B. thuringiensis. PhzF proteins are involved in the production of PZ compounds, increasingly reported to act as secondary metabolites able to regulate global gene expression patterns (PriceWhelan et al., 2006). The altered secretion of PZs detected in the non-swarming mutant of B. thuringiensis suggests that these molecules act as signals inducing the gene expression changes required for swarming (Salvetti et al., 2009). The finding that proteins involved in diverse physiological processes have a role in swarming motility underlines the complexity of the molecular mechanisms governing the swarming behaviour. Despite random mutagenesis was used to search genes specifically required for swarming (Salvetti et al., 2009), no mutant defective in regulators was identified. Thus, it remains unknown how swarming differentiation is regulated in B. cereus. Comparison with other swarming species, in particular B. subtilis, is difficult to explore, as genes coding for SigD, DegU/S, and SwrA have not been identified in B. cereus. In B. subtilis, these genes are evoked to play a role in swarming (Calvio et al., 2005; Calvio et al., 2008; Murray et al., 2009; Patrick and Kearns, 2009). Only for PlcR, a role has been proposed in regulating B. cereus swarming (Hsueh et al., 2007). However, although a DplcR mutant does not swarm, PlcR is a global regulator in B. cereus and the inability of mutant cells to undergo swarming differentiation in likely due to a reduced expression of flagellin, which in turn interferes with the production of hyperflagellated swarm cells. Finally, no gene governing surfactant biosynthesis was identified in B. cereus although production of a surfactant has been observed in this micro-organism (Hsueh et al., 2007). No role of this surfactant has been suggested in B. cereus swarming, and it is unlikely that secretion of this surfactant could have functions similar to those played by surfactin in B. subtilis. Indeed, while surfactin has been reported to play a role in swarming differentiation in B. subtilis (Patrick and Kearns, 2009), surfactant production by B. cereus appears to promote sliding rather than swarming motility (Hsueh et al., 2007). 5. Conclusions Described later than in other swarming-proficient bacteria, the ability to swarm by B. cereus is now recognised as a relatively widespread behaviour among environmental and clinical isolates. B. cereus produces hyperflagellated and elongated swarm cells under a wide range of growth conditions; this versatile behaviour is of great ecological relevance. Indeed, this ubiquitous bacterium may colonise by swarming migration different kinds of solid surfaces adapting swarming to environment changes. Swarming by B. cereus is also of considerable medical importance. Despite B. cereus may display a full saprophytic life cycle in soil, it behaves as successful opportunistic human pathogen that is increasingly recognised to be responsible for local and severe systemic infections. The pathogenicity B. cereus exerts largely depends on the secretion of a wide array of
748
S. Senesi et al. / Research in Microbiology 161 (2010) 743e749
tissue-destructive/reactive toxins and on the ability to swarm by the infectious strains. The colonisation of host tissues is significantly enhanced by the collective migration of swarm cells, which, in addition, secrete high levels of the pore-forming HBL at the site of colonisation/infection. The combined effect of tissue invasion by swarming and the rapid secretion of high levels of HBL may contribute to the severity and rapidity of disease progression. The genetics of the swarming process in B. cereus is less known than for Gram-negative bacteria. As expected, all mutants defective in the assembly/functionality of flagella resulted completely deficient in swarming. However, many of the non-swarming mutants analysed were characterised by a deletion in genes involved in several cellular activities and metabolic pathways, which were required for swarming but dispensable for vegetative growth. These data support the view that swarming requires a significant differential regulation of multiple integrated cellular functions. A comprehensive understanding of the physiology of swarm cells should help to identify genes that are required for inducing transition of vegetative swimmer cells into swarm cells and vice versa, and for maintaining the swarm-cell state during swarming migration. This basic knowledge will better define the molecular mechanisms whereby swarming differentiation contributes to enhance the virulence potential this opportunistic pathogen exerts. References Beecher, D.J., MacMillan, J.D., 1991. Characterization of the components of hemolysin BL from Bacillus cereus. Infect. Immun. 59, 1778e1784. Beecher, D.J., Wong, A.C., 1994. Improved purification and characterization of hemolysin BL, a hemolytic dermonecrotic vascular permeability factor from Bacillus cereus. Infect. Immun. 62, 980e986. Beecher, D.J., Schoeni, J.L., Wong, A.C., 1995. Enterotoxic activity of hemolysin BL from Bacillus cereus. Infect. Immun. 63, 4423e4428. Beecher, D.J., Olsen, T.W., Somers, E.B., Wong, A.C., 2000. Evidence for contribution of tripartite hemolysin BL, phosphatidylcholine-preferring phospholipase C, and collagenase to virulence of Bacillus cereus endophthalmitis. Infect. Immun. 68, 5269e5276. Belas, R., Goldman, M., Ashliman, K., 1995. Genetic analysis of Proteus mirabilis mutants defective in swarmer cell elongation. J. Bacteriol. 177, 823e828. Bischoff, D.S., Ordal, W.G., 1992. Identification and characterization of FliY, a novel component of the Bacillus subtilis flagellar switch complex. Mol. Microbiol. 18, 2715e2723. Bottone, E.J., 2010. Bacillus cereus: a volatile human pathogen. Clin. Microbiol. Rev. 23, 382e398. Bouillaut, L., Ramarao, N., Buisson, C., Gilois, N., Gohar, M., Lereclus, D., Nielsen-LeRoux, C., 2005. FlhA influences Bacillus thuringiensis PlcRregulated gene transcription, protein production, and virulence. Appl. Environ. Microbiol. 71, 8903e8910. Burkart, M., Toguchi, A., Harshey, R.M., 1998. The chemotaxis system, but not chemotaxis, is essential for swarming motility in Escherichia coli. Proc. Natl. Acad. Sci. USA 95, 2568e2573. Butler, M.T., Wang, Q., Harshey, R.M., 2010. Cell density and mobility protect swarming bacteria against antibiotics. Proc. Natl. Acad. Sci. USA 107, 3776e3781. Callegan, M.C., Booth, M.C., Jett, B.D., Gilmore, M.S., 1999. Pathogenesis of gram-positive bacterial endophthalmitis. Infect. Immun. 67, 3348e3356. Callegan, M.C., Novosad, B.D., Ramirez, R., Ghelardi, E., Senesi, S., 2006. Role of swarming migration in the pathogenesis of Bacillus endophthalmitis. Invest. Ophthalmol. Vis. Sci. 47, 4461e4467.
Calvio, C., Celandroni, F., Ghelardi, E., Amati, G., Salvetti, S., Ceciliani, F., Galizzi, A., Senesi, S., 2005. Swarming differentiation and swimming motility in Bacillus subtilis are controlled by swrA, a newly identified dicistronic operon. J. Bacteriol. 187, 5356e5366. Calvio, C., Osera, C., Amati, G., Galizzi, A., 2008. Autoregulation of swrAA and motility in Bacillus subtilis. J. Bacteriol. 190, 5720e5728. Carpenter, P.B., Hanlon, D.V., Ordal, G.W., 1992. flhF, a Bacillus subtilis flagellar gene that encodes a putative GTP-binding protein. Mol. Microbiol. 6, 2705e2713. Daniels, R., Vanderleyden, J., Michiels, J., 2004. Quorum sensing and swarming migration in bacteria. FEMS Microbiol. Rev. 28, 261e289. Drobniewski, F.A., 1993. Bacillus cereus and related species. Clin. Microbiol. Rev. 6, 324e338. Engelmann, S., Lindner, C., Hecker, M., 1995. Cloning, nucleotide sequence, and regulation of katE encoding a sigma B-dependent catalase in Bacillus subtilis. J. Bacteriol. 177, 5598e5605. Fraser, G.M., Hughes, C., 1999. Swarming motility. Curr. Opin. Microbiol. 2, 630e635. Ghelardi, E., Celandroni, F., Salvetti, S., Beecher, D.J., Gominet, M., Lereclus, D., Wong, A.C.L., Senesi, S., 2002. Requirement of flhA for swarming differentiation, flagellin export, and secretion of virulenceassociated proteins in Bacillus thuringiensis. J. Bacteriol. 184, 6424e6433. Ghelardi, E., Celandroni, F., Salvetti, S., Ceragioli, M., Beecher, D.J., Senesi, S., Wong, A.C.L., 2007. Swarming behavior of and hemolysin BL secretion by Bacillus cereus. Appl. Environ. Microbiol. 73, 4089e4093. Gominet, M., Slamti, L., Gilois, N., Rose, M., Lereclus, D., 2001. Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence. Mol. Microbiol. 40, 963e975. Green, J.C., Kahramanoglou, C., Rahman, A., Pender, A.M., Charbonnel, N., Fraser, G.M., 2009. Recruitment of the earliest component of the bacterial flagellum to the old cell division pole by a membrane-associated signal recognition particle family GTP-binding protein. J. Mol. Biol. 391, 679e690. Harshey, R.M., 2003. Bacterial motility on a surface: many ways to a common goal. Annu. Rev. Microbiol. 57, 249e273. } Hauser, G., 1885. Uber F. F. G. W. Vogel, Leipzig, Germany. Henrichsen, J., 1972. Bacterial surface translocation: a survey and a classification. Bacteriol. Rev. 36, 478e503. Hsueh, Y.H., Somers, E.B., Lereclus, D., Ghelardi, E., Wong, A.C.L., 2007. Biosurfactant production and surface translocation are regulated by PlcR in Bacillus cereus ATCC 14579 under low-nutrient conditions. Appl. Environ. Microbiol. 73, 7225e7231. Ko¨hler, T., Curty, L.K., Barja, F., van Delden, C., Peche`re, J.C., 2000. Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J. Bacteriol. 182, 5990e5996. Kearns, D.B., Losick, R., 2003. Swarming motility in undomesticated Bacillus subtilis. Mol. Microbiol. 49, 581e590. Kim, W., Surette, M.G., 2003. Swarming population of Salmonella represent a unique physiological state coupled to multiple mechanisms of antibiotic resistance. Biol. Proced. Online 5, 189e196. Kim, W., Surette, M.G., 2004. Metabolic differentiation in actively swarming Salmonella. Mol. Microbiol. 54, 702e714. Kinsinger, R.F., Shirk, M.C., Fall, R., 2003. Rapid surface motility in Bacillus subtilis is dependent on extracellular surfactin and potassium ion. J. Bacteriol. 185, 5627e5631. Kinsinger, R.F., Kearns, D.B., Hale, M., Fall, R., 2005. Genetic requirements for potassium ion-dependent colony spreading in Bacillus subtilis. J. Bacterial 187, 8462e8469. Lai, S., Tremblay, J., De´ziel, E., 2009. Swarming motility: a muticellular behaviour conferring antimicrobial resistance. Environ. Microbiol. 11, 126e136. Lechner, S., Mayr, R., Fransis, K.P., Pruss, B.M., Kaplan, T., WiebnerGunkel, E., Stewart, G.S., Scherer, A.B., 1998. Bacillus weihenstephanensis sp. nov. is a new psychrotolerant species of the Bacillus cereus group. Int. J. Syst. Bacteriol. 48, 1373e1382. Logan, N.A., Turnbull, P.C., 1999. In: Murray, P.R. (Ed.), Manual of Clinical Microbiology. American Society for Microbiology, Washington, DC, pp. 357e369.
S. Senesi et al. / Research in Microbiology 161 (2010) 743e749 Macfarlane, S., Hopkins, M.G., Macfarlane, G.T., 2001. Toxin synthesis and mucin breakdown are related to swarming phenomenon in Clostridium septicum. Infect. Immun. 69, 1120e1126. McCarter, L.L., 2004. Dual flagellar systems enable motility under different circumstances. J. Mol. Microbiol. Biotechnol. 7, 18e29. Minamino, T., Macnab, R.M., 1999. Components of the Salmonella flagellar export apparatus and classification of export substrates. J. Bacteriol. 181, 1388e1394. Murray, E.J., Kiley, T.B., Stanley-Wall, N.R., 2009. A pitoval role for the response regulator DegU in controlling multicellular behaviour. Microbiol 155, 1e8. Nakamura, L.K., 1998. Bacillus pseudomycoides sp. nov. Int. Syst. Bacteriol. 48, 1031e1034. Overhage, J., Bains, M., Brazas, M.D., Hancock, R.E., 2008. Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factors and antibiotic resistance. J. Bacteriol. 190, 2671e2679. Patrick, J.E., Kearns, D.B., 2009. Laboratory strains of Bacillus subtilis do not exhibit swarming motility. J. Bacteriol. 191, 7129e7133. Pearson, M.M., Rasko, D.A., Smith, S.N., Mobley, H.L., 2010. Transcriptome of swarming Proteus mirabilis. Infect. Immun. 78, 2834e2845. Price-Whelan, A., Dietrich, L.E.P., Newman, D.K., 2006. Rethinking “secondary” metabolism: physiological roles for phenazine antibiotics. Nat. Chem. Biol. 2, 71e78. Ramarao, N., Lereclus, D., 2006. Adhesion and cytotoxicity of Bacillus cereus and Bacillus thuringiensis to epithelial cells are FlhA and PlcR dependent, respectively. Microbes Infect. 8, 1483e1491. Rather, P.N., 2005. Swarmer cell differentiation in Proteus mirabilis. Environ. Microbiol. 7, 1065e1073. Salvetti, S., Ghelardi, E., Celandroni, F., Ceragioli, M., Giannessi, F., Senesi, S., 2007. FlhF, a signal recognition particle-like GTPase, is involved in the regulation of flagellar arrangement, motility behaviour and protein secretion in Bacillus cereus. Microbiology 153, 2541e2552. Salvetti, S., Celandroni, F., Ceragioli, M., Senesi, S., Ghelardi, E., 2009. Identification of non-flagellar genes involved in swarm cell differentiation using a Bacillus thuringiensis mini-Tn10 mutant library. Microbiology 155, 912e921.
749
Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. 62, 775e806. Schoeni, J.L., Wong, A.C.L., 2005. Bacillus cereus food poisoning and its toxins. J. Food Prot. 68, 636e648. Senesi, S., Ghelardi, E., 2010. Production, secretion and biological activity of Bacillus cereus enterotoxins. Toxins 2, 1690e1703. Senesi, S., Celandroni, F., Salvetti, S., Beecher, D.J., Wong, A.C.L., Ghelardi, E., 2002. Swarming motility in Bacillus cereus and characterization of a fliY mutant impaired in swarm cell differentiation. Microbiology 148, 1785e1794. Senesi, S., Ghelardi, E., Celandroni, F., Salvetti, S., Parisio, E., Galizzi, A., 2004. Surface-associated flagellum formation and swarming differentiation in Bacillus subtilis are controlled by ifm locus. J. Bacteriol. 186, 1158e1164. Shrout, J.D., Chopp, D.L., Just, C.L., Givskov, M., Parsek, M.R., 2006. The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional. Mol. Microbiol. 62, 1264e1277. Verstraeten, N., Braeken, K., Debkumari, B., Fauvart, M., Fransaer, J., Vermant, J., Michiels, J., 2008. Living on a surface: swarming and biofilm formation. Trends Microbiol. 16, 496e506. Vilain, S., Luo, Y., Hildreth, M.B., Bro¨zel, V.S., 2006. Analysis of the life cycle of the soil saprophyte Bacillus cereus in liquid soil extract and in soil. Appl. Environ. Microbiol. 72, 4970e4977. Wang, Q., Frye, J.G., McClelland, M., Harshey, R.M., 2004. Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes. Mol. Microbiol. 52, 169e187. Young, G.M., Smith, M.J., Minnich, S.A., Miller, V.L., 1999. The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility. J. Bacteriol. 181, 2823e2833. Zanen, G., Antelmann, H., Wester, H., Hecker, M., van Dijl, J.M., Quax, W.J., 2004. FlhF, the third signal recognition particle-GTPase of Bacillus subtilis, is dispensable for protein secretion. J. Bacteriol. 186, 5956e5960.