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Heterogeneity of intracellular replication of bacterial pathogens
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Heterogeneity of intracellular replication of bacterial pathogens Sophie Helaine and David W Holden Intracellular growth of bacterial pathogens is usually measured at the whole population level, which masks potential cell-to-cell variation. More direct measurements of replication using microscopy and Flow Cytometry have revealed extensive heterogeneity among populations of intracellular bacteria. Heterogeneity could result from differential exposure to nutritional deprivation and host cell antimicrobial activities, as well as variability in production or efficacy of virulence molecules. Furthermore, bacteria have evolved specific mechanisms to generate epigenetic variation. These include unequal partitioning of proteins during cell division, genetic phase variation and activation of toxin/antitoxin systems. An important aspect of heterogeneity concerns the generation of viable, non-replicating bacteria. These are predicted to confer tolerance to host-induced stress and antibiotics, and to be sources of persistent infection. Address MRC Centre for Molecular Bacteriology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, UK Corresponding author: Holden, David W ([email protected])
Current Opinion in Microbiology 2013, 16:184–191 This review comes from a themed issue on Cell regulation Edited by Bonnie Bassler and Jo¨rg Vogel For a complete overview see the Issue and the Editorial Available online 26th February 2013 1369-5274/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2012.12.004
Introduction Parameters that describe a microbial population are traditionally measured at the whole population level. As techniques have been developed which enable measurement of many of these parameters to be made at the single cell level, the heterogeneity of such processes has been revealed. These include cell division time [1], gene expression [2], competence for genetic transformation [3] and growth arrest [4]. To proliferate and cause disease, intracellular bacterial pathogens of animals rely on survival and replication within host cells where they avoid or subvert cellular defense mechanisms and are protected from detection and elimination by humoral immune mechanisms. Like many other bacterial processes, intracellular replication is heterogeneous: infection of tissue-cultured host cells with a clonal population of bacteria frequently results in variable numbers of bacteria in each host cell (e.g. [5]; Figure 1). This can be Current Opinion in Microbiology 2013, 16:184–191
caused by both bacterial and host factors, as well as the variable intracellular microenvironments inhabited by these pathogens. Heterogeneity is evident in the composition of vacuoles that contain bacteria, in processes mediating their growth inhibition and elimination and in bacterial replication itself. Here, we focus on heterogeneity of intracellular replication of bacterial pathogens and consider how it can be measured, how it is generated and its effect on bacterial pathogenesis.
Whole population measurement of intracellular replication The extent of intracellular bacterial proliferation is usually measured by the change in total bacterial numbers over time. This can be assessed by various techniques, and the most common continues to be quantification of colony forming units (CFU), after plating lysates of infected cells. This simple and useful method provides a measurement of the average net growth undergone by the total bacterial population. However, net growth reflects the combination of bacterial replication and killing by the host, together with possible bacterial migration into and from host cells. Thus, average values of growth provide no information on any underlying heterogeneity that might exist among individual bacteria (Figure 2). Several approaches have been developed to provide more direct measurements of bacterial replication. Each is on the basis of the principle of dilution of a non-propagated marker, which is partitioned at each bacterial cell division. By comparing rates of replication and overall net growth, it is possible to obtain a measurement of the extent of killing sustained by the bacterial population, and thereby define the components of net growth in terms of bacterial replication and killing. Early efforts exploited a superinfecting but non-proliferative phage of Salmonella Typhimurium to obtain division and death rates of this intracellular pathogen [6]. Because of its inability to propagate, the phage segregates to one daughter cell at each round of division and the proportion of bacteria carrying the phage is halved. The rate of dilution of the marker reveals the doubling time of the population. Subsequently, non-replicating plasmids have been used as ‘replication clocks’ to measure division rates of pathogens such as S. Typhimurium and more recently Mycobacterium tuberculosis, in vivo [7,8]. These techniques provide a much more accurate measurement of bacterial replication than CFU counts, but they do not reveal potential heterogeneity of replication within the population. www.sciencedirect.com
Heterogeneity of intracellular replication of bacterial pathogens Helaine and Holden 185
Figure 1
Micro-chambers of defined size enable isolation and subsequent analysis of large numbers of individual cells in parallel [16]. Furthermore, the composition of the environment within the micro-chambers can be controlled precisely. These devices were first used in microbiological studies to analyse bacterial chemotaxis by creating gradients of attractants and repellents within the microchannels [17]. They have been used since to analyse the growth of individual Escherichia coli cells before and during the course of antibiotic treatment [4]. As a result, antibiotic-tolerant persisters were shown to be non-replicating bacteria present in the population before antibiotic exposure. More recently, these devices were used to study mycobacterial growth at the single cell level. This revealed asymmetrical growth of the cells, which after division, generates two populations with different elongation rates [18]. Microfluidic devices are beginning to be applied to infected host cells [19]; they provide a powerful technological platform to study the behavior of intracellular bacteria at the single cell level. Flow Cytometry Current Opinion in Microbiology
Heterogeneity of intracellular bacterial replication. Mouse bone marrowderived macrophages were infected with gfp-expressing Salmonella for 17 hours. Variable numbers of bacteria are observed in each host cell.
Single cell-based methods Microscopy
Microscopic analyses provide insights into overall numbers of bacteria, their interactions with infected cells and how these parameters can vary from one infected cell to another [9]. Moreover, live imaging microscopy can reveal the dynamics of activities in real time. For example, heterogeneity of bacterial speed and trajectories within different subcellular locations of the infected cells, as well as fusion and fission events occurring on membranes of bacterial-containing vacuoles, have been measured at the single cell level by tracking individual bacteria over time [10,11,12]. Live imaging microscopy combined with a fluorescence resonance energy transfer (FRET)-based assay has provided detailed information on the kinetics of vacuole rupture by Shigella flexneri and mycobacteria [13,14]. A recent study combined the use of several techniques to analyse the physiological states of M. tuberculosis in primary macrophages. While CFU counts, a ‘replication clock’ and transcriptional profiling provided mean values for these parameters at the population level, electron microscopy revealed extensive heterogeneity of compartments containing bacteria [15]. Microfluidics
The development of transparent microfluidic devices, particularly through soft lithography, represents a very powerful approach for single-cell analyses. www.sciencedirect.com
The technique of choice to analyse heterogeneity in any cell population is Flow Cytometry. If the parameters to be measured can be associated with fluorescence, many thousands of individual cells can be reliably and quantifiably analysed in a few seconds. A fluorescent membrane dye (carboxyfluorescein diacetate succinimidyl ester, CFSE) that dilutes with cell division has been used extensively over the last 10 years to provide accurate measurements of mammalian cell division. Combined with Flow Cytometry, it represents a very powerful approach, enabling up to 10 cycles of cell division to be analysed at the single cell level [20]. The same approach using another membrane dye (PKH26), has been applied recently to measure the rate of bacterial replication in vitro [21]. To further exploit Flow Cytometry to assess bacterial replication, an alternative method has been developed in which plasmid-based GFP production is dependent on the presence of a homoserine lactone inducer. Upon removal of the inducer, dilution of the GFP occurs as the bacteria replicate in laboratory medium, enabling replicating and non-replicating E. coli to be differentiated [22]. We developed this fluorescence dilution (FD) technique further to quantify the rate of intracellular replication of S. Typhimurium at the single cell level in macrophages [23]. In our version, Salmonella cells contain a plasmid that encodes two fluorescent proteins. One fluorophore is constitutively produced while the synthesis of the second is under the control of an inducible promoter. After full induction, and before infection, the inducer is removed, thereby stopping production of the second fluorescent protein. The constitutively produced protein enables every viable bacterial cell to be detected by Flow Cytometry or microscopy, while the concentration of the second protein is halved at each bacterial cell division. The extent of Current Opinion in Microbiology 2013, 16:184–191
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Figure 2
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The power of single cell analysis. Bacterial growth in host cells is usually measured at the whole population level, revealing overall levels of proliferation or killing. Single cell analysis provides information on how bacteria act as individuals, and processes that contribute to net growth. Two possible scenarios are shown. (a) An increase in net bacterial load occurs over time. The ‘whole population’ level of analysis indicates the presence of actively dividing bacteria (green rods). However single-cell analysis reveals that the increase results from the combination of replication (green rods), killing by host defenses (grey rods), and the presence of dormant cells (red rod). Note that the dividing bacteria replicate at a higher rate than would be deduced from ‘whole population’ quantification. (b) A decrease in net bacterial load occurs over time. The ‘whole population’ level of analysis indicates the killing of part of the initial intracellular population (grey rods), and survival of the remainders (green rod). However, single-cell analysis reveals a combination of killing (grey rods), egress from the cell and active replication (green rods).
fluorescence dilution is quantified easily by Flow Cytometry and used as a read-out of the number of generations of division undergone by bacterial cells (Figure 3). In this way, six generations can be measured accurately. In another version of FD, production of two fluorophores is controlled by different inducers; sequential dilution of both fluorophores enables up to 10 generations to be measured [23]. These methods were used to show the importance of a type III secretion system in intracellular replication of Salmonella, as well as heterogeneity of bacterial replication in primary bone marrow-derived mouse macrophages (Figure 3c, [23]). Given its simplicity, the method should be readily applicable to other bacterial pathogens. The availability of instruments that combine Flow Cytometry and microscopy [24,25] should provide additional power in analyzing heterogeneity of Current Opinion in Microbiology 2013, 16:184–191
intracellular bacterial replication, by allowing examination of more subtle details such as bacterial morphology, recruitment of host proteins or subcellular localization.
Heterogeneity of intracellular bacterial replication Replication niche
Some intracellular pathogens replicate in the host cytosol, while others proliferate within membrane-bound phagosomes or vacuoles. However, several species are capable of colonizing more than one compartment. For example, upon invasion of epithelial cells, the majority of S. Typhimurium replicate inside Salmonella-containing vacuoles (SCVs). However a small proportion of vacuoles are ruptured, releasing bacteria into the cytoplasm. Although cytosolic bacteria represent less than 5% of www.sciencedirect.com
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Figure 3
(b) Number bacteria (% of max)
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Fluorescence dilution. (a) Salmonella cells constitutively produce GFP, while DsRed is under the control of an inducible promoter. After induction the inducer is removed and the concentration of DsRed is halved at each bacterial cell division, leading to fluorescence dilution that can be quantified by Flow Cytometry. (b) Fluorescence dilution profile indicating replication of Salmonella in bone marrow-derived macrophages after 22 hours of infection. The profile is spread over a broad range, reflecting heterogeneous replication. (c) Microscopic analysis of infected macrophages shows bacteria in the same cell that have undergone high (green), moderate (yellow) or no (red) replication. Scale bar represents 5 mm. (d) Replication of Salmonella in laboratory medium, monitored by dilution of red fluorescence at hourly intervals, produces overlapping, normally distributed curves, indicating homogeneous replication.
the total population at 10 hours post-invasion, they hyperreplicate and can contribute significantly to subsequent net growth [26,27]. Conversely, while Listeria monocytogenes is adapted to replicate in the host cell cytoplasm, a small proportion remains in vacuoles where they are capable of limited growth [28]. Electron microscopic analysis of macrophages infected with M. tuberculosis revealed both vacuolar and cytosolic bacteria [14,29]. However, cytosolic bacteria reportedly induce host cell death [14] and the relative contribution of these subpopulations to overall replication may depend on the experimental system being used. www.sciencedirect.com
Heterogeneity within compartments
Heterogeneity of replication can also be observed for bacteria within vacuoles. For example, although replication of Salmonella in macrophages occurs exclusively in vacuoles [30], use of FD showed that substantial heterogeneity exists in their replication rates, and microscopic analysis of these cells revealed heterogeneous replication of bacteria even within the same cell (Figure 3b,c). Uptake of non-pathogenic or dead bacteria results in phagosomes that mature by acidification and progressive acquisition and loss of various endosomal markers, culminating in the formation of a phago-lysosome. Many Current Opinion in Microbiology 2013, 16:184–191
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vacuolar pathogens interfere with this maturation pathway, allowing them to survive and replicate. However studies of vacuole composition show that they are frequently heterogeneous. For example, the luminal pH of vacuoles containing Salmonella or Legionella pneumophila is spread over a broad range of values [31,32], and both pathogens display considerable heterogeneity in acquisition of vesicles and tubules from the biosynthetic pathway of the host cell [33,34]. Me´resse and colleagues developed an elegant epitope-specific Flow Cytometric method to quantify the presence of markers on isolated SCVs [35,36]. These studies revealed considerable variability in the composition of SCVs at the same time point after bacterial internalization. Such differences in vacuole composition are very likely to influence the ability of bacteria to replicate and hence account for the observed heterogeneity in numbers of intracellular bacteria. Non-replicators
There is a widely held view that pathogenic bacteria can enter a viable but non-replicating state within host cells, where they can survive innate immune killing mechanisms and display tolerance to antibiotics. Indirect evidence for such non-proliferating or dormant bacteria has been obtained for several pathogens, including Mycobacterium tuberculosis [37], uropathogenic E. coli [38,39], Burkholderia cepacia [40], Bartonella [41], Chlamydia pneumonia [42], Campylobacter jujeni [43] and Haemophilus influenza [44]. While using FD to measure intra-macrophage replication of S. Typhimurium, we discovered that a substantial proportion of the infecting population entered a non-replicating but viable state that lasted for several days [23]. In some macrophages, non-replicating Salmonella represent the sole intracellular population while in others they were found together with replicating bacteria (Figure 3).
evolved specific mechanisms to generate epigenetic variation. Indeed it is now well-established that a clonal population of bacteria in a uniform environment displays phenotypic variation [45]. Such heterogeneity can be caused by stochastic molecular ‘noise’: random fluctuations in the number of molecules and reactions within a cell, and becomes more evident when molecules are present at low copy number in the cell and therefore have a greater probability of undergoing unequal partitioning during cell division [46,47]. For example, a recent quantification of the transcriptome and proteome in single E. coli cells revealed that half of the total pool of proteins is present at less than 10 copies per cell [48]. These data strongly suggest that the effects of unequal partitioning of low copy proteins could be significant, particularly if the protein has a regulatory function. It has also been shown that phenotypic variation is generated when some molecules, such as toxin components of toxin/antitoxin (TA) systems, exceed a threshold value in a subset of cells within a given population. TA loci are almost ubiquitous among bacteria and are involved in the generation of variability in replication by arresting growth in a small proportion of bacteria [49,50]. It will be very interesting to determine if these proteins account for nonreplicating intracellular bacteria. Noise can also be generated at the level of transcription and some promoters have been described as being noisier than others, thereby conferring higher levels of phenotypic variation [51]. This could be the case for certain virulence genes that are expressed at different levels by bacteria within the same host cell, such as an acid-inducible locus (aprBC) of M. tuberculosis in macrophages [52] and virulence genes by Staphyloccocus aureus [53] and S. Typhimurium [54]. The bacterial stress response is generally accompanied by a reduction in transcriptional and translational activities, which could increase molecular noise and thus phenotypic heterogeneity [45].
Generation of heterogeneity
Heterogeneity is likely to be generated by both bacterial and host cell factors and the combination of both probably amplifies the resulting variation. Replication of Salmonella in laboratory medium is a homogeneous process (Figure 3d, [23]). Therefore the heterogeneity of bacterial replication in macrophages is generated by the interaction between the host and the pathogen. The relationship between pathogenic bacteria and their host cell environment is highly dynamic and replication, even for a well-adapted pathogen, is likely to represent the successful outcome of possibly thousands of interactions between bacterial and the host cell molecules over a period of several hours. Each one of these interactions could be either conducive or unfavourable to bacterial replication. Thus, heterogeneity is due at least in part to differential exposure or sensitivity to nutritional deprivation or more toxic host processes, and the efficiency with which virulence molecules function to avoid, subvert or tolerate host defences. In addition, bacteria have Current Opinion in Microbiology 2013, 16:184–191
Phase variation is distinct from molecular noise as it results from reversible alterations in the genome sequence. This allows the variability to be heritable, even if the phase of expression is reversible between each generation. Phase variations occur in specific genomic loci at a relatively high frequency. They involve DNA inversion, slipped-strand mispairing or differential methylation events leading to ‘on/off’ switches, thereby causing bimodal gene expression, as well as more complex recombinational rearrangements resulting in multiphasic phenotypes [55,56]. Recently, phase variation has been proposed as a potential way of producing non-replicating subpopulations of S. Typhimurium in macrophages [57]. However, the switching frequency of the locus in question (involved in O-antigen length in LPS) is so low for bacteria grown in laboratory medium, that a non-replicating subpopulation would be a very minor proportion of the total. It would be interesting to investigate whether residency within the host cell altered the switching rate. www.sciencedirect.com
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Physiological significance of heterogeneity in intracellular replication In a phenotypically identical population of cells, a stress that kills one cell will in theory kill the entire population. In rapidly changing environments, this is likely to have provided a powerful selective pressure within asexually reproducing bacteria for mechanisms that generate phenotypic variation [58,59]. It is known that slowly replicating or non-replicating bacteria in stationary phase growth in vitro can be more resistant to oxidative stress, low pH and antimicrobial peptides [60,61]. These are all used by host cells to control bacterial growth. Therefore, it seems plausible that if non-replicating bacteria are induced inside host cells, then they would be better able to resist stresses imposed by their intracellular environment, as well as tolerating the effects of antibiotics. Many bacterial pathogens cause recurrent diseases, for example tuberculosis, typhoid fever, urinary tract infections and tonsillitis. The replication status of bacterial persisters during these longterm infections is not known but they may well be in a quiescent, non-replicating state. The development of techniques to study intracellular bacteria at the single cell level provides a means to greatly enhance our understanding of the effects and underlying causes of heterogeneity among these pathogens. Apart from the academic interest in understanding an important aspect of the physiology of bacterial pathogens, such studies are likely to have clinical relevance, given the intractability of treating chronic infections and the increasing spread of antibiotic resistance.
Acknowledgements S. Helaine is supported by a Junior Research Fellowship from Imperial College London. Work in DW Holden’s laboratory is supported by grants from the Medical Research Council, Wellcome Trust and Biotechnology and Biological Sciences Research Council (UK). We thank Nathalie Rolhion, Serge Mostowy and Andrew Ulijasz for critical reading of the manuscript. Space limitations prevented citation of all relevant literature and the emphasis on Salmonella reflects our own research interests.
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60. Battesti A, Majdalani N, Gottesman S: The rpos-mediated general stress response in Escherichia coli. Annu Rev Microbiol 2011, 65:189-213. 61. Barker HC, Kinsella N, Jaspe A, Friedrich T, O’Connor CD: Formate protects stationary-phase Escherichia coli and Salmonella cells from killing by a cationic antimicrobial peptide. Mol Microbiol 2000, 35: 1518-1529.
Current Opinion in Microbiology 2013, 16:184–191
Heterogeneity of intracellular replication of bacterial pathogens Sophie Helaine and David W Holden Intracellular growth of bacterial pathogens is usually measured at the whole population level, which masks potential cell-to-cell variation. More direct measurements of replication using microscopy and Flow Cytometry have revealed extensive heterogeneity among populations of intracellular bacteria. Heterogeneity could result from differential exposure to nutritional deprivation and host cell antimicrobial activities, as well as variability in production or efficacy of virulence molecules. Furthermore, bacteria have evolved specific mechanisms to generate epigenetic variation. These include unequal partitioning of proteins during cell division, genetic phase variation and activation of toxin/antitoxin systems. An important aspect of heterogeneity concerns the generation of viable, non-replicating bacteria. These are predicted to confer tolerance to host-induced stress and antibiotics, and to be sources of persistent infection. Address MRC Centre for Molecular Bacteriology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, UK Corresponding author: Holden, David W ([email protected])
Current Opinion in Microbiology 2013, 16:184–191 This review comes from a themed issue on Cell regulation Edited by Bonnie Bassler and Jo¨rg Vogel For a complete overview see the Issue and the Editorial Available online 26th February 2013 1369-5274/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2012.12.004
Introduction Parameters that describe a microbial population are traditionally measured at the whole population level. As techniques have been developed which enable measurement of many of these parameters to be made at the single cell level, the heterogeneity of such processes has been revealed. These include cell division time [1], gene expression [2], competence for genetic transformation [3] and growth arrest [4]. To proliferate and cause disease, intracellular bacterial pathogens of animals rely on survival and replication within host cells where they avoid or subvert cellular defense mechanisms and are protected from detection and elimination by humoral immune mechanisms. Like many other bacterial processes, intracellular replication is heterogeneous: infection of tissue-cultured host cells with a clonal population of bacteria frequently results in variable numbers of bacteria in each host cell (e.g. [5]; Figure 1). This can be Current Opinion in Microbiology 2013, 16:184–191
caused by both bacterial and host factors, as well as the variable intracellular microenvironments inhabited by these pathogens. Heterogeneity is evident in the composition of vacuoles that contain bacteria, in processes mediating their growth inhibition and elimination and in bacterial replication itself. Here, we focus on heterogeneity of intracellular replication of bacterial pathogens and consider how it can be measured, how it is generated and its effect on bacterial pathogenesis.
Whole population measurement of intracellular replication The extent of intracellular bacterial proliferation is usually measured by the change in total bacterial numbers over time. This can be assessed by various techniques, and the most common continues to be quantification of colony forming units (CFU), after plating lysates of infected cells. This simple and useful method provides a measurement of the average net growth undergone by the total bacterial population. However, net growth reflects the combination of bacterial replication and killing by the host, together with possible bacterial migration into and from host cells. Thus, average values of growth provide no information on any underlying heterogeneity that might exist among individual bacteria (Figure 2). Several approaches have been developed to provide more direct measurements of bacterial replication. Each is on the basis of the principle of dilution of a non-propagated marker, which is partitioned at each bacterial cell division. By comparing rates of replication and overall net growth, it is possible to obtain a measurement of the extent of killing sustained by the bacterial population, and thereby define the components of net growth in terms of bacterial replication and killing. Early efforts exploited a superinfecting but non-proliferative phage of Salmonella Typhimurium to obtain division and death rates of this intracellular pathogen [6]. Because of its inability to propagate, the phage segregates to one daughter cell at each round of division and the proportion of bacteria carrying the phage is halved. The rate of dilution of the marker reveals the doubling time of the population. Subsequently, non-replicating plasmids have been used as ‘replication clocks’ to measure division rates of pathogens such as S. Typhimurium and more recently Mycobacterium tuberculosis, in vivo [7,8]. These techniques provide a much more accurate measurement of bacterial replication than CFU counts, but they do not reveal potential heterogeneity of replication within the population. www.sciencedirect.com
Heterogeneity of intracellular replication of bacterial pathogens Helaine and Holden 185
Figure 1
Micro-chambers of defined size enable isolation and subsequent analysis of large numbers of individual cells in parallel [16]. Furthermore, the composition of the environment within the micro-chambers can be controlled precisely. These devices were first used in microbiological studies to analyse bacterial chemotaxis by creating gradients of attractants and repellents within the microchannels [17]. They have been used since to analyse the growth of individual Escherichia coli cells before and during the course of antibiotic treatment [4]. As a result, antibiotic-tolerant persisters were shown to be non-replicating bacteria present in the population before antibiotic exposure. More recently, these devices were used to study mycobacterial growth at the single cell level. This revealed asymmetrical growth of the cells, which after division, generates two populations with different elongation rates [18]. Microfluidic devices are beginning to be applied to infected host cells [19]; they provide a powerful technological platform to study the behavior of intracellular bacteria at the single cell level. Flow Cytometry Current Opinion in Microbiology
Heterogeneity of intracellular bacterial replication. Mouse bone marrowderived macrophages were infected with gfp-expressing Salmonella for 17 hours. Variable numbers of bacteria are observed in each host cell.
Single cell-based methods Microscopy
Microscopic analyses provide insights into overall numbers of bacteria, their interactions with infected cells and how these parameters can vary from one infected cell to another [9]. Moreover, live imaging microscopy can reveal the dynamics of activities in real time. For example, heterogeneity of bacterial speed and trajectories within different subcellular locations of the infected cells, as well as fusion and fission events occurring on membranes of bacterial-containing vacuoles, have been measured at the single cell level by tracking individual bacteria over time [10,11,12]. Live imaging microscopy combined with a fluorescence resonance energy transfer (FRET)-based assay has provided detailed information on the kinetics of vacuole rupture by Shigella flexneri and mycobacteria [13,14]. A recent study combined the use of several techniques to analyse the physiological states of M. tuberculosis in primary macrophages. While CFU counts, a ‘replication clock’ and transcriptional profiling provided mean values for these parameters at the population level, electron microscopy revealed extensive heterogeneity of compartments containing bacteria [15]. Microfluidics
The development of transparent microfluidic devices, particularly through soft lithography, represents a very powerful approach for single-cell analyses. www.sciencedirect.com
The technique of choice to analyse heterogeneity in any cell population is Flow Cytometry. If the parameters to be measured can be associated with fluorescence, many thousands of individual cells can be reliably and quantifiably analysed in a few seconds. A fluorescent membrane dye (carboxyfluorescein diacetate succinimidyl ester, CFSE) that dilutes with cell division has been used extensively over the last 10 years to provide accurate measurements of mammalian cell division. Combined with Flow Cytometry, it represents a very powerful approach, enabling up to 10 cycles of cell division to be analysed at the single cell level [20]. The same approach using another membrane dye (PKH26), has been applied recently to measure the rate of bacterial replication in vitro [21]. To further exploit Flow Cytometry to assess bacterial replication, an alternative method has been developed in which plasmid-based GFP production is dependent on the presence of a homoserine lactone inducer. Upon removal of the inducer, dilution of the GFP occurs as the bacteria replicate in laboratory medium, enabling replicating and non-replicating E. coli to be differentiated [22]. We developed this fluorescence dilution (FD) technique further to quantify the rate of intracellular replication of S. Typhimurium at the single cell level in macrophages [23]. In our version, Salmonella cells contain a plasmid that encodes two fluorescent proteins. One fluorophore is constitutively produced while the synthesis of the second is under the control of an inducible promoter. After full induction, and before infection, the inducer is removed, thereby stopping production of the second fluorescent protein. The constitutively produced protein enables every viable bacterial cell to be detected by Flow Cytometry or microscopy, while the concentration of the second protein is halved at each bacterial cell division. The extent of Current Opinion in Microbiology 2013, 16:184–191
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Figure 2
(a)
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The power of single cell analysis. Bacterial growth in host cells is usually measured at the whole population level, revealing overall levels of proliferation or killing. Single cell analysis provides information on how bacteria act as individuals, and processes that contribute to net growth. Two possible scenarios are shown. (a) An increase in net bacterial load occurs over time. The ‘whole population’ level of analysis indicates the presence of actively dividing bacteria (green rods). However single-cell analysis reveals that the increase results from the combination of replication (green rods), killing by host defenses (grey rods), and the presence of dormant cells (red rod). Note that the dividing bacteria replicate at a higher rate than would be deduced from ‘whole population’ quantification. (b) A decrease in net bacterial load occurs over time. The ‘whole population’ level of analysis indicates the killing of part of the initial intracellular population (grey rods), and survival of the remainders (green rod). However, single-cell analysis reveals a combination of killing (grey rods), egress from the cell and active replication (green rods).
fluorescence dilution is quantified easily by Flow Cytometry and used as a read-out of the number of generations of division undergone by bacterial cells (Figure 3). In this way, six generations can be measured accurately. In another version of FD, production of two fluorophores is controlled by different inducers; sequential dilution of both fluorophores enables up to 10 generations to be measured [23]. These methods were used to show the importance of a type III secretion system in intracellular replication of Salmonella, as well as heterogeneity of bacterial replication in primary bone marrow-derived mouse macrophages (Figure 3c, [23]). Given its simplicity, the method should be readily applicable to other bacterial pathogens. The availability of instruments that combine Flow Cytometry and microscopy [24,25] should provide additional power in analyzing heterogeneity of Current Opinion in Microbiology 2013, 16:184–191
intracellular bacterial replication, by allowing examination of more subtle details such as bacterial morphology, recruitment of host proteins or subcellular localization.
Heterogeneity of intracellular bacterial replication Replication niche
Some intracellular pathogens replicate in the host cytosol, while others proliferate within membrane-bound phagosomes or vacuoles. However, several species are capable of colonizing more than one compartment. For example, upon invasion of epithelial cells, the majority of S. Typhimurium replicate inside Salmonella-containing vacuoles (SCVs). However a small proportion of vacuoles are ruptured, releasing bacteria into the cytoplasm. Although cytosolic bacteria represent less than 5% of www.sciencedirect.com
Heterogeneity of intracellular replication of bacterial pathogens Helaine and Holden 187
Figure 3
(b) Number bacteria (% of max)
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Fluorescence dilution. (a) Salmonella cells constitutively produce GFP, while DsRed is under the control of an inducible promoter. After induction the inducer is removed and the concentration of DsRed is halved at each bacterial cell division, leading to fluorescence dilution that can be quantified by Flow Cytometry. (b) Fluorescence dilution profile indicating replication of Salmonella in bone marrow-derived macrophages after 22 hours of infection. The profile is spread over a broad range, reflecting heterogeneous replication. (c) Microscopic analysis of infected macrophages shows bacteria in the same cell that have undergone high (green), moderate (yellow) or no (red) replication. Scale bar represents 5 mm. (d) Replication of Salmonella in laboratory medium, monitored by dilution of red fluorescence at hourly intervals, produces overlapping, normally distributed curves, indicating homogeneous replication.
the total population at 10 hours post-invasion, they hyperreplicate and can contribute significantly to subsequent net growth [26,27]. Conversely, while Listeria monocytogenes is adapted to replicate in the host cell cytoplasm, a small proportion remains in vacuoles where they are capable of limited growth [28]. Electron microscopic analysis of macrophages infected with M. tuberculosis revealed both vacuolar and cytosolic bacteria [14,29]. However, cytosolic bacteria reportedly induce host cell death [14] and the relative contribution of these subpopulations to overall replication may depend on the experimental system being used. www.sciencedirect.com
Heterogeneity within compartments
Heterogeneity of replication can also be observed for bacteria within vacuoles. For example, although replication of Salmonella in macrophages occurs exclusively in vacuoles [30], use of FD showed that substantial heterogeneity exists in their replication rates, and microscopic analysis of these cells revealed heterogeneous replication of bacteria even within the same cell (Figure 3b,c). Uptake of non-pathogenic or dead bacteria results in phagosomes that mature by acidification and progressive acquisition and loss of various endosomal markers, culminating in the formation of a phago-lysosome. Many Current Opinion in Microbiology 2013, 16:184–191
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vacuolar pathogens interfere with this maturation pathway, allowing them to survive and replicate. However studies of vacuole composition show that they are frequently heterogeneous. For example, the luminal pH of vacuoles containing Salmonella or Legionella pneumophila is spread over a broad range of values [31,32], and both pathogens display considerable heterogeneity in acquisition of vesicles and tubules from the biosynthetic pathway of the host cell [33,34]. Me´resse and colleagues developed an elegant epitope-specific Flow Cytometric method to quantify the presence of markers on isolated SCVs [35,36]. These studies revealed considerable variability in the composition of SCVs at the same time point after bacterial internalization. Such differences in vacuole composition are very likely to influence the ability of bacteria to replicate and hence account for the observed heterogeneity in numbers of intracellular bacteria. Non-replicators
There is a widely held view that pathogenic bacteria can enter a viable but non-replicating state within host cells, where they can survive innate immune killing mechanisms and display tolerance to antibiotics. Indirect evidence for such non-proliferating or dormant bacteria has been obtained for several pathogens, including Mycobacterium tuberculosis [37], uropathogenic E. coli [38,39], Burkholderia cepacia [40], Bartonella [41], Chlamydia pneumonia [42], Campylobacter jujeni [43] and Haemophilus influenza [44]. While using FD to measure intra-macrophage replication of S. Typhimurium, we discovered that a substantial proportion of the infecting population entered a non-replicating but viable state that lasted for several days [23]. In some macrophages, non-replicating Salmonella represent the sole intracellular population while in others they were found together with replicating bacteria (Figure 3).
evolved specific mechanisms to generate epigenetic variation. Indeed it is now well-established that a clonal population of bacteria in a uniform environment displays phenotypic variation [45]. Such heterogeneity can be caused by stochastic molecular ‘noise’: random fluctuations in the number of molecules and reactions within a cell, and becomes more evident when molecules are present at low copy number in the cell and therefore have a greater probability of undergoing unequal partitioning during cell division [46,47]. For example, a recent quantification of the transcriptome and proteome in single E. coli cells revealed that half of the total pool of proteins is present at less than 10 copies per cell [48]. These data strongly suggest that the effects of unequal partitioning of low copy proteins could be significant, particularly if the protein has a regulatory function. It has also been shown that phenotypic variation is generated when some molecules, such as toxin components of toxin/antitoxin (TA) systems, exceed a threshold value in a subset of cells within a given population. TA loci are almost ubiquitous among bacteria and are involved in the generation of variability in replication by arresting growth in a small proportion of bacteria [49,50]. It will be very interesting to determine if these proteins account for nonreplicating intracellular bacteria. Noise can also be generated at the level of transcription and some promoters have been described as being noisier than others, thereby conferring higher levels of phenotypic variation [51]. This could be the case for certain virulence genes that are expressed at different levels by bacteria within the same host cell, such as an acid-inducible locus (aprBC) of M. tuberculosis in macrophages [52] and virulence genes by Staphyloccocus aureus [53] and S. Typhimurium [54]. The bacterial stress response is generally accompanied by a reduction in transcriptional and translational activities, which could increase molecular noise and thus phenotypic heterogeneity [45].
Generation of heterogeneity
Heterogeneity is likely to be generated by both bacterial and host cell factors and the combination of both probably amplifies the resulting variation. Replication of Salmonella in laboratory medium is a homogeneous process (Figure 3d, [23]). Therefore the heterogeneity of bacterial replication in macrophages is generated by the interaction between the host and the pathogen. The relationship between pathogenic bacteria and their host cell environment is highly dynamic and replication, even for a well-adapted pathogen, is likely to represent the successful outcome of possibly thousands of interactions between bacterial and the host cell molecules over a period of several hours. Each one of these interactions could be either conducive or unfavourable to bacterial replication. Thus, heterogeneity is due at least in part to differential exposure or sensitivity to nutritional deprivation or more toxic host processes, and the efficiency with which virulence molecules function to avoid, subvert or tolerate host defences. In addition, bacteria have Current Opinion in Microbiology 2013, 16:184–191
Phase variation is distinct from molecular noise as it results from reversible alterations in the genome sequence. This allows the variability to be heritable, even if the phase of expression is reversible between each generation. Phase variations occur in specific genomic loci at a relatively high frequency. They involve DNA inversion, slipped-strand mispairing or differential methylation events leading to ‘on/off’ switches, thereby causing bimodal gene expression, as well as more complex recombinational rearrangements resulting in multiphasic phenotypes [55,56]. Recently, phase variation has been proposed as a potential way of producing non-replicating subpopulations of S. Typhimurium in macrophages [57]. However, the switching frequency of the locus in question (involved in O-antigen length in LPS) is so low for bacteria grown in laboratory medium, that a non-replicating subpopulation would be a very minor proportion of the total. It would be interesting to investigate whether residency within the host cell altered the switching rate. www.sciencedirect.com
Heterogeneity of intracellular replication of bacterial pathogens Helaine and Holden 189
Physiological significance of heterogeneity in intracellular replication In a phenotypically identical population of cells, a stress that kills one cell will in theory kill the entire population. In rapidly changing environments, this is likely to have provided a powerful selective pressure within asexually reproducing bacteria for mechanisms that generate phenotypic variation [58,59]. It is known that slowly replicating or non-replicating bacteria in stationary phase growth in vitro can be more resistant to oxidative stress, low pH and antimicrobial peptides [60,61]. These are all used by host cells to control bacterial growth. Therefore, it seems plausible that if non-replicating bacteria are induced inside host cells, then they would be better able to resist stresses imposed by their intracellular environment, as well as tolerating the effects of antibiotics. Many bacterial pathogens cause recurrent diseases, for example tuberculosis, typhoid fever, urinary tract infections and tonsillitis. The replication status of bacterial persisters during these longterm infections is not known but they may well be in a quiescent, non-replicating state. The development of techniques to study intracellular bacteria at the single cell level provides a means to greatly enhance our understanding of the effects and underlying causes of heterogeneity among these pathogens. Apart from the academic interest in understanding an important aspect of the physiology of bacterial pathogens, such studies are likely to have clinical relevance, given the intractability of treating chronic infections and the increasing spread of antibiotic resistance.
Acknowledgements S. Helaine is supported by a Junior Research Fellowship from Imperial College London. Work in DW Holden’s laboratory is supported by grants from the Medical Research Council, Wellcome Trust and Biotechnology and Biological Sciences Research Council (UK). We thank Nathalie Rolhion, Serge Mostowy and Andrew Ulijasz for critical reading of the manuscript. Space limitations prevented citation of all relevant literature and the emphasis on Salmonella reflects our own research interests.
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