Abdelrahim Zoued

Andreas Diepold
email: andreas.diepold@bioch.ox.ac.uk

Defining assembly pathways by fluorescence microscopy

Keywords: Fluorescence microscopy, biogenesis, secretion systems, fluorescently labeled proteins, macromolecular complexes, epistasis experiments, subcellular localization Running head: Protein dynamics by fluorescence microscopy

Bacterial secretion systems are amongst the largest protein complexes in prokaryotes, and display remarkably complex architectures. Their assembly often follows clearly defined pathways. Deciphering these pathways not only reveals how bacteria accomplish to build these large functional complexes, but can provide crucial information on the interactions and subcomplexes within secretion systems, their distribution within the bacterium, and even functional insights. The emergence of fluorescent proteins has provided a new powerful tool for biological imaging, and the use of fluorescently labeled components presents an interesting method to accurately define the biogenesis of macromolecular complexes. Here, we describe the use of this method to decipher the assembly pathway of bacterial secretion systems.

Introduction

Bacterial secretion systems are macromolecular machines that mediate the transport of proteins between bacteria or from bacteria to eukaryotic cells [START_REF] Tseng | Protein secretion systems in bacterial-host associations, and their description in the Gene Ontology[END_REF][START_REF] Costa | Secretion systems in Gram-negative bacteria: structural and mechanistic insights[END_REF]. These complexes incorporate one or multiple copies of a large number of different proteins that are recruited in a hierarchical order. Important insights into the assembly of secretion systems were obtained by the purification and visualization of assembly intermediates, either in strains lacking certain components of the system, or upon overexpression of defined components [START_REF] Kimbrough | Contribution of Salmonella typhimurium type III secretion components to needle complex formation[END_REF][START_REF] Sukhan | Genetic analysis of assembly of the Salmonella enterica serovar Typhimurium type III secretion-associated needle complex[END_REF][START_REF] Kimbrough | Assembly of the type III secretion needle complex of Salmonella typhimurium[END_REF][START_REF] Ogino | Assembly of the type III secretion apparatus of enteropathogenic Escherichia coli[END_REF][START_REF] Fronzes | Structure of a type IV secretion system core complex[END_REF][START_REF] Schraidt | Topology and organization of the Salmonella typhimurium type III secretion needle complex components[END_REF][START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF][START_REF] Darbari | Structural Biology of Bacterial Type IV Secretion Systems[END_REF].

However, the often low number of stable intermediates and the difficulties in obtaining and visualizing these have limited the utilization of this approach. Increasingly, the assembly of secretion systems is therefore deciphered based on the localization of fluorescently labeled subunits. Variations of this approach have been applied for a variety of secretion systems including the Tat system [START_REF] Rose | Substrate-dependent assembly of the Tat translocase as observed in live Escherichia coli cells[END_REF]12), the type II secretion system (T2SS) [START_REF] Lybarger | Docking and assembly of the type II secretion complex of Vibrio cholerae[END_REF][START_REF] Johnson | Fluorescence microscopy and proteomics to investigate subcellular localization, assembly, and function of the type II secretion system[END_REF], the T3SS [START_REF] Diepold | Deciphering the assembly of the Yersinia type III secretion injectisome[END_REF][START_REF] Diepold | The assembly of the export apparatus (YscR,S,T,U,V) of the Yersinia type III secretion apparatus occurs independently of other structural components and involves the formation of an YscV oligomer[END_REF], the T4SS [START_REF] Aguilar | Agrobacterium type IV secretion system and its substrates form helical arrays around the circumference of virulence-induced cells[END_REF], or more recently the T6SS [START_REF] Durand | Biogenesis and structure of a type VI secretion membrane core complex[END_REF]. The rationale is that a fluorescently labeled component forms distinctive fluorescent foci where the secretion system resides, in wild-type cells and in strains lacking components that are not required for its assembly, but will have a diffuse fluorescent pattern when components responsible for its recruitment are missing. Prerequisites for this approach are the genetic amenability of the bacterium to create fluorescent fusion proteins, and a specific distribution of the secretion system(s) within the bacterium (see Note 1). Beyond studying the kinetics of secretion system assembly in live bacteria, this method can be used to obtain a detailed description of the assembly pathway by visualizing the labeled subunits in strains lacking other components of the secretion system.

In this chapter, we describe a generally applicable approach to decipher the assembly pathway of secretion systems using fluorescently labeled proteins. 2. Additional deletions of other components of the secretion system in the strain background mentioned above, to allow the investigation of the order of assembly.

Materials

3. Recommended: Untagged strain as a control for autofluorescence. 4. Recommended: Strain expressing the fluorescent protein in the cytosol from plasmid as a control (see Note 4).

Sample preparation

Methods

As the protocols for propagation of bacteria and induction of secretion systems greatly vary, we aim to provide a general protocol, which can be adapted to the specificities of the studied secretion system.

All buffers and solutions should be prepared using ultrapure water at room temperature.

3.1 Preparation of bacteria and setup of microscopy equipment 1. Streak bacteria from -80°C stock onto LB agar plates containing the required additives and antibiotics and incubate at the required temperature (e.g. 37°C), until single colonies are visible (usually 12-36 h).

2. Inoculate overnight cultures of bacteria with single colonies from agar plates and grow in shaking incubator at the required temperature and agitation.

3. On the next day, determine the optical density at 600 nm wavelength (OD 600 ) of the overnight culture and inoculate a main culture to OD 600 that is suitable for the expression of the analyzed secretion system (a culture volume of 5 mL is sufficient, and an OD 600 of 0.1 is a good starting point for many systems).

4. Incubate bacteria in a shaking incubator until they reach early stationary phase (usually one to two hours). 5. Induce secretion system according to standard conditions (e.g. temperature shift, addition of inducer).

6. In the meantime, prepare a 1.5% solution of low-fluorescence agarose in microscopy buffer (see Note 8). While less than 100 µL of agarose solution is required per strain, a higher volume (20-50 mL) is easier to prepare, and the agarose concentration is less influenced by evaporation. Add the agarose to the buffer, which is then carefully brought to boiling in a microwave oven. Be aware of the possibility of delays in boiling and use precaution. Check that the agarose is completely dissolved and allow to cool to approximately 55°C. After cooling, add any required additives (antibiotics, inducers) (see

Note 9)

7. Prepare a thin pad of 1.5% agarose on a microscopy slide (Figure 1) (see Note 10).

8. Harvest exponentially growing cells (OD 600 ~ 0.8-1) by centrifugation (2,400 × g, 4 min; these values depend on the bacterium). Resuspend in imaging buffer and recentrifuge once, using the same settings, before resuspending in imaging buffer to an OD 600 ~ 2 (see Note 11). 9. Remove cover slip from agarose patch prior to spotting the bacteria, and wait until no more liquid areas are visible on the surface of the agarose patch (usually 1-5 min). 3. Blurring of the image can be reduced by deconvolution, a mathematical post-imaging process which removes or reassigns the fraction of detected photons caused by out-offocus structures. This is especially useful when three-dimensional information from z stacks is available. Care should be taken not to mistake deconvolution artifacts for clustering, and negative controls are mandatory when applying deconvolution.

4. Detection and quantification of bacteria and fluorescent foci can be performed in ImageJ or using the Oufti software package (formerly known as MicrobeTracker, http://www.oufti.org/ (21)).

Determination of the assembly pathway

1. To build an assembly pathway from the aforementioned data, determine the presence and number of foci per cell in wild type and each mutant strains. Nucleating component(s) of the system are correctly localized in foci in strains lacking all other components of the secretion system. Subsequently assembled proteins correctly localize in foci in strains expressing all earlier assembled proteins. Finally, protein(s) that require the presence of all the other components of the system are recruited at the end of the assembly. Proteins which are recruited at the same time or interact together before being recruited to the system will show the same localization in mutant strains, and usually require each other to form foci.

Notes

Notes 1 to 4 correspond to the prerequisites for the applicability of this approach.

1. Most bacterial secretion systems have a sufficiently distinct localization within the bacterium that differs from the distribution of the free component. Should this not be the case, standard fluorescence microscopy cannot distinguish between the free and assembled state of the labeled component, and more sophisticated methods such as diffusion-based fluorescence correlation spectroscopy (FCS) or interaction-based Förster resonance energy transfer microscopy (FRET) have to be applied.

2. While most fluorescent proteins are relatively inert to interactions, their size of 25-30 kDa [START_REF] Shaner | A guide to choosing fluorescent proteins[END_REF] can lead to cleavage or degradation of the fusion protein, and impede assembly or functionality of the tagged protein. Smaller alternatives, such as tetracysteine tags [START_REF] Adams | New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications[END_REF][START_REF] Andresen | Short tetracysteine tags to beta-tubulin demonstrate the significance of small labels for live cell imaging[END_REF], require additional manipulation [START_REF] Enninga | Secretion of type III effectors into host cells in real time[END_REF] and may also disturb the function of the protein (own unpublished observations). It is therefore essential to test expression level and stability of the fusion protein (by immunoblot), as well as the functionality of secretion system in the corresponding strains (by a functional assay). While fusions that influence the function may be perfectly fine tools to decipher assembly, this has to be corroborated by independent experiments. To maximize the chances of obtaining a functional fusion protein, both termini of the protein as well as internal flexible loops should be considered. Flexible linkers between the fluorophore and the secretion system component (e.g. a stretch of 6 -15 amino acids with a high glycine content) have been shown to preserve functionality of the fusion protein.

Chromosomal fusion proteins are preferred to avoid mislocalization due to overproduction of the protein or wrong timing and order of the expression of subunits. Moreover, chromosomal fusions allow to analyze the secretion system under close-to-wild type conditions. However, especially for C-terminal fusions, care has to be taken not to disturb the expression of downstream genes in the same operon, and it has proven helpful to repeat the genetic region upstream of the following gene.

Concerning the choice of fluorescent protein, many variants of GFP have been produced

which vary in spectral properties, degrees of multimerization, folding rates and functionality in oxidizing environments, so it can be helpful to try different fusion proteins [START_REF] Shaner | A guide to choosing fluorescent proteins[END_REF]. Due to their fast folding, low multimerization tendency, and proper folding in the periplasm, sfGFP and mCherry are good starting points. mCherry has additionally been observed to retain functionality of the T3SS in cases where GFP fusions were non-functional (26 and unpublished results). Most bacteria also display considerably less autofluorescence in the red spectrum; however, mCherry is less photostable than GFP, which might make it less suitable for time course studies. 4. While these control strains are not absolutely required, especially in the presence of good controls for the protein of choice itself (i.e. deletion of a protein required for its localization), they are immensely valuable to set up and test the microscopy pipeline.

5. M9 and similar buffers have the advantage that due to their low autofluorescence, they can be directly used in microscopy. This ensures constant external conditions for the bacteria and may eliminate the need for the washing step described in step 8.

6. The choice of imaging buffer is crucial to obtain reproducible results, as it will influence the bacterial metabolism and possibly the state of the secretion system to be analyzed. While PBS is a popular and easy-to-obtain imaging buffer, some bacteria show visible alterations in cell morphology in PBS within less than an hour. Preliminary experiments can reveal whether cell morphology and the distribution of secretion systems are affected in different imaging buffers.

7. The microscope must have a sufficient resolution and, most importantly, a high sensitivity to visualize and resolve the assembled proteins. A 100× objective is required for most distributions of secretion systems, although the formation of a polar spot or the distribution of few membrane-bound foci in large bacteria can be detected with a 60× objective. Sensitivity of the microscope is crucial, especially for low-stoichiometry components. The labeled protein has to be present in multiple copies within the complex to be detectable. In our experience, sensitive widefield microscopes can detect about ten molecules within a diffraction-limited spot over low background. For single-molecule detection, more sensitive methods, such as total internal reflection microscopy (TIRF) [START_REF] Poulter | Advanced Fluorescence Microscopy Verveer PJ[END_REF] or photoactivated localization microscopy / stochastic optical reconstruction microscopy (PALM / STORM) [START_REF] Macdonald | Does super-resolution fluorescence microscopy obsolete previous microscopic approaches to protein co-localization?[END_REF] have to be applied.

8. The pad can also be done with agar (instead of agarose). This can be especially useful for longer time course experiments, where bacteria can be incubated for one hour at optimal growth temperature prior to microscopy acquisition to allow cell division on a plane surface. 9. The agarose solution should be prepared or re-dissolved freshly before the experiment. The solution will stay liquid in a 55°C water bath; small aliquots can be kept in a tabletop incubator shaker for 1.7 mL reaction tubes (vigorous shaking is required to prevent solidification of the agarose in this case).

10. The depth of the pad can vary; however, the surface should remain as smooth as possible.

The pad can be prepared using a microscopy slide and a cover slip, with spacers, using commercially available systems (e.g. GeneFrame) (Figure 1), alternatively using two microscope slides or prewarmed protein gel chambers for larger patches. The pad should be bubble free to facilitate the observation. Let the agarose solidify and dry at room temperature (>1 min, longer storage times are possible if the pad remains covered).

11. An OD of 2 leads to about 5% of the area being covered with bacteria (for E. coli; this obviously depends on the size of the bacterium), Increasing the OD will increase confluence leading to more cell-cell contact.

12. Depending on their surface and the properties of the agarose patch, bacteria may take some time to settle at this point. If a large part of the bacteria are still moving after some minutes, the volume of bacterial resuspension should be reduced and drying times increased. Don't dry the agarose pad at 4°C to avoid drifts during the observation. 13. To avoid saturation, strong photobleaching, or phototoxicity effects, all fluorescence images should be acquired with the minimal exposure time required to reach a sufficient signal/noise ratio. The optimal exposure time has to be determined for each protein; depending on the sensitivity of the microscope, exposure times of 20-100 ms for phase contrast or DIC, and 100 ms -2 s for fluorophores are good starting points. Narrow band microscopy filters can also reduce photobleaching.

14. For time-lapse experiments, many microscopy systems allow to define fields of view (x, y, z, focus offset) that are stored and then automatically accessed by a motorized stage. The fields of view should be sufficiently apart to avoid cross-photobleaching (run preliminary experiment with long exposures to determine the area of bleaching, if required). Ten fields of view at an OD of about 2 usually yield a sufficient number of bacteria for further analysis. [START_REF] Diepold | Deciphering the assembly of the Yersinia type III secretion injectisome[END_REF]. Z stacks allow a more complete coverage of the bacterium, ensuring images that comprise the region of interest (often the center of the bacterium). In addition, the three-dimensional data yields information about the spatial distribution of the secretion systems within the bacterium, and allows better deconvolution of the images. However, imaging z-stacks leads to stronger photobleaching, and is therefore generally avoided in kinetic experiments. For kinetic experiments, keeping the focus is of particular importance, and hardware-based focusing systems or sealed plates can be of advantage. 16. To avoid the loss of raw data, the original images files should be preserved. Keep the low and high boundary below the background value and above the highest measured intensity, respectively, to prevent misinterpratation of the data. Within an experiment, these values should be kept constant after background correction. and quickly covered with a cover slip (grey), which is then gently and evenly pressed onto the agarose to form an evenly distributed patch. This simple method is sufficient for quick imaging, but often leads to uneven or sloped agarose patches, which reduce image quality.

B,

To ensure a more level surface of the agarose patch, the covering cover slip can be supported by two flanking cover slips.

C, Double-sided tape or commercially available adhesives (e.g. "Gene Frame", ThermoFisher) (rippled pattern) can be used to permanently adhere the cover slip to the sample. This prevents evaporation during imaging, which is very useful for longer experiments. However, the decreasing oxygenation of the sample should be kept in mind in this case.

2. 1

 1 Strains 1. Strain(s) expressing a chromosomal fusion of a fluorescent protein to the target protein of interest, e.g. superfolder green fluorescent protein (sfGFP) fused either to the N-or Cterminus of the target protein (see Notes 2 and 3).

10 .

 10 Spot bacteria by either of the following two methods: a. Pipette 1-2 µL of resuspended bacteria into the center of the agarose or agar patch without damaging the patch itself, let dry for about one to two minutes (see Note 12), and carefully cover with a cover slip. b. Spot 1-2 µL of resuspended bacteria onto a cover slip and carefully cover with an agarose or agar pad (Figure2).

3. 2

 2 Microscopy1. Place a drop of immersion oil onto the center of the cover slip, and flip the slide, so that the cover slip faces the objective. Carefully insert the slide into the microscope and bring the lens into contact with the immersion oil. 2. In phase contrast or differential interference contrast (DIC) mode, slowly decrease the distance between the lens and the cover slip, until bacteria are visible (see Note 12 in case large numbers of detached or swimming bacteria are visible). 3. Adjust Koehler illumination of microscope for optimal phase contrast or DIC images (20). 4. To determine which phase contrast / DIC plane corresponds to the best fluorescence plane, run an automated z stack of phase contrast / DIC and fluorescence images (see Note 13). For bacteria with a diameter of about 1 µm, z stacks containing 10-20 planes with ∆z = 100 nm yield sufficient coverage. 5. Capture phase contrast and fluorescence micrographs: a. For kinetic studies: every 30 sec with a minimal exposure time to minimize bleaching and phototoxicity effects (see Note 14) b. For the determination of the assembly pathway: single micrograph or z stack image (see Note 15) in WT and mutant strains.

3. 3

 3 Image processing 1. Phase contrast and fluorescence images can be adjusted and merged using ImageJ or equivalent software (see Note 16). 2. Slight movements of the whole field during the time of the experiment can be corrected by registering individual frames using StackReg plug-in in ImageJ (http://fiji.sc/StackReg).

Figure 1 -

 1 Figure 1 -Methods for the preparation of agarose pads

Figure 2 -

 2 Figure 2 -Schematic representation of bacteria loaded onto cover slip and covered with agar pad.

see Note 7).

  Culture medium: standard culture medium and appropriate antibiotics for overnight incubation and growth of bacteria, e.g. Luria Broth (LB) or M9 minimal medium (see

	3. Note 5).
	4. Microscopy buffer: non-fluorescent minimal medium or imaging buffer, e.g. phosphate
	buffered saline (PBS) (see Note 6).
	2.3 Microscope slide preparation
	1. Low-melting agarose (or agar), commercially available.
	2. Microscopy slides and cover slips compatible with the used microscope. Standard sizes
	include 75*25*1 mm glass slides and 22*22 mm cover slips, with No.1 (0.13-0.16 mm)
	being the most commonly used thickness.
	3. Microwave oven to prepare agarose solution.
	2.4 Image acquisition
	1. Automated inverted epifluorescence microscope with 60× or 100× objective (2. Optical filters for visualizing fluorescence, e.g. ET-GFP filter set (Chroma 49002) for
	visualizing GFP fluorescence and ET-mCherry filter set (Chroma 49008) for visualizing
	mCherry fluorescence.
	3. Dichroic mirrors compatible with the used fluorophores and filter sets.
	4. Incubation chamber for microscope stage, where required.
	2.5 Software for image processing
	1. Proprietary software often pre-installed on microscopy controller, commercial software
	like Adobe Photoshop, or open-source solutions like ImageJ, a widely used and adaptable
	1. Incubator shaker open source image processing program (http://rsb.info.nih.gov/ij/) (19).
	2. Spectrophotometer


PhD Benoit Chassaing
email: bchassaing@gsu.edu

PhD Eric Cascales
email: cascales@imm.cnrs.fr

Commissioned review

Keywords: Microbiota, competition, type VI secretion system, bacteriocin, niche colonization

The intestinal microbiota plays an important role in health, particularly in promoting intestinal metabolic capacity and in maturing the immune system. The intestinal microbiota also mediates colonization resistance against pathogenic bacteria, hence protecting the host from infections. On the other hand, some bacterial pathogens deliver toxins that target phylogenetically related or distinct bacterial species in order to outcompete and establish within the microbiota. The most widely distributed weapons include bacteriocins, as well as contact-dependent growth inhibition and type VI secretion systems. In this review, we discuss important advances about the impact of such antibacterial systems on shaping the intestinal microbiota.

The intestinal microbiota: our best frenemy

The mammalian intestine is inhabited by a large and diverse community of microbes, referred to as the gut microbiota. The human gut microbiota, also referred to as a "microbial organ", weights 1-2 kg, and consists of approximately 100 trillion (10 14 ) bacteria representing 6-10 phyla, including two predominant phyla -Bacteroidetes and Firmicutes -, and about 500-1000 distinct species [START_REF] Lozupone | Diversity, stability and resilience of the human gut microbiota[END_REF]. This highly complex microbial community (see Glossary) is controlled by various factors, such as host genetics and environmental factors. Moreover, microbiota diversity and composition is influenced by host diet as well as by positive and antagonistic interactions between bacteria within the microbiota.

The intestinal microbiota has an overall beneficial impact on its host, by providing metabolic activities within the intestine and favoring the development of the intestinal immune system [START_REF] Wostmann | Dietary intake, energy metabolism, and excretory losses of adult male germfree Wistar rats[END_REF] (Figure 1). Exemplifying this notion is the observation that the immune responses in mice housed in germ-free conditions are abnormal compared to conventionallycolonized mice [START_REF] Mazmanian | An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system[END_REF][START_REF] Cahenzli | Intestinal microbial diversity during early-life colonization shapes long-term IgE levels[END_REF]. Therefore, early exposure to microbes in the intestine is a critical factor to modulate intestinal immune responses [START_REF] Ohnmacht | Mucosal immunology. The microbiota regulates type 2 immunity through RORgammat(+) T cells[END_REF], and a well-documented example of a single microbial member playing a central role in shaping the intestinal immune system is segmented filamentous bacteria (SFB), which can promote the robust differentiation of Th17 cells [START_REF] Ivanov | Induction of intestinal Th17 cells by segmented filamentous bacteria[END_REF][START_REF] Goto | Segmented filamentous bacteria antigens presented by intestinal dendritic cells drive mucosal Th17 cell differentiation[END_REF][START_REF] Geem | Specific microbiota-induced intestinal Th17 differentiation requires MHC class II but not GALT and mesenteric lymph nodes[END_REF]. Moreover, if not well managed, the gut microbiota can become deleterious, for example by inducing uncontrolled intestinal inflammation. In light of the benefits the microbiota confers and on its potential to harm its host, the gut microbiota has previously been referred as the host's best frenemy [START_REF] Chassaing | Gut microbiota, low-grade inflammation, and metabolic syndrome[END_REF].

Collectively, the microbiota and its derived metabolites are critical components for the maturation of host intestinal immunity, and research has accumulated on the central role played by the intestinal microbiota in the protection of the host intestine against pathogens, a phenomenon called colonization resistance [START_REF] Buffie | Microbiota-mediated colonization resistance against intestinal pathogens[END_REF][START_REF] Kim | The intestinal microbiota: antibiotics, colonization resistance, and enteric pathogens[END_REF][START_REF] Pickard | Gut microbiota: role in pathogen colonization, immune responses, and inflammatory disease[END_REF]. Bacterial competition occurs either by depleting nutrients from the milieu (exploitation competition) or by deploying antibacterial ! 4! weapons to specifically eliminate target cells (interference competition) (Figure 1). Many bacteria can directly prevent intestinal pathogens colonization or overgrowth by consuming common limited resources, hence inducing starvation of competing pathogens. One example highlighting this mechanism of exploitation competition is the finding that the commensal Bacteroides thetaiotaomicron consumes carbohydrates used by the pathogen Citrobacter rodentium, thus leading to a competitive exclusion of the pathogen from the intestine [START_REF] Kamada | Regulated virulence controls the ability of a pathogen to compete with the gut microbiota[END_REF][START_REF] Kamada | Control of pathogens and pathobionts by the gut microbiota[END_REF] (Figure 1). Through the production of specific metabolites, the intestinal microbiota can also modify the host environmental conditions, then compromising pathogen growth and/or virulence. Butyrate, a short-chain fatty acid (SCFA) produced by the intestinal microbiota, downregulates the expression of several virulence genes of Salmonella enterica serovar Enteritidis (S. Enteritidis) and Typhimurium (S. Typhimurium) [START_REF] Gantois | Butyrate specifically down-regulates Salmonella pathogenicity island 1 gene expression[END_REF] and inhibits the growth of enterohemorrhagic Escherichia coli (EHEC) [START_REF] Shin | Influence of intestinal anaerobes and organic acids on the growth of enterohaemorrhagic Escherichia coli O157:H7[END_REF] (Figure 1). Finally, members of the microbiota can affect the growth of other cells by producing and releasing inhibitory substances, such as antibiotics and peptide antibiotics, or by injecting antibacterial effectors into target cells. Here we will briefly describe the various mechanisms evolved by bacteria to destroy rivals and we will review recent studies on how these systems contribute to reshaping bacterial communities in vivo.

Bacterial weapons in the intestine

To combat competitors, bacteria deploy a broad arsenal of antibacterial weapons. These weapons vary in terms of mechanism of action, toxin targets, and mode of penetration, and hence limit the acquisition of mechanism of resistance by competitors. The most widely distributed weapons in Enterobacteria are microcins and bacteriocins [START_REF] Kleanthous | Swimming against the tide: progress and challenges in our understanding of colicin translocation[END_REF], contact-dependent growth inhibition (CDI) [START_REF] Ruhe | Bacterial contact-dependent growth inhibition[END_REF] and Type VI secretion systems (T6SS) [START_REF] Zoued | Architecture and assembly of the type VI secretion system[END_REF] (Figure 2). Other mechanisms, such as Type I, Type IV and Type VII secretion systems or outer membrane ! 5! exchange, have also been recently shown to mediate toxin delivery into competitors or to promote contact-dependent killing [START_REF] Souza | Bacterial killing via a type IV secretion system[END_REF][START_REF] García-Bayona | Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins[END_REF][START_REF] Cao | The type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria[END_REF][START_REF] Whitney | A broadly distributed toxin family mediates contact-dependent antagonism between gram-positive bacteria[END_REF][START_REF] Vassallo | Cell rejuvenation and social behaviors promoted by LPS exchange in myxobacteria[END_REF][START_REF] Vassallo | Infectious polymorphic toxins delivered by outer membrane exchange discriminate kin in myxobacteria[END_REF].

Bacteriocins are a group containing a large variety of proteinaceous antibiotics of various lengths: microcins are 15-60 amino-acid peptides, lantibiotics are peptide containing a modified amino-acid (lanthionine), whereas colicin-like proteins are > 40 kDa multidomain proteins and tailocins are high molecular weight multiprotein complexes resembling bacteriophage tails [START_REF] Cascales | Colicin biology[END_REF][START_REF] Rebuffat | Microcins in action: amazing defence strategies of Enterobacteria[END_REF][START_REF] Van Kraaij | Lantibiotics: biosynthesis, mode of action and applications[END_REF][START_REF] Scholl | Phage tail-like bacteriocins[END_REF]. Bacteriocins are produced upon stress conditions, and share a similar mechanism of action. After release, they bind to an outer membrane receptor and parasitize cell envelope components for penetration [START_REF] Cascales | Colicin biology[END_REF][START_REF] Rebuffat | Microcins in action: amazing defence strategies of Enterobacteria[END_REF][START_REF] Kim | Colicin import into E. coli cells: a model system for insights into the import mechanisms of bacteriocins[END_REF] (Figure 2). The exploitation of specific reception and translocation machineries restrict the action of these toxins to the same or phylogenetically related species. Cell toxicity is conferred by pore-forming activity that collapses the membrane potential at the inner membrane, by digestion or cleavage of nucleic acids or of cell wall precursors [START_REF] Cascales | Colicin biology[END_REF][START_REF] Kim | Colicin import into E. coli cells: a model system for insights into the import mechanisms of bacteriocins[END_REF]. Bacteriocins include S-type pyocins liberated by Pseudomonas species, and colicins that are produced by enteric strains such as E. coli, C. rodentium and Enterobacter species [START_REF] Cascales | Colicin biology[END_REF][START_REF] Kim | Colicin import into E. coli cells: a model system for insights into the import mechanisms of bacteriocins[END_REF].

By contrast to bacteriocins that are diffusible toxins, CDI and T6SSs are cell-cell contact-dependent mechanisms. CDI is a variant of two-partner secretion, a family of Type V secretion systems widely represented in Proteobacteria [START_REF] Hayes | Mechanisms and biological roles of contact-dependent growth inhibition systems[END_REF][START_REF] Danka | Are CDI systems multicolored, facultative, helping greenbeards?[END_REF][START_REF] Guerin | Two-partner secretion: combining efficiency and simplicity in the secretion of large proteins for bacteria-host and bacteria-bacteria interactions[END_REF]. It comprises two proteins: the CdiB outer membrane transporter translocates the CdiA protein to the cell surface. CdiA are usually elongated spring-like β-helical structures that carry a C-terminal toxin domain. The Cterminal domain mediates target cell recognition, penetration and toxicity (Figure 2). Similar to bacteriocins, the requirements for specific receptors and target cell factors restrict the action of CDI to close relatives [START_REF] Ruhe | Bacterial contact-dependent growth inhibition[END_REF][START_REF] Hayes | Mechanisms and biological roles of contact-dependent growth inhibition systems[END_REF][START_REF] Ruhe | Receptor polymorphism restricts contact-dependent growth inhibition to members of the same species[END_REF][START_REF] Willett | Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways[END_REF].

T6SS is a more complex machinery, broadly distributed in Proteobacteria and Bacteroidetes. Its assembly requires at least 13 components to form a speargun-like weapon [START_REF] Zoued | Architecture and assembly of the type VI secretion system[END_REF][START_REF] Kudryashev | Structure of the type VI secretion system contractile sheath[END_REF][START_REF] Basler | Type VI secretion system: secretion by a contractile nanomachine[END_REF]. T6SS could be considered as a bacteriophage-like contractile tail structure ! 6! anchored to the cell envelope [START_REF] Zoued | Architecture and assembly of the type VI secretion system[END_REF][START_REF] Kapitein | Deadly syringes: type VI secretion system activities in pathogenicity and interbacterial competition[END_REF][START_REF] Cianfanelli | Aim, load, fire: the type VI secretion system, a bacterial nanoweapon[END_REF]. The tail is constituted of an effector-loaded syringe wrapped by the sheath that is built under a metastable, extended conformation [START_REF] Zoued | Architecture and assembly of the type VI secretion system[END_REF][START_REF] Kudryashev | Structure of the type VI secretion system contractile sheath[END_REF][START_REF] Basler | Type VI secretion system: secretion by a contractile nanomachine[END_REF][START_REF] Basler | Type VI secretion requires a dynamic contractile phage tail-like structure[END_REF][START_REF] Ho | A view to a kill: the bacterial type VI secretion system[END_REF][START_REF] Brackmann | Using force to punch holes: mechanics of contractile nanomachines[END_REF]. Contraction of the sheath presumably propels the syringe towards the rival cell, and thus delivers effectors to cause cell damages [START_REF] Basler | Type VI secretion requires a dynamic contractile phage tail-like structure[END_REF][START_REF] Brackmann | Using force to punch holes: mechanics of contractile nanomachines[END_REF][START_REF] Basler | Type 6 secretion dynamics within and between bacterial cells[END_REF][START_REF] Basler | Tit-for-tat: type VI secretion system counterattack during bacterial cell-cell interactions[END_REF][START_REF] Brunet | Imaging type VI secretion-mediated bacterial killing[END_REF][START_REF] Leroux | Quantitative single-cell characterization of bacterial interactions reveals type VI secretion is a double-edged sword[END_REF] (Figure 2). How the target cell is sensed and how cell-cell contact triggers T6SS assembly or firing is largely unknown but in certain cases, transcriptional, post-transcriptional or post-translational regulatory cascades, cellenvelope damage or the presence of kin cell components in the milieu trigger a T6SS response to attacks [START_REF] Basler | Tit-for-tat: type VI secretion system counterattack during bacterial cell-cell interactions[END_REF][START_REF] Leroux | Quantitative single-cell characterization of bacterial interactions reveals type VI secretion is a double-edged sword[END_REF][START_REF] Mougous | Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa[END_REF][START_REF] Silverman | Structure and regulation of the type VI secretion system[END_REF][START_REF] Silverman | Separate inputs modulate phosphorylation-dependent andindependent type VI secretion activation[END_REF][START_REF] Lazzaro | A transcriptional regulatory mechanism finely tunes the firing of Type VI secretion system in response to bacterial enemies[END_REF][START_REF] Leroux | Kin cell lysis is a danger signal that activates antibacterial pathways of Pseudomonas aeruginosa[END_REF][START_REF] Leroux | Bacterial danger sensing[END_REF]. By contrast to bacteriocins and CDI, T6SSs do not exploit specific receptors, and hence target a broader range of bacteria, although no T6SS-dependent damages of Gram-positive bacteria have been observed [START_REF] Russell | Type VI secretion delivers bacteriolytic effectors to target cells[END_REF]. T6SS antibacterial effectors include peptidoglycan-acting enzymes (amidases, glycosyl hydrolases), membrane-targeting proteins (phospholipases and pore-forming), nucleases and NAD(P)(+) glycohydrolases [START_REF] Russell | Type VI secretion delivers bacteriolytic effectors to target cells[END_REF][START_REF] Durand | VgrG, Tae, Tle, and beyond: the versatile arsenal of type VI secretion effectors[END_REF][START_REF] Alcoforado Diniz | Molecular weaponry: diverse effectors delivered by the Type VI secretion system[END_REF][START_REF] Russell | A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism[END_REF][START_REF] Russell | A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach[END_REF][START_REF] Russell | Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors[END_REF][START_REF] Miyata | Vibrio cholerae requires the type VI secretion system virulence factor VasX to kill Dictyostelium discoideum[END_REF]. Interestingly, the T6SS is also capable of delivering effectors into eukaryotic cells, including single-celled microorganisms and animal tissues [START_REF] Durand | VgrG, Tae, Tle, and beyond: the versatile arsenal of type VI secretion effectors[END_REF][START_REF] Pukatzki | Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin[END_REF]. Phospholipases and nucleases can act as trans-kingdom effectors, but specialized toxins, such as those interfering with cytoskeleton or tubule dynamics, have been identified and characterized [START_REF] Pukatzki | Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin[END_REF][START_REF] Durand | Crystal structure of the VgrG1 actin cross-linking domain of the Vibrio cholerae type VI secretion system[END_REF][START_REF] Suarez | A type VI secretion system effector protein, VgrG1, from Aeromonas hydrophila that induces host cell toxicity by ADP ribosylation of actin[END_REF][START_REF] Jiang | A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells[END_REF][START_REF] Jiang | The Pseudomonas aeruginosa type VI secretion PGAP1-like effector induces host autophagy by activating endoplasmic reticulum stress[END_REF][START_REF] Sana | Internalization of Pseudomonas aeruginosa strain PAO1 into epithelial cells is promoted by interaction of a T6SS effector with the microtubule network[END_REF][START_REF] Aubert | A Burkholderia type VI effector deamidates Rho GTPases to activate the pyrin inflammasome and trigger inflammation[END_REF][START_REF] Bleves | Game of trans-kingdom effectors[END_REF].

For all these systems, attacker bacteria are protected from self-intoxication and intoxication by kin cells by the production of immunity proteins that usually bind with high affinity and inhibit the catalytic activity of the cognate effector [START_REF] Cascales | Colicin biology[END_REF][START_REF] Hayes | Mechanisms and biological roles of contact-dependent growth inhibition systems[END_REF][START_REF] Ruhe | Receptor polymorphism restricts contact-dependent growth inhibition to members of the same species[END_REF][START_REF] Durand | VgrG, Tae, Tle, and beyond: the versatile arsenal of type VI secretion effectors[END_REF][START_REF] Alcoforado Diniz | Molecular weaponry: diverse effectors delivered by the Type VI secretion system[END_REF][START_REF] Alcoforado Diniz | Intraspecies competition in Serratia marcescens is mediated by type VI-secreted Rhs effectors and a conserved effector-associated accessory protein[END_REF][START_REF] Russell | Type VI secretion system effectors: poisons with a purpose[END_REF].

In addition, some enteric pathogens can produce broadly bioactive small molecules such as antibiotics, non-ribosomal peptide antibiotics, sactibiotics and lantibiotics [START_REF] Marahiel | Working outside the protein-synthesis rules: insights into nonribosomal peptide synthesis[END_REF][START_REF] Marahiel | Nonribosomal peptide synthetases mechanistic and structural aspects of essential domains[END_REF][START_REF] Mathur | The sactibiotic subclass of bacteriocins: an update[END_REF], that may act toward other members of the intestinal microbiota and/or toward enteric pathogens. Finally, other extracellular contractile injection systems such as R-pyocins, antifeeding prophages (Afp) and Afp-like particles have antibacterial or antimicrobial activities [START_REF] Michel-Briand | The pyocins of Pseudomonas aeruginosa[END_REF][START_REF] Sarris | A phage tail-derived element with wide distribution among both prokaryotic domains: a comparative genomic and phylogenetic study[END_REF][START_REF] Bock | In situ architecture, function, and evolution of a contractile injection system[END_REF].

! 7!

Although the role of these systems in antibacterial competition is well documented in vitro, studies have only begun to investigate their contribution in in vivo animal models.

Role of bacterial competition in the intestinal microbiota

The gut microbiota is a very stable ecosystem [START_REF] Lozupone | Diversity, stability and resilience of the human gut microbiota[END_REF]. However, the invasion or overgrowth of pathogens induces dysbiosis, an instability that may alter the composition of the microbiota but also host physiology [START_REF] Kim | The intestinal microbiota: antibiotics, colonization resistance, and enteric pathogens[END_REF][START_REF] Wiles | Host gut motility promotes competitive exclusion within a model intestinal microbiota[END_REF][START_REF] De Muinck | Context-dependent competition in a model gut bacterial community[END_REF][START_REF] Weiss | Mechanisms and consequences of intestinal dysbiosis[END_REF]. It is now well appreciated that interference competition between members of the microbiota, in addition to indirect competition such as exploitation competition, plays a central role in microbiota ecology [START_REF] Weiss | Mechanisms and consequences of intestinal dysbiosis[END_REF].

Until recently, the role of interbacterial competition in shaping bacterial communities has been underestimated [START_REF] Sana | T6SS: The bacterial "fight club" in the host gut[END_REF]. However, antibacterial weapons are key players in the control of bacterial populations, and a summary of known examples of in vivo bacterial competition is presented in Table 1. The importance of these weapons in vivo is supported by the estimation that more than 10 9 T6SS firing events occur per minute per gram of colonic contents [START_REF] Wexler | Human symbionts inject and neutralize antibacterial toxins to persist in the gut[END_REF]. T6SS gene clusters are highly represented in Bacteroidales strains, which account for a large portion of the gut microbiota [START_REF] Coyne | Type VI secretion systems of human gut Bacteroidales segregate into three genetic architectures, two of which are contained on mobile genetic elements[END_REF][START_REF] Coyne | Evidence of extensive DNA transfer between bacteroidales species within the human gut[END_REF]. In addition, many Gram-negative enteric pathogens, including Vibrio cholerae, S. Typhimurium, C. rodentium, and Shigella sonnei utilize functional T6SSs to fire against other species in vitro [START_REF] Macintyre | The Vibrio cholerae type VI secretion system displays antimicrobial properties[END_REF][START_REF] Gueguen | Promoter swapping unveils the role of the Citrobacter rodentium CTS1 type VI secretion system in interbacterial competition[END_REF]. In agreement with the concept that T6SSs are important players in bacterial competition within the intestine, a number of enterobacterial T6SS gene clusters are upregulated in conditions encountered in the gut or when a threshold of cell density is reached [START_REF] Bernard | Nooks and crannies in type VI secretion regulation[END_REF]. The V. cholerae T6SS is activated by mucins and microbiota-modified bile salt [START_REF] Bachmann | Bile salts modulate the mucin-activated type VI secretion system of pandemic Vibrio cholerae[END_REF]; the S. Typhimurium T6SS is activated by bile salts [START_REF] Sana | Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut[END_REF]; and the enteroaggregative E. coli Sci-1 T6SS is responsive to iron starvation [START_REF] Brunet | An epigenetic switch involving overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster[END_REF].

V. cholerae, S. Typhimurium and S. sonnei deploy their T6SSs to kill or displace commensal bacteria, allowing a successful colonization of the host and an increased ! 8! persistence in experimental models [START_REF] Cheng | Vibrio cholerae response regulator VxrB controls colonization and regulates the type VI secretion system[END_REF][START_REF] Anderson | Shigella sonnei encodes a functional T6SS used for interbacterial competition and niche occupancy[END_REF][START_REF] Fast | Commensal-pathogen competition impacts host viability[END_REF][START_REF] Logan | The Vibrio cholerae type VI secretions system can modulate host intestinal mechanics to displace commensal gut bacteria[END_REF]. Using transposon insertion site sequencing (Tn-Seq) to identify V. cholerae mutants that exhibit a colonization defect in the rabbit intestine, Fu et al. found tsiV3, encoding immunity to the T6SS VgrG3 peptidoglycan hydrolase effector [START_REF] Fu | Tn-Seq analysis of Vibrio cholerae intestinal colonization reveals a role for T6SS-mediated antibacterial activity in the host[END_REF]. Interestingly, a recent study demonstrated that V. cholerae T6SS-mediated colonization specifically occurs in intestinal microenvironments, such as the middle small intestine, suggesting that T6SS effectors might target specific species [START_REF] Fu | Tracking Vibrio cholerae cell-cell interactions during infection reveals bacterial population dynamics within intestinal microenvironments[END_REF]. The antagonistic behavior of V. cholerae in the gut triggers intestinal colonization, virulence gene expression, and host innate immune response [START_REF] Zhao | Antagonism toward the intestinal microbiota and its effect on Vibrio cholerae virulence[END_REF]. In a recent study, Sana and collaborators demonstrated that the successful establishment of S. Typhimurium in the mouse intestine requires the T6SS Tae4 amidase effector [START_REF] Sana | Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut[END_REF]. Thus, the observation that bacterial-specific effectors are required for efficient colonization demonstrates that the T6SS mediates antagonistic interbacterial interactions during infection. It is not yet known whether T6SS specifically targets certain species, but the observation that S. Typhimurium targets Klebsiella oxytoca and has only weak impact on other species suggests that the T6SS does not fire randomly [START_REF] Sana | Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut[END_REF].

Interestingly, S. Typhimurium and K. oxytoca utilize the same carbon sources and thus the specific elimination of a metabolic competitor may provide a better access to the available nutritional resources [START_REF] Sana | T6SS: The bacterial "fight club" in the host gut[END_REF]. Another example of T6SS-mediated metabolic competition is the secretion of manganese-and zinc-scavenging enzymes by pathogens such as Burkholderia thailandensis [START_REF] Si | Manganese scavenging and oxidative stress response mediated by type VI secretion system in Burkholderia thailandensis[END_REF][START_REF] Si | The type VI secretion system engages a redox-regulated dualfunctional heme transporter for zinc acquisition[END_REF]. In addition, EHEC uses its T6SS to secrete catalases, thus providing a higher resistance to reactive oxygen species produced by the host [START_REF] Wan | Type VI secretion system contributes to Enterohemorrhagic Escherichia coli virulence by secreting catalase against host reactive oxygen species (ROS)[END_REF]. A recent example of the importance of the T6SS in intestinal colonization is related to the increased prevalence of S. sonnei infections over that of the close relative Shigella flexneri. By contrast to S. flexneri, the genome of S. sonnei encodes a functional T6SS that confers a competitive advantage by outcompeting S. flexneri in vitro as well as in the mouse gut [START_REF] Anderson | Shigella sonnei encodes a functional T6SS used for interbacterial competition and niche occupancy[END_REF]. Interestingly, S. sonnei also encodes the ColE1 colicin that enables E. coli elimination [START_REF] Anderson | Shigella sonnei encodes a functional T6SS used for interbacterial competition and niche occupancy[END_REF]. Those recent findings are in agreement with the observation that colicinogenic E. coli cells present an increased intestinal ! 9! persistence compared to the isogenic E. coli strain unable to produce colicins [START_REF] Gillor | Persistence of colicinogenic Escherichia coli in the mouse gastrointestinal tract[END_REF]100]. Other bacteriocins and R-pyocins have the ability to destroy rivals in biofilm or mixed communities, and to specifically eliminate bacterial species after therapeutic administration; and hence are Other commensals, such as those of the Bacteroidales order, antagonize gut microbiota using secreted antimicrobial proteins or T6SS [START_REF] Russell | A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism[END_REF][110][111][112][113]]. Interestingly, it has been shown that intense transfer of genetic material occurs between Bacteroidales species in the gut [START_REF] Coyne | Evidence of extensive DNA transfer between bacteroidales species within the human gut[END_REF].

As a consequence, Bacteroidales have accumulated genes encoding immunity proteins to T6SS effectors they do not encode [START_REF] Wexler | Human symbionts inject and neutralize antibacterial toxins to persist in the gut[END_REF], and thus maintain a stable balance in the microbiota by preventing their own elimination. In addition, the symbiotic Bacteroides fragilis strain was shown to use the T6SS to harm enterotoxigenic B. fragilis cells, demonstrating that the activity of Bacteroidales T6SSs may protect the host against intestinal inflammatory diseases [114]. Altogether, these data demonstrate that a broad range of competitive mechanisms occurs within the intestinal microbiota and plays a role in microbiota composition, establishment, stability, and evolution.

! 10!

Concluding Remarks

Future studies will be needed to further characterize mechanisms by which bacteria compete within the intestinal tract, with a particular focus on long-lasting consequences on microbiota composition and host physiology (see Outstanding Questions). Long-term experiments are also required to understand the real contribution of antibacterial weapons in shaping microbial communities. At present, it is not clear whether being well armed represents a true advantage, as antibacterial weapons have limited impact on well-structured communities [115]. In addition, microbial communities are subjected to a rock-paper-scissor game, in which the production of these weapons is energetically consuming, and hence attacker cells might be defeated by professional cheaters or by strains with a better fitness [116,117].

Another important field of research that needs further investigation is the impact that such antimicrobial systems may have on both microbiota composition and host physiology in the long term. An example highlighting the importance of interbacterial competition in long term dysbiosis is the observation that intestinal colonization of AIEC, an E. coli pathovar associated with Crohn's disease, causes alteration of microbiota composition and chronic colitis in mice, with both phenotypes persisting well beyond AIEC clearance [118]. Hence, in addition to their own virulence potential, AIEC bacteria are able to induce chronic inflammation by detrimentally altering the intestinal microbiota composition [118]. While the precise role played by antibacterial systems in such long-term alterations still needs to be investigated, they may further highlight the unappreciated importance of such bacteria/bacterial competition in microbiota stability and host physiology.

Finally, such antibacterial mechanisms may be tailored in a near future as an alternative or complementary approach to the use of antibiotics. A few bacteriocins, such as nisin, have been validated by the FDA and are used as food preservatives [119]. While such compounds are used to extend shelf life, we still ignore their impact on the intestinal ! 11! microbiota. In addition, colicins, R-pyocins, CdiA and T6SS effectors are modular proteins and hence might be genetically engineered to specifically target bacterial populations of interest. Modified colicins, R-pyocins or T6SS effectors have already been demonstrated to be efficiently delivered into target cells or to destroy specific species without affecting the gut microbiota diversity [START_REF] Sana | Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut[END_REF][START_REF] Zhao | Antagonism toward the intestinal microbiota and its effect on Vibrio cholerae virulence[END_REF][120][121][122][123][124]. One may predict that these initial attempts will be actively pursued to deliver toxins or CRISPR/Cas system into specific species, with the ultimate goal to prevent intestinal colonization or to beneficially reshape an altered microbial ecosystem.

Glossary

Dysbiosis: microbial population with an imbalanced composition, often associated with deleterious impact for the host. Dysbiosis has been identified as an important player of inflammation in inflammatory bowel diseases.

Intestinal immune system:

The complex population of cells and interactions inhabiting our intestine with the dual role of tolerance toward our commensal microbiota and protection against intestinal pathogen. Importantly, the intestine represents the largest compartment of the immune system, with the estimation that 70% of the mammalian immune system is hosted within the intestine. Microbial community: mixed population of bacteria and single-cell microorganisms.

Microbial communities include microbiota, but also biofilms (bacteria that adhere to a support) and consortiums (bacteria that exchange metabolic contents). Outstanding Questions Box -How do antibiotics and antibacterial molecules alter the intestinal microbiota composition and affect colonization resistance?

-What are the direct impacts of antibacterial weapons in the gut?

-What is the contribution of the antibacterial weapons of commensals in the protection of the host against pathogens?

-What is the target range of T6SS and what is the cost to produce antibacterial weapons?

-Is it a real benefit for the bacterium to be equipped with antibacterial weapons?

-How can antibacterial weapons be genetically modified for therapeutic purposes in order to manipulate the intestinal microbiota in beneficial ways?

-What are the long term impacts of such antibacterial systems on the microbiota community and host physiology?!

Trends Box

-The intestinal microbiota is a complex but stable ecosystem that plays a central role in human health, and disturbance of its composition and function is associated with many diseases.

-Within the intestinal microbiota, bacteria exchange material and information.

-The microbiota can be peaceful, but many bacteria fight with others to have a better access to their niche or nutrients.

-Different antibacterial weapons have been identified and characterized, and many bacterial pathogens use these weapons to establish themselves in the intestinal environment, whereas some commensals use these weapons to specifically target pathogens, leading to protection of the host.

!

  proposed to be viable alternatives to antibiotics [101-105]. While the above examples showed that antibacterial weapons are used by pathogens to colonize their hosts, the gut microbiota also exerts an important control to prevent colonization by pathogens [106, 107]. Indeed, many commensal strains produce those weapons and therefore protect the niche against the invasion of external microbes or against the overgrowth of indigenous pathogens. An elegant example is the recent observation that the probiotic strain E. coli Nissle uses microcins M and H47 to limit the expansion of competing Enterobacteriaceae, including pathogens such as adherent-invasive E. coli (AIEC) and S. Typhimurium during intestinal inflammation [108]. Another example is Bacillus thuringiensis, a bacterium able to secrete a bacteriocin (thuricin CD) that directly targets spore-forming Bacilli and Clostridia, including Clostridium difficile [109].

Figure 1 .

 1 Figure 1. Commensal microbiota-mediated colonization resistance in the intestine. The

Figure 2 .

 2 Figure 2. The arsenal of antibacterial weapons. The major antibacterial weapons used by

Table 1 . Summary of known in vivo bacterial competition.

 1

	Bacterium	Weapon	Target cell	Toxin / Activity	Reference
	Salmonella
	enterica	T6SS	Klebsiella oxytoca	Tae4 (amidase)	[87]
	Typhimurium
	Bacillus thuringiensis	Bacteriocin thuricin CD	Spore-forming Bacilli and Clostridia, including C. difficile	pore-forming	[109]
	Burkholderia thailandensis	T6SS	unknown	unknown	[96, 97]
	EHEC	T6SS	-	Catalases	[98]
	Shigella sonnei	T6SS	S. flexneri	Unknown	[90]
	Shigella sonnei	ColE1 colicin	E. coli	Pore-forming	[90]
	E. coli Nissle	Microcins M and H47	Enterobacteriaceae, including Typhimuirum pathogens such as AIEC and S.	unknown	[108]
	Bacteroides fragilis	T6SS	B. fragilis, gut microbiota and pathogenic bacteria	Bte2	[56, 110, 114]
	V. cholerae	T6SS	commensal E. coli	unknown	[95]

Acknowledgements

We thank the members of the Chassaing and Cascales laboratories for insightful discussions, and the anonymous reviewers for helpful comments. This review is dedicated to Arlette Darfeuille-Michaud, in loving memory. B.C. is a recipient of the Career Development Award from the Crohn's and Colitis Foundation and an Innovator Award from the Rainin Foundation. Work in E.C. laboratory is supported by the Aix-Marseille Université, the Centre National de la Recherche Scientifique and by grants from the Agence Nationale de la Recherche (ANR-14-CE14-0006-02 and ANR-15-CE11-0019-01). The authors declare no conflict of interest.