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- Bacterial Call to Arms for Warfare at the Infection SitePublication . Cabral, Vitor; Xavier, Karina BivarBacterial sensing is important for perceiving environmental cues and activating responses. In this issue of Cell Host & Microbe, Hertzog et al. (2018) show that group A Streptococcus can couple the ability to respond to host cues with autoinduction of a quorum sensing system, leading to killing of bacterial competitors.
- Diet leaves a genetic signature in a keystone member of the gut microbiotaPublication . Dapa, Tanja; Ramiro, Ricardo Serotte; Pedro, Miguel Filipe; Gordo, Isabel; Xavier, Karina BivarSwitching from a low-fat and high-fiber diet to a Western-style high-fat and high-sugar diet causes microbiota imbalances that underlay many pathological conditions (i.e., dysbiosis). Although the effects of dietary changes on microbiota composition and functions are well documented, their impact in gut bacterial evolution remains unexplored. We followed the emergence of mutations in Bacteroides thetaiotaomicron, a prevalent fiber-degrading microbiota member, upon colonization of the murine gut under different dietary regimens. B. thetaiotaomicron evolved rapidly in the gut and Western-style diet selected for mutations that promote degradation of mucin-derived glycans. Periodic dietary changes caused fluctuations in the frequency of such mutations and were associated with metabolic shifts, resulting in the maintenance of higher intraspecies genetic diversity compared to constant dietary regimens. These results show that dietary changes leave a genetic signature in microbiome members and suggest that B. thetaiotaomicron genetic diversity could be a biomarker for dietary differences among individuals.
- Escherichia coli adaptation to the gut environment: a constant fight for survivalPublication . Gordo, Isabel; Demengeot, Jocelyne; Xavier, Karina
- Identification of Functional LsrB-Like Autoinducer-2 ReceptorsPublication . Pereira, C. S.; de Regt, A. K.; Brito, P. H.; Miller, S. T.; Xavier, K. B.Although a variety of bacterial species have been reported to use the interspecies communication signal autoinducer-2 (AI-2) to regulate multiple behaviors, the molecular mechanisms of AI-2 recognition and signal transduction remain poorly understood. To date, two types of AI-2 receptors have been identified: LuxP, present in Vibrio spp., and LsrB, first identified in Salmonella enterica serovar Typhimurium. In S. Typhimurium, LsrB is the ligand binding protein of a transport system that enables the internalization of AI-2. Here, using both sequence analysis and structure prediction, we establish a set of criteria for identifying functional AI-2 receptors. We test our predictions experimentally, assaying key species for their abilities to import AI-2 in vivo, and test their LsrB orthologs for AI-2 binding in vitro. Using these experimental approaches, we were able to identify AI-2 receptors in organisms belonging to phylogenetically distinct families such as the Enterobacteriaceae, Rhizobiaceae, and Bacillaceae. Phylogenetic analysis of LsrB orthologs indicates that this pattern could result from one single origin of the functional LsrB gene in a gammaproteobacterium, suggesting possible posterior independent events of lateral gene transfer to the Alphaproteobacteria and Firmicutes. Finally, we used mutagenesis to show that two AI-2-interacting residues are essential for the AI-2 binding ability. These two residues are conserved in the binding sites of all the functional AI-2 binding proteins but not in the non-AI-2-binding orthologs. Together, these results strongly support our ability to identify functional LsrB-type AI-2 receptors, an important step in investigations of this interspecies signal.
- Identification of novel autoinducer-2 receptors in Clostridia reveals plasticity in the binding site of the LsrB receptor familyPublication . Torcato, Inês M.; Kasal, Meghann R.; Brito, Patrícia H.; Miller, Stephen T.; Xavier, Karina B.Autoinducer-2 (AI-2) is unique among quorum-sensing signaling molecules, as it is produced and recognized by a wide variety of bacteria and thus facilitates interspecies communication. To date, two classes of AI-2 receptors have been identified: the LuxP-type, present in the Vibrionales, and the LsrB-type, found in a number of phylogenetically distinct bacterial families. Recently, AI-2 was shown to affect the colonization levels of a variety of bacteria in the microbiome of the mouse gut, including members of the genus Clostridium, but no AI-2 receptor had been identified in this genus. Here, we identify a noncanonical, functional LsrB-type receptor in Clostridium saccharobutylicum. This novel LsrB-like receptor is the first one reported with variations in the binding-site amino acid residues that interact with AI-2. The crystal structure of the C. saccharobutylicum receptor determined at 1.35 Å resolution revealed that it binds the same form of AI-2 as the other known LsrB-type receptors, and isothermal titration calorimetry (ITC) assays showed that binding of AI-2 occurs at a submicromolar concentration. Using phylogenetic analysis, we inferred that the newly identified noncanonical LsrB receptor shares a common ancestor with known LsrB receptors and that noncanonical receptors are present in bacteria from different phyla. This led us to identify putative AI-2 receptors in bacterial species in which no receptors were known, as in bacteria belonging to the Spirochaetes and Actinobacteria phyla. Thus, this work represents a significant step toward understanding how AI-2-mediated quorum sensing influences bacterial interactions in complex biological niches.
- Klebsiella michiganensis transmission enhances resistance to Enterobacteriaceae gut invasion by nutrition competitionPublication . Oliveira, Rita A; Ng, Katharine M; Correia, Margarida B; Cabral, Vitor; Shi, Handuo; Sonnenburg, Justin L; Huang, Kerwyn Casey; Xavier, Karina BIntestinal microbiotas contain beneficial microorganisms that protect against pathogen colonization; treatment with antibiotics disrupts the microbiota and compromises colonization resistance. Here, we determine the impact of exchanging microorganisms between hosts on resilience to the colonization of invaders after antibiotic-induced dysbiosis. We assess the functional consequences of dysbiosis using a mouse model of colonization resistance against Escherichia coli. Antibiotics caused stochastic loss of members of the microbiota, but the microbiotas of co-housed mice remained more similar to each other compared with the microbiotas among singly housed animals. Strikingly, co-housed mice maintained colonization resistance after treatment with antibiotics, whereas most singly housed mice were susceptible to E. coli. The ability to retain or share the commensal Klebsiella michiganensis, a member of the Enterobacteriaceae family, was sufficient for colonization resistance after treatment with antibiotics. K. michiganensis generally outcompeted E. coli in vitro, but in vivo administration of galactitol-a nutrient that supports the growth of only E. coli-to bi-colonized gnotobiotic mice abolished the colonization-resistance capacity of K. michiganensis against E. coli, supporting the idea that nutrient competition is the primary interaction mechanism. K. michiganensis also hampered colonization of the pathogen Salmonella, prolonging host survival. Our results address functional consequences of the stochastic effects of microbiota perturbations, whereby microbial transmission through host interactions can facilitate reacquisition of beneficial commensals, minimizing the negative impact of antibiotics.
- LsrF, a coenzyme A-dependent thiolase, catalyzes the terminal step in processing the quorum sensing signal autoinducer-2Publication . Marques, João C.; Oh, Il Kyu; Ly, Daniel C.; Lamosa, Pedro; Ventura, M. Rita; Miller, Stephen T.; Xavier, Karina BivarThe quorum sensing signal autoinducer-2 (AI-2) regulates important bacterial behaviors, including biofilm formation and the production of virulence factors. Some bacteria, such as Escherichia coli, can quench the AI-2 signal produced by a variety of species present in the environment, and thus can influence AI-2-dependent bacterial behaviors. This process involves uptake of AI-2 via the Lsr transporter, followed by phosphorylation and consequent intracellular sequestration. Here we determine the metabolic fate of intracellular AI-2 by characterizing LsrF, the terminal protein in the Lsr AI-2 processing pathway. We identify the substrates of LsrF as 3-hydroxy-2,4-pentadione-5-phosphate (P-HPD, an isomer of AI-2-phosphate) and coenzyme A, determine the crystal structure of an LsrF catalytic mutant bound to P-HPD, and identify the reaction products. We show that LsrF catalyzes the transfer of an acetyl group from P-HPD to coenzyme A yielding dihydroxyacetone phosphate and acetyl-CoA, two key central metabolites. We further propose that LsrF, despite strong structural homology to aldolases, acts as a thiolase, an activity previously undescribed for this family of enzymes. With this work, we have fully characterized the biological pathway for AI-2 processing in E. coli, a pathway that can be used to quench AI-2 and control quorum-sensing-regulated bacterial behaviors.
- Maintenance of Microbial Cooperation Mediated by Public Goods in Single- and Multiple-Trait ScenariosPublication . Özkaya, Özhan; Xavier, Karina Bivar; Dionisio, Francisco; Balbontín, RobertoMicrobes often form densely populated communities, which favor competitive and cooperative interactions. Cooperation among bacteria often occurs through the production of metabolically costly molecules produced by certain individuals that become available to other neighboring individuals; such molecules are called public goods. This type of cooperation is susceptible to exploitation, since nonproducers of a public good can benefit from it while saving the cost of its production (cheating), gaining a fitness advantage over producers (cooperators). Thus, in mixed cultures, cheaters can increase in frequency in the population, relative to cooperators. Sometimes, and as predicted by simple game-theoretic arguments, such increases in the frequency of cheaters cause loss of the cooperative traits by exhaustion of the public goods, eventually leading to a collapse of the entire population. In other cases, however, both cooperators and cheaters remain in coexistence. This raises the question of how cooperation is maintained in microbial populations. Several strategies to prevent cheating have been studied in the context of a single trait and a unique environmental constraint. In this review, we describe current knowledge on the evolutionary stability of microbial cooperation and discuss recent discoveries describing the mechanisms operating in multiple-trait and multiple-constraint settings. We conclude with a consideration of the consequences of these complex interactions, and we briefly discuss the potential role of social interactions involving multiple traits and multiple environmental constraints in the evolution of specialization and division of labor in microbes.
- Manipulation of the Quorum Sensing Signal AI-2 Affects the Antibiotic-Treated Gut MicrobiotaPublication . Thompson, Jessica Ann; Oliveira, Rita Almeida; Djukovic, Ana; Ubeda, Carles; Xavier, Karina BivarThe mammalian gut microbiota harbors a diverse ecosystem where hundreds of bacterial species interact with each other and their host. Given that bacteria use signals to communicate and regulate group behaviors (quorum sensing), we asked whether such communication between different commensal species can influence the interactions occurring in this environment. We engineered the enteric bacterium, Escherichia coli, to manipulate the levels of the interspecies quorum sensing signal, autoinducer-2 (AI-2), in the mouse intestine and investigated the effect upon antibiotic-induced gut microbiota dysbiosis. E. coli that increased intestinal AI-2 levels altered the composition of the antibiotic-treated gut microbiota, favoring the expansion of the Firmicutes phylum. This significantly increased the Firmicutes/Bacteroidetes ratio, to oppose the strong effect of the antibiotic, which had almost cleared the Firmicutes. This demonstrates that AI-2 levels influence the abundance of the major phyla of the gut microbiota, the balance of which is known to influence human health.
- A Mutational Hotspot and Strong Selection Contribute to the Order of Mutations Selected for during Escherichia coli Adaptation to the GutPublication . Lourenço, Marta; Ramiro, Ricardo S; Güleresi, Daniela; Barroso-Batista, João; Xavier, Karina B; Gordo, Isabel; Sousa, AnaThe relative role of drift versus selection underlying the evolution of bacterial species within the gut microbiota remains poorly understood. The large sizes of bacterial populations in this environment suggest that even adaptive mutations with weak effects, thought to be the most frequently occurring, could substantially contribute to a rapid pace of evolutionary change in the gut. We followed the emergence of intra-species diversity in a commensal Escherichia coli strain that previously acquired an adaptive mutation with strong effect during one week of colonization of the mouse gut. Following this first step, which consisted of inactivating a metabolic operon, one third of the subsequent adaptive mutations were found to have a selective effect as high as the first. Nevertheless, the order of the adaptive steps was strongly affected by a mutational hotspot with an exceptionally high mutation rate of 10-5. The pattern of polymorphism emerging in the populations evolving within different hosts was characterized by periodic selection, which reduced diversity, but also frequency-dependent selection, actively maintaining genetic diversity. Furthermore, the continuous emergence of similar phenotypes due to distinct mutations, known as clonal interference, was pervasive. Evolutionary change within the gut is therefore highly repeatable within and across hosts, with adaptive mutations of selection coefficients as strong as 12% accumulating without strong constraints on genetic background. In vivo competitive assays showed that one of the second steps (focA) exhibited positive epistasis with the first, while another (dcuB) exhibited negative epistasis. The data shows that strong effect adaptive mutations continuously recur in gut commensal bacterial species.