Paired single-cell host profiling with multiplex-tagged bacterial mutants reveals intracellular virulence-immune networks.

Encounters between host cells and intracellular bacterial pathogens lead to complex phenotypes that determine the outcome of infection. Single-cell RNA sequencing (scRNA-seq) is increasingly used to study the host factors underlying diverse cellular phenotypes but has limited capacity to analyze the role of bacterial factors. Here, we developed scPAIR-seq, a single-cell approach to analyze infection with a pooled library of multiplex-tagged, barcoded bacterial mutants. Infected host cells and barcodes of intracellular bacterial mutants are both captured by scRNA-seq to functionally analyze mutant-dependent changes in host transcriptomes. We applied scPAIR-seq to macrophages infected with a library of Salmonella Typhimurium secretion system effector mutants. We analyzed redundancy between effectors and mutant-specific unique fingerprints and mapped the global virulence network of each individual effector by its impact on host immune pathways. ScPAIR-seq is a powerful tool to untangle bacterial virulence strategies and their complex interplay with host defense strategies that drive infection outcome.

Bacterial lifestyle switch in response to algal metabolites.

Unicellular algae, termed phytoplankton, greatly impact the marine environment by serving as the basis of marine food webs and by playing central roles in the biogeochemical cycling of elements. The interactions between phytoplankton and heterotrophic bacteria affect the fitness of both partners. It is becoming increasingly recognized that metabolic exchange determines the nature of such interactions, but the underlying molecular mechanisms remain underexplored. Here, we investigated the molecular and metabolic basis for the bacterial lifestyle switch, from coexistence to pathogenicity, in Sulfitobacter D7 during its interaction with Emiliania huxleyi, a cosmopolitan bloom-forming phytoplankter. To unravel the bacterial lifestyle switch, we analyzed bacterial transcriptomes in response to exudates derived from algae in exponential growth and stationary phase, which supported the Sulfitobacter D7 coexistence and pathogenicity lifestyles, respectively. In pathogenic mode, Sulfitobacter D7 upregulated flagellar motility and diverse transport systems, presumably to maximize assimilation of E. huxleyi-derived metabolites released by algal cells upon cell death. Algal dimethylsulfoniopropionate (DMSP) was a pivotal signaling molecule that mediated the transition between the lifestyles, supporting our previous findings. However, the coexisting and pathogenic lifestyles were evident only in the presence of additional algal metabolites. Specifically, we discovered that algae-produced benzoate promoted the growth of Sulfitobacter D7 and hindered the DMSP-induced lifestyle switch to pathogenicity, demonstrating that benzoate is important for maintaining the coexistence of algae and bacteria. We propose that bacteria can sense the physiological state of the algal host through changes in the metabolic composition, which will determine the bacterial lifestyle during interaction.

Immunometabolic crosstalk during bacterial infection.

Following detection of bacteria, macrophages switch their metabolism from oxidative respiration through the tricarboxylic acid cycle to high-rate aerobic glycolysis. This immunometabolic shift enables pro-inflammatory and antimicrobial responses and is facilitated by the accumulation of fatty acids, tricarboxylic acid-derived metabolites and catabolism of amino acids. Recent studies have shown that these immunometabolites are co-opted by pathogens as environmental cues for expression of virulence genes. We review mechanisms by which host immunometabolites regulate bacterial pathogenicity and discuss opportunities for the development of therapeutics targeting metabolic host-pathogen crosstalk.

A non-classical monocyte-derived macrophage subset provides a splenic replication niche for intracellular Salmonella.

Interactions between intracellular bacteria and mononuclear phagocytes give rise to diverse cellular phenotypes that may determine the outcome of infection. Recent advances in single-cell RNA sequencing (scRNA-seq) have identified multiple subsets within the mononuclear population, but implications to their function during infection are limited. Here, we surveyed the mononuclear niche of intracellular Salmonella Typhimurium (S.Tm) during early systemic infection in mice. We described eclipse-like growth kinetics in the spleen, with a first phase of bacterial control mediated by tissue-resident red-pulp macrophages. A second phase involved extensive bacterial replication within a macrophage population characterized by CD9 expression. We demonstrated that CD9+ macrophages induced pathways for detoxificating oxidized lipids, that may be utilized by intracellular S.Tm. We established that CD9+ macrophages originated from non-classical monocytes (NCM), and NCM-depleted mice were more resistant to S.Tm infection. Our study defines macrophage subset-specific host-pathogen interactions that determine early infection dynamics and infection outcome of the entire organism.

Host succinate is an activation signal for Salmonella virulence during intracellular infection.

Key to the success of intracellular pathogens is the ability to sense and respond to a changing host cell environment. Macrophages exposed to microbial products undergo metabolic changes that drive inflammatory responses. However, the role of macrophage metabolic reprogramming in bacterial adaptation to the intracellular environment has not been explored. Here, using metabolic profiling and dual RNA sequencing, we show that succinate accumulation in macrophages is sensed by intracellular Salmonella Typhimurium (S. Tm) to promote antimicrobial resistance and type III secretion. S Tm lacking the succinate uptake transporter DcuB displays impaired survival in macrophages and in mice. Thus, S Tm co-opts the metabolic reprogramming of infected macrophages as a signal that induces its own virulence and survival, providing an additional perspective on metabolic host-pathogen cross-talk.

The extracellular matrix protein TasA is a developmental cue that maintains a motile subpopulation within Bacillus subtilis biofilms.

In nature, bacteria form biofilms-differentiated multicellular communities attached to surfaces. Within these generally sessile biofilms, a subset of cells continues to express motility genes. We found that this subpopulation enabled Bacillus subtilis biofilms to expand on high-friction surfaces. The extracellular matrix (ECM) protein TasA was required for the expression of flagellar genes. In addition to its structural role as an adhesive fiber for cell attachment, TasA acted as a developmental signal stimulating a subset of biofilm cells to revert to a motile phenotype. Transcriptomic analysis revealed that TasA stimulated the expression of a specific subset of genes whose products promote motility and repress ECM production. Spontaneous suppressor mutations that restored motility in the absence of TasA revealed that activation of the biofilm-motility switch by the two-component system CssR/CssS antagonized the TasA-mediated reversion to motility in biofilm cells. Our results suggest that although mostly sessile, biofilms retain a degree of motility by actively maintaining a motile subpopulation.

A Sequence of Developmental Events Occurs Underneath Growing Bacillus subtilis Pellicles.

Biofilms are structured communities of bacteria that exhibit complex spatio-temporal dynamics. In liquid media, Bacillus subtilis produces an opaque floating biofilm, or a pellicle. Biofilms are generally associated with an interface, but the ability of Bacillus subtilis to swim means the bacteria are additionally able to reside within the liquid phase. However, due to imaging complications associated with the opacity of pellicles, the extent to which bacteria coexist within the liquid bulk as well as their behavior in the liquid is not well studied. We therefore develop a high-throughput imaging system to image underneath developing pellicles. Here we report a well-defined sequence of developmental events that occurs underneath a growing pellicle. Comparison with bacteria deficient in swimming and chemotaxis suggest that these properties enable collective bacterial swimming within the liquid phase which facilitate faster surface colonization. Furthermore, comparison to bacteria deficient in exopolymeric substances (EPS) suggest that the lack of a surface pellicle prevents further developmental steps from occurring within the liquid phase. Our results reveal a sequence of developmental events during pellicle growth, encompassing adhesion, conversion, growth, maturity, and detachment on the interface, which are synchronized with the bacteria in the liquid bulk increasing in density until the formation of a mature surface pellicle, after which the density of bacteria in the liquid drops.

Collective Vortex-Like Movement of Bacillus subtilis Facilitates the Generation of Floating Biofilms.

Bacteria in nature are usually found in complex multicellular structures, called biofilms. One common form of a biofilm is pellicle-a floating mat of bacteria formed in the water-air interphase. So far, our knowledge on the basic mechanisms underlying the formation of biofilms at air-liquid interfaces is not complete. In particular, the co-occurrence of motile cells and extracellular matrix producers has not been studied. In addition, the potential involvement of chemical communication in pellicle formation remained largely undefined. Our results indicate that vortex-like collective motility by aggregates of motile cells and EPS producers accelerate the formation of floating biofilms. Successful aggregation and migration to the water-air interphase depend on the chemical communication signal autoinducer 2 (AI-2). This ability of bacteria to form a biofilm in a preferable niche ahead of their potential rivals would provide a fitness advantage in the context of inter-species competition.

Effective optimization using sample persistence: A case study on quantum annealers and various Monte Carlo optimization methods.

We present and apply a general-purpose, multistart algorithm for improving the performance of low-energy samplers used for solving optimization problems. The algorithm iteratively fixes the value of a large portion of the variables to values that have a high probability of being optimal. The resulting problems are smaller and less connected, and samplers tend to give better low-energy samples for these problems. The algorithm is trivially parallelizable since each start in the multistart algorithm is independent, and could be applied to any heuristic solver that can be run multiple times to give a sample. We present results for several classes of hard problems solved using simulated annealing, path-integral quantum Monte Carlo, parallel tempering with isoenergetic cluster moves, and a quantum annealer, and show that the success metrics and the scaling are improved substantially. When combined with this algorithm, the quantum annealer's scaling was substantially improved for native Chimera graph problems. In addition, with this algorithm the scaling of the time to solution of the quantum annealer is comparable to the Hamze-de Freitas-Selby algorithm on the weak-strong cluster problems introduced by Boixo et al. Parallel tempering with isoenergetic cluster moves was able to consistently solve three-dimensional spin glass problems with 8000 variables when combined with our method, whereas without our method it could not solve any.

Toxin-Antitoxin systems eliminate defective cells and preserve symmetry in Bacillus subtilis biofilms.

Toxin-antitoxin modules are gene pairs encoding a toxin and its antitoxin, and are found on the chromosomes of many bacteria, including pathogens. Here, we characterize the specific contribution of the TxpA and YqcG toxins in elimination of defective cells from developing Bacillus subtilis biofilms. On nutrient limitation, defective cells accumulated in the biofilm breaking its symmetry. Deletion of the toxins resulted in accumulation of morphologically abnormal cells, and interfered with the proper development of the multicellular community. Dual physiological responses are of significance for TxpA and YqcG activation: nitrogen deprivation enhances the transcription of both TxpA and YqcG toxins, and simultaneously sensitizes the biofilm cells to their activity. Furthermore, we demonstrate that while both toxins when overexpressed affect the morphology of the developing biofilm, the toxin TxpA can act to lyse and dissolve pre-established B. subtilis biofilms.

Not so simple, not so subtle: the interspecies competition between Bacillus simplex and Bacillus subtilis and its impact on the evolution of biofilms.

Bacillus subtilis biofilms have a fundamental role in shaping the soil ecosystem. During this process, they unavoidably interact with neighbour bacterial species. We studied the interspecies interactions between biofilms of the soil-residing bacteria B. subtilis and related Bacillus species. We found that proximity between the biofilms triggered recruitment of motile B. subtilis cells, which engulfed the competing Bacillus simplex colony. Upon interaction, B. subtilis secreted surfactin and cannibalism toxins, at concentrations that were inert to B. subtilis itself, which eliminated the B. simplex colony, as well as colonies of Bacillus toyonensis. Surfactin toxicity was correlated with the presence of short carbon-tail length isomers, and synergistic with the cannibalism toxins. Importantly, during biofilm development and interspecies interactions a subpopulation in B. subtilis biofilm lost its native plasmid, leading to increased virulence against the competing Bacillus species. Overall, these findings indicate that genetic programs and traits that have little effect on biofilm development when each species is grown in isolation have a dramatic impact when different bacterial species interact.