Toxigenic Clostridium perfringens Isolated from At-Risk Paediatric Inflammatory Bowel Disease Patients.

BACKGROUND AND AIMS: This study aimed to identify microbial drivers of inflammatory bowel disease [IBD], by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from paediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying Clostridium perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in eight of nine patients' mucosal biopsies, correlating with haemolytic activity, but was not present in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults [18.7-27.1%] versus healthy controls [5.1%]. In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial cells, neuroblasts, and neutrophils, while the impact on epithelial cells was less pronounced, suggesting C. perfringens may be particularly damaging when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed perfringolysin O [PFO] toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.

Morphological and Transcriptional Responses to CRISPRi Knockdown of Essential Genes in Escherichia coli.

CRISPR interference (CRISPRi) has facilitated the study of essential genes in diverse organisms using both high-throughput and targeted approaches. Despite the promise of this technique, no comprehensive arrayed CRISPRi library targeting essential genes exists for the model bacterium Escherichia coli, or for any Gram-negative species. Here, we built and characterized such a library. Each of the ∼500 strains in our E. coli library contains an inducible, chromosomally integrated single guide RNA (sgRNA) targeting an essential (or selected nonessential) gene and can be mated with a pseudo-Hfr donor strain carrying a dcas9 cassette to create a CRISPRi knockdown strain. Using this system, we built an arrayed library of CRISPRi strains and performed population and single-cell growth and morphology measurements as well as targeted follow-up experiments. These studies found that inhibiting translation causes an extended lag phase, identified new modulators of cell morphology, and revealed that the morphogene mreB is subject to transcriptional feedback regulation, which is critical for the maintenance of morphology. Our findings highlight canonical and noncanonical roles for essential genes in numerous aspects of cellular homeostasis. IMPORTANCE Essential genes make up only ∼5 to 10% of the genetic complement in most organisms but occupy much of their protein synthesis and account for almost all antibiotic targets. Despite the importance of essential genes, their intractability has, until recently, hampered efforts to study them. CRISPRi has facilitated the study of essential genes by allowing inducible and titratable depletion. However, all large-scale CRISPRi studies in Gram-negative bacteria thus far have used plasmids to express CRISPRi components and have been constructed in pools, limiting their utility for targeted assays and complicating the determination of antibiotic effects. Here, we use a modular method to construct an arrayed library of chromosomally integrated CRISPRi strains targeting the essential genes of the model bacterium Escherichia coli. This library enables targeted studies of essential gene depletions and high-throughput determination of antibiotic targets and facilitates studies targeting the outer membrane, an essential component that serves as the major barrier to antibiotics.

Quantifying rapid bacterial evolution and transmission within the mouse intestine.

Due to limitations on high-resolution strain tracking, selection dynamics during gut microbiota colonization and transmission between hosts remain mostly mysterious. Here, we introduced hundreds of barcoded Escherichia coli strains into germ-free mice and quantified strain-level dynamics and metagenomic changes. Mutations in genes involved in motility and metabolite utilization are reproducibly selected within days. Even with rapid selection, coprophagy enforced similar barcode distributions across co-housed mice. Whole-genome sequencing of hundreds of isolates revealed linked alleles that demonstrate between-host transmission. A population-genetics model predicts substantial fitness advantages for certain mutants and that migration accounted for ∼10% of the resident microbiota each day. Treatment with ciprofloxacin suggests interplay between selection and transmission. While initial colonization was mostly uniform, in two mice a bottleneck reduced diversity and selected for ciprofloxacin resistance in the absence of drug. These findings highlight the interplay between environmental transmission and rapid, deterministic selection during evolution of the intestinal microbiota.

Effects of fixation on bacterial cellular dimensions and integrity.

Fixation facilitates imaging of subcellular localization and cell morphology, yet it remains unknown how fixation affects cellular dimensions and intracellular fluorescence patterns, particularly during long-term storage. Here, we characterized the effects of multiple fixatives on several bacterial species. Fixation generally reduced cell length by 5-15%; single-cell tracking in microfluidics revealed that the length decrease was an aggregate effect of many steps in the fixation protocol and that fluorescence of cytoplasmic GFP but not membrane-bound MreB-msfGFP was rapidly lost with formaldehyde-based fixatives. Cellular dimensions were preserved in formaldehyde-based fixatives for ≥4 days, but methanol caused length to decrease. Although methanol preserved cytoplasmic fluorescence better than formaldehyde-based fixatives, some Escherichia coli cells were able to grow directly after fixation. Moreover, methanol fixation caused lysis in a subpopulation of cells, with virtually all Bacillus subtilis cells lysing after one day. These findings highlight tradeoffs between maintenance of fluorescence and membrane integrity for future applications of fixation.

Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth.

Bacterial growth under nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over that of standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multiwell plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth.IMPORTANCE How starved bacteria adapt and multiply under replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allow for accurate growth rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

Bacterial Swarming Reduces Proteus mirabilis and Vibrio parahaemolyticus Cell Stiffness and Increases ß-Lactam Susceptibility.

Swarmer cells of the Gram-negative uropathogenic bacteria Proteus mirabilis and Vibrio parahaemolyticus become long (>10 to 100 µm) and multinucleate during their growth and motility on polymer surfaces. We demonstrated that the increasing cell length is accompanied by a large increase in flexibility. Using a microfluidic assay to measure single-cell mechanics, we identified large differences in the swarmer cell stiffness (bending rigidity) of P. mirabilis (5.5 × 10-22 N m2) and V. parahaemolyticus (1.0 × 10-22 N m2) compared to vegetative cells (1.4 × 10-20 N m2 and 2.2 × 10-22 N m2, respectively). The reduction in bending rigidity (∼2-fold to ∼26-fold) was accompanied by a decrease in the average polysaccharide strand length of the peptidoglycan layer of the cell wall from 28 to 30 disaccharides to 19 to 22 disaccharides. Atomic force microscopy revealed a reduction in P. mirabilis peptidoglycan thickness from 1.5 nm (vegetative cells) to 1.0 nm (swarmer cells), and electron cryotomography indicated changes in swarmer cell wall morphology. P. mirabilis and V. parahaemolyticus swarmer cells became increasingly sensitive to osmotic pressure and susceptible to cell wall-modifying antibiotics (compared to vegetative cells)-they were ∼30% more likely to die after 3 h of treatment with MICs of the ß-lactams cephalexin and penicillin G. The adaptive cost of "swarming" was offset by the increase in cell susceptibility to physical and chemical changes in their environment, thereby suggesting the development of new chemotherapies for bacteria that leverage swarming for the colonization of hosts and for survival.IMPORTANCEProteus mirabilis and Vibrio parahaemolyticus are bacteria that infect humans. To adapt to environmental changes, these bacteria alter their cell morphology and move collectively to access new sources of nutrients in a process referred to as "swarming." We found that changes in the composition and thickness of the peptidoglycan layer of the cell wall make swarmer cells of P. mirabilis and V. parahaemolyticus more flexible (i.e., reduce cell stiffness) and that they become more sensitive to osmotic pressure and cell wall-targeting antibiotics (e.g., ß-lactams). These results highlight the importance of assessing the extracellular environment in determining antibiotic doses and the use of ß-lactam antibiotics for treating infections caused by swarmer cells of P. mirabilis and V. parahaemolyticus.

Maspin binds to cardiolipin in mitochondria and triggers apoptosis.

A central question in cell biology is how cells respond to stress signals and biochemically regulate apoptosis. One critical pathway involves the change of mitochondrial function and release of cytochrome c to initiate apoptosis. In response to apoptotic stimuli, we found that maspin-a noninhibitory member of the serine protease inhibitor superfamily-translocates from the cytosol to mitochondria and binds to cardiolipin in the inner mitochondrial membrane. Biolayer interferometry assay revealed that recombinant maspin binds cardiolipin with an apparent Kd,of ∼15.8 µM and competes with cytochrome c (apparent Kd of ∼1.31 µM) for binding to cardiolipin-enriched membranes. A hydrophobic, lysine-rich domain in maspin consists of 27 aa, is located at position 268-294, and is responsible for the interaction of this protein with cardiolipin. Depletion of cardiolipin in cells significantly prevents maspin binding to the inner mitochondrial membrane and decreases cytochrome c release and apoptosis. Alteration to maspin's cardiolipin binding domain changes its ability to bind cardiolipin, and tumor cells expressing this mutant have a low frequency of apoptosis. We propose a model of apoptosis in which maspin binds to cardiolipin, displaces cytochrome c from the membrane, and facilitates its release to the cytoplasm.-Mahajan, N., Hoover, B., Rajendram, M., Shi, H. Y., Kawasaki, K., Weibel, D. B., Zhang, M. Maspin binds to cardiolipin in mitochondria and triggers apoptosis.

A Gut Commensal-Produced Metabolite Mediates Colonization Resistance to Salmonella Infection.

The intestinal microbiota provides colonization resistance against pathogens, limiting pathogen expansion and transmission. These microbiota-mediated mechanisms were previously identified by observing loss of colonization resistance after antibiotic treatment or dietary changes, which severely disrupt microbiota communities. We identify a microbiota-mediated mechanism of colonization resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) by comparing high-complexity commensal communities with different levels of colonization resistance. Using inbred mouse strains with different infection dynamics and S. Typhimurium intestinal burdens, we demonstrate that Bacteroides species mediate colonization resistance against S. Typhimurium by producing the short-chain fatty acid propionate. Propionate directly inhibits pathogen growth in vitro by disrupting intracellular pH homeostasis, and chemically increasing intestinal propionate levels protects mice from S. Typhimurium. In addition, administering susceptible mice Bacteroides, but not a propionate-production mutant, confers resistance to S. Typhimurium. This work provides mechanistic understanding into the role of individualized microbial communities in host-to-host variability of pathogen transmission.

Marine Mammal Microbiota Yields Novel Antibiotic with Potent Activity Against Clostridium difficile.

The recent explosion of research on the microbiota has highlighted the important interplay between commensal microorganisms and the health of their cognate hosts. Metabolites isolated from commensal bacteria have been demonstrated to possess a range of antimicrobial activities, and it is widely believed that some of these metabolites modulate host behavior, affecting predisposition to disease and pathogen invasion. Our access to the local marine mammal stranding network and previous successes in mining the fish microbiota poised us to test the hypothesis that the marine mammal microbiota is a novel source of commensal bacteria-produced bioactive metabolites. Examination of intestinal contents from five marine mammals led to the identification of a Micromonospora strain with potent and selective activity against a panel of Gram-positive pathogens and no discernible human cytotoxicity. Compound isolation afforded a new complex glycosylated polyketide, phocoenamicin, with potent activity against the intestinal pathogen Clostridium difficile, an organism challenging to treat in hospital settings. Use of our activity-profiling platform, BioMAP, clustered this metabolite with other known ionophore antibiotics. Fluorescence imaging and flow cytometry confirmed that phocoenamicin is capable of shifting membrane potential without damaging membrane integrity. Thus, exploration of gut microbiota in hosts from diverse environments can serve as a powerful strategy for the discovery of novel antibiotics against human pathogens.

Mechanical Genomics Identifies Diverse Modulators of Bacterial Cell Stiffness.

Bacteria must maintain mechanical integrity to withstand the large osmotic pressure differential across the cell membrane and wall. Although maintaining mechanical integrity is critical for proper cellular function, a fact exploited by prominent cell-wall-targeting antibiotics, the proteins that contribute to cellular mechanics remain unidentified. Here, we describe a high-throughput optical method for quantifying cell stiffness and apply this technique to a genome-wide collection of ∼4,000 Escherichia coli mutants. We identify genes with roles in diverse functional processes spanning cell-wall synthesis, energy production, and DNA replication and repair that significantly change cell stiffness when deleted. We observe that proteins with biochemically redundant roles in cell-wall synthesis exhibit different stiffness defects when deleted. Correlating our data with chemical screens reveals that reducing membrane potential generally increases cell stiffness. In total, our work demonstrates that bacterial cell stiffness is a property of both the cell wall and broader cell physiology and lays the groundwork for future systematic studies of mechanoregulation.

Ionic Hydrogen Bonds and Lipid Packing Defects Determine the Binding Orientation and Insertion Depth of RecA on Multicomponent Lipid Bilayers.

We describe a computational and experimental approach for probing the binding properties of the RecA protein at the surface of anionic membranes. Fluorescence measurements indicate that RecA behaves differently when bound to phosphatidylglycerol (PG)- and cardiolipin (CL)-containing liposomes. We use a multistage computational protocol that integrates an implicit membrane/solvent model, the highly mobile mimetic membrane model, and the full atomistic membrane model to study how different anionic lipids perturb RecA binding to the membrane. With anionic lipids studied here, the binding interface involves three key regions: the N-terminal helix, the DNA binding loop L2, and the M-M7 region. The nature of binding involves both electrostatic interactions between cationic protein residues and lipid polar/charged groups and insertion of hydrophobic residues. The L2 loop contributes more to membrane insertion than the N-terminal helix. More subtle aspects of RecA-membrane interaction are influenced by specific properties of anionic lipids. Ionic hydrogen bonds between the carboxylate group in phosphatidylserine and several lysine residues in the C-terminal region of RecA stabilize the parallel (∥) binding orientation, which is not locally stable on PG- and CL-containing membranes despite similarity in the overall charge density. Lipid packing defects, which are more prevalent in the presence of conical lipids, are observed to enhance the insertion depth of hydrophobic motifs. The computational finding that RecA binds in a similar orientation to PG- and CL-containing membranes is consistent with the fact that PG alone is sufficient to induce RecA polar localization, although CL might be more effective because of its tighter binding to RecA. The different fluorescence behaviors of RecA upon binding to PG- and CL-containing liposomes is likely due to the different structures and flexibility of the C-terminal region of RecA when it binds to different anionic phospholipids.

Anionic Phospholipids Stabilize RecA Filament Bundles in Escherichia coli.

We characterize the interaction of RecA with membranes in vivo and in vitro and demonstrate that RecA binds tightly to the anionic phospholipids cardiolipin (CL) and phosphatidylglycerol (PG). Using computational models, we identify two regions of RecA that interact with PG and CL: (1) the N-terminal helix and (2) loop L2. Mutating these regions decreased the affinity of RecA to PG and CL in vitro. Using 3D super-resolution microscopy, we demonstrate that depleting Escherichia coli PG and CL altered the localization of RecA foci and hindered the formation of RecA filament bundles. Consequently, E. coli cells lacking aPLs fail to initiate a robust SOS response after DNA damage, indicating that the membrane acts as a scaffold for nucleating the formation of RecA filament bundles and plays an important role in the SOS response.

Gyramides prevent bacterial growth by inhibiting DNA gyrase and altering chromosome topology.

Antibiotics targeting DNA gyrase have been a clinical success story for the past half-century, and the emergence of bacterial resistance has fueled the search for new gyrase inhibitors. In this paper we demonstrate that a new class of gyrase inhibitors, the gyramides, are bacteriostatic agents that competitively inhibit the ATPase activity of Escherichia coli gyrase and produce supercoiled DNA in vivo. E. coli cells treated with gyramide A have abnormally localized, condensed chromosomes that blocks DNA replication and interrupts chromosome segregation. The resulting alterations in DNA topology inhibit cell division through a mechanism that involves the SOS pathway. Importantly, gyramide A is a specific inhibitor of gyrase and does not inhibit the closely related E. coli enzyme topoisomerase IV. E. coli mutants with reduced susceptibility to gyramide A do not display cross-resistance to ciprofloxacin and novobiocin. The results demonstrate that the gyramides prevent bacterial growth by a mechanism in which the topological state of chromosomes is altered and halts DNA replication and segregation. The specificity and activity of the gyramides for inhibiting gyrase makes these compounds important chemical tools for studying the mechanism of gyrase and the connection between DNA topology and bacterial cell division.