Dietary carbohydrates regulate intestinal colonization and dissemination of Klebsiella pneumoniae.

Bacterial translocation from the gut microbiota is a source of sepsis in susceptible patients. Previous work suggests that overgrowth of gut pathobionts, including Klebsiella pneumoniae, increases the risk of disseminated infection. Our data from a human dietary intervention study found that, in the absence of fiber, K. pneumoniae bloomed during microbiota recovery from antibiotic treatment. We thus hypothesized that dietary nutrients directly support or suppress colonization of this gut pathobiont in the microbiota. Consistent with our study in humans, complex carbohydrates in dietary fiber suppressed the colonization of K. pneumoniae and allowed for recovery of competing commensals in mouse models. In contrast, through ex vivo and in vivo modeling, we identified simple carbohydrates as a limiting resource for K. pneumoniae in the gut. As proof of principle, supplementation with lactulose, a nonabsorbed simple carbohydrate and an FDA-approved therapy, increased colonization of K. pneumoniae. Disruption of the intestinal epithelium led to dissemination of K. pneumoniae into the bloodstream and liver, which was prevented by dietary fiber. Our results show that dietary simple and complex carbohydrates were critical not only in the regulation of pathobiont colonization but also disseminated infection, suggesting that targeted dietary interventions may offer a preventative strategy in high-risk patients.

Enterobacteriaceae Growth Promotion by Intestinal Acylcarnitines, a Biomarker of Dysbiosis in Inflammatory Bowel Disease.

BACKGROUND & AIMS: Altered plasma acylcarnitine levels are well-known biomarkers for a variety of mitochondrial fatty acid oxidation disorders and can be used as an alternative energy source for the intestinal epithelium when short-chain fatty acids are low. These membrane-permeable fatty acid intermediates are excreted into the gut lumen via bile and are increased in the feces of patients with inflammatory bowel disease (IBD). METHODS: Herein, based on studies in human subjects, animal models, and bacterial cultures, we show a strong positive correlation between fecal carnitine and acylcarnitines and the abundance of Enterobacteriaceae in IBD where they can be consumed by bacteria both in vitro and in vivo. RESULTS: Carnitine metabolism promotes the growth of Escherichia coli via anaerobic respiration dependent on the cai operon, and acetylcarnitine dietary supplementation increases fecal carnitine levels with enhanced intestinal colonization of the enteric pathogen Citrobacter rodentium. CONCLUSIONS: In total, these results indicate that the increased luminal concentrations of carnitine and acylcarnitines in patients with IBD may promote the expansion of pathobionts belonging to the Enterobacteriaceae family, thereby contributing to disease pathogenesis.

Colonization and Dissemination of Klebsiella pneumoniae is Dependent on Dietary Carbohydrates.

Dysbiosis of the gut microbiota is increasingly appreciated as both a consequence and precipitant of human disease. The outgrowth of the bacterial family Enterobacteriaceae is a common feature of dysbiosis, including the human pathogen Klebsiella pneumoniae . Dietary interventions have proven efficacious in the resolution of dysbiosis, though the specific dietary components involved remain poorly defined. Based on a previous human diet study, we hypothesized that dietary nutrients serve as a key resource for the growth of bacteria found in dysbiosis. Through human sample testing, and ex-vivo , and in vivo modeling, we find that nitrogen is not a limiting resource for the growth of Enterobacteriaceae in the gut, contrary to previous studies. Instead, we identify dietary simple carbohydrates as critical in colonization of K. pneumoniae . We additionally find that dietary fiber is necessary for colonization resistance against K. pneumoniae , mediated by recovery of the commensal microbiota, and protecting the host against dissemination from the gut microbiota during colitis. Targeted dietary therapies based on these findings may offer a therapeutic strategy in susceptible patients with dysbiosis.

Comparative Proteomic Analysis Revealing ActJ-Regulated Proteins in Sinorhizobium meliloti.

To adapt to different environmental conditions, Sinorhizobium meliloti relies on finely tuned regulatory networks, most of which are unexplored to date. We recently demonstrated that deletion of the two-component system ActJK renders an acid-vulnerable phenotype in S. meliloti and negatively impacts bacteroid development and nodule occupancy as well. To fully understand the role of ActJ in acid tolerance, S. meliloti wild-type and S. meliloti ΔactJ proteomes were compared in the presence or absence of acid stress by nanoflow ultrahigh-performance liquid chromatography coupled to mass spectrometry. The analysis demonstrated that proteins involved in the synthesis of exopolysaccharides (EPSs) were notably enriched in ΔactJ cells in acid pH. Total EPS quantification further revealed that although EPS production was augmented at pH 5.6 in both the ΔactJ and the parental strain, the lack of ActJ significantly enhanced this difference. Moreover, several efflux pumps were found to be downregulated in the ΔactJ strain. Promoter fusion assays suggested that ActJ positively modulated its own expression in an acid medium but not at under neutral conditions. The results presented here identify several ActJ-regulated genes in S. meliloti, highlighting key components associated with ActJK regulation that will contribute to a better understanding of rhizobia adaptation to acid stress.

Zwitterionic surface chemistry enhances detachment of bacteria under shear.

The ubiquitous nature of microorganisms, especially of biofilm-forming bacteria, makes biofouling a prevalent challenge in many settings, including medical and industrial environments immersed in liquid and subjected to shear forces. Recent studies have shown that zwitterionic groups are effective in suppressing bacteria and protein adhesion as well as biofilm growth. However, the effect of zwitterionic groups on the removal of surface-bound bacteria has not been extensively studied. Here we present a microfluidic approach to evaluate the effectiveness in facilitating bacteria detachment by shear of an antifouling surface treatment using (3-(dimethyl;(3-trimethoxysilyl)propyl)ammonia propane-1-sulfonate), a sulfobetaine silane (SBS). Control studies show that SBS-functionalized surfaces greatly increase protein (bovine serum albumin) removal upon rinsing. On the same surfaces, enhanced bacteria (Pseudomonas aeruginosa) removal is observed under shear. To quantify this enhancement a microfluidic shear device is employed to investigate how SBS-functionalized surfaces promote bacteria detachment under shear. By using a microfluidic channel with five shear zones, we compare the removal of bacteria from zwitterionic and glass surfaces under different shear rates. At times of 15 min, 30 min, and 60 min, bacteria adhesion on SBS-functionalized surfaces is reduced relative to the control surface (glass) under quiescent conditions. However, surface-associated bacteria on the SBS-functionalized glass and control show similar percentages of live cells, suggesting minimal intrinsic biocidal effect from the SBS-functionalized surface. Notably, when exposed to shear rates ranging from 104 to 105 s-1, significantly fewer bacteria remain on the SBS-functionalized surfaces. These results demonstrate the potential of zwitterionic sulfobetaine as effective antifouling coatings that facilitate the removal of bacteria under shear.

A commensal-encoded genotoxin drives restriction of Vibrio cholerae colonization and host gut microbiome remodeling.

SignificanceIn a polymicrobial battlefield where different species compete for nutrients and colonization niches, antimicrobial compounds are the sword and shield of commensal microbes in competition with invading pathogens and each other. The identification of an Escherichia coli-produced genotoxin, colibactin, and its specific targeted killing of enteric pathogens and commensals, including Vibrio cholerae and Bacteroides fragilis, sheds light on our understanding of intermicrobial interactions in the mammalian gut. Our findings elucidate the mechanisms through which genotoxins shape microbial communities and provide a platform for probing the larger role of enteric multibacterial interactions regarding infection and disease outcomes.

Thiol-based functional mimicry of phosphorylation of the two-component system response regulator ArcA promotes pathogenesis in enteric pathogens.

Pathogenic bacteria can rapidly respond to stresses such as reactive oxygen species (ROS) using reversible redox-sensitive oxidation of cysteine thiol (-SH) groups in regulators. Here, we use proteomics to profile reversible ROS-induced thiol oxidation in Vibrio cholerae, the etiologic agent of cholera, and identify two modified cysteines in ArcA, a regulator of global carbon oxidation that is phosphorylated and activated under low oxygen. ROS abolishes ArcA phosphorylation but induces the formation of an intramolecular disulfide bond that promotes ArcA-ArcA interactions and sustains activity. ArcA cysteines are oxidized in cholera patient stools, and ArcA thiol oxidation drives in vitro ROS resistance, colonization of ROS-rich guts, and environmental survival. In other pathogens, such as Salmonella enterica, oxidation of conserved cysteines of ArcA orthologs also promotes ROS resistance, suggesting a common role for ROS-induced ArcA thiol oxidation in modulating ArcA activity, allowing for a balance of expression of stress- and pathogenesis-related genetic programs.

Effects of Regulatory Network Organization and Environment on PmrD Connector Activity and Polymyxin Resistance in Klebsiella pneumoniae and Escherichia coli.

Polymyxins are a class of cyclic peptides with antimicrobial activity against Gram-negative bacteria. In Enterobacteriaceae, the PhoQ/PhoP and PmrB/PmrA two-component systems regulate many genes that confer resistance to both polymyxins and host antimicrobial peptides. The activities of these two-component systems are modulated by additional proteins that are conserved across Enterobacteriaceae, such as MgrB, a negative regulator of PhoQ, and PmrD, a "connector" protein that activates PmrB/PmrA in response to PhoQ/PhoP stimulation. Despite the conservation of many protein components of the PhoQ/PhoP-PmrD-PmrB/PmrA network, the specific molecular interactions and regulatory mechanisms vary across different genera. Here, we explore the role of PmrD in modulating this signaling network in Klebsiella pneumoniae and Escherichia coli We show that in K. pneumoniae, PmrD is not required for polymyxin resistance arising from mutation of mgrB-the most common cause of spontaneous polymyxin resistance in this bacterium-suggesting that direct activation of polymyxin resistance genes by PhoQ/PhoP plays a critical role in this resistance pathway. However, for conditions of low pH or intermediate iron concentrations, both of which stimulate PmrB/PmrA, we find that PmrD does contribute to resistance. We further show that in E. coli, PmrD functions as a connector between PhoQ/PhoP and PmrB/PmrA, in contrast with previous reports. In this case, activity also depends on PmrB/PmrA stimulation, or on very high activation of PhoQ/PhoP. Our results indicate that the importance of the PmrD connector in modulating the polymyxin resistance network depends on both the network organization and on the environmental conditions associated with PmrB stimulation.

The phosphohistidine phosphatase SixA dephosphorylates the phosphocarrier NPr.

Histidine phosphorylation is a posttranslational modification that alters protein function and also serves as an intermediate of phosphoryl transfer. Although phosphohistidine is relatively unstable, enzymatic dephosphorylation of this residue is apparently needed in some contexts, since both prokaryotic and eukaryotic phosphohistidine phosphatases have been reported. Here we identify the mechanism by which a bacterial phosphohistidine phosphatase dephosphorylates the nitrogen-related phosphotransferase system, a broadly conserved bacterial pathway that controls diverse metabolic processes. We show that the phosphatase SixA dephosphorylates the phosphocarrier protein NPr and that the reaction proceeds through phosphoryl transfer from a histidine on NPr to a histidine on SixA. In addition, we show that Escherichia coli lacking SixA are outcompeted by wild-type E. coli in the context of commensal colonization of the mouse intestine. Notably, this colonization defect requires NPr and is distinct from a previously identified in vitro growth defect associated with dysregulation of the nitrogen-related phosphotransferase system. The widespread conservation of SixA, and its coincidence with the phosphotransferase system studied here, suggests that this dephosphorylation mechanism may be conserved in other bacteria.

Functional determinants of a small protein controlling a broadly conserved bacterial sensor kinase.

The PhoQ/PhoP two-component system plays a vital role in the regulation of Mg2+ homeostasis, resistance to acid and hyperosmotic stress, cationic antimicrobial peptides, and virulence in Escherichia coli, Salmonella and related bacteria. Previous studies have shown that MgrB, a 47 amino acid membrane protein that is part of the PhoQ/PhoP regulon, inhibits the histidine kinase PhoQ. MgrB is part of a negative feedback loop modulating this two-component system that prevents hyperactivation of PhoQ and may also provide an entry point for additional input signals for the PhoQ/PhoP pathway. To explore the mechanism of action of MgrB, we have analyzed the effects of point mutations, C-terminal truncations and transmembrane region swaps on MgrB activity. In contrast with two other known membrane protein regulators of histidine kinases in E. coli, we find that the MgrB TM region is necessary for PhoQ inhibition. Our results indicate that the TM region mediates interactions with PhoQ and that W20 is a key residue for PhoQ/MgrB complex formation. Additionally, mutations of the MgrB cytosolic region suggest that the two N-terminal lysines play an important role in regulating PhoQ activity. Alanine scanning mutagenesis of the periplasmic region of MgrB further indicates that, with the exception of a few highly conserved residues, most residues are not essential for MgrB's function as a PhoQ inhibitor. Our results indicate that the regulatory function of the small protein MgrB depends on distinct contributions from multiple residues spread across the protein. Interestingly, the TM region also appears to interact with other non-cognate histidine kinases in a bacterial two-hybrid assay, suggesting a potential route for evolving new small protein modulators of histidine kinases.

Deciphering the Role of Colicins during Colonization of the Mammalian Gut by Commensal E. coli.

Colicins are specific and potent toxins produced by Enterobacteriaceae that result in the rapid elimination of sensitive cells. Colicin production is commonly found throughout microbial populations, suggesting its potential importance for bacterial survival in complex microbial environments. Nonetheless, as colicin biology has been predominately studied using synthetic models, it remains unclear how colicin production contributes to survival and fitness of a colicin-producing commensal strain in a natural environment. To address this gap, we took advantage of MP1, an E. coli strain that harbors a colicinogenic plasmid and is a natural colonizer of the murine gut. Using this model, we validated that MP1 is competent for colicin production and then directly interrogated the importance of colicin production and immunity for MP1 survival in the murine gut. We showed that colicin production is dispensable for sustained colonization in the unperturbed gut. A strain lacking colicin production or immunity shows minimal fitness defects and can resist displacement by colicin producers. This report extends our understanding of the role that colicin production may play for E. coli during gut colonization and suggests that colicin production is not essential for a commensal to persist in its physiologic niche in the absence of exogenous challenges.

Bacterial colonization reprograms the neonatal gut metabolome.

Initial microbial colonization and later succession in the gut of human infants are linked to health and disease later in life. The timing of the appearance of the first gut microbiome, and the consequences for the early life metabolome, are just starting to be defined. Here, we evaluated the gut microbiome, proteome and metabolome in 88 African-American newborns using faecal samples collected in the first few days of life. Gut bacteria became detectable using molecular methods by 16 h after birth. Detailed analysis of the three most common species, Escherichia coli, Enterococcus faecalis and Bacteroides vulgatus, did not suggest a genomic signature for neonatal gut colonization. The appearance of bacteria was associated with reduced abundance of approximately 50 human proteins, decreased levels of free amino acids and an increase in products of bacterial fermentation, including acetate and succinate. Using flux balance modelling and in vitro experiments, we provide evidence that fermentation of amino acids provides a mechanism for the initial growth of E. coli, the most common early colonizer, under anaerobic conditions. These results provide a deep characterization of the first microbes in the human gut and show how the biochemical environment is altered by their appearance.

Author Correction: Films of Bacteria at Interfaces (FBI): Remodeling of Fluid Interfaces by Pseudomonas aeruginosa.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Colistin-resistance-mediated bacterial surface modification sensitizes phage infection.

Colistin is a drug of last resort for the treatment of many multidrug resistant Gram-negative bacteria, including Klebsiella pneumoniae However, bacteria readily acquire resistance to this antibiotic via lipopolysaccharide modifications caused by spontaneous mutations or from enzymes acquired by lateral gene transfer. The fitness cost associated with these modifications remains poorly understood. In this study, we show that colistin-resistant K. pneumoniae are more susceptible to killing by a newly isolated lytic phage than the colistin sensitive parent strain. We observe this behavior for colistin-resistance conferred by a horizontally transferred mcr-1 containing plasmid and also from the inactivation of the chromosomal gene mgrB By measuring zeta potentials, we found that the phage particles were negatively charged at neutral pH and that colistin-resistant bacteria had less negative zeta potentials than did wildtype. These results suggest that the decreased negative surface charge of colistin-resistant cells lowers the electrostatic repulsion between the phage and bacteria, thereby promoting phage adherence and subsequent infection. To further explore this, we tested the effect of phage treatment on K. pneumoniae growing in several different environments. We found that colistin-resistant cells were more susceptible to phage than were the wildtype cells when growing in biofilms or infected moth larvae and when colonizing the mammalian gut. A better understanding of these fitness costs may lead to new treatment approaches that minimize the emergence and spread of colistin-resistant pathogens in human and environmental reservoirs.

Bacterial Killing Activity of Polymorphonuclear Myeloid-Derived Suppressor Cells Isolated From Tumor-Bearing Dogs.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are implicated in the progression and outcome of a variety of pathological states, from cancer to infection. Our previous work has identified three antimicrobial peptides differentially expressed by PMN-MDSCs compared to conventional neutrophils isolated from dogs, mice, and human patients with cancer. We therefore hypothesized that PMN-MDSCs in dogs with cancer possess antimicrobial activity. In the current work, we observed that exposure of PMN-MDSCs to Gram-negative bacteria (Escherichia coli) increased the expression of reactive oxygen species by the PMN-MDSCs, indicating that they are capable of initiating an anti-microbial response. Electron microscopy revealed that the PMN-MDSCs phagocytosed Gram-negative and Gram-positive (Staphylococcus aureus) bacterial species. Lysis of bacteria within some of the PMN-MDSCs suggested bactericidal activity, which was confirmed by the recovery of significantly lower numbers of bacteria of both species following exposure to PMN-MDSCs isolated from tumor-bearing dogs. Our data therefore indicate that PMN-MDSCs isolated from dogs with cancer, in common with PMNs, have phagocytic and bactericidal activity. This nexus of immunosuppressive and antimicrobial activity reveals a hitherto unrecognized function of MDSCs.

Phage integration alters the respiratory strategy of its host.

Temperate bacteriophages are viruses that can incorporate their genomes into their bacterial hosts, existing there as prophages that refrain from killing the host cell until induced. Prophages are largely quiescent, but they can alter host phenotype through factors encoded in their genomes (often virulence factors) or by disrupting host genes as a result of integration. Here we describe another mechanism by which a prophage can modulate host phenotype. We show that a temperate phage that integrates in Escherichia coli reprograms host regulation of an anaerobic respiratory system, thereby inhibiting a bet hedging strategy. The phage exerts this effect by upregulating a host-encoded signal transduction protein through transcription initiated from a phage-encoded promoter. We further show that this phenomenon occurs not only in a laboratory strain of E. coli, but also in a natural isolate that contains a prophage at this site.

tRNA Methylation Is a Global Determinant of Bacterial Multi-drug Resistance.

Gram-negative bacteria are intrinsically resistant to drugs because of their double-membrane envelope structure that acts as a permeability barrier and as an anchor for efflux pumps. Antibiotics are blocked and expelled from cells and cannot reach high-enough intracellular concentrations to exert a therapeutic effect. Efforts to target one membrane protein at a time have been ineffective. Here, we show that m1G37-tRNA methylation determines the synthesis of a multitude of membrane proteins via its control of translation at proline codons near the start of open reading frames. Decreases in m1G37 levels in Escherichia coli and Salmonella impair membrane structure and sensitize these bacteria to multiple classes of antibiotics, rendering them incapable of developing resistance or persistence. Codon engineering of membrane-associated genes reduces their translational dependence on m1G37 and confers resistance. These findings highlight the potential of tRNA methylation in codon-specific translation to control the development of multi-drug resistance in Gram-negative bacteria.

Encoding biological recognition in a bicomponent cell-membrane mimic.

Self-assembling dendrimers have facilitated the discovery of periodic and quasiperiodic arrays of supramolecular architectures and the diverse functions derived from them. Examples are liquid quasicrystals and their approximants plus helical columns and spheres, including some that disregard chirality. The same periodic and quasiperiodic arrays were subsequently found in block copolymers, surfactants, lipids, glycolipids, and other complex molecules. Here we report the discovery of lamellar and hexagonal periodic arrays on the surface of vesicles generated from sequence-defined bicomponent monodisperse oligomers containing lipid and glycolipid mimics. These vesicles, known as glycodendrimersomes, act as cell-membrane mimics with hierarchical morphologies resembling bicomponent rafts. These nanosegregated morphologies diminish sugar-sugar interactions enabling stronger binding to sugar-binding proteins than densely packed arrangements of sugars. Importantly, this provides a mechanism to encode the reactivity of sugars via their interaction with sugar-binding proteins. The observed sugar phase-separated hierarchical arrays with lamellar and hexagonal morphologies that encode biological recognition are among the most complex architectures yet discovered in soft matter. The enhanced reactivity of the sugar displays likely has applications in material science and nanomedicine, with potential to evolve into related technologies.

The SOS Response Mediates Sustained Colonization of the Mammalian Gut.

Bacteria have a remarkable ability to survive, persist, and ultimately adapt to environmental challenges. A ubiquitous environmental hazard is DNA damage, and most bacteria have evolved a network of genes to combat genotoxic stress. This network is known as the SOS response and aids in bacterial survival by regulating genes involved in DNA repair and damage tolerance. Recently, the SOS response has been shown to play an important role in bacterial pathogenesis, and yet the role of the SOS response in nonpathogenic organisms and in physiological settings remains underexplored. Using a commensal Escherichia coli strain, MP1, we showed that the SOS response plays a vital role during colonization of the murine gut. In an unperturbed environment, the SOS-off mutant is impaired for stable colonization relative to a wild-type strain, suggesting the presence of genotoxic stress in the mouse gut. We evaluated the possible origins of genotoxic stress in the mouse gut by examining factors associated with the host versus the competing commensal organisms. In a dextran sulfate sodium (DSS) colitis model, the SOS-off colonization defect persisted but was not exacerbated. In contrast, in a germ-free model, the SOS-off mutant colonized with efficiency equal to that seen with the wild-type strain, suggesting that competing commensal organisms might be a significant source of genotoxic stress. This report extends our understanding of the importance of a functional SOS response for bacterial fitness in the context of a complex physiological environment and highlights the SOS response as a possible mechanism that contributes to ongoing genomic changes, including potential antibiotic resistance, in the microbiome of healthy hosts.