Comparative analysis of reproductive tract microbiomes in modern and slower-growing broiler breeder lines.

Introduction: The reproductive tract microbiome in hens is of interest because bacteria in the reproductive tract could potentially affect fertilization and egg production, as well as integrate into the forming egg and vertically transmit to progeny. Methods: The reproductive tract microbiome of 37-week-old modern commercial Cobb breeding dams was compared with that of dams from a broiler Legacy line which has not undergone selection since 1986. All animals were kept together under the same management protocol from day of hatch to avoid confounders. Results: In regards to reproductive abilities, Cobb dams' eggs weighed more and the magnum section of their reproductive tract was longer. In regards to microbiome composition, it was found that the reproductive tract microbiomes of the two lines had a lot in common but also that the two breeds have unique reproductive tract microbiomes. Specifically, the order Pseudomonadales was higher in the magnum of Legacy dams, while Verrucomicrobiales was lower. In the infundibulum, Lactobacillales were higher in the Legacy dams while Verrucomicrobiales, Bacteroidales, RF32 and YS2 were lower. Discussion: our results show that breeding programs have modified not only the physiology of the reproductive tract but also the reproductive tract microbiome. Additional research is required to understand the implications of these changes in the reproductive tract microbiome on the chicken host.

The Effect of Oil-Based Cannabis Extracts on Metabolic Parameters and Microbiota Composition of Mice Fed a Standard and a High-Fat Diet.

The prevalence of obesity and obesity-related pathologies is lower in frequent cannabis users compared to non-users. It is well established that the endocannabinoid system has an important role in the development of obesity. We recently demonstrated that prolonged oral consumption of purified Δ-9 Tetrahydrocannabinol (THC), but not of cannabidiol (CBD), ameliorates diet-induced obesity and improves obesity-related metabolic complications in a high-fat diet mouse model. However, the effect of commercially available medical cannabis oils that contain numerous additional active molecules has not been examined. We tested herein the effects of THC- and CBD-enriched medical cannabis oils on obesity parameters and the gut microbiota composition of C57BL/6 male mice fed with either a high-fat or standard diet. We also assessed the levels of prominent endocannabinoids and endocannabinoid-like lipid mediators in the liver. THC-enriched extract prevented weight gain by a high-fat diet and attenuated diet-induced liver steatosis concomitantly with reduced levels of the lipid mediators palmitoyl ethanolamide (PEA) and docosahexaenoyl ethanolamide (DHEA) in the liver. In contrast, CBD-enriched extract had no effect on weight gain, but, on the contrary, it even exacerbated liver steatosis. An analysis of the gut microbiota revealed that mainly time but not treatment exerted a strong effect on gut microbiota alterations. From our data, we conclude that THC-enriched cannabis oil where THC is the main constituent exerts the optimal anti-obesity effects.

Vertical transmission of gut bacteria in commercial chickens is limited.

The existence of vertical transmission in chickens under commercial settings, where chicks are raised separately from adults, is unclear. To answer this question, the fecal microbiota of chicks hatched and grown separately was compared with their mothers' microbiota. Most amplicon sequence variants (ASVs) identified in hens were not detected at all in chicks up to two weeks of age by 16S rDNA sequencing, and those that were detected had a low incidence among the chicks. Nevertheless, a few ASVs that were common with the hens were highly prevalent among the chicks, implying that they were efficiently transmitted to chicks. These ASVs were culturable from the reproductive tract of hens and eggshells. Furthermore, interventions attempting to disrupt transmission resulted in a reduction in the prevalence of specific phylogenetic groups in chicks. To conclude, vertical transmission in commercial poultry grown separately from adults likely exists but is not efficient, possibly resulting in impairment of microbiota function. This implies that artificial exposure to adult bacterial strains might improve microbiota functioning.

Differences in gut bacterial community composition between modern and slower-growing broiler breeder lines: Implications of growth selection on microbiome composition.

In the last century broiler chicken lines have undergone an extensive breeding regime aimed primarily at growth and high meat yield. It is not known if breeding has also resulted in a change to the broiler breeder's associated gut microbiota. Here we compared the gut microbiota of 37-week-old commercial Cobb breeding dams with dams from a broiler Legacy line which has not undergone selection since 1986. The dams from both lines were kept together in the same shed under the same management protocol from day of hatch to avoid additional confounders. We chose this age to allow significant bacterial exchange, thus avoiding exposure dependent artifacts and so that we could compare dams at the same developmental state of adulthood and peak laying performance. Significant differences in the composition of the cecum bacterial communities were found. Bacteria of the genus Akkermansia, implicated in mucin degradation and associated with host metabolic health, accounted for 4.98% ± 5.04% of the Cobb cecum community, but were mostly absent from the ceca of the Legacy line dams. Inversely, Legacy dams had higher levels of Clostridiales, Lactobacillales and Aeromonadales. These results show that breeding has resulted in a change in the gut microbiota composition, likely by changing the physiological conditions in the mucosa. It remains unclear if changes in gut microbiota composition are a part of the mechanism affecting growth or are a secondary result of other physiological changes accelerating growth. Therefore, the identification of these changes opens the door to further targeted research.

Characterizing the chicken gut colonization ability of a diverse group of bacteria.

The development of probiotics for chickens is a rapidly expanding field. The main approach to probiotics is to administer the probiotic strain throughout the bird's life, usually through incorporation in the feed. However, probiotics which would utilize bacterial strains capable of permanently colonizing the gut after a single exposure are likely to have a greater impact on the developing gut community as well as on the host, would simplify probiotic use and also reduce costs in an industrial setting. Finally, very limited and conflicting information about the colonization ability of different bacterial strains has been reported. Here we report 2 colonization experiments using 14 different bacterial strains from diverse phylogenetic groups. In both experiments, groups of chicks were orally inoculated on the day of hatch with different bacterial strains that had been previously isolated from adult heavy breeders. In the first experiment, colonization of the bacterial strains in broiler chicks was determined 7 d after treatment. In the second experiment, colonization was followed in layer chicks until d 17. Ten of the bacterial strains, including Lactobacillales and Bacteroidales strains, were able to colonize chicks after a single exposure for the duration of the experiment. For a few of these strains, exposure had little effect compared to non-treated chicks due to natural background colonization. Only 4 strains failed to colonize the chicks. Moreover, it is shown that fecal samples are useful to identify and provide a dynamic view of colonization. We further analyzed the effects of artificial colonization on microbiota composition. Some of the strains used in this research were found to reduce Enterobacteriaceae family abundance, implying that they might be useful in reducing relevant pathogen levels. To conclude, our results show that the development of single exposure based probiotics is possible.

Characterizing 5-oxoproline sensing pathways of Salmonella enterica serovar typhimurium.

5-Oxoproline (5OP) is a poorly researched ubiquitous natural amino acid found in all life forms. We have previously shown that Salmonella enterica serovar Typhimurium (Salmonella) responds to 5OP exposure by reducing cyclic-di-GMP levels, and resultant cellulose dependent cellular aggregation in a YfeA and BcsA dependent manner. To understand if 5OP was specifically sensed by Salmonella we compared the interaction of Salmonella with 5OP to that of the chemically similar and biologically relevant molecule, L-proline. We show that L-proline but not 5OP can be utilized by Salmonella as a nutrient source. We also show that 5OP but not L-proline regulates cellulose dependent cellular aggregation. These results imply that 5OP is utilized by Salmonella as a specific signal. However, L-proline is a 5OP aggregation inhibitor implying that while it cannot activate the aggregation pathway by itself, it can inhibit 5OP dependent activation. We then show that in a L-proline transporter knockout mutant L-proline competition remain unaffected, implying sensing of 5OP is extracellular. Last, we identify a transcriptional effect of 5OP exposure, upregulation of the mgtCBR operon, known to be activated during host invasion. While mgtCBR is known to be regulated by both low pH and L-proline starvation, we show that 5OP regulation of mgtCBR is indirect through changes in pH and is not dependent on the 5OP chemical structure similarity to L-proline. We also show this response to be PhoPQ dependent. We further show that the aggregation response is independent of pH modulation, PhoPQ and MgtC and that the mgtCBR transcriptional response is independent of YfeA and BcsA. Thus, the two responses are mediated through two independent signaling pathways. To conclude, we show Salmonella responds to 5OP specifically to regulate aggregation and not specifically to regulate gene expression. When and where in the Salmonella life cycle does 5OP sensing takes place remains an open question. Furthermore, because 5OP inhibits c-di-GMP through the activation of an external sensor, and does not require an internalization step like many studied biofilm inhibitors, 5OP or derivatives might be developed into useful biofilm inhibitors.

Quantification of Microbial Fluorescent Sensors During Live Intracellular Infections.

The interaction of pathogens with their eukaryotic hosts during intracellular growth is critical to many diseases. However, the relative scarcity of pathogen biomolecules versus the abundant host biomolecule concentration can make quantitative evaluation of pathogen intracellular responses difficult. Recent years have seen an explosion in utilization of fluorescent proteins to serve as transcriptional reporters and biosensors for quantification of pathogen responses. Here, we describe a method to establish a fluorescent assay quantifying pathogen behavior during intracellular infection and to quantify these results at a single cell level. The sensitivity of these fluorescent assays permits the live observation of changing pathogen responses, while the ability to measure at a single cell level uncovers subpopulations of pathogens whose existence may be missed during the population-level assays often required to accumulate sufficient pathogen biomolecules for analysis.

Large Overlap Between the Intestinal and Reproductive Tract Microbiomes of Chickens.

Recent work characterized the chicken reproductive tract (oviduct) microbiome composition and its similarity to the egg and chick microbiomes. However, the origin of the oviduct microbiome has not been addressed yet. Here, we characterized the microbiome composition along the oviduct (infundibulum, magnum, and shell gland) as well as in the gut (jejunum and cecum) of broiler breeders at 37 weeks of age of the Cobb industrial breed. We found that while the microbiome composition along the oviduct is similar, the three sites, jejunum, cecum, and oviduct hold distinct microbiomes. However, there was also a large overlap in the composition of the gut and oviduct microbiomes, with 55 and 53% of amplicon sequence variants (ASVs) representing 96 and 90% of the total abundance in the jejunum and cecum, respectively, shared with the magnum. Furthermore, we identified a strong correlation between the relative abundance of ASVs in the gut and their probability to be found in the oviduct. These results suggest that material from the gut travels the full length of the oviduct. This is possibly the result of chicken physiology which includes the cloaca, a cavity to which both the intestinal and reproductive tracts open into. As the cloaca is common to birds, reptiles, amphibians, most fish, and monotremes, our finding may be relevant to many vertebrates. Importantly, these results indicate that mere presence in, and ascending of the oviduct are not virulence characteristics specific to pathogens, as commonly thought, but are the result of chicken physiology and characterize all gut bacteria. Furthermore, whereas a vertical transmission route from the hen to the chick has been suggested, our work starts laying a mechanistic foundation to this route, by describing the movement of gut bacteria to the oviduct, where they may be enclosed in the developing egg. Last, as our results show that gut material travels the full length of the oviduct, fertilization in poultry occurs in the presence of at least bacterial products if not live bacteria, and therefore food additives, probiotics, and diet possibly have a much more direct effect on reproduction and egg formation than previously considered.

Cyclic-di-GMP regulation promotes survival of a slow-replicating subpopulation of intracellular Salmonella Typhimurium.

Salmonella Typhimurium can invade and survive within macrophages where the bacterium encounters a range of host environmental conditions. Like many bacteria, S. Typhimurium rapidly responds to changing environments by the use of second messengers such as cyclic di-GMP (c-di-GMP). Here, we generate a fluorescent biosensor to measure c-di-GMP concentrations in thousands of individual bacteria during macrophage infection and to define the sensor enzymes important to c-di-GMP regulation. Three sensor phosphodiesterases were identified as critical to maintaining low c-di-GMP concentrations generated after initial phagocytosis by macrophages. Maintenance of low c-di-GMP concentrations by these phosphodiesterases was required to promote survival within macrophages and virulence for mice. Attenuation of S Typhimurium virulence was due to overproduction of c-di-GMP-regulated cellulose, as deletion of the cellulose synthase machinery restored virulence to a strain lacking enzymatic activity of the three phosphodiesterases. We further identified that the cellulose-mediated reduction in survival was constrained to a slow-replicating persister population of S. Typhimurium induced within the macrophage intracellular environment. As utilization of glucose has been shown to be required for S. Typhimurium macrophage survival, one possible hypothesis is that this persister population requires the glucose redirected to the synthesis of cellulose to maintain a slow-replicating, metabolically active state.

Breaking the population barrier by single cell analysis: one host against one pathogen.

Most of our understanding of the host-bacterium interaction has come from studies of bulk populations. In reality, highly adaptable and dynamic host cells and bacteria engage in complex, diverse interactions. This complexity necessarily limits the depth of understanding that can be gained with bulk population measurements. Here, we will review the merit of single cell analysis to characterize this diversity that can trigger heterogeneous outcomes. We will discuss heterogeneity of bacterial and host populations, differences in host microenvironments, technological advances that facilitate the analysis of rare subpopulations, and the potential relevance of these subpopulations to infection outcomes. We focus our discussion on intracellular bacterial pathogens and on methods that characterize and quantify RNA in single cells, aiming to highlight how novel methodologies have the potential to characterize the multidimensional process of infection and to provide answers to some of the most fundamental questions in the field.

Host cell attachment elicits posttranscriptional regulation in infecting enteropathogenic bacteria.

The mechanisms by which pathogens sense the host and respond by remodeling gene expression are poorly understood. Enteropathogenic Escherichia coli (EPEC), the cause of severe intestinal infection, employs a type III secretion system (T3SS) to inject effector proteins into intestinal epithelial cells. These effectors subvert host cell processes to promote bacterial colonization. We show that the T3SS also functions to sense the host cell and to trigger in response posttranscriptional remodeling of gene expression in the bacteria. We further show that upon effector injection, the effector-bound chaperone (CesT), which remains in the EPEC cytoplasm, antagonizes the posttranscriptional regulator CsrA. The CesT-CsrA interaction provokes reprogramming of expression of virulence and metabolic genes. This regulation is likely required for the pathogen's adaptation to life on the epithelium surface.

A direct screen for c-di-GMP modulators reveals a Salmonella Typhimurium periplasmic ʟ-arginine-sensing pathway.

Cyclic-di-GMP (c-di-GMP) is a bacterial second messenger that transduces internal and external signals and regulates bacterial motility and biofilm formation. Some organisms encode more than 100 c-di-GMP-modulating enzymes, but only for a few has a signal been defined that modulates their activity. We developed and applied a high-throughput, real-time flow cytometry method that uses a fluorescence resonance energy transfer (FRET)-based biosensor of free c-di-GMP to screen for signals that modulate its concentration within Salmonella Typhimurium. We identified multiple compounds, including glucose, N-acetyl-d-glucosamine, salicylic acid, and ʟ-arginine, that modulated the FRET signal and therefore the free c-di-GMP concentration. By screening a library of mutants, we identified proteins required for the c-di-GMP response to each compound. Furthermore, low micromolar concentrations of ʟ-arginine induced a rapid translation-independent increase in c-di-GMP concentrations and c-di-GMP-dependent cellulose synthesis, responses that required the regulatory periplasmic domain of the diguanylate cyclase STM1987. ʟ-Arginine signaling also required the periplasmic putative ʟ-arginine-binding protein ArtI, implying that ʟ-arginine sensing occurred in the periplasm. Among the 20 commonly used amino acids, S. Typhimurium specifically responded to ʟ-arginine with an increase in c-di-GMP, suggesting that ʟ-arginine may serve as a signal during S. Typhimurium infection. Our results demonstrate that a second-messenger biosensor can be used to identify environmental signals and define pathways that alter microbial behavior.

Structure of GrlR-GrlA complex that prevents GrlA activation of virulence genes.

The locus of enterocyte effacement (LEE) is essential for virulence of enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). The 41 genes of the LEE encode type III secretion system proteins and three associated regulators: Ler, GrlA and GrlR. Ler is a positive regulator for most of the LEE operons, including grlRA. GrlA controls the expression of ler, ehxCABD and flhDC operons. GrlR binds to GrlA and suppresses its function. Here we report the crystal structure of GrlR-GrlAΔ (aa 1-106) complex (2:1) and its functional characterization. We show that GrlR interacts with the Helix-Turn-Helix motif of GrlA. Moreover, GrlA binds to the promoter DNA fragments of ler, ehxCABD and flhDC, and GrlR outcompetes with these promoter DNA sequences for the Helix-Turn-Helix motif of GrlA. These findings provide mechanistic insight into a regulatory module for the virulence of EPEC and EHEC, two important pathogens that cause devastating diseases.

Dynamics of the type III secretion system activity of enteropathogenic Escherichia coli.

UNLABELLED: Type III secretion systems (TTSSs) are employed by pathogens to translocate host cells with effector proteins, which are crucial for virulence. The dynamics of effector translocation, behavior of the translocating bacteria, translocation temporal order, and relative amounts of each of the translocated effectors are all poorly characterized. To address these issues, we developed a microscopy-based assay that tracks effector translocation. We used this assay alongside a previously described real-time population-based translocation assay, focusing mainly on enteropathogenic Escherichia coli (EPEC) and partly comparing it to Salmonella. We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria. We also noted variability in host cell susceptibility, with some cells highly resistant to translocation. We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency. Further, we mapped the global effects of key TTSS-related components on TTSS activity. Our results provide a comprehensive description of the dynamics of the TTSS activity of EPEC and new insights into the mechanisms that control the dynamics. IMPORTANCE: EPEC and the closely related enterohemorrhagic Escherichia coli (EHEC) represent a global public health problem. New strategies to combat EPEC and EHEC infections are needed, and development of such strategies requires better understanding of their virulence machinery. The TTSS is a critical virulence mechanism employed by these pathogens, and by others, including Salmonella. In this study, we aimed at elucidating new aspects of TTSS function. The results obtained provide a comprehensive description of the dynamics of TTSS activity of EPEC and new insights into the mechanisms that control these changes. This knowledge sets the stage for further analysis of the system and may accelerate the development of new ways to treat EPEC and EHEC infections. Further, the newly described microscopy-based assay can be readily adapted to study the dynamics of TTSS activity in other pathogens.

The bacterial second messenger c-di-GMP: mechanisms of signalling.

Cyclic-di-GMP (c-di-GMP) regulates many important bacterial processes. Freely diffusible intracellular c-di-GMP is determined by the action of metabolizing enzymes that allow integration of numerous input signals. c-di-GMP specifically regulates multiple cellular processes by binding to diverse target molecules. This review highlights important questions in research into the mechanisms of c-di-GMP signalling and its role in bacterial physiology.

The crystal structure of Escherichia coli group 4 capsule protein GfcC reveals a domain organization resembling that of Wza.

We report the 1.9 Å resolution crystal structure of enteropathogenic Escherichia coli GfcC, a periplasmic protein encoded by the gfc operon, which is essential for assembly of group 4 polysaccharide capsule (O-antigen capsule). Presumed gene orthologs of gfcC are present in capsule-encoding regions of at least 29 genera of Gram-negative bacteria. GfcC, a member of the DUF1017 family, is comprised of tandem ß-grasp (ubiquitin-like) domains (D2 and D3) and a carboxyl-terminal amphipathic helix, a domain arrangement reminiscent of that of Wza that forms an exit pore for group 1 capsule export. Unlike the membrane-spanning C-terminal helix from Wza, the GfcC C-terminal helix packs against D3. Previously unobserved in a ß-grasp domain structure is a 48-residue helical hairpin insert in D2 that binds to D3, constraining its position and sequestering the carboxyl-terminal amphipathic helix. A centrally located and invariant Arg115 not only is essential for proper localization but also forms one of two mostly conserved pockets. Finally, we draw analogies between a GfcC protein fused to an outer membrane ß-barrel pore in some species and fusion proteins necessary for secreting biofilm-forming exopolysaccharides.

The type III secretion effector NleE inhibits NF-kappaB activation.

The complex host-pathogen interplay involves the recognition of the pathogen by the host's innate immune system and countermeasures taken by the pathogen. Detection of invading bacteria by the host leads to rapid activation of the transcription factor NF-kappaB, followed by inflammation and eradication of the intruders. In response, some pathogens, including enteropathogenic Escherichia coli (EPEC), acquired means of blocking NF-kappaB activation. We show that inhibition of NF-kappaB activation by EPEC involves the injection of NleE into the host cell. Importantly, we show that NleE inhibits NF-kappaB activation by preventing activation of IKKbeta and consequently the degradation of the NF-kappaB inhibitor, IkappaB. This NleE activity is enhanced by, but is not dependent on, a second injected effector, NleB. In conclusion, this study describes two effectors, NleB and NleE, with no similarity to other known proteins, used by pathogens to manipulate NF-kappaB signaling pathways.

Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli.

Bacteria use type III secretion systems (TTSS) to translocate effector proteins into host cells. Better understanding of the TTSS and its effectors' functions will require assays to measure their activities in vivo and in real time. We designed a real-time, high-throughput translocation assay that utilizes fusions of effector genes to the beta-lactamase reporter gene, positioned under the effector's native promoter and chromosomal location. Using this assay, we simultaneously and quantitatively analyzed the translocation kinetics of six core enteropathogenic E. coli effectors, EspF, EspG, EspH, EspZ, Map, and Tir. A distinct order in the efficiencies of effector translocation was observed. Translocation efficiency was determined by multiple factors, including the intrabacterial effector concentration, effector-chaperone interactions, the efficiency of bacterial attachment to the host cells, and possibly also by a translocation autoinhibition mechanism. The described real-time translocation assay could be easily adapted for varied applications in the study of bacterial pathogenesis.

Identification and characterization of NleI, a new non-LEE-encoded effector of enteropathogenic Escherichia coli (EPEC).

Enteropathogenic Escherichia coli (EPEC), a major gastrointestinal pathogen, causes infantile diarrhea in many developing countries. EPEC is an attaching and effacing (A/E) pathogen that utilizes a LEE-encoded type III secretion system (TTSS) to deliver effector proteins into host cells. These effectors have been identified as potential virulence factors in A/E pathogens including EPEC, enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR). We used a proteomics approach to identify a new non-LEE-encoded effector, NleI, from the EPEC sepL and sepD mutants. The nleI gene, located in a prophage-associated island with nleBCD, is also present in EHEC and CR but not in E. coli K-12. In EPEC, the transcription of nleI was increased upon sepD inactivation but remained unaffected in ler and sepL mutants. We demonstrated that NleI is secreted and translocated into HeLa cells in a TTSS-dependent manner and that the CesT chaperone is required for efficient NleI translocation. Nevertheless, under overexpression conditions, the first 20 amino acids of NleI are sufficient to support both secretion and translocation. After translocation, NleI can be detected in the cytoplasmic and membrane of HeLa cells.

Matefin, a Caenorhabditis elegans germ line-specific SUN-domain nuclear membrane protein, is essential for early embryonic and germ cell development.

Caenorhabditis elegans mtf-1 encodes matefin, which has a predicted SUN domain, a coiled-coil region, an anti-erbB-2 IgG domain, and two hydrophobic regions. We show that matefin is a nuclear membrane protein that colocalizes in vivo with Ce-lamin, the single nuclear lamin protein in C. elegans, and binds Ce-lamin in vitro but does not require Ce-lamin for its localization. Matefin is detected in all embryonic cells until midembryogenesis and thereafter only in germ-line cells. Embryonic matefin is maternally deposited, and matefin is the first nuclear membrane protein known to have germ line-restricted expression. Animals homozygous for an mtf-1 deletion allele show that matefin is essential for germ line maturation and survival. However, matefin is also required for embryogenesis because mtf-1 (RNAi) embryos die around the approximately 300-cell stage with defects in nuclear structure, DNA content, and chromatin morphology. Down-regulating matefin in mes-3 animals only slightly enhances embryonic lethality, and elimination of UNC-84, the only other SUN-domain gene in C. elegans, has no affect on mtf-1 (RNAi) animals. Thus, mtf-1 mediates a previously uncharacterized pathway(s) required for embryogenesis as well as germ line proliferation or survival.