Breck A. Duerkop

Immune Responses to the Microbiota at the Intestinal Mucosal Surface

The mammalian intestinal mucosal surface is continuously exposed to a complex and dynamic community of microorganisms. These microbes establish symbiotic relationships with their hosts, making important contributions to metabolism and digestive efficiency. The intestinal epithelial surface is the primary interface between the vast microbiota and internal host tissues. Given the enormous numbers of enteric bacteria and the persistent threat of opportunistic invasion, it is crucial that mammalian hosts monitor and regulate microbial interactions with intestinal epithelial surfaces. Here we discuss recent insights into how the innate and adaptive arms of the immune system collaborate to maintain homeostasis at the luminal surface of the intestinal host-microbial interface. These findings are also yielding a better understanding of how symbiotic host-microbial relationships can break down in inflammatory bowel disease.

A composite bacteriophage alters colonization by an intestinal commensal bacterium

The mammalian intestine is home to a dense community of bacteria and its associated bacteriophage (phage). Virtually nothing is known about how phages impact the establishment and maintenance of resident bacterial communities in the intestine. Here, we examine the phages harbored by Enterococcus faecalis, a commensal of the human intestine. We show that E. faecalis strain V583 produces a composite phage (ϕV1/7) derived from two distinct chromosomally encoded prophage elements. One prophage, prophage 1 (ϕV1), encodes the structural genes necessary for phage particle production. Another prophage, prophage 7 (ϕV7), is required for phage infection of susceptible host bacteria. Production of ϕV1/7 is controlled, in part, by nutrient availability, because ϕV1/7 particle numbers are elevated by free amino acids in culture and during growth in the mouse intestine. ϕV1/7 confers an advantage to E. faecalis V583 during competition with other E. faecalis strains in vitro and in vivo. Thus, we propose that E. faecalis V583 uses phage particles to establish and maintain dominance of its intestinal niche in the presence of closely related competing strains. Our findings indicate that bacteriophages can impact the dynamics of bacterial colonization in the mammalian intestinal ecosystem.

A structurally unrelated mimic of a Pseudomonas aeruginosa acyl-homoserine lactone quorum-sensing signal.

The pathogenic bacterium Pseudomonas aeruginosa uses acyl-homoserine lactone quorum-sensing signals to coordinate the expression of a battery of virulence genes in a cascade of regulatory events. The quorum-sensing signal that triggers the cascade is N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL), which interacts with two signal receptor-transcription factors, LasR and QscR. This signal is base labile, and it is degraded by mammalian PON lactonases. We have identified a structurally unrelated triphenyl mimic of 3OC12-HSL that is base-insensitive and PON-resistant. The triphenyl mimic seems to interact specifically with LasR but not with QscR. In silico analysis suggests that the mimic fits into the 3OC12-HSL-binding site of LasR and makes key contacts with LasR. The triphenyl mimic is an excellent scaffold for developing quorum-sensing inhibitors, and its stability and potency make it ideal for biotechnology uses such as heterologous gene expression.

Quorum-Sensing Control of Antibiotic Synthesis in Burkholderia thailandensis

The genome of Burkholderia thailandensis codes for several LuxR-LuxI quorum-sensing systems. We used B. thailandensis quorum-sensing deletion mutants and recombinant Escherichia coli to determine the nature of the signals produced by one of the systems, BtaR2-BtaI2, and to show that this system controls genes required for the synthesis of an antibiotic. BtaI2 is an acyl-homoserine lactone (acyl-HSL) synthase that produces two hydroxylated acyl-HSLs, N-3-hydroxy-decanoyl-HSL (3OHC(10)-HSL) and N-3-hydroxy-octanoyl-HSL (3OHC(8)-HSL). The btaI2 gene is positively regulated by BtaR2 in response to either 3OHC(10)-HSL or 3OHC(8)-HSL. The btaR2-btaI2 genes are located within clusters of genes with annotations that suggest they are involved in the synthesis of polyketide or peptide antibiotics. Stationary-phase cultures of wild-type B. thailandensis, but not a btaR2 mutant or a strain deficient in acyl-HSL synthesis, produced an antibiotic effective against gram-positive bacteria. Two of the putative antibiotic synthesis gene clusters require BtaR2 and either 3OHC(10)-HSL or 3OHC(8)-HSL for activation. This represents another example where antibiotic synthesis is controlled by quorum sensing, and it has implications for the evolutionary divergence of B. thailandensis and its close relatives Burkholderia pseudomallei and Burkholderia mallei.

Mutational Analysis of Burkholderia thailandensis Quorum Sensing and Self-Aggregation

Acyl-homoserine lactone (acyl-HSL) quorum-sensing signaling is common to many Proteobacteria. Acyl-HSLs are synthesized by the LuxI family of synthases, and the signal response is mediated by members of the LuxR family of transcriptional regulators. Burkholderia thailandensis is a member of a closely related cluster of three species, including the animal pathogens Burkholderia mallei and Burkholderia pseudomallei. Members of this group have similar luxI and luxR homologs, and these genes contribute to B. pseudomallei and B. mallei virulence. B. thailandensis possesses three pairs of luxI-luxR homologs. One of these pairs, BtaI2-BtaR2, has been shown to produce and respond to 3OHC(10)-HSL and to control the synthesis of an antibiotic. By using a markerless-exhange method, we constructed an assortment of B. thailandensis quorum-sensing mutants, and we used these mutants to show that BtaI1 is responsible for C(8)-HSL production and BtaI3 is responsible for 3OHC(8)-HSL production. We also show that a strain incapable of acyl-HSL production is capable of growth on the same assortment of carbon and nitrogen sources as the wild type. Furthermore, this mutant shows no loss of virulence compared to the wild type in mice. However, the wild type self-aggregates in minimal medium, whereas the quorum-sensing mutant does not. The wild-type aggregation phenotype is recovered by addition of the BtaI1-R1 HSL signal C(8)-HSL. We propose that the key function of the BtaR1-BtaI1 quorum-sensing system is to cause cells to gather into aggregates once a sufficient population has been established.

Quorum-sensing-regulated bactobolin production by Burkholderia thailandensis E264.

Bacterial acyl-homoserine lactones upregulated an uncharacterized gene cluster (bta) in Burkholderia thailandensis E264 to produce an uncharacterized polar antibiotic. The antibiotic is identified as a mixture of four bactobolins. Annotation of the bta cluster allows us to propose a biosynthetic scheme for bactobolin and reveals unusual enzymatic reactions for further study.

Evaluation of methods to purify virus-like particles for metagenomic sequencing of intestinal viromes

BackgroundViruses are a significant component of the intestinal microbiota in mammals. In recent years, advances in sequencing technologies and data analysis techniques have enabled detailed metagenomic studies investigating intestinal viromes (collections of bacteriophage and eukaryotic viral nucleic acids) and their potential contributions to the ecology of the microbiota. An important component of virome studies is the isolation and purification of virus-like particles (VLPs) from intestinal contents or feces. Several methods have been applied to isolate VLPs from intestinal samples, yet to our knowledge, the efficiency and reproducibility between methods have not been explored. A rigorous evaluation of methods for VLP purification is critical as many studies begin to move from descriptive analyses of virus diversity to studies striving to quantitatively compare viral abundances across many samples. Therefore, reproducible VLP purification methods which allow for high sample throughput are needed. Here we compared and evaluated four methods for VLP purification using artificial intestinal microbiota samples of known bacterial and viral composition.ResultsWe compared the following four methods of VLP purification from fecal samples: (i) filtration + DNase, (ii) dithiothreitol treatment + filtration + DNase, (iii) filtration + DNase + PEG precipitation and (iv) filtration + DNase + CsCl density gradient centrifugation. Three of the four tested methods worked well for VLP purification. We observed several differences between methods related to the removal efficiency of bacterial and host DNAs and biases against specific phages. In particular the CsCl density gradient centrifugation method, which is frequently used for VLP purification, was most efficient in removing host derived DNA, but also showed strong discrimination against specific phages and showed a lower reproducibility of quantitative results.ConclusionsBased on our data we recommend the use of methods (i) or (ii) for large scale studies when quantitative comparison of viral abundances across samples is required. The CsCl density gradient centrifugation method, while being excellently suited to achieve highly purified samples, in our opinion, should be used with caution when performing quantitative studies.

Octanoyl-Homoserine Lactone Is the Cognate Signal for Burkholderia mallei BmaR1-BmaI1 Quorum Sensing

Acyl-homoserine lactones (HSLs) serve as quorum-sensing signals for many Proteobacteria. Members of the LuxI family of signal generators catalyze the production of acyl-HSLs, which bind to a cognate receptor in the LuxR family of transcription factors. The obligate animal pathogen Burkholderia mallei produces several acyl-HSLs, and the B. mallei genome has four luxR and two luxI homologs, each of which has been established as a virulence factor. To begin to delineate the relevant acyl-HSL signals for B. mallei LuxR homologs, we analyzed the BmaR1-BmaI1 system. A comparison of acyl-HSL profiles from B. mallei ATCC 23344 and a B. mallei bmaI1 mutant indicates that octanoyl-HSL synthesis is BmaI1 dependent. Furthermore, octanoyl-HSL is the predominant acyl-HSL produced by BmaI1 in recombinant Escherichia coli. The synthesis of soluble BmaR1 in recombinant E. coli requires octanoyl-HSL or decanoyl-HSL. Insoluble aggregates of BmaR1 are produced in the presence of other acyl-HSLs and in the absence of acyl-HSLs. The bmaI1 promoter is activated by BmaR1 and octanoyl-HSL, and a 20-bp inverted repeat in the bmaI1 promoter is required for bmaI1 activation. Purified BmaR1 binds to this promoter region. These findings implicate octanoyl-HSL as the signal for BmaR1-BmaI1 quorum sensing and show that octanoyl-HSL and BmaR1 activate bmaI1 transcription.

Oxidant Generation by Single Infected Monocytes after Short-Term Fluorescence Labeling of a Protozoan Parasite

ABSTRACT Leishmania spp. are intracellular protozoa residing in mononuclear phagocytes. Leishmania organisms are susceptible to microbicidal responses generated in response to phagocytosis. Assuming that both phagocyte and parasite populations are heterogeneous, it is advantageous to examine the response of individual cells phagocytosing living parasites. Because Leishmania spp. lose virulence during the raising of transfectants, we developed a method to label live Leishmania chagasi short-term with fluorescent dyes. Up to six parasite divisions were detected by flow cytometry after labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE), dioctadecyl-tetramethylindo carbocyanine perchlorate, or chloromethyl tetramethylrhodamine. Labeled parasites entered mononuclear phagocytes as determined by confocal and time-lapse microscopy. Dihydroethidium (DHE) was used to detect macrophage-derived oxidants generated during phagocytosis. Presumably Leishmania organisms are opsonized with host serum/tissue components such as complement prior to phagocytosis. Therefore, we investigated the effects of opsonization and found that this increased the efficiency of CFSE-labeled parasite entry into monocytes (84.6% ± 8.8% versus 20.2% ± 3.8% monocytes infected; P < 0.001). Opsonization also increased the percentage of phagocytes undergoing a respiratory burst (66.0% ± 6.3% versus 41.0% ± 8.3% of monocytes containing CFSE-labeled parasites; P < 0.001) and the magnitude of oxidant generation by each infected monocyte. Inhibitor data indicated that DHE was oxidized by products of the NADPH oxidase. These data suggest that opsonized serum components such as complement lead to more efficient entry of Leishmania into their target cells but at the same time activate the phagocyte oxidase to generate microbicidal products in infected cells. The parasite must balance these positive and negative survival effects in order to initiate a viable infection.

The Burkholderia mallei BmaR3-BmaI3 Quorum-Sensing System Produces and Responds to N-3-Hydroxy-Octanoyl Homoserine Lactone

Burkholderia mallei has two acyl-homoserine lactone (acyl-HSL) signal generator-receptor pairs and two additional signal receptors, all of which contribute to virulence. We show that B. mallei produces N-3-hydroxy-octanoyl HSL (3OHC8-HSL) but a bmaI3 mutant does not. Recombinant Escherichia coli expressing BmaI3 produces hydroxylated acyl-HSLs, with 3OHC8-HSL being the most abundant compound. In recombinant E. coli, BmaR3 responds to 3OHC8-HSL but not to other acyl-HSLs. These data indicate that the signal for BmaR3-BmaI3 quorum sensing is 3OHC8-HSL.