Contribution of the Oral and Gastrointestinal Microbiomes to Bloodstream Infections in Leukemia Patients.

Bloodstream infections (BSIs) pose a significant mortality risk for acute myeloid leukemia (AML) patients. It has been previously reported that intestinal domination (>30% relative abundance [RA] attributed to a single taxon) with the infecting taxa often precedes BSI in stem cell transplant patients. Using 16S rRNA amplicon sequencing, we analyzed oral and stool samples from 63 AML patients with BSIs to determine the correlation between the infectious agent and microbiome composition. Whole-genome sequencing and antimicrobial susceptibilities were performed on all BSI isolates. Species-level detection of the infectious agent and presence of antibiotic resistance determinants in the stool (blaCTX-M-15, blaCTX-M-14, cfrA, and vanA) were confirmed via digital droplet PCR (ddPCR). Individuals with Escherichia coli (stool P < 0.001), Pseudomonas aeruginosa (oral P = 0.004, stool P < 0.001), and viridans group streptococci (VGS) (oral P = 0.001) bacteremia had a significantly higher relative abundance of those respective genera than other BSI patients, which appeared to be site specific. Although 78% of patients showed presence of the infectious genera in the stool and/or saliva, only 7 exhibited microbiome domination. ddPCR confirmed species specificity of the 16S data and detected the antibiotic resistance determinants found in the BSI isolates within concurrent stools. Although gastrointestinal (GI) domination by an infecting organism was not present at the time of most BSIs in AML, the pathogens, along with AMR elements, were detectable in the majority of patients. Thus, rapid genetic assessment of oral and stool samples for the presence of potential pathogens and AMR determinants might inform personalized therapeutic approaches in immunocompromised patients with suspected infection. IMPORTANCE A major cause of mortality in hematologic malignancy patients is BSI. Previous studies have demonstrated that bacterial translocation from the GI microbiome is a major source of BSIs and is often preceded by increased levels of the infectious taxa in the GI (>30% abundance by 16S rRNA sequencing). In this study, we sought to better understand how domination and abundance levels of the oral and gut microbiome relate to bacteremia occurrence in acute myeloid leukemia patients. We conclude that analyses of both oral and stool samples can help identify BSI and antimicrobial resistance determinants, thus potentially improving the timing and tailoring of antibiotic treatment strategies for high-risk patients.

Purification and characterization of Shiga toxin 2f, an immunologically unrelated subtype of Shiga toxin 2.

BACKGROUND: Shiga-like toxin 2 (Stx2) is one of the most important virulence factors in enterohaemorrhagic Escherichia coli (E. coli) strains such as O157H7. Subtypes of Stx2 are diverse with respect to their sequence, toxicity, and distribution. The most diverse Stx2 subtype, Stx2f, is difficult to detect immunologically, but is becoming more frequently associated with human illness. METHODS AND FINDINGS: A purification regimen was developed for the purification of Stx2f involving cation exchange, hydrophobic interaction, anion exchange, and gel filtration. The molecular weight of Stx2f B-subunit was approximately 5 kDa, which appeared significantly smaller than that of Stx2a (6 kDa) on a SDS-PAGE gel, although the size of the A subunit was similar to Stx2a (30 kDa). Stx2f was shown to be active in both cell-free and cell-based assays. The 50% cytotoxic dose in Vero cells was 3.4 or 1.7 pg (depending on the assay conditions), about 3-5 times higher than the archetypical Stx2a, while the activity of Stx2f and Stx2a in a cell-free rabbit reticulocyte system was similar. Stx2f bound to both globotriose-lipopolysaccharide (Gb3-LPS) and globotetraose-LPS (Gb4-LPS, mimics for globotriaosylceramide and globotetraosylceramide, respectively), but its ability to bind Gb4-LPS was much stronger than Stx2a. Stx2f was also much more stable at low pH and high temperature compared to Stx2a, suggesting the toxin itself may survive harsher food preparation practices. CONCLUSIONS: Here, we detail the purification, biochemical properties, and toxicity of Stx2f, from an E. coli strain isolated from a feral pigeon. Information obtained in this study will be valuable for characterizing Stx2f and explaining the differences of Stx2a and Stx2f in host specificity and cytotoxicity.

Development and characterization of monoclonal antibodies against Shiga toxin 2 and their application for toxin detection in milk.

Human infection by Shiga toxin producing Escherichia coli (STEC) is one of the most prevalent foodborne diseases. Shiga toxin type 2 (Stx2) is the major contributor to hemolytic-uremic syndrome (HUS) and other systemic complications caused by STEC. Although outbreaks of HUS due to the consumption of dairy products occur frequently, very few reports are available on assays for the detection of Stx2 in milk. In this study, we describe the development of five high-affinity monoclonal antibodies (dissociation constants below nM range) against Stx2 using a recombinant toxoid as an immunogen. These antibodies, designated Stx2-1, Stx2-2, Stx2-3, Stx2-4, and Stx2-5 are IgG1 or IgG2a heavy-chain subclass with kappa light-chains, did not cross-react with Stx1 and showed different preferences to variants of Stx2. Western blot analyses demonstrate that mAbs Stx2-2 and Stx2-5 bind both the A- and B-subunits, whereas the other 3 mAbs bind the A-subunit of Stx2a only. All antibodies bound stronger to the native than to the denatured Stx2a except the mAb Stx2-3, which bound equally well to both forms of the toxin. Of the five mAbs, Stx2-5 was capable of neutralizing Stx2a mediated cytotoxicity in Vero cells. Highly sensitive ELISA and immuno-PCR assays, capable of detecting 1 and 0.01 pg/mL of Stx2a in milk, were developed using mAb pair Stx2-1 and Stx2-2. Such assays are useful for routine diagnosis of Stx2 contamination in milk production process, thus reducing the risk of STEC outbreaks.

A single-step purification and molecular characterization of functional Shiga toxin 2 variants from pathogenic Escherichia coli.

A one-step affinity chromatography method was developed to purify Shiga toxin 2 variants (Stx2) Stx2a, Stx2c, Stx2d and Stx2g from bacterial culture supernatants. Analysis of the purified Stx2 variants by denaturing gel electrophoresis revealed 32 kDa and 7 kDa protein bands, corresponding to the Stx2A- and B-subunits, respectively. However, native gel electrophoresis indicated that purified Stx2c and Stx2d were significantly higher in molecular weight than Stx2a and Stx2g. In a cytotoxicity assay with Hela cells, the 50% cytotoxic dose of Stx2a and Stx2g were 100 pg and 10 pg, respectively, but 1 ng each for Stx2c and Stx2d. Interestingly, analysis of the 50% inhibitory dose in a cell-free translational system from rabbit reticulocyte lysates indicated that Stx2g had a lower capacity to inhibit protein synthesis than the other Stx2 variants. The cytotoxicities in Hela cells were neutralized with an anti-Stx2B antibody and were denatured at 80 °C for 1 h. These findings demonstrated that Stx2 variants exhibited different toxicities, holotoxin structure, and stabilities using distinct systems for assessing toxin activities. The development of a simple method for purification of Stx2 variants will enable further studies of Stx2-mediated toxicity in various model systems.

Comparison of a high-density genetic linkage map to genome features in the model grass Brachypodium distachyon.

The small annual grass Brachypodium distachyon (Brachypodium) is rapidly emerging as a powerful model system to study questions unique to the grasses. Many Brachypodium resources have been developed including a whole genome sequence, highly efficient transformation and a large germplasm collection. We developed a genetic linkage map of Brachypodium using single nucleotide polymorphism (SNP) markers and an F(2) mapping population of 476 individuals. SNPs were identified by targeted resequencing of single copy genomic sequences. Using the Illumina GoldenGate Genotyping platform we placed 558 markers into five linkage groups corresponding to the five chromosomes of Brachypodium. The unusually long total genetic map length, 1,598 centiMorgans (cM), indicates that the Brachypodium mapping population has a high recombination rate. By comparing the genetic map to genome features we found that the recombination rate was positively correlated with gene density and negatively correlated with repetitive regions and sites of ancestral chromosome fusions that retained centromeric repeat sequences. A comparison of adjacent genome regions with high versus low recombination rates revealed a positive correlation between interspecific synteny and recombination rate.

Sensitive detection of Shiga Toxin 2 and some of its variants in environmental samples by a novel immuno-PCR assay.

Shiga toxin-producing Escherichia coli (STEC) in the environment has been reported frequently. However, robust detection of STEC in environmental samples remains difficult because the numbers of bacteria in samples are often below the detection threshold of the method. We developed a novel and sensitive immuno-PCR (IPCR) assay for the detection of Shiga toxin 2 (Stx2) and Stx2 variants. The assay involves immunocapture of Stx2 at the B subunit and real-time PCR amplification of a DNA marker linked to a detection antibody recognizing the Stx2 A subunit. The qualitative detection limit of the assay is 0.1 pg/ml in phosphate-buffered saline (PBS), with a quantification range of 10 to 100,000 pg/ml. The IPCR method was 10,000-fold more sensitive than an analogue conventional enzyme-linked immunosorbent assay (ELISA) in PBS. Although the sensitivity of the IPCR for detection of Stx2 was affected by environmental sample matrices of feces, feral swine colons, soil, and water from watersheds, application of the IPCR assay to 23 enriched cultures of fecal, feral swine colon, soil, and watershed samples collected from the environment revealed that the IPCR detected Stx2 in all 15 samples that were shown to be STEC positive by real-time PCR and culture methods, demonstrating a 100% sensitivity and specificity. The modification of the sandwich IPCR we have described in this study will be a sensitive and specific screening method for evaluating the occurrence of STEC in the environment.

Ricin toxicokinetics and its sensitive detection in mouse sera or feces using immuno-PCR.

BACKGROUND: Ricin (also called RCA-II or RCA(60)), one of the most potent toxins and documented bioweapons, is derived from castor beans of Ricinus communis. Several in vitro methods have been designed for ricin detection in complex food matrices in the event of intentional contamination. Recently, a novel Immuno-PCR (IPCR) assay was developed with a limit of detection of 10 fg/ml in a buffer matrix and about 10-1000-fold greater sensitivity than other methods in various food matrices. METHODS AND FINDINGS: In order to devise a better diagnostic test for ricin, the IPCR assay was adapted for the detection of ricin in biological samples collected from mice after intoxication. The limit of detection in both mouse sera and feces was as low as 1 pg/ml. Using the mouse intravenous (iv) model for ricin intoxication, a biphasic half-life of ricin, with a rapid t(1/2)α of 4 min and a slower t(1/2)ß of 86 min were observed. The molecular biodistribution time for ricin following oral ingestion was estimated using an antibody neutralization assay. Ricin was detected in the blood stream starting at approximately 6-7 h post- oral intoxication. Whole animal histopathological analysis was performed on mice treated orally or systemically with ricin. Severe lesions were observed in the pancreas, spleen and intestinal mesenteric lymph nodes, but no severe pathology in other major organs was observed. CONCLUSIONS: The determination of in vivo toxicokinetics and pathological effects of ricin following systemic and oral intoxication provide a better understanding of the etiology of intoxication and will help in the future design of more effective diagnostic and therapeutic methods.

Development of a novel immuno-PCR assay for detection of ricin in ground beef, liquid chicken egg, and milk.

Reliable, sensitive, and high-throughput methods are essential for food defense, to detect foodborne contaminants and to facilitate remediation and recovery from potential toxin-related incidents. Ricin is a protein toxin that has been used for intentional contamination of foods in the past. In this study, we developed procedures for quantification of ricin in foods using immuno-PCR (IPCR). The direct adsorption of ricin onto the wells of a microtitration plate was compared with indirect immobilization via a capture antibody (sandwich IPCR). The latter procedure provided much greater sensitivity. We also compared a protocol with the immunoassay and PCR conducted in a single plate to a two-step procedure in which the PCR was conducted in a second plate, following release and transfer of the DNA marker. The two-step procedure proved 1,000-fold more sensitive for ricin detection, so this format was used to detect ricin in spiked samples of ground beef, chicken egg, and milk, and the results were compared with those obtained from enzyme-linked immunosorbent assay (ELISA). The IPCR had a limit of detection of 10 pg/ml in chicken egg and milk samples and 100 pg/ml in ground beef extracts. Comparable ELISA results were in the 1 to 10 ng/ml range. Thus, IPCR affords sensitivity that is 10-fold greater in the ground beef matrix, 100-fold greater in the milk, and 1,000-fold greater in the egg matrix than the sensitivity obtained by ELISA. Further optimization of the sandwich IPCR was performed by comparing various antibody combinations. Among the four formats investigated, the pAb-pAb combination yielded the lowest limit of detection (10 fg/ml).

Structural characterization of Brachypodium genome and its syntenic relationship with rice and wheat.

Brachypodium distachyon (Brachypodium) has been recently recognized as an emerging model system for both comparative and functional genomics in grass species. In this study, 55,221 repeat masked Brachypodium BAC end sequences (BES) were used for comparative analysis against the 12 rice pseudomolecules. The analysis revealed that approximately 26.4% of BES have significant matches with the rice genome and 82.4% of the matches were homologous to known genes. Further analysis of paired-end BES and approximately 1.0 Mb sequences from nine selected BACs proved to be useful in revealing conserved regions and regions that have undergone considerable genomic changes. Differential gene amplification, insertions/deletions and inversions appeared to be the common evolutionary events that caused variations of microcolinearity at different orthologous genomic regions. It was found that approximately 17% of genes in the two genomes are not colinear in the orthologous regions. Analysis of BAC sequences also revealed higher gene density (approximately 9 kb/gene) and lower repeat DNA content (approximately 13.1%) in Brachypodium when compared to the orthologous rice regions, consistent with the smaller size of the Brachypodium genome. The 119 annotated Brachypodium genes were BLASTN compared against the wheat EST database and deletion bin mapped wheat ESTs. About 77% of the genes retrieved significant matches in the EST database, while 9.2% matched to the bin mapped ESTs. In some cases, genes in single Brachypodium BACs matched to multiple ESTs that were mapped to the same deletion bins, suggesting that the Brachypodium genome will be useful for ordering wheat ESTs within the deletion bins and developing specific markers at targeted regions in the wheat genome.

Leukotriene D4 up-regulates furin expression through CysLT1 receptor signaling.

Leukotriene (LT)D(4) is suggested to play a role in airway remodeling, which is characterized by fibrogenesis and airway smooth muscle cell hyperplasia. In this study, we investigated the effects of LTD(4) on the expression of furin, a proprotein convertase involved in the maturation/activation of several substrates implicated in the remodeling processes. HEK293 cells stably transfected with the CysLT1 receptor were used to study the transcriptional regulation of furin by LTD(4). Stimulation of the cells with LTD(4) resulted in a time- and concentration-dependent induction of furin mRNA and protein expression. The study of furin gene (fur) promoters P1, P1A, and P1B revealed a selective transactivation of the P1 promoter by LTD(4). Mutations in the activator protein (AP)-1-binding element of the P1 promoter resulted in the partial loss of transactivation by LTD(4). Binding of AP-1 transcription factor to fur P1 promoter after stimulation with LTD(4) was demonstrated by electrophoretic mobility shift assay, and supershift assays indicated the formation of c-Jun/c-Fos complexes. LTD(4) induced the maturation of the furin substrates membrane-type 1 matrix metalloproteinase and transforming growth factor-beta1, which was inhibited by the furin inhibitor alpha1-PDX. Finally, LTD(4) induced furin gene expression in monocytic THP-1 cells, which was abrogated using a selective CysLT1 receptor antagonist and inhibitors of the mitogen-activated protein kinases MEK-1, p38, and JunK. Our data show for the first time that LTD(4), via the CysLT1 receptor, can transcriptionally activate furin production with consequent maturation of furin substrates relevant to airway remodeling. These findings suggest that CysLT1 is involved in remodeling processes through modulation of furin transcription.

Transforming growth factor beta1 induces hypoxia-inducible factor-1 stabilization through selective inhibition of PHD2 expression.

The hypoxia-inducible transcription factor-1 (HIF-1) is central to a number of pathological processes through the induction of specific genes such as vascular endothelial growth factor (VEGF). Even though HIF-1 is highly regulated by cellular oxygen levels, other elements of the inflammatory and tumor microenvironment were shown to influence its activity under normal oxygen concentration. Among others, recent studies indicated that transforming growth factor (TGF) beta increases the expression of the regulatory HIF-1alpha subunit, and induces HIF-1 DNA binding activity. Here, we demonstrate that TGFbeta acts on HIF-1alpha accumulation and activity by increasing HIF-1alpha protein stability. In particular, we demonstrate that TGFbeta markedly and specifically decreases both mRNA and protein levels of a HIF-1alpha-associated prolyl hydroxylase (PHD), PHD2, through the Smad signaling pathway. As a consequence, the degradation of HIF-1alpha was inhibited as determined by impaired degradation of a reporter protein containing the HIF-1alpha oxygen-dependent degradation domain encompassing the PHD-targeted prolines. Moreover, inhibition of the TGFbeta1 converting enzyme, furin, resulted in increased PHD2 expression, and decreased basal HIF-1alpha and VEGF levels, suggesting that endogenous production of bioactive TGFbeta1 efficiently regulates HIF-1-targeted genes. This was reinforced by results from HIF-1alpha knock-out or HIF-1alpha-inhibited cells that show impairment in VEGF production in response to TGFbeta. This study reveals a novel mechanism by which a growth factor controls HIF-1 stability, and thereby drives the expression of specific genes, through the regulation of PHD2 levels.

The CDX2 transcription factor regulates furin expression during intestinal epithelial cell differentiation.

CDX2, a member of the caudal family of transcription factors, is involved in enterocyte lineage specification. CDX2 activates many intestine-specific genes, such as sucrase-isomaltase and lactase-phlorizin hydrolase (LPH), and adhesion proteins, namely, LI-cadherin and claudin-2. In this study, we show that the proprotein convertase furin, involved in proteolytic maturation of proprotein substrates including LPH and cell surface proteins, is a CDX2 target. Indeed, expression of the rat furin homolog was induced 1.5-fold, as determined by microarray experiments that compared control with CDX2-expressing intestinal epithelial cells (IEC-6). As determined by transient transfection assays in Caco-2/15 cells, the furin P1 promoter 1.3-kb fragment between SacI and NheI was essential for CDX2 transcriptional activation. Electrophoretic mobility shift/supershift assays followed by site-specific mutagenesis and chromatin immunoprecipitation identified the CDX DNA-binding site (CBS)2 sequence from nt -1827 to -1821 as the major CBS involved in furin P1 promoter activation. Increased furin mRNA and protein expression correlated with both CDX2 expression and intestinal epithelial cell differentiation. In addition, furin mRNAs were detected predominantly in differentiated epithelial cells of the villus, as determined by in situ hybridization. Treatment of Caco-2/15 cells with a furin inhibitor led to inhibition of LPH activity. Morphological differentiation of enterocyte-like features in Caco-2/15 such as epithelial cell polarity and brush-border formation were strongly attenuated by furin inhibition. These results suggest that CDX2 regulates furin expression in intestinal epithelial cells. Furin may be important in modulating the maturation and/or activation of key factors involved in enterocyte differentiation.

Hypoxia-enhanced expression of the proprotein convertase furin is mediated by hypoxia-inducible factor-1: impact on the bioactivation of proproteins.

Hypoxia is a common tumorigenesis enhancer, mostly owing to its impact on gene expression of many angiogenic and invasion-related mediators, some of which are natural substrates for the proprotein convertase furin. Analysis of furin promoters revealed the presence of putative binding sites for hypoxia-inducible factor-1 (HIF-1), a transcription complex that plays a pivotal role in cellular adaptation to hypoxia. In fact, we demonstrate herein that the levels of fur mRNA, encoding furin, are remarkably increased upon hypoxic challenge. Cotransfection of a HIF-1alpha dominant negative form in wild-type (WT) cells or transfection of a furin promoter-reporter gene in HIF-1-deficient cells indicated the requirement of HIF-1 for furin promoter activation by hypoxia. Direct HIF-1 action on the furin promoter was identified as a canonical hypoxia-responsive element site with enhancer capability. The hypoxic/HIF-1 regulation of furin correlated with an increased proteolytic activation of the substrates membrane-type 1 matrix metalloproteinase and transforming growth factor-beta1. Our findings unveil a new facet of the physiological consequences of hypoxia/HIF-1, through enhanced furin-induced proteolytic processing/activation of proproteins known to be involved in tumorigenesis.

Alternative pathway for the role of furin in tumor cell invasion process. Enhanced MMP-2 levels through bioactive TGFbeta.

The mammalian convertase furin plays a significant role in tumorigenesis and its overexpression was observed in a number of different cancer types. To date, however, few mechanisms of action have been described. Most of the information available concerns the invasion step and designates MT1-MMP, through the activation of MMP-2, as the bona fide substrate mediating furin activity. However, recent reports indicate furin-independent pathways for MT1-MMP activation. To gain further insights into the role of furin in the invasion process, we studied the in vitro invasive capacity of LoVo cells, a furin-deficient adenocarcinoma cell line transfected with wild-type furin. Furin complementation resulted in an increased cell invasiveness that correlated with their capacity to produce MMP-2. Chemical blockage of MMPs activity with BB-3103 or MMP-2-specific antibodies revealed that the increased invasive capacity of furin-complemented cells was mediated by MMP-2. Unexpectedly, furin complementation did not change the status of MT1-MMP expression or activation, but instead resulted in the production of mature and bioactive TGFbeta1. Western blot-analysis of TGFbeta1 fragmentation species indicated that TGFbeta maturation step required furin activity, whereas results from TGFbeta-inducible reporter assays in the presence of MMP inhibitors or exogenous MMP-2 suggested that the activation step was under MMP influence. In addition, blockage with TGFbeta neutralizing antibodies revealed that furin-induced invasiveness was mediated by endogenous production of TGFbeta. Taken together, our findings established the existence of a novel alternative/complementary pathway by which furin increases tumor cell invasion through an amplification/activation loop between MMP-2 and TGFbeta.

TACE/ADAM-17 maturation and activation of sheddase activity require proprotein convertase activity.

Proprotein convertases (PCs) have been proposed to play a role in tumor necrosis factor-alpha converting enzyme (TACE) processing/activation. Using the furin-deficient LoVo cells, as well as the furin-proficient synoviocytes and HT1080 cells expressing the furin inhibitor alpha(1)-PDX, we demonstrate that furin activity alone is not sufficient for effective maturation and activation of the TACE enzyme. Data from in vitro and in vivo cleavage assays indicate that PACE-4, PC5/PC6, PC1 and PC2 can directly cleave the TACE protein and/or peptide. PC inhibition in macrophages reduced the release of soluble TNF-alpha from transmembrane pro-TNF-alpha. We therefore conclude that furin, in addition to other candidate PCs, is involved in TACE maturation and activation.