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Infections caused by antimicrobial-resistant bacterial pathogens are fast becoming an important global health issue. Strains of Escherichia coli are common causal agents of urinary tract infection and can carry multiple resistance genes. This includes the gene bla(CTX-M-15), which encodes an extended-spectrum beta-lactamase (ESBL). While studying antimicrobial resistance (AMR) in the environment, we isolated several strains of E. coli ST131 downstream of a wastewater treatment plan (WWTP) in a local river. These isolates were surviving in the river sediment, and characterization proved that a multiresistant phenotype was evident. Here, we show that E. coli strain 48 (river isolate ST131) provided a protective effect against a third-generation cephalosporin (cefotaxime) for susceptible E. coli strain 33 (river isolate ST3576) through secretion of a functional ESBL into the growth medium. Furthermore, extracellular ESBL activity was stable for at least 24 h after secretion. Proteomic and molecular genetic analyses identified CTX-M-15 as the major secreted ESBL responsible for the observed protective effect. In contrast to previous studies, outer membrane vesicles (OMVs) were not the route for CTX-M-15 secretion. Indeed, mutation of the type I secretion system led to a significant reduction in the growth of the ESBL-producing strain as well as a significantly reduced ability to confer protective effect. We speculate that CTX-M-15 secretion, mediated through active secretion using molecular machinery, provides a public goods service by facilitating the survival of otherwise susceptible bacteria in the presence of cefotaxime.

The nuclear lamina supports many functions, including maintaining nuclear structure and gene expression control, and correct spatio-temporal assembly is vital to meet these activities. Recently, multiple lamina systems have been described that, despite independent evolutionary origins, share analogous functions. In trypanosomatids the two known lamina proteins, NUP-1 and NUP-2, have molecular masses of 450 and 170 kDa, respectively, which demands a distinct architecture from the ∼60 kDa lamin-based system of metazoa and other lineages. To uncover organizational principles for the trypanosome lamina we generated NUP-1 deletion mutants to identify domains and their arrangements responsible for oligomerization. We found that both the N- and C-termini act as interaction hubs, and that perturbation of these interactions impacts additional components of the lamina and nuclear envelope. Furthermore, the assembly of NUP-1 terminal domains suggests intrinsic organizational capacity. Remarkably, there is little impact on silencing of telomeric variant surface glycoprotein genes. We suggest that both terminal domains of NUP-1 have roles in assembling the trypanosome lamina and propose a novel architecture based on a hub-and-spoke configuration., Summary: The arrangement of the lamina in trypanosomatids has architectural differences to the metazoan system.

Infections caused by antimicrobial resistant bacterial pathogens are fast becoming an important global health issue. Strains of Escherichia coli are common causal agents of urinary tract infection and can carry multiple resistance genes. This includes the gene blaCTX-M-15 that encodes for an extended spectrum beta-lactamase (ESBL). While studying antimicrobial resistance (AMR) in the environment we isolated several strains of E. coli ST131 downstream of a WWTP in a local river. These isolates were surviving in the river sediment and characterisation proved that a multi-resistant phenotype was evident. Here, we show that E. coli strain 48 (river isolate ST131), provided a protective effect against a third-generation cephalosporin (cefotaxime) for a susceptible E. coli strain 33 (river isolate ST3576) through secretion of a functional ESBL into the growth medium. Furthermore, extracellular ESBL activity was stable for at least 24 h after secretion. Proteomic and molecular genetic analyses identified CTX-M-15 as the major secreted ESBL responsible for the observed protective effect. In contrast to previous studies, OMVs were not the sole route for CTX-M-15 secretion. Indeed, mutation of the Type I secretion system led to a significant reduction in the growth of the ESBL-producing strain as well as a significantly reduced ability to confer protective effect. We speculate that CTX-M-15 secretion, mediated through active secretion using molecular machinery provides a public goods service by facilitating the survival of otherwise susceptible bacteria in the presence of cefotaxime.Abstract importanceInfections caused by antimicrobial resistant bacterial pathogens have become an important global health issue. Wastewater treatment plants (WWTPs) have been identified as hotspots for the dissemination of antimicrobial resistant genes/bacteria into the environment. In this study, we investigated resistance enzyme secretion by a multi-drug resistant human pathogenic E. coli, isolated from a UK river, downstream of a WWTP. We present evidence that the resistant strain actively secreted an important resistance enzyme into the surrounding medium which degraded the antibiotic cefotaxime. This research provided evidence for the mechanism for secretion of this enzyme which could indicate a new target to tackle antibiotic resistance pathogens.

Recent evidence suggests that anthropogenic activity can increase the levels of antimicrobial resistance (AMR) in the environment. Rivers and waterways are significant examples of environmental settings that have become repositories of antibiotics and antibiotic resistance genes (ARGs). Our recent study quantified drug concentrations in freshwater samples taken at a range of sites located on the Thames catchment; the highest levels of antibiotics and other drugs were recorded downstream of waste water treatment plants (WWTPs). One specific antibiotic: Trimethoprim (TMP) was shown at elevated concentrations reaching 2000ng/L at particular sites. We have also shown a correlative relationship between the residue of TMP and the prevalence of sulfonamide antibiotic resistance genes such as sul1. Despite this, there is still no evidence of a causative relationship between TMP concentrations and the prevalence of the ARGs at river sites. The aim of the current study was to conduct in-depth analysis using a combination of large metagenomic, geospatial and chemical datasets, in order to conduct a comparison between those sites with the highest TMP and lowest TMP levels across the Thames catchment. We aimed to establish the proximity of these sites to WWTPs, their population equivalence (PE) and land coverage. A secondary aim was to investigate seasonal variation in TMP and ARGs. Exploring these factors will help to decipher the clinical relevance of ARG accumulation at river sites. A significant correlation was shown between TMP levels at river sites and their distance downstream from a WWTP. Three sites located on the Rivers Cut and Ray showed significantly higher TMP concentrations in winter compared to summer. The population equivalence (PE) for sites with the highest TMP levels was significantly higher than those with the lowest levels. The land coverage of sites with the highest TMP levels was significantly more urban/suburban than sites with the lowest TMP concentrations, which were found to be significantly more arable. Five ARGs relevant to TMP and sulfonamides were identified across the Thames catchment. The most prevalent ARG was sul1, which was significantly more prevalent in winter compared to summer. By contrast sul2 was found to be significantly more prevalent in summer compared to winter at a site on the River Coln. The prevalence of the class 1 integron marker gene (inti1) did not differ significantly by season or between sites with the highest/lowest TMP levels.

In previous work ([Davison et al., 2016][1]), we used both genetic mapping and a chemical knockdown to show that a frameshift mutation in one copy of a duplicated formin gene is most likely the mutation that causes changes in left-right (LR) asymmetry, or chirality, in the pond snail Lymnaea

The nuclear pore complex is an ancient component of the eukaryotic cell. We show here that an FG nucleoporin, TbNup53b, in trypanosomes has an association with the splicing machinery and roles in gene expression, indicating that moonlighting roles for nucleoporins are highly ancient and present in the earliest eukaryotes., Components of the nuclear periphery coordinate a multitude of activities, including macromolecular transport, cell-cycle progression, and chromatin organization. Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport, mRNA processing, and transcriptional regulation, and NPC components can define regions of high transcriptional activity in some organisms at the nuclear periphery and nucleoplasm. Lineage-specific features underpin several core nuclear functions and in trypanosomatids, which branched very early from other eukaryotes, unique protein components constitute the lamina, kinetochores, and parts of the NPCs. Here we describe a phenylalanine-glycine (FG)-repeat nucleoporin, TbNup53b, that has dual localizations within the nucleoplasm and NPC. In addition to association with nucleoporins, TbNup53b interacts with a known trans-splicing component, TSR1, and has a role in controlling expression of surface proteins including the nucleolar periphery-located, procyclin genes. Significantly, while several nucleoporins are implicated in intranuclear transcriptional regulation in metazoa, TbNup53b appears orthologous to components of the yeast/human Nup49/Nup58 complex, for which no transcriptional functions are known. These data suggest that FG-Nups are frequently co-opted to transcriptional functions during evolution and extend the presence of FG-repeat nucleoporin control of gene expression to trypanosomes, suggesting that this is a widespread and ancient eukaryotic feature, as well as underscoring once more flexibility within nucleoporin function.

The nuclear pore complex (NPC) is the sole mediator of bidirectional nucleo-cytoplasmic transport and is also an important scaffold for chromatin organization and transcriptional regulation. Proteomic studies of numerous diverse eukaryotic species initially characterized the NPC as built with a number of remarkably similar structural features, suggesting its status as an ancient and conserved eukaryotic cell component. However, further detailed analyses now suggest that several key specific NPC features have a more convoluted evolutionary history than initially assumed. Recently we reported on TbNup92, a component in trypanosomes of one such conserved structural feature, a basket-like structure on the nuclear face of the NPC. We showed that TbNup92 has similar roles to nuclear basket proteins from yeasts and animals (Mlp and Tpr, respectively) in interacting with both the NPC and the mitotic spindle. However, comparative genomics suggests that TbNup92 and Mlp/Tpr may be products of distinct evolutionary histories, raising the possibility that these gene products are analogs rather than direct orthologs. Taken together with recent evidence for divergence in the nuclear lamina and kinetochores, it is apparent that the trypanosome nucleus functions by employing several novel or highly divergent protein complexes in parallel with conserved elements. These findings have major implications for how the trypanosomatid nucleus operates and the evolution of hierarchical nuclear organization.

Most trypanosomatid parasites have both arthropod and mammalian or plant hosts, and the ability to survive and complete a developmental program in each of these very different environments is essential for life cycle progression and hence being a successful pathogen. For African trypanosomes, where the mammalian stage is exclusively extracellular, this presents specific challenges and requires evasion of both the acquired and innate immune systems, together with adaptation to a specific nutritional environment and resistance to mechanical and biochemical stresses. Here we consider the basis for these adaptations, the specific features of the mammalian infective trypanosome that are required to meet these challenges, and how these processes both inform on basic parasite biology and present potential therapeutic targets.

NUP1, the first example of a nuclear lamin analog in nonmetazoans, performs roles similar to those of lamins in maintaining the structure and organization of the nucleus in Trypanosoma brucei., A unifying feature of eukaryotic nuclear organization is genome segregation into transcriptionally active euchromatin and transcriptionally repressed heterochromatin. In metazoa, lamin proteins preserve nuclear integrity and higher order heterochromatin organization at the nuclear periphery, but no non-metazoan lamin orthologues have been identified, despite the likely presence of nucleoskeletal elements in many lineages. This suggests a metazoan-specific origin for lamins, and therefore that distinct protein elements must compose the nucleoskeleton in other lineages. The trypanosomatids are highly divergent organisms and possess well-documented but remarkably distinct mechanisms for control of gene expression, including polycistronic transcription and trans-splicing. NUP-1 is a large protein localizing to the nuclear periphery of Trypanosoma brucei and a candidate nucleoskeletal component. We sought to determine if NUP-1 mediates heterochromatin organization and gene regulation at the nuclear periphery by examining the influence of NUP-1 knockdown on morphology, chromatin positioning, and transcription. We demonstrate that NUP-1 is essential and part of a stable network at the inner face of the trypanosome nuclear envelope, since knockdown cells have abnormally shaped nuclei with compromised structural integrity. NUP-1 knockdown also disrupts organization of nuclear pore complexes and chromosomes. Most significantly, we find that NUP-1 is required to maintain the silenced state of developmentally regulated genes at the nuclear periphery; NUP-1 knockdown results in highly specific mis-regulation of telomere-proximal silenced variant surface glycoprotein (VSG) expression sites and procyclin loci, indicating a disruption to normal chromatin organization essential to life-cycle progression. Further, NUP-1 depletion leads to increased VSG switching and therefore appears to have a role in control of antigenic variation. Thus, analogous to vertebrate lamins, NUP-1 is a major component of the nucleoskeleton with key roles in organization of the nuclear periphery, heterochromatin, and epigenetic control of developmentally regulated loci., Author Summary Eukaryotes—fungi, plants, animals, and many unicellular organisms—are defined by the presence of a cell nucleus that contains the chromosomes and is enveloped by a lipid membrane lined on the inner face with a protein network called the lamina. Among other functions, the lamina serves as an anchorage site for the ends of chromosomes. In multicellular animals (metazoa), the lamina comprises a few related proteins called lamins, which are very important for many functions related to the nucleus; abnormal lamins result in multiple nuclear defects and diseases, including inappropriate gene expression and premature aging. Until now, however, lamins had been found only in metazoa; no protein of equivalent function had been identified in plants, fungi, or unicellular organisms. Here, we describe a protein from African trypanosomes—the single-cell parasites that cause sleeping sickness—that fulfils many lamin-like roles, including maintaining nuclear structure and organizing the chromosomes of this organism. We show that this protein, which we call NUP-1 for nuclear periphery protein-1, is vital for the antigenic variation mechanisms that allow the parasite to escape the host immune response. We propose that NUP-1 is a lamin analogue that performs similar functions in trypanosomes to those of authentic lamins in metazoa. These findings, we believe, have important implications for understanding the evolution of the nucleus.

Background: Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year.\ud Methodology/Principal Findings: We report a multi-operator blinded trial for a\ud rigorous comparison of five DNA extraction methods from a variety of soil and\ud faecal samples to assess recovery of M. bovis via real-time PCR detection. The\ud methods included four commercial kits: the QIAamp Stool Mini kit with a pretreatment\ud step, the FastDNA® Spin kit, the UltraCleanTM and PowerSoilTM soil kits\ud and a published manual method based on phenol:chloroform purification, termed\ud Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and\ud evaluated using a specific real-time PCR assay. A novel inhibition control assay was\ud used alongside spectrophotometric ratios to monitor the level of inhibitory\ud compounds affecting PCR, DNA yield, and purity. There were statistically\ud significant differences in M. bovis detection between methods of extraction and types\ud of environmental samples; no significant differences were observed between\ud operators. Processing times and costs were also evaluated. To improve M. bovis\ud detection further, the two best performing methods, FastDNA® Spin kit and\ud Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was\ud adopted, leading to improved sensitivities.\ud \ud Conclusions: M. bovis was successfully detected in all environmental samples; DNA\ud extraction using FastDNA® Spin kit was the most sensitive method with highest\ud recoveries from all soil types tested. For troublesome faecal samples, we have used\ud and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25 x 105 cells g-1 using Griffiths and 4.25 x 106 cells g-1 using\ud FastDNA® Spin kit.

There is a need for standardization and simplification of the existing methods for molecular detection of Leishmania infantum in the canine reservoir host. The commercially available OligoC-TesT kit incorporates standardized PCR reagents with rapid oligochromatographic dipstick detection of PCR products and is highly sensitive for use in humans but not yet independently validated for use in dogs. Here we compare the sensitivity of OligoC-TesT with those of nested kinetoplast DNA (kDNA) PCR, nested internal transcribed spacer 1 (ITS-1) PCR, and a PCR-hybridization protocol, using longitudinal naturally infected canine bone marrow samples whose parasite burdens were measured by real-time quantitative PCR (qPCR). The sensitivity of OligoC-TesT for infected dogs was 70% (95% confidence interval [CI], 63 to 78%), similar to that of kDNA PCR (72%; 95% CI, 65 to 80%; P = 0.69) but significantly greater than those of PCR-hybridization (61%; 95% CI, 53 to 69%; P = 0.007) and ITS-1 nested PCR (54%; 95% CI, 45 to 62%; P < 0.001); real-time qPCR had the highest sensitivity (91%; 95% CI, 85 to 95%; P < 0.001). OligoC-TesT sensitivity was greater for polysymptomatic and oligosymptomatic dogs than for asymptomatic dogs (93%, 74%, and 61%, respectively; P = 0.005), a trend also observed for the other qualitative PCR methods tested ( P ≤ 0.05). Test positivity increased with increasing parasite burdens, as measured by real-time qPCR: OligoC-TesT and kDNA PCR detected 100% and 99% of positive samples when parasite burdens exceeded 74 and 49 parasites/ml, respectively. OligoC-TesT has high sensitivity for detection of canine Leishmania infections; its ease of operation and ease of interpretation are further advantages for veterinary diagnostic laboratories and for large-scale survey work in developing countries.