Mechanisms Involved in the Active Secretion of CTX-M-15 ß-Lactamase by Pathogenic Escherichia coli ST131.

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, 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.

A hub-and-spoke nuclear lamina architecture in trypanosomes.

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.

Gene duplication drives genome expansion in a major lineage of Thaumarchaeota.

Ammonia-oxidising archaea of the phylum Thaumarchaeota are important organisms in the nitrogen cycle, but the mechanisms driving their radiation into diverse ecosystems remain underexplored. Here, existing thaumarchaeotal genomes are complemented with 12 genomes belonging to the previously under-sampled Nitrososphaerales to investigate the impact of lateral gene transfer (LGT), gene duplication and loss across thaumarchaeotal evolution. We reveal a major role for gene duplication in driving genome expansion subsequent to early LGT. In particular, two large LGT events are identified into Nitrososphaerales and the fate of these gene families is highly lineage-specific, being lost in some descendant lineages, but undergoing extensive duplication in others, suggesting niche-specific roles. Notably, some genes involved in carbohydrate transport or coenzyme metabolism were duplicated, likely facilitating niche specialisation in soils and sediments. Overall, our results suggest that LGT followed by gene duplication drives Nitrososphaerales evolution, highlighting a previously under-appreciated mechanism of genome expansion in archaea.

Involvement in surface antigen expression by a moonlighting FG-repeat nucleoporin in trypanosomes.

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.

Formin Is Associated with Left-Right Asymmetry in the Pond Snail and the Frog.

While components of the pathway that establishes left-right asymmetry have been identified in diverse animals, from vertebrates to flies, it is striking that the genes involved in the first symmetry-breaking step remain wholly unknown in the most obviously chiral animals, the gastropod snails. Previously, research on snails was used to show that left-right signaling of Nodal, downstream of symmetry breaking, may be an ancestral feature of the Bilateria [1 and 2]. Here, we report that a disabling mutation in one copy of a tandemly duplicated, diaphanous-related formin is perfectly associated with symmetry breaking in the pond snail. This is supported by the observation that an anti-formin drug treatment converts dextral snail embryos to a sinistral phenocopy, and in frogs, drug inhibition or overexpression by microinjection of formin has a chirality-randomizing effect in early (pre-cilia) embryos. Contrary to expectations based on existing models [3, 4 and 5], we discovered asymmetric gene expression in 2- and 4-cell snail embryos, preceding morphological asymmetry. As the formin-actin filament has been shown to be part of an asymmetry-breaking switch in vitro [6 and 7], together these results are consistent with the view that animals with diverse body plans may derive their asymmetries from the same intracellular chiral elements [8].

Touching from a distance.

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.

Nuclear pore complex evolution: a trypanosome Mlp analogue functions in chromosomal segregation but lacks transcriptional barrier activity.

The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organization. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialized regions of active transcription, whereas Mlp2 associates with the mitotic spindle/NPC in a cell cycle-dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity of telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analogue of Mlp/Tpr proteins, and, together with the lamina analogue NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina.

NUP-1 Is a large coiled-coil nucleoskeletal protein in trypanosomes with lamin-like functions.

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.

Specializations in a successful parasite: what makes the bloodstream-form African trypanosome so deadly?

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.

Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition.

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. METHODOLOGY/PRINCIPAL FINDINGS: We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities. CONCLUSIONS: M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×10(5) cells g(-1) using Griffiths and 4.25×10(6) cells g(-1) using FastDNA® Spin kit.

Comparison of Leishmania OligoC-TesT PCR with conventional and real-time PCR for Diagnosis of canine Leishmania infection.

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

Does capture-recapture analysis provide more reliable estimates of the incidence and prevalence of leg ulcers in the community?

OBJECTIVE: To determine whether capture-recapture analysis provides more reliable estimates of the cumulative incidence and prevalence of leg ulcers in Auckland, New Zealand. METHODS: A population-based, cross-sectional study was conducted in the Central and North Auckland health districts of New Zealand in 1998. Cases were identified through health professional referral and by self-notification. All ages and ulcer types were investigated. Both traditional and capture-recapture methods of analysis were used to estimate the cumulative incidence and prevalence of leg ulcers in the study population. RESULTS: Four hundred and twenty-six people with current leg ulcers were identified during the 12-month study period. Using traditional methods of analysis, the annual cumulative incidence rate of leg ulcers in Auckland was 32 per 100,000, with a point prevalence of 39 per 100,000 and a period prevalence of 79 per 100,000 per year. Results from capture-recapture analysis, however, suggest an annual cumulative incidence rate of 252 per 100,000, with a point prevalence of 248 per 100,000 and a period prevalence of 530 per 100,000 per year. CONCLUSIONS: The traditional method of calculating cumulative incidence and prevalence clearly under-estimates the frequency of leg ulcers in the Auckland region. Capture-recapture analysis provides a more reliable estimate of disease frequency, since cases that remain unidentified in the population are considered.