Transcriptional alterations in bladder epithelial cells in response to infection with different morphological states of uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) may undergo a cyclic cascade of morphological alterations that are believed to enhance the potential of UPEC to evade host responses and re-infect host cell. However, knowledge on the pathogenic potential and host activation properties of UPEC during the morphological switch is limited. Microarray analysis was performed on mRNA isolated from human bladder epithelial cells (HBEP) after exposure to three different morphological states of UPEC (normal coliform, filamentous form and reverted form). Cells stimulated with filamentous bacteria showed the lowest number of significant gene alterations, although the number of enriched gene ontology classes was high suggesting diverse effects on many different classes of host genes. The normal coliform was in general superior in stimulating transcriptional activity in HBEP cells compared to the filamentous and reverted form. Top-scored gene entities activated by all three morphological states included IL17C, TNFAIP6, TNF, IL20, CXCL2, CXCL3, IL6 and CXCL8. The number of significantly changed canonical pathways was lower in HBEP cells stimulated with the reverted form (32 pathways), than in cells stimulated with the coliform (83 pathways) or filamentous bacteria (138 pathways). A host cell invasion assay showed that filamentous bacteria were unable to invade bladder cells, and that the number of intracellular bacteria was markedly lower in cells infected with the reverted form compared to the coliform. In conclusion, the morphological state of UPEC has major impact on the host bladder response both when evaluating the number and the identity of altered host genes and pathways.

Exogenous Vimentin Supplementation Transiently Affects Early Steps during HPV16 Pseudovirus Infection

Understanding and modulating the early steps in oncogenic Human Papillomavirus (HPV) infection has great cancer-preventative potential, as this virus is the etiological agent of virtually all cervical cancer cases and is associated with many other anogenital and oropharyngeal cancers. Previous work from our laboratory has identified cell-surface-expressed vimentin as a novel HPV16 pseudovirus (HPV16-PsVs)-binding molecule modulating its infectious potential. To further explore its mode of inhibiting HPV16-PsVs internalisation, we supplemented it with exogenous recombinant human vimentin and show that only the globular form of the molecule (as opposed to the filamentous form) inhibited HPV16-PsVs internalisation in vitro. Further, this inhibitory effect was only transient and not sustained over prolonged incubation times, as demonstrated in vitro and in vivo, possibly due to full-entry molecule engagement by the virions once saturation levels have been reached. The vimentin-mediated delay of HPV16-PsVs internalisation could be narrowed down to affecting multiple steps during the virus’ interaction with the host cell and was found to affect both heparan sulphate proteoglycan (HSPG) binding as well as the subsequent entry receptor complex engagement. Interestingly, decreased pseudovirus internalisation (but not infection) in the presence of vimentin was also demonstrated for oncogenic HPV types 18, 31 and 45. Together, these data demonstrate the potential of vimentin as a modulator of HPV infection which can be used as a tool to study early mechanisms in infectious internalisation. However, further refinement is needed with regard to vimentin’s stabilisation and formulation before its development as an alternative prophylactic means.

The isolation and characterization of Taphrina betulina and other yeasts residing in the Betula pendula phylloplane.

The phylloplane is an important microbial habitat and a reservoir of organisms that affect plant health, both positively and negatively. Taphrina betulina is the causative agent of birch witches′ broom disease. Taphrina are dimorphic, invading theirs hosts in a filamentous form and residing in the host phyllosphere in their non-infectious yeast form. As such, they are widely accepted to be found a resident yeasts on their hosts, even on healthy tissues; however, there is little experimental data to support this. With the aim of exploring the local infection ecology of T. betulina, we have isolated yeasts from the phylloplane of birch, using three classes of samples; from infected symptom bearing leaves inside brooms, healthy leaves from branches away from brooms on broom bearing trees, and symptom-free leaves from symptom-free trees. Isolations yielded 224 yeast strains, representing 11 taxa, including T. betulina, which was the most common isolate and was found in all sample classes, including asymptomatic leaves. Genotyping with two genetic markers revealed genetic diversity among these T. betulina isolates, with seven distinct genotype differentiated by the markers used. Of the 57 T. betulina strains, 22 representative strains were selected for further studies and preliminarily characterized, revealing differences in size and the ability to produced compounds with activity to activate the signalling pathway for the plant hormone auxin.

Intravital Imaging of Candida albicans Identifies Differential In Vitro and In Vivo Filamentation Phenotypes for Transcription Factor Deletion Mutants

Candida albicans is one of the most common causes of fungal infections in humans. C. albicans undergoes a transition from a round yeast form to a filamentous form during infection, which is critical for its ability to cause disease. Although this transition has been studied in the laboratory for years, methods to do so in an animal model of infection have been limited.

#45: Phenotypic heterogeneity among isolates of Candida albicans from specific anatomical niches in VLBW premature infants

Background Disseminated candidiasis is associated with high morbidity and mortality among very low birth weight (VLBW) infants. Research about C. albicans traits which lead to colonization versus invasive disease is lacking. Ability to grow in a filamentous form is a known virulence trait of C. albicans. Nevertheless, significant heterogeneity in filamentous growth in C. albicans clinical isolates has been documented (Hirakawa et. al). However, the clinical isolates investigated were obtained from varied tissue niches from hosts with differing risk factors, which could account for the variation in filamentation. A collection of C. albicans isolates prospectively collected from VLBW infants and their mothers demonstrated vertical transmission of C. albicans from mother to infant (Bliss et. al). We used the infants’ isolates from this collection to investigate filamentation amongst isolates obtained from the same tissue niche and host type. Methods The standard C. albicans reference strain in the field, SC5314 was chosen for a positive control as it demonstrates strong filamentation and high virulence. A strain lacking Efg1, a regulatory transcription factor that promotes filamentation, produces hypofilamentous colonies and was used as the negative control. Reference strains and the clinical isolates were grown on two types of stimulation media at 37 C for 7 days after which filamentation was scored. Results Filamentation development varied widely among isolates. Of the strains which were acquired through vertical transmission, 5 of 11 strains showed strong filamentation, 5 of 11 had an intermediate phenotype, and 1 did not display any filamentation. Three strains were acquired through horizontal transmission (RO10, RO54, and RO56). Of these, one showed strong filamentation whereas the other two failed to form filaments. None of the neonatal isolates showed colony wrinkling, a prominent phenotype in the reference strain SC5314. Strain RO61, an isolate associated with candidemia showed strong filamentation. In contrast, strain RO39, which was associated with dermatitis, was non-filamentous. Conclusions We observed phenotypic heterogeneity with respect to filament development among clinical isolates obtained from the same niche (rectal) in similar hosts (VLBW premature infants). This is consistent with the variation seen in Hirakawa et al and indicates that phenotypic variation is not a function of host risk factors nor clinical niche. Filamentation was not required for vertical transmission. No conclusions can be drawn based on this observation with only 2 strains, but it is interesting to note that the isolate which caused candidemia and death of the patient was highly filamentous whereas the isolate that caused dermatitis was not. Further investigation of these strains, including genomic sequencing and additional phenotypic evaluations, will explore the underlying factors leading to transmission and virulence of C. albicans in VLBW premature infants.

Constructing a DNAzyme Delivery and Expression System for Escherichia coli: A Prototype for Phage Therapy

Antisense oligonucleotides exhibit high potential for use as therapeutic agents. '10-23' DNAzymes are antisense molecules with a high chemical stability and catalytic efficiency. In the present study, we developed a phagemid containing a DNAzyme expression system regulated by two promoters. One of these promoters, pA1, promotes constitutive expression of Moloney murine leukemia virus reverse transcriptase (MoMuLV-RT). The other promoter, plac, regulates transcription of the RNA substrate from which MoMuLV-RT produces the DNAzyme by reverse transcription. The ftsZ DNAzyme was used to validate this expression system in the phagemid, named pDESCP. ftsZ DNAzyme expression altered the morphological pattern of Escherichia coli from a bacillary to filamentous form. In E. coli FtsZ is the primary component of the cell division apparatus, forming a structure known as Z-ring, which is the place of division. It is suggested that the DNAzyme ftsZ is decreasing the translation of this protein. Delivery of pDESCP into F+ strain of E. coli cells, using VCSM13, and the possible insertion of other DNAzymes into the cassette makes this phagemid an important prototype for phage therapy.

Stressful Conditions Give Rise to a Novel and Cryptic Filamentous Form of Caulerpa cylindracea

Morphological plasticity can enable algae to adapt to environmental change and increase their invasibility when introduced into new habitats. Nevertheless, there is still a lack of knowledge on how such plasticity can affect the invasion process of an invasive species. In this context, the high plasticity in the genus Caulerpa is well documented. However, after an extremely hot summer, a previously unreported filamentous morphology of Caulerpa cylindracea was detected; indeed, this morphology could only be confirmed taxonomically after in-depth morphological characterization and molecular analysis with the genetic marker tufA. We describe an ex situ culture experiment which showed that stressful conditions, such as high temperatures, can trigger this morphological change. Almost all of the thalli maintained at a constant extreme temperature of 29°C died, but after being returned to optimum temperature conditions, the filamentous morphology began to develop from the surviving microscopic tissue. In contrast, thalli at a control temperature of 21°C maintained the regular morphology throughout the experiment. When C. cylindracea develops this filamentous morphology, it may act as a cryptic invader because it is difficult to detect in the field. Furthermore, the filaments likely improve C. cylindracea’s invasive capabilities with regard to resistance, persistence and dispersion and may have an important role in the re-colonization process, after a population disappears following a period of stressful conditions. Possibly, C. cylindracea’s ability to respond plastically to stressful conditions might explain its remarkable success as an invasive species.

Inhibition of the filamentation of Candida albicans by Borojoa patinoi silver nanoparticles

Candida albicans is fungus capable of changing from yeast to filamentous form when it’s transformed from a normal commensal to an opportunistic pathogen. The development of alternatives that interfere with this transition could be an effective way to reduce candidiasis. In this regard, evaluate the inhibitory effect of two Borojoa patinoi silver nanoparticles (AgNPs) produced by green synthesis at 5 °C and 25 °C on the process of filamentation of Candida albicans. The percentage of inhibition of filamentous forms of C. albicans ATCC10231 and C. albicans SC5314 with AgNPs was determined. Results showed that temperature of synthesis affected both the shape and size of silver nanoparticles synthesized using Borojoa patinoi extracts. The inhibition percentage of filamentous forms of Candida albicans ATCC10231 when treated with silver nanoparticles synthesized at 5 °C was 85.9% and at 25 °C it was 40%. C. albicans SC5314 when treated with AgNP synthesized at 5 °C was 97.2% and at 25 °C it was 64%. Cell toxicity assay showed that at 100ng/ml, AgNPs synthesized at 25 °C were safe in MES-OV CRL-3272 cell line. Our results showed that the silver nanoparticles obtained from Borojoa patinoi are inhibitors of the filamentous process of C. albicans.

Identification of a filamentous form of kunitz protease inhibitor in Asteraceae

Filamentous structures were observed in purified extracts from chrysanthemum, gerbera, sunflower and zinnia. When purified filament proteins were subjected to SDS-PAGE, the major protein associated with filaments from all three species has an apparent molecular mass of ≈25 kDa. Protein bands from chrysanthemum, gerbera, and zinnia were subjected to N-terminal protein sequencing while proteins from sunflower were sequenced by CID MS/MS. All of the sequences shared highest similarity to the kunitz trypsin inhibitor family. The sequencing results indicated that the proteins lacked the signal sequences. We tested the gerbera filament protein for glycosylation and found that it was a glycoprotein. Together these results indicate that the filaments are composed of mature KTI protein. This is the first report of a KTI assembling into filaments and the first report of a filament forming Asteraceae enzyme.

Visualization of endogenous nuclear F-actin in mouse embryos reveals abnormal actin assembly after somatic cell nuclear transfer

Actin in the nucleus, referred to as nuclear actin, is involved in a variety of nuclear events. Nuclear actin is present as a globular (G-actin) and filamentous form (F-actin), and dynamic assembly/disassembly of nuclear actin profoundly affects nuclear functions. However, it is still challenging to observe endogenous nuclear F-actin. Here, we present a condition to visualize endogenous nuclear F-actin of mouse zygotes using different fixation methods. Zygotes fixed with paraformaldehyde and treated with fluorescently conjugated phalloidin show both short and long actin filaments in their pronuclei. Short nuclear actin filaments are characteristic of phalloidin staining, rather than the consequence of severing actin filaments by the fixation process, since long nuclear actin filaments probed with the nuclear actin chromobody are not disassembled into short filaments after fixation with paraformaldehyde. Furthermore, we find that nuclear actin assembly is impaired after somatic cell nuclear transfer (SCNT), suggesting abnormal nucleoskeleton structures in SCNT embryos. Taken together, our presented method for visualizing nuclear F-actin with phalloidin can be used to observe the states of nuclear actin assembly, and revealed improper reprogramming of actin nucleoskeleton structures in cloned mouse embryos.