Super-Resolution Fluorescence Microscopy Study of the Production of K1 Capsules by Escherichia coli: Evidence for the Differential Distribution of the Capsule at the Poles and the Equator of the Cell.

The production of Escherichia coli K1 serotype capsule was investigated using direct stochastic optical reconstruction microscopy with live bacteria and graphene oxide-coated coverslips, overcoming many morphological artifacts found in other high-resolution imaging techniques. Super-resolution fluorescence images showed that the K1 capsular polysaccharide is not uniformly distributed on the cell surface, as previously thought. These studies demonstrated that on the cell surfaces the K1 capsule at the poles had bimodal thicknesses of 238 ± 41 and 323 ± 62 nm, whereas at the equator, there was a monomodal thickness of 217 ± 29 nm. This bimodal variation was also observed in high-pressure light-scattering chromatography measurements of purified K1 capsular polysaccharide. Particle tracking demonstrated that the formation of the capsule was dominated by the expansion of lyso-phosphatidylglycerol (lyso-PG) rafts that anchor the capsular polysaccharide in the outer membrane, and the expansion of these rafts across the cell surface was driven by new material transported through the capsular biosynthesis channels. The discovery of thicker capsules at the poles of the cell will have implications in mediating interactions between the bacterium and its immediate environment.

Bacterial Surfaces: Front Lines in Host-Pathogen Interaction.

All bacteria are bound by at least one membrane that acts as a barrier between the cell's interior and the outside environment. Surface components within and attached to the cell membrane are essential for ensuring that the overall homeostasis of the cell is maintained. However, many surface components of the bacterial cell also have an indispensable role mediating interactions of the bacteria with their immediate environment and as such are essential to the pathogenesis of infectious disease. During the course of an infection, bacterial pathogens will encounter many different ecological niches where environmental conditions such as salinity, temperature, pH, and the availability of nutrients fluctuate. It is the bacterial cell surface that is at the front-line of these host-pathogen interactions often protecting the bacterium from hostile surroundings but at the same time playing a critical role in the adherence to host tissues promoting colonization and subsequent infection. To deal effectively with the changing environments that pathogens may encounter in different ecological niches within the host many of the surface components of the bacterial cell are subject to phenotypic variation resulting in heterogeneous subpopulations of bacteria within the clonal population. This dynamic phenotypic heterogeneity ensures that at least a small fraction of the population will be adapted for a particular circumstance should it arise. Diversity within the clonal population has often been masked by studies on entire bacterial populations where it was often assumed genes were expressed in a uniform manner. This chapter, therefore, aims to highlight the non-uniformity in certain cell surface structures and will discuss the implication of this heterogeneity in bacterial-host interaction. Some of the recent advances in studying bacterial surface structures at the single cell level will also be reviewed.

Phenotypic Heterogeneity in Expression of the K1 Polysaccharide Capsule of Uropathogenic Escherichia coli and Downregulation of the Capsule Genes during Growth in Urine.

Uropathogenic Escherichia coli (UPEC) is the major causative agent of uncomplicated urinary tract infections (UTI). The K1 capsule on the surface of UPEC strains is a key virulence factor, and its expression may be important in the onset and progression of UTI. In order to understand capsule expression in more detail, we analyzed its expression in the UPEC strain UTI89 during growth in rich medium (LB medium) and urine and during infection of a bladder epithelial cell line. Comparison of capsule gene transcription using a chromosomal gfp reporter fusion showed a significant reduction in transcription during growth in urine compared to that during growth in LB medium. When examined at the single-cell level, following growth in both media, capsule gene expression appears to be heterogeneous, with two distinct green fluorescent protein (GFP)-expressing populations. Using anti-K1 antibody, we showed that this heterogeneity in gene expression results in two populations of encapsulated and unencapsulated cells. We demonstrated that the capsule hinders attachment to and invasion of epithelial cells and that the unencapsulated cells within the population preferentially adhere to and invade bladder epithelial cells. We found that once internalized, UTI89 starts to produce capsule to aid in its intracellular survival and spread. We propose that this observed phenotypic diversity in capsule expression is a fitness strategy used by the bacterium to deal with the constantly changing environment of the urinary tract.

Lamellipodin Is Important for Cell-to-Cell Spread and Actin-Based Motility in Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen capable of invading a broad range of cell types and replicating within the host cell cytoplasm. This paper describes the colocalization of host cell lamellipodin (Lpd) with intracellular L. monocytogenes detectable 6 h postinfection of epithelial cells. The association was mediated via interactions between both the peckstrin homology (PH) domain in Lpd and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2] on the bacterial surface and by interactions between the C-terminal EVH1 (Ena/VASP [vasodilator-stimulated phosphoprotein] homology domain 1) binding domains of Lpd and the host VASP (vasodilator-stimulated phosphoprotein) recruited to the bacterial cell surface by the listerial ActA protein. Depletion of Lpd by short interfering RNA (siRNA) resulted in reduced plaque size and number, indicating a role for Lpd in cell-to-cell spread. In contrast, overexpression of Lpd resulted in an increase in the number of L. monocytogenes-containing protrusions (listeriopods). Manipulation of the levels of Lpd within the cell also affected the intracellular velocity of L. monocytogenes, with a reduction in Lpd corresponding to an increase in intracellular velocity. These data, together with the observation that Lpd accumulated at the interface between the bacteria and the developing actin tail at the initiation of actin-based movement, indicate a possible role for Lpd in the actin-based movement and the cell-to-cell spread of L. monocytogenes.

Three tandem promoters, together with IHF, regulate growth phase dependent expression of the Escherichia coli kps capsule gene cluster.

In this study we characterise three tandem promoters (PR1-1, PR1-2 and PR1-3) within the PR1 regulatory region of the Escherichia coli kps capsule gene cluster. Transcription from promoter PR1-2 was dependent on the activity of the upstream promoter PR1-1, which activated PR1-2 via transcription coupled DNA supercoiling. During growth at 37 °C a temporal pattern of transcription from all three promoters was observed with maximum transcriptional activity evident during mid-exponential phase followed by a sharp decrease in activity as the cells enter stationary phase. The growth phase dependent transcription was regulated by Integration Host Factor (IHF), which bound within the PR1 region to repress transcription from PR1-2 and PR1-3. This pattern of transcription was mirrored by growth phase dependent expression of the K1 capsule. Overall these data reveal a complex pattern of transcriptional regulation for an important virulence factor with IHF playing a role in regulating growth phase expression.

Sense of self and anorexia nervosa: A grounded theory.

OBJECTIVES: The aim of this study was to explore the nature of the relationship between the self and the eating disorder in individuals with a lifetime history of anorexia nervosa (AN). DESIGN: A qualitative design was used, given the exploratory nature of the study and the need to gain rich and in-depth data regarding the topic under investigation. METHOD: Semi-structured interviews were conducted with 11 women with a lifetime history of AN. Interview transcripts were analysed using constructivist grounded theory methodology. RESULTS: A theoretical framework of the nature of the relationship between the self and AN was developed, which included five related categories: AN taking over the self, AN protecting the self, sharing the self with AN, being no one without AN, and discovering the real me (accepting the fear). CONCLUSION: Participants described a process of the self being taken over by AN to the point where it was shared with the eating disorder. This led participants to fear being no one without AN and to be unable to let go of the disorder, appreciating AN's ability to protect the self. To recover from AN, participants had to discover the real self, by accepting the fear of the unknown and separating the self from AN. The findings have important implications for the target of therapeutic interventions to improve recovery rates. PRACTITIONER POINTS: The self is shared with the eating disorder in AN, and separating the self from AN is crucial to recover from the disorder. Therapeutic interventions for AN need to target the enmeshed relationship between the self and the eating disorder, as opposed to focusing exclusively on weight and shape concerns.

Functional outcome of botulinum toxin A injections to the lower limbs in cerebral palsy.

We evaluated gross motor function following botulinum toxin A (BTX-A) injections in the lower limbs of children with spastic cerebral palsy in a randomized clinical trial, using a cross-over design. Forty-nine children (24 males, 25 females, age range 22 to 80 months) were randomly allocated to two groups: group 1 received BTX-A and physiotherapy, and group 2 received physiotherapy alone for 6 months. At the end of this period, group 2 received BTX-A and physiotherapy and group 1 continued with physiotherapy alone. Assessment measures were the Gross Motor Function Measure (GMFM), the Vulpe Assessment Battery (VAB), joint range of movement, the Modified Ashworth Scale, and a parental questionnaire. Sustained gains in gross motor function were found in both groups of children but the only additional benefit found in group 1 was a significant increase in fine motor rating on the VAB. By contrast, parents rated the benefit of treatment highly. It is likely that assessment at 3 and 6 months post injection was too late to demonstrate peak gross motor function response and that changes in GMFM are not sustained over 6 months with a single dose. Further studies should investigate changes over shorter time periods and consider covariables such as BTX-A dosage, number of injection sites, and the role of repeated injections combined with other interventions such as casting.