Increased toxin expression in a Clostridium difficile mfd mutant

BACKGROUND: The symptoms of Clostridium difficile infection are mediated primarily by two toxins, TcdA and TcdB, the expression of which is governed by a multitude of factors including nutrient availability, growth phase and cell stress. Several global regulators have been implicated in the regulation of toxin expression, such as CcpA and CodY. RESULTS: During attempts to insertionally inactivate a putative secondary cell wall polysaccharide synthesis gene, we obtained several mutants containing off-target insertions. One mutant displayed an unusual branched colony morphology and was investigated further. Marker recovery revealed an insertion in mfd, a gene encoding a transcription-coupled repair factor. The mfd mutant exhibited pleiotropic effects, in particular increased expression of both toxin A and B (TcdA and TcdB) compared to the parental strain. Western blotting and cellular cytotoxicity assays revealed increased expression across all time points over a 24 h period, with inactivation of mfd resulting in at least a 10 fold increase in cell cytotoxicity. qRT-PCR demonstrated the upregulation of both toxins occurred on a transcriptional level. All effects of the mfd mutation were complemented by a plasmid-encoded copy of mfd, showing the effects are not due to polar effects of the intron insertion or to second site mutations. CONCLUSIONS: This study adds Mfd to the repertoire of factors involved in regulation of toxin expression in Clostridium difficile. Mfd is known to remove RNA polymerase molecules from transcriptional sites where it has stalled due to repressor action, preventing transcriptional read through. The consistently high levels of toxin in the C. difficile mfd mutant indicate this process is inefficient leading to transcriptional de-repression

Spatial organization of Clostridium difficile S-layer biogenesis

Surface layers (S-layers) are protective protein coats which form around all archaea and most bacterial cells. Clostridium difficile is a Gram-positive bacterium with an S-layer covering its peptidoglycan cell wall. The S-layer in C. difficile is constructed mainly of S-layer protein A (SlpA), which is a key virulence factor and an absolute requirement for disease. S-layer biogenesis is a complex multi-step process, disruption of which has severe consequences for the bacterium. We examined the subcellular localization of SlpA secretion and S-layer growth; observing formation of S-layer at specific sites that coincide with cell wall synthesis, while the secretion of SlpA from the cell is relatively delocalized. We conclude that this delocalized secretion of SlpA leads to a pool of precursor in the cell wall which is available to repair openings in the S-layer formed during cell growth or following damage

The impact of a family-based assessment and intervention healthy lifestyle programme on health knowledge and beliefs of children with obesity and their families

Objective: To determine the impact of a family-based assessment-and-intervention healthy lifestyle programme on health knowledge and beliefs of children and families affected by obesity. Second, to compare the health knowledge of the programme cohort to those of a national cohort in Aotearoa/New Zealand (NZ). Design: This mixed-methods study collected health knowledge and health belief data in a questionnaire at baseline and 12-, 24-, and 60-month follow-up assessments. Health knowledge over time was compared with baseline knowledge and with data from a nationally representative survey. A data-driven subsumption approach was used to analyse open-text responses to health belief questions across the study period. Setting: Taranaki region, a mixed urban–rural setting in NZ. Participants: Participants (caregiver/child dyads) from the Whānau Pakari randomised trial. Results: A greater proportion of the cohort correctly categorised foods and drinks as healthy or unhealthy at 12 months compared to baseline for most questionnaire items. Retention of this health knowledge was evident at 24- and 60-month follow-ups. More than twice as many participants correctly reported physical activity recommendations at follow-up compared to baseline (p < 0.001). Health knowledge of participants was similar to the national survey cohort at baseline, but surpassed it at 12 and 24 months. Participant beliefs around healthy lifestyles related to physical functioning, mental and emotional wellbeing, and enhancement of appearance, and gained greater depth and detail over time. Conclusions: This study demonstrates the important role that community-level healthy lifestyle programmes can have in knowledge-sharing and health promotion

Early infections are associated with increased risk for celiac disease : an incident case-referent study

BACKGROUND: Celiac disease is defined as a 'chronic small intestinal immune-mediated enteropathy precipitated by exposure to dietary gluten in genetically predisposed individuals'. Sweden has experienced an "epidemic" of celiac disease in children below two years of age. Celiac disease etiology is considered multifactorial; however, little is known regarding potential risk- or protecting factors. We present data on the possible association between early infectious episodes and celiac disease, including their possible contribution to the Swedish celiac disease epidemic. METHODS: A population-based incident case-referent study (475 cases, 950 referents) with exposure information obtained via a questionnaire (including family characteristics, infant feeding, and the child's general health) was performed. Celiac disease cases were diagnosed before two years of age, fulfilling the diagnostic criteria of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition. Referents were randomly selected from the national population register after fulfilling matching criteria. The final analyses included 954 children, 373 (79%) cases and 581 (61%) referents, with complete information on main variables of interest in a matched set of one case with one or two referents. RESULTS: Having three or more parental-reported infectious episodes, regardless of type of infection, during the first six months of life was associated with a significantly increased risk for later celiac disease, and this remained after adjusting for infant feeding and socioeconomic status (odds ratio [OR] 1.5; 95% confidence interval [CI], 1.1-2.0; P=0.014). The celiac disease risk increased synergistically if, in addition to having several infectious episodes, infants were introduced to dietary gluten in large amounts, compared to small or medium amounts, after breastfeeding was discontinued (OR 5.6; 95% CI, 3.1-10; P&lt;0.001). CONCLUSION: This study suggests that having repeated infectious episodes early in life increases the risk for later celiac disease. In addition, we found a synergistic effect between early infections and daily amount of gluten intake, more pronounced among infants for whom breastfeeding had been discontinued prior to gluten introduction. Regarding contribution to the Swedish celiac disease epidemic, which partly was attributed to concurrent changes in infant feeding, early infections probably made a minor contribution via the synergistic effect with gluten amount

Interaction effects between estrogen receptor α and vitamin D receptor genes on age at menarche in Chinese women

Aim: To evaluate whether estrogen receptor α(ER-α) and vitamin D receptor (VDR) genes are associated with the age at menarche in Chinese women. Methods: A total of 390 pre-menopausal Chinese women were genotyped at the ER-α PvuII, XbaI, and VDR ApaI loci using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Results: Neither the ER-α gene nor the VDR gene individually had significant effects on the age at menarche in our subjects (P>0.10). However, evidence of interaction effects between the two genes were observed: with the aa genotype at the VDR ApaI locus, subjects with haplotype PX at the ER-α gene had, on average, 6 months later onset of menarche than the non-carriers (P=0.01). Conclusion: We found that neither the ER-α gene or the VDR gene had a significant association with the age at menarche individually. However, potential interaction effects between the two genes were observed in Chinese women. ©2005 CPS and SIMM.link_to_subscribed_fulltex