Conditional growth defect of Bordetella pertussis and Bordetella bronchiseptica ferric uptake regulator (fur) mutants

Outer-membrane vesicles (OMVs) are promising tools in the development of novel vaccines against the respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica. Unfortunately, vesiculation by bordetellae is too low for cost-effective vaccine production. In other bacteria, iron limitation or inactivation of the fur gene has been shown to increase OMV production, presumably by downregulation of the mla genes, which encode machinery for maintenance of lipid asymmetry in the outer membrane. Here, we followed a similar approach in bordetellae. Whereas a fur mutant was readily obtained in B. bronchiseptica, a B. pertussis fur mutant could only be obtained in iron-deplete conditions, indicating that a fur mutation is conditionally lethal in this bacterium. The fur mutants displayed a growth defect in iron-replete media, presumably because constitutive expression of iron-uptake systems resulted in iron intoxication. Accordingly, expression of the Escherichia coli ferritin FtnA to sequester intracellularly accumulated iron rescued the growth of the mutants in these media. The fur mutations led to the constitutive expression of novel vaccine candidates, such as the TonB-dependent receptors FauA for the siderophore alcaligin and BhuR for heme. However, neither inactivation of fur nor growth under iron limitation improved vesiculation, presumably because the expression of the mla genes appeared unaffected

Experiences of Dutch maternity care professionals during the first wave of COVID-19 in a community based maternity care system

Background and objective During the COVID-19 pandemic the organization of maternity care changed drastically; this study into the experiences of maternity care professionals with these changes provides suggestions for the organization of care during and after pandemics. Design An online survey among Dutch midwives, obstetricians and obstetric residents. Multinomial logistic regression analyses were used to investigate associations between the respondents' characteristics and answers. Results Reported advantages of the changes were fewer prenatal and postpartum consultations (50.1%). The necessity and safety of medical interventions and ultrasounds were considered more critically (75.9%); 14.8% of community midwives stated they referred fewer women to the hospital for decreased fetal movements, whereas 64.2% of the respondents working in hospital-based care experienced fewer consultations for this indication. Respondents felt that women had more confidence in giving birth at home (57.5%). Homebirths seemed to have increased according to 38.5% of the community midwives and 65.3% of the respondents working in hospital-based care. Respondents appreciated the shift to more digital consultations rather than face-to-face consultations. Mentioned disadvantages were that women had appointments alone, (71.1%) and that the community midwife was not allowed to join a woman to obstetric-led care during labour and subsequently stay with her (56.8%). Fewer postpartum visits by family and friends led to more tranquility (59.8%). Overall, however, 48.0% of the respondents felt that the safety of maternity care was compromised due to policy changes. Conclusions Maternity care professionals were positive about the decrease in routine care and the increased confidence of women in home birth, but also felt that safety in maternity care was sometimes compromised. According to the respondents in a future crisis situation it should be possible for community midwives to continue to deliver a personal handover after the referral of women to the hospital, and to stay with them

Inactivation of the Mla system and outer-membrane phospholipase A results in disrupted outer-membrane lipid asymmetry and hypervesiculation in Bordetella pertussis

Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. With the goal of improving the production of outer-membrane vesicles (OMVs), we studied here the mechanisms that are involved in maintaining lipid asymmetry in the outer membrane of this organism. We identified homologues of the phospholipid (PL)-transport systems Mla and Pqi and of outer-membrane phospholipase A (OMPLA). Inactivation of mlaF, encoding the ATPase of the Mla system, together with pldA, which encodes OMPLA, resulted in an accumulation of PLs at the cell surface as demonstrated by the binding of a phosphatidylethanolamine-specific fluorescent probe to intact cells of this strain. The corresponding single mutations did hardly or not affect binding of the probe. These results are consistent with a retrograde transport directionality of the Mla system in B. pertussis and indicate that PLs accumulating at the cell surface in the mlaF mutant are degraded by OMPLA. Consequently, the mlaF mutant showed a conditional growth defect due to the production of free fatty acids by OMPLA, which could be compensated by inactivation of OMPLA or by sequestration of the produced fatty acids with starch. The mlaF pldA double mutant showed markedly increased OMV production, and representative antigens were detected in these OMVs as in wild-type OMVs. Further phenotypic characterization showed that the barrier function of the outer membrane of the mlaF pldA mutant was compromised as manifested by increased susceptibility to SDS and to several antibiotics. Moreover, inactivation of mlaF alone or together with pldA resulted in increased biofilm formation, which was, however, not directly related to increased vesiculation as the addition of purified OMVs to the wild-type strain decreased biofilm formation. We conclude that the absence of MlaF together with OMPLA results in PL accumulation in the outer leaflet of the outer membrane, and the increased vesiculation of the mutant could be useful in the development of novel, OMV-based pertussis vaccines

Substrate specificity of the pyrophosphohydrolase LpxH determines the asymmetry of Bordetella pertussis lipid A

Lipopolysaccharides are anchored to the outer membrane of Gram-negative bacteria by a hydrophobic moiety known as lipid A, which potently activates the host innate immune response. Lipid A of Bordetella pertussis, the causative agent of whooping cough, displays unusual structural asymmetry with respect to the length of the acyl chains at the 3 and 3′ positions, which are 3OH-C10 and 3OH-C14 chains, respectively. Both chains are attached by the acyltransferase LpxA, the first enzyme in the lipid A biosynthesis pathway, which, in B. pertussis, has limited chain length specificity. However, this only partially explains the strict asymmetry of lipid A. In attempts to modulate the endotoxicity of B. pertussis lipid A, here we expressed the gene encoding LpxA from Neisseria meningitidis, which specifically attaches 3OH-C12 chains, in B. pertussis. This expression was lethal, suggesting that one of the downstream enzymes in the lipid A biosynthesis pathway in B. pertussis cannot handle precursors with a 3OH-C12 chain. We considered that the UDP-diacylglucosamine pyrophosphohydrolase LpxH could be responsible for this defect as well as for the asymmetry of B. pertussis lipid A. Expression of meningococcal LpxH in B. pertussis indeed resulted in new symmetric lipid A species with 3OH-C10 or 3OH-C14 chains at both the 3 and 3′ positions, as revealed by MS analysis. Furthermore, co-expression of meningococcal lpxH and lpxA resulted in viable cells that incorporated 3OH-C12 chains in B. pertussis lipid A. We conclude that the asymmetry of B. pertussis lipid A is determined by the acyl chain length specificity of LpxH

Regulated Expression of lpxC Allows for Reduction of Endotoxicity in Bordetella pertussis

The Gram-negative bacterium Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. Previously developed whole-cell pertussis vaccines were effective, but appeared to be too reactogenic mainly due to the presence of lipopolysaccharide (LPS, also known as endotoxin) in the outer membrane (OM). Here, we investigated the possibility of reducing endotoxicity by modulating the LPS levels. The promoter of the lpxC gene, which encodes the first committed enzyme in LPS biosynthesis, was replaced by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter. The IPTG was essential for growth, even when the construct was moved into a strain that should allow for the replacement of LPS in the outer leaflet of the OM with phospholipids by defective phospholipid transporter Mla and OM phospholipase A. LpxC depletion in the absence of IPTG resulted in morphological changes of the cells and in overproduction of outer-membrane vesicles (OMVs). The reduced amounts of LPS in whole-cell preparations and in isolated OMVs of LpxC-depleted cells resulted in lower activation of Toll-like receptor 4 in HEK-Blue reporter cells. We suggest that, besides lipid A engineering, also a reduction in LPS synthesis is an attractive strategy for the production of either whole-cell- or OMV-based vaccines, with reduced reactogenicity for B. pertussis and other Gram-negative bacteria

Cell-type-specific transcriptomics reveals that root hairs and endodermal barriers play important roles in beneficial plant-rhizobacterium interactions

Growth- and health-promoting bacteria can boost crop productivity in a sustainable way. Pseudomonas simiae WCS417 is such a bacterium that efficiently colonizes roots, modifies the architecture of the root system to increase its size, and induces systemic resistance to make plants more resistant to pests and pathogens. Our previous work suggested that WCS417-induced phenotypes are controlled by root cell-type-specific mechanisms. However, it remains unclear how WCS417 affects these mechanisms. In this study, we transcriptionally profiled five Arabidopsis thaliana root cell types following WCS417 colonization. We found that the cortex and endodermis have the most differentially expressed genes, even though they are not in direct contact with this epiphytic bacterium. Many of these genes are associated with reduced cell wall biogenesis, and mutant analysis suggests that this downregulation facilitates WCS417-driven root architectural changes. Furthermore, we observed elevated expression of suberin biosynthesis genes and increased deposition of suberin in the endodermis of WCS417-colonized roots. Using an endodermal barrier mutant, we showed the importance of endodermal barrier integrity for optimal plant-beneficial bacterium association. Comparison of the transcriptome profiles in the two epidermal cell types that are in direct contact with WCS417—trichoblasts that form root hairs and atrichoblasts that do not—implies a difference in potential for defense gene activation. While both cell types respond to WCS417, trichoblasts displayed both higher basal and WCS417-dependent activation of defense-related genes compared with atrichoblasts. This suggests that root hairs may activate root immunity, a hypothesis that is supported by differential immune responses in root hair mutants. Taken together, these results highlight the strength of cell-type-specific transcriptional profiling to uncover “masked” biological mechanisms underlying beneficial plant-microbe associations

Elements of organisation of integrated maternity care and their associations with outcomes: a scoping review protocol

Introduction Integrated care is seen as an enabling strategy in organising healthcare to improve quality, finances, personnel and sustainability. Developments in the organisation of maternity care follow this trend. The way care is organised should support the general aims and outcomes of healthcare systems. Organisation itself consists of a variety of smaller ‘elements of organisation’. Various elements of organisation are implemented in different organisations and networks. We will examine which elements of integrated maternity care are associated with maternal and neonatal health outcomes, experiences of women and professionals, healthcare spending and care processes. Methods and analysis We will conduct this review using the JBI methodology for scoping reviews and the reporting guideline PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analysis extension for Scoping Reviews). We will undertake a systematic search in the databases PubMed, Scopus, Cochrane and PsycINFO. A machine learning tool, ASReview, will be used to select relevant papers. These papers will be analysed and classified thematically using the framework of the Rainbow Model of Integrated Care (RMIC). The Population Concept Context framework for scoping reviews will be used in which ‘Population’ is defined as elements of the organisation of integrated maternity care, ‘Context’ as high-income countries and ‘Concepts’ as outcomes stated in the objective of this review. We will include papers from 2012 onwards, in Dutch or English language, which describe both ‘how the care is organised’ (elements) and ‘outcomes’. Ethics and dissemination Since this is a scoping review of previously published summary data, ethical approval for this study is not needed. Findings will be published in a peer-reviewed international journal, discussed in a webinar and presented at (inter)national conferences and meetings of professional associations. The findings of this scoping review will give insight into the nature and effectiveness of elements of integrated care and will generate hypotheses for further research

Cell-type specific transcriptomics reveals roles for root hairs and endodermal barriers in interaction with beneficial rhizobacterium

Growth-promoting bacteria can boost crop productivity in a sustainable way. Pseudomonas simiae WCS417 is a well-studied bacterium that promotes growth of many plant species. Upon colonization, WCS417 affects root system architecture resulting in an expanded root system. Both immunity and root system architecture, are controlled by root-cell-type specific biological mechanisms, but it is unknown how WCS417 affects these mechanisms. Therefore, here, we transcriptionally profiled five Arabidopsis thaliana root cell types following WCS417 colonization. The cortex and endodermis displayed the most differentially expressed genes, even though they were not in direct contact with this epiphytic bacterium. Many of these genes are associated with reduced cell wall biogenesis, possibly facilitating the root architectural changes observed in WCS417-colonized roots. Comparison of the transcriptome profiles in the two epidermal cell types that were in direct contact with WCS417 -- trichoblasts that form root hairs and atrichoblasts that do not -- imply functional specialization. Whereas basal expression levels of nutrient uptake-related genes and defense-related genes are highest in trichoblasts and atrichoblasts, respectively, upon exposure to WCS417 these roles revert. This suggests that root hairs participate in the activation of root immunity, further supported by attenuation of immunity in a root hairless mutant. Furthermore, we observed elevated expression of suberin biosynthesis genes and increased deposition of suberin in the endodermis in WCS417-colonized roots. Using an endodermal barrier mutant we show the importance of endodermal barrier integrity for optimal plant-beneficial bacterium association. Altogether, we highlight the strength of cell-type-specific transcriptional profiling to uncover masked biological mechanisms underlying successful plant-microbe associations

Motivation and treatment engagement intervention trial (MotivaTe-IT): The effects of motivation feedback to clinicians on treatment engagement in patients with severe mental illness

Background: Treatment disengagement and non-completion poses a major problem for the successful treatment of patients with severe mental illness. Motivation for treatment has long been proposed as a major determinant of treatment engagement, but exact mechanisms remain unclear. This current study serves three purposes: 1) to determine whether a feedback intervention based on the patients' motivation for treatment is effective at improving treatment engagement (TE) of severe mentally ill patients in outpatient psychiatric treatment, 2) to gather insight into motivational processes and pos

Biogenesis of outer-membrane vesicles in Bordetella species

The Gram-negative bacterium Bordetella pertussis causes whooping cough, also known as pertussis, which is an infection of the respiratory tract in humans. Pertussis is most severe in newborns. Currently, babies are vaccinated against pertussis, but these vaccines do not protect sufficiently as appears from the increase in pertussis cases over the last two decades. A closely related bacterium, Bordetella bronchiseptica, is the causative agent of respiratory diseases in animals, such as atrophic rhinitis in pigs and kennel cough in dogs. As for pertussis, the effectiveness of the available vaccines against B. bronchiseptica is under debate. Therefore, new vaccines against these pathogens need to be developed. A promising strategy is the use of outer-membrane vesicles (OMVs). These vesicles are naturally shed by Gram-negative bacteria. OMVs are safe because they are unable to replicate, they contain the most relevant antigens, and they are small which facilitates their uptake by immune cells. Unfortunately, spontaneous OMV production by Bordetella is too low for cost-effective vaccine production. In the work described in this thesis, we investigated the formation of OMVs in Bordetella and explored ways to increase OMV production. We show that OMV production can be increased by applying a heat shock to the bacteria and by genetically modifying the composition of the outer membrane or reducing the anchoring of the outer membrane to the underlying layers. The different approaches to increase OMV production resulted in OMVs with different compositions. In the future, the protection induced by the different OMVs needs to be determined