Prevalence and social determinants of anxiety and depression among adults in Ghana: a systematic review and meta-analysis protocol.

INTRODUCTION: Anxiety and depression pose a significant global health challenge, especially affecting adults in low-income and middle-income countries. In many low-income and middle-income countries, including those in sub-Saharan Africa, social determinants such as access to affordable health services, conflict, food insecurity, and poverty may be associated with the prevalence of anxiety and depression, further contributing to health disparities. To mitigate the burden of anxiety and depression in sub-Saharan Africa, it is essential to develop country-level tailored mental health policies and strategies. For example, Ghana is working towards improving mental health via its 12 year Mental Health policy launched in 2021. However, the prevalence of anxiety and depression among adults in Ghana, along with associated social determinants remains largely unknown, posing challenges for mental health planning, resource allocation and developing targeted interventions. This systematic review seeks to (1) examine the prevalence of anxiety and depression among adults in Ghana and (2) explore social determinants potentially associated with anxiety and depression. METHODS AND ANALYSIS: Electronic databases (eg, African Index Medicus, CINAHL, EMBASE, MEDLINE, and PsycINFO) will be searched with all screening steps conducted by two independent reviewers. Secondary search strategies, including grey literature searches, will be used. Studies reporting on the prevalence of anxiety, depression and/or a combined symptom measure (ie, psychological distress) among adults in Ghana, using validated instruments will be included. If data allows, random-effects-meta-analyses will be performed to estimate pooled prevalence rates of anxiety and depression. Potential clinical and methodological moderators will be examined using subgroup analyses and meta-regression. A narrative synthesis will explore social determinants potentially associated with anxiety and depression among adults in Ghana. ETHICS AND DISSEMINATION: Ethical approval is not required as no primary data will be collected. Results will be disseminated via a peer-reviewed publication and presentations at academic conferences. Plain language summaries will be provided to relevant non-governmental organisations working in Ghana. PROSPERO REGISTRATION NUMBER: CRD42023463078.

"Systems seem to get in the way": a qualitative study exploring experiences of accessing and receiving support among informal caregivers of people living with chronic kidney disease.

BACKGROUND: The well-being of informal caregivers of people living with chronic kidney disease is influenced by their experiences with support, however, few studies have focused on exploring these experiences. This study aimed to explore informal caregivers' experiences accessing and receiving support while caring for someone living with chronic kidney disease. METHODS: Informal caregivers of people living with chronic kidney disease (n = 13) in the United Kingdom were primarily recruited via community organisations and social media adverts to participate in semi-structured interviews. Interviews explored support needs, experiences of receiving support from different groups (e.g. healthcare professionals, family/friends), and barriers and facilitators to accessing support. Support was understood as including emotional, practical, and informational support. Data were analysed using reflexive thematic analysis. RESULTS: Three themes were generated: (1) "Systems seem to get in the way" - challenges within support systems, illustrating the challenges informal caregivers encountered when navigating complex support systems; (2) Relying on yourself, describing how informal caregivers leveraged their existing skills and networks to access support independently, while recognising the limitations of having to rely on yourself to find support; and (3) Support systems can "take the pressure off", showing how support systems were able to help informal caregivers cope with the challenges they experienced if certain conditions were met. CONCLUSIONS: In response to the challenges informal caregivers experienced when seeking support, improvements are needed to better consider informal caregiver needs within healthcare systems, and to develop interventions tailored to informal caregiver needs and context. Within the healthcare system, informal caregivers may benefit from system navigation support and better integration within healthcare teams to ensure their informational support needs are met. New interventions developed to support informal caregivers should fit within their existing support systems and incorporate the qualities of support, such as empathy, that were valued. Additionally, use of an equity framework and user-centered design approaches during intervention development could help ensure interventions are accessible and acceptable.

Potential Implementers' Perspectives on the Development and Implementation of an e-Mental Health Intervention for Caregivers of Adults With Chronic Kidney Disease: Qualitative Interview Study.

BACKGROUND: e-Mental health interventions can improve access to mental health support for caregivers of people living with chronic kidney disease (CKD). However, implementation challenges often prevent effective interventions from being put into practice. To develop an e-mental health intervention for caregivers of people living with CKD that is optimized for future implementation, it is important to engage professionals that may endorse or deliver the intervention (ie, potential implementers) during intervention development. OBJECTIVE: This study aims to explore the perspectives of potential implementers working in kidney care, in mental health care, or at nonprofit organizations regarding the design and implementation of an e-mental health intervention for caregivers of people living with CKD. METHODS: Potential implementers (N=18) were recruited via National Health Service Trusts, email, and social media advertisements to participate in semistructured video interviews. Interview questions were informed by the Consolidated Framework for Implementation Research (CFIR). Data were analyzed using a deductive analysis approach using the CFIR, with inductive coding applied to relevant data not captured by the framework. RESULTS: A total of 29 generic categories, related to 17 CFIR constructs, were identified. The perceived fit between the intervention and implementation context (ie, existing service delivery models and work routines) and existing social networks among potential implementers were perceived as important factors in enhancing implementation potential. However, a need for capacity building among potential implementers to create systems to support the identification and referral of caregivers to an e-mental health intervention was identified. Equity concerns were raised regarding the intervention, highlighting the importance of incorporating an equity lens during intervention design to enhance accessibility and adoption. CONCLUSIONS: Potential implementers provided valuable insights into key design and implementation factors to help inform the development of an e-mental health intervention for caregivers of people living with CKD. Incorporating their feedback can help ensure the intervention is acceptable and inform the selection of future implementation strategies to enhance the implementation potential of the intervention. Potential implementers should continue to be engaged throughout intervention development.

Cognitive behavioural therapy self-help intervention preferences among informal caregivers of adults with chronic kidney disease: an online cross-sectional survey.

BACKGROUND: Informal caregivers (i.e. family and friends) provide essential support to people with chronic kidney disease (CKD). Many informal caregivers experience mental health problems such as anxiety and depression due to the caregiving role, and commonly have unmet psychological support needs. One potential solution is cognitive behavioural therapy (CBT) self-help interventions that are less reliant on extensive involvement of healthcare professionals, which may increase access. Within the intervention development phase of the MRC framework, the study's primary objective was to examine informal caregivers' self-help intervention preferences (e.g. delivery format, content). Secondary objectives were to describe the informal caregiver's situation (e.g. type of care activities) and mental health (symptoms of depression, anxiety, and stress). METHODS: An online cross-sectional survey conducted in the United Kingdom. Informal caregivers of adults living with CKD were recruited via social media, websites, newsletters, magazine articles, a podcast episode, and paid Facebook advertisements. The survey examined: informal caregiver characteristics; care recipient characteristics; self-help intervention preferences; and informal caregiver's mental health using the DASS-21. Data were analysed using descriptive statistics. RESULTS: Sixty-five informal caregivers participated. The majority (85%) were female, caring for a male (77%) spouse/partner (74%). Responses indicated 58% of informal caregivers were experiencing at least mild depression. In total, 48% indicated they were likely to use a CBT self-help intervention, preferring an intervention provided via internet (e.g. website) (64%), workbook (56%), or individually in-person (54%). Regarding content, interventions should cover a wide range of topics including living with CKD, support services, informal caregiver's physical health, and diet. Overall, 48% reported a preference for a supported intervention, with support delivered in-person or via email by a trained professional at a community organisation. CONCLUSIONS: Results suggest CBT self-help interventions may be an acceptable way to provide psychological support to informal caregivers, however the study is limited by the small sample size. A wide range of intervention preferences were identified indicating a need to tailor intervention content and delivery to enhance acceptability and engagement. Results will inform development of a CBT self-help intervention for informal caregivers of people with CKD.

Structural Determinants and Functional Significance of Dimerization for Osmosensing Transporter ProP in Escherichia coli.

Osmosensing transporter ProP forestalls cellular dehydration by detecting environments with high osmotic pressure and mediating the accumulation of organic osmolytes by bacterial cells. It is composed of 12 transmembrane helices with cytoplasmic N- and C-termini. In Escherichia coli, dimers form when the C-terminal domains of ProP molecules form homodimeric, antiparallel, α-helical coiled coils. No dominant negative effect was detected when inactive and active ProP molecules formed heterodimers in vivo. Purification of ProP in detergent dodecylmaltoside yielded monomers, which were functional after reconstitution in proteoliposomes. With other evidence, this suggests that ProP monomers function independently whether in the monomeric or dimeric state. Amino acid replacements that disrupted or reversed the coiled coil did not prevent in vivo dimerization of ProP detected with a bacterial two-hybrid system. Maleimide labeling detected no osmolality-dependent variation in the reactivities of cysteine residues introduced to transmembrane helix (TM) XII. In contrast, coarse-grained molecular dynamic simulations detected deformation of the lipid around TMs III and VI, on the lipid-exposed protein surface opposite to TM XII. This suggests that the dimer interface of ProP includes the surfaces of TMs III and VI, not of TM XII as previously suggested by crosslinking data. Homology modeling suggested that coiled-coil formation and dimerization via such an interface are not mutually exclusive. In previous work, alterations to the C-terminal coiled coil blocked co-localization of ProP with phospholipid cardiolipin at E. coli cell poles. Thus, dimerization may contribute to ProP targeting, adjust its lipid environment, and hence indirectly modify its osmotic stress response.

Psychological interventions for symptoms of depression among informal caregivers of older adult populations: A systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Symptoms of depression are commonly experienced by informal caregivers of older adults, however there is uncertainty concerning effectiveness of psychological interventions targeting symptoms of depression in this population. Further, there is uncertainty concerning important clinical moderators, including intervention type and care recipient health condition. This review examined the effectiveness of psychological interventions targeting symptoms of depression in informal caregivers of older adults. METHODS: PubMed, CINAHL, Embase, PsycINFO, Cochrane Library and Web of Science were searched. Risk of bias was assessed using the Cochrane Risk of Bias tool version 2. RESULTS: Fifteen studies were identified and twelve (1270 participants) provided data for the meta-analysis. Interventions included cognitive behavioral therapy (4 studies), problem-solving therapy (4 studies); non-directive supportive therapy (4 studies) and behavioral activation (3 studies). A small effect size favouring the intervention was found for symptoms of depression (g = -0.49, CI = -0.79, -0.19, I2 = 83.42 %) and interventions were effective in reducing incidence of major depression (OR = 0.177, CI = 0.08, 0.38), caregiver burden (g = -0.35, CI = -0.55, -0.15) and psychological distress (g = -0.49, CI = -0.70, -0.28). Given high heterogeneity, findings should be interpreted with caution. Overall risk of bias was high. LIMITATIONS: Studies were limited to those in English or Swedish. CONCLUSION: Psychological interventions may be effective in reducing symptoms of depression among informal caregivers of older adults. However, evidence is inconclusive due to heterogeneity, high risk of bias, and indirectness of evidence.

Implementation of e-Mental Health Interventions for Informal Caregivers of Adults With Chronic Diseases: Mixed Methods Systematic Review With a Qualitative Comparative Analysis and Thematic Synthesis.

BACKGROUND: Informal caregivers commonly experience mental health difficulties related to their caregiving role. e-Mental health interventions provide mental health support in a format that may be more accessible to informal caregivers. However, e-mental health interventions are seldom implemented in real-world practice. OBJECTIVE: This mixed methods systematic review aimed to examine factors associated with the effectiveness and implementation of e-mental health interventions for informal caregivers of adults with chronic diseases. To achieve this aim, two approaches were adopted: combinations of implementation and intervention characteristics sufficient for intervention effectiveness were explored using qualitative comparative analysis, and barriers to and facilitators of implementation of e-mental health interventions for informal caregivers were explored using thematic synthesis. METHODS: We identified relevant studies published from January 1, 2007, to July 6, 2022, by systematically searching 6 electronic databases and various secondary search strategies. Included studies reported on the effectiveness or implementation of e-mental health interventions for informal caregivers of adults with cancer, chronic obstructive pulmonary disease, dementia, diabetes, heart disease, or stroke. Randomized controlled trials reporting on caregivers' mental health outcomes were included in a crisp-set qualitative comparative analysis. We assessed randomized controlled trials for bias using the Risk of Bias 2.0 tool, and we assessed how pragmatic or explanatory their trial design was using the Pragmatic Explanatory Continuum Indicator Summary 2 tool. Studies of any design reporting on implementation were included in a thematic synthesis using the Consolidated Framework for Implementation Research to identify barriers to and facilitators of implementation. RESULTS: Overall, 53 reports, representing 29 interventions, were included in the review. Most interventions (27/29, 93%) focused on informal cancer or dementia caregivers. In total, 14 reports were included in the qualitative comparative analysis, exploring conditions including the presence of peer or professional support and key persuasive design features. Low consistency and coverage prevented the determination of condition sets sufficient for intervention effectiveness. Overall, 44 reports were included in the thematic synthesis, and 152 barriers and facilitators were identified, with the majority related to the intervention and individual characteristic domains of the Consolidated Framework for Implementation Research. Implementation barriers and facilitators in the inner setting (eg, organizational culture) and outer setting (eg, external policies and resources) domains were largely unexplored. CONCLUSIONS: e-Mental health interventions for informal caregivers tend to be well-designed, with several barriers to and facilitators of implementation identified related to the intervention and individual user characteristics. Future work should focus on exploring the views of stakeholders involved in implementation to determine barriers to and facilitators of implementing e-mental health interventions for informal caregivers, focusing on inner and outer setting barriers and facilitators. TRIAL REGISTRATION: PROSPERO (International Prospective Register of Systematic Reviews) CRD42020155727; https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020155727. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1136/bmjopen-2019-035406.

The effects of self-management interventions on depressive symptoms in adults with chronic physical disease(s) experiencing depressive symptomatology: a systematic review and meta-analysis.

BACKGROUND: Chronic diseases are the leading cause of death worldwide. It is estimated that 20% of adults with chronic physical diseases experience concomitant depression, increasing their risk of morbidity and mortality. Low intensity psychosocial interventions, such as self-management, are part of recommended treatment; however, no systematic review has evaluated the effects of depression self-management interventions for this population. The primary objective was to examine the effect of self-management interventions on reducing depressive symptomatology in adults with chronic disease(s) and co-occurring depressive symptoms. Secondary objectives were to evaluate the effect of these interventions on improving other psychosocial and physiological outcomes (e.g., anxiety, glycemic control) and to assess potential differential effect based on key participant and intervention characteristics (e.g., chronic disease, provider). METHODS: Studies comparing depression self-management interventions to a control group were identified through a) systematic searches of databases to June 2018 [MEDLINE (1946 -), EMBASE (1996 -), PsycINFO (1967 -), CINAHL (1984 -)] and b) secondary 'snowball' search strategies. The methodological quality of included studies was critically reviewed. Screening of all titles, abstracts, and full texts for eligibility was assessed independently by two authors. Data were extracted by one author and verified by a second. RESULTS: Fifteen studies were retained: 12 for meta-analysis and three for descriptive review. In total, these trials included 2064 participants and most commonly evaluated interventions for people with cancer (n = 7) or diabetes (n = 4). From baseline to < 6-months (T1), the pooled mean effect size was - 0.47 [95% CI -0.73, - 0.21] as compared to control groups for the primary outcome of depression and - 0.53 [95% CI -0.91, - 0.15] at ≥ 6-months (T2). Results were also significant for anxiety (T1) and glycemic control (T2). Self-management skills of decision-making and taking action were significant moderators of depression at T1. CONCLUSION: Self-management interventions show promise in improving depression and anxiety in those with concomitant chronic physical disease. The findings may contribute to the development of future Self-management interventions and delivering evidence-based care to this population. Further high-quality RCTs are needed to identify sources of heterogeneity and investigate key intervention components.

Adapting a guided low-intensity behavioural activation intervention for people with dementia and depression in the Swedish healthcare context (INVOLVERA): a study protocol using codesign and participatory action research.

INTRODUCTION: Dementia is a worldwide health concern with incident rates continuing to increase. While depression prevalence is high in people with dementia and psychological interventions such as cognitive behavioural therapy (CBT) are effective, access to psychological interventions remains limited. Reliance on traditional CBT for people with dementia and depression may present difficulties given it is a complex psychological approach, costly to deliver, and professional training time is lengthy. An alternative approach is behavioural activation (BA), a simpler psychological intervention for depression. The present study seeks to work with people with dementia, informal caregivers, community stakeholders, and healthcare professionals, to adapt a guided low-intensity BA intervention for people with dementia and depression, while maximising implementation potential within the Swedish healthcare context. METHODS AND ANALYSIS: A mixed methods study using codesign, principles from participatory action research (PAR) and normalisation process theory to facilitate the cultural relevance, appropriateness and implementation potential of the intervention. The study will consist of four iterative PAR phases, using focus groups with healthcare professionals and community stakeholders, and semi-structured interviews with people with dementia and informal caregivers. A content analysis approach will be adopted to analyse the transcribed focus groups and semi-structured interviews recordings. ETHICS AND DISSEMINATION: The study will be conducted in accordance with the Declaration of Helsinki and data handled according to General Data Protection Regulation. Written informed consent will be obtained from all study participants. In accordance with the Swedish Health and Medical Services Act, capacity to consent will be examined by a member of the research team. Ethical approval has been obtained from the Swedish Ethical Review Authority (Dnr: 2020-05542 and Dnr: 2021-00925). Findings will be published in an open access peer-reviewed journal, presented at academic conferences, and disseminated among lay and healthcare professional audiences.

Non-pharmacological Interventions for Caregivers with Depression and Caregivers of Care Recipients with Co-morbid Depression: Systematic Review and Meta-analysis.

BACKGROUND: Caregivers experiencing depression or caring for people experiencing depression are at risk of high burden. This systematic review examined the effect of non-pharmacological interventions for caregivers that (a) target improving caregivers' depressive symptoms, (b) help caregivers manage the depressive symptoms of the person for whom they provide care, or (c) both (a) and (b). METHODS: Eligible trials published between January 1, 1985, and May 30, 2019 were retrieved from five electronic databases. The studies' methodological quality was assessed against 15 criteria. Pooled effect sizes (ESs) were calculated, and heterogeneity assessed using the Higgin's I2 statistic. Meta-regressions were also conducted to identify significant moderators (participant sub-group analyses) and mediators (identify how the interventions worked). RESULTS: Sixteen studies evaluating 18 interventions were included for review. These studies included a total of 2178 participants (mean = 94, SD = 129.18, range 25-518). The most common condition (n = 10/16) of the care recipient was dementia. The average methodological score was in the moderate range (8.76/15). Interventions had a moderate effect on caregivers' depression in the short term (ES = - 0.62, 95% CI - 0.81, - 0.44), but the effect dissipated over time (ES = - 0.19; 95% CI - 0.29, - 0.09). A similar pattern was noted for anxiety. The moderator analysis was not significant, and of the mediators examined, significant ones were self-management skills of taking action, problem solving, and decision-making. DISCUSSION: Non-pharmacological interventions are associated with improvement of depression and anxiety in caregivers, particularly in the short term. The main recommendation for future interventions is to include the self-management skills taking action, problem-solving, and decision-making. Enhancing the effect of these interventions will need to be the focus of future studies, particularly examining the impact of booster sessions. More research is needed on non-dementia caregiving and dyadic approaches.

Building the capacity for psycho-Oncology research: a survey of the research barriers and training needs within the International Psycho-Oncology Society.

BACKGROUND: The International Psycho-Oncology Society (IPOS) is a multidisciplinary professional network that aims to improve psychosocial care for individuals impacted by cancer. IPOS encourages research activity, recognizing that a high-quality evidence base is essential to provide best-practice, data-driven clinical care. This study aimed to determine the barriers to research involvement and the training needs and priorities of IPOS members, with the goal of facilitating the development of training resources tailored to the needs of IPOS members. METHODS: A link to an online, cross-sectional survey was disseminated to all registered members of IPOS via email. The online survey platform SimpleSurvey was used, and questions included demographic characteristics and items related to research interests, involvement, and training needs. High priority research training needs were identified as research tasks respondents rated as highly important, yet possessed a low perceived skill level in. RESULTS: Thirty-two percent of IPOS members (n = 142) completed the survey. Participants represented 49 countries and were at a variety of career stages. Overall, participants reported spending an average of 17.3 hours per week on research (range 0-80 hours per week), with 69% of respondents wanting to increase their research involvement. The main barriers to research participation included lack of research funding (80%) and lack of protected time (63%). IPOS members identified 5 high-priority training needs: preparing successful grant applications; preparing research budgets; community-based participatory research; working with decision makers; and finding collaborators or expert consultants. Participants suggested funding access, statistical advisors, and networking and mentorship opportunities as ways to enhance research involvement. Members preferred online training modules (39%) and mentorship programs (19%) as methods by which IPOS could provide research support. IPOS was viewed as being able to contribute to many aspects of research capacity building such as networking, training, and dissemination of research findings. CONCLUSIONS: IPOS has an important role in encouraging research capacity building among members. This survey provides an agenda for workshops and training opportunities. Mainly, for respondents it was less about training in research methods and more about training in how to prepare successful grant applications, including budgets, and receiving mentorship on this as well as having opportunities to collaborate with other researchers.

Implementation of e-mental health interventions for informal caregivers of adults with chronic diseases: a protocol for a mixed-methods systematic review with a qualitative comparative analysis.

INTRODUCTION: Informal caregivers provide the majority of care to individuals with chronic health conditions, benefiting the care recipient and reducing use of formal care services. However, providing informal care negatively impacts the mental health of many caregivers. E-mental health interventions have emerged as a way to provide accessible mental healthcare to caregivers. Much attention has been given to reviewing the effectiveness and efficacy of such interventions, however, factors related to implementation have received less consideration. Therefore, this mixed-methods systematic review will aim to examine factors associated with the effectiveness and implementation of e-mental health interventions for caregivers. METHODS AND ANALYSIS: Eligible studies published since 1 January 2007 will be searched for in several electronic databases (CINAHL Plus with Full Text, the Cochrane Library, EMBASE, PsycINFO, PubMed and Web of Science), clinical trial registries and OpenGrey, with all screening steps conducted by two independent reviewers. Studies will be included if they focus on the implementation or effectiveness of e-mental health interventions designed for informal adult caregivers of adults with cancer, heart disease, stroke, diabetes, dementia or chronic obstructive pulmonary disease. Pragmatic randomised controlled trials quantitatively reporting on caregiver anxiety, depression, psychological distress or stress will be used for a qualitative comparative analysis to identify combinations of conditions that result in effective interventions. Qualitative and quantitative data on implementation of e-mental health interventions for caregivers will be integrated in a thematic synthesis to identify barriers and facilitators to implementation. These results will inform future development and implementation planning of e-mental health interventions for caregivers. ETHICS AND DISSEMINATION: Ethical approval is not required for this study as no primary data will be collected. Results will be disseminated in the form of a scientific publication and presentations at academic conferences and plain language summaries for various stakeholders. PROSPERO REGISTRATION NUMBER: CRD42020155727.

The direct and indirect financial costs of informal cancer care: A scoping review.

Informal caregivers are the primary source of support for cancer patients, providing assistance with household tasks, medical care and emotional support. These responsibilities often result in high levels of emotional, physical, social and financial burden for the caregiver. The aim of this study was to perform a scoping review exploring what is known regarding the financial costs experienced by caregivers and identify gaps in the literature. Seven databases were searched for articles published between May 2008 and May 2018 related to direct and indirect costs of informal cancer care. Included articles reported on the costs incurred by cancer caregivers as a dollar value, relied on caregiver-reported costs and were peer reviewed. A total of 19 studies met the inclusion criteria. These studies reported out of pocket costs' opportunity costs of informal care time and caregiver time loss from paid employment. Care time was the largest source of cancer caregiver costs, averaging $4,809 per month when valued using the proxy good method or $2,877 per month when the opportunity cost approach was used. Caregiver costs were highest when the care recipient was in the palliative phase of the disease. There was an absence of literature reporting costs for cancer caregivers in low and middle income countries and none of the included studies considered costs related to the caregivers' medical expenditures. There were many challenges when comparing the costs across studies due to variations in the type of expenses reported and the methods used to value expenses. Quantifying the financial costs associated with being an informal caregiver can facilitate the communication of the financial burden caregivers experience, potentially spurring the development of policies and programs to reduce their financial burden and better support cancer caregivers.

Unique Regions of the Polysaccharide Copolymerase Wzz2 from Pseudomonas aeruginosa Are Essential for O-Specific Antigen Chain Length Control.

The outer leaflet of the outer membrane of nearly all Gram-negative bacteria contains lipopolysaccharide (LPS). The distal end of LPS may be capped with O antigen, a long polysaccharide that can range from a few to hundreds of sugars in length. The chain length of the polysaccharide has many implications for bacterial survival and consequently is tightly controlled. In the Wzx/Wzy-dependent route of O antigen synthesis, one or more Wzz proteins determine the chain length via an unknown mechanism. To gain insight into this mechanism, we identified and characterized important regions of two Wzz proteins in Pseudomonas aeruginosa serotype O13, which confer the production of "long" (Wzz1) and "very long" (Wzz2) chain lengths, respectively. We found that compared to Wzz1, Wzz2 has distinct amino acid insertions in the central α-helices (insα6 and insα7) and in membrane-distal (insL4) and -proximal (insIL) loops. When these regions were deleted in Wzz2, the mutant proteins conferred drastically shortened chain lengths. Within these regions we identified several conserved amino acid residues that were then targeted for site-directed mutagenesis. Our results implicate an RTE motif in loop 4 and a "hot spot" of charged and polar residues in insα7 in the function of Wzz2 We present evidence that the functionally important residues of insα7 are likely involved in stabilizing Wzz through coiled-coil interactions.IMPORTANCE O antigen is an important virulence factor presented on the cell surface of Gram-negative bacteria that is critical for bacterial physiology and pathogenesis. However, some aspects of O antigen biosynthesis, such as the mechanisms for determining polysaccharide chain length, are poorly understood. In this study, we identified unique regions in the O antigen chain length regulators (termed Wzz) of the problematic opportunistic pathogen Pseudomonas aeruginosa We show that these regions are critical for determining O antigen chain length, which provides new insight into the model of the Wzz mechanism. Ultimately, our work adds knowledge toward understanding an important step in the biosynthesis of this virulence factor, which is applicable to a wide range of Gram-negative pathogens.

Cardiolipin synthase A colocalizes with cardiolipin and osmosensing transporter ProP at the poles of Escherichia coli cells.

Osmosensing by transporter ProP is modulated by its cardiolipin (CL)-dependent concentration at the poles of Escherichia coli cells. Other contributors to this phenomenon were sought with the BACterial Two-Hybrid System (BACTH). The BACTH-tagged variants T18-ProP and T25-ProP retained ProP function and localization. Their interaction confirmed the ProP homo-dimerization previously established by protein crosslinking. YdhP, YjbJ and ClsA were prominent among the putative ProP interactors identified by the BACTH system. The functions of YdhP and YjbJ are unknown, although YjbJ is an abundant, osmotically induced, soluble protein. ClsA (CL Synthase A) had been shown to determine ProP localization by mediating CL synthesis. Unlike a deletion of clsA, deletion of ydhP or yjbJ had no effect on ProP localization or function. All three proteins were concentrated at the cell poles, but only ClsA localization was CL-dependent. ClsA was shown to be N-terminally processed and membrane-anchored, with dual, cytoplasmic, catalytic domains. Active site amino acid replacements (H224A plus H404A) inactivated ClsA and compromised ProP localization. YdhP and YjbJ may be ClsA effectors, and interactions of YdhP, YjbJ and ClsA with ProP may reflect their colocalization at the cell poles. Targeted CL synthesis may contribute to the polar localization of CL, ClsA and ProP.

YehZYXW of Escherichia coli Is a Low-Affinity, Non-Osmoregulatory Betaine-Specific ABC Transporter.

Transporter-mediated osmolyte accumulation stimulates the growth of Escherichia coli in high-osmolality environments. YehZYXW was predicted to be an osmoregulatory transporter because (1) osmotic and stationary phase induction of yehZYXW is mediated by RpoS, (2) the Yeh proteins are homologous to the components of known osmoregulatory ABC transporters (e.g., ProU of E. coli), and (3) YehZ models based on the structures of periplasmic betaine-binding proteins suggested that YehZ retains key betaine-binding residues. The betaines choline-O-sulfate, glycine betaine, and dimethylsulfoniopropionate bound YehZ and ProX with millimolar and micromolar affinities, respectively, as determined by equilibrium dialysis and isothermal titration calorimetry. The crystal structure of the YehZ apoprotein, determined at 1.5 Å resolution (PDB ID: 4WEP ), confirmed its similarity to other betaine-binding proteins. Small and nonpolar residues in the hinge region of YehZ (e.g., Gly223) pack more closely than the corresponding residues in ProX, stabilizing the apoprotein. Betaines bound YehZ-Gly223Ser an order of magnitude more tightly than YehZ, suggesting that weak substrate binding in YehZ is at least partially due to apo state stabilization. Neither ProX nor YehZ bound proline. Assays based on osmoprotection or proline auxotrophy failed to detect YehZYXW-mediated uptake of proline, betaines, or other osmolytes. However, transport assays revealed low-affinity glycine betaine uptake, mediated by YehZYXW, that was inhibited at high salinity. Thus, YehZYXW is a betaine transporter that shares substrate specificity, but not an osmoregulatory function, with homologues like E. coli ProU. Other work suggests that yehZYXW may be an antivirulence locus whose expression promotes persistent, asymptomatic bacterial infection.

Analysis of strains lacking known osmolyte accumulation mechanisms reveals contributions of osmolytes and transporters to protection against abiotic stress.

Osmolyte accumulation and release can protect cells from abiotic stresses. In Escherichia coli, known mechanisms mediate osmotic stress-induced accumulation of K(+) glutamate, trehalose, or zwitterions like glycine betaine. Previous observations suggested that additional osmolyte accumulation mechanisms (OAMs) exist and their impacts may be abiotic stress specific. Derivatives of the uropathogenic strain CFT073 and the laboratory strain MG1655 lacking known OAMs were created. CFT073 grew without osmoprotectants in minimal medium with up to 0.9 M NaCl. CFT073 and its OAM-deficient derivative grew equally well in high- and low-osmolality urine pools. Urine-grown bacteria did not accumulate large amounts of known or novel osmolytes. Thus, CFT073 showed unusual osmotolerance and did not require osmolyte accumulation to grow in urine. Yeast extract and brain heart infusion stimulated growth of the OAM-deficient MG1655 derivative at high salinity. Neither known nor putative osmoprotectants did so. Glutamate and glutamine accumulated after growth with either organic mixture, and no novel osmolytes were detected. MG1655 derivatives retaining individual OAMs were created. Their abilities to mediate osmoprotection were compared at 15°C, 37°C without or with urea, and 42°C. Stress protection was not OAM specific, and variations in osmoprotectant effectiveness were similar under all conditions. Glycine betaine and dimethylsulfoniopropionate (DMSP) were the most effective. Trimethylamine-N-oxide (TMAO) was a weak osmoprotectant and a particularly effective urea protectant. The effectiveness of glycine betaine, TMAO, and proline as osmoprotectants correlated with their preferential exclusion from protein surfaces, not with their propensity to prevent protein denaturation. Thus, their effectiveness as stress protectants correlated with their ability to rehydrate the cytoplasm.