High-resolution structure and strain comparison of infectious mammalian prions.

Within the extensive range of self-propagating pathologic protein aggregates of mammals, prions are the most clearly infectious (e.g., ∼109 lethal doses per milligram). The structures of such lethal assemblies of PrP molecules have been poorly understood. Here we report a near-atomic core structure of a brain-derived, fully infectious prion (263K strain). Cryo-electron microscopy showed amyloid fibrils assembled with parallel in-register intermolecular ß sheets. Each monomer provides one rung of the ordered fibril core, with N-linked glycans and glycolipid anchors projecting outward. Thus, single monomers form the templating surface for incoming monomers at fibril ends, where prion growth occurs. Comparison to another prion strain (aRML) revealed major differences in fibril morphology but, like 263K, an asymmetric fibril cross-section without paired protofilaments. These findings provide structural insights into prion propagation, strains, species barriers, and membrane pathogenesis. This structure also helps frame considerations of factors influencing the relative transmissibility of other pathologic amyloids.

Evaluation of Ergot Resistance in Hard Red Spring Wheat in North Dakota.

Higher levels of ergot (Claviceps purpurea (Fr.)) Tul. were reported in North Dakota hard red spring wheat (HRSW) in 2018, leading to questions pertaining to management and cultivar resistance. To better understand pathogen and HRSW cultivar responses, greenhouse experiments were conducted from 2020 to 2021 to evaluate aggressiveness of nine C. purpurea isolates and ergot resistance in 21 HRSW cultivars. Results from the aggressiveness assay indicated significant cultivar by isolate interactions for total weight of sclerotia produced and ergot incidence. Mean data across all cultivar by isolate combinations suggested isolates CC-3 and IA-Tim were the most aggressive and subsequently used in ergot resistance experiments. Results from ergot resistance screening indicated none of the HRSW cultivars were immune to C. purpurea as all cultivars produced sclerotia. However, differences in ergot incidence, kernel incidence, aborted kernel incidence, total sclerotia weight, sclerotia length, and sclerotia width occurred among cultivars. Both 'ND-Frohberg' and 'TCG-Spitfire' had the lowest ergot incidence values and were among the lowest in total sclerotia weight. 'Waldron' and 'LCS-Trigger' had the highest ergot incidence and the highest total sclerotia weight. Given that most concerns with ergot occur post-harvest, we suggest two categories to describe ergot resistance: host resistance (fate of inoculation for a stigma) and logistical resistance (size characteristics of a sclerotium that influence its ability to remain with a seed lot after harvest and cleaning). This research provides a strong foundation on our understanding of HRSW resistance to ergot that will influence variety decisions in ergot-prone areas in North Dakota.

Effect of Fungicide and Timing of Application on Management of Phoma Black Stem of Cultivated Sunflowers in the United States.

Phoma black stem (PBS), caused by Phoma macdonaldii Boerema (teleomorph Leptosphaeria lindquistii Frezzi), is the most common stem disease of sunflower (Helianthus annuus L.) in the northern Great Plains region of the United States. However, the impact of PBS on sunflower yield in the United States is unclear, and a near complete absence of information on the impact of fungicides on disease management exists. The objectives of this study were to determine the impact of PBS on sunflower yield, the efficacy of available fungicides, the optimal fungicide application timing, and the economic viability of fungicides as a management tool. Fungicide timing efficacy was evaluated by applying single and/or sequential applications of pyraclostrobin fungicide at three sunflower growth stages in 10 field trials between 2017 and 2019. Efficacy of 10 fungicides from the Fungicide Resistance Action Committee (FRAC) groups 3, 7, and 11 were evaluated in four field trials between 2018 and 2019. The impact of treatments on PBS were evaluated by determination of incidence, severity, maximum lesion height, disease severity index (DSI), and harvested yield. Nine of the 10 fungicides evaluated and all fungicide timings that included an early bud application resulted in disease reductions when compared with the nontreated controls. The DSI was negatively correlated to sunflower yield in high-yield environments (P = 0.0004; R2 = 0.3425) but not in low- or moderate-yield environments. Although FRAC 7 fungicides were generally most efficacious, the sufficient efficacy and lower cost of FRAC 11 fungicides make them more economically viable in high-yielding environments at current market conditions.

The Impact of Measurement-Based Care in Psychiatry: An Integrative Review.

BACKGROUND: The quality of mental health treatment has not progressed to the same extent as physical health treatment. Suboptimal mental health outcomes may be attributed to the lack of a systematic approach to tracking and measuring patient progress. In psychiatry, Measurement-Based Care (MBC) offers an objective, systematic approach to monitor clinical progress, evaluate treatment efficacy, and inform clinical decisions, yet remains seldom used in daily practice. AIMS: To identify the clinical impact of using MBC in psychiatry. METHODS: Using the Johns Hopkins Nursing Evidence-Based Practice Model as a guideline, this integrative review focused on the research query, "What is the clinical impact of Measurement-Based Care when treating patients with mental illness?" RESULTS: A total of nine articles met the inclusion criteria for this integrative review. The articles ranged from 2008 to 2021. The following themes were identified from the analysis of these articles: (1) the clinical impact of MBC, (2) provider attitudes about MBC, and (3) barriers to MBC implementation. CONCLUSIONS: MBC, augmenting usual standard care, provides an objective, systematic approach using quantifiable data to monitor symptom severity and treatment effectiveness. This review highlights the clinical benefits of MBC, including increased remission rates, lower risk of relapse, improved medication adherence, and strengthening the therapeutic alliance. Although this review provides an overview of the benefits of MBC in psychiatry, there remains a substantial practice gap warranting further investigation. Strategies must be developed to address barriers at the individual and organizational levels which impede the successful adoption of MBC.

Increase of Bacterial Leaf Streak in Hard Red Spring Wheat in North Dakota and Yield Loss Considerations.

Bacterial leaf streak (BLS), caused by Xanthomonas translucens pv. undulosa, has increased in both prevalence and severity in the major hard red spring wheat (HRSW)-producing state North Dakota. The disease is readily observed after flag leaf emergence and can quickly lead to defoliation and severe yield loss. The objectives of this research were to document the prevalence and incidence of BLS in North Dakota and provide estimations of yield and economic losses. Trained field scouts determined the incidence and prevalence of BLS in ND on HRSW plants between Feekes growth stage (FGS) 8 and FGS 11.2 from 2015 to 2021, and data were used to determine BLS-affected hectares. Yield data in combination with BLS ratings were obtained from HRSW performance trials to estimate the impact of BLS on yield. The combination of variety identity, hectarage data, BLS-affected hectarage estimates, and yield loss estimates was used to estimate economic losses from BLS in 2019 and 2020. Our data suggest that BLS-affected hectares ranged from 747 to 141,680 between 2015 and 2021. Yield loss was observed at multiple HRSW performance trial locations, with estimated yield losses as high as 60% on susceptible varieties. The amount of BLS-affected hectares was the highest in 2019 and 2020, and direct economic losses for North Dakota HRSW producers were estimated to be as high as $4.7 and $8.0 million, respectively. These data highlight the importance of BLS in HRSW and the need to procure resources for breeding efforts and grower education on management of BLS.

HPV immunization among young adults (HIYA!) in family practice: A quality improvement project.

AIMS: Thousands of anogenital and oropharyngeal cancers every year might be prevented through increased receipt of vaccination against the human papillomavirus (HPV). This project aimed to (1) increase the rate of HPV vaccination status assessment, and (2) improve HPV vaccine initiation and completion rates among 18 to 26-year-old patients in the family practice setting. DESIGN: Quality improvement project, pre/post intervention design. METHODS: This project implemented the HIYA! (HPV Immunization among Young Adults) Intervention in a private sports and family practice in central New Jersey. HIYA! implemented 10 pre-, during, and post-visit outcome measures during every family medicine visit with an 18 to 26-year-old patient for HPV. Data collection involved retrospective chart review of every eligible patient during the 12-week implementation period from 17 August to 06 November 2020 and during the same 12-week control period in 2019. RESULTS/FINDINGS: One hundred sixteen charts from 2019 and 129 charts from 2020 were reviewed for assessment of HPV vaccination status and HPV vaccine initiation and/or completion. Multivariable logistic regression analysis demonstrated that participants in the control group were 84% less likely to be assessed for HPV vaccination status and were 91% less likely to initiate and complete HPV vaccination compared with the intervention group. CONCLUSION: This QI project found HIYA! to be an effective and feasible strategy to improve HPV vaccination rates among 18 to 26-year-old patients in a family practice setting. IMPACT: The human papillomavirus (HPV) is the most common sexually transmitted disease in the United States, and causes thousands of cancers annually. Although vaccination against HPV can prevent these cancers, vaccination rates remain low, particularly among young adults ages 18 to 26 years. The positive impact of HIYA! was evident based on its success despite the unique challenges presented during the implementation period due to the COVID-19 pandemic.

Strategies to improve human papillomavirus vaccination rates among adolescents in family practice settings in the United States: A systematic review.

AIMS AND OBJECTIVES: To explore the interventions aimed at increasing human papillomavirus (HPV) vaccination rates among adolescents in family practice settings. BACKGROUND: HPV is the most common sexually transmitted disease in the United States, and the cause of thousands of anogenital and oropharyngeal cancers annually. Although HPV infection can be prevented with recommended vaccination during adolescence, national HPV vaccine rates remain low. DESIGN: Systematic review. METHODS: Four databases (MEDLINE, CINAHL, EMBASE and the Cochrane Library) were searched. The search was guided by PRISMA and by the question, 'What are targeted interventions that improve HPV vaccination rates among adolescents in family practice settings?' Articles were reviewed for study characteristics and appraised for quality using the revised Cochrane risk of bias tools. RESULTS: Eleven studies met inclusion and exclusion criteria. Individual study size samples ranged from 749-147,294, with a combined total from all included studies of 276,205; the largest sample reviewed to date from family practice settings. Interventions used to increase HPV vaccination rates included reminder systems; provider and staff education; sensory incentives such as hitting a gong or petting a puppy; and iPad tailored messaging programmes. Studies that employed interventions pre-, during and postvisit were most effective in increasing HPV vaccination rates. CONCLUSIONS: This review provides the largest data supporting multimodal strategies to increase HPV vaccination rates among adolescent populations. It provides strong evidence to suggest that vaccination rates can be improved using measures at varying times of the patient visit. RELEVANCE TO CLINICAL PRACTICE: Adolescents seek health care in various settings. Many studies have examined interventions to increase HPV vaccination in paediatric settings, but few have examined interventions in family practice settings. This review suggests that family practices should implement multimodal measures before, during and after visits to increase HPV vaccination among adolescent patients.

Interprofessional education and collaboration among healthcare students and professionals: a systematic review and call for action.

Interprofessional education (IPE) is a prerequisite to building a collaborative practice environment and optimizing patient care. The purpose of this systematic review was to assess the impact of IPE on outcomes related to health-care pre-licensure learners and professionals, including: changes in attitudes/perceptions; acquisition of knowledge regarding other disciplines' roles and development of collaborative skills; and change in collaborative behavior. We searched PubMed, CINAHL, Embase, and ERIC for studies published from 2007 to 2017 in English; 19 studies were eligible. The Joanna Briggs Institute appraisal tool was used to assess the quality of the studies. Thirteen studies used a quasi-experimental design. The studies varied in terms of setting, teaching methods, assessment measures, and quality. Seventeen of the 19 studies (89%) that assessed change in attitudes toward other disciplines and value placed on a team-based approach for improving patient care, found statistically significant improvements. All seven of the studies that assessed change in collaborative behavior found statistically significant improvements. Among the 12 studies that assessed the development of collaborative skills, there were mixed results. Future directions include: conducting more studies among health-care professionals, assessing the long-term impact of IPE, objectively assessing change in collaborative behavior, and assessing the impact of IPE on patient-centered outcomes.

Molecular Fingerprinting of On-Off Direction-Selective Retinal Ganglion Cells Across Species and Relevance to Primate Visual Circuits.

The ability to detect moving objects is an ethologically salient function. Direction-selective neurons have been identified in the retina, thalamus, and cortex of many species, but their homology has remained unclear. For instance, it is unknown whether direction-selective retinal ganglion cells (DSGCs) exist in primates and, if so, whether they are the equivalent to mouse and rabbit DSGCs. Here, we used a molecular/circuit approach in both sexes to address these issues. In mice, we identify the transcription factor Satb2 (special AT-rich sequence-binding protein 2) as a selective marker for three RGC types: On-Off DSGCs encoding motion in either the anterior or posterior direction, a newly identified type of Off-DSGC, and an Off-sustained RGC type. In rabbits, we find that expression of Satb2 is conserved in On-Off DSGCs; however, it has evolved to include On-Off DSGCs encoding upward and downward motion in addition to anterior and posterior motion. Next, we show that macaque RGCs express Satb2 most likely in a single type. We used rabies virus-based circuit-mapping tools to reveal the identity of macaque Satb2-RGCs and discovered that their dendritic arbors are relatively large and monostratified. Together, these data indicate Satb2-expressing On-Off DSGCs are likely not present in the primate retina. Moreover, if DSGCs are present in the primate retina, it is unlikely that they express Satb2.SIGNIFICANCE STATEMENT The ability to detect object motion is a fundamental feature of almost all visual systems. Here, we identify a novel marker for retinal ganglion cells encoding directional motion that is evolutionarily conserved in mice and rabbits, but not in primates. We show in macaque monkeys that retinal ganglion cells (RGCs) that express this marker comprise a single type and are morphologically distinct from mouse and rabbit direction-selective RGCs. Our findings indicate that On-Off direction-selective retinal neurons may have evolutionarily diverged in primates and more generally provide novel insight into the identity and organization of primate parallel visual pathways.

Psychometric Properties of the Person-Centered Version of the Alcohol and Alcohol Problems Perceptions Questionnaire (PC-AAPPQ).

AIMS: Given the importance of addressing provider attitudes toward individuals with unhealthy alcohol use and the current emphasis on person-centered language to help decrease stigma and mitigate negative attitudes, the aim of this study was to evaluate the psychometric properties of a contemporary version of the Alcohol and Alcohol Problems Perception Questionnaire (AAPPQ) that uses person-centered language and addresses the spectrum of alcohol use. METHODS: The authors created a person-centered version of the AAPPQ (PC-AAPPQ) and conducted a cross-sectional study of its psychometric properties in academic settings in the Northeastern United States. The PC-AAPPQ was administered to 651 nursing students. Reliability analysis of the new instrument was performed using the total sample. Only surveys with complete data (n = 637) were randomly split into two datasets, one used for the exploratory factor analysis (EFA) (n = 310) and the other for confirmatory factor analysis (CFA) (n = 327). RESULTS: Compared to all the models generated from the EFA, neither the original six-factor structure nor the five-factor structure was superior to any of the other models. The results indicate that a seven-factor structure with all 30 items is the best fit for the PC-AAPPQ. CONCLUSIONS: The PC-AAPPQ represents a positive effort to modernize the four-decade-old AAPPQ. This 30-item instrument, which adds one additional subscale, offers a means to assess providers' attitudes using respectful wording that avoids perpetuating negative biases and reinforces efforts to affirm the worth and dignity of the population being treated.

Enigmatic origin of the poxvirus membrane from the endoplasmic reticulum shown by 3D imaging of vaccinia virus assembly mutants.

The long-standing inability to visualize connections between poxvirus membranes and cellular organelles has led to uncertainty regarding the origin of the viral membrane. Indeed, there has been speculation that viral membranes form de novo in cytoplasmic factories. Another possibility, that the connections are too short-lived to be captured by microscopy during a normal infection, motivated us to identify and characterize virus mutants that are arrested in assembly. Five conserved vaccinia virus proteins, referred to as Viral Membrane Assembly Proteins (VMAPs), that are necessary for formation of immature virions were found. Transmission electron microscopy studies of two VMAP deletion mutants had suggested retention of connections between viral membranes and the endoplasmic reticulum (ER). We now analyzed cells infected with each of the five VMAP deletion mutants by electron tomography, which is necessary to validate membrane continuity, in addition to conventional transmission electron microscopy. In all cases, connections between the ER and viral membranes were demonstrated by 3D reconstructions, supporting a role for the VMAPs in creating and/or stabilizing membrane scissions. Furthermore, coexpression of the viral reticulon-like transmembrane protein A17 and the capsid-like scaffold protein D13 was sufficient to form similar ER-associated viral structures in the absence of other major virion proteins. Determination of the mechanism of ER disruption during a normal VACV infection and the likely participation of both viral and cell proteins in this process may provide important insights into membrane dynamics.

Delirium point prevalence studies in inpatient settings: A systematic review and meta-analysis.

AIMS: To examine the delirium point prevalence studies conducted in different inpatient settings and to discuss the implication of the findings for delirium screening, assessment, prevention and management. BACKGROUND: Delirium-a common and distressing condition manifesting as an acute decline of attention and cognition-is frequently overlooked, misdiagnosed or treated inappropriately. This neuropsychiatric syndrome manifests as changes in attention, cognition and awareness, with resultant impact on behaviour, function and emotions. Delirium is recognised as a patient management challenge in the inpatient setting, and there is a need to understand the current point prevalence and assessment practices of delirium. DESIGN: A systematic review and meta-analysis. METHODS: A systematic review of published delirium prevalence studies in inpatient settings was conducted and the implications of findings for delirium screening, assessment, prevention and management identified. The random-effects meta-analysis was conducted among studies measuring delirium point prevalence. The PRISMA statement was used to report systematic review and meta-analysis. RESULTS: Nine studies were included in the review, with sample sizes ranging from 47-1867. Delirium point prevalence ranged from 9%-32%. Hypoactive delirium was the most common subtype, ranging from 23%-78%. Fifteen delirium screening tools or assessment or diagnostic methods were used. Comorbid dementia was present in up to 50% of inpatients. CONCLUSIONS: Gaining a consensus on effective delirium instruments, the time windows for assessment and measurement will be crucial in driving benchmarking and quality improvement studies. RELEVANCE TO CLINICAL PRACTICE: Consistent identification of high-risk patients and treatment settings with elevated risk, accompanied by the implementation of effective preventive and management strategies, are critical to addressing delirium-a frequent and burdensome condition, that adversely affects patient outcomes.

Nurse-Led Delivery of Brief Interventions for At-Risk Alcohol Use: An Integrative Review.

BACKGROUND: Nurses are in key positions to reduce the global burden associated with alcohol, yet many are ill-prepared to screen for alcohol use and intervene accordingly. The purpose of this integrative review was to identify best practices for educating nurses to work with patients who are at risk for alcohol-related adverse consequences, implement alcohol screening, and deliver alcohol brief interventions (ABIs). AIMS: To identify and synthesize findings from randomized control trials of ABIs delivered by nurses to patients identified through screening to be at risk because of alcohol use. METHOD: The results of 11 published randomized control trials identified from a multi-database search were synthesized. RESULTS: The Alcohol Use Disorder Identification Test was used for alcohol screening in more than half of the studies. Most of the ABIs were based on motivational interviewing and delivered in 30 minutes or less. While there was limited information on the characteristics of nurses who delivered the interventions and how nurses were prepared to deliver the ABIs, the exemplar was a full day workshop teaching nurses on an evidence-based framework for the ABI. All studies measured alcohol consumption as an outcome, yet few used rigorous methods for obtaining this self-reported data. CONCLUSIONS: A 1-day workshop is recommended as an educational modality to prepare nurses to implement the Alcohol Use Disorder Identification Test for identification of persons who are at risk because of alcohol use, deliver a structured brief intervention in less than 30 minutes, and utilize a standard measure of alcohol consumption for evaluation.

Representation of Rapid Image Sequences in V4 Networks.

Natural viewing often consists of sequences of brief fixations to image patches of different structure. Whether and how briefly presented sequential stimuli are encoded in a temporal-position manner is poorly understood. Here, we performed multiple-electrode recordings in the visual cortex (area V4) of nonhuman primates (Macaca mulatta) viewing a sequence of 7 briefly flashed natural images, and measured correlations between the cue-triggered population response in the presence and absence of the stimulus. Surprisingly, we found significant correlations for images occurring at the beginning and the end of a sequence, but not for those in the middle. The correlation strength increased with stimulus exposure and favored the image position in the sequence rather than image identity. These results challenge the commonly held view that images are represented in visual cortex exclusively based on their informational content, and indicate that, in the absence of sensory information, neuronal populations exhibit reactivation of stimulus-evoked responses in a way that reflects temporal position within a stimulus sequence.

Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L.

Coxiella burnetii is a gram-negative intracellular bacterium that forms a large, lysosome-like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking and signalling are unknown. Oxysterol-binding protein-related protein 1 long (ORP1L) is a mammalian lipid-binding protein that plays a dual role in cholesterol-dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 h of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signalling pathways. The ORP1L N-terminal ankyrin repeats were necessary and sufficient for PV localization, indicating that ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co-localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L-depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest that ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.

Focal Changes to Human Electrocorticography With Drowsiness: A Novel Measure of Local Sleep.

Drowsiness may be defined as the progressive loss of cortical processing efficiency that occurs with time passing while awake. This loss of cortical processing efficiency is reflected in focal changes to the electroencephalogram, including islands of increased delta power concurrent with drop-offs in neuronal activity (i.e., focal cortical inactivity). The authors hypothesized that these focal changes are evidenced at individual electrodes by combination of increased instantaneous amplitude in delta band and decreased instantaneous frequency in theta-alpha band, permitting their categorization as "active" and "inactive." An analysis of records from six patients with refractory epilepsy undergoing video-electrocorticographic monitoring was conducted. Feature extraction and state classification on multiple recordings revealed focal changes consistent with the hypothesis, as well as progressively increased numbers of inactive electrodes with time awake. The implications of these findings on the study of sleep, and particularly local sleep, are discussed.

Reactivation of visual-evoked activity in human cortical networks.

In the absence of sensory input, neuronal networks are far from being silent. Whether spontaneous changes in ongoing activity reflect previous sensory experience or stochastic fluctuations in brain activity is not well understood. Here we demonstrate reactivation of stimulus-evoked activity that is distributed across large areas in the human brain. We performed simultaneous electrocorticography recordings from occipital, parietal, temporal, and frontal areas in awake humans in the presence and absence of sensory stimulation. We found that, in the absence of visual input, repeated exposure to brief natural movies induces robust stimulus-specific reactivation at individual recording sites. The reactivation sites were characterized by greater global connectivity compared with those sites that did not exhibit reactivation. Our results indicate a surprising degree of short-term plasticity across multiple networks in the human brain as a result of repeated exposure to unattended information.

Cross-neutralizing human anti-poliovirus antibodies bind the recognition site for cellular receptor.

Most structural information about poliovirus interaction with neutralizing antibodies was obtained in the 1980s in studies of mouse monoclonal antibodies. Recently we have isolated a number of human/chimpanzee anti-poliovirus antibodies and demonstrated that one of them, MAb A12, could neutralize polioviruses of both serotypes 1 and 2. This communication presents data on isolation of an additional cross-neutralizing antibody (F12) and identification of a previously unknown epitope on the surface of poliovirus virions. Epitope mapping was performed by sequencing of antibody-resistant mutants and by cryo-EM of complexes of virions with Fab fragments. The results have demonstrated that both cross-neutralizing antibodies bind the site located at the bottom of the canyon surrounding the fivefold axis of symmetry that was previously shown to interact with cellular poliovirus receptor CD155. However, the same antibody binds to serotypes 1 and 2 through different specific interactions. It was also shown to interact with type 3 poliovirus, albeit with about 10-fold lower affinity, insufficient for effective neutralization. Antibody interaction with the binding site of the cellular receptor may explain its broad reactivity and suggest that further screening or antibody engineering could lead to a universal antibody capable of neutralizing all three serotypes of poliovirus.

Bacterial colonization of host cells in the absence of cholesterol.

Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24(-/-) mouse embryonic fibroblasts (MEFs). DHCR24(-/-) MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24(-/-) MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24(-/-) MEFs. In contrast, C. burnetii entry was significantly decreased in -cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated α(V)ß(3) integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24(-/-) MEFs lacked the CD63-positive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions.

The Annexin A2/p11 complex is required for efficient invasion of Salmonella Typhimurium in epithelial cells.

The facultative intracellular pathogen, Salmonella enterica, triggers its own uptake into non-phagocytic epithelial cells. Invasion is dependent on a type 3 secretion system (T3SS), which delivers a cohort of effector proteins across the plasma membrane where they induce dynamic actin-driven ruffling of the membrane and ultimately, internalization of the bacteria into a modified phagosome. In eukaryotic cells, the calcium- and phospholipid-binding protein Annexin A2 (AnxA2) functions as a platform for actin remodelling in the vicinity of dynamic cellular membranes. AnxA2 is mostly found in a stable heterotetramer, with p11, which can interact with other proteins such as the giant phosphoprotein AHNAK. We show here that AnxA2, p11 and AHNAK are required for T3SS-mediated Salmonella invasion of cultured epithelial cells and that the T3SS effector SopB is required for recruitment of AnxA2 and AHNAK to Salmonella invasion sites. Altogether this work shows that, in addition to targeting Rho-family GTPases, Salmonella can intersect the host cell actin pathway via AnxA2.