Dual species transcriptomics reveals conserved metabolic and immunologic processes in interactions between human neutrophils and Neisseria gonorrhoeae.

Neisseria gonorrhoeae (the gonococcus, Gc) causes the sexually transmitted infection gonorrhea. Gc is a prominent threat to human health by causing severe lifelong sequelae, including infertility and chronic pelvic pain, which is amplified by the emergence of "superbug" strains resistant to all current antibiotics. Gc is highly adapted to colonize human mucosal surfaces, where it survives despite initiating a robust inflammatory response and influx of polymorphonuclear leukocytes (PMNs, neutrophils) that typically clear bacteria. Here, dual-species RNA-sequencing was used to define Gc and PMN transcriptional profiles alone and after infection. Core host and bacterial responses were assessed for two strains of Gc and three human donors' PMNs. Comparative analysis of Gc transcripts revealed overlap between Gc responses to PMNs, iron, and hydrogen peroxide; 98 transcripts were differentially expressed across both Gc strains in response to PMN co-culture, including iron-responsive and oxidative stress response genes. We experimentally determined that the iron-dependent TbpB is suppressed by PMN co-culture, and iron-limited Gc have a survival advantage when cultured with PMNs. Analysis of PMN transcripts modulated by Gc infection revealed differential expression of genes driving cell adhesion, migration, inflammatory responses, and inflammation resolution pathways. Production of pro-inflammatory cytokines, including IL1B and IL8, the adhesion factor ICAM1, and prostaglandin PGE2 were induced in PMNs in response to Gc. Together, this study represents a comprehensive and experimentally validated dual-species transcriptomic analysis of two isolates of Gc and primary human PMNs that gives insight into how this bacterium survives innate immune onslaught to cause disease.

Pharmacy staff-reported adaptations to naloxone provision and over-the-counter (OTC) syringe sales during the COVID-19 pandemic: Experiences across multiple states and 2 pharmacy chains.

BACKGROUND: Access to harm reduction materials was greatly disrupted during coronavirus disease 2019 (COVID-19). Community pharmacies often continued provision of harm reduction materials as part of their usual operations during the pandemic, but little is known about what, if any, adaptations were made and the perceived impact of these actions from the perspective of pharmacy staff. OBJECTIVES: We explored how pharmacy staff across 4 states in 2 major pharmacy chains adapted to the COVID-19 pandemic for ongoing naloxone and over-the-counter (OTC) syringe access and how staff perceived the pandemic affected drug use in the community they served and their pharmacy's volume of syringe sales and naloxone provision. METHODS: We analyzed 134 pharmacy staff responses to a 12-month follow-up assessment for an educational intervention conducted in 2 pharmacy chains in Oregon, Washington, Massachusetts, and New Hampshire. Respondents answered closed- and open-ended questions collected online from July 2020 to February 2022. Questions measured prevalence of adaptations and perceived uptake of naloxone and OTC syringe services. Descriptive statistics summarized adaptations and perceived impact and chi-square tests explored differences by state and pharmacy chain. Open-ended responses were reviewed and analyzed to identify summary points and themes. RESULTS: With few differences by state or pharmacy chain detected, pharmacy staff reported more naloxone mailing, requests by phone, streamlined counseling, and drive-thru provision adaptations to OTC syringe sales and naloxone provision during the pandemic. Most staff perceived adaptations as increasing or maintaining naloxone provision and OTC syringe sales. Respondents described specific aspects of the pharmacy that contributed to successful adaptations, including tailoring to specific product demand, inventory levels, drive-thru access, and a perception of extraordinary public health need at a time of and in places affected by the opioid crisis. CONCLUSIONS: Pharmacy OTC syringe and naloxone access continued during the COVID-19 pandemic through streamlining workflows and innovating no-contact harm reduction services, reinforcing pharmacy's public health role.

Evaluating vaccine-elicited antibody activities against Neisseria gonorrhoeae: cross-protective responses elicited by the 4CMenB meningococcal vaccine.

The bacterial pathogen Neisseria gonorrhoeae is an urgent global health problem due to increasing numbers of infections, coupled with rampant antibiotic resistance. Vaccines against gonorrhea are being prioritized to combat drug-resistant N. gonorrhoeae. Meningococcal serogroup B vaccines such as four-component meningococcal B vaccine (4CMenB) are predicted by epidemiology studies to cross-protect individuals from natural infection with N. gonorrhoeae and elicit antibodies that cross-react with N. gonorrhoeae. Evaluation of vaccine candidates for gonorrhea requires a suite of assays for predicting efficacy in vitro and in animal models of infection, including the role of antibodies elicited by immunization. Here, we present the development and optimization of assays to evaluate antibody functionality after immunization of mice: antibody binding to intact N. gonorrhoeae, serum bactericidal activity, and opsonophagocytic killing activity using primary human neutrophils [polymorphonuclear leukocytes (PMNs)]. These assays were developed with purified antibodies against N. gonorrhoeae and used to evaluate serum from mice that were vaccinated with 4CMenB or given alum as a negative control. Results from these assays will help prioritize gonorrhea vaccine candidates for advanced preclinical to early clinical studies and will contribute to identifying correlates and mechanisms of immune protection against N. gonorrhoeae.

"Like it was just everyday business": A qualitative study of pharmacy-based naloxone and syringe customer experience.

BACKGROUND: As rates of overdoses involving opioids continue to rise in the United States, community pharmacies are uniquely positioned as a central access point of care for individuals to access harm reduction supplies, such as naloxone and nonprescription syringes (NPS). OBJECTIVES: This study aimed to identify the facilitators and barriers of obtaining naloxone and NPS at community pharmacies that participated in Respond to Prevent (R2P), a multicomponent intervention to increase dispensing rates of naloxone, buprenorphine, and NPS. METHODS: Pharmacy customers were recruited to participate in semistructured qualitative interviews conducted immediately after they obtained, or attempted to obtain, naloxone and NPS (when applicable) from R2P-participating pharmacies. Thematic analysis was conducted on the transcribed interviews, and content coding was applied to ethnographic notes and text messages from participants. RESULTS: Of the 32 participants, most (n = 28, 88%) successfully obtained naloxone and most of those seeking NPS successfully (n = 14, 82%) purchased them as well. Participants reported positive overall experiences at the community pharmacies. Participants described using the intervention advertising materials, as designed, to facilitate the request for naloxone. Many participants shared that they felt respected by pharmacists and that they valued naloxone counseling sessions that were tailored to meet their needs and allowed space for them to ask questions. Barriers included experiences where the intervention did not address structural challenges that prohibited the purchase of naloxone and where certain types of staff lacked knowledge, treated participants poorly, or did not adequately provide expected naloxone counseling. CONCLUSION: Pharmacy customer experiences obtaining naloxone and NPS in R2P-participating pharmacies identify facilitators and barriers to access that may be used to reform implementation and future interventions. Barriers identified can help enhance strategies or inform policies to improve pharmacy-based harm reduction supply distribution not addressed through existing interventions.

Readiness of community pharmacies to implement an opioid safety intervention.

BACKGROUND: We report on efforts to measure readiness to adopt opioid safety initiatives in community pharmacies within 2 large chains. Previous studies identified lack of knowledge, confidence, or enthusiasm in addressing harm reduction efforts. We implemented an intervention that provided training to improve opioid safety. The goal was to increase naloxone prescribing and nonprescription syringe sales, reduce stigma, and decrease opioid overdoses among patients and customers. OBJECTIVES: To assess pharmacy readiness for intervention delivery, by characterizing pharmacy culture around opioid safety; describing current practices and challenges interacting with patients and customers on naloxone, nonprescription syringe sales, and buprenorphine; and determining pharmacy defined goals for implementing the intervention. METHODS: The sample included pharmacy managers and staff pharmacists from 2 large chains who completed a brief phone interview. Interviews consisted of Likert-scale and open-ended, theoretically driven questions. Questions focused on workplace culture, patient engagement, naloxone and buprenorphine prescribing, nonprescription syringe sales, and intervention goals. Coding categories for the open-ended questions were derived using a thematic review of responses. RESULTS: A total of 163 respondents described both workplace culture and how they encourage patient opioid safety as including public health awareness, patient engagement, and naloxone prescribing. Sale of nonprescription syringes exhibited high variability: no sales barriers (53.9%), sales with barriers (21.5%), and no sales (20.9%). Half of pharmacists (50.3%) interacted with buprenorphine prescribers outside of medication fills. Most respondents (68.7%) endorsed being ready to promote the intervention. Pharmacists named goals in adopting the intervention of wanting more knowledge and educational materials, talking points with patients, and best practices for offering naloxone. CONCLUSION: Community pharmacists, before implementation, described awareness of and receptiveness to opioid safety initiatives, with substantial barriers around nonprescription syringe sales. Assessed knowledge level, culture, and identified barriers that emerged in the readiness assessments can be used to tailor future pharmacy-specific programming.

Implementation of an academic detailing intervention to increase naloxone distribution and foster engagement in harm reduction from the community clinician.

BACKGROUND: Respond to Prevent (R2P) is a randomized clinical trial which sought to accelerate distribution of naloxone and other harm reduction materials from community pharmacies. R2P combined an online continuing education course with in-store materials, specifically designed for use in community pharmacies, and then supported implementation through the one-on-one educational technique of academic detailing. OBJECTIVE: The objective of this paper is to describe and synthesize our experiences providing academic detailing as part of the R2P randomized trial. METHODS: Closed-ended items from standardized post detailing questionnaires were analyzed with descriptive statistics. Open-ended items were content analyzed for key themes using immersion-crystallization qualitative methods. RESULTS: A total of 176 pharmacies participated in R2P with 175 receiving their initial academic detailing visit between August 2019 and May 2021. Initial visits were in-person and lasted a median of 35 minutes (interquartile range, 20-45 minutes). The R2P naloxone guide was the most common topic covered (n = 162, 92.6%). Following a fidelity check to assess adequacy of the R2P program implementation, 80 pharmacies (45.7%) required secondary academic detailing. Secondary detailing was more targeted and most frequently focused on the sale of nonprescription syringes (n = 28; 35.2%) or disposal container distribution (n = 30; 37.5%). Analysis of the open-ended items identified factors that the detailers perceived to affect the quality of academic detailing sessions, including the pharmacy environment, participant knowledge of and attitudes toward the subject matter, and ability of the detailer to remain flexible yet consistent. CONCLUSION: R2P provided a standardized process to foster naloxone distribution and engagement in harm reduction with demonstrated implementation in 175 community pharmacies across 4 states. Academic detailing was perceived to be well-received and effective at providing education and promoting distribution of naloxone and nonprescription syringes in community pharmacies. Additional research is needed to confirm these perceptions through evaluation post-intervention behavioral and attitude changes.

Study protocol for the Respond to Prevent Study: a multi-state randomized controlled trial to improve provision of naloxone, buprenorphine and nonprescription syringes in community pharmacies.

Access to the opioid antidote naloxone is a critical component of addressing the opioid crisis. Naloxone is a population-level prevention intervention associated with substantial reductions in overdose mortality and reduction of nonfatal overdose. Pharmacies' pivotal role in dispensing medications like buprenorphine for the treatment of opioid use disorder and selling nonprescription syringes places them at the crossroads of opioid access and risk mitigation methods like naloxone provision. Testing ways to optimize pharmacy-based naloxone provision will be key as the country expands the implementation of naloxone through the medical system. In the Respond to Prevent Study, we conducted a large, practical study of a pharmacy-focused intervention in a sample of Washington, Oregon, Massachusetts and New Hampshire community chain pharmacies to increase naloxone dispensing and improve opioid safety. The intervention integrated two evidence-based educational toolkits and streamlined materials to enhance the focus on naloxone policy, stigma reduction, and patient communications around naloxone, nonprescription syringes and buprenorphine access. The real-world study implemented a stepped wedge, clustered randomized trial design across 175 community chain pharmacies to evaluate the effectiveness of the Respond to Prevent intervention in increasing: (a) pharmacy based naloxone distribution rates, naloxone-related patient engagement, and pharmacist and technicians' attitudes, knowledge, perceived behavioral control and self-efficacy toward naloxone; and (b) pharmacy nonprescription syringe sales, and pharmacist and technicians' attitudes, knowledge, perceived behavioral control and self-efficacy toward dispensing buprenorphine for opioid use disorder (secondary outcomes). This commentary provides a brief narrative about the study and presents insights on the design and adaptations to our study protocol, including those adopted during the unprecedented COVID-19 pandemic further compounded by Western wildfires in 2020.

Harm Reduction and Adaptations Among PWUD in Rural Oregon During COVID-19.

Coronavirus Disease 2019 (COVID-19) may influence HIV/HCV transmission risk behaviors in rural communities. We conducted semi-structured qualitative interviews with people who use drugs (PWUD) in five rural Oregon counties and asked about COVID-19 impact on substance use and harm reduction practices and their advice for improving public health responses. Participants (n = 36) reported using only methamphetamine (52.8%), only heroin (16.7%), or both (30.6%); 75% of participants reported recent injection. Three thematic categories emerged: SSP adaptations and accessibility, PWUD harm reduction practices, and policy suggestions. Participants noted the importance of SSPs to COVID-19 prevention and wellbeing, though some experienced increased barriers, leading to increased risky injection practices. Participants suggested need-based rather than one-for-one exchange, increasing syringe delivery services, encouraging secondary exchange by PWUD, and peers as trusted voices for information exchange. Rapid implementation of policy and practice changes are urgently required to improve SSP access, reinforce safer use, and prevent HIV/HCV and COVID-19 transmission.

Capturing COVID-19-Like Symptoms at Scale Using Banner Ads on an Online News Platform: Pilot Survey Study.

BACKGROUND: Identifying new COVID-19 cases is challenging. Not every suspected case undergoes testing, because testing kits and other equipment are limited in many parts of the world. Yet populations increasingly use the internet to manage both home and work life during the pandemic, giving researchers mediated connections to millions of people sheltering in place. OBJECTIVE: The goal of this study was to assess the feasibility of using an online news platform to recruit volunteers willing to report COVID-19-like symptoms and behaviors. METHODS: An online epidemiologic survey captured COVID-19-related symptoms and behaviors from individuals recruited through banner ads offered through Microsoft News. Respondents indicated whether they were experiencing symptoms, whether they received COVID-19 testing, and whether they traveled outside of their local area. RESULTS: A total of 87,322 respondents completed the survey across a 3-week span at the end of April 2020, with 54.3% of the responses from the United States and 32.0% from Japan. Of the total respondents, 19,631 (22.3%) reported at least one symptom associated with COVID-19. Nearly two-fifths of these respondents (39.1%) reported more than one COVID-19-like symptom. Individuals who reported being tested for COVID-19 were significantly more likely to report symptoms (47.7% vs 21.5%; P<.001). Symptom reporting rates positively correlated with per capita COVID-19 testing rates (R2=0.26; P<.001). Respondents were geographically diverse, with all states and most ZIP Codes represented. More than half of the respondents from both countries were older than 50 years of age. CONCLUSIONS: News platforms can be used to quickly recruit study participants, enabling collection of infectious disease symptoms at scale and with populations that are older than those found through social media platforms. Such platforms could enable epidemiologists and researchers to quickly assess trends in emerging infections potentially before at-risk populations present to clinics and hospitals for testing and/or treatment.

Examining nonprescription syringe sales in Massachusetts and Rhode Island community pharmacies.

BACKGROUND: The role pharmacies play in addressing the opioid crisis and drug-related risks such as injection drug use is evolving. Estimating the prevalence of injection drug use at the community level is challenging because of the stigma of drug use. Many community pharmacies sell nonprescription sterile syringes; thus, pharmacy-level sales of injection equipment may be an indicator of drug-related harms and unmet needs of high-risk populations. OBJECTIVES: To describe, compare, and assess the convergent validity of staff-reported nonprescription syringe (NPS) sales volume and NPS administrative sales data from community pharmacies in Massachusetts (MA) and Rhode Island (RI). METHODS: This study employed both prospective cross-sectional survey data collection and utilization of administrative pharmacy sales data. Between November 2017 and January 2018, we administered a telephone-based survey to estimate average weekly NPS type and volume for 191 chain pharmacies (CVS Health) located in communities experiencing fatal opioid overdoses above the state's 2015 annual median rate. For the same time period, we obtained NPS sales data from surveyed pharmacies and all CVS Health pharmacies in the 2 states. We calculated Spearman correlations to assess convergence of average weekly volume between pharmacy staff reports and sales data. RESULTS: All pharmacies responded to the survey. Most (98.4%) pharmacies surveyed sold NPS, but 42.0% reported running out of stock monthly or more frequently. Pharmacy staff tended to under-report syringe sales. Staff-reported weekly NPS sales volume was 67,922 versus 70,962 syringes from administrative pharmacy sales data. Spearman correlation between reported and actual NPS sales was 0.40 (95% CI 0.27-0.51). CONCLUSION: The counts of administrative pharmacy syringe sales data in MA and RI indicate high need, substantial volume, and notable access at community pharmacies. Future research should use NPS sales data rather than self-report data to track emerging trends and tailor local responses.

Effect of Lipidation on the Localization and Activity of a Lysozyme Inhibitor in Neisseria gonorrhoeae.

The Gram-negative pathogen Neisseria gonorrhoeae (gonococcus [Gc]) colonizes lysozyme-rich mucosal surfaces. Lysozyme hydrolyzes peptidoglycan, leading to bacterial lysis. Gc expresses two proteins, SliC and NgACP, that bind and inhibit the enzymatic activity of lysozyme. SliC is a surface-exposed lipoprotein, while NgACP is found in the periplasm and also released extracellularly. Purified SliC and NgACP similarly inhibit lysozyme. However, whereas mutation of ngACP increases Gc susceptibility to lysozyme, the sliC mutant is only susceptible to lysozyme when ngACP is inactivated. In this work, we examined how lipidation contributes to SliC expression, cellular localization, and resistance of Gc to killing by lysozyme. To do so, we mutated the conserved cysteine residue (C18) in the N-terminal lipobox motif of SliC, the site for lipid anchor attachment, to alanine. SliC(C18A) localized to soluble rather than membrane fractions in Gc and was not displayed on the bacterial surface. Less SliC(C18A) was detected in Gc lysates compared to the wild-type protein. This was due in part to some release of the C18A mutant, but not wild-type, protein into the extracellular space. Surprisingly, Gc expressing SliC(C18A) survived better than SliC (wild type)-expressing Gc after exposure to lysozyme. We conclude that lipidation is not required for the ability of SliC to inhibit lysozyme, even though the lipidated cysteine is 100% conserved in Gc SliC alleles. These findings shed light on how members of the growing family of lysozyme inhibitors with distinct subcellular localizations contribute to bacterial defense against lysozyme.IMPORTANCENeisseria gonorrhoeae is one of many bacterial species that express multiple lysozyme inhibitors. It is unclear how inhibitors that differ in their subcellular localization contribute to defense from lysozyme. We investigated how lipidation of SliC, an MliC (membrane-bound lysozyme inhibitor of c-type lysozyme)-type inhibitor, contributes to its localization and lysozyme inhibitory activity. We found that lipidation was required for surface exposure of SliC and yet was dispensable for protecting the gonococcus from killing by lysozyme. To our knowledge, this is the first time the role of lipid anchoring of a lysozyme inhibitor has been investigated. These results help us understand how different lysozyme inhibitors are localized in bacteria and how this impacts resistance to lysozyme.

Endocervical and Neutrophil Lipoxygenases Coordinate Neutrophil Transepithelial Migration to Neisseria gonorrhoeae.

Background: Infection with Neisseria gonorrhoeae (GC) is characterized by robust neutrophil influx that is insufficient to clear the bacteria. Sustained neutrophilic inflammation contributes to serious clinical sequelae that particularly affect women, including pelvic inflammatory disease and infertility. Methods: We established a 3-component system using GC, End1 polarized human endocervical cells, and primary human neutrophils to investigate neutrophil transepithelial migration following infection. Results: Neutrophil migration across endocervical monolayers increased with the infectious dose and required GC-epithelial cell contact. Epithelial protein kinase C, cytosolic phospholipase A2, 12R-lipoxygenase (LOX), and eLOX3 hepoxilin synthase were required for neutrophil transmigration to GC, and migration was abrogated by blocking the MRP2 efflux pump and by adding recombinant soluble epoxide hydrolase. These results are all consistent with epithelial cell production of the neutrophil chemoattractant hepoxilin A3 (HXA3). Neutrophil transmigration was also accompanied by increasing apical concentrations of leukotriene B4 (LTB4). Neutrophil 5-lipoxygenase and active BLT1 receptor were required for apical LTB4 and neutrophil migration. Conclusions: Our data support a model in which GC-endocervical cell contact infection stimulates HXA3 production, driving neutrophil migration that is amplified by neutrophil-derived LTB4. Therapeutic targeting of these pathways could limit inflammation and deleterious clinical sequelae in women with gonorrhea.

Evaluation of Adenylate Cyclase Toxoid Antigen in Acellular Pertussis Vaccines by Using a Bordetella pertussis Challenge Model in Mice.

Bordetella pertussis is the primary causative agent of pertussis (whooping cough), which is a respiratory infection that leads to a violent cough and can be fatal in infants. There is a need to develop more effective vaccines because of the resurgence of cases of pertussis in the United States since the switch from the whole-cell pertussis vaccines (wP) to the acellular pertussis vaccines (aP; diphtheria-tetanus-acellular-pertussis vaccine/tetanus-diphtheria-pertussis vaccine). Adenylate cyclase toxin (ACT) is a major virulence factor of B. pertussis that is (i) required for establishment of infection, (ii) an effective immunogen, and (iii) a protective antigen. The C-terminal repeats-in-toxin domain (RTX) of ACT is sufficient to induce production of toxin-neutralizing antibodies. In this study, we characterized the effectiveness of vaccines containing the RTX antigen against experimental murine infection with B. pertussis RTX was not protective as a single-antigen vaccine against B. pertussis challenge, and adding RTX to 1/5 human dose of aP did not enhance protection. Since the doses of aP used in murine studies are not proportionate to mouse/human body masses, we titrated the aP from 1/20 to 1/160 of the human dose. Mice receiving 1/80 human aP dose had bacterial burden comparable to those of naive controls. Adding RTX antigen to the 1/80 aP base resulted in enhanced bacterial clearance. Inclusion of RTX induced production of antibodies recognizing RTX, enhanced production of anti-pertussis toxin, decreased secretion of proinflammatory cytokines, such as interleukin-6, and decreased recruitment of total macrophages in the lung. This study shows that adding RTX antigen to an appropriate dose of aP can enhance protection against B. pertussis challenge in mice.

Bordetella adenylate cyclase toxin interacts with filamentous haemagglutinin to inhibit biofilm formation in vitro.

Bordetella pertussis, the causative agent of whooping cough, secretes and releases adenylate cyclase toxin (ACT), which is a protein bacterial toxin that targets host cells and disarms immune defenses. ACT binds filamentous haemagglutinin (FHA), a surface-displayed adhesin, and until now, the consequences of this interaction were unknown. A B. bronchiseptica mutant lacking ACT produced more biofilm than the parental strain; leading Irie et al. to propose the ACT-FHA interaction could be responsible for biofilm inhibition. Here we characterize the physical interaction of ACT with FHA and provide evidence linking that interaction to inhibition of biofilm in vitro. Exogenous ACT inhibits biofilm formation in a concentration-dependent manner and the N-terminal catalytic domain of ACT (AC domain) is necessary and sufficient for this inhibitory effect. AC Domain interacts with the C-terminal segment of FHA with ∼650 nM affinity. ACT does not inhibit biofilm formation by Bordetella lacking the mature C-terminal domain (MCD), suggesting the direct interaction between AC domain and the MCD is required for the inhibitory effect. Additionally, AC domain disrupts preformed biofilm on abiotic surfaces. The demonstrated inhibition of biofilm formation by a host-directed protein bacterial toxin represents a novel regulatory mechanism and identifies an unprecedented role for ACT.

Mutations Preventing Regulated Exon Skipping in MET Cause Osteofibrous Dysplasia.

The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to the understanding of bone development within the epiphyseal growth plate, factors that regulate periosteal osteogenesis have not been studied as intensively. Osteofibrous dysplasia (OFD) is a congenital disorder of osteogenesis and is typically sporadic and characterized by radiolucent lesions affecting the cortical bone immediately under the periosteum of the tibia and fibula. We identified germline mutations in MET, encoding a receptor tyrosine kinase, that segregate with an autosomal-dominant form of OFD in three families and a mutation in a fourth affected subject from a simplex family and with bilateral disease. Mutations identified in all families with dominant inheritance and in the one simplex subject with bilateral disease abolished the splice inclusion of exon 14 in MET transcripts, which resulted in a MET receptor (MET(Δ14)) lacking a cytoplasmic juxtamembrane domain. Splice exclusion of this domain occurs during normal embryonic development, and forced induction of this exon-exclusion event retarded osteoblastic differentiation in vitro and inhibited bone-matrix mineralization. In an additional subject with unilateral OFD, we identified a somatic MET mutation, also affecting exon 14, that substituted a tyrosine residue critical for MET receptor turnover and, as in the case of the MET(Δ14) mutations, had a stabilizing effect on the mature protein. Taken together, these data show that aberrant MET regulation via the juxtamembrane domain subverts core MET receptor functions that regulate osteogenesis within cortical diaphyseal bone.

Albumin, in the Presence of Calcium, Elicits a Massive Increase in Extracellular Bordetella Adenylate Cyclase Toxin.

Pertussis (whooping cough), caused by Bordetella pertussis, is resurging in the United States and worldwide. Adenylate cyclase toxin (ACT) is a critical factor in establishing infection with B. pertussis and acts by specifically inhibiting the response of myeloid leukocytes to the pathogen. We report here that serum components, as discovered during growth in fetal bovine serum (FBS), elicit a robust increase in the amount of ACT, and ≥90% of this ACT is localized to the supernatant, unlike growth without FBS, in which ≥90% is associated with the bacterium. We have found that albumin, in the presence of physiological concentrations of calcium, acts specifically to enhance the amount of ACT and its localization to the supernatant. Respiratory secretions, which contain albumin, promote an increase in amount and localization of active ACT that is comparable to that elicited by serum and albumin. The response to albumin is not mediated through regulation of ACT at the transcriptional level or activation of the Bvg two-component system. As further illustration of the specificity of this phenomenon, serum collected from mice that lack albumin does not stimulate an increase in ACT. These data, demonstrating that albumin and calcium act synergistically in the host environment to increase production and release of ACT, strongly suggest that this phenomenon reflects a novel host-pathogen interaction that is central to infection with B. pertussis and other Bordetella species.

Modulation of Pertussis and Adenylate Cyclase Toxins by Sigma Factor RpoE in Bordetella pertussis.

Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions.

The Bordetella adenylate cyclase repeat-in-toxin (RTX) domain is immunodominant and elicits neutralizing antibodies.

The adenylate cyclase toxin (ACT) is a multifunctional virulence factor secreted by Bordetella species. Upon interaction of its C-terminal hemolysin moiety with the cell surface receptor αMß2 integrin, the N-terminal cyclase domain translocates into the host cell cytosol where it rapidly generates supraphysiological cAMP concentrations, which inhibit host cell anti-bacterial activities. Although ACT has been shown to induce protective immunity in mice, it is not included in any current acellular pertussis vaccines due to protein stability issues and a poor understanding of its role as a protective antigen. Here, we aimed to determine whether any single domain could recapitulate the antibody responses induced by the holo-toxin and to characterize the dominant neutralizing antibody response. We first immunized mice with ACT and screened antibody phage display libraries for binding to purified ACT. The vast majority of unique antibodies identified bound the C-terminal repeat-in-toxin (RTX) domain. Representative antibodies binding two nonoverlapping, neutralizing epitopes in the RTX domain prevented ACT association with J774A.1 macrophages and soluble αMß2 integrin, suggesting that these antibodies inhibit the ACT-receptor interaction. Sera from mice immunized with the RTX domain showed similar neutralizing activity as ACT-immunized mice, indicating that this domain induced an antibody response similar to that induced by ACT. These data demonstrate that RTX can elicit neutralizing antibodies and suggest it may present an alternative to ACT.

High temporal resolution of glucosyltransferase dependent and independent effects of Clostridium difficile toxins across multiple cell types.

BACKGROUND: Clostridium difficile toxins A and B (TcdA and TcdB), considered to be essential for C. difficile infection, affect the morphology of several cell types with different potencies and timing. However, morphological changes over various time scales are poorly characterized. The toxins' glucosyltransferase domains are critical to their deleterious effects, and cell responses to glucosyltransferase-independent activities are incompletely understood. By tracking morphological changes of multiple cell types to C. difficile toxins with high temporal resolution, cellular responses to TcdA, TcdB, and a glucosyltransferase-deficient TcdB (gdTcdB) are elucidated. RESULTS: Human umbilical vein endothelial cells, J774 macrophage-like cells, and four epithelial cell lines (HCT8, T84, CHO, and immortalized mouse cecal epithelial cells) were treated with TcdA, TcdB, gdTcdB. Impedance across cell cultures was measured to track changes in cell morphology. Metrics from impedance data, developed to quantify rapid and long-lasting responses, produced standard curves with wide dynamic ranges that defined cell line sensitivities. Except for T84 cells, all cell lines were most sensitive to TcdB. J774 macrophages stretched and increased in size in response to TcdA and TcdB but not gdTcdB. High concentrations of TcdB and gdTcdB (>10 ng/ml) greatly reduced macrophage viability. In HCT8 cells, gdTcdB did not induce a rapid cytopathic effect, yet it delayed TcdA and TcdB's rapid effects. gdTcdB did not clearly delay TcdA or TcdB's toxin-induced effects on macrophages. CONCLUSIONS: Epithelial and endothelial cells have similar responses to toxins yet differ in timing and degree. Relative potencies of TcdA and TcdB in mouse epithelial cells in vitro do not correlate with potencies in vivo. TcdB requires glucosyltransferase activity to cause macrophages to spread, but cell death from high TcdB concentrations is glucosyltransferase-independent. Competition experiments with gdTcdB in epithelial cells confirm common TcdA and TcdB mechanisms, yet different responses of macrophages to TcdA and TcdB suggest different, additional mechanisms or targets in these cells. This first-time, precise quantification of the response of multiple cell lines to TcdA and TcdB provides a comparative framework for delineating the roles of different cell types and toxin-host interactions.