EF-Hand Calcium Sensor, EfhP, Controls Transcriptional Regulation of Iron Uptake by Calcium in Pseudomonas aeruginosa.

The human pathogen Pseudomonas aeruginosa poses a major risk for a range of severe infections, particularly lung infections in patients suffering from cystic fibrosis (CF). As previously reported, the virulent behavior of this pathogen is enhanced by elevated levels of Ca 2+ that are commonly present in CF nasal and lung fluids. In addition, a Ca 2+ -binding EF-hand protein, EfhP (PA4107), was partially characterized and shown to be critical for the Ca 2+ -regulated virulence in P. aeruginosa . Here we describe the rapid (10 min, 60 min), and adaptive (12 h) transcriptional responses of PAO1 to elevated Ca 2+ detected by genome-wide RNA sequencing and show that efhP deletion significantly hindered both rapid and adaptive Ca 2+ regulation. The most differentially regulated genes included multiple Fe sequestering mechanisms, a large number of extracytoplasmic function sigma factors (ECFσ) and several virulence factors, such as production of pyocins. The Ca 2+ regulation of Fe uptake was also observed in CF clinical isolates and appeared to involve the global regulator Fur. In addition, we showed that the efhP transcription is controlled by Ca 2+ and Fe, and this regulation required Ca 2+ -dependent two-component regulatory system CarSR. Furthermore, the efhP expression is significantly increased in CF clinical isolates and upon pathogen internalization into epithelial cells. Overall, the results established for the first time that Ca 2+ controls Fe sequestering mechanisms in P. aeruginosa and that EfhP plays a key role in the regulatory interconnectedness between Ca 2+ and Fe signaling pathways, the two distinct and important signaling pathways that guide the pathogen's adaptation to host. IMPORTANCE: Pseudomonas aeruginosa ( Pa ) poses a major risk for severe infections, particularly in patients suffering from cystic fibrosis (CF). For the first time, kinetic RNA sequencing analysis identified Pa rapid and adaptive transcriptional responses to Ca 2+ levels consistent with those present in CF respiratory fluids. The most highly upregulated processes include iron sequestering, iron starvation sigma factors, and self-lysis factors pyocins. An EF-hand Ca 2+ sensor, EfhP, is required for at least 1/3 of the Ca 2+ response, including all the iron uptake mechanisms and production of pyocins. Transcription of efhP itself is regulated by Ca 2+ , Fe, and increases during interactions with host epithelial cells, suggesting the protein's important role in Pa infections. The findings establish the regulatory interconnectedness between Ca 2+ and iron signaling pathways that shape Pa transcriptional responses. Therefore, understanding Pa's transcriptional response to Ca 2+ and associated regulatory mechanisms will serve the development of future therapeutics targeting Pa dangerous infections.

Evaluation of a Clinic-Based, Electronic Social Determinants of Health Screening and Intervention in Primary Care Pediatrics.

OBJECTIVE: Social determinants of health (SDOH) significantly affect individuals' health outcomes, yet universal electronic SDOH screening is not standard in primary care. Our study explores the implementation of an electronic SDOH screening in the electronic health record (EHR) and follow-up intervention among primary care pediatric patients within an academic clinic. METHODS: Beginning in August of 2022, patients and their families determined to have at least one SDOH need qualified for an in-clinic referral to a coordinated care team member. We assessed the overall efficacy and feasibility of the implementation. RESULTS: Over the 4-month pilot, 1473 of 2064 (71.4%) eligible patients were screened, with 472 (32%) patients screening positive on at least one SDOH domain. Of the 472 screened positive, 48 (10.2%) declined a referral. Two hundred and forty-seven of the 424 (58.3%) received a referral to a care coordination team member. CONCLUSIONS: This study demonstrates the feasibility of a universal electronic SDOH screening tool within the EHR within an urban, academic-based clinic.

Women Empowering Women: Assessing the American College of Surgeons Women in Surgery Committee Mentorship Program.

BACKGROUND: Mentorship is an important factor for career promotion and professional development. The Women in Surgery Committee developed a mentorship program that matched early career female surgeons to senior female surgeons for 1 year. We hypothesized participation in the program would empower junior surgeons by providing opportunities to network and hone skills necessary to attain their career goals. METHODS: Survey was sent 4 to 6 weeks after program completion. Statements about mentorship and value of the Women in Surgery Committee program were ranked on a 5-point Likert scale ranging from strongly disagree (1) to strongly agree (5). Participants were compared based on frequency of encounters using Student's t-test. RESULTS: A total of 105 pairs were identified; response rate was 60%. Results reported as (mean ± SD). Participants believed mentorship was essential for young surgeons (4.5 ± 1.0), and limiting the program to female surgeons added value (4.4 ± 0.6). When compared with mentees who met less than 4 times in a year, those who met 4 or more times perceived the program as beneficial (4.4 ± 0.82, p < 0.001). Mentees who met 4 or more times in a year benefitted from creating and achieving goals (4.3 ± 0.75, p < 0.001), setting expectations (4.5 ± 0.6, p < 0.001), providing networking opportunities (4.1 ± 1.1, p < 0.05), and developing professional skills (3.9 ± 0.98). CONCLUSION: The Women in Surgery Committee Mentorship Program provides an opportunity for young female surgeons; however, perceived benefit is dependent on mentee engagement.

EF-hand protein, EfhP, specifically binds Ca2+ and mediates Ca2+ regulation of virulence in a human pathogen Pseudomonas aeruginosa.

Calcium (Ca2+) is well known as a second messenger in eukaryotes, where Ca2+ signaling controls life-sustaining cellular processes. Although bacteria produce the components required for Ca2+ signaling, little is known about the mechanisms of bacterial Ca2+ signaling. Previously, we have identified a putative Ca2+-binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca2+ regulation of virulence factors production and infectivity in Pseudomonas aeruginosa, a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca2+ with 13.7 µM affinity, and that mutations at the +X and -Z positions within each or both EF-hand motifs abolished Ca2+ binding. We also show that the hydrophobicity of EfhP increased in a Ca2+-dependent manner, however no such response was detected in the mutated proteins. 15 N-NMR showed Ca2+-dependent chemical shifts in EfhP confirming Ca2+-binding triggered structural rearrangements in the protein. Deletion of efhP impaired P. aeruginosa survival in macrophages and virulence in vivo. Disabling EfhP Ca2+ binding abolished Ca2+ induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca2+, which triggers its structural changes required for the Ca2+ regulation of P. aeruginosa virulence, thus establishing the role of EfhP as a Ca2+ sensor.