Is it time to reduce the length of postgraduate training for physician-scientists in internal medicine?

Physician-scientists play a crucial role in advancing medical knowledge and patient care, yet the long periods of time required to complete training may impede expansion of this workforce. We examined the relationship between postgraduate training and time to receipt of NIH or Veterans Affairs career development awards (CDAs) for physician-scientists in internal medicine. Data from NIH RePORTER were analyzed for internal medicine residency graduates who received specific CDAs (K08, K23, K99, or IK2) in 2022. Additionally, information on degrees and training duration was collected. Internal medicine residency graduates constituted 19% of K awardees and 28% of IK2 awardees. Of MD-PhD internal medicine-trained graduates who received a K award, 92% received a K08 award; of MD-only graduates who received a K award, a majority received a K23 award. The median time from medical school graduation to CDA was 9.6 years for K awardees and 10.2 years for IK2 awardees. The time from medical school graduation to K or IK2 award was shorter for US MD-PhD graduates than US MD-only graduates. We propose that the time from medical school graduation to receipt of CDAs must be shortened to accelerate training and retention of physician-scientists.

Interventions to support fellowship application success among predoctoral physician-scientists.

A critical element of physician-scientist training is the development and practice of core competencies that promote success in research careers. The ability to develop compelling training and research proposals is one such foundational skill. The NIH Ruth L. Kirschstein National Research Service Award (NRSA) individual fellowship for dual-degree students (F30, F31, or F31-Diversity) creates an ideal opportunity to provide formal instruction in grant-writing skills to physician-scientists early in training. In the guided process of preparing a predoctoral fellowship application, students learn to formulate clear short- and long-term research and training goals; construct a comprehensive, well-reasoned, and rigorous proposal; become familiar with funding agency priorities; and gain strategic insights into the peer review system. Beyond building scientific writing skills, the application process for an NRSA F30 or F31 is an opportunity for trainees to strengthen mentor-mentee relationships, identify learning opportunities key to their scientific development, and build effective research and mentoring teams. These skills also apply to developing future postdoctoral mentored K applications or faculty research program grants. Here, we outline key features of the structured proposal development training developed for students in the Yale MD-PhD Program and review outcomes associated with its implementation.

Non-genetic adaptation by collective migration.

Collective behaviors require coordination of individuals. Thus, a population must adjust its phenotypic distribution to adapt to changing environments. How can a population regulate its phenotypic distribution? One strategy is to utilize specialized networks for gene regulation and maintaining distinct phenotypic subsets. Another involves genetic mutations, which can be augmented by stress-response pathways. Here, we studied how a migrating bacterial population regulates its phenotypic distribution to traverse across diverse environments. We generated isogenic Escherichia coli populations with varying distributions of swimming behaviors and observed their phenotype distributions during migration in liquid and porous environments. Surprisingly, we found that during collective migration, the distributions of swimming phenotypes adapt to the environment without mutations or gene regulation. Instead, adaptation is caused by the dynamic and reversible enrichment of high-performing swimming phenotypes within each environment. This adaptation mechanism is supported by a recent theoretical study, which proposed that the phenotypic composition of a migrating population results from a balance between cell growth generating diversity and collective migration eliminating the phenotypes that are unable to keep up with the migrating group. Furthermore, by examining chemoreceptor abundance distributions during migration towards different attractants, we found that this mechanism acts on multiple chemotaxis-related traits simultaneously. Our findings reveal that collective migration itself can enable cell populations with continuous, multi-dimensional phenotypes to flexibly and rapidly adapt their phenotypic composition to diverse environmental conditions. Significance statement: Conventional cell adaptation mechanisms, like gene regulation and random phenotypic switching, act swiftly but are limited to a few traits, while mutation-driven adaptations unfold slowly. By quantifying phenotypic diversity during bacterial collective migration, we discovered an adaptation mechanism that rapidly and reversibly adjusts multiple traits simultaneously. By dynamically balancing the elimination of phenotypes unable to keep pace with generation of diversity through growth, this process enables populations to tune their phenotypic composition based on the environment, without the need for gene regulation or mutations. Given the prevalence of collective migration in microbes, cancers, and embryonic development, non-genetic adaptation through collective migration may be a universal mechanism for populations to navigate diverse environments, offering insights into broader applications across various fields.

Structural insights into the career path between pre- and postgraduate physician-scientist training programs.

The growing complexities of clinical medicine and biomedical research have clouded the career path for physician-scientists. In this perspective piece, we address one of the most opaque career stage transitions along the physician-scientist career path, the transition from medical school to research-focused internal medicine residency programs, or physician-scientist training programs (PSTPs). We present the perspectives of medical scientist training program (MSTP) and PSTP directors on critical features of PSTPs that can help trainees proactively align their clinical and scientific training for successful career development. We aim to provide both trainees and MSTP directors with a conceptual framework to better understand and navigate PSTPs. We also offer interview-specific questions to help trainees gather data and make informed decisions in choosing a residency program that best supports their career.

Identification of efflux substrates using a riboswitch-based reporter in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is intrinsically resistant to many classes of antibiotics, reflecting the restrictive nature of its outer membrane and the action of its numerous efflux systems. However, the dynamics of compound uptake, retention and efflux in this bacterium remain incompletely understood. Here, we exploited the sensor capabilities of a Z-nucleotide sensing riboswitch to create an experimental system able to identify physicochemical and structural properties of compounds that permeate the bacterial cell, avoid efflux, and perturb the folate cycle or de novo purine synthesis. In a first step, a collection of structurally diverse compounds enriched in antifolate drugs was screened for ZTP riboswitch reporter activity in efflux-deficient P. aeruginosa , allowing us to identify compounds that entered the cell and disrupted the folate pathway. These initial hits were then rescreened using isogenic efflux-proficient bacteria, allowing us to separate efflux substrates from efflux avoiders. We confirmed this categorization by measuring intracellular levels of select compounds in the efflux-deficient and - proficient strain using high resolution LC-MS. This simple yet powerful method, optimized for high throughput screening, enables the discovery of numerous permeable compounds that avoid efflux and paves the way for further refinement of the physicochemical and structural rules governing efflux in this multi-drug resistant Gram-negative pathogen. Importance: Treatment of Pseudomonas aeruginosa infections has become increasingly challenging. The development of novel antibiotics against this multi-drug resistant bacterium is a priority, but many drug candidates never achieve effective concentrations in the bacterial cell due due to its highly restrictive outer membrane and the action of multiple efflux pumps. Here, we develop a robust and simple reporter system in P. aeruginosa to screen chemical libraries and identify compounds that either enter the cell and remain inside, or enter the cell and are exported by efflux systems. This approach enables developing rules of compound uptake and retention in P. aeruginosa that will lead to more rational design of novel antibiotics.

Identification of Efflux Substrates Using a Riboswitch-Based Reporter in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is intrinsically resistant to many classes of antibiotics, reflecting the restrictive nature of its outer membrane and the action of its numerous efflux systems. However, the dynamics of compound uptake, retention, and efflux in this bacterium remain incompletely understood. Here, we exploited the sensor capabilities of a Z-nucleotide-sensing riboswitch to create an experimental system able to identify physicochemical and structural properties of compounds that permeate the bacterial cell, avoid efflux, and perturb the folate cycle or de novo purine synthesis. In the first step, a collection of structurally diverse compounds enriched in antifolate drugs was screened for ZTP (5-aminoimidazole-4-carboxamide riboside 5'-triphosphate) riboswitch reporter activity in efflux-deficient P. aeruginosa, allowing us to identify compounds that entered the cell and disrupted the folate pathway. These initial hits were then rescreened using isogenic efflux-proficient bacteria, allowing us to separate efflux substrates from efflux avoiders. We confirmed this categorization by measuring intracellular levels of select compounds in the efflux-deficient and -proficient strain using high-resolution liquid chromatography-mass spectrometry (LC-MS). This simple yet powerful method, optimized for high-throughput screening, enables the discovery of numerous permeable compounds that avoid efflux and paves the way for further refinement of the physicochemical and structural rules governing efflux in this multidrug-resistant Gram-negative pathogen. IMPORTANCE Treatment of Pseudomonas aeruginosa infections has become increasingly challenging. The development of novel antibiotics against this multidrug-resistant bacterium is a priority, but many drug candidates never achieve effective concentrations in the bacterial cell due to its highly restrictive outer membrane and the action of multiple efflux pumps. Here, we develop a robust and simple reporter system in P. aeruginosa to screen chemical libraries and identify compounds that either enter the cell and remain inside or enter the cell and are exported by efflux systems. This approach enables the development of rules of compound uptake and retention in P. aeruginosa that will lead to more rational design of novel antibiotics.

Assessment of polymicrobial interactions in bacterial isolates from transfused platelet units associated with sepsis.

BACKGROUND: In 2019 the Centers for Disease Control and Prevention (CDC) reported a series of 4 transfusion reactions that resulted from contamination of apheresis platelet products. Products involved in all 4 cases were contaminated with Acinetobacter calcoaceticus-baumannii complex (ACBC) and in 3 products Staphylococcus saprophyticus was found as well. CDC investigation found that bacterial isolates from the cases were genetically related and suggested a common source of contamination. The contamination of blood products with ACBC is rare and polymicrobial contamination of blood products even less common. ACBC and S. saprophyticus have been observed to adhere to one another and sediment out of suspension in vitro, a process referred to as coaggregation, and we hypothesized that there was an interaction between the strains from these cases that contributed to their co-contamination of blood products. STUDY DESIGN AND METHODS: To test the hypothesis of bacterial interaction, we performed coaggregation experiments and observed the growth characteristics of ACBC and S. saprophyticus strains recovered from contaminated blood products involved in a subset of the CDC cases. RESULTS: An increase in S. saprophyticus CFU concentration was observed after several days of co-culture with ACBC in LB and plasma; however, no other findings suggested coaggregation or augmentative growth interaction between the bacterial strains. CONCLUSION: Ultimately, an interaction between ACBC and S. saprophyticus that could help explain their co-occurrence and growth in contaminated platelet units was not found; however future studies of potential interactions may be warranted.

Pearls of wisdom for aspiring physician-scientist residency applicants and program directors.

Postgraduate physician-scientist training programs (PSTPs) enhance the experiences of physician-scientist trainees following medical school graduation. PSTPs usually span residency and fellowship training, but this varies widely by institution. Applicant competitiveness for these programs would be enhanced, and unnecessary trainee anxiety relieved, by a clear understanding of what factors define a successful PSTP matriculant. Such information would also be invaluable to PSTP directors and would allow benchmarking of their admissions processes with peer programs. We conducted a survey of PSTP directors across the US to understand the importance they placed on components of PSTP applications. Of 41 survey respondents, most were from internal medicine and pediatrics residency programs. Of all components in the application, two elements were considered very important by a majority of PSTP directors: (a) having one or more first-author publications and (b) the thesis advisor's letter. Less weight was consistently placed on factors often considered more relevant for non-physician-scientist postgraduate applicants - such as US Medical Licensing Examination scores, awards, and leadership activities. The data presented here highlight important metrics for PSTP applicants and directors and suggest that indicators of scientific productivity and commitment to research outweigh traditional quantitative measures of medical school performance.

The Enemy of my Enemy: Bacterial Competition in the Cystic Fibrosis Lung.

Bacterial competition within host-associated polymicrobial communities shapes their composition, often with far-reaching consequences for human health. In this issue of Cell Host & Microbe, Perault et al. reveal how competition between two opportunistic pathogens could account for the epidemiology of chronic lung infections in people with cystic fibrosis.

Hfq and sRNA 179 Inhibit Expression of the Pseudomonas aeruginosa cAMP-Vfr and Type III Secretion Regulons.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen causing skin and soft tissue, respiratory, and bloodstream infections. The type III secretion system (T3SS) is one important virulence factor. Production of the T3SS is controlled by ExsA, a transcription factor that activates expression of the entire T3SS regulon. Global regulators including Vfr, RsmA, and Hfq also contribute to regulation of the T3SS. Vfr is a cAMP-responsive transcription factor that activates exsA transcription. RsmA, an RNA-binding protein, inversely controls expression of the T3SS and the type VI secretion system (T6SS). Hfq is an RNA chaperone that functions by stabilizing small noncoding RNAs (sRNAs) and/or facilitating base pairing between sRNAs and mRNA targets. A previous study identified sRNA 1061, which directly targets the exsA mRNA and likely inhibits ExsA synthesis. In this study, we screened an sRNA expression library and identified sRNA 179 as an Hfq-dependent inhibitor of T3SS gene expression. Further characterization revealed that sRNA 179 inhibits the synthesis of both ExsA and Vfr. The previous finding that RsmA stimulates ExsA and Vfr synthesis suggested that sRNA 179 impacts the Gac/Rsm system. Consistent with that idea, the inhibitory activity of sRNA 179 is suppressed in a mutant lacking rsmY and rsmZ, and sRNA 179 expression stimulates rsmY transcription. RsmY and RsmZ are small noncoding RNAs that sequester RsmA from target mRNAs. Our combined findings show that Hfq and sRNA 179 indirectly regulate ExsA and Vfr synthesis by reducing the available pool of RsmA, leading to reduced expression of the T3SS and cAMP-Vfr regulons.IMPORTANCE Control of gene expression by small noncoding RNA (sRNA) is well documented but underappreciated. Deep sequencing of mRNA preparations from Pseudomonas aeruginosa suggests that >500 sRNAs are generated. Few of those sRNAs have defined roles in gene expression. To address that knowledge gap, we constructed an sRNA expression library and identified sRNA 179 as a regulator of the type III secretion system (T3SS) and the cAMP-Vfr regulons. The T3SS- and cAMP-Vfr-controlled genes are critical virulence factors. Increased understanding of the signals and regulatory mechanisms that control these important factors will enhance our understanding of disease progression and reveal potential approaches for therapeutic intervention.

Global chemical effects of the microbiome include new bile-acid conjugations.

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1-9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11-13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.

Modulation of flagellar rotation in surface-attached bacteria: A pathway for rapid surface-sensing after flagellar attachment.

Attachment is a necessary first step in bacterial commitment to surface-associated behaviors that include colonization, biofilm formation, and host-directed virulence. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa can initially attach to surfaces via its single polar flagellum. Although many bacteria quickly detach, some become irreversibly attached and express surface-associated structures, such as Type IV pili, and behaviors, including twitching motility and biofilm initiation. P. aeruginosa that lack the GTPase FlhF assemble a randomly placed flagellum that is motile; however, we observed that these mutant bacteria show defects in biofilm formation comparable to those seen for non-motile, aflagellate bacteria. This phenotype was associated with altered behavior of ΔflhF bacteria immediately following surface-attachment. Forward and reverse genetic screens led to the discovery that FlhF interacts with FimV to control flagellar rotation at a surface, and implicated cAMP signaling in this pathway. Although cAMP controls many transcriptional programs in P. aeruginosa, known targets of this second messenger were not required to modulate flagellar rotation in surface-attached bacteria. Instead, alterations in switching behavior of the motor appeared to result from direct or indirect effects of cAMP on switch complex proteins and/or the stators associated with them.

New Twists and Turns in Bacterial Locomotion and Signal Transduction.

Prokaryotic organisms occupy the most diverse set of environments and conditions on our planet. Their ability to sense and respond to a broad range of external cues remain key research areas in modern microbiology, central to behaviors that underlie beneficial and pathogenic interactions of bacteria with multicellular organisms and within complex ecosystems. Advances in our understanding of the one- and two-component signal transduction systems that underlie these sensing pathways have been driven by advances in imaging the behavior of many individual bacterial cells, as well as visualizing individual proteins and protein arrays within living cells. Cryo-electron tomography continues to provide new insights into the structure and function of chemosensory receptors and flagellar motors, while advances in protein labeling and tracking are applied to understand information flow between receptor and motor. Sophisticated microfluidics allow simultaneous analysis of the behavior of thousands of individual cells, increasing our understanding of how variance between individuals is generated, regulated and employed to maximize fitness of a population. In vitro experiments have been complemented by the study of signal transduction and motility in complex in vivo models, allowing investigators to directly address the contribution of motility, chemotaxis and aggregation/adhesion on virulence during infection. Finally, systems biology approaches have demonstrated previously uncharted areas of protein space in which novel two-component signal transduction pathways can be designed and constructed de novo These exciting experimental advances were just some of the many novel findings presented at the 15th Bacterial Locomotion and Signal Transduction conference (BLAST XV) in January 2019.

In Situ Structures of Polar and Lateral Flagella Revealed by Cryo-Electron Tomography.

The bacterial flagellum is a sophisticated self-assembling nanomachine responsible for motility in many bacterial pathogens, including Pseudomonas aeruginosa, Vibrio spp., and Salmonella enterica The bacterial flagellum has been studied extensively in the model systems Escherichia coli and Salmonella enterica serovar Typhimurium, yet the range of variation in flagellar structure and assembly remains incompletely understood. Here, we used cryo-electron tomography and subtomogram averaging to determine in situ structures of polar flagella in P. aeruginosa and peritrichous flagella in S Typhimurium, revealing notable differences between these two flagellar systems. Furthermore, we observed flagellar outer membrane complexes as well as many incomplete flagellar subassemblies, which provide additional insight into mechanisms underlying flagellar assembly and loss in both P. aeruginosa and S Typhimurium.IMPORTANCE The bacterial flagellum has evolved as one of the most sophisticated self-assembled molecular machines, which confers locomotion and is often associated with virulence of bacterial pathogens. Variation in species-specific features of the flagellum, as well as in flagellar number and placement, results in structurally distinct flagella that appear to be adapted to the specific environments that bacteria encounter. Here, we used cutting-edge imaging techniques to determine high-resolution in situ structures of polar flagella in Pseudomonas aeruginosa and peritrichous flagella in Salmonella enterica serovar Typhimurium, demonstrating substantial variation between flagella in these organisms. Importantly, we observed novel flagellar subassemblies and provided additional insight into the structural basis of flagellar assembly and loss in both P. aeruginosa and S Typhimurium.

Should I Stay or Should I Go? Pseudomonas Just Can't Decide.

Surface sensing is a fundamental yet poorly understood behavior of swimming bacteria. In this issue of Cell Host & Microbe, Laventie et al. (2019) describe a cyclic-di-GMP-dependent pathway used by the opportunistic pathogen Pseudomonas aeruginosa to respond to surface binding on a several second timescale.

Host suppression of quorum sensing during catheter-associated urinary tract infections.

Chronic bacterial infections on medical devices, including catheter-associated urinary tract infections (CAUTI), are associated with bacterial biofilm communities that are refractory to antibiotic therapy and resistant to host immunity. Previously, we have shown that Pseudomonas aeruginosa can cause CAUTI by forming a device-associated biofilm that is independent of known biofilm exopolysaccharides. Here, we show by RNA-seq that host urine alters the transcriptome of P. aeruginosa by suppressing quorum sensing regulated genes. P. aeruginosa produces acyl homoserine lactones (AHLs) in the presence of urea, but cannot perceive AHLs. Repression of quorum sensing by urine implies that quorum sensing should be dispensable during infection of the urinary tract. Indeed, mutants defective in quorum sensing are able to colonize similarly to wild-type in a murine model of CAUTI. Quorum sensing-regulated processes in clinical isolates are also inhibited by urea. These data show that urea in urine is a natural anti-quorum sensing mechanism in mammals.

Synthesis of [32P]-c-di-GMP for Diguanylate Cyclase and Phosphodiesterase Activity Determinations.

Diguanylate cyclases that synthesize and phosphodiesterases that hydrolyze the second messenger cyclic-di-GMP (c-di-GMP) are at the center of bacterial signaling pathways that control behaviors relevant to all aspects of microbial physiology and pathogenesis (Romling et al., Microbiol Mol Biol Rev 77(1):1-52, 2013). Bioinformatics tools can easily predict the presence of the diguanylate cyclase GGDEF domain, or the EAL and HD-GYP domains associated with phosphodiesterase activity. However, experimental confirmation of enzymatic activity is still necessary, as many proteins contain degenerate domains that lack catalytic activity but nonetheless function as c-di-GMP receptors.

Determining Phosphodiesterase Activity (Radioactive Assay).

Cyclic-di-GMP phosphodiesterases (PDEs) catalyze the hydrolysis of the bacterial second messenger c-di-GMP. This protocol describes a sensitive radioactive assay for PDE activity in which substrate and product can be quickly and easily separated by thin-layer chromatography.

Determining Diguanylate Cyclase Activity (Radioactive Assay).

Bioinformatics approaches can identify sequence motifs associated with diguanylate cyclases (DGCs), but experimental demonstration of DGC enzymatic activity is often desired. This protocol describes a sensitive radioactive assay for DGC activity in which substrate and product are quickly and easily separated by thin-layer chromatography.

Interaction of the cyclic-di-GMP binding protein FimX and the Type 4 pilus assembly ATPase promotes pilus assembly.

Type IVa pili (T4P) are bacterial surface structures that enable motility, adhesion, biofilm formation and virulence. T4P are assembled by nanomachines that span the bacterial cell envelope. Cycles of T4P assembly and retraction, powered by the ATPases PilB and PilT, allow bacteria to attach to and pull themselves along surfaces, so-called "twitching motility". These opposing ATPase activities must be coordinated and T4P assembly limited to one pole for bacteria to show directional movement. How this occurs is still incompletely understood. Herein, we show that the c-di-GMP binding protein FimX, which is required for T4P assembly in Pseudomonas aeruginosa, localizes to the leading pole of twitching bacteria. Polar FimX localization requires both the presence of T4P assembly machine proteins and the assembly ATPase PilB. PilB itself loses its polar localization pattern when FimX is absent. We use two different approaches to confirm that FimX and PilB interact in vivo and in vitro, and further show that point mutant alleles of FimX that do not bind c-di-GMP also do not interact with PilB. Lastly, we demonstrate that FimX positively regulates T4P assembly and twitching motility by promoting the activity of the PilB ATPase, and not by stabilizing assembled pili or by preventing PilT-mediated retraction. Mutated alleles of FimX that no longer bind c-di-GMP do not allow rapid T4P assembly in these assays. We propose that by virtue of its high-affinity for c-di-GMP, FimX can promote T4P assembly when intracellular levels of this cyclic nucleotide are low. As P. aeruginosa PilB is not itself a high-affinity c-di-GMP receptor, unlike many other assembly ATPases, FimX may play a key role in coupling T4P mediated motility and adhesion to levels of this second messenger.