CREST-UK: Real-world effectiveness, safety and outpatient delivery of CPX-351 for first-line treatment of newly diagnosed therapy-related AML and AML with myelodysplasia-related changes in the UK.

Favourable outcomes with CPX-351 versus conventional 7 + 3 were demonstrated in the pivotal phase III trial in adults aged 60-75 years with newly diagnosed, highrisk/secondary acute myeloid leukaemia (AML). As a complement to the clinical trial and to address important data gaps, the CPX-351 Real-World Effectiveness and SafeTy (CREST-UK; NCT05169307) study evaluated the use of CPX-351 in routine clinical practice in the UK, in 147 patients with newly diagnosed therapy-related AML or AML with myelodysplasia-related changes. Best response of complete remission or complete remission with incomplete platelet or neutrophil recovery was achieved by 53% of evaluable patients. Kaplan-Meier median overall survival (OS) was 12.8 months (95% confidence interval 9.2-15.3). Fifty (34%) patients proceeded to haematopoietic cell transplantation (HCT); median OS landmarked from the HCT date was not reached. There were no new safety concerns with CPX-351 identified in CREST-UK. Patients treated with CPX-351 in the outpatient setting spent an average of 24.4, 16.7, 28.2, and 27.7 fewer days on the ward compared with inpatients during first induction, second induction, first consolidation, and second consolidation, respectively. The results from CREST-UK provide valuable insights into the effectiveness, safety, and outpatient delivery of CPX-351 in routine clinical practice in the UK.

The origin, dissemination, and molecular networks of HIV-1 CRF65_cpx strain in Hainan Island, China.

BACKGROUND: HIV-1 CRF65_cpx strain carries drug-resistant mutations, which raises concerns about its potential for causing virologic failure. The CRF65_cpx ranks as the fourth most prevalent on Hainan Island, China. However, the origin and molecular epidemiology of CRF65_cpx strains in this area remain unclear. This study aims to estimate the spatial origins and dissemination patterns of HIV-1 CRF65_cpx in this specific region. METHODS: Between 2018 and 2021, a total of 58 pol sequences of the CRF65_cpx were collected from HIV-positive patients on Hainan Island. The available CRF65_cpx pol sequences from public databases were compiled. The HIV-TRACE tool was used to construct transmission networks. The evolutionary history of the introduction and dissemination of HIV-1 CRF65_cpx on Hainan Island were analyzed using phylogenetic analysis and the Bayesian coalescent-based approach. RESULTS: Among the 58 participants, 89.66% were men who have sex with men (MSM). The median age was 25 years, and 43.10% of the individuals had a college degree or above. The results indicated that 39 (67.24%) sequences were interconnected within a single transmission network. A consistent expansion was evident from 2019 to 2021, with an incremental annual addition of four sequences into the networks. Phylodynamic analyses showed that the CRF65_cpx on Hainan Island originated from Beijing (Bayes factor, BF = 17.4), with transmission among MSM on Hainan Island in 2013.2 (95%HPD: 2012.4, 2019.5), subsequently leading to an outbreak. Haikou was the local center of the CRF65_cpx epidemic. This strain propagated from Haikou to other locations, including Sanya (BF > 1000), Danzhou (BF = 299.3), Chengmai (BF = 27.0) and Tunchang (BF = 16.3). The analyses of the viral migration patterns between age subgroups and risk subgroups revealed that the viral migration directions were from "25-40 years old" to "17-24 years old" (BF = 14.6) and to "over 40 years old" (BF = 17.6), and from MSM to heterosexuals (BF > 1000) on Hainan Island. CONCLUSION: Our analyses elucidate the transmission dynamics of CRF65_cpx strain on Hainan Island. Haikou is identified as the potential hotspot for CRF65_cpx transmission, with middle-aged MSM identified as the key population. These findings suggest that targeted interventions in hotspots and key populations may be more effective in controlling the HIV epidemic.

A 3' UTR-Derived Small RNA Provides the Regulatory Noncoding Arm of the Inner Membrane Stress Response.

Small RNAs (sRNAs) from conserved noncoding genes are crucial regulators in bacterial signaling pathways but have remained elusive in the Cpx response to inner membrane stress. Here we report that an alternative biogenesis pathway releasing the conserved mRNA 3' UTR of stress chaperone CpxP as an ∼60-nt sRNA provides the noncoding arm of the Cpx response. This so-called CpxQ sRNA, generated by general mRNA decay through RNase E, acts as an Hfq-dependent repressor of multiple mRNAs encoding extracytoplasmic proteins. Both CpxQ and the Cpx pathway are required for cell survival under conditions of dissipation of membrane potential. Our discovery of CpxQ illustrates how the conversion of a transcribed 3' UTR into an sRNA doubles the output of a single mRNA to produce two factors with spatially segregated functions during inner membrane stress: a chaperone that targets problematic proteins in the periplasm and a regulatory RNA that dampens their synthesis in the cytosol.

NlpE Is an OmpA-Associated Outer Membrane Sensor of the Cpx Envelope Stress Response.

Gram-negative bacteria utilize several envelope stress responses (ESRs) to sense and respond to diverse signals within a multilayered cell envelope. The CpxRA ESR responds to multiple stresses that perturb envelope protein homeostasis. Signaling in the Cpx response is regulated by auxiliary factors, such as the outer membrane (OM) lipoprotein NlpE, an activator of the response. NlpE communicates surface adhesion to the Cpx response; however, the mechanism by which NlpE accomplishes this remains unknown. In this study, we report a novel interaction between NlpE and the major OM protein OmpA. Both NlpE and OmpA are required to activate the Cpx response in surface-adhered cells. Furthermore, NlpE senses OmpA overexpression and the NlpE C-terminal domain transduces this signal to the Cpx response, revealing a novel signaling function for this domain. Mutation of OmpA peptidoglycan-binding residues abrogates signaling during OmpA overexpression, suggesting that NlpE signaling from the OM through the cell wall is coordinated via OmpA. Overall, these findings reveal NlpE to be a versatile envelope sensor that takes advantage of its structure, localization, and cooperation with other envelope proteins to initiate adaptation to diverse signals. IMPORTANCE The envelope is not only a barrier that protects bacteria from the environment but also a crucial site for the transduction of signals critical for colonization and pathogenesis. The discovery of novel complexes between NlpE and OmpA contributes to an emerging understanding of the key contribution of OM ß-barrel protein and lipoprotein complexes to envelope stress signaling. Overall, our findings provide mechanistic insight into how the Cpx response senses signals relevant to surface adhesion and biofilm growth to facilitate bacterial adaptation.

Catalytic Atroposelective C7 Functionalisation of Indolines and Indoles.

Axially chiral atropisomeric compounds are widely applied in asymmetric catalysis and medicinal chemistry. In particular, axially chiral indole- and indoline-based frameworks have been recognised as important heterobiaryl classes because they are the core units of bioactive natural alkaloids, chiral ligands and bioactive compounds. Among them, the synthesis of C7-substituted indole biaryls and the analogous indoline derivatives is particularly challenging, and methods for their efficient synthesis are in high demand. Transition-metal catalysis is considered one of the most efficient methods to construct atropisomers. Here, we report the enantioselective synthesis of C7-indolino- and C7-indolo biaryl atropisomers by means of C-H functionalisation catalysed by chiral RhJasCp complexes.

Older Patients with Acute Myeloid Leukemia Deserve Individualized Treatment.

PURPOSE OF REVIEW: Treatment of elderly patients with acute myeloid leukemia is a known challenge for hematologists due to patient diversity, heterogeneous disease biology, and a rapidly evolving treatment landscape. Here, we highlight the importance of determining fitness, review the latest therapeutic developments, and discuss clinical scenarios to provide guidance on individualized treatment for older AML patients. RECENT FINDINGS: Several factors, like age, performance status, and comorbidities, play a role in fitness and are associated with outcome. Comorbidity scoring systems and geriatric assessments are tools to help physicians select the most appropriate treatment for each patient. The addition of venetoclax, targeted therapy with IDH1/2 and FLT3 inhibitors, and enhanced formulas of existing drugs like CPX-351 and oral azacitidine have improved responses and outcomes. New drugs and combination therapies have increased the therapeutic options for elderly AML patients but determination of fitness and disease biology is essential to select patient-tailored treatments.

Developing a best practice framework for clinical competency education in the traditional East-Asian medicine curriculum.

BACKGROUND: While clinical competency is crucial for traditional East-Asian medical education, available studies on the educational system for fostering clinical performance are scarce. This study aimed to review the educational system, curriculum, facilities, and management of current traditional East-Asian medicine in a well-established university of Korea and develop a Best Practice Framework (BPF) of clinical competency education. METHODS: The clinical competency education system in Pusan National University School of Korean Medicine was systematically described through 5 steps of governance of the educational system, competency of the graduates, educational resources, assessment strategies and tools, and gaps in the curriculum. We also reviewed the experiences in education and the points to be improved. RESULTS: The Office of Traditional Korean Medicine Education governs the development, implementation, and evaluation of the educational curriculum for cultivating students' clinical competency. Medical students have undertaken 39 modules of clinical biomedicine and 21 of traditional medicine during the clinical clerkship courses in an affiliated hospital, Clinical Skill Practice Center, clinical research center, practice lab for medical herb, and other locations. After training, 15 modules of simulated clinical training using standardized patients, students' clinical competency are evaluated by a Clinical Performance Test using a Clinical Performance Examination (CPX) and an Objective Structured Clinical Examination (OSCE) for biomedical and traditional medical skills. CONCLUSIONS: A clinical competency framework is required for a qualified physician of traditional East-Asian medicine. This study reviewed the current well-organized educational system of Korean traditional medicine in detail, which can be used for the BPF of competency-based clinical education. We expect the current study to be a representative reference for establishing an educational system of traditional medicine such as acupuncture and medical herbs in other countries.

Envelope Stress Responses: An Interconnected Safety Net.

The Escherichia coli cell envelope is a protective barrier at the frontline of interaction with the environment. Fidelity of envelope biogenesis must be monitored to establish and maintain a contiguous barrier. Indeed, the envelope must also be repaired and modified in response to environmental assaults. Envelope stress responses (ESRs) sense envelope damage or defects and alter the transcriptome to mitigate stress. Here, we review recent insights into the stress-sensing mechanisms of the σE and Cpx systems and the interaction of these ESRs. Small RNAs (sRNAs) are increasingly prominent regulators of the transcriptional response to stress. These fast-acting regulators also provide avenues for inter-ESR regulation that could be important when cells face multiple contemporaneous stresses, as is the case during infection.

How the assembly and protection of the bacterial cell envelope depend on cysteine residues.

The cell envelope of Gram-negative bacteria is a multilayered structure essential for bacterial viability; the peptidoglycan cell wall provides shape and osmotic protection to the cell, and the outer membrane serves as a permeability barrier against noxious compounds in the external environment. Assembling the envelope properly and maintaining its integrity are matters of life and death for bacteria. Our understanding of the mechanisms of envelope assembly and maintenance has increased tremendously over the past two decades. Here, we review the major achievements made during this time, giving central stage to the amino acid cysteine, one of the least abundant amino acid residues in proteins, whose unique chemical and physical properties often critically support biological processes. First, we review how cysteines contribute to envelope homeostasis by forming stabilizing disulfides in crucial bacterial assembly factors (LptD, BamA, and FtsN) and stress sensors (RcsF and NlpE). Second, we highlight the emerging role of enzymes that use cysteine residues to catalyze reactions that are necessary for proper envelope assembly, and we also explain how these enzymes are protected from oxidative inactivation. Finally, we suggest future areas of investigation, including a discussion of how cysteine residues could contribute to envelope homeostasis by functioning as redox switches. By highlighting the redox pathways that are active in the envelope of Escherichia coli, we provide a timely overview of the assembly of a cellular compartment that is the hallmark of Gram-negative bacteria.

Revisiting long-chain fatty acid metabolism in Escherichia coli: integration with stress responses.

Long-chain fatty acids (LCFAs) are a tremendous source of metabolic energy, an essential component of membranes, and important effector molecules that regulate a myriad of cellular processes. As an energy-rich nutrient source, the role of LCFAs in promoting bacterial survival and infectivity is well appreciated. LCFA degradation generates a large number of reduced cofactors that may confer redox stress; therefore, it is imperative to understand how bacteria deal with this paradoxical situation. Although the LCFA utilization pathway has been studied in great detail, especially in Escherichia coli, where the earliest studies date back to the 1960s, the interconnection of LCFA degradation with bacterial stress responses remained largely unexplored. Recent work in E. coli shows that LCFA degradation induces oxidative stress and also impedes oxidative protein folding. Importantly, both issues arise due to the insufficiency of ubiquinone, a lipid-soluble electron carrier in the electron transport chain. However, to maintain redox homeostasis, bacteria induce sophisticated cellular responses. Here, we review these findings in light of our current knowledge of the LCFA metabolic pathway, metabolism-induced oxidative stress, the process of oxidative protein folding, and stress combat mechanisms. We discuss probable mechanisms for the activation of defense players during LCFA metabolism and the likely feedback imparted by them. We suggest that besides defending against intrinsic stresses, LCFA-mediated upregulation of stress response pathways primes bacteria to adapt to harsh external environments. Collectively, the interplay between LCFA metabolism and stress responses is likely an important factor that underlies the success of LCFA-utilizing bacteria in the host.

3+7 Combined Chemotherapy for Acute Myeloid Leukemia: Is It Time to Say Goodbye?

PURPOSE OF REVIEW: With the recent approval of multiple new drugs for the treatment of acute myeloid leukemia (AML), the relevance of conventional treatment approaches, such as daunorubicin and cytarabine ("3+7") induction chemotherapy, has been challenged. We review the AML risk stratification, the efficacy of the newly approved drugs, and the role of "3+7". RECENT FINDINGS: Treatment of AML is becoming more niched with specific subtypes more appropriately treated with gemtuzumab, midostaurin, and CPX-351. Although lower intensity therapies can yield high response rates, they are less efficient at preventing relapses. The only curative potential for poor-risk AML is still an allogeneic stem cell transplant. The number of AML subtypes where 3+7 alone is an appropriate therapeutic option is shrinking. However, it remains the backbone for combination therapy with newer agents in patients suitable for intensive chemotherapy.

A Relapsing Meningeal Acute Myeloid Leukaemia FLT3-ITD+ Responding to Gilteritinib.

A patient with a therapy-related acute myeloid leukaemia (AML), NPM1mut, and FLT3-ITD+ was treated with induction and consolidation with CPX-351, obtaining a complete response (CR) but minimal residual disease persisted positive. Later, she complained progressive burning leg pain, weakening of the right hand and leg muscles, associated with absence of osteotendinous leg reflexes. Examination of cerebrospinal fluid (CSF) showed a meningeal relapse of AML. Moreover, a magnetic resonance imaging (MRI) showed 2 right meningeal implants of myeloid sarcoma and bone marrow revealed haematologic relapse of disease. She was treated with medicated lumbar punctures (LPs) followed by an FLA-Ida scheme, and she achieved a 2nd CR. Unfortunately, the patient developed hyperleucocytosis and reappearance of meningeal myeloid sarcoma at MRI. For this reason, a monotherapy with gilteritinib (an FLT3 inhibitor) was started: after 3 months of therapy, central nervous system (CNS)-disease shrunken and then faded, while AML in the bone marrow achieved only a partial response. This is the 1st report of a positive biological effect of gilteritinib on CNS (meningeal) myeloid sarcoma. There are no studies of gilteritinib concentration into CSF and penetration of gilteritinib into the blood-brain barrier should be further studied, given the paucity of drugs active on CNS relapse of AML. In patients receiving CPX-351 only, diagnostic LP should be considered after induction.

[Progress on the function and regulatory mechanisms of bacterial Cpx signal transduction system].

The Cpx (conjugative pilus expression) two-component signal transduction system is a complex envelope stress response system in Gram-negative bacteria, which can sense a variety of extracellular stimuli that enter the signaling pathway at different points. The phosphorylation of the CpxR, the cytoplasmic cognate response regulator of the Cpx system, can lead to changes in the expression of genes encoding proteins involved in inner and outer membrane functions. Activation of the Cpx system contributes to bacterial resistance/tolerance to certain antibiotics and acidic stress. In this review, we summarize the composition, and the mechanisms of signal detection, and the transcriptional regulation of the Cpx system, with a goal to provide guidance for the study of the regulatory network of the Cpx system and its important regulatory roles in bacterial physiology.

Characterization of the TyrR Regulon in the Rhizobacterium Enterobacter ludwigii UW5 Reveals Overlap with the CpxR Envelope Stress Response.

The TyrR transcription factor controls the expression of genes for the uptake and biosynthesis of aromatic amino acids in Escherichia coli In the plant-associated and clinically significant proteobacterium Enterobacter ludwigii UW5, the TyrR orthologue was previously shown to regulate genes that encode enzymes for synthesis of the plant hormone indole-3-acetic acid and for gluconeogenesis, indicating a broader function for the transcription factor. This study aimed to delineate the TyrR regulon of E. ludwigii by comparing the transcriptomes of the wild type and a tyrR deletion strain. In E. ludwigii, TyrR positively or negatively regulates the expression of over 150 genes. TyrR downregulated expression of envelope stress response regulators CpxR and CpxP through interaction with a DNA binding site in the intergenic region between divergently transcribed cpxP and cpxR Repression of cpxP was alleviated by tyrosine. Methyltransferase gene dmpM, which is possibly involved in antibiotic synthesis, was strongly activated in the presence of tyrosine and phenylalanine by TyrR binding to its promoter region. TyrR also regulated expression of genes for aromatic catabolism and anaerobic respiration. Our findings suggest that the E. ludwigii TyrR regulon has diverged from that of E. coli to include genes for survival in the diverse environments that this bacterium inhabits and illustrate the expansion and plasticity of transcription factor regulons.IMPORTANCE Genome-wide RNA sequencing revealed a broader regulatory role for the TyrR transcription factor in the ecologically versatile bacterium Enterobacter ludwigii beyond that of aromatic amino acid synthesis and transport that constitute the role of the TyrR regulon of E. coli In E. ludwigii, a plant symbiont and human gut commensal, the TyrR regulon is expanded to include genes that are beneficial for plant interactions and response to stresses. Identification of the genes regulated by TyrR provides insight into the mechanisms by which the bacterium adapts to its environment.

Natural presence of the V179D and K103R/V179D mutations associated with resistance to nonnucleoside reverse transcriptase inhibitors in HIV-1 CRF65_cpx strains.

BACKGROUND: There is increasing evidence that HIV-1 genetic diversity can have an impact on drug resistance. The aim of this study is to investigate the epidemiological situation of CRF65_cpx and the impact of natural polymorphisms of this variant on genotypic resistance. METHODS: We used the BLAST search program followed by phylogenetic analysis to identify additional CRF65_cpx pol sequences from the Los Alamos HIV Sequence Database. Maximum likelihood phylogeny was estimated to clarify the epidemiological relationship of CRF65_cpx strains. Genotypic resistance was determined by submitting sequences to the Stanford HIV Drug Resistance Database. RESULTS: A total of 32 CRF65_cpx pol sequences were obtained. The CRF65_cpx strains were detected in seven provinces with large geographic distance. Yunnan CRF65_cpx sequences were mainly derived from a heterosexual risk group, whereas the CRF65_cpx sequences in other provinces were almost exclusively derived from an MSM population. With one exception of V179E, the other 31 strains harbored V179D mutation. The combination of V179D and K103R, conferring intermediate resistance to EFV and NVP, was detected in seven treatment-naive MSM patients. CONCLUSIONS: This study confirmed the expansion CRF65_cpx in China. Furthermore, we found the natural presence of the V179D and K103R/V179D mutations associated with resistance to NNRTIs in HIV-1 CRF65_cpx. Our findings highlight the contribution of polymorphic mutations to drug resistance and underscore the challenges in treating patients harboring CRF65_cpx strains.

Treatment of Relapsed Acute Myeloid Leukemia.

OPINION STATEMENT: Relapse is still a common scenario in acute myeloid leukemia (AML) treatment and occurs in 40-50% of younger and the great majority of elderly patients. The prognosis in relapsed AML patients is generally poor but depends largely on the timing of relapse (early versus late) and the possibility of allogeneic hematopoietic stem cell transplantation (HSCT). At the time of relapse, we again perform a mutational screening and cytogenetic analysis in all AML patients as clonal evolution of disease is frequent. Clinical trials should be first priority in all relapsed patients. In fit patients without prior transplant, we aim to perform HSCT after salvage therapy. In AML patients relapsing after HSCT and good performance status, intensive therapy can be considered with subsequent cellular therapy such as donor lymphocyte infusion (DLI) or a second HSCT. However, less than 20% of these patients are alive after 5 years. For those patients that are unfit, the therapeutic aim is to prolong life with acceptable quality of life. Here, hypomethylating agents (HMA), low-dose AraC (LDAC), and solely cytoreductive therapy with hydroxurea are options depending on first-line therapy. For those patients that have not been treated with venetoclax in first line, the combination therapy of venetoclax with demethylating agents achieves encouraging response rates. Venetoclax is currently also studied in combination with intensive salvage therapy. Importantly, for patients with isocitrate dehydrogenase (IDH) 1/2-mutated AML, ivosidenib, an IDH1 inhibitor, and enasidenib, an IDH2 inhibitor, present well-tolerated options in the setting of refractory or relapsed (r/r) disease even in elderly and heavily pre-treated patients with response rates of 30-40%. Both substances have been approved by the U.S. Food and Drug Administration (FDA) for r/r AML patients with IDH1/2 mutations (but not yet by the European Medicines Agency (EMA)). For patients with FMS-like tyrosine kinase 3 (FLT3) mutations, treatment with the selective FLT3 inhibitor gilteritinib is well tolerated and leads to improved outcome compared with standard salvage therapy. The approval has been granted by the FDA and the EMA. Generally, we would recommend targeted therapy for IDH1/2- and FLT3-mutated AML if available. In order to improve outcome in relapsed AML, it will be important to intelligently combine novel substances with each other as well as chemotherapy in prospective clinical trials. The development of therapies with bispecific antibodies or chimeric antigen receptor T cells (CAR-T) are still in early development.

Reduced exercise capacity and clinical outcomes following acute myocardial infarction.

Reduced exercise capacity is known to be an important predictor of poor prognosis and disability in patients with cardiovascular diseases and chronic heart failure, and even members of the general population. However, data about exercise capacity assessed by cardiopulmonary exercise testing (CPX) in acute myocardial infarction (AMI) patients who underwent primary percutaneous coronary intervention (PCI) is scarce. Among 594 consecutive AMI patients who underwent primary PCI, we examined 136 patients (85.3% men, 64.9 ± 11.9 years) who underwent CPX during hospitalization for AMI. CPX was usually performed 5 days after the onset of AMI. Reduced exercise capacity was defined as peak VO2 ≤ 12. Clinical outcomes including all-cause death, myocardial infarction, and hospitalization due to heart failure were followed. Among 136 patients, reduced exercise capacity (peak VO2 ≤ 12) was seen in 38 patients (28%). Patients with reduced exercise capacity were older, more likely to have hypertension, and had lower renal function. In echocardiography, patients with reduced exercise capacity had higher E/e' and larger left atrial dimension. Multivariate logistic analysis showed that E/e' (OR 1.19, 95% CI 1.09-1.31, p < 0.001) was an independent predictor of reduced exercise capacity (peak VO2 ≤ 12). Median follow-up term was 12 months (IQR 9-22). The occurrence of composite endpoints of all-cause death, myocardial infarction, and hospitalization due to heart failure was significantly higher in patients with peak VO2 ≤ 12 than those with peak VO2 > 12 (p < 0.001). Reduced exercise capacity following primary PCI in AMI patients is associated with diastolic dysfunction and may lead to poorer clinical outcomes.

Pharmacokinetics, drug metabolism, and tissue distribution of CPX-351 in animals.

CPX-351, a liposomal encapsulation of cytarabine and daunorubicin at a synergistic 5:1 molar ratio, is indicated for adults with newly diagnosed, therapy-related acute myeloid leukemia or acute myeloid leukemia with myelodysplasia-related changes. In preclinical species, this article demonstrated (1) similar release of cytarabine and daunorubicin by CPX-351 in plasma; (2) similar patterns of metabolism of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal cytarabine/daunorubicin combination; (3) prolonged tissue exposure to CPX-351; (4) dramatically different tissue distribution of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal combination (tissue:plasma ratios generally <1 versus >1, respectively); and (5) dramatically lower unbound plasma and tissue concentrations of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal combination. Together, these results provide insight into the safety profile of CPX-351, as well as mechanisms that drive the improved efficacy observed for CPX-351 versus the conventional 7 + 3 cytarabine/daunorubicin regimen in clinical studies.

Redefining the essential trafficking pathway for outer membrane lipoproteins.

The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins' key function is to prevent lethal accumulation of mislocalized lipoproteins.

The Lipoprotein NlpE Is a Cpx Sensor That Serves as a Sentinel for Protein Sorting and Folding Defects in the Escherichia coli Envelope.

The envelope of Gram-negative bacteria is a complex compartment that is essential for viability. To ensure survival of the bacterial cells in fluctuating environments, several signal transduction systems, called envelope stress response systems (ESRSs), exist to monitor envelope biogenesis and homeostasis. The Cpx two-component system is an extensively studied ESRS in Escherichia coli that is active during exposure to a vast array of stresses and protects the envelope under those harmful circumstances. Overproduction of NlpE, a two-domain outer membrane lipoprotein of unclear function, has been used in numerous studies as a molecular trigger to turn on the system artificially. However, the mechanism of Cpx activation by NlpE, as well as its physiological relevance, awaited further investigation. In this paper, we provide novel insights into the role played by NlpE in the Cpx system. We found that, among all outer membrane lipoproteins in E. coli, NlpE is sufficient to induce Cpx when lipoprotein trafficking is perturbed. Under such conditions, fitness is increased by the presence of NlpE. Moreover, we show that NlpE, through its N-terminal domain, physically interacts with the Cpx sensor kinase CpxA. Our data suggest that NlpE also serves to activate the Cpx system during oxidative folding defects in the periplasm and that its C-terminal domain is involved in the sensing mechanism. Overall, our data demonstrate that NlpE acts as a sentinel for two important envelope biogenesis processes, namely, lipoprotein sorting and oxidative folding, and they further establish NlpE as a bona fide member of the Cpx two-component system.IMPORTANCE Bacteria rely on a sophisticated envelope to shield them against challenging environmental conditions and therefore need to ensure correct envelope assembly and integrity. A major signaling pathway that performs this role in Gram-negative species is the Cpx system. An outer membrane lipoprotein of unclear function, NlpE, has long been exploited as a research tool to study Cpx in E. coli, since it triggers this system when overproduced or mislocalized; however, the mechanism and physiological relevance of the NlpE-Cpx connection have awaited further investigation. We elucidate a new function for NlpE by showing that it physically interacts with the Cpx sensor CpxA and acts as a sentinel that specifically monitors two essential envelope biogenesis processes, namely, lipoprotein sorting and oxidative folding.