Proteomics Analysis of Interactions between Drug-Resistant and Drug-Sensitive Cancer Cells: Comparative Studies of Monoculture and Coculture Cell Systems.

Cell-cell interactions, which allow cells to communicate with each other through molecules in their microenvironment, are critical for the growth, health, and functions of cells. Previous studies show that drug-resistant cells can interact with drug-sensitive cells to elevate their drug resistance level, which is partially responsible for cancer recurrence. Studying protein targets and pathways involved in cell-cell communication provides essential information for fundamental cell biology studies and therapeutics of human diseases. In the current studies, we performed direct coculture and indirect coculture of drug-resistant and drug-sensitive cell lines, aiming to investigate intracellular proteins responsible for cell communication. Comparative studies were carried out using monoculture cells. Shotgun bottom-up proteomics results indicate that the P53 signaling pathway has a strong association with drug resistance mechanisms, and multiple TP53-related proteins were upregulated in both direct and indirect coculture systems. In addition, cell-cell communication pathways, including the phagosome and the HIF-signaling pathway, contribute to both direct and indirect coculture systems. Consequently, AK3 and H3-3A proteins were identified as potential targets for cell-cell interactions that are relevant to drug resistance mechanisms. We propose that the P53 signaling pathway, in which mitochondrial proteins play an important role, is responsible for inducing drug resistance through communication between drug-resistant and drug-sensitive cancer cells.

Decoding Arabidopsis thaliana CPK/SnRK Superfamily Kinase Client Signaling Networks Using Peptide Library and Mass Spectrometry.

Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay-a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay-to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets.

Comparative Seed Proteome Profile Reveals No Alternation of Major Allergens in High-Yielding Mung Bean Cultivars.

Mung bean contains up to 32.6% protein and is one of the great sources of plant-based protein. Because many allergens also function as defense-related proteins, it is important to determine their abundance levels in the high-yielding, disease-resistant cultivars. In this study, for the first time, we compared the seed proteome of high-yielding mung bean cultivars developed by a conventional breeding approach. Using a label-free quantitative proteomic platform, we successfully identified and quantified a total of 1373 proteins. Comparative analysis between the high-yielding disease-resistant cultivar (MC5) and the other three cultivars showed that a total of 69 common proteins were significantly altered in their abundances across all cultivars. Bioinformatic analysis of these altered proteins demonstrated that PDF1 (a defensin-like protein) exhibited high sequence similarity and epitope matching with the established peanut allergens, indicating a potential mung bean allergen that showed a cultivar-specific response. Conversely, known mung bean allergen proteins such as PR-2/PR-10 (Vig r 1), Vig r 2, Vig r 4, LTP1, ß-conglycinin, and glycinin G4 showed no alternation in the MC5 compared to other cultivars. Taken together, our findings suggest that the known allergen profiles may not be impacted by the conventional plant breeding method to develop improved mung bean cultivars.

Extent of Virulence and Antibiotic Resistance Genes in Helicobacter pylori and Campylobacteria.

Helicobacter pylori, a member of the clade campylobacteria, is the leading cause of chronic gastritis and gastric cancer. Virulence and antibiotic resistance of H. pylori are of great concern to public health. However, the relationship between virulence and antibiotic resistance genes in H. pylori in relation to other campylobacteria remains unclear. Using the virulence and comprehensive antibiotic resistance databases, we explored all available 354 complete genomes of H. pylori and compared it with 90 species of campylobacteria for virulence and antibiotic resistance genes/proteins. On average, H. pylori had 129 virulence genes, highest among Helicobacter spp. and 71 antibiotic resistance genes, one of the lowest among campylobacteria. Just 2.6% of virulence genes were shared by all campylobacterial members, whereas 9.4% were unique to H. pylori. The cytotoxin-associated genes (cags) seemed to be exclusive to H. pylori. Majority of the isolates from Asia and South America were cag2-negative and many antibiotic resistance genes showed isolate-specific patterns of occurrence. Just 15 (8.8%) antibiotic resistance genes, but 103 (66%) virulence genes including 25 cags were proteomically identified in H. pylori. Arcobacterial members showed large variation in the number of antibiotic resistance genes and there was a positive relation with the genome size. Large repository of antibiotic resistance genes in campylobacteria and a unique set of virulence genes might have important implications in shaping the course of virulence and antibiotic resistance in H. pylori.

Urine proteome profile of firefighters with exposure to emergency fire-induced smoke: A pilot study to identify potential carcinogenic effects.

Firefighters are frequently exposed to a variety of chemicals formed from smoke, which pose a risk for numerous diseases, including cancer. Comparative urine proteome profiling could significantly improve our understanding of the early detection of potential cancer biomarkers. In this study, for the first time, we conducted a comparative protein profile analysis of 20 urine samples collected from ten real-life firefighters prior to and following emergency fire-induced smoke. Using a label-free quantitative proteomics platform, we identified and quantified 1325 unique protein groups, of which 45 proteins showed differential expressions in abundance in response to fire-smoke exposure (post) compared to the control (pre). Pathway analysis showed proteins associated with epithelium development (e.g., RHCG, HEG1, ADAMTSL2) and Alzheimer's disease (SORL1) were significantly increased in response to smoke exposure samples. A protein-protein-network study showed a possible link between these differentially abundant proteins and the known cancer gene (TP53). Moreover, a cross-comparison analysis revealed that seven proteins-ALDH1A1, APCS, POMC, COL2A1, RDX, DDAH2, and SDC4 overlapped with the previously published urine cancer proteome datasets, suggesting a potential cancer risk. Our findings demonstrated that the discovery proteomic platform is a promising analytical technique for identifying potential non-invasive biomarkers associated with fire-smoke exposure in firefighters that may be related to cancer.

Identification of mitogen-activated protein kinases substrates in Arabidopsis using kinase client assay.

Mitogen-activated protein kinase (MPK) cascades are essential signal transduction components that control a variety of cellular responses in all eukaryotes. MPKs convert extracellular stimuli into cellular responses by the phosphorylation of downstream substrates. Although MPK cascades are predicted to be very complex, only limited numbers of MPK substrates have been identified in plants. Here, we used the kinase client (KiC) assay to identify novel substrates of MPK3 and MPK6. Recombinant MPK3 or MPK6 were tested against a large synthetic peptide library representing in vivo phosphorylation sites, and phosphorylated peptides were identified by high-resolution tandem mass spectrometry. From this screen, we identified 23 and 21 putative client peptides of MPK3 and MPK6, respectively. To verify the phosphorylation of putative client peptides, we performed in vitro kinase assay with recombinant fusion proteins of isolated client peptides. We found that 13 and 9 recombinant proteins were phosphorylated by MPK3 and MPK6. Among them, 11 proteins were proven to be the novel substrates of two MPKs. This study suggests that the KiC assay is a useful method to identify new substrates of MPKs.

Antimicrobial Activity of Polycaprolactone Nanofiber Coated with Lavender and Neem Oil Nanoemulsions against Airborne Bacteria.

The development of efficient, eco-friendly antimicrobial agents for air purification and disinfection addresses public health issues connected to preventing airborne pathogens. Herein, the antimicrobial activity of a nanoemulsion (control, 5%, 10%, and 15%) containing neem and lavender oils with polycaprolactone (PCL) was investigated against airborne bacteria, including Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. Various parameters such as the physicochemical properties of the nanoemulsion, pH, droplet size, the polydispersity index (PDI), the minimum inhibitory concentration (MIC), the minimum bacterial concentration (MBC), and the color measurement of the emulsion have been evaluated and optimized. Our results showed that the antimicrobial activity of PCL combined with neem and lavender oil was found to be the highest MIC and MBC against all tested bacteria. The droplet sizes for lavender oil are 21.86-115.15 nm, the droplet sizes for neem oil are 23.92-119.15 nm, and their combination is 25.97-50.22 nm. The range of pH and viscosity of nanoemulsions of various concentrations was found to be 5.8 to 6.6 pH and 0.372 to 2.101 cP. This study highlights the potential of nanotechnology in harnessing the antimicrobial properties of natural essential oils, paving the way for innovative and sustainable solutions in the fight against bacterial contamination.

Blocking of doublecortin-like kinase 1-regulated SARS-CoV-2 replication cycle restores cell signaling network.

IMPORTANCE: Severe COVID-19 and post-acute sequelae often afflict patients with underlying co-morbidities. There is a pressing need for highly effective treatment, particularly in light of the emergence of SARS-CoV-2 variants. In a previous study, we demonstrated that DCLK1, a protein associated with cancer stem cells, is highly expressed in the lungs of COVID-19 patients and enhances viral production and hyperinflammatory responses. In this study, we report the pivotal role of DCLK1-regulated mechanisms in driving SARS-CoV-2 replication-transcription processes and pathogenic signaling. Notably, pharmacological inhibition of DCLK1 kinase during SARS-CoV-2 effectively impedes these processes and counteracts virus-induced alternations in global cell signaling. These findings hold significant potential for immediate application in treating COVID-19.

Orbitrap Mass Spectrometry and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Enable the in-Depth Analysis of Human Serum Proteoforms.

Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches that cannot recapitulate the original proteoform content of samples. Clinical laboratories have been slow to adopt top-down analysis, also due to higher sample handling requirements. In this study, we describe a straightforward protocol for intact proteoform sample preparation based on the depletion of albumin and immunoglobulins, followed by simplified protein fractionation via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography-tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to further simplify serum proteoform mixtures. This LC-FAIMS-MS2 method led to the identification of over 1000 serum proteoforms < 30 kDa, outperforming traditional LC-MS2 data acquisition and more than doubling the number of proteoforms identified in previous studies.

Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum.

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%-19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.

The Raf-like MAPKKK INTEGRIN-LINKED KINASE 5 regulates purinergic receptor-mediated innate immunity in Arabidopsis.

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in eukaryotic defense against various pathogens. Activation of the extracellular ATP (eATP) receptor P2K1 triggers MAP kinase 3 and 6 (MPK3/6) phosphorylation, which leads to an elevated plant defense response. However, the mechanism by which P2K1 activates the MAPK cascade is unclear. In this study, we show that in Arabidopsis thaliana, P2K1 phosphorylates the Raf-like MAP kinase kinase kinase (MAPKKK) INTEGRIN-LINKED KINASE 5 (ILK5) on serine 192 in the presence of eATP. The interaction between P2K1 and ILK5 was confirmed both in vitro and in planta and their interaction was enhanced by ATP treatment. Similar to P2K1 expression, ILK5 expression levels were highly induced by treatment with ATP, flg22, Pseudomonas syringae pv. tomato DC3000, and various abiotic stresses. ILK5 interacts with and phosphorylates the MAP kinase MKK5. Moreover, phosphorylation of MPK3/6 was significantly reduced upon ATP treatment in ilk5 mutant plants, relative to wild-type (WT). The ilk5 mutant plants showed higher susceptibility to P. syringae pathogen infection relative to WT plants. Plants expressing only the mutant ILK5S192A protein, with decreased kinase activity, did not activate the MAPK cascade upon ATP addition. These results suggest that eATP activation of P2K1 results in transphosphorylation of the Raf-like MAPKKK ILK5, which subsequently triggers the MAPK cascade, culminating in activation of MPK3/6 associated with an elevated innate immune response.

Proteomic Assessment of Hypoxia-Pre-Conditioned Human Bone Marrow Mesenchymal Stem Cell-Derived Extracellular Vesicles Demonstrates Promise in the Treatment of Cardiovascular Disease.

Human bone marrow mesenchymal stem cell derived-extracellular vesicles (HBMSC-EV) are known for their regenerative and anti-inflammatory effects in animal models of myocardial ischemia. However, it is not known whether the efficacy of the EVs can be modulated by pre-conditioning of HBMSC by exposing them to either starvation or hypoxia prior to EV collection. HBMSC-EVs were isolated following normoxia starvation (NS), normoxia non-starvation (NNS), hypoxia starvation (HS), or hypoxia non-starvation (HNS) pre-conditioning. The HBMSC-EVs were characterized by nanoparticle tracking analysis, electron microscopy, Western blot, and proteomic analysis. Comparative proteomic profiling revealed that starvation pre-conditioning led to a smaller variety of proteins expressed, with the associated lesser effect of normoxia versus hypoxia pre-conditioning. In the absence of starvation, normoxia and hypoxia pre-conditioning led to disparate HBMSC-EV proteomic profiles. HNS HBMSC-EV was found to have the greatest variety of proteins overall, with 74 unique proteins, the greatest number of redox proteins, and pathway analysis suggestive of improved angiogenic properties. Future HBMSC-EV studies in the treatment of cardiovascular disease may achieve the most therapeutic benefits from hypoxia non-starved pre-conditioned HBMSC. This study was limited by the lack of functional and animal models of cardiovascular disease and transcriptomic studies.

Proximity proteomics reveals role of Abelson interactor 1 in the regulation of TAK1/RIPK1 signaling.

Dysregulation of the adaptor protein Abelson interactor 1 (ABI1) is linked to malignant transformation. To interrogate the role of ABI1 in cancer development, we mapped the ABI1 interactome using proximity-dependent labeling (PDL) with biotin followed by mass spectrometry. Using a novel PDL data filtering strategy, considering both peptide spectral matches and peak areas of detected peptides, we identified 212 ABI1 proximal interactors. These included WAVE2 complex components such as CYFIP1, NCKAP1, or WASF1, confirming the known role of ABI1 in the regulation of actin-polymerization-dependent processes. We also identified proteins associated with the TAK1-IKK pathway, including TAK1, TAB2, and RIPK1, denoting a newly identified function of ABI1 in TAK1-NF-κB inflammatory signaling. Functional assays using TNFα-stimulated, ABI1-overexpressing or ABI1-deficient cells showed effects on the TAK1-NF-kB pathway-dependent signaling to RIPK1, with ABI1-knockout cells being less susceptible to TNFα-induced, RIPK1-mediated, TAK1-dependent apoptosis. In sum, our PDL-based strategy enabled mapping of the ABI1 proximal interactome, thus revealing a previously unknown role of this adaptor protein in TAK1/RIPK1-based regulation of cell death and survival.

Reduction in mitochondrial ROS improves oxidative phosphorylation and provides resilience to coronary endothelium in non-reperfused myocardial infarction.

Recent studies demonstrated that mitochondrial antioxidant MnSOD that reduces mitochondrial (mito) reactive oxygen species (ROS) helps maintain an optimal balance between sub-cellular ROS levels in coronary vascular endothelial cells (ECs). However, it is not known whether EC-specific mito-ROS modulation provides resilience to coronary ECs after a non-reperfused acute myocardial infarction (MI). This study examined whether a reduction in endothelium-specific mito-ROS improves the survival and proliferation of coronary ECs in vivo. We generated a novel conditional binary transgenic animal model that overexpresses (OE) mitochondrial antioxidant MnSOD in an EC-specific manner (MnSOD-OE). EC-specific MnSOD-OE was validated in heart sections and mouse heart ECs (MHECs). Mitosox and mito-roGFP assays demonstrated that MnSOD-OE resulted in a 50% reduction in mito-ROS in MHEC. Control and MnSOD-OE mice were subject to non-reperfusion MI surgery, echocardiography, and heart harvest. In post-MI hearts, MnSOD-OE promoted EC proliferation (by 2.4 ± 0.9 fold) and coronary angiogenesis (by 3.4 ± 0.9 fold), reduced myocardial infarct size (by 27%), and improved left ventricle ejection fraction (by 16%) and fractional shortening (by 20%). Interestingly, proteomic and Western blot analyses demonstrated upregulation in mitochondrial complex I and oxidative phosphorylation (OXPHOS) proteins in MnSOD-OE MHECs. These MHECs also showed increased mitochondrial oxygen consumption rate (OCR) and membrane potential. These findings suggest that mito-ROS reduction in EC improves coronary angiogenesis and cardiac function in non-reperfused MI, which are associated with increased activation of OXPHOS in EC-mitochondria. Activation of an energy-efficient mechanism in EC may be a novel mechanism to confer resilience to coronary EC during MI.

Hyperoxia impairs intraflagellar transport and causes dysregulated metabolism with resultant decreased cilia length.

Supplemental oxygen is a lifesaving measure in infants born premature to facilitate oxygenation. Unfortunately, it may lead to alveolar simplification and loss of proximal airway epithelial cilia. Little is known about the mechanism by which hyperoxia causes ciliary dysfunction in the proximal respiratory tract. We hypothesized that hyperoxia causes intraflagellar transport (IFT) dysfunction with resultant decreased cilia length. Differentiated basal human airway epithelial cells (HAEC) were exposed to hyperoxia or air for up to 48 h. Neonatal mice (<12 h old) were exposed to hyperoxia for 72 h and recovered in room air until postnatal day (PND) 60. Cilia length was measured from scanning electron microscopy images using a MATLAB-derived program. Proteomics and metabolomics were carried out in cells after hyperoxia. After hyperoxia, there was a significant time-dependent reduction in cilia length after hyperoxia in HAEC. Proteomic analysis showed decreased abundance of multiple proteins related to IFT including dynein motor proteins. In neonatal mice exposed to hyperoxia, there was a significant decrease in acetylated α tubulin at PND10 followed by recovery to normal levels at PND60. In HAEC, hyperoxia decreased the abundance of multiple proteins associated with complex I of the electron transport chain. In HAEC, hyperoxia increased levels of malate, fumarate, and citrate, and reduced the ATP/ADP ratio at 24 h with a subsequent increase at 36 h. Exposure to hyperoxia reduced cilia length, and this was associated with aberrant IFT protein expression and dysregulated metabolism. This suggests that hyperoxic exposure leads to aberrant IFT protein expression in the respiratory epithelium resulting in shortened cilia.

The germline factor DDX4 contributes to the chemoresistance of small cell lung cancer cells.

Human cancers often re-express germline factors, yet their mechanistic role in oncogenesis and cancer progression remains unknown. Here we demonstrate that DEAD-box helicase 4 (DDX4), a germline factor and RNA helicase conserved in all multicellular organisms, contributes to increased cell motility and cisplatin-mediated drug resistance in small cell lung cancer (SCLC) cells. Proteomic analysis suggests that DDX4 expression upregulates proteins related to DNA repair and immune/inflammatory response. Consistent with these trends in cell lines, DDX4 depletion compromised in vivo tumor development while its overexpression enhanced tumor growth even after cisplatin treatment in nude mice. Further, the relatively higher DDX4 expression in SCLC patients correlates with decreased survival and shows increased expression of immune/inflammatory response markers. Taken together, we propose that DDX4 increases SCLC cell survival, by increasing the DNA damage and immune response pathways, especially under challenging conditions such as cisplatin treatment.

Back From the Dead: The Atypical Kinase Activity of a Pseudokinase Regulator of Cation Fluxes During Inducible Immunity.

Pseudokinases are thought to lack phosphotransfer activity due to altered canonical catalytic residues within their kinase domain. However, a subset of pseudokinases maintain activity through atypical phosphotransfer mechanisms. The Arabidopsis ILK1 is a pseudokinase from the Raf-like MAP3K family and is the only known plant pseudokinase with confirmed protein kinase activity. ILK1 activity promotes disease resistance and molecular pattern-induced root growth inhibition through its stabilization of the HAK5 potassium transporter with the calmodulin-like protein CML9. ILK1 also has a kinase-independent function in salt stress suggesting that it interacts with additional proteins. We determined that members of the ILK subfamily are the sole pseudokinases within the Raf-like MAP3K family and identified 179 novel putative ILK1 protein interactors. We also identified 70 novel peptide targets for ILK1, the majority of which were phosphorylated in the presence of Mn2+ instead of Mg2+ in line with modifications in ILK1's DFG cofactor binding domain. Overall, the ILK1-targeted or interacting proteins included diverse protein types including transporters (HAK5, STP1), protein kinases (MEKK1, MEKK3), and a cytokinin receptor (AHK2). The expression of 31 genes encoding putative ILK1-interacting or phosphorylated proteins, including AHK2, were altered in the root and shoot in response to molecular patterns suggesting a role for these genes in immunity. We describe a potential role for ILK1 interactors in the context of cation-dependent immune signaling, highlighting the importance of K+ in MAMP responses. This work further supports the notion that ILK1 is an atypical kinase with an unusual cofactor dependence that may interact with multiple proteins in the cell.

Lentil allergens identification and quantification: An update from omics perspective.

Among legumes, the lentil (Lens culinaris) is a major dietary component in many Mediterranean and Asian countries due to its high nutritional value, especially protein. However, allergic reactions triggered by lentil consumption have also been documented in many countries. Complete allergens profiling is critical for better management of lentil food allergies. Earlier studies suggested Len c 1, a 47 kDa vicilin, Len c 2, a seed-specific-biotinylated 66-kDa protein, and Len c 3, low molecular weight lipid transfer proteins (LTPs) were major allergenic proteins in lentils. Recently, mass-spectrometry-based proteomic platforms successfully identified proteins from lentil samples homologous to known plant allergens. Furthermore, in silico analysis using 337 protein sequences revealed lentil allergens that have not previously been identified as potential allergens in lentil. Herein, we discuss the feasibility of omics platforms utilized for lentil allergens profiling and quantification. In addition, we propose some future strategies that might be beneficial for profiling and development of precise assays for lentil allergens and could facilitate identification of the low allergen-containing lentil cultivars.

CpxA Phosphatase Inhibitor Activates CpxRA and Is a Potential Treatment for Uropathogenic Escherichia coli in a Murine Model of Infection.

CpxRA is an envelope stress response system that is highly conserved in the Enterobacteriaceae. CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR (CpxR-P), a transcription factor. In response to membrane stress, CpxR-P is produced and upregulates genes involved in membrane repair and downregulates genes that encode virulence factors that are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and in uropathogenic Escherichia coli (UPEC) are attenuated in murine models. We hypothesized that pharmacologic activation of CpxR could serve as an antimicrobial/antivirulence strategy and recently showed that 2,3,4,9-tetrahydro-1H-carbazol-1-amines activate the CpxRA system by inhibiting CpxA phosphatase activity. Here, we tested the ability of a series of three CpxRA-activating compounds with increasing potency to clear UPEC stain CFT073 in a murine urinary tract infection model. We show that these compounds are well tolerated and achieve sufficient levels to activate CpxR in the kidneys, bladder, and urine. Although the first two compounds were ineffective in promoting clearance of CFT073 in the murine model, the most potent derivative, compound 26, significantly reduced bacterial recovery in the urine and trended toward reducing bacterial recovery in the bladder and kidneys, with efficacy similar to ciprofloxacin. Treatment of CFT073 cultured in human urine with compound 26 fostered accumulation of CpxR-P and decreased the expression of proteins involved in siderophore biosynthesis and binding, heme degradation, and flagellar movement. These studies suggest that chemical activation of CpxRA may present a viable strategy for treating infections due to UPEC. IMPORTANCE The increasing prevalence of urinary tract infections (UTIs) due to antibiotic-resistant uropathogenic Escherichia coli (UPEC) is a major public health concern. Bacteria contain proteins that sense their environment and have no human homologs and, thus, are attractive drug targets. CpxRA is a conserved sensing system whose function is to reduce stress in the bacterial cell membrane; activation of CpxRA reduces the expression of virulence determinants, which must cross the cell membrane to reach the bacterial surface. We previously identified a class of compounds that activate CpxRA. We show in a mouse UTI model that our most potent compound significantly reduced recovery of UPEC in the urine, trended toward reducing bacterial recovery in the bladder and kidneys, did not kill UPEC, and downregulated multiple proteins involved in UPEC virulence. Since these compounds do not act by a killing mechanism, they have potential to treat UTIs caused by antibiotic-resistant bacteria.

Evolutionary Dynamics of Indels in SARS-CoV-2 Spike Glycoprotein.

SARS-CoV-2, responsible for the current COVID-19 pandemic that claimed over 5.0 million lives, belongs to a class of enveloped viruses that undergo quick evolutionary adjustments under selection pressure. Numerous variants have emerged in SARS-CoV-2, posing a serious challenge to the global vaccination effort and COVID-19 management. The evolutionary dynamics of this virus are only beginning to be explored. In this work, we have analysed 1.79 million spike glycoprotein sequences of SARS-CoV-2 and found that the virus is fine-tuning the spike with numerous amino acid insertions and deletions (indels). Indels seem to have a selective advantage as the proportions of sequences with indels steadily increased over time, currently at over 89%, with similar trends across countries/variants. There were as many as 420 unique indel positions and 447 unique combinations of indels. Despite their high frequency, indels resulted in only minimal alteration of N-glycosylation sites, including both gain and loss. As indels and point mutations are positively correlated and sequences with indels have significantly more point mutations, they have implications in the evolutionary dynamics of the SARS-CoV-2 spike glycoprotein.