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Organs in the abdominal cavity are covered by a peritoneal membrane, which is comprised of a monolayer of mesothelial cells (MC). Diseases involving the peritoneal membrane include peritonitis, primary cancer (mesothelioma), and metastatic cancers (ovarian, pancreatic, colorectal). These diseases have gender- and/or age-related pathologies; however, the impact of gender and age on the peritoneal MC is not well evaluated. To address this, we identified and characterized gender- and age-related differences in the proteomes of murine primary peritoneal MC. Primary peritoneal MC were isolated from young female (FY) or male (MY) mice (3-6 months) and aged female (FA) or male (MA) mice (20-23 months), lysed, trypsin digested using S-Traps, then subjected to bottom-up proteomics using an LC-Orbitrap mass spectrometer. In each cohort, we identified >1000 protein groups. Proteins were categorized using Gene Ontology and pairwise comparisons between gender and age cohorts were conducted. This study establishes baseline information for studies on peritoneal MC in health and disease at two physiologic age/gender points. Segregation of the data by gender and age could reveal novel factors to specific disease states involving the peritoneum. [This in vitro primary cell model has utility for future studies on the interaction between the mesothelium and foreign materials.]., Competing Interests: CONFLICT OF INTEREST The authors have no financial or commercial conflicts of interest to declare.

Synonymous codons were originally viewed as interchangeable, with no phenotypic consequences. However, substantial evidence has now demonstrated that synonymous substitutions can perturb a variety of gene expression and protein homeostasis mechanisms, including translational efficiency, translational fidelity, and cotranslational folding of the encoded protein. To date, most studies of synonymous codon-derived perturbations have focused on effects within a single gene. Here, we show that synonymous codon substitutions made far within the coding sequence of Escherichia coli plasmid-encoded chloramphenicol acetyltransferase ( cat ) can significantly increase expression of the divergent upstream tetracycline resistance gene, tetR . In four out of nine synonymously recoded cat sequences tested, expression of the upstream tetR gene was significantly elevated due to transcription of a long antisense RNA (asRNA) originating from a transcription start site within cat . Surprisingly, transcription of this asRNA readily bypassed the native tet transcriptional repression mechanism. Even more surprisingly, accumulation of the TetR protein correlated with the level of asRNA, rather than total tetR RNA. These effects of synonymous codon substitutions on transcription and translation of a neighboring gene suggest that synonymous codon usage in bacteria may be under selection to both preserve the amino acid sequence of the encoded gene and avoid DNA sequence elements that can significantly perturb expression of neighboring genes. Avoiding such sequences may be especially important in plasmids and prokaryotic genomes, where genes and regulatory elements are often densely packed. Similar considerations may apply to the design of genetic circuits for synthetic biology applications., Competing Interests: Competing interests statement:The authors declare no competing interest.

Quenching digestions in proteomics prior to analysis is routine in order to eliminate residual protease activity. Residual activity leads to overdigestion, nonspecific star-activity, and back-exchange in isotopic 18 O quantitation. Chemical and isobaric labeling (e.g., TMT/iTRAQ) of proteins or peptides for mass spectrometry-based proteomics is generally incompatible with ubiquitous postdigestion acidification. This necessitates buffer exchange and pH adjustments. We demonstrate that quenching is unnecessary with peptides generated from protein filter-traps, as trypsin activity and intact trypsin are negligible in the eluate from these preparations. Labeling can be directly performed on enzymatic digests from these methods, improving recovery, throughput, and ease of automation.

Phagosomal lysis is a key aspect of mycobacterial infection of host macrophages. Acetylation is a protein modification mediated enzymatically by N-acetyltransferases (NATs) that impacts bacterial pathogenesis and physiology. To identify NATs required for lytic activity, we leveraged Mycobacterium marinum, a nontubercular pathogen and an established model for M. tuberculosis. M. marinum hemolysis is a proxy for phagolytic activity. We generated M. marinum strains with deletions in conserved NAT genes and screened for hemolytic activity. Several conserved lysine acetyltransferases (KATs) contributed to hemolysis. Hemolysis is mediated by the ESX-1 secretion system and by phthiocerol dimycocerosate (PDIM), a virulence lipid. For several strains, the hemolytic activity was restored by the addition of second copy of the ESX-1 locus. Using thin-layer chromatography (TLC), we found a single NAT required for PDIM and phenolic glycolipid (PGL) production. MbtK is a conserved KAT required for mycobactin siderophore synthesis and virulence. Mycobactin J exogenously complemented PDIM/PGL production in the Δ mbtK strain. The Δ mbtK M. marinum strain was attenuated in macrophage and Galleria mellonella infection models. Constitutive expression of either eis or papA5, which encode a KAT required for aminoglycoside resistance and a PDIM/PGL biosynthetic enzyme, rescued PDIM/PGL production and virulence of the Δ mbtK strain. Eis N-terminally acetylated PapA5 in vitro , supporting a mechanism for restored lipid production. Overall, our study establishes connections between the MbtK and Eis NATs, and between iron uptake and PDIM and PGL synthesis in M. marinum . Our findings underscore the multifunctional nature of mycobacterial NATs and their connection to key virulence pathways., Significance Statement: Acetylation is a modification of protein N-termini, lysine residues, antibiotics and lipids. Many of the enzymes that promote acetylation belong to the GNAT family of proteins. M. marinum is a well-established as a model to understand how M. tuberculosis causes tuberculosis. In this study we sought to identify conserved GNAT proteins required for early stages of mycobacterial infection. Using M. marinum, we determined that several GNAT proteins are required for the lytic activity of M. marinum. We uncovered previously unknown connections between acetyl-transferases required for iron uptake and antimicrobial resistance, and the production of the unique mycobacterial lipids, PDIM and PGLOur data support that acetyl-transferases from the GNAT family are interconnected, and have activities beyond those previously reported.

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (K D  < 0.5 μM) include PBPs (PBP1a, K D  = 0.07 μM; PBP5 = 0.4 μM); other lytic transglycosylases (SltB2, K D  = 0.09 μM; RlpA, K D  = 0.4 μM); a type VI secretion system effector (Tse5, K D  = 0.3 μM); and a regulatory protease for alginate biosynthesis (AlgO, K D  < 0.4 μM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm., (© 2024 The Protein Society.)

The mycobacterial cell envelope is a major virulence determinant in pathogenic mycobacteria. Specific outer lipids play roles in pathogenesis, modulating the immune system and promoting the secretion of virulence factors. ESX-1 (ESAT-6 system-1) is a conserved protein secretion system required for mycobacterial pathogenesis. Previous studies revealed that mycobacterial strains lacking the outer lipid PDIM have impaired ESX-1 function during laboratory growth and infection. The mechanisms underlying changes in ESX-1 function are unknown. We used a proteo-genetic approach to measure phthiocerol dimycocerosate (PDIM)- and phenolic glycolipid (PGL)-dependent protein secretion in M. marinum, a non-tubercular mycobacterial pathogen that causes tuberculosis-like disease in ectothermic animals. Importantly, M. marinum is a well-established model for mycobacterial pathogenesis. Our findings showed that M. marinum strains without PDIM and PGL showed specific, significant reductions in protein secretion compared to the WT and complemented strains. We recently established a hierarchy for the secretion of ESX-1 substrates in four (I-IV) groups. Loss of PDIM differentially impacted secretion of Group III and IV ESX-1 substrates, which are likely the effectors of pathogenesis. Our data suggest that the altered secretion of specific ESX-1 substrates is responsible for the observed ESX-1-related effects in PDIM-deficient strains.IMPORTANCE Mycobacterium tuberculosis, the cause of human tuberculosis, killed an estimated 1.3 million people in 2022. Non-tubercular mycobacterial species cause acute and chronic human infections. Understanding how these bacteria cause disease is critical. Lipids in the cell envelope are essential for mycobacteria to interact with the host and promote disease. Strains lacking outer lipids are attenuated for infection, but the reasons are unclear. Our research aims to identify a mechanism for attenuation of mycobacterial strains without the PDIM and PGL outer lipids in M. marinum . These findings will enhance our understanding of the importance of lipids in pathogenesis and how these lipids contribute to other established virulence mechanisms., Competing Interests: The authors declare no conflict of interest.

N-terminal acetylation in Mycobacterium tuberculosis is correlated with pathogenic activity. We used genomics and bottom-up proteomics to identify protein Emp1 as the sole acetyltransferase responsible for acetylation of EsxA, a known virulence factor. Using custom data analysis, we screened the proteome to identify 22 additional putative substrates of Emp1., Competing Interests: M.M.C. is an editor at MRA and is in conflict as a potential editor for this revised submission.

Bacterial pathogens use protein secretion systems to transport virulence factors and regulate gene expression. Among pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum , the ESAT-6 system 1 (ESX-1) secretion is crucial for host interaction. Secretion of protein substrates by the ESX-1 secretion system disrupts phagosomes, allowing mycobacteria cytoplasmic access during macrophage infections. Deletion or mutation of the ESX-1 system attenuates mycobacterial pathogens. Pathogenic mycobacteria respond to the presence or absence of the ESX-1 system in the cytoplasmic membrane by altering transcription. Under laboratory conditions, the EspM repressor and WhiB6 activator control transcription of specific ESX-1-responsive genes, including the ESX-1 substrate genes. However, deleting the espM or whiB6 gene does not phenocopy the deletion of the ESX-1 substrate genes during macrophage infection by M. marinum . In this study, we identified EspN, a critical transcription factor whose activity is masked by the EspM repressor under laboratory conditions. In the absence of EspM, EspN activates transcription of whiB6 and ESX-1 genes during both laboratory growth and macrophage infection. EspN is also independently required for M. marinum growth within and cytolysis of macrophages, similar to the ESX-1 genes, and for disease burden in a zebrafish larval model of infection. These findings suggest that EspN and EspM coordinate to counterbalance the regulation of the ESX-1 system and support mycobacterial pathogenesis.IMPORTANCEPathogenic mycobacteria, which are responsible for tuberculosis and other long-term diseases, use the ESX-1 system to transport proteins that control the host response to infection and promote bacterial survival. In this study, we identify an undescribed transcription factor that controls the expression of ESX-1 genes and is required for both macrophage and animal infection. However, this transcription factor is not the primary regulator of ESX-1 genes under standard laboratory conditions. These findings identify a critical transcription factor that likely controls expression of a major virulence pathway during infection, but whose effect is not detectable with standard laboratory strains and growth conditions., Competing Interests: The authors declare no conflict of interest.

Amino acid substitutions (AASs) alter proteins from their genome-expected sequences. Accumulation of substitutions in proteins underlies numerous diseases and antibiotic mechanisms. Accurate global detection of AASs and their frequencies is crucial for understanding these mechanisms. Shotgun proteomics provides an untargeted method for measuring AASs but introduces biases when extrapolating from the genome to identify AASs. To characterize these biases, we created a "ground-truth" approach using the similarities between Escherichia coli and Salmonella typhimurium to model the complexity of AAS detection. Shotgun proteomics on mixed lysates generated libraries representing ∼100,000 peptide-spectra and 4161 peptide sequences with a single AAS and defined stoichiometry. Identifying S. typhimurium peptide-spectra with only the E. coli genome resulted in 64.1% correctly identified library peptides. Specific AASs exhibit variable identification efficiencies. There was no inherent bias from the stoichiometry of the substitutions. Short peptides and AASs localized near peptide termini had poor identification efficiency. We identify a new class of "scissor substitutions" that gain or lose protease cleavage sites. Scissor substitutions also had poor identification efficiency. This ground-truth AAS library reveals various sources of bias, which will guide the application of shotgun proteomics to validate AAS hypotheses.

Capillary zone electrophoresis (CZE) is a powerful tool for high resolution chemical separations. Applying CZE to microbial samples may facilitate a deeper understanding of bacterial physiology and behavior. However, the study of complex microbial samples has been limited by the uncontrolled hetero-aggregation of bacterial cells under an applied electric field. We tested a wide range of sample buffers and buffer additives for the optimization of bacterial CZE separations using a 20 mM Tris-HCl background electrolyte. By modifying the sample buffer, but not the background electrolyte, we retain constant separation conditions, which aids in the comparison of the sample buffer additives. We report optimized methods for automated CZE separation and simultaneous fractionation of Escherichia coli B, which is one of the two most widely used wild-type strains. A modified sample buffer containing neutral salts and the addition of glycerol produced a 20-fold increase in loading capacity and a reduction in peak width/broadening of 86% in comparison to previously reported work. In addition, the glycerol-modified sample buffer appears to reduce the persistent aggregation and adhesion to the capillary walls during electrophoretic separations of complex environmental microbiota., (© 2023. The Author(s).)

Importance: N-terminal acetylation is a protein modification that broadly impacts basic cellular function and disease in higher organisms. Although bacterial proteins are N-terminally acetylated, little is understood how N-terminal acetylation impacts bacterial physiology and pathogenesis. Mycobacterial pathogens cause acute and chronic disease in humans and in animals. Approximately 15% of mycobacterial proteins are N-terminally acetylated, but the responsible enzymes are largely unknown. We identified a conserved mycobacterial protein required for the N-terminal acetylation of 23 mycobacterial proteins including the EsxA virulence factor. Loss of this enzyme from M. marinum reduced macrophage killing and spread of M. marinum to new host cells. Defining the acetyltransferases responsible for the N-terminal protein acetylation of essential virulence factors could lead to new targets for therapeutics against mycobacteria., Competing Interests: The authors declare no conflict of interest.

Bacterial ribonucleoprotein bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here, we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We find 111 BR-body-enriched proteins showing that BR-bodies are more complex than previously assumed. We identify five BR-body-enriched proteins that undergo RNA-dependent phase separation in vitro with a complex network of condensate mixing. We observe that some RNP condensates co-assemble with preferred directionality, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body-associated proteins have the capacity to dissolve the condensate. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The 11 lytic transglycosylases of Pseudomonas aeruginosa have overlapping activities in the turnover of the cell-wall peptidoglycan. Rare lipoprotein A (RlpA) is distinct among the 11 by its use of only peptidoglycan lacking peptide stems. The spatial localization of RlpA and its interactome within P. aeruginosa are unknown. We employed suppression of introduced amber codons at sites in the rlpA gene for the introduction of the unnatural-amino-acids Ν ζ -[(2-azidoethoxy)carbonyl]-l-lysine (compound 1) and N ζ -[[[3-(3-methyl-3H-diazirin-3-yl)propyl]amino]carbonyl]-l-lysine (compound 2). In live P. aeruginosa, full-length RlpA incorporating compound 1 into its sequence was fluorescently tagged using strained-promoted alkyne-azide cycloaddition and examined by fluorescence microscopy. RlpA is present at low levels along the sidewall length of the bacterium, and at higher levels at the nascent septa of replicating bacteria. In intact P. aeruginosa, UV photolysis of full-length RlpA having compound 2 within its sequence generated a transient reactive carbene, which engaged in photoaffinity capture of neighboring proteins. Thirteen proteins were identified. Three of these proteins-PBP1a, PBP5, and MreB-are members of the bacterial divisome. The use of the complementary methodologies of non-canonical amino-acid incorporation, photoaffinity proximity analysis, and fluorescent microscopy confirm a dominant septal location for the RlpA enzyme of P. aeruginosa, as a divisome-associated activity. This accomplishment adds to the emerging recognition of the value of these methodologies for identification of the intracellular localization of bacterial proteins., (© 2023 The Protein Society.)

The pathological accumulation of cholesterol is a signature feature of Niemann-Pick type C (NPC) disease, in which excessive lipid levels induce Purkinje cell death in the cerebellum. NPC1 encodes a lysosomal cholesterol-binding protein, and mutations in NPC1 drive cholesterol accumulation in late endosomes and lysosomes (LE/Ls). However, the fundamental role of NPC proteins in LE/L cholesterol transport remains unclear. Here, we demonstrate that NPC1 mutations impair the projection of cholesterol-containing membrane tubules from the surface of LE/Ls. A proteomic survey of purified LE/Ls identified StARD9 as a novel lysosomal kinesin responsible for LE/L tubulation. StARD9 contains an N-terminal kinesin domain, a C-terminal StART domain, and a dileucine signal shared with other lysosome-associated membrane proteins. Depletion of StARD9 disrupts LE/L tubulation, paralyzes bidirectional LE/L motility and induces accumulation of cholesterol in LE/Ls. Finally, a novel StARD9 knock-out mouse recapitulates the progressive loss of Purkinje cells in the cerebellum. Together, these studies identify StARD9 as a microtubule motor protein responsible for LE/L tubulation and provide support for a novel model of LE/L cholesterol transport that becomes impaired in NPC disease., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Complete enzymatic digestion of proteins for bottom-up proteomics is substantially improved by use of detergents for denaturation and solubilization. Detergents however, are incompatible with many proteases and highly detrimental to LC-MS/MS. Recently; filter-based methods have seen wide use due to their capacity to remove detergents and harmful reagents prior to digestion and mass spectrometric analysis. We hypothesized that non-specific protein binding to negatively charged silica-based filters would be enhanced by addition of lyotropic salts, similar to DNA purification. We sought to exploit these interactions and investigate if low-cost DNA purification spin-filters, 'Minipreps,' efficiently and reproducibly bind proteins for digestion and LC-MS/MS analysis. We propose a new method, Miniprep Assisted Proteomics (MAP), for sample preparation. We demonstrate binding capacity, performance, recovery and identification rates for proteins and whole-cell lysates using MAP. MAP recovered equivalent or greater protein yields from 0.5-50 μg analyses benchmarked against commercial trapping preparations. Nano UHPLC-MS/MS proteome profiling of lysates of Escherichia coli had 99.3% overlap vs. existing approaches and reproducibility of replicate minipreps was 98.8% at the 1% FDR protein level. Label Free Quantitative proteomics was performed and 91.2% of quantified proteins had a %CV <20% (2044/2241). Miniprep Assisted Proteomics can be performed in minutes, shows low variability, high recovery and proteome depth. This suggests a significant role for adventitious binding in developing new proteomics sample preparation techniques. MAP represents an efficient, ultra-low-cost alternative for sample preparation in a commercially obtainable device that costs ∼$0.50 (USD) per miniprep.

Bottom-up proteomics (BUP) produces rich data, but visualization and analysis are time-consuming and often require programming skills. Many tools analyze these data at the proteome-level, but fewer options exist for individual proteins. Sequence coverage maps are common, but do not proportion peptide intensity. Abundance-based visualization of sequence coverage facilitates detection of protein isoforms, domains, potential truncation sites, peptide "hot-spots", and localization of post-translational modifications (PTMs). Redundant stacked-sequence coverage is an important tool in designing hydrogen-deuterium exchange (HDX) experiments. Visualization tools often lack graphical and tabular-export of processed data which complicates publication of results. Quantitative peptide abundance across amino acid sequences is an essential and missing tool in proteomics toolkits. Here we created PrIntMap-R, an online application that only requires peptide files from a database search and FASTA protein sequences. PrIntMap-R produces a variety of plots for quantitative visualization of coverage; annotation of specific sequences, PTM's, and comparisons of one or many samples overlaid with calculated fold-change or several intensity metrics. We show use-cases including protein phosphorylation, identification of glycosylation, and the optimization of digestion conditions for HDX experiments. PrIntMap-R is freely available, open source, and can run online with no installation, or locally by downloading source code from GitHub.

Public health efforts to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic rely on accurate information on the spread of the disease in the community. Acute and surveillance testing has been primarily used to characterize the extent of the disease. However, obtaining a representative sample of the human population is challenging because of limited testing capacity and incomplete testing compliance. Wastewater-based epidemiology is an agnostic alternative to surveillance testing that provides an average sample from the population served by the treatment facility. We compare the performance of reverse transcription quantitative PCR (RT-qPCR) and reverse transcription digital droplet PCR (RT-dPCR) for analysis of SARS-CoV-2 RNA in a regional wastewater treatment facility in northern Indiana, USA from the earliest stages of the pandemic. 1-L grab samples of wastewater were clarified and concentrated. Nucleic acids were extracted from aliquots and analyzed in parallel using the two methods. Synthetic viral nucleic acids were used for method development and generation of add-in standard-curves. Both methods were highly sensitive in detecting SARS-CoV-2 in wastewater, with detection limits as low as 1 copy per 500 mL wastewater. RT-qPCR and RT-dPCR provided essentially identical coefficients of variation (s/[Formula: see text] = 0.15) for triplicate measurements made on wastewater samples taken on 16 days. We also observed a sevenfold decrease in viral load from a grab sample that was frozen at - 80 °C for 92 days compared to results obtained without freezing. Freezing samples before analysis should be discouraged. Finally, we found that treatment with a glycine release buffer resulted in a fourfold inhibition in RT-qPCR signal; treatment with a glycine release buffer also should be discouraged. Despite their prevalence and convenience in wastewater analysis, glycine release and freezing samples severely and additively (~ tenfold) degraded recovery and detection of SARS-CoV-2., (© 2022. The Author(s).)

The protein networks of cell-wall-biosynthesis assemblies are largely unknown. A key class of enzymes in these assemblies is the lytic transglycosylases (LTs), of which eleven exist in P. aeruginosa. We have undertaken a pulldown strategy in conjunction with mass-spectrometry-based proteomics to identify the putative binding partners for the eleven LTs of P. aeruginosa. A total of 71 putative binding partners were identified for the eleven LTs. A systematic assessment of the binding partners of the rare lipoprotein A (RlpA), one of the pseudomonal LTs, was made. This 37-kDa lipoprotein is involved in bacterial daughter-cell separation by an unknown process. RlpA participates in both the multi-protein and multi-enzyme divisome and elongasome assemblies. We reveal an extensive protein-interaction network for RlpA involving at least 19 proteins. Their kinetic parameters for interaction with RlpA were assessed by microscale thermophoresis, surface-plasmon resonance, and isothermal-titration calorimetry. Notable RlpA binding partners include PBP1b, PBP4, and SltB1. Elucidation of the protein-interaction networks for each of the LTs, and specifically for RlpA, opens opportunities for the study of their roles in the complex protein assemblies intimately involved with the cell wall as a structural edifice critical for bacterial survival., (© 2022. The Author(s).)

The human pathogen Mycobacterium tuberculosis typically causes lung disease but can also disseminate to other tissues. We identified a M. tuberculosis (Mtb) outbreak presenting with unusually high rates of extrapulmonary dissemination and bone disease. We found that the causal strain carried an ancestral full-length version of the type VII-secreted effector EsxM rather than the truncated version present in other modern Mtb lineages. The ancestral EsxM variant exacerbated dissemination through enhancement of macrophage motility, increased egress of macrophages from established granulomas, and alterations in macrophage actin dynamics. Reconstitution of the ancestral version of EsxM in an attenuated modern strain of Mtb altered the migratory mode of infected macrophages, enhancing their motility. In a zebrafish model, full-length EsxM promoted bone disease. The presence of a derived nonsense variant in EsxM throughout the major Mtb lineages 2, 3, and 4 is consistent with a role for EsxM in regulating the extent of dissemination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The ESX-1 (ESAT-6-system-1) system and the protein substrates it transports are essential for mycobacterial pathogenesis. The precise ways that ESX-1 substrates contribute to virulence remains unknown. Several known ESX-1 substrates are also required for the secretion of other proteins. We used a proteo-genetic approach to construct high-resolution dependency relationships for the roles of individual ESX-1 substrates in secretion and virulence in Mycobacterium marinum, a pathogen of humans and animals. Characterizing a collection of M. marinum strains with in-frame deletions in each of the known ESX-1 substrate genes and the corresponding complementation strains, we demonstrate that ESX-1 substrates are differentially required for ESX-1 activity and for virulence. Using isobaric-tagged proteomics, we quantified the degree of requirement of each substrate on protein secretion. We conclusively defined distinct contributions of ESX-1 substrates in protein secretion. Our data reveal a hierarchy of ESX-1 substrate secretion, which supports a model for the composition of the extracytoplasmic ESX-1 secretory machinery. Overall, our proteo-genetic analysis demonstrates discrete roles for ESX-1 substrates in ESX-1 function and secretion in M. marinum.

Microbial adhesion to medical devices is common for hospital-acquired infections, particularly for urinary catheters. If not properly treated these infections cause complications and exacerbate antimicrobial resistance. Catheter use elicits bladder inflammation, releasing host serum proteins, including fibrinogen (Fg), into the bladder, which deposit on the urinary catheter. Enterococcus faecalis uses Fg as a scaffold to bind and persist in the bladder despite antibiotic treatments. Inhibition of Fg-pathogen interaction significantly reduces infection. Here, we show deposited Fg is advantageous for uropathogens E. faecalis , Escherichia coli , Pseudomonas aeruginosa , K. pneumoniae , A. baumannii , and C. albicans , suggesting that targeting catheter protein deposition may reduce colonization creating an effective intervention for catheter-associated urinary tract infections (CAUTIs). In a mouse model of CAUTI, host-protein deposition was reduced, using liquid-infused silicone catheters, resulting in decreased colonization on catheters, in bladders, and dissemination in vivo. Furthermore, proteomics revealed a significant decrease in deposition of host-secreted proteins on liquid-infused catheter surfaces. Our findings suggest targeting microbial-binding scaffolds may be an effective antibiotic-sparing intervention for use against CAUTIs and other medical device infections., Competing Interests: MA, CF, AL, JM, AM, MC, CH, AF No competing interests declared, (© 2022, Andersen et al.)

Most bacterial ORFs are identified by automated prediction algorithms. However, these algorithms often fail to identify ORFs lacking canonical features such as a length of >50 codons or the presence of an upstream Shine-Dalgarno sequence. Here, we use ribosome profiling approaches to identify actively translated ORFs in Mycobacterium tuberculosis . Most of the ORFs we identify have not been previously described, indicating that the M. tuberculosis transcriptome is pervasively translated. The newly described ORFs are predominantly short, with many encoding proteins of ≤50 amino acids. Codon usage of the newly discovered ORFs suggests that most have not been subject to purifying selection, and hence are unlikely to contribute to cell fitness. Nevertheless, we identify 90 new ORFs (median length of 52 codons) that bear the hallmarks of purifying selection. Thus, our data suggest that pervasive translation of short ORFs in Mycobacterium tuberculosis serves as a rich source for the evolution of new functional proteins., Competing Interests: CS, JC, AW, MC, KD, TG No competing interests declared, JW Reviewing editor, eLife

Commercial systems for capillary electrophoresis are designed for the unattended analysis of several samples, and are usually large, complex, and expensive. We report a compact system for manual injection of a single sample in capillary electrophoresis, which is ideal for method development and for student training. The injector consists of two parts that are manufactured by three-dimensional printing (STL and STEP files are included as ESI). One part is immobile and holds an electrode for powering electrophoresis and a gas line for pressurized injection and pumping fluids through the capillary. The second part is removable and is used to hold washing solutions, separation electrolyte, or sample. Conventional machining is used to tap holes to hold the electrode, separation capillary, gas line, and safety interlock. The system is used for either pressure or electrokinetic sample injection, and can be used to pump fluids through the capillary for changing background electrolytes and reconditioning the capillary between runs. We coupled the injection system to our high-dynamic range laser-induced fluorescence detector and evaluated the system by performing capillary zone electrophoresis on solutions of fluorescein. Electrokinetic injection produced a linear response across five orders of magnitude dynamic range (slope of the log-log calibration curve was 1.02), concentration detection limits of 5 pM, and mass detection limits of 1 zmol. Pressure injection produced a linear response across at least four orders of magnitude (slope of the log-log calibration curve was 0.92), concentration detection limits of 2 pM, and mass detection limits of 10 zmol.

The Balbiani body (Bb), an organelle comprised of mitochondria, ER, and RNA, is found in the oocytes of most organisms. In Xenopus , the structure is initially positioned immediately adjacent to the nucleus, extends toward the vegetal pole, and eventually disperses, leaving behind a region highly enriched in mitochondria. This area is later transversed by RNP complexes that are being localized to the vegetal cortex. Inhibition of mitochondrial ATP synthesis prevents perinuclear formation of the transport complexes that can be reversed by a nonhydrolyzable ATP analog, indicating the nucleotide is acting as a hydrotrope. The protein composition, sensitivity to hexanediol, and coalescence in the absence of transport provide evidence that the transport RNP complexes are biocondensates. The breakdown of the Bb engenders regions of clustered mitochondria that are used not to meet extraordinary energy demands, but rather to promote a liquid-liquid phase separation., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Small proteins of up to ∼50 amino acids are an abundant class of biomolecules across all domains of life. Yet due to the challenges inherent in their size, they are often missed in genome annotations, and are difficult to identify and characterize using standard experimental approaches. Consequently, we still know few small proteins even in well-studied prokaryotic model organisms. Mass spectrometry (MS) has great potential for the discovery, validation, and functional characterization of small proteins. However, standard MS approaches are poorly suited to the identification of both known and novel small proteins due to limitations at each step of a typical proteomics workflow, i.e., sample preparation, protease digestion, liquid chromatography, MS data acquisition, and data analysis. Here, we outline the major MS-based workflows and bioinformatic pipelines used for small protein discovery and validation. Special emphasis is placed on highlighting the adjustments required to improve detection and data quality for small proteins. We discuss both the unbiased detection of small proteins and the targeted analysis of small proteins of interest. Finally, we provide guidelines to prioritize novel small proteins, and an outlook on methods with particular potential to further improve comprehensive discovery and characterization of small proteins.

Mycobacterial pathogens pose a sustained threat to human health. There is a critical need for new diagnostics, therapeutics, and vaccines targeting both tuberculous and nontuberculous mycobacterial species. Understanding the basic mechanisms used by diverse mycobacterial species to cause disease will facilitate efforts to design new approaches toward detection, treatment, and prevention of mycobacterial disease. Molecular, genetic, and biochemical approaches have been widely employed to define fundamental aspects of mycobacterial physiology and virulence. The recent expansion of genetic tools in mycobacteria has further increased the accessibility of forward genetic approaches. Proteomics has also emerged as a powerful approach to further our understanding of diverse mycobacterial species. Detection of large numbers of proteins and their modifications from complex mixtures of mycobacterial proteins is now routine, with efforts of quantification of these datasets becoming more robust. In this review, we discuss the "genetic proteome," how the power of genetics, molecular biology, and biochemistry informs and amplifies the quality of subsequent analytical approaches and maximizes the potential of hypothesis-driven mycobacterial research. Published proteomics datasets can be used for hypothesis generation and effective post hoc supplementation to experimental data. Overall, we highlight how the integration of proteomics, genetic, molecular, and biochemical approaches can be employed successfully to define fundamental aspects of mycobacterial pathobiology., Competing Interests: The authors have declared that no competing interests exist.

Digestion of proteins separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) remains a popular method for protein identification using mass-spectrometry based proteomics. Although robust and routine, the in-gel digestion procedure is laborious and time-consuming. Electroblotting to a capture membrane prior to digestion reduces preparation steps but requires on-membrane digestion that yields fewer peptides than in-gel digestion. This paper develops direct electroblotting through a trypsin-containing membrane to a capture membrane to simplify extraction and digestion of proteins separated by SDS-PAGE. Subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) identifies the extracted peptides. Analysis of peptides from different capture membrane pieces shows that electrodigestion does not greatly disturb the spatial resolution of a standard protein mixture separated by SDS-PAGE. Electrodigestion of an Escherichia coli (E. coli) cell lysate requires four hours of total sample preparation and results in only 13% fewer protein identifications than in-gel digestion, which can take 24 h. Compared to simple electroblotting and protein digestion on a poly(vinylidene difluoride) (PVDF) capture membrane, adding a trypsin membrane to the electroblot increases the number of protein identifications by 22%. Additionally, electrodigestion experiments using capture membranes coated with polyelectrolyte layers identify a higher fraction of small proteolytic peptides than capture on PVDF or in-gel digestion.

Stable heavy-isotope labeling is commonly used in quantitative proteomics. Several common techniques incorporate deuterium ( 2 H) as the heavy isotopic label using reductive amination with formaldehyde. Compared with alternatives, dimethyl labeling reagents are inexpensive and the labeling chemistry is simple and rapid. However, the substitution of hydrogen by deuterium can introduce subtle changes in peptides' polarities, leading to a shift in chromatographic retention times between deuterated and nondeuterated peptides that can lead to quantification deviations. Capillary zone electrophoresis has emerged as a complementary separation for ESI-MS-based proteomics, including targeted and quantitative approaches. The extent to which the deuterium isotope effect impacts CZE-based proteomics, which separates peptides based on their S/N ratios, has not been investigated. To address this issue, CZE was used to analyze dimethyl labeled E. coli tryptic digests in 100 min single-shot analyses. The median migration time shift was 0.1 s for light versus heavy labeled peptides, which is 2.5% of the peak width. For comparison, nUHPLC-ESI-MS/MS was used to analyze the same sample. In UPLC, deuterated peptides tended to elute earlier than nondeuterated peptides, with a retention shift of 3 s for light versus heavy labeled peptides, which is roughly half the peak width. This shift in separation time did not have a significant effect on quantitation for either method for equal mixing ratios of the light-intermediate-heavy isotope labeled samples., (© 2020 The Authors. Electrophoresis published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Genome-wide transcriptomic analyses have revealed abundant expressed short open reading frames (ORFs) in bacteria. Whether these short ORFs, or the small proteins they encode, are functional remains an open question. One quarter of mycobacterial mRNAs are leaderless, beginning with a 5'-AUG or GUG initiation codon. Leaderless mRNAs often encode unannotated short ORFs as the first gene of a polycistronic transcript. Here, we show that polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression. Detailed mutational analysis shows that one polycysteine short ORF controls expression of the downstream genes. Our data indicate that ribosomes stalled in the polycysteine tract block mRNA structures that otherwise sequester the ribosome-binding site of the 3'gene. We assessed endogenous proteomic responses to cysteine limitation in Mycobacterium smegmatis using mass spectrometry. Six cysteine metabolic loci having unannotated polycysteine-encoding leaderless short ORF architectures responded to cysteine limitation, revealing widespread cysteine-responsive attenuation in mycobacteria. Individual leaderless short ORFs confer independent operon-level control, while their shared dependence on cysteine ensures a collective response mediated by ribosome pausing. We propose the term ribulon to classify ribosome-directed regulons. Regulon-level coordination by ribosomes on sensory short ORFs illustrates one utility of the many unannotated short ORFs expressed in bacterial genomes., (© 2020 John Wiley & Sons Ltd.)

Glycans are known to be involved in many biological processes, while little is known about the expression of N-glycans during vertebrate development. We now report the first quantitative studies of both the expression of N-linked glycans at six early development stages and the expression of N-glycosylated peptides at two early development stages in Xenopus laevis, the African clawed frog. N-Glycans were labeled with isobaric tandem mass tags, pooled, separated by capillary electrophoresis, and characterized using tandem mass spectrometry. We quantified 110 N-glycan compositions that spanned four orders of magnitude in abundance. Capillary electrophoresis was particularly useful in identifying charged glycans; over 40% of the observed glycan compositions were sialylated. The glycan expression was relatively constant until the gastrula-neurula transition (developmental stage 13), followed by massive reprogramming. An increase in oligomannosidic and a decrease in the paucimannosidic and phosphorylated oligomannosidic glycans were observed at the late tailbud stage (developmental stage 41). Two notable and opposing regulation events were detected for sialylated glycans. LacdiNAc and Lewis antigen features distinguished down-regulated sialylation from up-regulated species. The level of Lewis antigen decreased at later stages, which was validated by Aleuria aurantia lectin (AAL) and Ulex europaeus lectin (UEA-I) blots. We also used HPLC coupled with tandem mass spectrometry to identify 611 N-glycosylation sites on 350 N-glycoproteins at the early stage developmental stage 1 (fertilized egg), and 1682 N-glycosylation sites on 1023 N-glycoproteins at stage 41 (late tailbud stage). Over two thirds of the N-glycoproteins identified in the late tailbud stage are associated with neuron projection morphogenesis, suggesting a vital role of the N-glycome in neuronal development.

Diabetic foot ulcers are characterized by hypoxia. For many patients, hyperbaric oxygen (HBO) therapy is the last recourse for saving the limb from amputation, for which the molecular basis is not understood. We previously identified the active form of matrix metalloproteinase-9 (MMP-9) as responsible for diabetic foot ulcer's recalcitrance to healing. Transcription of mmp-9 to the inactive zymogen is upregulated during hypoxia. Activation of the zymogen is promoted by proteases and reactive oxygen species (ROS). We hypothesized that the dynamics of these two events might lead to a lowering of active MMP-9 levels in the wounded tissue. We employed the full-thickness excisional db/db mouse model to study wound healing, and treated the mice to 3.0 atm of molecular oxygen for 90 minutes, 5 days per week for 10 days in an HBO research chamber. Treatment with HBO accelerated diabetic wound healing compared to untreated mice, with more completed and extended reepithelialization. We imaged the wounds for ROS in vivo with a luminol-based probe and found that HBO treatment actually decreases ROS levels. The levels of superoxide dismutase, catalase, and glutathione peroxidase-enzymes that turn over ROS-increased after HBO treatment, hence the observation of decreased ROS. Since ROS levels are lowered, we explored the effect that this would have on activation of MMP-9. Quantitative analysis with an affinity resin that binds and pulls down the active MMPs exclusively, coupled with proteomics, revealed that HBO treatment indeed reduces the active MMP-9 levels. This work for the first time demonstrates that diminution of active MMP-9 is a contributing factor and a mechanism for enhancement of diabetic wound healing by HBO therapy., (© 2019 by the Wound Healing Society.)

Pathogenic mycobacteria encounter multiple environments during macrophage infection. Temporally, the bacteria are engulfed into the phagosome, lyse the phagosomal membrane, and interact with the cytosol before spreading to another cell. Virulence factors secreted by the mycobacterial ESX-1 (ESAT-6-system-1) secretion system mediate the essential transition from the phagosome to the cytosol. It was recently discovered that the ESX-1 system also regulates mycobacterial gene expression in Mycobacterium marinum (R. E. Bosserman, T. T. Nguyen, K. G. Sanchez, A. E. Chirakos, et al., Proc Natl Acad Sci U S A 114:E10772-E10781, 2017, https://doi.org/10.1073/pnas.1710167114), a nontuberculous mycobacterial pathogen, and in the human-pathogenic species M. tuberculosis (A. M. Abdallah, E. M. Weerdenburg, Q. Guan, R. Ummels, et al., PLoS One 14:e0211003, 2019, https://doi.org/10.1371/journal.pone.0211003). It is not known how the ESX-1 system regulates gene expression. Here, we identify the first transcription factor required for the ESX-1-dependent transcriptional response in pathogenic mycobacteria. We demonstrate that the gene divergently transcribed from the whiB6 gene and adjacent to the ESX-1 locus in mycobacterial pathogens encodes a conserved transcription factor ( MMAR&#95;5438 , Rv3863 , now espM ). We prove that EspM from both M. marinum and M. tuberculosis directly and specifically binds the whiB6-espM intergenic region. We show that EspM is required for ESX-1-dependent repression of whiB6 expression and for the regulation of ESX-1 -associated gene expression. Finally, we demonstrate that EspM functions to fine-tune ESX-1 activity in M. marinum Taking the data together, this report extends the esx-1 locus, defines a conserved regulator of the ESX-1 virulence pathway, and begins to elucidate how the ESX-1 system regulates gene expression. IMPORTANCE Mycobacterial pathogens use the ESX-1 system to transport protein substrates that mediate essential interactions with the host during infection. We previously demonstrated that in addition to transporting proteins, the ESX-1 secretion system regulates gene expression. Here, we identify a conserved transcription factor that regulates gene expression in response to the ESX-1 system. We demonstrate that this transcription factor is functionally conserved in M. marinum , a pathogen of ectothermic animals; M. tuberculosis , the human-pathogenic species that causes tuberculosis; and M. smegmatis , a nonpathogenic mycobacterial species. These findings provide the first mechanistic insight into how the ESX-1 system elicits a transcriptional response, a function of this protein transport system that was previously unknown., (Copyright © 2020 Sanchez et al.)

The ESX-1 (ESAT-6 system 1) secretion system plays a conserved role in the virulence of diverse mycobacterial pathogens, including the human pathogen Mycobacterium tuberculosis and M. marinum , an environmental mycobacterial species. The ESX-1 system promotes the secretion of protein virulence factors to the extracytoplasmic environment. The secretion of these proteins triggers the host response by lysing the phagosome during macrophage infection. Using proteomic analyses of the M. marinum secretome in the presence and absence of a functional ESX-1 system, we and others have hypothesized that MMAR&#95;2894, a PE family protein, is a potential ESX-1 substrate in M. marinum We used genetic and quantitative proteomic approaches to determine if MMAR&#95;2894 is secreted by the ESX-1 system, and we defined the requirement of MMAR&#95;2894 for ESX-1-mediated secretion and virulence. We show that MMAR&#95;2894 is secreted by the ESX-1 system in M. marinum and is itself required for the optimal secretion of the known ESX-1 substrates in M. marinum Moreover, we found that MMAR&#95;2894 was differentially required for hemolysis and cytolysis of macrophages, two lytic activities ascribed to the M. marinum ESX-1 system. IMPORTANCE Both Mycobacterium tuberculosis , the cause of human tuberculosis (TB), and Mycobacterium marinum , a pathogen of ectotherms, use the ESX-1 secretion system to cause disease. There are many established similarities between the ESX-1 systems in M. tuberculosis and in M. marinum Yet the two bacteria infect different hosts, hinting at species-specific functions of the ESX-1 system. Our findings demonstrate that MMAR&#95;2894 is a PE protein secreted by the ESX-1 system of M. marinum We show that MMAR&#95;2894 is required for the optimal secretion of mycobacterial proteins required for disease. Because the MMAR&#95;2894 gene is not conserved in M. tuberculosis , our findings demonstrate that MMAR&#95;2894 may contribute to a species-specific function of the ESX-1 system in M. marinum , providing new insight into how the M. marinum and M. tuberculosis systems differ., (Copyright © 2019 American Society for Microbiology.)

Capillary zone electrophoresis (CZE) can produce high-resolution separations of biological samples, including microbial mixtures. The study of complex populations of microorganisms using CZE is limited because most detectors have limited sensitivity, are destructive, and provide limited information for microbial identification. To address these issues, we developed an integrated capillary zone electrophoresis apparatus to fractionate bacteria from complex mixtures. We deposited fractions onto nutrient agar in a Petri dish for microbial culturing, and we subjected the observed colonies to Sanger sequencing of a phylogenetic marker, the 16S rRNA gene, for microbial identification. We separated and cultured both a single bacteria species, the model Gram-negative organism Escherichia coli, and a complex environmental isolate of primary sewage effluent. Sequence analysis of the 16S rRNA genes from this mixture identified 15 ± 5 distinct bacterial species per run. This approach requires minimal manipulation of microbial populations and combines electrophoretic fractionation of bacterial cells with automated collection for accurate identification of species. This approach should be applicable to microorganisms in general and may enable discrimination of physiologically different cells in complex assemblages, such as in microbiome samples.

Multiple Myeloma (MM) is highly sensitive to disruptions in cellular protein homeostasis. Proteasome inhibitors (PIs) are initially effective in the treatment of MM, although cures are not achievable and the emergence of resistance limits the durability of responses. New therapies are needed for refractory patients, and those that combat resistance to standard of care agents would be particularly valuable. Screening of multiple chemical libraries for PI re-sensitizing compounds identified E61 as a potent enhancer of multiple PIs and MM specific activity. Using a tandem approach of click chemistry and peptide mass fingerprinting, we identified multiple protein disulfide isomerase (PDI) family members as the primary molecular targets of E61. PDIs mediate oxidative protein folding, and E61 treatment induced robust ER and oxidative stress responses as well as the accumulation of ubiquitinylated proteins. A chemical optimization program led to a new structural class of indene (exemplified by lead E64FC26), which are highly potent pan-style inhibitors of PDIs. In mice with MM, E64FC26 improved survival and enhanced the activity of bortezomib without any adverse effects. This work demonstrates the potential of E64FC26 as an early drug candidate and the strategy of targeting multiple PDI isoforms for the treatment of refractory MM and beyond.

Diabetic foot ulcers (DFUs) are a significant health problem. A single existing FDA-approved drug for this ailment, becaplermin, is not standard-of-care. We previously demonstrated that upregulation of active matrix metalloproteinase (MMP)-9 is the reason that the diabetic wound in mice is recalcitrant to healing and that MMP-8 participates in wound repair. In the present study, we validate the target MMP-9 by identifying and quantifying active MMP-8 and MMP-9 in human diabetic wounds using an affinity resin that binds exclusively to the active forms of MMPs coupled with proteomics. Furthermore, we synthesize and evaluate enantiomerically pure ( R)- and ( S)-ND-336, as inhibitors of the detrimental MMP-9, and show that the ( R)-enantiomer has superior efficacy in wound healing over becaplermin. Our results reveal that the mechanisms of pathology and repair are similar in diabetic mice and diabetic humans and that ( R)-ND-336 holds promise for the treatment of DFUs as a first-in-class therapeutic.

Potent immunosuppressive mechanisms within the tumor microenvironment contribute to the resistance of aggressive human cancers to immune checkpoint blockade (ICB) therapy. One of the main mechanisms for myeloid-derived suppressor cells (MDSCs) to induce T cell tolerance is through secretion of reactive nitrogen species (RNS), which nitrates tyrosine residues in proteins involved in T cell function. However, so far very few nitrated proteins have been identified. Here, using a transgenic mouse model of prostate cancer and a syngeneic cell line model of lung cancer, we applied a nitroproteomic approach based on chemical derivation of 3-nitrotyrosine and identified that lymphocyte-specific protein tyrosine kinase (LCK), an initiating tyrosine kinase in the T cell receptor signaling cascade, is nitrated at Tyr394 by MDSCs. LCK nitration inhibits T cell activation, leading to reduced interleukin 2 (IL2) production and proliferation. In human T cells with defective endogenous LCK, wild type, but not nitrated LCK, rescues IL2 production. In the mouse model of castration-resistant prostate cancer (CRPC) by prostate-specific deletion of Pten , p53 , and Smad4 , CRPC is resistant to an ICB therapy composed of antiprogrammed cell death 1 (PD1) and anticytotoxic-T lymphocyte-associated protein 4 (CTLA4) antibodies. However, we showed that ICB elicits strong anti-CRPC efficacy when combined with an RNS neutralizing agent. Together, these data identify a previously unknown mechanism of T cell inactivation by MDSC-induced protein nitration and illuminate a clinical path hypothesis for combining ICB with RNS-reducing agents in the treatment of CRPC., Competing Interests: The authors declare no conflict of interest.

N-terminal acetylation (NTA) is a post-transcriptional modification of proteins that is conserved from bacteria to humans. In bacteria, the enzymes that mediate protein NTA also promote antimicrobial resistance. In pathogenic mycobacteria, which cause human tuberculosis and other chronic infections, NTA has been linked to pathogenesis and stress response, yet the fundamental biology underlying NTA of mycobacterial proteins remains unclear. We enriched, defined, and quantified the NT-acetylated populations of both cell-associated and secreted proteins from both the human pathogen, Mycobacterium tuberculosis, and the nontuberculous opportunistic pathogen, Mycobacterium marinum. We used a parallel N-terminal enrichment strategy from proteolytic digests coupled to charge-based selection and stable isotope ratio mass spectrometry. We show that NTA of the mycobacterial proteome is abundant, diverse, and primarily on Thr residues, which is unique compared with other bacteria. We isolated both the acetylated and unacetylated forms of 256 proteins, indicating that NTA of mycobacterial proteins is homeostatic. We identified 16 mycobacterial proteins with differential levels of NTA on the cytoplasmic and secreted forms, linking protein modification and localization. Our findings reveal novel biology underlying the NTA of mycobacterial proteins, which may provide a basis to understand NTA in mycobacterial physiology, pathogenesis, and antimicrobial resistance.

Chronic wounds are a complication of diabetes. Treatment for diabetic foot ulcers is complex with little clinical recourse, resulting in 108,000 lower-limb amputations annually in the United States alone. Matrix metalloproteinases (MMPs) play important roles in the pathology and in the repair of chronic wounds. We previously identified active MMP-8 and MMP-9 in wounds of diabetic mice and determined that MMP-8 accelerates wound repair, while MMP-9 is the culprit for the diabetic wound being refractory to healing. Aclerastide, a peptide analog of angiotensin II, recently failed in phase III clinical trials for treatment of diabetic foot ulcers. We demonstrate herein that treatment of wounds of diabetic mice with aclerastide results in elevated levels of reactive oxygen species and of active MMP-9, which is likely an important contributor to the failure of aclerastide in clinical trials., (Copyright © 2018 Elsevier B.V. All rights reserved.)

ESX (ESAT-6 system) export systems play diverse roles across mycobacterial species. Interestingly, genetic disruption of ESX systems in different species does not result in an accumulation of protein substrates in the mycobacterial cell. However, the mechanisms underlying this observation are elusive. We hypothesized that the levels of ESX substrates were regulated by a feedback-control mechanism, linking the levels of substrates to the secretory status of ESX systems. To test this hypothesis, we used a combination of genetic, transcriptomic, and proteomic approaches to define export-dependent mechanisms regulating the levels of ESX-1 substrates in Mycobacterium marinum WhiB6 is a transcription factor that regulates expression of genes encoding ESX-1 substrates. We found that, in the absence of the genes encoding conserved membrane components of the ESX-1 system, the expression of the whiB6 gene and genes encoding ESX-1 substrates were reduced. Accordingly, the levels of ESX-1 substrates were decreased, and WhiB6 was not detected in M. marinum strains lacking genes encoding ESX-1 components. We demonstrated that, in the absence of EccCb 1 , a conserved ESX-1 component, substrate gene expression was restored by constitutive, but not native, expression of the whiB6 gene. Finally, we found that the loss of WhiB6 resulted in a virulent M. marinum strain with reduced ESX-1 secretion. Together, our findings demonstrate that the levels of ESX-1 substrates in M. marinum are fine-tuned by negative feedback control, linking the expression of the whiB6 gene to the presence, not the functionality, of the ESX-1 membrane complex., Competing Interests: The authors declare no conflict of interest.

The earliest stages of animal development are largely controlled by changes in protein phosphorylation mediated by signaling pathways and cyclin-dependent kinases. In order to decipher these complex networks and to discover new aspects of regulation by this post-translational modification, we undertook an analysis of the X. laevis phosphoproteome at seven developmental stages beginning with stage VI oocytes and ending with two-cell embryos. Concurrent measurement of the proteome and phosphoproteome enabled measurement of phosphosite occupancy as a function of developmental stage. We observed little change in protein expression levels during this period. We detected the expected phosphorylation of MAP kinases, translational regulatory proteins, and subunits of APC/C that validate the accuracy of our measurements. We find that more than half the identified proteins possess multiple sites of phosphorylation that are often clustered, where kinases work together in a hierarchical manner to create stretches of phosphorylated residues, which may be a means to amplify signals or stabilize a particular protein conformation. Conversely, other proteins have opposing sites of phosphorylation that seemingly reflect distinct changes in activity during this developmental timeline.

Mycobacterial pathogens use the ESAT-6 system 1 (Esx-1) exporter to promote virulence. Previously, we used gene disruption and complementation to conclude that the MMAR&#95;0039 gene in Mycobacterium marinum is required to promote Esx-1 export. Here we applied molecular genetics, proteomics, and whole-genome sequencing to demonstrate that the MMAR&#95;0039 gene is not required for Esx-1 secretion or virulence. These findings suggest that we initially observed an indirect mechanism of genetic complementation. We identified a spontaneous nonsense mutation in a known Esx-1-associated gene which causes a loss of Esx-1 activity. We show that the Esx-1 function was restored by nonsense suppression. Moreover, we identified a polar mutation in the ppsC gene which reduced cellular impermeability but did not impact cytotoxicity in macrophages. Our studies reveal insight into Esx-1 export, nonsense suppression, and cell envelope lipid biogenesis., (Copyright © 2017 American Society for Microbiology.)

DNA methylation in prokaryotes is widespread. The most common modification of the genome is the methylation of adenine at the N-6 position. In Escherichia coli K-12 and many gammaproteobacteria, this modification is catalyzed by DNA adenine methyltransferase (Dam) at the GATC consensus sequence and is known to modulate cellular processes including transcriptional regulation of gene expression, initiation of chromosomal replication, and DNA mismatch repair. While studies thus far have focused on the motifs associated with methylated adenine (meA), the frequency of meA across the genome, and temporal dynamics during early periods of incubation, here we conduct the first study on the temporal dynamics of adenine methylation in E. coli by Dam throughout all five phases of the bacterial life cycle in the laboratory. Using single-molecule real-time sequencing, we show that virtually all GATC sites are significantly methylated over time; nearly complete methylation of the chromosome was confirmed by mass spectroscopy analysis. However, we also detect 66 sites whose methylation patterns change significantly over time within a population, including three sites associated with sialic acid transport and catabolism, suggesting a potential role for Dam regulation of these genes; differential expression of this subset of genes was confirmed by quantitative real-time PCR. Further, we show significant growth defects of the dam mutant during long-term stationary phase (LTSP). Together these data suggest that the cell places a high premium on fully methylating the chromosome and that alterations in methylation patterns may have significant impact on patterns of transcription, maintenance of genetic fidelity, and cell survival. IMPORTANCE While it has been shown that methylation remains relatively constant into early stationary phase of E. coli , this study goes further through death phase and long-term stationary phase, a unique time in the bacterial life cycle due to nutrient limitation and strong selection for mutants with increased fitness. The absence of methylation at GATC sites can influence the mutation frequency within a population due to aberrant mismatch repair. Therefore, it is important to investigate the methylation status of GATC sites in an environment where cells may not prioritize methylation of the chromosome. This study demonstrates that chromosome methylation remains a priority even under conditions of nutrient limitation, indicating that continuous methylation at GATC sites could be under positive selection.

Mass spectrometry (MS) for the detection of proteins is an indispensable tool for evaluating the biological processes of the proteome. Proteomics frequently requires proteolysis of proteins into peptide fragments. Proteins can be refractory to ideal proteolysis at the sequence level rendering them difficult to analyze by routine proteomics methods. EsxA (ESAT-6, Early Secreted Antigen, 6kDa) is a major virulence determinant of Mycobacterium tuberculosis, the cause of human tuberculosis. EsxA is routinely used to evaluate mycobacterial virulence in the laboratory and as a biomarker for tuberculosis in humans. The sequence of EsxA hinders deeper MS analysis beyond routine detection. Here we engineer the sequence of EsxA to add desirable tryptic properties aimed at improving complex MS analysis. We demonstrate that EsxA variants are amenable to MS analysis and remain functional in established in vitro and ex vivo assays of Esx-1-function. We provide the first demonstration of molecular engineering to specifically improve MS analysis of individual microbial proteins.

The periplasm of Gram-negative bacteria includes a variety of molecular chaperones that shepherd the folding and targeting of secreted proteins. A central player of this quality control network is DegP, a protease also suggested to have a chaperone function. We serendipitously discovered that production of the Bordetella pertussis autotransporter virulence protein pertactin is lethal in Escherichia coli ΔdegP strains. We investigated specific contributions of DegP to secretion of pertactin as a model system to test the functions of DegP in vivo. The DegP chaperone activity was sufficient to restore growth during pertactin production. This chaperone dependency could be relieved by changing the pertactin signal sequence: an E. coli signal sequence leading to co-translational inner membrane (IM) translocation was sufficient to suppress lethality in the absence of DegP, whereas an E. coli post-translational signal sequence was sufficient to recapitulate the lethal phenotype. These results identify a novel connection between the DegP chaperone and the mechanism used to translocate a protein across the IM. Lethality coincided with loss of periplasmic proteins, soluble σE, and proteins regulated by this essential stress response. These results suggest post-translational IM translocation can lead to the formation of toxic periplasmic folding intermediates, which DegP can suppress., Competing Interests: The authors have declared that no competing interests exist.

Modern mass spectrometry-based methods provide an exciting opportunity to characterize protein expression in the developing embryo. We have employed an isotopic labeling technology to quantify the expression dynamics of nearly 6000 proteins across six stages of development in Xenopus laevis from the single stage zygote through the mid-blastula transition and the onset of organogenesis. Approximately 40% of the proteins show significant changes in expression across the development stages. The expression changes for these proteins naturally falls into six clusters corresponding to major events that mark early Xenopus development. A subset of experiments in this study have quantified protein expression differences between single embryos at the same stage of development, showing that, within experimental error, embryos at the same developmental stage have identical protein expression levels., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Capillary zone electrophoresis (CZE) is emerging as a useful tool in proteomic analysis. Interest arises from dramatic improvements in performance that result from improvements in the background electrolyte used for the separation, the incorporation of advanced sample injection methods, the development of robust and sensitive electrospray interfaces, and the coupling with Orbitrap mass spectrometers with high resolution and sensitivity. The combination of these technologies produces performance that is rapidly approaching the performance of UPLC-based methods for microgram samples and exceeds the performance of UPLC-based methods for mid- to low nanogram samples. These systems now produce over 10 000 peptide IDs in a single 100-min analysis of the HeLa proteome., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)