Integrated Immunopeptidomics and Proteomics Study of SARS-CoV-2-Infected Calu-3 Cells Reveals Dynamic Changes in Allele-specific HLA Abundance and Antigen Presentation.

We present an integrated immunopeptidomics and proteomics study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection to comprehensively decipher the changes in host cells in response to viral infection. Immunopeptidomics analysis identified viral antigens presented by host cells through both class I and class II MHC system for recognition by the adaptive immune system. The host proteome changes were characterized by quantitative proteomics and glycoproteomics and from these data, the activation of toll-like receptor 3-interferon pathway was identified. Glycosylation analysis of human leukocyte antigen (HLA) proteins from the elution and flow-through of immunoprecipitation revealed that SARS-CoV-2 infection changed the glycosylation pattern of certain HLA alleles with different HLA alleles, showing distinct dynamic changes in relative abundance. The difference in the glycosylation and abundance of HLA alleles changed the number of strong binding antigens each allele presented, suggesting the impact of SARS-CoV-2 infection on antigen presentation is allele-specific. These results could be further exploited to explain the imbalanced response from innate and adaptive immune system in coronavirus disease 2019 cases, which would be helpful for the development of therapeutics and vaccine for coronavirus disease 2019 and preparation for future pandemic.

Collateral sensitivity profiling in drug-resistant Escherichia coli identifies natural products suppressing cephalosporin resistance.

The rapid emergence of antimicrobial resistance presents serious health challenges to the management of infectious diseases, a problem that is further exacerbated by slowing rates of antimicrobial drug discovery in recent years. The phenomenon of collateral sensitivity (CS), whereby resistance to one drug is accompanied by increased sensitivity to another, provides new opportunities to address both these challenges. Here, we present a high-throughput screening platform termed Collateral Sensitivity Profiling (CSP) to map the difference in bioactivity of large chemical libraries across 29 drug-resistant strains of E. coli. CSP screening of 80 commercial antimicrobials demonstrated multiple CS interactions. Further screening of a 6195-member natural product library revealed extensive CS relationships in nature. In particular, we report the isolation of known and new analogues of borrelidin A with potent CS activities against cephalosporin-resistant strains. Co-dosing ceftazidime with borrelidin A slows broader cephalosporin resistance with no recognizable resistance to borrelidin A itself.

Safety and uptake of fully oxidized ß-carotene.

Spontaneous oxidation of ß-carotene yields a polymer-rich product (OxBC) together with minor amounts of many apocarotenoids. OxBC's activity extends ß-carotene's benefits beyond vitamin A, finding utility in supporting health in livestock, pets, and humans. Although the naturally occurring form of OxBC is consumed in foods and feeds, a direct demonstration of synthetic OxBC's safety provides additional support for its usage. A toxicological study in rats showed a maximum tolerated single oral dose of 5000 mg/kg, an LD50 of more than 10,000 mg/kg, and a NOAEL of 1875 mg/kg body weight. A repeat-dose 90-day oral toxicity study showed no adverse physiological or pathological effects. A study of OxBC uptake by mice over 2-5 days showed OxBC already was naturally present. The highest levels were in liver, lung, and hamstring. Dosing did not increase levels in liver, kidney, lung, and muscle. Increases occurred in urine, intestinal content, plasma, feces, spleen, and cecum with preferential elimination of polymer, consistent with processing of OxBC. Compared to the 4:1 polymer: apocarotenoid ratio of OxBC, polymer was enriched in liver and spleen and depleted in lung, kidney, hamstring, and abdominal muscle. The apparent control of OxBC in major tissues further supports its safety.

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility.

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

Characterization of Extracellular Vesicles Secreted in Lentiviral Producing HEK293SF Cell Cultures.

Lentiviral vectors (LVs) are a powerful tool for gene and cell therapy and human embryonic kidney cells (HEK293) have been extensively used as a platform for production of these vectors. Like most cells and cellular tissues, HEK293 cells release extracellular vesicles (EVs). EVs released by cells share similar size, biophysical characteristics and even a biogenesis pathway with cell-produced enveloped viruses, making it a challenge to efficiently separate EVs from LVs. Thus, EVs co-purified with LVs during downstream processing, are considered "impurities" in the context of gene and cell therapy. A greater understanding of EVs co-purifying with LVs is needed to enable improved downstream processing. To that end, EVs from an inducible lentivirus producing cell line were studied under two conditions: non-induced and induced. EVs were identified in both conditions, with their presence confirmed by transmission electron microscopy and Western blot. EV cargos from each condition were then further characterized by a multi-omic approach. Nineteen proteins were identified by mass spectrometry as potential EV markers to differentiate EVs in LV preparations. Lipid composition of EV preparations before and after LV induction showed similar enrichment in phosphatidylserine. RNA cargos in EVs showed enrichment in transcripts involved in viral processes and binding functions. These findings provide insights on the product profile of lentiviral preparations and could support the development of improved separation strategies aimed at removing co-produced EVs.

IDENTIFICATION OF MHC PEPTIDES USING MASS SPECTROMETRY FOR NEOANTIGEN DISCOVERY AND CANCER VACCINE DEVELOPMENT.

Immunotherapy with neoantigens presented by major histocompatibility complex (MHC) is one of the most promising approaches in cancer treatment. Using this approach, cancer vaccines can be designed to target tumor-specific mutations that are not found in normal tissues. Clinical trials have demonstrated an increased immune response and eradication of tumors after injecting synthetic peptides selected from the immunopeptidome. Although the sequence of MHC binding peptides can be predicted from genome sequencing and prediction algorithms, this approach results in large numbers of predicted peptides, requiring the confirmation by mass spectrometry (MS) analysis. Identification of MHC peptides by direct MS analysis of immunopeptidome is accurate and sensitive, with tens of thousands of unique peptides potentially identified from either cancer cell line or tumor tissue. Peptides with mutations can also be identified with patient-specific protein databases constructed from genome or transcriptome sequencing data. MS analysis also enables the characterization of the post-translational modifications (PTMs) of those antigens that cannot be predicted. Moreover, PTMs were found to be more efficient in triggering an immune response. In addition to reviewing recent advances in the identification of neoantigens using MS, the techniques for cancer vaccine candidate selection and formulation, vaccine delivery systems, and the potential for use in combination with other therapeutics are also discussed. It is anticipated that MS-based techniques will play an important role in future cancer vaccine development. © 2019 John Wiley & Sons Ltd. Mass Spec Rev 40:110-125, 2021.

Thiomicrorhabdus streamers and sulfur cycling in perennial hypersaline cold springs in the Canadian high Arctic.

The Gypsum Hill (GH) springs on Axel Heiberg Island in the Canadian high Arctic are host to chemolithoautotrophic, sulfur-oxidizing streamers that flourish in the high Arctic winter in water temperatures from -1.3 to 7°C with ~8% salinity in a high Arctic winter environment with air temperatures commonly less than -40°C and an average annual air temperature of -15°C. Metagenome sequencing and binning of streamer samples produced a 96% complete Thiomicrorhabdus sp. metagenome-assembled genome representing a possible new species or subspecies. This is the most cold- and salt-extreme source environment for a Thiomicrorhabdus genome yet described. Metaproteomic and metatranscriptomic analysis attributed nearly all gene expression in the streamers to the Thiomicrorhabdus sp. and suggested that it is active in CO2 fixation and oxidation of sulfide to elemental sulfur. In situ geochemical and isotopic analyses of the fractionation of multiple sulfur isotopes determined the biogeochemical transformation of sulfur from its source in Carboniferous evaporites to biotic processes occurring in the sediment and streamers. These complementary molecular tools provided a functional link between the geochemical substrates and the collective traits and activity that define the microbial community's interactions within a unique polar saline habitat where Thiomicrorhabdus-dominated streamers form and flourish.

Polar Flagella Glycosylation in Aeromonas: Genomic Characterization and Involvement of a Specific Glycosyltransferase (Fgi-1) in Heterogeneous Flagella Glycosylation.

Polar flagella from mesophilic Aeromonas strains have previously been shown to be modified with a range of glycans. Mass spectrometry studies of purified polar flagellins suggested the glycan typically includes a putative pseudaminic acid like derivative; while some strains are modified with this single monosaccharide, others modified with a heterologous glycan. In the current study, we demonstrate that genes involved in polar flagella glycosylation are clustered in highly polymorphic genomic islands flanked by pseudaminic acid biosynthetic genes (pse). Bioinformatic analysis of mesophilic Aeromonas genomes identified three types of polar flagella glycosylation islands (FGIs), denoted Group I, II and III. FGI Groups I and III are small genomic islands present in Aeromonas strains with flagellins modified with a single monosaccharide pseudaminic acid derivative. Group II were large genomic islands, present in strains found to modify polar flagellins with heterogeneous glycan moieties. Group II, in addition to pse genes, contained numerous glycosyltransferases and other biosynthetic enzymes. All Group II strains shared a common glycosyltransferase downstream of luxC that we named flagella glycosylation island 1, fgi-1, in A. piscicola AH-3. We demonstrate that Fgi-1 transfers the first sugar of the heterogeneous glycan to the pseudaminic acid derivative linked to polar flagellins and could be used as marker for polysaccharidic glycosylation of Aeromonas polar flagella.

Immunoproteomics Methods and Techniques.

The varied landscape of the adaptive immune response is determined by the peptides presented by immune cells, derived from viral or microbial pathogens or cancerous cells. The study of immune biomarkers or antigens is not new, and classical methods such as agglutination, enzyme-linked immunosorbent assay, or Western blotting have been used for many years to study the immune response to vaccination or disease. However, in many of these traditional techniques, protein or peptide identification has often been the bottleneck. Recent progress in genomics and mass spectrometry have led to many of the rapid advances in proteomics approaches. Immunoproteomics describes a rapidly growing collection of approaches that have the common goal of identifying and measuring antigenic peptides or proteins. This includes gel-based, array-based, mass spectrometry-based, DNA-based, or in silico approaches. Immunoproteomics is yielding an understanding of disease and disease progression, vaccine candidates, and biomarkers. This review gives an overview of immunoproteomics and closely related technologies that are used to define the full set of protein antigens targeted by the immune system during disease.

Classical Immunoproteomics: Serological Proteome Analysis (SERPA) for Antigen Identification.

The study of the humoral immune response to infectious and chronic diseases is important for understanding the disease progression, identification of protective antigens, vaccine development, and discovery of biomarkers for early diagnosis. Proteomic approaches, including serological proteome analysis (SERPA), have been used to identify the repertoire of immunoreactive proteins in various diseases. In this chapter, we provide an outline of the SERPA approach, using the analysis of sera from mice vaccinated with a live attenuated tularemia vaccine as an example.

Enriching for Low-Abundance Serum Proteins Using ProteoMiner™ and Protein-Level HPLC.

The mammalian immune system acts to protect the body from harmful diseases ranging from cancer to infection. Differentially expressed proteins as a result of such an immune response can shed light on the mechanism of disease or serve as biomarkers. These biomarkers can be used in a diagnostic capacity or as correlates of protection following vaccination. Protein levels in the circulatory system are considered representative of the system as a whole, making serum an ideal matrix for surveilling immune responses. However, serum proteomics using mass spectrometry is extremely challenging due to the complexity of the matrix and the dynamic range of protein concentration. This chapter will describe two orthogonal enrichment strategies that can be used sequentially or in isolation to improve the identification of low-abundance serum proteins by mass spectrometry.

Online porous graphic carbon chromatography coupled with tandem mass spectrometry for post-translational modification analysis.

RATIONALE: Porous graphic carbon chromatography (PGC) has a different mechanism in the retention of tryptic peptides compared with reversed-phase chromatography and in this study we show that coupling PGC with tandem mass spectrometry offer advantages for the quantitation of phosphorylation stoichiometry and characterization of site-specific glycosylation. METHODS: Digests of protein standards (horse myoglobin, bovine fetuin and ß-casein) were analyzed with a capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) system by coupling an Agilent 1100 HPLC system to a Synapt G2-Si HDMS (Waters). Peptides were separated using a HyperCarb PGC column (300 µm i.d. × 100 mm) packed with 3 µm particles. MS/MS data were collected in data-dependent mode and three MS/MS scans were acquired after the full MS scan. RAW data were transformed to .mgf by PLGS (Waters) and searched against the Swissprot database by Mascot. Chromatograms and MS/MS spectra of identified compounds were extracted with Masslynx (Waters) and imported to Origin for analysis. Glycan composition and peptide sequence were manually annotated. RESULTS: PGC/MS/MS enabled accurate quantitation of the stoichiometry of specific phosphorylation sites from ß-casein by efficient separation of the phosphopeptide and its non-phosphorylated counterpart, which cannot be achieved by reversed-phase chromatography. PGC/MS/MS also enabled comprehensive characterization of protein sialoglycosylation as isomeric glycopeptides with different combinations of α2-3- and α2-6-linked sialic acids can be separated and the ratios of each combination were verified by exoglycosidase digestion. CONCLUSIONS: PGC has demonstrated superior separation of peptides with phosphorylation and glycosylation and can be used as an alternative in the proteomic characterization of post-translational modifications (PTMs) by polar groups.

Chemical Derivatization Strategy for Extending the Identification of MHC Class I Immunopeptides.

Neoantigen-based therapeutic vaccines have a high potential impact on tumor eradication and patient survival. Mass spectrometry (MS)-based immunopeptidomics has the capacity to identify tumor-associated epitopes and pinpoint mutation-bearing major histocompatibility complex (MHC)-binding peptides. This approach presents several challenges, including the identification of low-abundance peptides. In addition, MHC peptides have much lower MS/MS identification rates than tryptic peptides due to their shorter sequence and lack of basic amino acid at C-termini. In this study, we report the development and application of a novel chemical derivatization strategy that combines the analysis of native, dimethylated, and alkylamidated peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to expand the coverage of the MHC peptidome. The results revealed that dimethylation increases hydrophobicity and ionization efficiency of MHC class I peptides, while alkylamidation significantly improves the fragmentation by producing more y-ions during MS/MS fragmentation. Thus, the combination of dimethylation and alkylamidation enabled the identification of peptides that could not be identified from the analysis of their native form. Using this strategy, we identified 3148 unique MHC I peptides from HCT 116 cell lines, compared to only 1388 peptides identified in their native form. Among these, 10 mutation-bearing peptides were identified with high confidence, indicating that this chemical derivatization strategy is a promising approach for neoantigen discovery in clinical applications.

Further Characterization of the Capsule-Like Complex (CLC) Produced by Francisella tularensis Subspecies tularensis: Protective Efficacy and Similarity to Outer Membrane Vesicles.

Francisella tularensis is the etiologic agent of tularemia, and subspecies tularensis (type A) is the most virulent subspecies. The live vaccine strain (LVS) of subspecies holarctica produces a capsule-like complex (CLC) that consists of a large variety of glycoproteins. Expression of the CLC is greatly enhanced when the bacteria are subcultured in and grown on chemically defined medium. Deletion of two genes responsible for CLC glycosylation in LVS results in an attenuated mutant that is protective against respiratory tularemia in a mouse model. We sought to further characterize the CLC composition and to determine if a type A CLC glycosylation mutant would be attenuated in mice. The CLCs isolated from LVS extracted with 0.5% phenol or 1 M urea were similar, as determined by gel electrophoresis and Western blotting, but the CLC extracted with urea was more water-soluble. The CLC extracted with either 0.5% phenol or 1 M urea from type A strains was also similar to the CLC of LVS in antigenic properties, electrophoretic profile, and by transmission electron microscopy (TEM). The solubility of the CLC could be further enhanced by fractionation with Triton X-114 followed by N-Lauroylsarcosine detergents; the largest (>250 kDa) molecular size component appeared to be an aggregate of smaller components. Outer membrane vesicles/tubules (OMV/T) isolated by differential centrifugation and micro-filtration appeared similar to the CLC by TEM, and many of the proteins present in the OMV/T were also identified in soluble and insoluble fractions of the CLC. Further investigation is warranted to assess the relationship between OMV/T and the CLC. The CLC conjugated to keyhole limpet hemocyanin or flagellin was highly protective against high-dose LVS intradermal challenge and partially protective against intranasal challenge. A protective response was associated with a significant rise in cytokines IL-12, IL-10, and IFN-γ. However, a type A CLC glycosylation mutant remained virulent in BALB/c mice, and immunization with the CLC did not protect mice against high dose respiratory challenge with type A strain SCHU S4.

Characterizing bacterial glycoproteins with LC-MS.

INTRODUCTION: Though eukaryotic glycoproteins have been studied since their discovery in the 1930s, the first bacterial glycoprotein was not identified until the 1970s. As a result, their role in bacterial pathogenesis is still not well understood and they remain an understudied component of bacterial virulence. In recent years, mass spectrometry has emerged as a leading technology for the study of bacterial glycoproteins, largely due to its sensitivity and versatility. Areas covered: Identification and comprehensive characterization of bacterial glycoproteins usually requires multiple complementary mass spectrometry approaches, including intact protein analysis, top-down analysis, and bottom-up methods used in combination with specialized liquid chromatography. This review provides an overview of liquid chromatography separation technologies, as well as current and emerging mass spectrometry approaches used specifically for bacterial glycoprotein identification and characterization. Expert commentary: Bacterial glycoproteins may have significant clinical utility as a result of their unique structures and exposure on the surface of the cells. Better understanding of these glycoconjugates is an essential first step towards that goal. These often unique structures, and by extension the key enzymes involved in their synthesis, represent promising targets for novel antimicrobials, while unique carbohydrate structures may be used as antigens in vaccines or as biomarkers.

Clinical applications of bacterial glycoproteins.

There is an ongoing race between bacterial evolution and medical advances. Pathogens have the advantages of short generation times and horizontal gene transfer that enable rapid adaptation to new host environments and therapeutics that currently outpaces clinical research. Antibiotic resistance, the growing impact of nosocomial infections, cancer-causing bacteria, the risk of zoonosis, and the possibility of biowarfare all emphasize the increasingly urgent need for medical research focussed on bacterial pathogens. Bacterial glycoproteins are promising targets for alternative therapeutic intervention since they are often surface exposed, involved in host-pathogen interactions, required for virulence, and contain distinctive glycan structures. The potential exists to exploit these unique structures to improve clinical prevention, diagnosis, and treatment strategies. Translation of the potential in this field to actual clinical impact is an exciting prospect for fighting infectious diseases.

Polar Glycosylated and Lateral Non-Glycosylated Flagella from Aeromonas hydrophila Strain AH-1 (Serotype O11).

Polar and but not lateral flagellin proteins from Aeromonas hydrophila strain AH-1 (serotype O11) were found to be glycosylated. Top-down mass spectrometry studies of purified polar flagellins suggested the presence of a 403 Da glycan of mass. Bottom-up mass spectrometry studies showed the polar flagellin peptides to be modified with 403 Da glycans in O-linkage. The MS fragmentation pattern of this putative glycan was similar to that of pseudaminic acid derivative. Mutants lacking the biosynthesis of pseudaminic acid (pseB and pseI homologues) were unable to produce polar flagella but no changes were observed in lateral flagella by post-transcriptional regulation of the flagellin. Complementation was achieved by reintroduction of the wild-type pseB and pseI. We compared two pathogenic features (adhesion to eukaryotic cells and biofilm production) between the wild-type strain and two kinds of mutants: mutants lacking polar flagella glycosylation and lacking the O11-antigen lipopolysaccharide (LPS) but with unaltered polar flagella glycosylation. Results suggest that polar flagella glycosylation is extremely important for A. hydrophila AH-1 adhesion to Hep-2 cells and biofilm formation. In addition, we show the importance of the polar flagella glycosylation for immune stimulation of IL-8 production via toll-"like" receptor 5 (TLR5).

Next Generation Vaccine Biomarkers workshop October 30-31, 2014--Ottawa, Canada.

Vaccine biomarkers are critical to many aspects of vaccine development and licensure, including bridging findings in pre-clinical studies to clinical studies, predicting potential adverse events, and predicting vaccine efficacy. Despite advances in our understanding of various biological pathways, and advances in systems analyses of the immune response, there remains much to learn about qualitative and quantitative aspects of the human host response to vaccination. To stimulate discussion and identify opportunities for collaborative ways to advance the field of vaccine biomarkers, A Next Generation Vaccine Biomarker workshop was held in Ottawa. The two day workshop, sponsored by the National Research Council Canada, Canadian Institutes of Health Research, Public Health Agency of Canada, Pfizer, and Medicago, brought together stakeholders from Canadian and international industry, government and academia. The workshop was grouped in themes, covering vaccine biomarker challenges in the pre-clinical and clinical spaces, veterinary vaccines, regulatory challenges, and development of biomarkers for adjuvants and cancer vaccines. The use of case studies allowed participants to identify the needs and gaps requiring innovation. The workshop concluded with a discussion on opportunities for vaccine biomarker discovery, the Canadian context, and approaches for moving forward. This article provides a synopsis of these discussions and identifies steps forward for advancing vaccine biomarker research in Canada.

The polar and lateral flagella from Plesiomonas shigelloides are glycosylated with legionaminic acid.

Plesiomonas shigelloides is the unique member of the Enterobacteriaceae family able to produce polar flagella when grow in liquid medium and lateral flagella when grown in solid or semisolid media. In this study on P. shigelloides 302-73 strain, we found two different gene clusters, one exclusively for the lateral flagella biosynthesis and the other one containing the biosynthetic polar flagella genes with additional putative glycosylation genes. P. shigelloides is the first Enterobacteriaceae were a complete lateral flagella cluster leading to a lateral flagella production is described. We also show that both flagella in P. shigelloides 302-73 strain are glycosylated by a derivative of legionaminic acid (Leg), which explains the presence of Leg pathway genes between the two polar flagella regions in their biosynthetic gene cluster. It is the first bacterium reported with O-glycosylated Leg in both polar and lateral flagella. The flagella O-glycosylation is essential for bacterial flagella formation, either polar or lateral, because gene mutants on the biosynthesis of Leg are non-flagellated. Furthermore, the presence of the lateral flagella cluster and Leg O-flagella glycosylation genes are widely spread features among the P. shigelloides strains tested.

Small-molecule inhibitors of the pseudaminic acid biosynthetic pathway: targeting motility as a key bacterial virulence factor.

Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 µM concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 µM concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structure-activity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC50s) of approximately 14 µM, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella.