Fluorescent sensors of siderophores produced by bacterial pathogens.

Siderophores are iron-chelating molecules that solubilize Fe3+ for microbial utilization and facilitate colonization or infection of eukaryotes by liberating host iron for bacterial uptake. By fluorescently labeling membrane receptors and binding proteins, we created 20 sensors that detect, discriminate, and quantify apo- and ferric siderophores. The sensor proteins originated from TonB-dependent ligand-gated porins (LGPs) of Escherichia coli (Fiu, FepA, Cir, FhuA, IutA, BtuB), Klebsiella pneumoniae (IroN, FepA, FyuA), Acinetobacter baumannii (PiuA, FepA, PirA, BauA), Pseudomonas aeruginosa (FepA, FpvA), and Caulobacter crescentus (HutA) from a periplasmic E. coli binding protein (FepB) and from a human serum binding protein (siderocalin). They detected ferric catecholates (enterobactin, degraded enterobactin, glucosylated enterobactin, dihydroxybenzoate, dihydroxybenzoyl serine, cefidericol, MB-1), ferric hydroxamates (ferrichromes, aerobactin), mixed iron complexes (yersiniabactin, acinetobactin, pyoverdine), and porphyrins (hemin, vitamin B12). The sensors defined the specificities and corresponding affinities of the LGPs and binding proteins and monitored ferric siderophore and porphyrin transport by microbial pathogens. We also quantified, for the first time, broad recognition of diverse ferric complexes by some LGPs, as well as monospecificity for a single metal chelate by others. In addition to their primary ferric siderophore ligands, most LGPs bound the corresponding aposiderophore with ∼100-fold lower affinity. These sensors provide insights into ferric siderophore biosynthesis and uptake pathways in free-living, commensal, and pathogenic Gram-negative bacteria.

Examining the activity of cefepime-taniborbactam against Burkholderia cepacia complex and Burkholderia gladioli isolated from cystic fibrosis patients in the United States.

The novel clinical-stage ß-lactam-ß-lactamase inhibitor combination, cefepime-taniborbactam, demonstrates promising activity toward many Gram-negative bacteria producing class A, B, C, and/or D ß-lactamases. We tested this combination against a panel of 150 Burkholderia cepacia complex (Bcc) and Burkholderia gladioli strains. The addition of taniborbactam to cefepime shifted cefepime minimum inhibitory concentrations toward the provisionally susceptible range in 59% of the isolates tested. Therefore, cefepime-taniborbactam possessed similar activity as first-line agents, ceftazidime and trimethoprim-sulfamethoxazole, supporting further development.

In Vitro Activity of Cefepime-Taniborbactam and Comparators against Clinical Isolates of Gram-Negative Bacilli from 2018 to 2020: Results from the Global Evaluation of Antimicrobial Resistance via Surveillance (GEARS) Program.

Taniborbactam is a novel cyclic boronate ß-lactamase inhibitor in clinical development in combination with cefepime. We assessed the in vitro activity of cefepime-taniborbactam and comparators against a 2018-2020 collection of Enterobacterales (n = 13,731) and Pseudomonas aeruginosa (n = 4,619) isolates cultured from infected patients attending hospitals in 56 countries. MICs were determined by CLSI broth microdilution. Taniborbactam was tested at a fixed concentration of 4 µg/mL. Isolates with cefepime-taniborbactam MICs of ≥16 µg/mL underwent whole-genome sequencing. ß-lactamase genes were identified in meropenem-resistant isolates by PCR/Sanger sequencing. Against Enterobacterales, taniborbactam reduced the cefepime MIC90 value by >64-fold (from >16 to 0.25 µg/mL). At ≤16 µg/mL, cefepime-taniborbactam inhibited 99.7% of all Enterobacterales isolates; >97% of isolates with multidrug-resistant (MDR) and ceftolozane-tazobactam-resistant phenotypes; ≥90% of isolates with meropenem-resistant, difficult-to-treat-resistant (DTR), meropenem-vaborbactam-resistant, and ceftazidime-avibactam-resistant phenotypes; 100% of VIM-positive, AmpC-positive, and KPC-positive isolates; 98.7% of extended-spectrum ß-lactamase (ESBL)-positive; 98.8% of OXA-48-like-positive; and 84.6% of NDM-positive isolates. Against P. aeruginosa, taniborbactam reduced the cefepime MIC90 value by 4-fold (from 32 to 8 µg/mL). At ≤16 µg/mL, cefepime-taniborbactam inhibited 97.4% of all P. aeruginosa isolates; ≥85% of isolates with meropenem-resistant, MDR, and meropenem-vaborbactam-resistant phenotypes; >75% of isolates with DTR, ceftazidime-avibactam-resistant, and ceftolozane-tazobactam-resistant phenotypes; and 87.4% of VIM-positive isolates. Multiple potential mechanisms, including carriage of IMP, certain alterations in PBP3, permeability (porin) defects, and possibly, upregulation of efflux were present in most isolates with cefepime-taniborbactam MICs of ≥16 µg/mL. We conclude that cefepime-taniborbactam exhibited potent in vitro activity against Enterobacterales and P. aeruginosa and inhibited most carbapenem-resistant isolates, including those carrying serine carbapenemases or NDM/VIM metallo-ß-lactamases (MBLs).

Iron Acquisition Systems of Gram-negative Bacterial Pathogens Define TonB-Dependent Pathways to Novel Antibiotics.

Iron is an indispensable metabolic cofactor in both pro- and eukaryotes, which engenders a natural competition for the metal between bacterial pathogens and their human or animal hosts. Bacteria secrete siderophores that extract Fe3+ from tissues, fluids, cells, and proteins; the ligand gated porins of the Gram-negative bacterial outer membrane actively acquire the resulting ferric siderophores, as well as other iron-containing molecules like heme. Conversely, eukaryotic hosts combat bacterial iron scavenging by sequestering Fe3+ in binding proteins and ferritin. The variety of iron uptake systems in Gram-negative bacterial pathogens illustrates a range of chemical and biochemical mechanisms that facilitate microbial pathogenesis. This document attempts to summarize and understand these processes, to guide discovery of immunological or chemical interventions that may thwart infectious disease.

Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes.

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPSs) on the outer leaflet and phospholipids (PLs) on the inner leaflet. The loss of this asymmetry due to mutations in the LPS biosynthesis or transport pathways causes the externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we used metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase expression in Escherichia coli allowed for the incorporation of exogenous propargylcholine into phosphatidyl(propargyl)choline and exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC), as confirmed by LC/MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool for detecting a compromised bacterial OM, revealing aberrant PL externalization, and identifying or characterizing novel cell-active inhibitors of LPS biosynthesis or transport.

VNRX-5133 (Taniborbactam), a Broad-Spectrum Inhibitor of Serine- and Metallo-ß-Lactamases, Restores Activity of Cefepime in Enterobacterales and Pseudomonas aeruginosa.

As shifts in the epidemiology of ß-lactamase-mediated resistance continue, carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) are the most urgent threats. Although approved ß-lactam (BL)-ß-lactamase inhibitor (BLI) combinations address widespread serine ß-lactamases (SBLs), such as CTX-M-15, none provide broad coverage against either clinically important serine-ß-lactamases (KPC, OXA-48) or clinically important metallo-ß-lactamases (MBLs; e.g., NDM-1). VNRX-5133 (taniborbactam) is a new cyclic boronate BLI that is in clinical development combined with cefepime for the treatment of infections caused by ß-lactamase-producing CRE and CRPA. Taniborbactam is the first BLI with direct inhibitory activity against Ambler class A, B, C, and D enzymes. From biochemical and structural analyses, taniborbactam exploits substrate mimicry while employing distinct mechanisms to inhibit both SBLs and MBLs. It is a reversible covalent inhibitor of SBLs with slow dissociation and a prolonged active-site residence time (half-life, 30 to 105 min), while in MBLs, it behaves as a competitive inhibitor, with inhibitor constant (Ki ) values ranging from 0.019 to 0.081 µM. Inhibition is achieved by mimicking the transition state structure and exploiting interactions with highly conserved active-site residues. In microbiological testing, taniborbactam restored cefepime activity in 33/34 engineered Escherichia coli strains overproducing individual enzymes covering Ambler classes A, B, C, and D, providing up to a 1,024-fold shift in the MIC. Addition of taniborbactam restored the antibacterial activity of cefepime against all 102 Enterobacterales clinical isolates tested and 38/41 P. aeruginosa clinical isolates tested with MIC90s of 1 and 4 µg/ml, respectively, representing ≥256- and ≥32-fold improvements, respectively, in antibacterial activity over that of cefepime alone. The data demonstrate the potent, broad-spectrum rescue of cefepime activity by taniborbactam against clinical isolates of CRE and CRPA.

The sialic acid transporter NanT is necessary and sufficient for uptake of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and its azido analog in Escherichia coli.

3-Deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an essential component of lipopolysaccharides (LPS) in the Gram-negative bacterial outer membrane. Metabolic labeling of Escherichia coli LPS with 8-azido-3,8-dideoxy-d-manno-oct-2-ulosonic acid (Kdo-N3 ) has been reported but is inefficient. For optimization, it is important to understand how exogenous Kdo-N3 enters the cytoplasm. Based on similarities between Kdo and sialic acids, we proposed and verified that the sialic acid transporter NanT imports exogenous Kdo-N3 into E. coli. We demonstrated that E. coli ΔnanT were not labeled with Kdo-N3 , while expression of NanT in the ΔnanT mutant restored Kdo-N3 incorporation. Induced NanT expression in a strain lacking Kdo biosynthesis led to higher exogenous Kdo incorporation and restoration of full-length core-LPS, suggesting that NanT also transports Kdo. While Kdo-N3 incorporation was observed in strains having NanT, it was not detected in Pseudomonas aeruginosa and Acinetobacter baumannii, which lack nanT. However, heterologous expression of E. coli NanT in P. aeruginosa enabled Kdo-N3 incorporation and labeling, though this led to abnormal morphology and growth arrest. NanT seems to define which bacteria can be labeled with Kdo-N3 , provides opportunities to enhance Kdo-N3 labeling efficiency and spectrum, and raises the possibility of Kdo biosynthetic bypass where exogenous Kdo is present, perhaps even in vivo.

Defects in Efflux (oprM), ß-Lactamase (ampC), and Lipopolysaccharide Transport (lptE) Genes Mediate Antibiotic Hypersusceptibility of Pseudomonas aeruginosa Strain Z61.

Antibiotic hypersensitive bacterial mutants (e.g., Escherichia coliimp) are used to investigate intrinsic resistance and are exploited in antibacterial discovery to track weak antibacterial activity of novel inhibitor compounds. Pseudomonas aeruginosa Z61 is one such drug-hypersusceptible strain generated by chemical mutagenesis, although the genetic basis for hypersusceptibility is not fully understood. Genome sequencing of Z61 revealed nonsynonymous single-nucleotide polymorphisms in 153 genes relative to its parent strain, and three candidate mutations (in oprM, ampC, and lptE) predicted to mediate hypersusceptibility were characterized. The contribution of these mutations was confirmed by genomic restoration of the wild-type sequences, individually or in combination, in the Z61 background. Introduction of the lptE mutation or genetic inactivation of oprM and ampC genes alone or together in the parent strain recapitulated drug sensitivities. This showed that disruption of oprM (which encodes a major outer membrane efflux pump channel) increased susceptibility to pump substrate antibiotics, that inactivation of the inducible ß-lactamase gene ampC contributed to ß-lactam susceptibility, and that mutation of the lipopolysaccharide transporter gene lptE strongly altered the outer membrane permeability barrier, causing susceptibility to large antibiotics such as rifampin and also to ß-lactams.

Molecular characterization and verification of azido-3,8-dideoxy-d-manno-oct-2-ulosonic acid incorporation into bacterial lipopolysaccharide.

3-Deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an essential component of LPS in the outer leaflet of the Gram-negative bacterial outer membrane. Although labeling of Escherichia coli with the chemical reporter 8-azido-3,8-dideoxy-d-manno-oct-2-ulosonic acid (Kdo-N3) has been reported, its incorporation into LPS has not been directly shown. We have now verified Kdo-N3 incorporation into E. coli LPS at the molecular level. Using microscopy and PAGE analysis, we show that Kdo-N3 is localized to the outer membrane and specifically incorporates into rough and deep-rough LPS. In an E. coli strain lacking endogenous Kdo biosynthesis, supplementation with exogenous Kdo restored full-length core-LPS, which suggests that the Kdo biosynthetic pathways might not be essential in vivo in the presence of sufficient exogenous Kdo. In contrast, exogenous Kdo-N3 only restored a small fraction of core LPS with the majority incorporated into truncated LPS. The truncated LPS were identified as Kdo-N3-lipid IVA and (Kdo-N3)2-lipid IVA by MS analysis. The low level of Kdo-N3 incorporation could be partly explained by a 6-fold reduction in the specificity constant of the CMP-Kdo synthetase KdsB with Kdo-N3 compared with Kdo. These results indicate that the azido moiety in Kdo-N3 interferes with its utilization and may limit its utility as a tracer of LPS biosynthesis and transport in E. coli We propose that our findings will be helpful for researchers using Kdo and its chemical derivatives for investigating LPS biosynthesis, transport, and assembly in Gram-negative bacteria.

Subcellular Chemical Imaging of Antibiotics in Single Bacteria Using C60-Secondary Ion Mass Spectrometry.

The inherent difficulty of discovering new and effective antibacterials and the rapid development of resistance particularly in Gram-negative bacteria, illustrates the urgent need for new methods that enable rational drug design. Here we report the development of 3D imaging cluster Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) as a label-free approach to chemically map small molecules in aggregated and single Escherichia coli cells, with ∼300 nm spatial resolution and high chemical sensitivity. The feasibility of quantitative analysis was explored, and a nonlinear relationship between treatment dose and signal for tetracycline and ampicillin, two clinically used antibacterials, was observed. The methodology was further validated by the observation of reduction in tetracycline accumulation in an E. coli strain expressing the tetracycline-specific efflux pump (TetA) compared to the isogenic control. This study serves as a proof-of-concept for a new strategy for chemical imaging at the nanoscale and has the potential to aid discovery of new antibacterials.

Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis.

UNLABELLED: Lipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. Some Acinetobacter baumannii strains can tolerate the complete loss of lipopolysaccharide (LPS) resulting from the inactivation of early LPS pathway genes such as lpxC. Here, we characterized a mutant deleted for lptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lacking lptD had a growth defect comparable to that of an lpxC deletion mutant under the growth conditions tested but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from the loss of LPS synthesis. Consistent with this, ATP leakage and N-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact of lptD deletion than of lpxC deletion on inner and outer membrane permeability, respectively. Targeted liquid chromatography-mass spectrometry (LCMS) analysis of LPS intermediates from UDP-3-O-R-3-hydroxylauroyl-N-acetyl-α-d-glucosamine through lipid IV(A) showed that the loss of LptD caused an accumulation of lipid IV(A). This suggested that pathway intermediate accumulation or mislocalization caused by the blockage of later LPS pathway steps impacts envelope integrity. Supporting this notion, chemical inhibition of lipid A precursor enzymes, including LpxC and FabB/F, in the lptD deletion strain partially rescued growth and permeability defects. IMPORTANCE: New antibiotics to treat Gram-negative bacterial infections are urgently needed. Inhibition of LPS biosynthesis is attractive because this would impact viability and cell permeability. Therefore, a better understanding of this pathway is important, especially in strains such as A. baumannii ATCC 19606, where LPS biosynthesis is not essential in vitro. We show that ATCC 19606 also survives the loss of the final translocation of LPS into the OM (lptD deletion). Intriguingly, this impaired cell envelope integrity more than the loss of LPS biosynthesis (lpxC deletion), presumably due to the accumulation of toxic intermediates. Supporting this, chemical inhibition of LPS biosynthesis partially reversed this permeability defect. This extends our understanding of the LPS machinery and provides insights into potential interrelationships of the target steps along this important pathway.

Complex transcriptional and post-transcriptional regulation of an enzyme for lipopolysaccharide modification.

The PhoQ/PhoP two-component system activates many genes for lipopolysaccharide (LPS) modification when cells are grown at low Mg(2+) concentrations. An additional target of PhoQ and PhoP is MgrR, an Hfq-dependent small RNA that negatively regulates expression of eptB, also encoding a protein that carries out LPS modification. Examination of LPS confirmed that MgrR effectively silences EptB; the phosphoethanolamine modification associated with EptB is found in ΔmgrR::kan but not mgrR(+) cells. Sigma E has been reported to positively regulate eptB, although the eptB promoter does not have the expected Sigma E recognition motifs. The effects of Sigma E and deletion of mgrR on levels of eptB mRNA were independent, and the same 5' end was found in both cases. In vitro transcription and the behaviour of transcriptional and translational fusions demonstrate that Sigma E acts directly at the level of transcription initiation for eptB, from the same start point as Sigma 70. The results suggest that when Sigma E is active, synthesis of eptB transcript outstrips MgrR-dependent degradation; presumably the modification of LPS is important under these conditions. Adding to the complexity of eptB regulation is a second sRNA, ArcZ, which also directly and negatively regulates eptB.

Deletion of the ß-acetoacetyl synthase FabY in Pseudomonas aeruginosa induces hypoacylation of lipopolysaccharide and increases antimicrobial susceptibility.

The ß-acetoacetyl-acyl carrier protein synthase FabY is a key enzyme in the initiation of fatty acid biosynthesis in Pseudomonas aeruginosa. Deletion of fabY results in an increased susceptibility of P. aeruginosa in vitro to a number of antibiotics, including vancomycin and cephalosporins. Because antibiotic susceptibility can be influenced by changes in membrane lipid composition, we determined the total fatty acid profile of the ΔfabY mutant, which suggested alterations in the lipid A region of the lipopolysaccharide. The majority of lipid A species in the ΔfabY mutant lacked a single secondary lauroyl group, resulting in hypoacylated lipid A. Adding exogenous fatty acids to the growth media restored the wild-type antibiotic susceptibility profile and the wild-type lipid A fatty acid profile. We suggest that incorporation of hypoacylated lipid A species into the outer membrane contributes to the shift in the antibiotic susceptibility profile of the ΔfabY mutant.

Density gradient enrichment of Escherichia coli lpxL mutants.

We previously described enrichment of conditional Escherichia coli msbA mutants defective in lipopolysaccharide export using Ludox density gradients (Doerrler WT (2007) Appl Environ Microbiol 73; 7992-7996). Here, we use this approach to isolate and characterize temperature-sensitive lpxL mutants. LpxL is a late acyltransferase of the pathway of lipid A biosynthesis (The Raetz Pathway). Sequencing the lpxL gene from the mutants revealed the presence of both missense and nonsense mutations. The missense mutations include several in close proximity to the enzyme's active site or conserved residues (E137K, H132Y, G168D). These data demonstrate that Ludox gradients can be used to efficiently isolate conditional E. coli mutants with defects in lipopolysaccharide biosynthesis and provide insight into the enzymatic mechanism of LpxL.

Pathogenicity of Yersinia pestis synthesis of 1-dephosphorylated lipid A.

Synthesis of Escherichia coli LpxL, which transfers a secondary laurate chain to the 2' position of lipid A, in Yersinia pestis produced bisphosphoryl hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Our previous observations also indicated that strain χ10015(pCD1Ap) (ΔlpxP32::P(lpxL) lpxL) stimulated a strong inflammatory reaction but sickened mice before recovery and retained virulence via intranasal (i.n.) infection. The development of live, attenuated Y. pestis vaccines may be facilitated by detoxification of its lipopolysaccharide (LPS). Heterologous expression of the lipid A 1-phosphatase, LpxE, from Francisella tularensis in Y. pestis yields predominantly 1-dephosphorylated lipid A, as confirmed by mass spectrometry. Results indicated that expression of LpxE on top of LpxL provided no significant reduction in virulence of Y. pestis in mice when it was administered i.n. but actually reduced the 50% lethal dose (LD(50)) by 3 orders of magnitude when the strain was administered subcutaneously (s.c.). Additionally, LpxE synthesis in wild-type Y. pestis KIM6+(pCD1Ap) led to slight attenuation by s.c. inoculation but no virulence change by i.n. inoculation in mice. In contrast to Salmonella enterica, expression of LpxE does not attenuate the virulence of Y. pestis.

Salmonella synthesizing 1-dephosphorylated [corrected] lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity.

The development of safe live, attenuated Salmonella vaccines may be facilitated by detoxification of its LPS. Recent characterization of the lipid A 1-phosphatase, LpxE, from Francisella tularensis allowed us to construct recombinant, plasmid-free strains of Salmonella that produce predominantly 1-dephosphorylated lipid A, similar to the adjuvant approved for human use. Complete lipid A 1-dephosphorylation was also confirmed under low pH, low Mg(2+) culture conditions, which induce lipid A modifications. LpxE expression in Salmonella reduced its virulence in mice by five orders of magnitude. Moreover, mice inoculated with these detoxified strains were protected against wild-type challenge. Candidate Salmonella vaccine strains synthesizing pneumococcal surface protein A (PspA) were also confirmed to possess nearly complete lipid A 1-dephosphorylation. After inoculation by the LpxE/PspA strains, mice produced robust levels of anti-PspA Abs and showed significantly improved survival against challenge with wild-type Streptococcus pneumoniae WU2 compared with vector-only-immunized mice, validating Salmonella synthesizing 1-dephosphorylated lipid A as an Ag-delivery system.

Phosphate groups of lipid A are essential for Salmonella enterica serovar Typhimurium virulence and affect innate and adaptive immunity.

Lipid A is a key component of the outer membrane of Gram-negative bacteria and stimulates proinflammatory responses via the Toll-like receptor 4 (TLR4)-MD2-CD14 pathway. Its endotoxic activity depends on the number and length of acyl chains and its phosphorylation state. In Salmonella enterica serovar Typhimurium, removal of the secondary laurate or myristate chain in lipid A results in bacterial attenuation and growth defects in vitro. However, the roles of the two lipid A phosphate groups in bacterial virulence and immunogenicity remain unknown. Here, we used an S. Typhimurium msbB pagL pagP lpxR mutant, carrying penta-acylated lipid A, as the parent strain to construct a series of mutants synthesizing 1-dephosphorylated, 4'-dephosphorylated, or nonphosphorylated penta-acylated lipid A. Dephosphorylated mutants exhibited increased sensitivity to deoxycholate and showed increased resistance to polymyxin B. Removal of both phosphate groups severely attenuated the mutants when administered orally to BALB/c mice, but the mutants colonized the lymphatic tissues and were sufficiently immunogenic to protect the host from challenge with wild-type S. Typhimurium. Mice receiving S. Typhimurium with 1-dephosphorylated or nonphosphorylated penta-acylated lipid A exhibited reduced levels of cytokines. Attenuated and dephosphorylated Salmonella vaccines were able to induce adaptive immunity against heterologous (PspA of Streptococcus pneumoniae) and homologous antigens (lipopolysaccharide [LPS] and outer membrane proteins [OMPs]).

Metabolic Incorporation of Azido-Sugars into LPS to Enable Live-Cell Fluorescence Imaging.

Metabolic labeling of lipopolysaccharides (LPS) with the exogenous azido analog of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) or Kdo-N3 allows for both live-cell and molecular analysis of the outer membrane composition and biosynthesis in different Gram-negative bacteria. Here, we describe Kdo-N3 incorporation into bacterial cells, followed by click labeling with a fluorescent dye. The fluorescently labeled LPS can be analyzed from lysed cells by SDS-PAGE and from intact cells by microscopy and flow cytometry. These methods have been applied to the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae, which possess the sialic acid transporter NanT that is also capable of transporting exogenous Kdo and Kdo analogs into the cytoplasm for incorporation into nascent LPS.

Palmitoylation state impacts induction of innate and acquired immunity by the Salmonella enterica serovar typhimurium msbB mutant.

Lipopolysaccharide (LPS), composed of lipid A, core, and O-antigen, is a major virulence factor of Salmonella enterica serovar Typhimurium, with lipid A being a major stimulator to induce the proinflammatory response via the Toll-like receptor 4 (TLR4)-MD2-CD14 pathway. While Salmonella msbB mutants lacking the myristate chain in lipid A were investigated widely as an anticancer vaccine, inclusion of the msbB mutation in a Salmonella vaccine to deliver heterologous antigens has not yet been investigated. We introduced the msbB mutation alone or in combination with mutations in other lipid A acyl chain modification genes encoding PagL, PagP, and LpxR into wild-type S. enterica serovar Typhimurium. The msbB mutation reduced virulence, while the pagL, pagP, and lpxR mutations did not affect virulence in the msbB mutant background when administered orally to BALB/c mice. Also, all mutants exhibited sensitivity to polymyxin B but did not display sensitivity to deoxycholate. LPS derived from msbB mutants induced less inflammatory responses in human Mono Mac 6 and murine macrophage RAW264.7 cells in vitro. However, an msbB mutant did not decrease the induction of inflammatory responses in mice compared to the levels induced by the wild-type strain, whereas an msbB pagP mutant induced less inflammatory responses in vivo. The mutations were moved to an attenuated Salmonella vaccine strain to evaluate their effects on immunogenicity. Lipid A modification caused by the msbB mutation alone and in combination with pagL, pagP, and lpxR mutations led to higher IgA production in the vaginal tract but still retained the same IgG titer level in serum to PspA, a test antigen from Streptococcus pneumoniae, and to outer membrane proteins (OMPs) from Salmonella.

The Next-Generation ß-Lactamase Inhibitor Taniborbactam Restores the Morphological Effects of Cefepime in KPC-Producing Escherichia coli.

Gram-negative bacteria producing carbapenemases are resistant to a variety of ß-lactam antibiotics and pose a significant health risk. Given the dearth of new antibiotics, combinations of new broad-spectrum ß-lactamase inhibitors (BLIs) with approved ß-lactams have provided treatment options for resistant bacterial infections. Taniborbactam (formerly VNRX-5133) is an investigational BLI that is effective against both serine- and metallo-ß-lactamases, including the serine carbapenemase KPC. In the current study, we assessed the effectiveness of taniborbactam to restore antibacterial activity of cefepime against KPC-3-producing Escherichia coli by inhibiting the KPC-3-dependent hydrolysis of cefepime. Time-lapse microscopy revealed that cells treated with greater than 1× MIC of cefepime (128 µg/ml) and cefepime-taniborbactam (4 µg/ml cefepime and 4 µg/ml taniborbactam) exhibited significant elongation, whereas cells treated with taniborbactam alone did not owing to a lack of standalone antibacterial activity of the BLI. The elongated cells also had frequent cellular voids thought to be formed by attempted cell divisions and pinching of the cytoplasmic membrane. Additionally, the effect of taniborbactam continued even after its removal from the growth medium. Pretreatment with 4 µg/ml taniborbactam helped to restore the antibacterial action of cefepime by neutralizing the effect of the KPC-3 ß-lactamase. IMPORTANCE ß-lactam (BL) antibiotics are the most prescribed antimicrobial class. The efficacy of ß-lactams is threatened by the production of ß-lactamase enzymes, the predominant resistance mechanism impacting these agents in Gram-negative bacterial pathogens. This study visualizes the effects of a combination treatment of taniborbactam, a broad spectrum ß-lactamase inhibitor (BLI), and the BL antibiotic cefepime on a carbapenemase-producing E. coli strain. While this treatment has been described in the context of other cephalosporin-resistant bacteria, this is the first description of a microscopic evaluation of a KPC-3-producing strain of E. coli challenged by this BL-BLI combination. Live-cell microscopy analysis of cells treated with taniborbactam and cefepime demonstrated the antimicrobial effects on cellular morphology and highlighted the long-lasting inhibition of ß-lactamases by taniborbactam even after it was removed from the medium. This research speaks to the importance of taniborbactam in fighting BL-mediated antibiotic resistance.