Journal: The American Journal of Tropical Medicine and Hygiene

Article Title: Susceptibility of Clinical Isolates of Burkholderia pseudomallei to a Lipid A Biosynthesis Inhibitor

doi: 10.4269/ajtmh.16-0858

Figure Lengend Snippet: Time-kill curves for Burkholderia pseudomallei K96243. ( A ) LpxC-4 was tested at 0.5×, 1×, 2×, 4×, and 8× minimum inhibitory concentrations (MICs). ( B ) LpxC-4 and ceftazidime (CAZ) were tested individually at 4× MIC or in combination (4× MIC for each drug). Error bars represent standard deviation.

Article Snippet: Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as controls for CAZ and SXT., , Susceptibility to an LpxC inhibitor, LpxC-4, was examined using a broth microdilution test according to the Clinical and Laboratory Standards Institute guidelines.

Techniques: Standard Deviation

Journal: The American Journal of Tropical Medicine and Hygiene

Article Title: Susceptibility of Clinical Isolates of Burkholderia pseudomallei to a Lipid A Biosynthesis Inhibitor

doi: 10.4269/ajtmh.16-0858

Figure Lengend Snippet: Susceptibility of LpxC-4 to three groups of Burkholderia pseudomallei isolates: ceftazidime (CAZ)/trimethoprim-sulfamethoxazole (SXT) susceptible, CAZ-resistant, and SXT resistant. Box plots represent the 25th and 75th percentile boundaries in the box, with the median line indicated within the box; the whiskers indicate the 10th and 90th percentiles. The plots show the ( A ) minimum inhibitory concentration (MIC) and ( B ) minimum bactericidal concentration (MBC) for each group of isolates.

Article Snippet: Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as controls for CAZ and SXT., , Susceptibility to an LpxC inhibitor, LpxC-4, was examined using a broth microdilution test according to the Clinical and Laboratory Standards Institute guidelines.

Techniques: Concentration Assay

Journal: Journal of Bacteriology

Article Title: Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth

doi: 10.1128/jb.00308-23

Figure Lengend Snippet: Models of enterobacterial LPS regulation involving the PbgA-LapB complex and LPS sensing at the IM. ( A ) Prevailing models suppose that when nutrients are abundant and bacteria are in logarithmic-phase growth (log phase), the coupled processes of bacterial LPS synthesis and transport proceed rapidly and PbgA is unbound to LPS molecules yet bound to LapB. When unbound to LPS, the PbgA-LapB complex is conformationally restricted for LpxC and FtsH binding, which prevents LapB from driving LpxC proteolysis. Two conflicting models exist for PbgA-LapB interplay when the bacterial environment deteriorates. ( B ) In response to limiting nutrients, such as during stationary-phase growth, PbgA signals for LPS downregulation by binding LPS molecules that accumulate on the periplasm-IM interface. In the “dissociation model,” PbgA-LPS binding releases LapB so that LapB can bind LpxC and FtsH to promote LpxC proteolysis. ( C ) We present data to contradict the “dissociation model” and instead provide evidence for what we term the “constitutive-complex model,” whereby PbgA-LapB forms a complex during both log- and stationary-phase growth that differentially binds LpxC to facilitate regulated proteolysis.

Article Snippet: To blot chromosomal LpxC and DnaK, polyclonal antibodies to LpxC (MyBioSource Cat. # MBS1488471), DnaK (MyBioSource Cat. # MBS565041) were diluted 1:10,000 in TBS-T and incubated at room temperature for 4 h. Anti-PbgA 191-586 antibodies were obtained previously and cleared from rabbit-antisera as described ( ).

Techniques: Bacteria, Binding Assay

Journal: Journal of Bacteriology

Article Title: Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth

doi: 10.1128/jb.00308-23

Figure Lengend Snippet: LapB levels increase in the stationary phase while LpxC levels decrease, and lapB is necessary for S . Typhimurium growth, OM integrity, and LPS composition. ( A ) S . Typhimurium lapB is required for growth and thermo-tolerance in nutrient-rich broth media. ( B ) Δ lapB activates the wza-lacZ gene reporter of OM integrity as measured by β-galactosidase activity and quantified as Miller units. ( C ) lapB mutants accumulate lipid A-core molecules and are depleted for LPS molecules with L and VL O-antigens relative to the wild type when cultured to the stationary phase. LPS samples were extracted from normalized cell culture densities [optical density (OD 600 )]. ( D ) Immunoblot depicting the requirement of lapB to limit 34 kDa LpxC levels (α-LpxC, upper panel, demarcated by asterisk) in comparison to a loading control (α-DnaK, lower panel). ( E ) LapB Flag-C levels were monitored in the log and stationary growth phases and semi-quantified using an α-FLAG antibody (top panel) in comparison to a loading control (α-DnaK, bottom panel). The increase in LapB Flag-C levels in stationary-phase bacteria versus log-phase bacteria was statistically significant ( * P = 0.037) by paired Student’s t -test. ( F ) LpxC expression decreases as LapB expression increases during the log-to-stationary phase growth transition. Immunoblots of lapB FLAG-C from whole-cell lysates of bacteria induced (+0.1% ARA) to express a plasmid-borne copy of an amino-terminal streptavidin tagged LpxC (LpxC Strep-N ). OD 600 readings were used to define the log phase as OD 600 of 0.6–0.8 and the stationary phase as the culture density obtained after incubation at 37°C for 16 h. Data are reported as an average of three biological replicates and represent the standard error of the mean (±SEM).

Article Snippet: To blot chromosomal LpxC and DnaK, polyclonal antibodies to LpxC (MyBioSource Cat. # MBS1488471), DnaK (MyBioSource Cat. # MBS565041) were diluted 1:10,000 in TBS-T and incubated at room temperature for 4 h. Anti-PbgA 191-586 antibodies were obtained previously and cleared from rabbit-antisera as described ( ).

Techniques: Activity Assay, Cell Culture, Western Blot, Comparison, Control, Bacteria, Expressing, Plasmid Preparation, Incubation

Journal: Journal of Bacteriology

Article Title: Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth

doi: 10.1128/jb.00308-23

Figure Lengend Snippet: Transcomplementation of the lapB deletion mutant with LapB restores S . Typhimurium growth, LpxC levels, and LPS composition. ( A ) Arabinose induction of LapB in trans is sufficient to restore the lapB mutant growth defect, while glucose repression results in an intermediate growth phenotype. Bacterial growth was assessed by measuring the terminal OD 600 of stationary-phase bacteria. Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test comparing the mean of each column with the mean of every column ( * P < 0.0332, ** P < 0.0021, *** P < 0.0002, and **** P < 0.0001) ( n = 3, ±SEM). ( B ) LapB induction (0.1% ARA) increases LapB expression levels and decreases LpxC levels while repressing LapB levels (0.2% GLU) increases LpxC levels. For reference, the estimated size of LapB is 44 kDa, LpxC is 34 kDa, and the cross-reactive non-specific protein band routinely detected by the polyclonal LpxC antisera is 70 kDa (denoted by ***, served as loading control). ( C ) LapB induction (0.1% ARA) restores the levels of lipid A-core and L- and VL-LPS molecules to lapB mutant S . Typhimurium, while glucose repression (0.2% GLU) resembles the LPS profile of the lapB mutant without the vector. ( D ) Female and male C57BL/6J mice were intraperitoneally injected with ~5 × 10 5 cfu of the wild type, the lapB mutant, and the transcomplemented lapB mutant post-culturing for 16 h at 37°C in Luria-Bertani/lysogeny broth (LB). After 2 days post-infection, the mice were euthanized, and colony counts were enumerated from liver and spleen homogenates. Data are shown as the mean number of cfu per gram of liver ±SEM and spleen ±SEM. Each genotype was assessed in at least three mice ( n = 3). Statistical significance was calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test using lapB + :pEMPTY as a control group ( * P < 0.0332, ** P < 0.0021).

Article Snippet: To blot chromosomal LpxC and DnaK, polyclonal antibodies to LpxC (MyBioSource Cat. # MBS1488471), DnaK (MyBioSource Cat. # MBS565041) were diluted 1:10,000 in TBS-T and incubated at room temperature for 4 h. Anti-PbgA 191-586 antibodies were obtained previously and cleared from rabbit-antisera as described ( ).

Techniques: Mutagenesis, Bacteria, Expressing, Control, Plasmid Preparation, Injection, Infection

Journal: Journal of Bacteriology

Article Title: Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth

doi: 10.1128/jb.00308-23

Figure Lengend Snippet: S . Typhimurium requires LapB’s transmembrane to limit LpxC expression, but overexpression of the cytosolic domain is sufficient to limit lipid A-core biosynthesis and alter LPS production. ( A ) Terminal liquid culture density (16 h, 37°C, LB, +0.1% ARA) of lapB + :pEMPTY, ∆ lapB mutant, a lapB mutant transcomplemented with full-length LapB ( lapB + , FL), or transmembrane-deficient LapB ( lapB ∆TM , ∆TM) was recorded and analyzed ( n = 3, ±SEM). ( B ) LPS analysis comparing wild-type, ∆ lapB , ∆ lapB : p lapB + , ∆ lapB : p lapB ∆TM . LapB and LpxC immunoblot derived from whole-cell lysates. α-His antibody was used to verify LapB presence alongside polyclonal α-LpxC in comparison to α-GAPDH as a loading control.

Article Snippet: To blot chromosomal LpxC and DnaK, polyclonal antibodies to LpxC (MyBioSource Cat. # MBS1488471), DnaK (MyBioSource Cat. # MBS565041) were diluted 1:10,000 in TBS-T and incubated at room temperature for 4 h. Anti-PbgA 191-586 antibodies were obtained previously and cleared from rabbit-antisera as described ( ).

Techniques: Expressing, Over Expression, Mutagenesis, Western Blot, Derivative Assay, Comparison, Control

Journal: Journal of Bacteriology

Article Title: Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth

doi: 10.1128/jb.00308-23

Figure Lengend Snippet: LapB overexpression diminishes LpxC levels and perturbs S . Typhimurium growth, while overexpressing LapB without the TM segment (LapB ΔTM ) causes antimicrobial resistance to decrease and LpxC to accumulate. ( A ) LapB induction in wild-type S . Typhimurium ( lapB + ) limits bacterial growth in a manner that requires the TM segment. The lapB + :pEMPTY and lapB + bacteria carrying a plasmid-borne copy of wild type (WT) (pLapB 6xHisC ) or mutant (pLapB ∆TM6xHisC ) were cultured to the log phase, 0.1% arabinose was added to the media, and bacteria growth was monitored as a function of time. ( B ) Arabinose induction of lapB + :pLapB 6xHisC limits bacterial growth on solidified LB agar while lapB + :pLapB ΔTM6xHisC does not. ( C ) Overexpressing LapB 6xHisC or LapB ΔTM6xHisC activates the wza-lacZ reporter of OM integrity damage as measured by β-galactosidase activity and quantified as Miller units. Statistical significance was calculated via ordinary one-way ANOVA followed by Tukey’s multiple comparisons test ( * P < 0.0332, ** P < 0.0021, *** P < 0.002, and **** P < 0.0001) ( n = 3, ±SEM). ( D ) Induction of pLapB ∆TM6xHisC increases S . Typhimurium sensitivity to rifampin and bile salts. ( E ) Induction of pLapB 6xHisC elevates LapB levels, but no detectable LpxC was present, while induction of pLapB ∆TM6xHisC results in elevated levels of LapB and LpxC ( n = 3).

Article Snippet: To blot chromosomal LpxC and DnaK, polyclonal antibodies to LpxC (MyBioSource Cat. # MBS1488471), DnaK (MyBioSource Cat. # MBS565041) were diluted 1:10,000 in TBS-T and incubated at room temperature for 4 h. Anti-PbgA 191-586 antibodies were obtained previously and cleared from rabbit-antisera as described ( ).

Techniques: Over Expression, Bacteria, Plasmid Preparation, Mutagenesis, Cell Culture, Activity Assay

Journal: Journal of Bacteriology

Article Title: Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth

doi: 10.1128/jb.00308-23

Figure Lengend Snippet: LapB alone forms a homotrimeric complex, and LapB-LpxC forms a heterotrimeric complex of one LpxC molecule and two LapB molecules. ( A ) Linear schematic of the engineered proteins encoded onto pACYCDuet-1, co-overexpressed, and purified. The LapB TM was removed to avoid detergent use and replaced with an N-terminal polyhistidine tag ( 6xHisN LapB ∆TM ). The LpxC C-terminal tail was removed to increase protein stability and replaced with an S-Tag (LpxC ∆293-305-S-Tag ). ( B ) LapB and LpxC co-overexpression followed by affinity purification and gel filtration results in co-elution of LapB and LpxC. SDS-PAGE analysis of the elution fractions from a two-step affinity purification using cobalt resin (Co 2+ ) and gel filtration analysis with a Superdex 75 column (SEC 75 ). ( C ) LapB forms a homotrimeric complex while LapB-LpxC forms a heterotrimeric complex of two LapB molecules and one LpxC molecule. Calibrated gel filtration chromatograms of the purified LapB or the LapB-LpxC complexes followed by ( D ) SDS-PAGE analysis of the peak fractions corresponding to the 11–12-mL elution volumes. A protein standard mix consisting of bovine thyroglobulin (670 kDa), bovine gamma-globulin (158 kDa), ovalbumin (44 kDa), myoglobulin (17 kDa), and vitamin B12 (1.35 kDa) was used to generate a standard curve and estimate molecular weight (gray dotted line above the peaks in the representative chromatogram). ( E ) The LapB complex is larger than the LapB-LpxC complex. Native-PAGE analysis of 5 µg of purified LapB or co-purified LapB-LpxC. Protein presence was verified by Coomassie staining (upper images) or Western blotting (lower images) with antibodies against the affinity tags, α-His and α-S-tag.

Article Snippet: To blot chromosomal LpxC and DnaK, polyclonal antibodies to LpxC (MyBioSource Cat. # MBS1488471), DnaK (MyBioSource Cat. # MBS565041) were diluted 1:10,000 in TBS-T and incubated at room temperature for 4 h. Anti-PbgA 191-586 antibodies were obtained previously and cleared from rabbit-antisera as described ( ).

Techniques: Purification, Over Expression, Affinity Purification, Filtration, Co-Elution Assay, SDS Page, Molecular Weight, Clear Native PAGE, Staining, Western Blot

Journal: Journal of Bacteriology

Article Title: Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth

doi: 10.1128/jb.00308-23

Figure Lengend Snippet: LapB binds LpxC using TPRs 2–4 proximal to the amino-terminal TM. ( A ) Surface representation of AlphaFold2 multimer-predicted LapB-LpxC complex. ( B ) Linear schematic depicting sequential amino-terminal LapB TPR deletion and carboxyl-terminal TPR-Rdx deletions used to identify the LapB-LpxC interaction interface. ( C ) LapB and LpxC co-elute following microscale affinity purification. SDS-PAGE analysis of fractions collected from the purification of 6xHisN LapB ∆TM and LpxC Δ293-385-S-Tag (left panel) or LpxC Δ293-385-S-Tag alone (right panel) using cobalt-coated microbeads. Sample fractions were normalized to 2.5 µg of protein and equally loaded and resolved on a 10% SDS-PAGE gel and immunoblotted using affinity tag-specific antibodies: α-His for LapB and α-S-tag for LpxC. [L] refers to ladder, [I] refers to input, [FT] refers to flow-through, [W] refers to wash, and [E] refers to elution. Single expression and purification of LpxC Δ293-385-S-Tag were used as a control to exclude non-specific binding to the magnetic beads (right panel). ( D ) LapB TPR1 is not required for the interaction with LpxC, while TPR2 is necessary. Representative co-purification of LapB ∆TM-TPR1 (left panel) and LapB ∆TM-TPR2 (right panel). ( E ) LapB TPRs 1–4 are sufficient for LapB to interact with LpxC while TPRs 5–9 and the Rdx are dispensable. Each purification is representative of one of three biological replicates.

Article Snippet: To blot chromosomal LpxC and DnaK, polyclonal antibodies to LpxC (MyBioSource Cat. # MBS1488471), DnaK (MyBioSource Cat. # MBS565041) were diluted 1:10,000 in TBS-T and incubated at room temperature for 4 h. Anti-PbgA 191-586 antibodies were obtained previously and cleared from rabbit-antisera as described ( ).

Techniques: Affinity Purification, SDS Page, Purification, Expressing, Control, Binding Assay, Magnetic Beads, Copurification

Journal: Journal of Bacteriology

Article Title: Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth

doi: 10.1128/jb.00308-23

Figure Lengend Snippet: LapB interacts with PbgA in log- and stationary-phase bacteria and PbgA-LapB as well as LapB-LpxC interactions are enriched in stationary-phase salmonellae. ( A ) Immunoprecipitation of LapB from the membranes of log- and stationary-phase S . Typhimurium results in the co-elution of PbgA, not LpxC. Representative immunoprecipitation assay with anti-Flag loaded magnetic beads. Equal amounts of isolated membranes from S . Typhimurium lapB + or lapB Flag-C cultured to either the log or stationary phase were incubated with the beads, washed, and eluted. Fifteen microliters of the elution fraction was analyzed by SDS-PAGE and immunoblotting. The following proteins were examined: LapB FLAG-C (47 kDa) using anti-Flag monoclonal antibody, LpxC (34 kDa) using anti-LpxC polyclonal antibody, and PbgA (67 kDa) using anti-PbgA polyclonal antibody. ( B ) Immunoprecipitation of LapB from S . Typhimurium whole-cell lysates collected from log- and stationary-phase bacteria results in the co-elution of PbgA in both growth phases, while both PbgA and LpxC are enriched in the elution fractions of stationary-phase bacteria. A representative immunoprecipitation assay with anti-Flag loaded magnetic beads from whole-cell lysates was prepared from 0.1% arabinose-supplemented cultures of lapB Flag-C harboring a plasmid-borne copy of PbgA 6xHis-C or an empty vector, which had been harvested in the log or stationary growth phase. Equal amounts of bacterial lysates were incubated with the magnetic beads, washed, and eluted. Fifteen microliters of the elution fraction was analyzed by SDS-PAGE and Western blotting. The same proteins were assessed as in panel ( A ) apart from PbgA 6xHis-C (68 kDa), which was visualized using an anti-His monoclonal antibody. The lines tooled around the marker lanes are drawn to distinguish between two different image files of the same membrane. The left most image file of the ladder is the colorimetric image. In some instances, the secondary antibody interacts with the protein markers in the ladder; therefore, the chemiluminescent image alone cannot always be used.

Article Snippet: To blot chromosomal LpxC and DnaK, polyclonal antibodies to LpxC (MyBioSource Cat. # MBS1488471), DnaK (MyBioSource Cat. # MBS565041) were diluted 1:10,000 in TBS-T and incubated at room temperature for 4 h. Anti-PbgA 191-586 antibodies were obtained previously and cleared from rabbit-antisera as described ( ).

Techniques: Bacteria, Immunoprecipitation, Co-Elution Assay, Magnetic Beads, Isolation, Cell Culture, Incubation, SDS Page, Western Blot, Plasmid Preparation, Marker, Membrane

Journal: Journal of Bacteriology

Article Title: Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth

doi: 10.1128/jb.00308-23

Figure Lengend Snippet: PbgA interacts with LpxC in S . Typhimurium during the logarithmic and stationary growth phase. ( A ) Immunoprecipitation of LpxC using magnetic beads loaded with anti-Strep antibodies from whole-cell lysates of 0.1% arabinose-supplemented cultures of lapB + and lapB Flag-C harboring a plasmid-borne copy of LpxC ∆293-305-Strep-C harvested in the logarithmic (Log) or stationary (Stat) growth phase. Equal amounts of bacterial lysates were incubated with the magnetic beads, washed, and then eluted. Fifteen microliters of the elution fractions was analyzed by SDS-PAGE and subjected to Western blotting for LapB (anti-Flag), LpxC (anti-Strep), and PbgA (anti-PbgA). The data are representative of three independent experiments. ( B ) Immunoprecipitation of PbgA using magnetic beads loaded with anti-HA antibodies from whole-cell lysates of 0.1% arabinose-supplemented cultures of lapB + or lapB Flag-C harboring a plasmid-borne copy of PbgA HA-C or an empty vector harvested in the logarithmic (Log) or stationary (Stat) growth phase. Equal amounts of bacterial lysates were incubated with the magnetic beads, washed, and then eluted. Fifteen microliters of the elution fractions was analyzed by SDS-PAGE and subjected to Western blotting for LapB (anti-Flag), LpxC (anti-LpxC), and PbgA (anti-HA). The data are representative of three independent experiments. The lines tooled around the marker lanes are drawn to distinguish between two different image files of the same membrane. The left most image file of the ladder is the colorimetric image. In some instances, the secondary antibody interacts with the protein markers in the ladder; therefore, the chemiluminescent image alone cannot always be used.

Article Snippet: To blot chromosomal LpxC and DnaK, polyclonal antibodies to LpxC (MyBioSource Cat. # MBS1488471), DnaK (MyBioSource Cat. # MBS565041) were diluted 1:10,000 in TBS-T and incubated at room temperature for 4 h. Anti-PbgA 191-586 antibodies were obtained previously and cleared from rabbit-antisera as described ( ).

Techniques: Immunoprecipitation, Magnetic Beads, Plasmid Preparation, Incubation, SDS Page, Western Blot, Marker, Membrane