Penicillin-Binding Proteins in
            <i>Leptospira interrogans</i>

Brenot, Audrey; Trott, Daren; Girons, Isabelle Saint; Zuerner, Richard L.

doi:10.1128/aac.45.3.870-877.2001

Cited by 15 publications

(8 citation statements)

References 23 publications

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“…Previous studies have shown that Triton X-114 solubilizes the outer membrane of L. kirschneri, leaving behind the protoplasmic cylinder consisting of the cytoplasm surrounded by the inner membrane (27,56). As expected, these studies localized the penicillin-binding proteins to the protoplasmic cylinder of Leptospira (10,27). Furthermore, when the temperature of the solubilized material is raised above the clouding point of the detergent to 37°C, LPS and outer membrane proteins LipL32, LipL36, LipL41, and OmpL1 partition into the detergent phase and periplasmic proteins partition into the aqueous phase (24,25,45).…”

Section: Vol 70 2002supporting

confidence: 58%

Novel 45-Kilodalton Leptospiral Protein That Is Processed to a 31-Kilodalton Growth-Phase-Regulated Peripheral Membrane Protein

et al. 2002

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Leptospiral protein antigens are of interest as potential virulence factors and as candidate serodiagnostic and immunoprotective reagents. We identified leptospiral protein antigens by screening a genomic expression library with serum from a rabbit hyperimmunized with formalin-killed, virulent Leptospira kirschneri serovar grippotyphosa. Genes expressing known outer membrane lipoproteins LipL32 and LipL41, the heat shock protein GroEL, and the ␣, ␤, and ␤ subunits of RNA polymerase were isolated from the library. In addition, a new leptospiral gene that in Escherichia coli expressed a 45-kDa antigen with an amino-terminal signal peptide followed by the spirochetal lipobox Val ؊4 -Phe ؊3 -Asn ؊2 -Ala ؊1 2Cys ؉1 was isolated. We designated this putative lipoprotein LipL45. Immunoblot analysis of a panel of Leptospira strains probed with LipL45 antiserum demonstrated that many low-passage strains expressed LipL45. In contrast, LipL45 was not detected in high-passage, culture-attenuated strains, suggesting that LipL45 is a virulence-associated protein. In addition, all leptospiral strains tested, irrespective of culture passage, expressed a 31-kDa antigen that was recognized by LipL45 antiserum. Southern blot and peptide mapping studies indicated that this 31-kDa antigen was derived from the carboxy terminus of LipL45; therefore, it was designated P31 LipL45 . Membrane fractionation studies demonstrated that P31 LipL45 is a peripheral membrane protein. Finally, we found that P31 LipL45 levels increased as Leptospira entered the stationary phase, indicating that P31 LipL45 levels were regulated. Hamsters infected with L. kirschneri formed an antibody response to LipL45, indicating that LipL45 was expressed during infection. Furthermore, the immunohistochemistry of kidneys from infected hamsters indicated that LipL45 was expressed by L. kirschneri that colonized the renal tubule. These observations suggest that expression of LipL45 responds to environmental cues, including those encountered during infection of a mammalian host.

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Section: Vol 70 2002supporting

confidence: 58%

Novel 45-Kilodalton Leptospiral Protein That Is Processed to a 31-Kilodalton Growth-Phase-Regulated Peripheral Membrane Protein

et al. 2002

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“…The mechanism of the synergistic effect is still unknown. But the role of penicillin is inhibition of peptidoglycan formation by binding to transpeptidases [42,43]. For γ-mangostin, it may work synergy with penicillin G in breakdown of bacterial membrane.…”

Section: Discussionmentioning

confidence: 99%

Antileptospiral activity of xanthones from Garcinia mangostanaand synergy of gamma-mangostin with penicillin G

Seesom

Jaratrungtawee

Suksamrarn

et al. 2013

BMC Complement Altern Med

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BackgroundLeptospirosis, one of the most widespread zoonotic infectious diseases worldwide, is caused by spirochetes bacteria of the genus Leptospira. The present study examined inhibitory activity of purified xanthones and crude extracts from Garcinia mangostana against both non-pathogenic and pathogenic leptospira. Synergy between γ-mangostin and penicillin G against leptospires was also determined.MethodsMinimal inhibitory concentrations (MIC) of crude extracts and purified xanthones from G. mangostana and penicillin G for a non-pathogenic (L. biflexa serovar Patoc) and pathogenic (L. interrogans serovar Bataviae, Autumnalis, Javanica and Saigon) leptospires were determined by using broth microdilution method and alamar blue. The synergy was evaluated by calculating the fractional inhibitory concentration (FIC) index.ResultsThe results of broth microdilution test demonstrated that the crude extract and purified xanthones from mangosteen possessed antileptospiral activities. The crude extracts were active against all five serovars of test leptospira with MICs ranging from 200 to ≥ 800 μg/ml. Among the crude extracts and purified xanthones, garcinone C was the most active compound against both of pathogenic (MIC =100 μg/ml) and non-pathogenic leptospira (MIC = 200 μg/ml). However, these MIC values were higher than those of traditional antibiotics. Combinations of γ-mangostin with penicillin G generated synergistic effect against L. interrogans serovars Bataviae, Autumnalis and Javanica (FIC = 0.52, 0.50, and 0.04, respectively) and no interaction against L. biflexa serovar Patoc (FIC =0.75). However, antagonistic activity (FIC = 4.03) was observed in L. interrogans serovar Saigon.ConclusionsCrude extracts and purified xanthones from fruit pericarp of G. mangostana with significant antibacterial activity may be used to control leptospirosis. The combination of xanthone with antibiotic enhances the antileptospiral efficacy.

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“…Other PC components that could potentially contaminate the leptospiral OM fractions are the endoflagella, and we have included immunoblots probed with antibody specific for endoflagellar sheath protein (40). Probing of immunoblots with antisera to these proteins is a more rapid and sensitive approach to detection of the PC than the use of radiolabeled or digoxigenin-labeled penicillin binding proteins (6,20).…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Leptospiral Outer Membrane and Description of Three Novel Leptospiral Membrane Proteins

2002

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The outer membrane (OM) of the mammalian pathogen Leptospira kirschneri was isolated in the form of membrane vesicles by alkaline plasmolysis and separated from the protoplasmic cylinder by sucrose density gradient ultracentrifugation. All four components of the alkaline plasmolysis buffer, including 1.0 M NaCl, 27% sucrose (wt/vol), 2 mM EDTA, and 10 mM Tris (pH 9), were required for efficient OM release, as judged by recovery of leptospiral lipopolysaccharide. Two populations of OM vesicles (OMVs) were recovered, with peak concentrations found in the sucrose gradient at densities of 1.16 and 1.18 g/ml. Transmission electron microscopy revealed that the more buoyant OMV population was smaller (<0.1 m in diameter) than the denser OMV population (0.2 to 0.3 m in diameter). The densities of both populations of OMVs were distinct from that of the protoplasmic-cylinder material, which was found in the sucrose gradient at a density of 1.20 g/ml. The OMV fractions were free of protoplasmic-cylinder material, as judged by immunoblotting with antibodies to the endoflagellar sheath protein, heat shock protein GroEL, and two novel cytoplasmic membrane proteins, lipoprotein LipL31 and transmembrane protein ImpL63. The protein components of the OMVs were characterized by one-and two-dimensional immunoblotting and found to include previously described OM proteins (OMPs), including the porin OmpL1; the lipoproteins LipL32, LipL36, and LipL41; and the peripheral membrane protein P31 LipL45 . A number of less well-characterized OMPs were also identified, including those with molecular masses of 16, 21, 21.5, 22, 31, 36, 44, 48, 90, and 116 kDa. The 48-kDa OMP was identified as a novel OM lipoprotein designated LipL48. The use of membrane-specific markers in OM isolation techniques facilitates an accurate description of the leptospiral OM and its components.Identification and characterization of outer membrane (OM) components are essential in the development of a molecular understanding of bacterial structure and function. Development of techniques for isolation of the OM from Leptospira species and other spirochetes has been difficult because of their unique architecture and a lack of sensitive markers for the cytoplasmic membrane. Like enteric gram-negative bacteria, spirochetes have both an OM and a cytoplasmic membrane, separated by a periplasmic space. However, spirochetal architecture differs significantly from that of gram-negative bacteria in that the peptidoglycan layer of spirochetes is associated with the cytoplasmic membrane rather than the OM (22,24). For this reason, techniques developed for isolation of the gram-negative OM from components of the underlying cell wall and cytoplasmic membrane are unlikely to be useful in spirochetes.Several approaches have been used in the isolation of the leptospiral OM, including sodium dodecyl sulfate (SDS) treatment of salt-altered cells (2,7,25,28), Sarkosyl (27), and Triton X-114 extraction and phase partitioning (20,45). On the basis of these approaches, three classes of lep...

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Penicillin-Binding Proteins in Leptospira interrogans

Cited by 15 publications

References 23 publications

Novel 45-Kilodalton Leptospiral Protein That Is Processed to a 31-Kilodalton Growth-Phase-Regulated Peripheral Membrane Protein

Novel 45-Kilodalton Leptospiral Protein That Is Processed to a 31-Kilodalton Growth-Phase-Regulated Peripheral Membrane Protein

Antileptospiral activity of xanthones from Garcinia mangostanaand synergy of gamma-mangostin with penicillin G

Characterization of the Leptospiral Outer Membrane and Description of Three Novel Leptospiral Membrane Proteins

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