ABSTRACT
Pathogenic Leptospira species adapt to a wide range of environmental conditions during disease transmission and infection. While the proteome of in vitro cultivated Leptospira has been characterized in several studies to date, relatively little is known of the proteome as expressed by Leptospira during disease processes. Isolates of Leptospira obtained from patients suffering the severe pulmonary form of leptospirosis cause acute lethal infection in guinea pigs and chronic asymptomatic infection in rats. Recent studies have demonstrated that protein and lipopolysaccharide constituents of Leptospira recovered from acutely infected guinea pig tissue differ from that of Leptospira in chronically infected rat tissue and in vitro cultivated Leptospira (J. E. Nally, E. Chow, M. C. Fishbein, D. R. Blanco, and M. A. Lovett, Infect. Immun. 73:3251-3260, 2005). In the current study, the proteome of Leptospira expressed during disease processes was characterized relative to that of in vitro cultivated Leptospira (IVCL) after enrichment for hydrophobic membrane proteins with Triton X-114. Protein samples were separated by two-dimensional gel electrophoresis, and antigens expressed during infection were identified by immunoblotting with monospecific antiserum and convalescent rat serum in addition to mass spectrometry. Results suggest a significant increase in the expression of the outer membrane protein Loa22 during acute infection of guinea pigs relative to other outer membrane proteins, whose expression is generally diminished relative to expression in IVCL. Significant amounts of LipL32 are also expressed by Leptospira during acute infection of guinea pigs.
Subject(s)
Bacterial Proteins/analysis , Leptospira interrogans/chemistry , Leptospirosis/microbiology , Membrane Proteins/analysis , Proteome/analysis , Adaptation, Physiological , Animals , Bacterial Proteins/isolation & purification , Disease Models, Animal , Electrophoresis, Gel, Two-Dimensional , Gene Expression Regulation, Bacterial , Guinea Pigs , Immunoblotting , Leptospira interrogans/genetics , Male , Mass Spectrometry , Membrane Proteins/isolation & purification , Proteome/isolation & purificationABSTRACT
Leptospira interrogans serovar Copenhageni strain RJ16441, a blood isolate from humans with the severe pulmonary form of leptospirosis, has previously been shown to cause fatal pulmonary hemorrhage in guinea pigs and asymptomatic chronic renal tubular colonization with urinary shedding in rats. In this study, RJ16441 caused lethal infection of both C3H/HeJ and C3H/SCID mice, but no hemorrhagic phenomena were observed.
Subject(s)
Bacteremia/microbiology , Kidney/microbiology , Leptospira interrogans/pathogenicity , Leptospirosis/pathology , Liver/microbiology , Animals , Bacteremia/pathology , Kidney/pathology , Leptospira interrogans/isolation & purification , Leptospirosis/microbiology , Liver/pathology , Mice , Mice, Inbred C3H , Mice, SCIDABSTRACT
Protection in experimental rabbit syphilis has been previously assessed by lesion development following intradermal challenge with Treponema pallidum. We have recently reported that passive immunization using monoclonal antibody M131 conveys partial protection as evidenced by significant lesion delays following intradermal challenge. To determine whether such delays in time to lesion appearance corresponded to decreases in the numbers of spirochetes, we used real-time PCR to quantitate T. pallidum genomic DNA copy numbers in lesion biopsies taken throughout the course of lesion development. Three groups of animals were given one prechallenge passive immunization with immune rabbit serum (IRS), M131, or control monoclonal antibody (CMAb) and then challenged with treponemal admixtures of IRS or monoclonal antibody in normal rabbit serum (NRS). As compared to the CMAb NRS controls, delays in the mean time to lesion appearance of 5.8 days for IRS and 8.8 days for M131 were observed. At the earliest time point (10 days postchallenge), real-time PCR showed a mean T. pallidum DNA copy number per mug of rabbit DNA in the CMAb NRS group of 7.65 x 10(3) copies, while no T. pallidum DNA could be detected in the M131 group. At approximately the mean time to lesion appearance in the IRS and M131 groups (17 and 20 days, respectively), the numbers of T. pallidum DNA copies were still 5- and 30-fold less, respectively, than those in the control group at these times. By 30 days postchallenge, the T. pallidum DNA copy numbers were similar in all three groups. These findings indicate that the delays in appearance of syphilitic lesions conferred by IRS and M131 corresponded to a marked decrease in treponemal numbers during the course of lesion development.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Immunization, Passive , Syphilis/immunology , Syphilis/prevention & control , Animals , Chemokines, CC , Disease Models, Animal , Gene Dosage , Polymerase Chain Reaction , Rabbits , Treponema pallidum/genetics , Treponema pallidum/immunology , Viral ProteinsABSTRACT
We have recently shown that a monoclonal antibody, designated M131, that binds a surface phosphorylcholine epitope on Treponema pallidum possesses complement-dependent killing activity and confers partial protection in rabbits following passive immunization (Blanco et al., 2005, Infect. Immun. 73:3083-3095). In this study, the protective potential of M131 was further tested using the rabbit skin protection assay of Titus and Weiser. Both M131 and infection-derived immune rabbit serum resulted in significant lesion delays corresponding to at least a 90% reduction of the treponemal challenge inoculum. The skin protection assay provides a way to assess the protective potential of specific immunogens while using far less antibody than in passive immunization protocols.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Antibody Specificity , Epitopes/immunology , Immunization, Passive/methods , Skin/immunology , Syphilis/prevention & control , Treponema pallidum/immunology , Animals , Antibodies, Bacterial/administration & dosage , Antibodies, Bacterial/immunology , Antibodies, Monoclonal/immunology , Antigens, Surface/immunology , Humans , Immune Sera/administration & dosage , Immune Sera/immunology , Male , Rabbits , Skin/microbiology , Skin/pathology , Syphilis/immunology , Syphilis/pathology , Treponema pallidum/pathogenicityABSTRACT
Leptospirosis is the most geographically widespread zoonotic disease in the world. A severe pulmonary form of leptospirosis (SPFL) is being recognized with increased frequency. We have reported that human SPFL isolates of Leptospira cause acute lethal infection with prominent pulmonary hemorrhage in guinea pigs. We have found that the same SPFL strains cause asymptomatic infection and chronic renal shedding in rats, where infection is restricted to the renal tubules. To address the antigenic composition of host tissue-derived Leptospira (HTL), motile leptospires were purified from guinea pig liver by centrifugation on Percoll density gradients and compared to Percoll-purified in vitro-cultivated Leptospira (IVCL). The lipopolysaccharide O antigen (Oag) content of guinea pig liver-derived HTL was markedly reduced compared to that of IVCL, as demonstrated both by immunoblotting with a monoclonal antibody that was serovar specific for Oag and by periodate-silver staining. Confocal microscopy of HTL in guinea pig liver and kidney with the Oag-specific monoclonal antibody provided further evidence that diminution of the Oag content occurred in situ during lethal infection. In contrast, the Oag content of HTL in chronically infected rat renal tubules was indistinguishable from that of IVCL. These findings suggest that there may be regulation of Oag synthesis by Leptospira specific to the animal host infected. The hypothesis that the Oag content is related to whether lethal infection or chronic renal tubular colonization occurs remains to be tested.
Subject(s)
Leptospira interrogans , Leptospirosis/immunology , O Antigens/analysis , Acute Disease , Animals , Chronic Disease , Guinea Pigs , Kidney/microbiology , Leptospirosis/microbiology , Leptospirosis/pathology , Lipopolysaccharides/analysis , Liver/microbiology , Male , Microscopy, Confocal , Rats , Rats, Sprague-DawleyABSTRACT
Immunization with purified Treponema pallidum outer membrane vesicles (OMV) has previously resulted in high-titer complement-dependent serum bactericidal activity. In this study, OMV immunization resulted in the isolation of a monoclonal antibody, M131, with complement-dependent killing activity. Passive immunization of rabbits with M131 administered intravenously conferred significant immunity demonstrated by the failure of syphilitic lesions to appear at 29% of intradermal challenge sites (7/24) and a mean delay of approximately 8 days to lesion appearance at the remaining sites (17/24). M131 not only bound to OMV and to the surfaces of intact motile T. pallidum cells but also bound to organisms whose outer membranes were removed, indicating both surface and subsurface locations for the killing target. This target was determined to be a T. pallidum lipid. Lipid extracted from T. pallidum and made into liposomes bound M131. Reverse-phase high-pressure liquid chromatography separation and fraction collection mass spectrometry (LC-MS+) of T. pallidum lipid showed that the target of M131 was phosphorylcholine. M131 binding required both liposome formation and a critical concentration of phospholipid containing phosphorylcholine, suggesting that the epitope has both a conformational and a compositional requirement. M131 did not react with red blood cells, which have phosphorylcholine-containing lipids in their exterior membrane leaflets, or with Venereal Disease Research Laboratory antigen that also contains phosphorylcholine, further indicating the specificity of M131. This is the first physical demonstration of an antigen on the T. pallidum surface and indication that such a surface antigen can be a target of immunity.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antigens, Surface/immunology , Phosphorylcholine/immunology , Syphilis/prevention & control , Treponema pallidum/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/metabolism , Antibody Specificity , Antigens, Surface/metabolism , Epitopes/immunology , Humans , Immunization, Passive , Mice , Mice, Inbred BALB C , Phosphorylcholine/metabolism , Polymerase Chain Reaction , Rabbits , Syphilis/immunology , Syphilis/microbiology , Treponema pallidum/geneticsABSTRACT
The severe pulmonary form of leptospirosis (SPFL) is an especially serious and rapid disease process characterized by alveolar hemorrhage and acute respiratory failure. The outer membrane of Leptospira facilitates direct interactions with the environs and likely contains important constituents involved during infection, transmission, survival, and adaptation to environmental conditions, including putative vaccinogen and diagnostic candidates. Outer membrane vesicles (OMVs) were purified by incubation in low-pH citrate buffer, treatment in a French press, and centrifugation over a continuous sucrose gradient. OMVs characterized by two-dimensional gel electrophoresis (2-DE) contained the previously described outer membrane proteins OmpL1, Qlp42, LipL32, LipL41, LipL36 and Loa22. In addition, unknown, hypothetical and putative outer membrane proteins were identified. High-performance liquid chromatography (HPLC) coupled with mass spectrometry and fraction collection (LC-MS+) measured the intact mass profile of the major outer membrane protein, LipL32, and the putative lipoprotein Qlp42. In contrast to a predicted molecularmass of 27,653.5 Da for LipL32 after cleavage of its signal peptide, intact mass proteomics measured the mass as ranging from 28,468 to 28,583 Da, consistent with lipidation of LipL32. In contrast to a predicted molecular mass of 39.8 kDa for Qlp42, the actual mass was measured as 24,811 and 26,461 Da consistent with a 30 kDa doublet observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and processing of the N-terminus of the mature protein. These studies indicate that purified OMVs are highly compatible with proteomics technologies including 2-DE and intact mass proteomics using LC-MS+ that facilitates definition of actual molecular masses of intact outer membrane proteins, and heterogeneity associated with them.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Leptospira interrogans/chemistry , Amino Acid Sequence , Bacterial Outer Membrane Proteins/isolation & purification , Centrifugation, Density Gradient , Chromatography, High Pressure Liquid , Electrophoresis, Gel, Two-Dimensional , Mass Spectrometry , Molecular Sequence Data , ProteomicsABSTRACT
The numbers of host-adapted Borrelia burgdorferi (HAB) organisms in rabbit skin were assessed by real-time PCR over the first 3 weeks of infection. Maximal numbers were found at day 11, while spirochete numbers decreased by more than 30-fold by day 21. The antigenic composition of HAB in skin biopsy samples was determined by use of a procedure termed hydrophobic antigen tissue Triton extraction. Immune sera from rabbits, sera from chronically infected mice, and monospecific antiserum to the antigenic variation protein, VlsE, were used to probe parallel two-dimensional immunoblots representing each time point. Individual proteins were identified using either specific antisera or by matching protein spots to mass spectrometry-identified protein spots from in vitro-cultivated Borrelia. There were significant changes in the relative expression of a variety of known and previously unrecognized HAB antigens during the 21-day period. OspC and the outer membrane proteins OspA and OspB were prominent at the earliest time point, day 5, when the antigenic variation protein VlsE was barely detected. OspA and OspB were not detected after day 5. OspC was not detected after day 9. VlsE was the most prominent antigen from day 7 through day 21. BmpA, ErpN, ErpP, LA7, OppA-2, DbpA, and an unidentified 15-kDa protein were also detected from day 7 through day 21. Immunoblot analysis using monospecific anti-VlsE revealed the presence of prominent distinct VlsE lower forms in HAB at days 9, 11, and 14; however, these lower forms were no longer detected at day 21. This marked diminution in VlsE lower forms paralleled the clearance of the spirochete from skin.
Subject(s)
Antigens, Bacterial/analysis , Borrelia burgdorferi/immunology , Lyme Disease/microbiology , Animals , Antigens, Bacterial/immunology , Bacterial Proteins/analysis , Hydrophobic and Hydrophilic Interactions , Immune Sera/immunology , Lipoproteins/analysis , Lyme Disease/immunology , Male , Mice , Rabbits , Skin/microbiology , Time FactorsABSTRACT
Human patients suffering from leptospirosis present with a diverse array of clinical manifestations, including the more severe and often fatal pulmonary form of the disease. The etiology of pulmonary hemorrhage is unclear. Isolates of Leptospira acquired from patients suffering from pulmonary hemorrhage were used to develop a guinea pig model of pulmonary hemorrhage. Gross findings post-infection confirmed extensive hemorrhage in the lungs and on peritoneal surfaces as the likely cause of death. Immunohistochemistry confirmed the presence of large numbers of leptospires in kidney, liver, intestinal tissues, and spleen, but few inflammatory cells were seen. In marked contrast, few leptospires were detected in infected hemorrhagic lung tissue. Blood chemistries and hematology did not reveal the etiology of the hemorrhage observed. There was no chemical or microscopic evidence for disseminated intravascular coagulation. To ascertain an immunopathologic role during disease, immunofluorescence was performed on infected lung tissues and confirmed the presence of IgM, IgG, IgA, and C3 along the alveolar basement membrane. This suggests that an autoimmune process may be the etiology of fatal pulmonary hemorrhage in leptospirosis.
Subject(s)
Autoimmune Diseases/complications , Disease Models, Animal , Hemorrhage/etiology , Leptospirosis/immunology , Pulmonary Alveoli/pathology , Animals , Complement System Proteins/immunology , Complement System Proteins/metabolism , Guinea Pigs , Hemorrhage/pathology , Immunoglobulins/immunology , Immunoglobulins/metabolism , Immunohistochemistry , Intestines/immunology , Intestines/microbiology , Intestines/pathology , Kidney/immunology , Kidney/microbiology , Kidney/pathology , Leptospira/immunology , Leptospirosis/pathology , Liver/immunology , Liver/microbiology , Liver/pathology , Lung Diseases/immunology , Lung Diseases/microbiology , Lung Diseases/pathology , Male , Microscopy, Electron , Pulmonary Alveoli/immunology , Pulmonary Alveoli/ultrastructure , Spleen/immunology , Spleen/microbiology , Spleen/pathologyABSTRACT
The general concept that during infection of mice the Borrelia burgdorferi surface protein composition differs profoundly from that of tick-borne or in vitro-cultivated spirochetes is well established. Specific knowledge concerning the differences is limited because the small numbers of spirochetes present in tissue have not been amenable to direct compositional analysis. In this report we describe novel means for studying the antigenic composition of host-adapted Borrelia (HAB). The detergent Triton X-114 was used to extract the detergent-phase HAB proteins from mouse ears, ankles, knees, and hearts. Immunoblot analysis revealed a profile distinct from that of in vitro-cultivated Borrelia (IVCB). OspA and OspB were not found in the tissues of SCID mice 17 days after infection. The amounts of antigenic variation protein VlsE and the relative amounts of its transcripts were markedly increased in ear, ankle, and knee tissues but not in heart tissue. VlsE existed as isoforms having both different unit sizes and discrete lower molecular masses. The hydrophobic smaller forms of VlsE were also found in IVCB. The amounts of the surface protein (OspC) and the decorin binding protein (DbpA) were increased in ear, ankle, knee, and heart tissues, as were the relative amounts of their transcripts. Along with these findings regarding VlsE, OspC, and DbpA, two-dimensional immunoblot analysis with immune sera also revealed additional details of the antigenic composition of HAB extracted from ear, heart, and joint tissues. A variety of novel antigens, including antigens with molecular masses of 65 and 30 kDa, were found to be upregulated in mouse tissues. Extraction of hydrophobic B. burgdorferi antigens from tissue provides a powerful tool for determining the antigenic composition of HAB.