Mao Yf

Leptospira interrogans Induces Apoptosis in Macrophages via Caspase-8- and Caspase-3-Dependent Pathways

ABSTRACT Apoptosis of host cells plays an important role in modulating the pathogenesis of many infectious diseases. It has been reported that Leptospira interrogans, the causal agent of leptospirosis, induces apoptosis in macrophages and hepatocytes. However, the molecular mechanisms responsible for host cell death remained largely unknown. Here we demonstrate that L. interrogans induced apoptosis in a macrophage-like cell line, J774A.1, and primary murine macrophages in a time- and dose-dependent manner. Apoptosis was associated with the activation of cysteine aspartic acid-specific proteases (caspase-3, caspase-6, and caspase-8), the increased expression of Fas-associated death domain (FADD), and the cleavage of the caspase substrates poly(ADP-ribose) polymerase (PARP) and nuclear lamina protein (lamin A and lamin C). Caspase-9 was activated to a lesser extent, whereas no release of cytochrome c from mitochondria was detectable. Inhibition of caspase-8 impaired L. interrogans-induced caspase-3 and -6 activation, as well as PARP and lamin A/C cleavage and apoptosis, suggesting that apoptosis is initiated via caspase-8 activation. Furthermore, caspase-3 was required for the activation of caspase-6 and seemed to be involved in caspase-9 activation through a feedback amplification loop. These data indicate that L. interrogans-induced apoptosis in macrophages is mediated by caspase-3 and -6 activation through a FADD-caspase-8-dependent pathway, independently of mitochondrial cytochrome c-caspase-9-dependent signaling.

Characterization of the ompL1 gene of pathogenic Leptospira species in China and cross-immunogenicity of the OmpL1 protein

BackgroundThe usefulness of available vaccine and serological tests for leptospirosis is limited by the low cross-reactivity of antigens from numerous serovars of pathogenic Leptospira spp. Identification of genus-specific protein antigens (GP-Ag) of Leptospira would be important for development of universal vaccines and serodiagnostic methods. OmpL1, a transmembrane porin of pathogenic leptospires, was identified as a possible GP-Ag, but its sequence diversity and immune cross-reactivity among different serovars of pathogenic leptospires remains largely unknown.ResultsPCR analysis demonstrated that the ompL1 gene existed in all 15 official Chinese standard strains as well as 163 clinical strains of pathogenic leptospires isolated in China. In the standard strains, the ompL1 gene could be divided into three groups (ompL1/1, ompL1/2 and ompL1/3) according to their sequence identities. Immune electron microscopy demonstrated that all products of the different gene types of ompL1 are located on the surface of leptospires. The microscopic agglutination test revealed extensive yet distinct cross-immunoagglutination among the antisera against recombinant OmpL1 (rOmpL1) and leptospiral strains belonging to different ompL1 gene types. These cross-immunoreactions were further verified by ELISAs using the OmpL1 proteins as the coated antigens in serum samples from 385 leptospirosis patients. All the antisera against rOmpL1 proteins could inhibit L. interrogans strain Lai from adhering to J774A.1 cells. Furthermore, immunization of guinea pigs with each of the rOmpL1 proteins could cause cross-immunoprotection against lethal challenge with leptospires from different ompL1 gene types.ConclusionThree types of the ompL1 gene are present in pathogenic leptospires in China. OmpL1 is an immunoprotective GP-Ag which should be considered in the design of new universal vaccines and serodiagnostic methods against leptospirosis.

Pathogenesis of leptospirosis: interaction of Leptospira interrogans with in vitro cultured mammalian cells

Interactions of virulent Leptospira interrogans with murine monocyte-macrophage-like J774A.1 cells and Vero (African green monkey kidney fibroblasts) cells from attachment to internalization were investigated by a series of morphological analysis. Fontana silver staining revealed that only the pathogenic leptospires were able to attach to host cells and the attachment pattern varied depending on cell types that they interacted with. Transmission electron microscopy (TEM) analysis confirmed the formation of the leptospires-containing phagosomes and their colocalization with lysosomes in macrophages were verified by confocal microscopic analysis. Results of F-actin rearrangements examination indicated that virulent leptospires invaded host cells via a microfilament-independent pathway.

Protein typing of major outer membrane lipoproteins from Chinese pathogenic Leptospira spp. and characterization of their immunogenicity

Leptospirosis, caused by different Leptospira species, is one of the most widespread zoonotic infections worldwide. Here we expressed three major leptospiral lipoproteins and examined their immunogenicity. All the pathogenic Leptospira strains tested possess the lipL21, lipL32 and lipL41 genes, but the latter two can be further divided into different gene types (lipL32-1, lipL32-2, lipL41-1, lipL41-2). Microscopic agglutination test revealed that rLipLs antisera had extensive cross-immunoagglutination among the 178 leptospiral strains in which rLipL32-1 contributed the highest agglutination titer. The rLipLs-based ELISAs established in this study demonstrated that in the sera of 385 leptospirosis patients infected with different serovars of Leptospira interrogans, rLipL32-1 had the highest positive rates for IgG and IgM (89.4-98.7%), followed by the IgG/IgM positive rates of rLipL21 (87.0-96.1%) and rLipL32-2 (86.5-96.9%), while the two rLipL41s presented the lowest IgG/IgM positive rates (69.9-83.9%). The immunoprotective levels in guinea pigs of rLipL32-1 (58.3% and 66.7%) were the highest, compared to those of the other rLipLs (25.0-58.3%). Multiple different rLipLs would increase immunoprotective levels (from 58.3% and 66.7% to 83.3% and 91.7%). The data suggest that all the rLipLs are the genus-specific superficial antigens of pathogenic Leptospira species and should be considered in designing universal vaccines against leptospirosis.

Identification of immunodominant B- and T-cell combined epitopes in outer membrane lipoproteins LipL32 and LipL21 of Leptospira interrogans.

ABSTRACT Leptospirosis is a serious infectious disease caused by pathogenic Leptospira. B- and T-cell-mediated immune responses contribute to the mechanisms of Leptospira interrogans infection and immune intervention. LipL32 and LipL21 are the conserved outer membrane lipoproteins of L. interrogans and are considered vaccine candidates. In this study, we identified B- and T-cell combined epitopes within LipL32 and LipL21 to further develop a novel vaccine. By using a computer prediction algorithm, two B- and T-cell combined epitopes of LipL21 and four of LipL32 were predicted. All of the predicted epitopes were expressed in a phage display system. Four epitopes, LipL21 residues 97 to 112 and 176 to 184 (LipL2197-112 and LipL21176-184, respectively) and LipL32133-160 and LipL32221-247 of LipL32 were selected as antigens by Western blotting and enzyme-linked immunosorbent assay. These selected epitopes were also recognized by CD4+ T lymphocytes derived from LipL21- or LipL32-immunized BALB/c (H-2d) mice and mainly polarized the immune response toward a Th1 phenotype. The identification of epitopes that have both B- and T-cell immune reactivities is of value for studying the immune mechanisms in response to leptospiral infection and for designing an effective vaccine for leptospirosis.

Sensitive and specific ELISA coated by TpN15-TpN17-TpN47 fusion protein for detection of antibodies to Treponema pallidum.

Abstract Background: We constructed an artificial fusion gene tpN15-17-47 and then used the prokaryotic expression fusion protein rTpN15-17-47 as the coated antigen to establish a new enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of syphilis. Methods: tpN15, tpN17, and tpN47 genes were amplified separately by polymerase chain reaction (PCR) and then assembled into a fusion gene coding a trigeminy protein antigen by primer linking PCR. The target recombinant protein antigens rTpN15, rTpN17, rTpN-47, and rTpN15-17-47 were expressed and then purified antigens were immobilized on the surface of microplate wells for detecting Treponema pallidum-specific antibodies by ELISAs. Results: The relative positive rate of rTpN15-17-47-ELISA in 965 serum specimens of syphilis patients was 99.5%, which was higher than that of the T. pallidum hemagglutination assay (TPHA) (98.3%) (p<0.05) and much higher than that of the rTpN15-ELISA (83.1%), the rTpN17-ELISA (84.4%), the rTpN47-ELISA (82.1%), and the toluidine red unheated serum test (TRUST) (72.2%) (p<0.01). All the ELISAs and the TPHA in detecting serum specimens from 62 cases with systemic lupus erythematosus (SLE), 86 cases with rheumatic arthritis (RA), and 250 healthy cases were negative, but the TRUST was positive in five cases with SLE, seven cases with RA, and two healthy cases. Conclusions: The rTpN15-17-47-ELISA is a sensitive and specific serological screening or a diagnostic method for syphilis in the clinical setting. Clin Chem Lab Med 2009;47:321–6.

Recombinant SpaO and H1a as immunogens for protection of mice from lethal infection with Salmonella paratyphi A: implications for rational design of typhoid fever vaccines

The Vi capsular polysaccharide vaccine is one of two vaccines against typhoid recommended worldwide and is the vaccine generally used in China. However, in recent years a Salmonella paratyphi A strain that is naturally devoid of capsule has caused frequent outbreaks of typhoid fever in Southern China, leading to the need for identification of additional antigens that could be incorporated into new vaccines. SpaO acts as a major invasion factor of Salmonella enterica spp. and H1a is the unique flagellin subunit ofS. paratyphi A. In this study, the two prokaryotic recombinant antigens, rSpaO and rH1a, were expressed and their immunogenicity was demonstrated by the slide agglutination test and Western blot assays. Using PCR and sequencing analysis as well as ELISA, we find that the spaO and h1a genes are widely distributed in 196 S. paratyphi A isolates (97.5 and 100%, respectively), with high expression frequencies for the SpaO (98.0%) and H1a (100%) antigens. The two genes also show high sequence conservation (similarities from 99.31 to 99.88% for both genes). In sera from 172 paratyphoid A patients, anti-SpaO and anti-H1a IgGs were detectable by ELISA, in 94.8 and 98.8% of patients, respectively. Furthermore, 41.7-66.7% of mice immunized with rSpaO or rH1a alone were protected against subsequent infection, and the protection rate rose to 75.0-91.7% in mice co-immunized with the two antigens. As the spaO and h1a genes of S. paratyphi A are sequence conserved, extensively distributed and highly expressed, the rSpaO and rH1a immunogens should be considered in the development of novel vaccines to prevent S. paratyphi A-caused typhoid fever.

Predominant porB1A and porB1B genotypes and correlation of gene mutations with drug resistance in Neisseria gonorrhoeae isolates in Eastern China

BackgroundVariations of porB1A and porB1B genes and their serotypes exist in Neisseria gonorrhoeae isolates from different geographical areas, and some site mutations in the porB1B gene correlate with drug resistance.MethodsThe β-lactamase production of N. gonorrhoeae isolates was determined by paper acidometric test and nitrocefin discs. The porB1A and porB1B genes of 315 non-penicillinase-producting N. gonorrhoeae (non-PPNG) strains were amplified by PCR for sequencing to determine serotypes and site mutations. A duplex PCR was designed to simultaneously detect both porB1A and porB1B genes. Penicillin and tetracycline resistance was assessed by an in vitro drug sensitivity test.ResultsOf the N. gonorrhoeae isolates, 31.1% tested positive for porB1A and 68.9% for porB1B genes. All the 98 porB1A+ isolates belonging to IA6 serotype with either no mutation at the 120 and 121 sites (88.8%) or a D120G (11.2%) mutation and were no resistance to both penicillin and tetracycline. Among the 217 porB1B+ isolates, 26.7%, 22.6% and 11.5% belonged to IB3, IB3/6 and IB4 serotypes, respectively. Particularly, two novel chimeric serotypes, IB3/6-IB2 and IB2-IB4-IB2, were found in 77 and 8 porB1B+ isolates. Two hundred and twelve (97.7%) of the porB1B+ isolates were presented G120 and/or A121 mutations with 163 (76.9%) at both sites. Interestingly, within the 77 porB1B+ isolates belonging to IB3/6-IB2 serotype, 15 were discovered to possess novel deletions at both A121 and N122 sites. All the replacement mutations at these sites in PorB1B were correlated with resistance and the deletion mutation showed the highest resistance.ConclusionN. gonorrhoeae isolates circulating in Eastern China include a sole PorB1A serotype (IA6) and five PorB1B serotypes. Multiple mutations in porB1B genes, including novel A121 and N122 deletions, are correlated with high levels of penicillin and tetracycline resistance.