Trachoma and LGV biovars of Chlamydia trachomatis share the same glycosaminoglycan-dependent mechanism for infection of eukaryotic cells

A sulphated glycosaminoglycan-dependent mechanism of microbial infection for mammailan cells was characterized for the Chlamydia trachomatis trachoma and lymphogranuloma venereum (LGV) biovars. We demonstrated that the trachoma and LGV biovars compete for the same receptor(s) on host cells and that their infectivity was inhibited by heparin or heparan sulphate. Using a specific heparan suiphate lyase (heparitinase) to treat organisms, the Infectivity of both biovars was abolished. Furthermore, exogenous heparan sulphate rescued chlamydial infectivity following treatment with heparitinase and the restored infectivity was neutralized by an anti-heparan sulphate monoclonal antibody. These data suggest that heparan sulphate-like-mediated Interactions between C. trachomatis and eukaryotic cells are essential for infectivity.     

Physiological studies on the utilization of oleic acid by Saccharomyces cerevisiae in relation to microbody development

Twenty one Chlamydia trachomatis reference strains and 40 clinical isolates belonging to the lymphogranuloma venerum (LGV) and trachoma biovars were genotyped by differential restriction mapping of the major-outer-membrane-protein gene (MOMP) obtained by the polymerase-chain reaction (PCR). AluI digestion of the PCR product distinguishes eight MOMP-genotypes corresponding to 8 serovars. Six additional enzymes (NlaIII, CfoI, EcoRI, HinfI, DdeI and FokI) further permit the discrimination of 10 MOMP-genotypes corresponding to the 10 remaining serovars of the species. AluI alone allows direct typing of 78% of the clinical isolates. AluI digestion patterns of mouse C. trachomatis biovar, a C. pneumoniae and two C. psittaci strains, studied for comparison, were clearly distinguishable from one another and from the C. trachomatis LGV and trachoma strains. These results indicate that MOMP genotyping by PCR is a valuable molecular tool for studying C. trachomatis epidemiology.     

Diversity of the Chlamydia trachomatis common plasmid in biovars with different pathogenicity

The 7.5-kb plasmid of Chlamydia trachomatis (CT) is believed to encode essential genes and might have a role in CT pathogenicity. Accordingly, analysis of plasmid-linked mutations in isolates from biovars with different pathogenic properties should help in identifying which plasmid-encoded genes, if any, may be involved in modulating virulence. For this purpose, the plasmid present in a low-virulence isolate (trachoma biovar, serotype D) was cloned and sequenced. Nucleotide changes were experimentally checked against the sequence of the plasmid variant from the highly virulent strain L2/434/Bu (LGV biovar). By aligning our data with two published sequences of different trachoma and LGV variants a general consensus structure was determined, comprising eight major open reading frames (ORF) and a number of points where there is consensus only between isolates of the same biovar (biovar-specific mutations). The degree of variation between different isolates is less than 1%. In particular, comparison of serotype-D and -L2 plasmids shows mutations which are generally silent or lead to few (one to four), often conservative, amino acid changes in ORFs 1, 2, 4, 5, 6, and 7. The protein encoded by ORF8 is completely conserved. In contrast, the polypeptide variants encoded by ORF3 show nine amino acid changes, seven of which are due to biovar-specific mutations.     

Chlamydia trachomatis Mip-like protein

A 27 kDa Chlamydia trachomatis Mip-like protein with homology of a 175-amino-acid C-terminal fragment to the surface-exposed Legionella pneumophila mip-gene product has previously been described. In this paper the entire chlamydia Mip-like sequence of C. trachomatis serovar L2 (lymphogranuloma venereum (LGV) biovar) is presented. The sequence shows high similarity to the legionella Mip protein and its C-terminal region, like that of the legionella Mip, has high amino acid similarity to eukaryotic and prokaryotic FK506-binding proteins. The chlamydial mip-like gene was detected by polymerase chain reaction (PCR) in other C. trachomatis serovars and by sequencing of the mip-like genes of serovars B and E (trachoma biovar) was shown to be highly conserved within the two major biovars of C. trachomatis. Monoclonal and polyclonal antibodies raised against the recombinant Mip-like protein failed to demonstrate surface-exposed epitopes on infectious elementary bodies or reproductive reticulate body forms either by immunofluorescence or immuno-gold electron microscopy. However, a complement-dependent inhibition of up to 91% of infectivity for cell cultures was observed with antibodies to the N-terminal fragment of the Mip-like protein suggesting that antibody-accessible epitopes are present on infectious EBs.     

Effects of human peripheral blood monocytes,monocyte-derived macrophages,and spleen mononuclear phagocytes on Toxoplasma gondii

In vitro effects of human peripheral blood monocytes, peripheral blood monocyte-derived macrophages, and spleen mononuclear phagocytes on Toxoplasma gondii were studied. In almost all instances, over 80% of human monocytes and monocyte-derived macrophages infected with Toxoplasma in vitro destroyed the organism. Degeneration of intracellular Toxoplasma was not due to decreased viability of organisms in the challenge inoculum. Human monocytes did not elaborate into the culture medium substances which altered the capacity of Toxoplasma to survive and replicate within mouse macrophages. The early reduction in intracellular Toxoplasma was not affected by inhibitors of various intracellular processes or by diseases associated with altered cellular immunity (sarcoidosis, infectious mononucleosis, or lymphoma.) The Toxoplasma that remained after 6 hr within human monocytes and macrophages multiplied. This multiplication was observed both microscopically and in a radioassay which detects uptake of [³H]uracil or [³H]deoxyuridine into nucleic acids of intracellular Toxoplasma. Intracellular Toxoplasma in monocytes cultured with poly(I:C) or in monocyte-derived macrophages cultured with lymphokines showed decreased uptake of radiolabeled precursors into nucleic acids of intracellular Toxoplasma. Treatment of monocytes with endotoxin did not alter nucleic acid synthesis of surviving intracellular Toxoplasma. These results suggest that human mononuclear phagocytes in peripheral blood and in tissue (spleen) have the capacity to eliminate a large percentage of the Toxoplasma that they ingest or that invade them. The inhibition of nucleic acid synthesis of remaining Toxoplasma by exposure of monocyte-derived macrophages to lymphokines suggests that lymphocyte products may be important for elimination of the Toxoplasma that remain and multiply within a small proportion of mononuclear phagocytes.     

Cysteine-rich outer membrane proteins of Chlamydia trachomatis display compensatory sequence changes between biovariants

Two cysteine-rich proteins of Chlamydia trachomatis are essential structural components of the unique outer membrane of the infectious elementary body. These 58,000 (outer membrane protein 2; OMP2) and 15,000 (OMP3) proteins also differ structurally and chemically between biovariants that differ in invasive capability. We have identified the gene for OMP3 and sequenced both trachoma and lymphogranuloma venereum (LGV) omp3 genes. We have previously sequenced omp2 from the LGV biovar and now describe the omp2 sequence for a trachoma biovariant. Amino acid sequence differences between biovariants were few but, significantly, these changes have altered the charge of both OMP2 and OMP3 such that the net charge of each protein differs between biovariants. These compensatory charge alterations have implications for the outer membrane organization of these proteins. In addition, examination of the OMP3 sequence suggests that OMP3 may be a lipoprotein.     

The mediator of cellular immunity. X. Interaction of macrophages and specifically sensitized lymphocytes.

Specifically sensitized lymphocytes have a focusing influence on mononuclear phagocytes that is expressed in the local accumulation and division of macrophages in bacteria-induced exudates. This was demonstrated by injecting Listeria monocytogenes into the peritoneal cavity of normal rats immediately before the animals were transfused with thoracic duct lymphocytes from either Listeria-immune donors or donors that had been infected with the unrelated parasite, Francisella tularensis. Sensitized lymphocytes originally present in the intravenous inocula were found later in the exudates. The arrival in the inflamed peritoneal cavity of specifically sensitized lymphocytes was associated with an exuberant influx of newly formed host cells and a local proliferative response that involved both immunoblasts and macrophages. These cytokinetic observations provide a plausible explanation of the delayed inflammatory response induced by the parasite, and imply that sensitized lymphocytes contribute to the host's defence by encouraging the prompt and purposeful deployment of monocyte-derived macrophages in centers of infection. In addition to their focusing influence on mononuclear phagocytes, specifically sensitized lymphocytes or their products can enhance the metabolic and microbicidal activity of macrophages. This activation process was revealed in the ability of rats infected with L. monocytogenes or BCG to control the growth of F. tularensis at a challenge site in the testis. But resistance was expressed only when the challenge organisms were injected with killed bacteria against which the recipients had been specifically immunized. The results accord with the view that macrophages are functionally activated by an immunological mechanism, and imply that the process is triggered locally by sensitized lymphocytes which are recruited from the blood.     

沙眼衣原体血清型L2体外培养影响因素的研究

研究沙眼衣原体（Chlamydia trachomatis,Ct）血清型LGV L2在体外培养的繁殖规律及其影响因素,确定血清型LGV L2在体外培养的最佳生长发育条件。用沙眼衣原体血清型LGV L2分别感染HEp-2细胞、HeLa229细胞、HepG-2细胞、SGC-7901细胞和Vero细胞,在荧光显微镜下计数包涵体形成单位（inclusion fig-ure unity,IFU）和观察培养不同时间后包涵体的形态,比较不同细胞对血清型LGV L2的敏感性及血清型LGVL2在不同细胞内的生长情况。同时分别设DEAE葡聚糖处理组与未处理组,含放线菌酮培养组与不含放线菌酮培养组,比较培养12、24、36和48 h后血清型LGV L2包涵体形态、IFU和Real-Time PCR定量检测血清型LGV L2的核酸量,判断DEAE葡聚糖和放线菌酮对沙眼衣原体血清型LGV L2生长的影响。在感染20 h后,显微镜下观察发现HEp-2、Vero、HepG-2、HeLa和SGC-7901细胞均不同程度肿胀,5种细胞内均可见包涵体,大约40~48 h后包涵体占据整个胞浆。IFU计数和Real-Time PCR结果显示5种细胞中HeLa细胞感染率最高,HepG-2细胞感染率最低,血清型LGV L2在HeLa细胞中生长速度最快。荧光显微镜下计数IFU,发现DE-AE葡聚糖预处理组和对照组中血清型LGV L2的感染率和生长发育没有明显区别,而含放线菌酮培养组中各细胞内血清型LGV L2生长速度较对照组快,Real-Time PCR检测结果显示放线菌酮组各细胞内血清型LGVL2核酸量较对照组高。血清型LGV L2在HeLa细胞中的感染率最高,DEAE葡聚糖对血清型LGV L2的感染没有明显影响,而体外培养时添加放线菌酮有利于血清型LGV L2的生长发育。     

溶脲脲原体及其两个生物群与非淋菌性尿道炎相关性的研究

目的 阐明溶脲脲原体及其2个生物群与非淋菌性尿道炎的关系。方法 使用通用引物-PCR-毛细管电泳法对淋菌性尿道炎组,非淋菌性尿道炎组和对照组中的溶脲脲原体的2个生物群进行检测。结果 溶脲脲原体生物群2在非淋菌性尿道炎中的检出率高于对照组（P〈0．05）,溶脲脲原体生物群1在淋菌性尿道炎中的检出率低于对照组（P〈0．05）,而在非淋菌性尿道炎和对照组中,溶脲脲原体生物群1的检出率差异无显著性（P〉0．05）。结论 溶脲脲原体生物群2是和非淋菌性尿道炎有一定关系的,溶脲脲原体生物群2可能才是引起非淋尊性尿道炎的病原体之一,而生物群1不引起非淋菌性尿道炎,淋球菌的增殖有可能抑制尿道中的溶脲脲原体生物群1的生长。     

Production of penicillinase by staphylococcal strains of different origins

A total of 319 strains of S. aureus and 729 strains of S. epidermidis belonging to different biovars isolated from the skin and nasal mucosa of 349 persons representing 8 independent groups were tested. On the whole production of penicillinase was more often observed in the strains of S. aureus than in the strains of S. epidermidis. Within the first species this property was more often detected in the strains of biovar I as compared to the other biovars. However, the frequency of the penicillinase-producing strains within S. aureus and the biovars of S. epidermidis markedly varied.     

Antigenic specificity of human antibody to chlamydia in trachoma and lymphogranuloma venereum

An understanding of the molecular basis of the humoral immune response to chlamydial infections in man requires the identification of target antigens to which antibodies are directed. The antigenic specificity of antibody from patients with lymphogranuloma venereum (LGV) or trachoma was therefore assessed by Western blotting. Surface polypeptides were first identified using purified chlamydial outer membrane complex as antigen. Antibodies in sera from patients with LGV but not from control negative sera reacted with a wide range of chlamydial surface polypeptides with molecular masses of 19, 29, 41, 58, 63 and 65 kDa. The major component of the antibody response detected by both immunoblotting and immunoprecipitation assay was directed against the major outer membrane protein (MOMP). Antibody to MOMP was species-specific on Western blotting, whereas antibody to several other polypeptides recognized common immunodeterminants on polypeptides of C. psittaci Cal-10 of equivalent molecular mass. Immunologically C. psittaci Cal-10 was more closely related to LGV strains of C. trachomatis than a guinea pig inclusion conjunctivitis strain of C. psittaci. Trachoma sera collected from a village in southern Iran showed predominantly type-specific antibody on micro-immunofluorescence to serotype A or B trachoma agents. These sera showed a weak immune response to MOMP, a pronounced response to a polypeptide of 36 kDa and much less widespread reactivity with other chlamydial polypeptides. The lack of an immune response to SDS-stable immunodeterminants on MOMP might contribute to the susceptibility of trachoma patients to repeated cycles of ocular infection with chlamydiae.     

Vimentin expressed on Mycobacterium tuberculosis-infected human monocytes is involved in binding to the NKp46 receptor

We previously showed that human NK cells used the NKp46 receptor to lyse Mycobacterium tuberculosis H37Ra-infected monocytes. To identify ligands on H37Ra-infected human mononuclear phagocytes, we used anti-NKp46 to immunoprecipitate NKp46 from NK cells bound to its ligand(s) on H37Ra-infected monocytes. Mass spectrometry analysis identified a 57-kDa molecule, vimentin, as a putative ligand for NKp46. Vimentin expression was significantly up-regulated on the surface of infected monocytes, compared with uninfected cells, and this was confirmed by fluorescence microscopy. Anti-vimentin antiserum inhibited NK cell lysis of infected monocytes, whereas antiserum to actin, another filamentous protein, did not. CHO-K1 cells transfected with a vimentin construct were lysed much more efficiently by NK cells than cells transfected with a control plasmid. This lysis was inhibited by mAb-mediated masking of NKp46 (on NK cells) or vimentin (on infected monocytes). ELISA and Far Western blotting showed that recombinant vimentin bound to a NKp46 fusion protein. These results indicate that vimentin is involved in binding of NKp46 to M. tuberculosis H37Ra-infected mononuclear phagocytes.     

Species of Pseudomonas obtained at 7°C and 30°C during aerobic storage of lamb carcasses

A total of 268 strains of Pseudomonas isolated during storage life of lamb carcasses was identified to species level. One-hundred and thirteen strains obtained at 30°C were Ps.fragi (51), Ps. lundensis (17), Ps. fluorescens biovars I (10), III (9) and VI (1), Ps. putida biovar A (8 strains) and unidentified (17 strains). Species and biovars isolated at 7°C (155) were Ps. fragi (101), Ps. lundensis (32), Ps. fluorescens biovar I (6), Ps. putida biovar A (8) and unidentified (8). Numerical analysis (82% S _SM, UPGMA) of 'psychrotrophic' and 'mesophilic' strains resulted in the formation of nine and eight clusters respectively. The dendrograms obtained exhibited similar structures. Most of the strains of Ps. lundensis and Ps. fragi clustered together. Strains of this latter species also joined the type strain of Ps. testosteroni and appeared included with phenons containing the Ps. putida strains. There were clusters made up exclusively of strains assigned to one biovar or group ( Ps. fluorescens biovars I and II and unidentified). A high level of similarity was observed between clusters of Ps. fluorescens biovar I and those containing the Ps. fragi-Ps. lundensis complex (>74% S _SM) and Ps. lundensis (>80%). The recovery of pseudomonads seemed to be affected by the sampling day.     

Transfer of Eimeria nieschulzi using extraintestinal tissue

The issue of extraintestinal infection by Eimeria nieschulzi in the rat was addressed by transferring various tissues from infected to uninfected rats by mouth. All 6 rats receiving liver, spleen, or small intestine from rats killed at 3 or 8 hr postinoculation (PI), and all 5 rats receiving spleen and small intestine from rats killed 8 days PI, showed infections. Rats receiving tissues from rats killed at 8 days PI showed infections 24 hr later, indicating that fourth-generation merozoites were transferred. This is the first demonstration of an extraintestinal rodent eimerian.     

The intracellular survival and growth of gonococci in human phagocytes.

In reassessment of previous tests for intracellular survival, results have been confirmed and additional evidence obtained indicating that some gonococci can survive and multiply in human phagocytes. Use was made of the ability of penicillin to penetrate phagocytes and to kill only actively growing organisms. In microscopic counts on 33 urethral exudate smears, an average of 49% of gonococci were associated with polymorphonuclear phagocytes. The organisms were unevenly distributed amongst the phagocytes, with most cells uninfected and some containing large numbers. Many phagocytes also remained uninfected in tests in vitro with low gonococcal inocula although experiments with large inocula showed that most phagocytes could ingest gonococci. It is proposed that ingestion of one gonococcus may stimulate the phagocytes to take up more. Phagocytes were killed and disintegrated after ingesting large numbers of gonococci and similar effect in vivo may be responsible for the large clumps of organisms seen in urethral exudate. These results underline the probable importance in the pathogenesis of gonorrhoea of intracellular survival in phagocytes.     

Genome Sequence of Brucella melitensis Strain 128, an Isolate of Biovar 3 of Sequence Type 8

Brucella melitensis is the most common Brucella species causing human brucellosis. B. melitensis is divided into 3 biovars. Here, we report the complete genome sequence of B. melitensis strain 128, a strain of biovar 3 of sequence type 8, which is prevalent in China.     

Cytotoxicity of Neisseria gonorrhoeae for human peripheral blood phagocytes

The toxicity of gonococci [strain BS4 (agar)] for human peripheral blood polymorphonuclear phagocytes, infected in vitro, was assessed by light microscopic examination of Giemsa stained cell deposits of polymorphonuclear phagocytes which had ingested these bacteria. The cytotoxicity elicited by viable gonococci, assessed by percentage lysis and concomitant reduction in the number of polymorphonuclear phagocytes increased as the ratio of gonococci to phagocytes in the original suspension mixture was raised. Pretreatment of viable gonococci with antiserum raised to whole organisms increased the cytotoxic effect produced by the organisms. Killed (heat or UV irradiation) gonococci caused little or no cytotoxicity, even when the organisms were pretreated with specific antiserum. Hence, the lysis of polymorphonuclear phagocytes appears to be caused by a factor or factors produced by viable gonococci and not by LPS per se.     

Species of Pseudomonas obtained at 7 degrees C and 30 degrees C during aerobic storage of lamb carcasses.

A total of 268 strains of Pseudomonas isolated during storage life of lamb carcasses was identified to species level. One-hundred and thirteen strains obtained at 30 degrees C were Ps. fragi (51), Ps. lundensis (17), Ps. fluorescens biovars I (10), III (9) and VI (1), Ps. putida biovar A (8 strains) and unidentified (17 strains). Species and biovars isolated at 7 degrees C (155) were Ps. fragi (101), Ps. lundensis (32), Ps. fluorescens biovar I (6), Ps. putida biovar A (8) and unidentified (8). Numerical analysis (82% SSM, UPGMA) of 'psychrotrophic' and 'mesophilic' strains resulted in the formation of nine and eight clusters respectively. The dendrograms obtained exhibited similar structures. Most of the strains of Ps. lundensis and Ps. fragi clustered together. Strains of this latter species also joined the type strain of Ps. testosteroni and appeared included with phenons containing the Ps. putida strains. There were clusters made up exclusively of strains assigned to one biovar or group (Ps. fluorescens biovars I and II and unidentified). A high level of similarity was observed between clusters of Ps. fluorescens biovar I and those containing the Ps. fragi-Ps. lundensis complex (> 74% SSM) and Ps. lundensis (> 80%). The recovery of pseudomonads seemed to be affected by the sampling day.     

Interferon-gamma-activated human monocytes inhibit the intracellular multiplication of Legionella pneumophila

We have examined the interaction between interferon-gamma (IFN-gamma)-activated human monocytes and Legionella pneumophila, the agent of Legionnaires' disease. Human monocytes activated with human recombinant IFN-gamma inhibit the intracellular multiplication of L. pneumophila. The degree of inhibition is proportional to the concentration of IFN-gamma, and maximal inhibition consistently occurs with greater than or equal to 2 micrograms/ml. Monoclonal anti-IFN-gamma antibody completely neutralizes the capacity of IFN-gamma to activate monocytes. Monocytes infected 24 hr after explantation maximally inhibit L. pneumophila multiplication if treated with IFN-gamma before infection or up to 2 hr after infection; treatment 6 hr or more after infection results in submaximal inhibition. Monocytes infected 48 hr after explantation inhibit L. pneumophila multiplication maximally if treated with IFN-gamma up to 12 hr before infection, but submaximally if treated at the time of infection. Once activated, monocytes inhibit L. pneumophila multiplication in the absence of IFN-gamma in the culture. Strikingly, monocytes maximally inhibit L. pneumophila multiplication after treatment with IFN-gamma for as briefly as 1 hr before infection. In the absence of anti-L. pneumophila antibody, neither IFN-gamma-activated monocytes nor nonactivated monocytes kill L. pneumophila. In the presence of specific antibody and complement, IFN-gamma-activated monocytes kill a proportion (0.5 log) of an inoculum but not more than nonactivated monocytes. L. pneumophila forms a specialized phagosome in IFN-gamma-activated monocytes that does not differ ultrastructurally from the L. pneumophila phagosome in nonactivated monocytes. These results demonstrate that IFN-gamma can activate human monocytes to exert a potent antimicrobial effect against a highly virulent intracellular bacterial pathogen. These findings extend previous observations on interactions between activated mononuclear phagocytes and L. pneumophila, and additionally support the hypothesis that cell-mediated immunity plays a major role in host defense against L. pneumophila.     

Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus Calmette-Guérin. An in vitro comparison of leprosy vaccine components

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.