The lipopolysaccharide (R type) as a common antigen of Neisseria gonorrhoeae. I. Immunizing properties.

The ability of R-type lipopolysaccharide (LPS), isolated from Neisseria gonorrhoeae colony type 4, to protect against infection with N. gonorrhoea colony type 1 (T1 isolates) in the mouse and chicken embryo was investigated. C57 black mice were immunized intraperitoneally with 50 microgram of LPS, and challenged intracerebrally with 10-20 LD50's of N. gonorrhoeae colony type 1. Immunized mice were significantly protected (P less than 0.01 to less than 0.05) against challenge with different T1 isolates of N. gonorrhoeae when compared with non-immunized mice. Mice, injected with succinylated or alkali-treated LPS were not protected against gonococcal challenges. In a second animal model, leghorn hens were immunized intravenously with three injections of 500 microgram of LPS followed by a booster of 2.5 mg 2 weeks later. Embryonated eggs obtained from immunized hens were protected against challenge with 5 x 10(3) - 1 x 10(4) LD50's of three different T1 isolates. When hens were injected with the chemically modified LPS, the embryos were not resistant to gonococcal challenge. The results of this study demonstrate the ability of R-type gonococcal LPS to provide protection against different T1 isolates of N. gonorrhoeae.     

Streptococcus pneumoniae multiplication in the allantoic cavity of developing chick embryos

The possibility of the active multiplication of S. pneumoniae (serotypes 1, 3, 6 and 19) in the allantoic cavity of chick embryos has been demonstrated. This multiplication is accompanied by the development of characteristic changes whose intensity and time of manifestation have been found to depend on the infective dose and the age of the embryo. The accumulation of S. pneumoniae in the allantoic cavity of chick embryos in the absence of visible changes in the biological properties of the infective agent after 5 successive subcultures makes it possible to recommend chick embryos as a model for the study of experimental pneumococcal infection.     

Asymptomatic gonorrhoea and chlamydial infection in rural Tanzanian men.

OBJECTIVE: To measure the prevalence of urethritis due to Neisseria gonorrhoeae and Chlamydial infection trachomatis in rural Tanzanian men DESIGN: About 500 men aged 15-54 years were selected from each of 12 rural communities by random cluster sampling; interviewed concerning past or present symptoms of sexually transmitted diseases; and asked to provide a first catch urine specimen, which was tested for pyuria with a leucocyte esterase dipstick test. Subjects with symptoms or with a positive result on testing were examined, and urethral swabs were taken for detection of N gonorrhoeae by gram stain and of C trachomatis by antigen detection immunoassay. SETTING: Mwanza region, north western Tanzania. SUBJECTS: 5876 men aged 15-54 years. MAIN OUTCOME MEASURES: Prevalence of urethral symptoms, observed urethral discharge, pyuria, urethritis ( > 4 pus cells per high power field on urethral smear), N gonorrhoeae infection (intracellular gram negative diplococci), and C trachomatis infection (IDEIA antigen detection assay). RESULTS: 1618 (28%) subjects reported ever having a urethral discharge. Current discharge was reported by 149 (2.5%) and observed on examination in 207 (3.5%). Gonorrhoea was found in 128 subjects (2.2%) and chlamydial infection in 39 (0.7%). Only 24 of 158 infected subjects complained of urethral discharge at the time of interview (15%). CONCLUSION: Infection with N gonorrhoeae and C trachomatis is commonly asymptomatic among men in this rural African population. This has important implications for the design of control programmes for sexually transmitted disease.     

Physiology of rickettsiae. VII. Amino acid incorporation by Coxiella burnetii and by infected hosts

Protein synthesis, in terms of (14)C-labeled amino acid incorporation into a hot trichloroacetic acid fraction, was studied in cell-free preparations of Coxiella burnetii, and in uninfected and Q fever-infected guinea pig and chick embryo hosts. Purified and disrupted suspensions of C. burnetii incorporated (14)C-labeled l-leucine, l-phenylalanine and algal hydrolysate. Livers of infected guinea pigs and chick embryos had a greater incorporation rate at the height of infection than comparable preparations from uninfected animals. As chick embryonic development continued during infection, the rate of incorporation progressively decreased below that of uninfected embryos.     

Leishmania in the chick embryo: IV. Effects of embryo age and hatching,and behavior of L. donovani in cultures of chick fibroblasts

On incubation Days 9, 11, 12, 14, or 15, chick embryos were injected intravenously with 4.0 × 10⁶L. donovani amastigotes. Embryos were incubated at 33 C immediately after infection. Numbers of amastigotes found in the liver 1 hr after injection increased as the age of embryo recipients increased. Most 14- or 15-day infected embryos hatched when allowed to do so, but many younger embryos were unable to survive at 33 C. Numbers of amastigotes in the liver of chicks, hatched after infection as embryos, decreased as the cloacal temperature of the chicks increased. Despite a 31 C incubation temperature, chicks exhibited a mean 38.3 C cloacal temperature 1 day after hatching.Chick fibroblast cultures were initiated as explants of embryo brain and infected with amastigotes from hamster spleen. Only amastigotes were seen in cultures kept at 37 C, but extracellular promastigotes and intracellular amastigotes were present in cultures at 33 C. Although promastigotes increased in number in the medium overlay at 33 C, amastigotes decreased in number at 33 C and 37 C. One intracellular amastigote was seen in a culture which had been incubated at 25 C after inoculation with promastigotes.     

5-Methoxyindole-2-carboxylic acid is a potent inhibitor of respiration and potassium ion transport in the archaebacterium Haloferax volcanii

Abstract The chorioallantoic membrane (CAM) inoculated chick embryo model was used to study the effect of host lineage on the virulence of Campylobacter jejuni and Campylobacter coli . LD₅₀ values were used to compare the susceptibilities of chick embryos from eight inbred chicken lines to infection by four strains of C. jejuni and one strain of C. coli . Differences in susceptibility were found between inbred chicken lines. These were shown not to be due to maternal antibody status, not transfer of antibody to the developing embryo. Susceptibility to infection was also found to vary according to the Campylobacter strain used. These results indicate that both the bacterial strain and host lineage of the chicken line used affect resistance to infection in the CAM inoculated chick embryo model.     

The role of complement receptor 3 (CR3) in Neisseria gonorrhoeae infection of human cervical epithelia

Neisseria gonorrhoeae is an important sexually transmitted pathogen and a major cofactor in HIV-1 infection. This organism uses different mechanisms to infect male and female genital tract epithelia. Receptor-mediated endocytosis of N. gonorrhoeae is the principle mechanism of entry into male urethral epithelial cells. Infection in men leads to a pronounced inflammatory response. In contrast, N. gonorrhoeae infection in women induces ruffling of the cervical epithelia, allowing a macropinocytic mechanism of entry. Infection in women is frequently asymptomatic, suggesting suppression of the inflammatory response. N. gonorrhoeae -induced membrane ruffling and inflammation suppression are consistent with the ability of this bacterium to enter cervical epithelial cells, in vitro and in vivo , by interaction with complement receptor 3 (CR3), a receptor that does not trigger an inflammatory response. This receptor is present on cervical epithelial cells but not on male urogenital tract epithelia. N. gonorrhoeae engagement of CR3 initiates a unique mechanism of bacterial-induced membrane ruffling and internalization. These studies explain why the pathology of N. gonorrhoeae infection differs between males and females. Additionally, the observation that this receptor is present on cervical epithelia may provide insight into the pathogenesis of other sexually transmitted pathogens     

Leishmania in the Chick Embryo. II. Effects of Inoculum Size,Strain Origin,and Stage Injected and Infectivity of Embryo-Derived Parasites for Hamsters*

SYNOPSIS. Amastigotes of Leishmani donovani strains 2S, 3S, 3K, Hm, Gm, and Et were inoculated intravenously into 14-day chick embryos. The course of infection was followed by examinations of liver impression smears. With strain 33 at 33 C incubation, there was a 29-fold increase at 6 days postinfection when the inoculum contained ～4 × 10⁶ amastigotes, but only a ～6.3-fold increase when ～64 × 10⁶ parasites were injected. Infection courses of several geographic strains were compared at 30, 33, and 35 C incubation. Although the results were variable, Sudan strains 2S and 3S appeared to be separate isolations of a single strain. The Burma (Et), Kenya (3K), and Mediterranean (Hm, Gm) strains appeared to be distinct, but confirming evidence of their distinctness should be sought using serologic, epidemiologic, clinical, and biochemical criteria. Strains 2S and 3S multiplied best at 33 C or below, but the embryos usually failed to survive at 28 or 30 C. Multiplication was inhibited partially at 35 C and completely at 37 C. Inoculation of strain 3S promastigotes into chick embryos resulted in a loss of parasites in 1 hr to 2 days postinfection. Only amastigotes were seen in embryos incubated at 28 and 33 C for 4 days. Hamsters infected with parasites passaged once in chick embryos died at median postinoculation times that were closely comparable to those noted among hosts infected with amastigotes from hamster spleen.     

Inhibition of Neisseria gonorrhoeae by a Factor Produced by Candida albicans

When Candida albicans is present on Transgrow specimens, Neisseria gonorrhoeae is detected less frequently or else can be seen in Gram stains but cannot be readily cultured. When C. albicans and N. gonorrhoeae are grown together on Transgrow, the gonococcal cells die off much more readily than N. gonorrhoeae grown on Transgrow alone. By use of a cross-streaking technique on agar plates, it has been demonstrated that C. albicans produces a soluble substance inhibitory to N. gonorrhoeae, although not to other microorganisms tested. Preliminary results indicate that this inhibitory factor can be extracted by the use of tertiary butanol. Since approximately one-third of the Transgrow specimens with growth contains yeasts, of which C. albicans is by far the most frequent, this factor presents an important complication in the diagnosis of gonorrhea in women.     

Biosynthesis of prolyl hydroxylase: evidence for two separate dolichol-media pathways of glycosylation

Prolyl hydroxylase is a glycoprotein containing two nonidentical subunits, alpha and beta. The alpha subunit of prolyl hydroxylase isolated from 13-day-old chick embryos contains a single high mannose oligosaccharide having seven mannosyl residues. Two forms of alpha subunit have been shown to exist in enzyme purified from tendon cells of 17-day-old chick embryos, one of which (alpha) appears to be identical in molecular weight and carbohydrate content with the single alpha of enzyme from 13-day-old chick embryos, as well as another form (alpha') that contains two oligosaccharides, each containing eight mannosyl units [see Kedersha, N. L., Tkacz, J. S., & Berg, R. A. (1985) Biochemistry (preceding paper in this issue)]. Biosynthetic labeling studies were performed with chick tendon cells using [2-3H]mannose, [6-3H]glucosamine, [14C(U)]mannose, and [14C(U)]glucose. Analysis of the labeled products using polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that only the oligosaccharides on alpha' incorporated measurable mannose or glucosamine isotopes; however, both alpha subunits incorporated 14C amino acid mix and [14C(U)]glucose [metabolically converted to [14C(U)]mannose] under similar conditions. Pulse-chase labeling studies using 14C amino acid mix demonstrated that both glycosylated polypeptide chains alpha and alpha' were synthesized simultaneously and that no precursor product relationship between alpha and alpha' was apparent. In the presence of tunicamycin, neither alpha nor alpha' was detected; a single polypeptide of greater mobility appeared instead. Incubation of the cells with inhibitory concentrations of glucosamine partially depressed the glycosylation of alpha' but allowed the glycosylation of alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Draft genome sequence of a dominant, multidrug-resistant Neisseria gonorrhoeae strain, TCDC-NG08107, from a sexual group at high risk of acquiring human immunodeficiency virus infection and syphilis

Neisseria gonorrhoeae infection is the second major cause of sexually transmitted diseases worldwide. Development of resistance to multiple classes of antimicrobials in N. gonorrhoeae has compromised treatment and disease control. Herein, we report the availability of the draft genome sequence of a multidrug-resistant N. gonorrhoeae isolate, TCDC-NG08107, which spread in groups of men who have sex with men (MSM) in Taiwan.     

Ng-MIP, a surface-exposed lipoprotein of Neisseria gonorrhoeae, has a peptidyl-prolyl cis/trans isomerase (PPIase) activity and is involved in persistence in macrophages

Macrophage infectivity potentiators (MIPs) are a family of surface-exposed virulence factors of intracellular microorganisms such as Legionella, Chlamydia and Trypanosoma. These proteins display peptidyl-prolyl cis/trans isomerase (PPIase) activity that is inhibited by immunosuppressants FK506 and rapamycin. Here we describe the identification and characterization in Neisseria gonorrhoeae of Ng-MIP, a surface-exposed lipoprotein with high homology to MIPs. The protein is an homodimer with rapamycin-inhibited PPIase activity confirming that it is a functional member of the MIP family. A knock-out strain, generated by deletion of the mip gene in N. gonorrhoeae F62 strain, was evaluated for its role in infection of mouse and human macrophages. We show that Ng-MIP promotes the intracellular survival of N. gonorrhoeae in macrophages, highlighting a possible role of this protein in promoting the persistence of gonococcal infection.     

70株淋球菌的质粒及对青霉素敏感性的检测

本文检测的70株淋球菌含三种质粒,质粒总检出率为95.7％(67株)。均含2.6Md者有67株,占94.29％,可能是隐蔽质粒。含3.2Md及23.1Md者20株均对青霉素耐药,故含此两种质粒的淋球菌有可能成为哈市耐青霉素的流行株。提取2.6Md质粒有希望制备诊断的探针。     

Synthesis of glucuronoglucans in chicken cartilage cultured in synthetic medium.

Cartilaginous femur and tibia rudiments from 10-day-old chick embryos were grown in vitro for 4 days in Parker's solution without protein added, and subsequently fixed and extracted successively with 0-2 N HClO4 at 4 degrees C (fraction A), 5 per cent trichloracetic acid (TCA) at 4 degrees C (fraction B), and 5 per cent TCA at 90 degrees C (fraction C). The residue after extraction was dissolved in 1 N NaOH at room temperature (fraction D). Fraction C containing most of hexuronic acids and aminosugars of the cartilage was used to study the quantitative changes of glucuronoglucans throughout the culture period. The amount of hexuronic acids and aminosugars was increased after 24, 48, 72 and 96 hours of culture. After 96 hours the level of hexuronic acids was twice that found prior to establishing the culture. The increment was statistically significant.     

The effects of collagen synthesis inhibitory drugs on somitogenesis and myogenin expression in cultured chick and mouse embryos

The role of fibrillar collagen on myogenic differentiation has previously been studied in tissue culture cell lines but has not been studied in situ. We treated cultured chick and mouse embryos with collagen synthesis inhibitors to determine the role of fibrillar collagen on somitogenesis and on myogenic differentiation in vivo. Stage 12 chick embryos and 8.7 dpc mouse embryos were cultured in control medium or a range of concentrations of the collagen synthesis inhibitors ethyl-3,4-dihydroxybenzoate (EDHB) or cis-hydroxy-proline (CHP). Chick embryos were cultured for 24 h and mouse embryos were cultured for 30 h. Both collagen synthesis inhibitors produced a range of somite abnormalities including formation of fewer and irregular somites in both chick and mouse at high drug concentrations, as well as formation of double somites in EDHB-treated chick embryos. Examination of EDHB-treated mouse embryos by scanning electron microscopy demonstrated a dosage-dependent loss of fibrillar collagen and associated extracellular matrix. Expression of myogenin in EDHB-treated mouse embryos, examined by whole-mount in situ hybridization, was suppressed at higher dosage levels. This study suggests that inhibition of fibrillar collagen production and/or loss of fibrillar collagen in the developing avian and mammalian embryo results in abnormal somite formation and perturbed myogenic differentiation.     

Targeted infection of endothelial cells by avian influenza virus A/FPV/Rostock/34 (H7N1) in chicken embryos

The tissue tropism and spread of infection of the highly pathogenic avian influenza virus A/FPV/Rostock/34 (H7N1) (FPV) were analyzed in 11-day-old chicken embryos. As shown by in situ hybridization, the virus caused generalized infection that was strictly confined to endothelial cells in all organs. Studies with reassortants of FPV and the apathogenic avian strain A/chick/Germany/N/49 (H10N7) revealed that endotheliotropism was linked to FPV hemagglutinin (HA). To further analyze the factors determining endotheliotropism, the HA-activating protease furin was cloned from chicken tissue. Ubiquitous expression of furin and other proprotein convertases in the chick embryo indicated that proteolytic activation of HA was not responsible for restriction of infection to the endothelium. To determine the expression of virus receptors in embryonic tissues, histochemical analysis of alpha2,3- and alpha2,6-linked neuraminic acid was carried out by lectin-binding assays. These receptors were found on endothelial cells and on several epithelial cells, but not on tissues surrounding endothelia. Finally, we analyzed the polarity of virus maturation in endothelial cells. Studies on cultured human endothelial cells employing confocal laser scanning microscopy revealed that HA is specifically targeted to the apical surface of these cells, and electron microscopy of embryonic tissues showed that virus maturation occurs also at the luminar side. Taken together, these observations indicate that endotheliotropism of FPV in the chicken embryo is determined, on one hand, by the high cleavability of HA, which mediates virus entry into the vascular system, and, on the other hand, by restricted receptor expression and polar budding, which prevent spread of infection into tissues surrounding endothelia.     

Neisseria gonorrhoeae infection induces altered amphiregulin processing and release

Adhesion of the human pathogen Neisseria gonorrhoeae has established effects on the host cell and evokes a variety of cellular events including growth factor activation. In the present study we report that infection with N. gonorrhoeae causes altered amphiregulin processing and release in human epithelial cells. Amphiregulin is a well-studied growth factor with functions in various cell processes and is upregulated in different forms cancer and proliferative diseases. The protein is prototypically cleaved on the cell surface in response to external stimuli. We demonstrate that upon infection, a massive upregulation of amphiregulin mRNA is seen. The protein changes its subcellular distribution and is also alternatively cleaved at the plasma membrane, which results in augmented release of an infection-specific 36 kDa amphiregulin product from the surface of human cervical epithelial cells. Further, using antibodies directed against different domains of the protein we could determine the impact of infection on pro-peptide processing. In summary, we present data showing that the infection of N. gonorrhoeae causes an alternative amphiregulin processing, subcellular distribution and release in human epithelial cervical cells that likely contribute to the predisposition cellular abnormalities and anti-apoptotic features of N. gonorrhoeae infections.