Compared to urogenital infections, little is known of serovar distribution in rectal chlamydial infection. The aim of this study was to explore possible relations between demographics, sexual behaviour, clinical manifestations, rectal symptoms, and chlamydial serovars including L2 (lymphogranuloma venereum).
Genotyping was done prospectively in all rectal chlamydial infections since the outbreak of proctitis caused by lymphogranuloma venereum in February 2003. 33 (15.1%) rectal Chlamydia trachomatis infections from the years 2001 and 2002 were genotyped retrospectively.
Of all 219 rectal chlamydial infections, detected in the period July 2001 to August 2005, a total of 149 (68.0%) were successfully genotyped including 21 (14.1%) infections with serovar L2. In univariable and multivariable analyses, L2 serovar positive patients were significantly more often HIV positive (p = 0.002; OR: 6.5; 95% CI: 2.0 to 21.1), and had had sex in the past 6 months with more partners compared to other serovars. Furthermore, patients with L2 proctitis presented far more often with self reported rectal symptoms (p<0.005; OR: 19.4; 95% CI: 4.9 to 77.0) and clinical manifestations (p<0.005; OR: 15.4; 95% CI: 4.5 to 52.5).
Chlamydial infections with serovar L2 show a different clinical and epidemiological pattern compared to serovar D‐K. LGV proctitis is significantly associated with HIV positivity and a high number of sexual partners and causes more rectal symptoms and clinical manifestations. Neither young age nor ethnicity were identified as risk factors for any of the serovars investigated in this study.
; rectal infection; lymphogranuloma venereum; serovars
We developed a murine model of systemic infection with Chlamydia trachomatis biovar lymphogranuloma venereum (LGV). The pathological features of this infection resemble those of human LGV infection since both are characterized by granuloma formation. Mice developed resistance to reinfection with LGV, and this resistance was based on cellular immune mechanisms since it was transferable with immune spleen cells but not with immune serum. Resistance required viable organisms for induction. We compared LGV biovar infection with trachoma biovar infection. Trachoma biovar produced similar but less marked microbiological and pathological features. Cross-immunity was less apparent between serovars from trachoma and LGV biovars than it was between serovars within the same biovar. This model of systemic C. trachomatis infection will be useful in exploring virulence features of LGV.
Chlamydia trachomatis is an obligate intracellular bacterium that causes a diversity of severe and debilitating diseases worldwide. Sporadic and ongoing outbreaks of lymphogranuloma venereum (LGV) strains among men who have sex with men (MSM) support the need for research on virulence factors associated with these organisms. Previous analyses have been limited to single genes or genomes of laboratory-adapted reference strain L2/434 and outbreak strain L2b/UCH-1/proctitis. We characterized an unusual LGV strain, termed L2c, isolated from an MSM with severe hemorrhagic proctitis. L2c developed nonfusing, grape-like inclusions and a cytotoxic phenotype in culture, unlike the LGV strains described to date. Deep genome sequencing revealed that L2c was a recombinant of L2 and D strains with conserved clustered regions of genetic exchange, including a 78-kb region and a partial, yet functional, toxin gene that was lost with prolonged culture. Indels (insertions/deletions) were discovered in an ftsK gene promoter and in the tarp and hctB genes, which encode key proteins involved in replication, inclusion formation, and histone H1-like protein activity, respectively. Analyses suggest that these indels affect gene and/or protein function, supporting the in vitro and disease phenotypes. While recombination has been known to occur for C. trachomatis based on gene sequence analyses, we provide the first whole-genome evidence for recombination between a virulent, invasive LGV strain and a noninvasive common urogenital strain. Given the lack of a genetic system for producing stable C. trachomatis mutants, identifying naturally occurring recombinants can clarify gene function and provide opportunities for discovering avenues for genomic manipulation.
Lymphogranuloma venereum (LGV) is a prevalent and debilitating sexually transmitted disease in developing countries, although there are significant ongoing outbreaks in Australia, Europe, and the United States among men who have sex with men (MSM). Relatively little is known about LGV virulence factors, and only two LGV genomes have been sequenced to date. We isolated an LGV strain from an MSM with severe hemorrhagic proctitis that was morphologically unique in tissue culture compared with other LGV strains. Bioinformatic and statistical analyses identified the strain as a recombinant of L2 and D strains with highly conserved clustered regions of genetic exchange. The unique culture morphology and, more importantly, disease phenotype could be traced to the genes involved in recombination. The findings have implications for bacterial species evolution and, in the case of ongoing LGV outbreaks, suggest that recombination is a mechanism for strain emergence that results in significant disease pathology.
We present the case of a 26 year old HIV positive homosexual man who was managed for suspected Crohn's disease for over 1 year before lymphogranuloma venereum (LGV) was clinically diagnosed. He had presented with constipation, secondary to acute haemorrhagic proctitis, and subsequently had two chlamydia negative rectal smears, using direct fluorescent antibody (DFA) Chlamydia trachomatis staining. Positive chlamydial serology guided retrospective testing of an early rectal biopsy, which was found to have C trachomatis by polymerase chain reaction (Roche Cobas) and identified as LGV serovar L2 by the Sexually Transmitted Bacteria Reference Laboratory (STBRL), Health Protection Agency (HPA), Colindale, London. Chlamydial serology may have a role in identifying late stage LGV infection. Although no standardised test currently exists, consideration should be given to evaluating the role of chlamydial serology in establishing a diagnosis of LGV.
lymphogranuloma venereum; chlamydia; Crohn's disease; serology; proctitis
Chlamydia trachomatis is a human bacterial pathogen that multiplies only within an intracellular membrane-bound vacuole, the inclusion. C. trachomatis includes ocular and urogenital strains, usually causing infections restricted to epithelial cells of the conjunctiva and genital mucosa, respectively, and lymphogranuloma venereum (LGV) strains, which can infect macrophages and spread into lymph nodes. However, C. trachomatis genomes display >98% identity at the DNA level. In this work, we studied whether C. trachomatis Inc proteins, which have a bilobed hydrophobic domain that may mediate their insertion in the inclusion membrane, could be a factor determining these different types of infection and tropisms. Analyses of polymorphisms and phylogeny of 48 Inc proteins from 51 strains encompassing the three disease groups showed significant amino acid differences that were mainly due to variations between Inc proteins from LGV and ocular or urogenital isolates. Studies of the evolutionary dynamics of inc genes suggested that 10 of them are likely under positive selection and indicated that most nonsilent mutations are LGV specific. Additionally, real-time quantitative PCR analyses in prototype and clinical strains covering the three disease groups identified three inc genes with LGV-specific expression. We determined the transcriptional start sites of these genes and found LGV-specific nucleotides within their promoters. Thus, subtle variations in the amino acids of a subset of Inc proteins and in the expression of inc genes may contribute to the unique tropism and invasiveness of C. trachomatis LGV strains.
The kinetics of attachment and ingestion of Chlamydia trachomatis serotype L1 by monolayers of McCoy cells were studied by using a method that discriminated between attachment and uptake. When about 1% of the McCoy cells was infected, the proteinase K-resistant chlamydial fraction, regarded as ingested chlamydiae, reached a constant value after about 3 h of incubation at 37 degrees C. Uptake of chlamydiae at 4 degrees C could not be demonstrated. The attached and ingested chlamydial fractions were constant over an eightfold increase in chlamydial inoculum. Chitobiose and chitotriose, the di- and trisaccharides of N-acetyl-D-glucosamine, reduced the association of C. trachomatis serotype L1 with McCoy cells. Higher concentrations of chitobiose also selectively inhibited ingestion of chlamydiae. A corresponding effect of chitobiose was also observed on the number of chlamydial inclusions. Wheat germ agglutinin, specific for N-acetyl-D-glucosamine residues, reduced the association of chlamydiae when incubated at 4 degrees C, but not at 37 degrees C. A small inhibiting effect of concanavalin A on association of chlamydiae, but no effect of the corresponding carbohydrates, indicates a nonspecific effect on chlamydial attachment of this lectin. These results suggest that beta 1 leads to 4-linked oligomers of N-acetyl-D-glucosamine are important in the specificity of attachment of C. trachomatis to McCoy cells.
The lymphogranuloma venereum (LGV) and trachoma biovars of Chlamydia trachomatis exhibit differences in biological properties both in vivo and in vitro. To identify analogous biochemical differences, we studied the molecular charges of chlamydial outer membrane proteins (OMPs) by means of isoelectric focusing and nonequilibrium pH gradient electrophoresis. Analysis of proteins of whole elementary bodies biosynthetically labeled with L-[35S]cysteine revealed that most chlamydial proteins were neutral or acidic. The major OMPs (MOMPs) of all strains tested were acidic and had apparent isoelectric points (pIs) that varied within narrow limits (approximately 5.3 to 5.5) despite differences in molecular mass of up to 3,000 daltons (Da). However, a low-molecular-mass cysteine-rich OMP analogous to that previously described for Chlamydia psittaci varied consistently in molecular mass (12,500 versus 12,000 Da) and pI (5.4 versus 6.9) between LGV strains and trachoma strains, respectively. OMPs with a molecular mass of 60,000 Da in the trachoma biovar strains had pIs in the 7.3 to 7.7 range. However, analogous OMPs in the LGV strains existed as a doublet with a molecular mass of about 60,000 Da. Both members of the doublet were basic (pIs greater than 8.5). Both proteins of this basic doublet in LGV strains and the neutral analog in trachoma strains bound a species-specific monoclonal antibody in an immunoblot assay. These data indicate substantial differences in biochemical characteristics of analogous OMPs in the LGV and trachoma biovars. Such differences are the first structural differences described between LGV and trachoma strains which support their distinction into separate biovars and may be related to some of their biological differences.
As arguably the most successful parasite, Chlamydia is an obligate intracellular bacterium replicating inside a vacuole of eukaryotic host cells. The chlamydial vacuole does not fuse with the defense cell organelle lysosome. We previously showed that chlamydial infection increases markers of autophagy, an innate antimicrobial activity requiring lysosomal function. However, the work presented here demonstrates that p62, an autophagy protein that is degraded in lysosomes, either remained unchanged or increased in chlamydia-infected human epithelial, mouse fibroblast, and mouse macrophage cell lines. In addition, the activities of three lysosomal enzymes analyzed were diminished in chlamydia-infected macrophages. Bafilomycin A1 (BafA), a specific inhibitor of vacuolar ATPase (vATPase) required for lysosomal function, increased the growth of the human pathogen Chlamydia trachomatis (L2) in wild-type murine fibroblasts and macrophages but inhibited growth in the autophagy-deficient ATG5−/− fibroblasts. BafA exhibited only slight inhibition or no effect on L2 growth in multiple human genital epithelial cell lines. In contrast to L2, the mouse pathogen Chlamydia muridarum (MoPn) was consistently inhibited by BafA in all cell lines examined, regardless of species origin and autophagy status. Finally, L2 but not MoPn grew more efficiently in the ATG5−/− cells than in wild-type cells. These results suggest that there are two types of vATPase-bearing organelles that regulate chlamydial infection: one supports chlamydial infection, while the other plays a defensive role through autophagy when cells are artificially infected with certain chlamydiae that have not been adapted to the host species.
Glycogen metabolism of monkey kidney (LLC-MK-2) cells and HeLa 229 cells infected with a Chlamydia trachomatis lymphogranuloma venereum 440 L (LGV) was studied. The growth cycle of LGV in both host cells was similar; however, a greater number of infectious organism developed intracellularly and were released into the medium during LGV infection of HeLa 229 cells than MK-2 cells. A rapid infection accompanied by a high rate of glycogen synthesis and a short period of accumulation was found in GeLa 229 cells infected with LGV. LGV infected MK-2 cells started to accumulate glycogen about the same time as HeLa 229 cells; however, the rate of glycogen synthesis was lower and the period of accumulation was longer. The LGV agent grew in cycloheximide-treated cells in the absence of host cell protein synthesis. Protein synthesis associated with LGV throughout the developmental cycle was similar in both cell types and could be abolished by chloramphenicol. The continued synthesis of glycogen in the presence of cycloheximide suggested that the synthesis of glycogen was directed by the organism in both MK-2 cells and HeLa 229 cells.
To determine the prevalence of lymphogranuloma venereum (LGV) and non-LGV associated serovars of urethral and rectal Chlamydia trachomatis (CT) infection in men who have sex with men (MSM).
Multicentre cross-sectional survey.
Four genitourinary medicine clinics in the United Kingdom from 2006–7.
4825 urethral and 6778 rectal samples from consecutive MSM attending for sexual health screening.
Urethral swabs or urine and rectal swabs were tested for CT using standard nucleic acid amplification tests. Chlamydia-positive specimens were sent to the reference laboratory for serovar determination.
Positivity for both LGV and non-LGV associated CT serovars; proportion of cases that were symptomatic.
The positivity (with 95% confidence intervals) in rectal samples was 6.06% (5.51% to 6.66%) for non-LGV CT and 0.90% (0.69% to 1.16%) for LGV; for urethral samples 3.21% (2.74% to 3.76%) for non-LGV CT and 0.04% (0.01% to 0.16%) for LGV. The majority of LGV was symptomatic (95% of rectal, one of two urethral cases); non-LGV chlamydia was mostly symptomatic in the urethra (68%) but not in the rectum (16%).
Chlamydial infections are common in MSM attending for sexual health screening, and the majority are non-LGV associated serovars. We did not identify a large reservoir of asymptomatic LGV in the rectum or urethra. Testing for chlamydia from the rectum and urethra should be included for MSM requesting a sexual health screen, but serovar-typing is not indicated in the absence of symptoms. We have yet to identify the source of most cases of LGV in the UK.
A system for measuring chlamydial lipid synthesis was developed with mouse L cells grown in serum-free modified Waymouth 752/l medium in a shaker culture. Host lipid synthesis was reduced approximately 90% when cells were incubated for 24 h in medium containing cycloheximide (2 micrograms/ml). Lipid metabolism was monitored by measuring the incorporation of [3H]isoleucine into the total lipid of normal and infected cells. The results suggested that lipid synthesis of Chlamydia trachomatis lymphogranuloma venereum (LGV-404L) was not inhibited by cycloheximide treatment when the chlamydiae were grown in L cells, whereas host lipid synthesis was inhibited. Chlamydial lipid metabolism began about 6 to 12 h after infection when the noninfectious reticulate body was found and continually increased until the beginning of the appearance of intracellular infectious elementary bodies at 24 to 30 h.
The obligate intracellular bacterium Chlamydia trachomatis possesses a biphasic developmental cycle that is manifested by differentiation of infectious, metabolically inert elementary bodies (EBs) to larger, metabolically active reticulate bodies (RBs). The cycle is completed by asynchronous differentiation of dividing RBs back to a population of dormant EBs that can initiate further rounds of infection upon lysis of the host cell. Chlamydiae express a type III secretion system (T3SS) which is presumably employed to establish and maintain the permissive intracellular niche by secretion of anti-host proteins. We hypothesize that T3SS activity is essential for chlamydial development and pathogenesis. However, the lack of a genetic system has confounded efforts to establish any role of the T3SS. We therefore employed the small molecule Yersinia T3SS inhibitor ′N′-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide, designated compound 1 (C1), to examine the inter-dependence of the chlamydial T3SS and development. C1-treatment inhibited C. trachomatis but not T4SS-expressing Coxiella burnetii development in a dose-dependent manner. Although chlamydiae remained viable and metabolically active, they failed to divide significantly and RB to EB differentiation was inhibited. These effects occurred in the absence of host cell cytotoxicity and were reversible by washing out C1. We further demonstrate that secretion of T3S substrates is perturbed in C1-treated chlamydial cultures. We have therefore provided evidence that C1 can inhibit C. trachomatis development and T3SS activity and present a model in which progression of the C. trachomatis developmental cycle requires a fully functional T3SS.
Chlamydia; type III secretion; Compound 1
Chlamydia trachomatis is a major human pathogen with a unique obligate intracellular developmental cycle that takes place inside a modified cytoplasmic structure known as an inclusion. Following entry into a cell, the infectious elementary body (EB) differentiates into a non - infectious replicative form known as a reticulate body (RB). RBs divide by binary fission and at the end of the cycle they redifferentiate into EBs. Treatment of C.trachomatis with penicillin prevents maturation of RBs which survive and enlarge to become aberrant RBs within the inclusion in a non - infective persistent state. Persistently infected individuals may be a reservoir for chlamydial infection. The C.trachomatis genome encodes the enzymes for peptidoglycan (PG) biosynthesis but a PG sacculus has never been detected. This coupled to the action of penicillin is known as the chlamydial anomaly. We have applied video microscopy and quantitative DNA assays to the chlamydial developmental cycle to assess the effects of penicillin treatment and establish a framework for investigating penicillin induced chlamydial persistence.
Addition of penicillin at the time of cell infection does not prevent uptake and the establishment of an inclusion. EB to RB transition occurs but bacterial cytokinesis is arrested by the second binary fission. RBs continue to enlarge but not divide in the presence of penicillin. The normal developmental cycle can be recovered by the removal of penicillin although the large, aberrant RBs do not revert to the normal smaller size but remain present to the completion of the developmental cycle. Chromosomal and plasmid DNA replication is unaffected by the addition of penicillin but the arrest of bacterial cytokinesis under these conditions results in RBs accumulating multiple copies of the genome.
We have applied video time lapse microscopy to the study of the chlamydial developmental cycle. Linked with accurate measures of genome replication this provides a defined framework to analyse the developmental cycle and to investigate and provide new insights into the effects of antibiotic treatments. Removal of penicillin allows recovery of the normal developmental cycle by 10–20 hrs and the process occurs by budding from aberrant RBs.
Lipid metabolism of monkey kidney (LLC-MK-2) cells and cells infected with a Chlamydia trachomatis strain lymphogranuloma venereum (LGV) was studied. The protein-to-lipid ratio of normal MK-2 cells was found to increase linearly over a 60-h period of incubation. The protein-to-lipid ratio of the infected cells was similar to that in normal cells until 36 h after infection, when a plateau in the ratio was observed. Lipid synthesis of the infected cells was found to be inhibited after 48 h of infection. Turnover of host lipids did not appear to be markedly altered by infection with LGV over a 48-h period of incubation. An anteiso branched chain of 15:0 fatty acid was found in infected cells but not in normal cells. The appearance of this fatty acid, correlated with a rise in the infectivity of LGV, suggests that synthesis of specific lipids was associated with the infection.
Coxiella burnetii and Chlamydia trachomatis are bacterial obligate intracellular parasites that occupy distinct vacuolar niches within eucaryotic host cells. We have employed immunofluorescence, cytochemistry, fluorescent vital stains, and fluid-phase markers in conjunction with electron, confocal, and conventional microscopy to characterize the vacuolar environments of these pathogens. The acidic nature of the C. burnetii-containing vacuole was confirmed by its acquisition of the acidotropic base acridine orange (AO). The presence of the vacuolar-type (H+) ATPase (V-ATPase) within the Coxiella vacuolar membrane was demonstrated by indirect immunofluorescence, and growth of C. burnetii was inhibited by bafilomycin A1 (Baf A), a specific inhibitor of the V-ATPase. In contrast, AO did not accumulate in C. trachomatis inclusions nor was the V-ATPase found in the inclusion membrane. Moreover, chlamydial growth was not inhibited by Baf A or the lysosomotropic amines methylamine, ammonium chloride, and chloroquine. Vacuoles harboring C. burnetii incorporated the fluorescent fluid- phase markers, fluorescein isothiocyanate-dextran (FITC-dex) and Lucifer yellow (LY), indicating trafficking between that vacuole and the endocytic pathway. Neither FITC-dex nor LY was sequestered by chlamydial inclusions. The late endosomal-prelysosomal marker cation-independent mannose 6-phosphate receptor was not detectable in the vacuolar membranes encompassing either parasite. However, the lysosomal enzymes acid phosphatase and cathepsin D and the lysosomal glycoproteins LAMP-1 and LAMP-2 localized to the C. burnetii vacuole but not the chlamydial vacuole. Interaction of C. trachomatis inclusions with the Golgi-derived vesicles was demonstrated by the transport of sphingomyelin, endogenously synthesized from C6-NBD-ceramide, to the chlamydial inclusion and incorporation into the bacterial cell wall. Similar trafficking of C-NBD-ceramide was not evident in C. burnetii-infected cells. Collectively, the data indicate that C. trachomatis replicates within a nonacidified vacuole that is disconnected from endosome-lysosome trafficking but may receive lipid from exocytic vesicles derived from the trans-Golgi network. These observations are in sharp contrast to those for C. burnetii, which by all criteria resides in a typical phagolysosome.
Chlamydia trachomatis is a Gram-negative obligate intracellular pathogen that remains the leading cause of bacterial sexually transmitted disease worldwide, despite the availability of efficacious antimicrobial therapy. Given that chlamydial infections cause severe pathological sequelae in the upper genital tract, a licensed vaccine to prevent infection and disease would be an ideal solution. Chlamydial protease-like activity factor (CPAF) is a protein secreted in considerable amounts into the cytosol of infected cells and released into the extracellular milieu upon cellular lysis, which therefore is accessible to the host immune system. This is further substantiated by the observation that CPAF is immunodominant among other antigens in Chlamydia sero-positive humans. The efficacy of vaccination with CPAF against genital chlamydial challenge has been evaluated extensively in the murine model. This review will discuss important insights into the potential of CPAF as a component of an anti-chlamydial vaccine.
Chlamydia trachomatis is an obligate intracellular parasite of eucaryotic cells. Little is known about the role of the host in supporting chlamydial replication beyond the facts that host cells provide ATP and that de novo host protein synthesis is not required for bacterial growth. To further explore potential contributions of host nuclear function to chlamydial development, we questioned whether murine C. trachomatis could grow in mouse L cells that had been enucleated with cytochalasin B. Following enucleation, cells were infected with chlamydiae and analyzed morphologically and biochemically. Late in infection, substantial numbers of chlamydiae of all developmental stages were seen within large cytoplasmic inclusions that were indistinguishable from those seen in infected intact cells. Normal numbers of infectious progeny particles were produced from enucleated cultures. We conclude that active host cell nuclear function is not required to support the growth of chlamydiae.
The synthesis and accumulation of Chlamydia trachomatis outer membrane proteins within infected HeLa 229 host cells were monitored by assessing the uptake of [35S]cysteine into chlamydial proteins during the 48-h growth cycle of a lymphogranuloma venereum strain, L2/434/Bu. Synthesis of the major outer membrane protein, a protein that accounts for about 60% of the outer membrane protein mass of elementary bodies (EB), was first detected between 12 and 18 h after infection. The uptake of [35S]cysteine into the 60,000-molecular-weight doublet (60K doublet) and 12.5K cysteine-rich proteins was not observed until 30 h after infection, when the intracellularly dividing reticulate bodies were beginning to transform into infectious EBs. By using a more sensitive immunoblotting method in conjunction with monoclonal antibodies specific for the 60K doublet proteins, synthesis of these proteins was detected even earlier, by 18 h after infection. These data suggest that the time and extent of synthesis of these outer membrane proteins are regulated by processes that coincide in time with the transformation of reticulate bodies into EBs. Additional studies were performed to determine the extent of disulfide cross-linking of outer membrane proteins during the growth cycle. Both the major outer membrane protein and the 12.5K protein became progressively cross-linked to about 60% during the last 24 h of the growth cycle, whereas the 60K doublet proteins were extensively cross-linked during most of the cycle. These data may indicate an intracellular cross-linking mechanism, possibly enzymatic, that exists in addition to an auto-oxidation mechanism that occurs upon host cell lysis and exposure to the extracellular environment.
To investigate whether serological titres of species‐specific IgA and IgG antibodies in patients with rectal chlamydial infection could discriminate between infection with serovar L2 lymphogranuloma venereum (LGV) and infection with non‐LGV serovars.
A total of 39 male patients with chlamydial infection of the rectum were tested for titres of IgA and IgG antibodies within 14 days after detection of the infection and 6 and 12 months after adequate treatment. Data were collected regarding demographics, sexual orientation, HIV serostatus, history of chlamydial infection, concomitant sexually transmitted infection (STI) or HIV infection, hepatitis C virus antibodies and new STIs during follow‐up.
Between May 2003 and November 2005, 24 men with confirmed L2 proctitis and 15 men with non‐LGV rectal chlamydial infection were recruited. In multivariable analyses, both high titre of IgA within 14 days after detection of the infection and older age of the individual were found significantly associated with L2 proctitis (p<0.001 and p = 0.001, respectively). A total sum score of seven times IgA titre and individual's age ⩾50 years resulted in an overall sensitivity of 92% and specificity of 100%. This total sum score was highly accurate for detection of LGV proctitis, with an area under the curve in a receiver operating characteristic curve of 0.989.
An increased IgA antibody response and the age of the infected individual are of possible diagnostic value for (early) detection of LGV proctitis.
The effect of human polymorphonuclear leukocytes (PMNs) on Chlamydia trachomatis was studied. Both trachoma (B/TW-5/OT) and lymphogranuloma venereum (L2/434/Bu) biotypes were rapidly inactivated by exposure to human PMNs. A decrease of 3 to 3.5 logs in viable count was observed after 60 min of incubation at a chlamydia-to-PMN ratio of 1:10. Both chlamydial biotypes were also rapidly inactivated by the cell-free myeloperoxidase-H2O2-halide system. A decrease in infectivity titer of 4 to 5 logs for TW-5 and complete inactivation of 434 were seen after 30 min of incubation. The microbicidal effect was prevented by the deletion of each component of the system or by the addition of the peroxidase inhibitors cyanide or azide. PMNs from myeloperoxidase-deficient patients inactivated chlamydiae normally, whereas PMNs from patients with chronic granulomatous disease, although strongly chlamydicidal, were less effective than normal PMNs in the activation of TW-5 (2-log drop in viable organisms versus a 3 to 3.5-log drop). The chlamydicidal activity of PMNs from patients with chronic granulomatous disease and normal PMNs were comparable against the 434 biotype. These studies suggest that the myeloperoxidase system, or indeed oxygen-dependent antimicrobial systems, are not essential for the chlamydicidal activity of PMNs.
Chlamydia trachomatis, an obligate intracellular bacterial pathogen of mucosal surfaces, is a major cause of preventable blindness and sexually transmitted diseases for which vaccines are badly needed. Despite considerable effort, antichlamydial vaccines have proven to be elusive using conventional immunization strategies. We report the use of murine bone marrow–derived dendritic cells (DC) pulsed ex vivo with killed chlamydiae as a novel approach to vaccination against chlamydial infection. Our results show that DC efficiently phagocytose chlamydiae, secrete IL-12 p40, and present chlamydial antigen(s) to infection sensitized CD4+ T cells. Mice immunized intravenously with chlamydial-pulsed DC produce protective immunity against chlamydial infection of the female genital tract equal to that obtained after infection with live organisms. Immunized mice shed ∼3 logs fewer infectious chlamydiae and are protected from genital tract inflammatory and obstructive disease. Protective immunity is correlated with a chlamydial-specific Th1-biased response that closely mimics the immune response produced after chlamydial infection. Thus, ex vivo antigen-pulsed DC represent a powerful tool for the study of protective immunity to chlamydial mucosal infection and for the identification of chlamydial protective antigens through reconstitution experiments. Moreover, these findings might impact the design of vaccine strategies against other medically important sexually transmitted diseases for which vaccines are sought but which have proven difficult to develop.
Chlamydia; pulsed dendritic cells; immunization; CD4+ T cells; mucosal protective immunity
The infectivity of trachoma-inclusion conjunctivitis (TRIC) organisms (TW-5) was enhanced by pretreatment of HeLa cell monolayers before inoculation with diethylaminoethyl (DEAE)-dextran (30 μg/ml) and poly-l-lysine (10 μg/ml) and inhibited by dextran sulphate (250 μg/ml), fetuin (4%), ovomucoid (5%), N-acetyl neuraminic acid (0.5%), and Cholera vibrio neuraminidase (100 U/ml). The infectivity of lymphogranuloma venereum organisms (434) was not affected by DEAE-dextran, fetuin, and neuraminidase, was slightly inhibited by poly-l-lysine, and was inhibited by dextran-sulphate, ovomucoid, and N-acetyl neuraminic acid. The study suggested that sialic acid residues on the cell surface may be specific receptors for TRIC organisms. The receptors for TRIC organisms (TW-5 and TW-3) could be specifically blocked with inactivated (56 C for 30 min) TRIC organisms at the ratio of one live to 100 inactivated TRIC organisms, but not by inactivated lymphogranuloma venereum (434) or influenza virus (A2/Jap 305).
Chlamydia organisms are obligate intracellular bacterial pathogens responsible for a range of human diseases. Persistent infection or reinfection with Chlamydia trachomatis leads to scarring of ocular or genital tissues, and Chlamydia pneumoniae infection is associated with the development of atherosclerosis. We demonstrate that C. trachomatis and C. pneumoniae infection in vitro elicits the externalization of the lipid phosphatidylserine on the surface of human epithelial, endothelial, granulocytic, and monocytic cells. Phosphatidylserine externalization is associated with cellular development, differentiation, and death. Infection-induced phosphatidylserine externalization was immediate, transient, calcium dependent, and infectious dose dependent and was unaffected by a broad-spectrum caspase inhibitor. Chlamydia-infected cells accelerated plasma clotting and increased the macrophage phagocytosis of infected cells that was phosphatidylserine dependent. The rapid externalization of phosphatidylserine by infected cells may be an important factor in the pathogenesis of chlamydial infections.
Chlamydia trachomatis is an obligate intracellular bacteria and the infectious agent responsible for the sexually transmitted disease Chlamydia. Infection with Chlamydia can lead to serious health sequelae such as pelvic inflammatory disease and reproductive tract scarring contributing to infertility and ectopic pregnancies. Additionally, chlamydial infections have been epidemiologically linked to cervical cancer in patients with a prior human papilomavirus (HPV) infection. Chlamydial infection of cultured cells causes multinucleation, a potential pathway for chromosomal instability. Two mechanisms that are known to initiate multinucleation are cell fusion and cytokinesis failure. This study demonstrates that multinucleation of the host cell by Chlamydia is entirely due to cytokinesis failure. Moreover, cytokinesis failure is due in part to the chlamydial effector CPAF acting as an anaphase promoting complex mimic causing cells to exit mitosis with unaligned and unattached chromosomes. These lagging and missegregated chromosomes inhibit cytokinesis by blocking abscission, the final stage of cytokinesis.
Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ2-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ2-NAD(P)H increase inside the chlamydial inclusion. τ2-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ2-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity.
Separate analysis of host and pathogen metabolic changes in intracellular C. trachomatis infections is arduous and has not been comprehensively realized so far. A more detailed understanding about the metabolic activity and needs of C. trachomatis and its specific interactions with the host cell would be the basis for the development of novel treatment strategies. We therefore applied fluorescence lifetime imaging (FLIM) of the metabolic coenzymes NAD(P)H using two-photon microscopy to directly visualize metabolic changes of host cells and pathogens in living cells. NAD(P)H fluorescence was detected both on the chlamydial inclusion membrane and inside the inclusion. Interestingly, changes in chlamydial growth and progeny induced by glucose starvation and IFNγ treatment were directly linked to significant changes of the NAD(P)H fluorescence lifetimes inside the inclusions. Furthermore, measurement of the NAD(P)H fluorescence lifetime in the host cell nucleus revealed that infected cells were programmed for starvation during the metabolically active phase of intracellular chlamydial growth. Our findings highlight for the first time a direct interaction between host and pathogen metabolism in intracellular bacterial infections that exceeds sole competition for nutrients. In conclusion, fluorescence lifetime imaging of NAD(P)H by two-photon microscopy enables real-time analysis of metabolic host-pathogen interactions in intracellular infections with high spatial and temporal resolution.