RESUMEN
Chlamydia muridarum and Chlamydia caviae have equivalent growth rates in mouse epithelial cells but only C. muridarum replicates inside mouse macrophages, while C. caviae does not. Macrophages infected with C. muridarum or C. caviae were used to address the hypothesis that the early signaling pathways initiated during infection depend on the fate of chlamydiae in the host cell. Transmission electron microscopy of C. muridarum-infected macrophages showed intact chlamydial elementary bodies and reticulate bodies 2 h postinfection in compact vacuoles. Conversely, in macrophages infected with C. caviae, chlamydiae were observed in large phagocytic vacuoles. Furthermore, C. caviae infections failed to develop into inclusions or produce viable bacteria. Expression of proinflammatory cytokines TNFα, IL-1ß and MMP13 was similar in C. caviae- or C. muridarum-infected macrophages at 3 h postinfection, indicating that chlamydial survival is not required for initiation of these responses. IL-1ß secretion, dependent on inflammasome activation, occurred in C. caviae-infected macrophages despite no chlamydial growth. Conversely, IFNß mRNA was observed only in C. muridarum- but not in C. caviae-infected macrophages. These data demonstrate that differential signaling events are initiated during a productive versus nonproductive chlamydial infection in a macrophage.
Asunto(s)
Infecciones por Chlamydia/metabolismo , Infecciones por Chlamydia/microbiología , Chlamydia/fisiología , Espacio Intracelular/microbiología , Macrófagos/metabolismo , Macrófagos/microbiología , Transducción de Señal , Animales , Línea Celular , Chlamydia/crecimiento & desarrollo , Chlamydia/ultraestructura , Infecciones por Chlamydia/genética , Infecciones por Chlamydia/patología , Endosomas/metabolismo , Endosomas/ultraestructura , Regulación de la Expresión Génica , Inflamación/genética , Interleucina-1beta , Macrófagos/patología , Macrófagos/ultraestructura , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Microbial interactions represent an understudied facet of human health and disease. In this study, the interactions that occur between Chlamydia trachomatis and the opportunistic fungal pathogen, Candida albicans were investigated. Candida albicans is a common component of the oral and vaginal microbiota responsible for thrush and vaginal yeast infections. Normally, Candida exist in the body as yeast. However, disruptions to the microbiota create conditions that allow expanded growth of Candida, conversion to the hyphal form, and tissue invasion. Previous studies have shown that a myriad of outcomes can occur when Candida albicans interacts with pathogenic bacteria. To determine if C. trachomatis physically interacts with C. albicans, we incubated chlamydial elementary bodies (EB) in medium alone or with C. albicans yeast or hyphal forms for 1 h. Following incubation, the samples were formaldehyde-fixed and processed for immunofluorescence assays using anti-chlamydial MOMP or anti- chlamydial LPS antibodies. Replicate samples were replenished with culture medium and incubated at 35°C for 0-120 h prior to fixation for immunofluorescence analysis or collection for EB infectivity assays. Data from this study indicates that both C. trachomatis serovar E and C. muridarum EB bind to C. albicans yeast and hyphal forms. This interaction was not blocked by pre-incubation of EB with the Candida cell wall components, mannan or ß-glucans, suggesting that EB interact with a Candida cell wall protein or other structure. Bound EB remained attached to C. albicans for a minimum of 5 days (120 h). Infectivity assays demonstrated that EB bound to C. albicans are infectious immediately following binding (0h). However, once bound to C. albicans, EB infectivity decreased at a faster rate than EB in medium alone. At 6h post binding, 40% of EB incubated in medium alone remained infectious compared to only 16% of EB bound to C. albicans. Likewise, pre-incubation of EB with laminarin, a soluble preparation of ß-glucan, alone or in combination with other fungal cell wall components significantly decreases chlamydial infectivity in HeLa cells. These data indicate that interactions between EB and C. albicans inhibit chlamydial infectivity, possibly by physically blocking EB interactions with host cell receptors.
RESUMEN
Chlamydia trachomatis infections represent the predominant cause of bacterial sexually transmitted infections. As an obligate intracellular bacterium, C. trachomatis is dependent on the host cell for survival, propagation, and transmission. Thus, factors that affect the host cell, including nutrition, cell cycle, and environmental signals, have the potential to impact chlamydial development. Previous studies have demonstrated that activation of Wnt/ß-catenin signaling benefits C. trachomatis infections in fallopian tube epithelia. In cervical epithelial cells chlamydiae sequester ß-catenin within the inclusion. These data indicate that chlamydiae interact with the Wnt signaling pathway in both the upper and lower female genital tract (FGT). However, hormonal activation of canonical and non-canonical Wnt signaling pathways is an essential component of cyclic remodeling in another prominent area of the FGT, the endometrium. Given this information, we hypothesized that Wnt signaling would impact chlamydial infection in endometrial epithelial cells. To investigate this hypothesis, we analyzed the effect of Wnt inhibition on chlamydial inclusion development and elementary body (EB) production in two endometrial cell lines, Ishikawa (IK) and Hec-1B, in nonpolarized cell culture and in a polarized endometrial epithelial (IK)/stromal (SHT-290) cell co-culture model. Inhibition of Wnt by the small molecule inhibitor (IWP2) significantly decreased inclusion size in IK and IK/SHT-290 cultures (p < 0.005) and chlamydial infectivity (p ≤ 0.01) in both IK and Hec-1B cells. Confocal and electron microscopy analysis of chlamydial inclusions revealed that Wnt inhibition caused chlamydiae to become aberrant in morphology. EB formation was also impaired in IK, Hec-1B and IK/SHT-290 cultures regardless of whether Wnt inhibition occurred throughout, in the middle (24 hpi) or late (36 hpi) during the development cycle. Overall, these data lead us to conclude that Wnt signaling in the endometrium is a key host pathway for the proper development of C. trachomatis.
Asunto(s)
Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/fisiología , Endometrio/microbiología , Células Epiteliales/microbiología , Interacciones Huésped-Patógeno , Vía de Señalización Wnt , Línea Celular , Técnicas de Cocultivo , Femenino , Humanos , Cuerpos de Inclusión/microbiología , Microscopía Confocal , Microscopía Electrónica , Modelos BiológicosRESUMEN
Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).
Asunto(s)
Enterocitos/metabolismo , Mucosa Intestinal/metabolismo , Metabolismo de los Lípidos/fisiología , Transporte Biológico , Células CACO-2 , Enterocitos/citología , Expresión Génica , Humanos , Absorción Intestinal , Lipoproteínas/metabolismo , Microscopía Electrónica de Transmisión , TransfecciónRESUMEN
Interaction of Herpes Simplex Virus (HSV) glycoprotein D (gD) with the host cell surface during Chlamydia trachomatis/HSV co-infection stimulates chlamydiae to become persistent. During viral entry, gD interacts with one of 4 host co-receptors: HVEM (herpes virus entry mediator), nectin-1, nectin-2 and 3-O-sulfated heparan sulfate. HVEM and nectin-1 are high-affinity entry receptors for both HSV-1 and HSV-2. Nectin-2 mediates HSV-2 entry but is inactive for HSV-1, while 3-O-sulfated heparan sulfate facilitates HSV-1, but not HSV-2, entry. Western blot and RT-PCR analyses demonstrate that HeLa and HEC-1B cells express nectin-1 and nectin-2, but not HVEM. Because both HSV-1 and HSV-2 trigger persistence, these data suggest that nectin-1 is the most likely co-receptor involved. Co-infections with nectin-1 specific HSV-1 mutants stimulate chlamydial persistence, as evidenced by aberrant body (AB) formation and decreased production of elementary bodies (EBs). These data indicate that nectin-1 is involved in viral-induced chlamydial persistence. However, inhibition of signal transduction molecules associated with HSV attachment and entry does not rescue EB production during C. trachomatis/HSV-2 co-infection. HSV attachment also does not activate Cdc42 in HeLa cells, as would be expected with viral stimulated activation of nectin-1 signaling. Additionally, immunofluorescence assays confirm that HSV infection decreases nectin-1 expression. Together, these observations suggest that gD binding-induced loss of nectin-1 signaling negatively influences chlamydial growth. Chlamydial infection studies in nectin-1 knockdown (NKD) HeLa cell lines support this hypothesis. In NKD cells, chlamydial inclusions are smaller in size, contain ABs, and produce significantly fewer infectious EBs compared to C. trachomatis infection in control HeLa cells. Overall, the current study indicates that the actions of host molecule, nectin-1, are required for successful C. trachomatis development.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Infecciones por Chlamydia/metabolismo , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/clasificación , Chlamydia trachomatis/fisiología , Interacciones Huésped-Patógeno , Animales , Moléculas de Adhesión Celular/genética , Línea Celular , Infecciones por Chlamydia/genética , Coinfección , Cricetinae , Expresión Génica , Técnicas de Inactivación de Genes , Células HeLa , Herpesvirus Humano 1 , Humanos , Cuerpos de Inclusión Viral , Nectinas , Estrés Oxidativo , Miembro 14 de Receptores del Factor de Necrosis Tumoral/genética , Miembro 14 de Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Simplexvirus/fisiología , Acoplamiento Viral , Internalización del VirusRESUMEN
The small intestine generally transports dietary fats to circulation in triglyceride (TG)-rich lipoproteins. The two main intestinal lipoproteins are chylomicron (CM) and very low-density lipoprotein (VLDL). Unfortunately, studies on the CM biogenesis and intestinal transport of dietary fats have been hampered by the lack of an adequate in vitro model. In this study, we investigated the possible factors that might increase the efficiency of CM production by Caco-2 cells. We utilized sequential NaCl gradient ultracentrifugation to isolate the CMs that were secreted by the Caco-2 cells. To confirm the successful isolation of the CMs, we performed Fat Red 7B staining, TG reading, apolipoprotein B (ApoB) measurement, and transmission electron microcopy (TEM) analysis. We then tested the effects of cell differentiation, oleic acid, mono-olein, egg lecithin, incubation time, and collagen matrix on CM secretion. We found that cell differentiation, oleic acid, and lecithin were critical for CM secretion. Using the Transwell system, we further confirmed that the CMs produced by our Caco-2 cells contained significant amount of TGs and ApoB-48 such that they could be detected without the use of isotope labeling. In conclusion, when fully differentiated Caco-2 were challenged with oleic acid, lecithin, and sodium taurocholate, 21% of their total number of lipoproteins were CMs with the diameter of 80-200 nm.
RESUMEN
Chlamydia trachomatis, the most common bacterial sexually transmitted disease agent worldwide, enters a viable, non-dividing and non-infectious state (historically termed persistence and more recently referred to as the chlamydial stress response) when exposed to penicillin G in culture. Notably, penicillin G-exposed chlamydiae can reenter the normal developmental cycle upon drug removal and are resistant to azithromycin-mediated killing. Because penicillin G is less frequently prescribed than other ß-lactams, the clinical relevance of penicillin G-induced chlamydial persistence/stress has been questioned. The goal of this study was to determine whether more commonly used penicillins also induce C. trachomatis serovar E persistence/stress. All penicillins tested, as well as clavulanic acid, induced formation of aberrant, enlarged reticulate bodies (RB) (called aberrant bodies or AB) characteristic of persistent/stressed chlamydiae. Exposure to the penicillins and clavulanic acid also reduced chlamydial infectivity by >95%. None of the drugs tested significantly reduced chlamydial unprocessed 16S rRNA or genomic DNA accumulation, indicating that the organisms were viable, though non-infectious. Finally, recovery assays demonstrated that chlamydiae rendered essentially non-infectious by exposure to ampicillin, amoxicillin, carbenicillin, piperacillin, penicillin V, and clavulanic acid recovered infectivity after antibiotic removal. These data definitively demonstrate that several commonly used penicillins induce C. trachomatis persistence/stress at clinically relevant concentrations.
Asunto(s)
Antibacterianos/farmacología , Chlamydia trachomatis/efectos de los fármacos , Chlamydia trachomatis/fisiología , Estrés Fisiológico/efectos de los fármacos , beta-Lactamas/farmacología , Línea Celular , Células Cultivadas , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/ultraestructura , ADN Bacteriano/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Penicilinas/farmacología , ARN Ribosómico 16S/efectos de los fármacos , ARN Ribosómico 16S/genéticaRESUMEN
The oestrogen receptor (ER) α-ß+ HEC-1B and the ERα+ß+ Ishikawa (IK) cell lines were investigated to dissect the effects of oestrogen exposure on several parameters of Chlamydia trachomatis infection. Antibody blockage of ERα or ERß alone or simultaneously significantly decreased C. trachomatis infectivity (45-68%). Addition of the ERß antagonist, tamoxifen, to IK or HEC-1B prior to or after chlamydial infection caused a 30-90% decrease in infectivity, the latter due to disrupted eukaryotic organelles. In vivo, endometrial glandular epithelial cells are stimulated by hormonally influenced stromal signals. Accordingly, chlamydial infectivity was significantly increased by 27% and 21% in IK and HEC-1B cells co-cultured with SHT-290 stromal cells exposed to oestrogen. Endometrial stromal cell/epithelial cell co-culture revealed indirect effects of oestrogen on phosphorylation of extracellular signal-regulated kinase and calcium-dependant phospholipase A2 and significantly increased production of interleukin (IL)-8 and IL-6 in both uninfected and chlamydiae-infected epithelial cells. These results indicate that oestrogen and its receptors play multiple roles in chlamydial infection: (i) membrane oestrogen receptors (mERs) aid in chlamydial entry into host cells, and (ii) mER signalling may contribute to inclusion development during infection. Additionally, enhancement of chlamydial infection is affected by hormonally influenced stromal signals in conjunction with direct oestrogen stimulation of the human epithelia.
Asunto(s)
Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/crecimiento & desarrollo , Chlamydia trachomatis/patogenicidad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Estrógenos/metabolismo , Línea Celular , Técnicas de Cocultivo , Perfilación de la Expresión Génica , Humanos , Microscopía Electrónica de Transmisión , Modelos Biológicos , Receptores de Estrógenos/metabolismoRESUMEN
We utilized a recently developed model of intracervical infection with Chlamydia muridarum in the mouse to elicit a relatively synchronous infection during the initial developmental cycle in order to examine at the ultrastructural level the development of both the chlamydial inclusion and the onset of the inflammatory response. At 18 h after infection, only a few elementary bodies attached to cells were visible, as were an occasional intracellular intermediate body and reticulate body. By 24 h, inclusions had 2 to 5 reticulate bodies and were beginning to fuse. A few polymorphonuclear leukocytes (PMNs) were already present in the epithelium in the vicinity of and directly adjacent to infected cells. By 30 h, the inclusions were larger and consisted solely of reticulate bodies, but by 36 to 42 h, they contained intermediate bodies and elementary bodies as well. Many PMNs were adjacent to or actually inside infected cells. Chlamydiae appeared to exit the cell either (i) through disintegration of the inclusion membrane and rupture of the cell, (ii) by dislodgement of the cell from the epithelium by PMNs, or (iii) by direct invasion of the infected cell by the PMNs. When PMNs were depleted, the number of released elementary bodies was significantly greater as determined both visually and by culture. Interestingly, depletion of PMNs revealed the presence of inclusions containing aberrant reticulate bodies, reminiscent of effects seen in vitro when chlamydiae are incubated with gamma interferon. In vivo evidence for the contact-dependent development hypothesis, a potential mechanism for triggering the conversion of reticulate bodies to elementary bodies, and for translocation of lipid droplets into the inclusion is also presented.
Asunto(s)
Chlamydia muridarum/inmunología , Chlamydia muridarum/ultraestructura , Cuerpos de Inclusión/microbiología , Cuerpos de Inclusión/ultraestructura , Neutrófilos/microbiología , Neutrófilos/ultraestructura , Animales , Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/microbiología , Modelos Animales de Enfermedad , Células Epiteliales/microbiología , Ratones , Ratones Endogámicos C57BL , Enfermedades de los Roedores/inmunología , Enfermedades de los Roedores/microbiología , Factores de TiempoRESUMEN
The initial host response in a primary chlamydial infection is the onset of acute inflammation. However, we still know very little about the early temporal events in the induction of the acute inflammatory response and how these events relate to the initial chlamydial developmental cycle in an actual genital infection. Because it was critical to initiate a synchronous infection in the endocervix in the first 24 h to evaluate the sequential expression of the host response, we developed the surgical methodology of depositing Chlamydia muridarum directly on the endocervix. Cervical tissue was collected at 3, 12, and 24 h after inoculation and the expression array of chemokines, cytokines, and receptors was assessed to characterize the response during the initial developmental cycle. Polymorphonuclear leukocyte (PMN) infiltration was first observed at 12 h after inoculation, and a few PMNs could be seen in the epithelium at 24 h. Electron microscopic analysis at 24 h showed that virtually all inclusions were at the same stage of development, indicating a synchronous infection. Several chemokine and cytokine genes were expressed as early as 3 h after infection, but by 12 h, 41 genes were expressed. Thus, activation of the host response occurs both with the introduction of elementary bodies into the host and early replication of reticulate bodies. No significant response was observed when UV-inactivated organisms were inoculated into the cervix at any time interval. This model provides an ideal opportunity to investigate the mechanisms by which the early inflammatory response is induced in vivo.
Asunto(s)
Cuello del Útero/metabolismo , Infecciones por Chlamydia/metabolismo , Chlamydia muridarum/fisiología , Citocinas/metabolismo , Enfermedades del Cuello del Útero/microbiología , Animales , Femenino , Inflamación/metabolismo , Ratones , Factores de Tiempo , Enfermedades del Cuello del Útero/metabolismoRESUMEN
While much is known about the attachment of the chlamydiae to the host cell and intracellular events during the developmental cycle, little is known about the mechanism(s) by which elementary bodies exit the cell. In this report, we use the guinea-pig conjunctival model of Chlamydia caviae infection to present in vivo ultrastructural evidence supporting two mechanisms for release of chlamydiae from the mucosal epithelia. Four days after infection, histopathologic observation shows an intense infiltration of polymorphonuclear leukocytes (PMN) in the conjunctival epithelium. Using transmission electron microscopy, a gradient-directed PMN response to chlamydiae-infected epithelial cells was observed. As PMN infiltration intensifies, epithelial hemidesmosome/integrin/focal adhesion adherence with the basal lamina is disconnected and PMNs literally lift off and release infected superficial epithelia from the mucosa. Many of these infected cells appear to be healthy with intact microvilli, nuclei, and mitochondria. While lysis of some infected cells occurs with release of chlamydiae into the extracellular surface milieu, the majority of infected cells are pushed off the epithelium. We propose that PMNs play an active role in detaching infected cells from the epithelium and that these infected cells eventually die releasing organisms but, in the process, move to new tissue sites via fluid dynamics.
Asunto(s)
Chlamydia/patogenicidad , Conjuntiva/inmunología , Conjuntivitis de Inclusión/transmisión , Células Epiteliales/microbiología , Neutrófilos/inmunología , Animales , Adhesión Celular , Quimiotaxis de Leucocito , Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/microbiología , Infecciones por Chlamydia/transmisión , Conjuntiva/citología , Conjuntiva/microbiología , Conjuntiva/ultraestructura , Conjuntivitis de Inclusión/inmunología , Conjuntivitis de Inclusión/microbiología , Modelos Animales de Enfermedad , Células Epiteliales/inmunología , Células Epiteliales/ultraestructura , Femenino , Cobayas , Humanos , Microscopía Electrónica de Transmisión , Especificidad de ÓrganosRESUMEN
A common model for studying Chlamydia trachomatis and growing chlamydial stocks uses Lymphogranuloma venereum serovar L2 and non-polarized HeLa cells. However, recent publications indicate that the growth rate and progeny yields can vary considerably for a particular strain depending on the cell line/type used, and seem to be partially related to cell tropism. In the present study, the growth of invasive serovar L2 was compared in endometrial HEC-1B and endocervical HeLa cells polarized on collagen-coated microcarrier beads, as well as in HeLa cells grown in tissue culture flasks. Microscopy analysis revealed no difference in chlamydial attachment/entry patterns or in inclusion development throughout the developmental cycle between cell lines. Very comparable growth curves in both cell lines were also found using real-time PCR analysis, with increases in chlamydial DNA content of 400-500-fold between 2 and 36 h post-inoculation. Similar progeny yields with comparable infectivity were recovered from HEC-1B and HeLa cell bead cultures, and no difference in chlamydial growth was found in polarized vs. non-polarized HeLa cells. In conclusion, unlike other C. trachomatis strains such as urogenital serovar E, invasive serovar L2 grows equally well in physiologically different endometrial and endocervical environments, regardless of the host cell polarization state.
Asunto(s)
Chlamydia trachomatis/crecimiento & desarrollo , Endometrio/microbiología , Chlamydia trachomatis/aislamiento & purificación , ADN Bacteriano/análisis , Endometrio/ultraestructura , Femenino , Células HeLa , Humanos , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Microesferas , Reacción en Cadena de la PolimerasaRESUMEN
Chlamydial 60-kDa heat shock proteins (cHsp60s) are known to play a prominent role in the immunopathogenesis of disease. It is also known that several stress-inducing growth conditions, such as heat, iron deprivation, or exposure to gamma interferon, result in the development of persistent chlamydial forms that often exhibit enhanced expression of cHsp60. We have shown previously that the expression of cHsp60 is greatly enhanced in Chlamydia trachomatis serovar E propagated in an iron-deficient medium. The objective of this work was to determine which single cHsp60 or combination of the three cHsp60 homologs encoded by this organism responds to iron limitation. Using monospecific polyclonal peptide antisera that recognize only cHsp60-1, cHsp60-2, or cHsp60-3, we found that expression of cHsp60-2 is responsive to iron deprivation. Overall, our studies suggest that the expression of cHsp60 homologs differs among the mechanisms currently known to induce persistence.
Asunto(s)
Chaperonina 60/metabolismo , Chlamydia trachomatis/crecimiento & desarrollo , Endometrio/microbiología , Células Epiteliales/microbiología , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Chaperonina 60/genética , Chlamydia trachomatis/clasificación , Chlamydia trachomatis/patogenicidad , Endometrio/citología , Femenino , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , SerotipificaciónRESUMEN
Epidemiological studies have demonstrated that co-infections of herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occur in vivo. Data from a tissue culture model of C. trachomatis/HSV-2 co-infection indicate that viral co-infection stimulates the formation of persistent chlamydiae. Transmission electron microscopic (TEM) analyses demonstrated that in both HeLa and HEC-1B cells, co-infection caused developing chlamydiae to exhibit swollen, aberrantly shaped reticulate bodies (RBs), characteristically observed in persistence. Additionally, HSV-2 co-infection suppressed production of infectious chlamydial elementary bodies (EBs) in both host cell types. Co-infection with HSV type 1 (HSV-1) produced similar morphologic alterations and abrogated infectious EB production. These data indicate that virus-induced chlamydial persistence was neither host cell- nor virus strain-specific. Purification of crude HSV-2 stocks demonstrated that viral particles were required for coinfection-induced chlamydial persistence to occur. Finally, co-infection with either UV-inactivated, replication-incompetent virus or replication-competent HSV-2 in the presence of cyclohexamide reduced chlamydial infectivity without altering chlamydial genomic DNA accumulation. These data demonstrate that productive viral replication is not required for the induction of chlamydial persistence and suggest that HSV attachment and entry can provide the necessary stimulus to alter C. trachomatis development.
Asunto(s)
Chlamydia trachomatis/efectos de los fármacos , Cicloheximida/farmacología , Herpesvirus Humano 2/efectos de los fármacos , Animales , Línea Celular , Chlamydia trachomatis/fisiología , Chlorocebus aethiops , Células Epiteliales/microbiología , Células Epiteliales/ultraestructura , Células Epiteliales/virología , Células HeLa , Herpesvirus Humano 2/fisiología , Humanos , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Inhibidores de la Síntesis de la Proteína/farmacología , Factores de Tiempo , Células VeroRESUMEN
In vitro studies of obligate intracellular chlamydia biology and pathogenesis are highly dependent on the use of experimental models and growth conditions that mimic the mucosal architecture and environment these pathogens encounter during natural infections. In this study, the growth of Chlamydia trachomatis genital serovar E was monitored in mouse fibroblast McCoy cells and compared to more relevant host human epithelial endometrium-derived HEC-1B and cervix-derived HeLa cells, seeded and polarized on collagen-coated microcarrier beads, using a three-dimensional culture system. Microscopy analysis of these cell lines prior to infection revealed morphological differences reminiscent of their in vivo architecture. Upon infection, early chlamydial inclusion distribution was uniform in McCoy cells but patchy in both epithelial cell lines. Although no difference in chlamydial attachment to or entry into the two genital epithelial cell lines was noted, active bacterial genome replication and transcription, as well as initial transformation of elementary bodies to reticulate bodies, were detected earlier in HEC-1B than in HeLa cells, suggesting a faster growth, which led to higher progeny counts and titers in HEC-1B cells upon completion of the developmental cycle. Chlamydial development in the less relevant McCoy cells was very similar to that in HeLa cells, although higher progeny counts were obtained. In conclusion, this three-dimensional bead culture system represents an improved model for harvesting large quantities of infectious chlamydia progeny from their more natural polarized epithelial host cells.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Chlamydia trachomatis/crecimiento & desarrollo , Células Epiteliales/microbiología , Animales , Línea Celular , Polaridad Celular , Citoplasma/microbiología , Replicación del ADN , ADN Bacteriano/análisis , Endometrio/citología , Células Epiteliales/citología , Femenino , Fibroblastos/microbiología , Células HeLa , Humanos , Cuerpos de Inclusión , Ratones , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Microesferas , ARN Bacteriano/análisis , Transcripción GenéticaRESUMEN
Modification of the phosphate groups of lipid A with amine-containing substituents, such as phosphoethanolamine, reduces the overall net negative charge of gram-negative bacterial lipopolysaccharide, thereby lowering its affinity to cationic antimicrobial peptides. Modification of the 1 position of Helicobacter pylori lipid A is a two-step process involving the removal of the 1-phosphate group by a lipid A phosphatase, LpxEHP (Hp0021), followed by the addition of a phosphoethanolamine residue catalyzed by EptAHP (Hp0022). To demonstrate the importance of modifying the 1 position of H. pylori lipid A, we generated LpxEHP-deficient mutants in various H. pylori strains by insertion of a chloramphenicol resistance cassette into lpxEHP and examined the significance of LpxE with respect to cationic antimicrobial peptide resistance. Using both mass spectrometry analysis and an in vitro assay system, we showed that the loss of LpxEHP activity in various H. pylori strains resulted in the loss of modification of the 1 position of H. pylori lipid A, thus confirming the function of LpxEHP. Due to its unique lipid A structure, H. pylori is highly resistant to the antimicrobial peptide polymyxin (MIC > 250 microg/ml). However, disruption of lpxEHP in H. pylori results in a dramatic decrease in polymyxin resistance (MIC, 10 microg/ml). In conclusion, we have characterized the first gram-negative LpxE-deficient mutant and have shown the importance of modifying the 1 position of H. pylori lipid A for resistance to polymyxin.
Asunto(s)
Helicobacter pylori/enzimología , Monoéster Fosfórico Hidrolasas/fisiología , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/ultraestructura , Farmacorresistencia Bacteriana , Etanolaminas , Helicobacter pylori/efectos de los fármacos , Lípido A/química , Lípido A/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Polimixinas/farmacologíaRESUMEN
Several chlamydial antigens have been detected in the infected epithelial cell cytosol and on the host cell surface prior to their presumed natural release at the end of the 72-96 h developmental cycle. These extra-inclusion antigens are proposed to influence vital host cell functions, antigen trafficking and presentation and, ultimately, contribute to a prolonged inflammatory response. To begin to dissect the mechanisms for escape of these antigens from the chlamydial inclusion, which are enhanced on exposure to antibiotics, polarized endometrial epithelial cells (HEC-1B) were infected with Chlamydia trachomatis serovar E for 36 h or 48 h. Infected cells were then exposed to chemotactic human polymorphonuclear neutrophils not loaded or pre-loaded in vitro with the antibiotic azithromycin. Viewed by electron microscopy, the azithromycin-mediated killing of chlamydiae involved an increase in chlamydial outer membrane blebbing followed by the appearance of the blebs in larger vesicles (i) everting from but still associated with the inclusion as well as (ii) external to the inclusion. Evidence that the vesicles originated from the chlamydial inclusion membrane was shown by immuno-localization of inclusion membrane proteins A, F, and G on the vesicular membranes. Chlamydial heat shock protein 60 (chsp60) copies 2 and 3, but not copy 1, were released from RB and incorporated into the everted inclusion membrane vesicles and delivered to the infected cell surface. These data represent direct evidence for one mechanism of early antigen delivery, albeit membrane-bound, beyond the confines of the chlamydial inclusion.
Asunto(s)
Antígenos Bacterianos/inmunología , Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/inmunología , Chlamydia trachomatis/ultraestructura , Vesículas Citoplasmáticas/ultraestructura , Secuencia de Aminoácidos , Antibacterianos/farmacología , Azitromicina/farmacología , Proteínas Bacterianas/metabolismo , Línea Celular , Chaperonina 60/metabolismo , Infecciones por Chlamydia/tratamiento farmacológico , Infecciones por Chlamydia/microbiología , Vesículas Citoplasmáticas/inmunología , Endometrio/citología , Endometrio/inmunología , Endometrio/microbiología , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Femenino , Aparato de Golgi/inmunología , Aparato de Golgi/ultraestructura , Humanos , Cuerpos de Inclusión/efectos de los fármacos , Cuerpos de Inclusión/inmunología , Cuerpos de Inclusión/microbiología , Cuerpos de Inclusión/ultraestructura , Proteínas de la Membrana/metabolismo , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Datos de Secuencia MolecularRESUMEN
Mast cells are bone marrow-derived cells that are widely distributed in the tissue. They are found predominantly in the subepithelial tissue near blood vessels and nerves and usually are sprinkled diffusely without forming clusters. In tissue sections stained with hematoxylin and eosin, normal mast cells usually display a round-to-oval nucleus with clumped chromatin and indistinct or no nucleoli. They have moderately abundant cytoplasm and are oval, spindle, or polygonal in shape. The cytoplasm is amphophilic, and sometimes small slightly eosinophilic granules may be visible. Hematoxylin and eosin staining is not a specific or reliable method for detecting mast cells in tissue sections because of variable cellular morphology. For confirmation of mast cells, special stains, such as mast cell tryptase or CD117, are required.
Asunto(s)
Mastocitos/química , Mastocitos/ultraestructura , Coloración y Etiquetado/métodos , Microscopía Inmunoelectrónica/métodos , Proteínas Proto-Oncogénicas c-kit/análisis , Serina Endopeptidasas/análisis , Fijación del Tejido/métodos , TriptasasRESUMEN
Epidemiological and clinical studies have shown that double infection with herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occurs in vivo. We hypothesized that co-infection would alter replication of these agents. To test this hypothesis, HeLa cells were infected with C. trachomatis serovar E, followed 24 h later by HSV-2 strain 333. Transmission electron microscopic (TEM) analyses indicated that, by 10 h after HSV addition, reticulate bodies (RBs) in co-infected cells were swollen, aberrantly shaped and electron-lucent. In infectious titre assays, HSV-2 co-infection abrogated production of infectious chlamydial progeny. Western blot analyses indicated that accumulation of chlamydial major outer membrane protein (MOMP) was decreased by HSV co-infection while accumulation of chlamydial heat-shock protein 60-1 (HSP60-1) was increased. Polymerase chain reaction (PCR) experiments indicated that chlamydial genome copy number was unaltered by HSV-2 superinfection. Semi-quantitative, reverse transcription PCR (RT-PCR) experiments demonstrated that levels of chlamydial groEL, ftsK, ftsW, dnaA and unprocessed 16S rRNA transcripts were not changed by HSV-2 super-infection. These data indicate that HSV-2 superinfection drives chlamydia into a viable but non-cultivable state, which is the hallmark of persistence. Because chlamydial HSP60-1 has been associated with immunopathology in vivo, these results also suggest that disease severity might be increased in co-infected individuals.
Asunto(s)
Chlamydia trachomatis/fisiología , Herpesvirus Humano 2/fisiología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Chaperonina 60/metabolismo , Infecciones por Chlamydia/complicaciones , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/genética , Chlamydia trachomatis/ultraestructura , Dosificación de Gen , Células HeLa , Herpes Genital/complicaciones , Herpes Genital/virología , Herpesvirus Humano 2/ultraestructura , Humanos , Microscopía Electrónica de Transmisión , ARN Bacteriano/metabolismo , ARN Ribosómico 16S/metabolismo , Sobreinfección/complicaciones , Sobreinfección/virologíaRESUMEN
Chlamydial attachment and infectivity in vitro and ascending disease and sequelae in vivo have been reported to be enhanced/modulated by estrogen. Endometrial carcinoma cell lines Ishikawa and HEC-1B and the breast cancer lines MCF-7 and HCC-1806 were examined for Chlamydia trachomatis E infectivity. Estrogen receptor (ER) presence was confirmed by Western blot and qRT-PCR analyses. FACS analysis was used to determine the percent of plasma membrane-localized ERs (mERs), and their activity was tested by estrogen binding and competitive estrogen antagonists assays. Chlamydiae grew in all cell lines with HEC (90%) >> MCF-7 (57%)>Ishikawa (51%) >> HCC-1806 (20%). The cell line ER isoform composition was re-defined as: ERalpha + ERbeta + for MCF-7, HCC-1806 and Ishikawa; and ERbeta only for HEC-1B. HeLa cells were also tested and found to express ERbeta, but not ERalpha. A small percentage of both ERs were surface-exposed and functionally active. The endometrium-predominant ERbeta isoform was found in all cell lines, including those most representative of the common sites of C. trachomatis infection. Thus, the role of chlamydial attachment/infectivity will now be analyzed in ERbeta+and-isogenic HEC-1B cells.