RESUMO
The antibiotic, fosmidomycin (FSM) targets the methylerythritol phosphate (MEP) pathway of isoprenoid synthesis by inhibiting the essential enzyme, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) and is lethal to intracellular parasites and bacteria. The obligate intracellular bacterial pathogen, Chlamydia trachomatis, alternates between two developmental forms: the extracellular, infectious elementary body (EB), and the intracellular, replicative form called the reticulate body (RB). Several stressful growth conditions including iron deprivation halt chlamydial cell division and cause development of a morphologically enlarged, but viable form termed an aberrant body (AB). This phenotype constitutes the chlamydial developmental state known as persistence. This state is reversible as removal of the stressor allows the chlamydiae to re-enter and complete the normal developmental cycle. Bioinformatic analysis indicates that C. trachomatis encodes a homolog of Dxr, but its function and the requirement for isoprenoid synthesis in chlamydial development is not fully understood. We hypothesized that chlamydial Dxr (DxrCT) is functional and that the methylerythritol phosphate (MEP) pathway is required for normal chlamydial development. Thus, FSM exposure should be lethal to C. trachomatis. Overexpression of chlamydial Dxr (DxrCT) in Escherichia coli under FSM exposure and in a conditionally lethal dxr mutant demonstrated that DxrCT functions similarly to E. coli Dxr. When Chlamydia-infected cultures were exposed to FSM, EB production was significantly reduced. However, titer recovery assays, electron microscopy, and peptidoglycan labeling revealed that FSM inhibition of isoprenoid synthesis is not lethal to C. trachomatis, but instead induces persistence. Bactoprenol is a critical isoprenoid required for peptidoglycan precursor assembly. We therefore conclude that FSM induces persistence in Chlamydia by preventing bactoprenol production necessary for peptidoglycan precursor assembly and subsequent cell division.
Assuntos
Antibacterianos/farmacologia , Chlamydia trachomatis/efeitos dos fármacos , Fosfomicina/análogos & derivados , Peptidoglicano/biossíntese , Terpenos/metabolismo , Aldose-Cetose Isomerases/antagonistas & inibidores , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Linhagem Celular Tumoral , Infecções por Chlamydia/patologia , Chlamydia trachomatis/enzimologia , Chlamydia trachomatis/fisiologia , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfomicina/farmacologia , Células HeLa , HumanosRESUMO
Peptidoglycan, the sugar-amino acid polymer that composes the bacterial cell wall, requires a significant expenditure of energy to synthesize and is highly immunogenic. To minimize the loss of an energetically expensive metabolite and avoid host detection, bacteria often recycle their peptidoglycan, transporting its components back into the cytoplasm, where they can be used for subsequent rounds of new synthesis. The peptidoglycan-recycling substrate binding protein (SBP) MppA, which is responsible for recycling peptidoglycan fragments in Escherichia coli, has not been annotated for most intracellular pathogens. One such pathogen, Chlamydia trachomatis, has a limited capacity to synthesize amino acids de novo and therefore must obtain oligopeptides from its host cell for growth. Bioinformatics analysis suggests that the putative C. trachomatis oligopeptide transporter OppABCDF (OppABCDF Ct ) encodes multiple SBPs (OppA1 Ct , OppA2 Ct , and OppA3 Ct ). Intracellular pathogens often encode multiple SBPs, while only one, OppA, is encoded in the E. coliopp operon. We hypothesized that the putative OppABCDF transporter of C. trachomatis functions in both oligopeptide transport and peptidoglycan recycling. We coexpressed the putative SBP genes (oppA1Ct , oppA2Ct , oppA3Ct ) along with oppBCDFCt in an E. coli mutant lacking the Opp transporter and determined that all three chlamydial OppA subunits supported oligopeptide transport. We also demonstrated the in vivo functionality of the chlamydial Opp transporter in C. trachomatis Importantly, we found that one chlamydial SBP, OppA3 Ct , possessed dual substrate recognition properties and is capable of transporting peptidoglycan fragments (tri-diaminopimelic acid) in E. coli and in C. trachomatis These findings suggest that Chlamydia evolved an oligopeptide transporter to facilitate the acquisition of oligopeptides for growth while simultaneously reducing the accumulation of immunostimulatory peptidoglycan fragments in the host cell cytosol. The latter property reflects bacterial pathoadaptation that dampens the host innate immune response to Chlamydia infection.
Assuntos
Chlamydia trachomatis/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oligopeptídeos/metabolismo , Peptidoglicano/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico/genética , Transporte Biológico/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Parede Celular/genética , Parede Celular/metabolismo , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/genética , Citosol/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos/genética , Células HeLa , Humanos , Imunidade Inata/genética , Proteínas de Membrana Transportadoras/genética , Oligopeptídeos/genética , Óperon/genética , Peptidoglicano/genéticaRESUMO
The Chlamydia trachomatis genome encodes multiple bifunctional enzymes, such as DapF, which is capable of both diaminopimelic acid (DAP) epimerase and glutamate racemase activity. Our previous work demonstrated the bifunctional activity of chlamydial DapF in vitro and in a heterologous system (Escherichia coli). In the present study, we employed a substrate competition strategy to demonstrate DapF Ct function in vivo in C. trachomatis We reasoned that, because DapF Ct utilizes a shared substrate-binding site for both racemase and epimerase activities, only one activity can occur at a time. Therefore, an excess of one substrate relative to another must determine which activity is favored. We show that the addition of excess l-glutamate or meso-DAP (mDAP) to C. trachomatis resulted in 90% reduction in bacterial titers, compared to untreated controls. Excess l-glutamate reduced in vivo synthesis of mDAP by C. trachomatis to undetectable levels, thus confirming that excess racemase substrate led to inhibition of DapF Ct DAP epimerase activity. We previously showed that expression of dapFCt in a murI (racemase) ΔdapF (epimerase) double mutant of E. coli rescues the d-glutamate auxotrophic defect. Addition of excess mDAP inhibited growth of this strain, but overexpression of dapFCt allowed the mutant to overcome growth inhibition. These results confirm that DapF Ct is the primary target of these mDAP and l-glutamate treatments. Our findings demonstrate that suppression of either the glutamate racemase or epimerase activity of DapF compromises the growth of C. trachomatis Thus, a substrate competition strategy can be a useful tool for in vivo validation of an essential bifunctional enzyme.
Assuntos
Isomerases de Aminoácido/metabolismo , Chlamydia trachomatis/fisiologia , Peptidoglicano/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Infecções por Chlamydia/microbiologia , Ácido Diaminopimélico/metabolismo , Regulação Bacteriana da Expressão Gênica , Ácido Glutâmico/metabolismo , Interações Hospedeiro-Patógeno , HumanosRESUMO
The role of a slaughterhouse worker (SHW) involves the authorized killing of living beings, yet there is limited understanding of the consequences this behavior has on their well-being. The purpose of this systematic review is to collate and evaluate the current literature on the psychological impact of slaughterhouse employment. Fourteen studies met the specific a priori inclusion criteria. The findings from this review were demarcated by the focus of studies: (1) the prevalence of mental health disorders, (2) the types of coping mechanisms used, and (3) the link between slaughterhouse employment and crime perpetration. It was found that SHWs have a higher prevalence rate of mental health issues, in particular depression and anxiety, in addition to violence-supportive attitudes. Furthermore, the workers employ a variety of both adaptive and maladaptive strategies to cope with the workplace environment and associated stressors. Finally, there is some evidence that slaughterhouse work is associated with increased crime levels. The research reviewed has shown a link between slaughterhouse work and antisocial behavior generally and sexual offending specifically. There was no support for such an association with violent crimes, however. Based on existing research, we suggest future directions for research (i.e., applying more methodological rigor) but highlight key findings for practitioners and policymakers that warrant attention.
Assuntos
Matadouros , Violência , Humanos , Crime , Transtornos de Ansiedade , EmpregoRESUMO
Chlamydia trachomatis/HSV-2 vaginal co-infections are seen clinically, suggesting that these sexually transmitted pathogens may interact. We previously established an intravaginal Chlamydia muridarum/HSV-2 super-infection model and observed that chlamydial pre-infection protects mice from a subsequent lethal HSV-2 challenge. However, the mechanism of protection remains unknown. The type I interferon, IFN-ß, binds to the type I interferon receptor (IFNR), elicits a host cellular antiviral response and inhibits HSV replication in vitro and in vivo. Previous studies have demonstrated that C. muridarum infection stimulates genital tract (GT) IFN-ß production; therefore, we hypothesized that chlamydial pre-infection protects mice from HSV-2 challenge via the IFN-ß/IFNR-induced antiviral response. To test this prediction, we quantified IFN-ß levels in vaginal swab samples. Detection of IFN-ß in C. muridarum singly infected, but not in mock-infected animals, prompted the use of the super-infection model in IFNR knockout (IFNR-/-) mice. We observed that C. muridarum pre-infection reduces HSV-2-induced mortality by 40% in wild-type mice and by 60% IFNR-/- mice. Severity of HSV-2 disease symptoms and viral shedding was also similarly reduced by C. muridarum pre-infection. These data indicate that, while chlamydial infection induces GT production of IFN-ß, type I IFN-induced antiviral responses are likely not required for the observed protective effect.
Assuntos
Infecções por Chlamydia/complicações , Herpes Genital/prevenção & controle , Receptor de Interferon alfa e beta/metabolismo , Superinfecção/prevenção & controle , Animais , Chlamydia muridarum/imunologia , Modelos Animais de Doenças , Feminino , Herpesvirus Humano 2/imunologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vagina/imunologiaRESUMO
The Chlamydiaceae are widespread pathogens of both humans and animals. Chlamydia trachomatis infection causes blinding trachoma and reproductive complications in humans. Chlamydia pneumoniae causes human respiratory tract infections and atypical pneumonia. Chlamydia suis infection is associated with conjunctivitis, diarrhea, and failure to gain weight in domestic swine. Chlamydial infections in humans and domesticated animals are generally controlled by antibiotic treatment-particularly macrolides (usually azithromycin) and tetracyclines (tetracycline and doxycycline). Tetracycline-containing feed has also been used to limit infections and promote growth in livestock populations, although its use has decreased because of growing concerns about antimicrobial resistance development. Because Sandoz and Rockey published an elegant review of chlamydial anti-microbial resistance in 2010, we will review the following: (i) antibiotic resistance in C. suis, (ii) recent evidence for acquired resistance in human chlamydial infections, and (iii) recent non-genetic mechanisms of antibiotic resistance that may contribute to treatment failure.
RESUMO
Chlamydia trachomatis and Herpes Simplex Virus-2 (HSV-2) genital tract co-infections have been reported in humans and studied in vitro but the clinical consequences are unknown. Limited epidemiologic evidence suggests that these co-infections could be more severe than single infections of either pathogen, but the host-pathogen interactions during co-infection remain uncharacterized. To determine whether disease progression and/or pathogen shedding differs between singly-infected and super-infected animals, we developed an in vivo super-infection model in which female BALB/c mice were vaginally infected with Chlamydia muridarum (Cm) followed later by HSV-2. Pre-infection with Chlamydia 3 or 9 days prior to HSV-2 super-infection conferred significant protection from HSV-2-induced neurologic disease and significantly reduced viral recovery compared to HSV-2 singly-infected controls. Neither protection from mortality nor reduced viral recovery were observed when mice were i) super-infected with HSV-2 on day 27 post Cm; ii) infected with UV-irradiated Cm and super-infected with HSV-2; or iii) azithromycin-treated prior to HSV-2 super-infection. Therefore, protection from HSV-2-induced disease requires active infection with viable chlamydiae and is not observed after chlamydial shedding ceases, either naturally or due to antibiotic treatment. Thus, Chlamydia-induced protection is transient and requires the continued presence of chlamydiae or their components. These data demonstrate that chlamydial pre-infection can alter progression of subsequent HSV-2 infection, with implications for HSV-2 transmission from co-infected humans.
Assuntos
Infecções por Chlamydia/complicações , Chlamydia trachomatis/fisiologia , Herpes Genital/prevenção & controle , Herpesvirus Humano 2/fisiologia , Interações Hospedeiro-Patógeno , Superinfecção , Vaginose Bacteriana/complicações , Animais , Azitromicina/uso terapêutico , Infecções por Chlamydia/tratamento farmacológico , Infecções por Chlamydia/microbiologia , Infecções por Chlamydia/virologia , Chlamydia trachomatis/isolamento & purificação , Chlamydia trachomatis/efeitos da radiação , Coinfecção , Progressão da Doença , Feminino , Herpes Genital/complicações , Herpes Genital/microbiologia , Herpes Genital/virologia , Herpesvirus Humano 2/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Paraplegia/etiologia , Paraplegia/virologia , Fatores de Tempo , Vaginose Bacteriana/tratamento farmacológico , Vaginose Bacteriana/microbiologia , Vaginose Bacteriana/virologia , Carga ViralRESUMO
Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen, but more than 70% of patients fail to seek treatment due to the asymptomatic nature of these infections. Women suffer from numerous complications from chronic chlamydial infections, which include pelvic inflammatory disease and infertility. We previously demonstrated in culture that host cell nectin-1 knockdown significantly reduced chlamydial titers and inclusion size. Here, we sought to determine whether nectin-1 was required for chlamydial development in vivo by intravaginally infecting nectin-1-/- mice with Chlamydia muridarum and monitoring chlamydial shedding by chlamydial titer assay. We observed a significant reduction in chlamydial shedding in female nectin-1-/- mice compared to nectin-1+/+ control mice, an observation that was confirmed by PCR. Immunohistochemical staining in mouse cervical tissue confirmed that there are fewer chlamydial inclusions in Chlamydia-infected nectin-1-/- mice. Notably, anorectal chlamydial infections are becoming a substantial health burden, though little is known regarding the pathogenesis of these infections. We therefore established a novel male murine model of rectal chlamydial infection, which we used to determine whether nectin-1 is required for anorectal chlamydial infection in male mice. In contrast to the data from vaginal infection, no difference in rectal chlamydial shedding was observed when male nectin-1+/+ and nectin-1-/- mice were compared. Through the use of these two models, we have demonstrated that nectin-1 promotes chlamydial infection in the female genital tract but does not appear to contribute to rectal infection in male mice. These models could be used to further characterize tissue and sex related differences in chlamydial infection.
Assuntos
Moléculas de Adesão Celular/fisiologia , Infecções por Chlamydia/genética , Doenças dos Genitais Femininos/genética , Doenças Retais/genética , Infecções do Sistema Genital/genética , Animais , Moléculas de Adesão Celular/genética , Chlamydia muridarum/crescimento & desenvolvimento , Chlamydia muridarum/patogenicidade , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydia trachomatis/patogenicidade , Feminino , Predisposição Genética para Doença , Doenças dos Genitais Femininos/microbiologia , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nectinas , Doenças Retais/microbiologiaRESUMO
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.