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2.
Sex Transm Dis ; 47(5): 329-331, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32149960

RESUMEN

Identifying pathogen-specific signs or symptoms of nongonococcal urethritis could improve syndromic management accuracy. We evaluated nongonococcal urethritis signs and symptoms in 220 men with single-pathogen infections (Chlamydia trachomatis, Mycoplasma genitalium, Trichomonas vaginalis, or Ureaplasma urealyticum) or idiopathic urethritis. No individual sign or symptom accurately predicted the infectious etiology.

3.
PLoS One ; 15(2): e0228467, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32040516

RESUMEN

Urethritis, or inflammation of the urethra, is one of the most common reasons men seek clinical care. Sexually transmitted pathogens including Neisseria gonorrhoeae are responsible for over half of the symptomatic urethritis cases in U.S. men. Recently, clinics in Indianapolis, Columbus, Atlanta, and other U.S. cities began to note increasing numbers of men presenting with urethritis and Gram-negative intracellular diplococci in their urethral smears who test negative for N. gonorrhoeae. Many of these discordant cases, which have periodically reached highs of more than 25% of presumed gonococcal cases in some sexually transmitted infection clinics in the U.S. Midwest, are infected with strains in a novel urethrotropic clade of Neisseria meningitidis ST-11 (US_NmUC). However, no cultivation-independent tests are available for the US_NmUC strains, and prior studies relied on microbial culture and genome sequencing to identify them. Here, we describe a PCR test that can identify the US_NmUC strains and distinguish them from commensal and invasive N. meningitidis strains as well as N. gonorrhoeae. Our SimpleProbe®-based real-time PCR assay targets a conserved nucleotide substitution in a horizontally acquired region of US_NmUC strain genomes. We applied the assay to 241 urine specimens whose microbial compositions had previously been determined by deep shotgun metagenomic sequencing. The assay detected the single US_NmUC positive case in this cohort, with no false positives. Overall, our simple and readily adaptable assay could facilitate investigation of the pathogenesis and epidemiology of the US_NmUC clade.


Asunto(s)
Neisseria meningitidis/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Uretritis/microbiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Reacciones Falso Positivas , Gonorrea/diagnóstico , Gonorrea/epidemiología , Gonorrea/microbiología , Gonorrea/orina , Humanos , Masculino , Persona de Mediana Edad , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/aislamiento & purificación , Neisseria meningitidis/clasificación , Neisseria meningitidis/aislamiento & purificación , Polimorfismo de Nucleótido Simple , Enfermedades Bacterianas de Transmisión Sexual/diagnóstico , Enfermedades Bacterianas de Transmisión Sexual/epidemiología , Enfermedades Bacterianas de Transmisión Sexual/microbiología , Enfermedades Bacterianas de Transmisión Sexual/orina , Estados Unidos/epidemiología , Uretra/microbiología , Uretra/patología , Uretritis/diagnóstico , Urinálisis/métodos , Secuenciación Completa del Genoma , Adulto Joven
4.
Sex Transm Infect ; 96(4): 306-311, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-31515293

RESUMEN

OBJECTIVES: Chlamydia trachomatis (CT) and Mycoplasma genitalium (MG) cause the majority of non-gonococcal urethritis (NGU). The role of Ureaplasma urealyticum (UU) in NGU is unclear. Prior case-control studies that examined the association of UU and NGU may have been confounded by mixed infections and less stringent criteria for controls. The objective of this case-control study was to determine the prevalence and aetiology of mixed infections in men and assess if UU monoinfection is associated with NGU. METHODS: We identified 155 men with NGU and 103 controls. Behavioural and clinical information was obtained and men were tested for Neisseria gonorrhoeae and CT, MG, UU and Trichomonas vaginalis (TV). Men who were five-pathogen negative were classified as idiopathic urethritis (IU). RESULTS: Twelve per cent of NGU cases in which a pathogen was identified had mixed infections, mostly UU coinfections with MG or CT; 27% had IU. In monoinfected NGU cases, 34% had CT, 17% had MG, 11% had UU and 2% had TV. In controls, pathogens were rarely identified, except for UU, which was present in 20%. Comparing cases and controls, NGU was associated with CT and MG monoinfections and mixed infections. UU monoinfection was not associated with NGU and was almost twice as prevalent in controls. Men in both the case and control groups who were younger and who reported no prior NGU diagnosis were more likely to have UU (OR 0.97 per year of age, 95% CI 0.94 to 0.998 and OR 6.3, 95% CI 1.4 to 28.5, respectively). CONCLUSIONS: Mixed infections are common in men with NGU and most of these are UU coinfections with other pathogens that are well-established causes of NGU. UU monoinfections are not associated with NGU and are common in younger men and men who have never previously had NGU. Almost half of NGU cases are idiopathic.

5.
Front Physiol ; 10: 1278, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31649556

RESUMEN

Fbxo7 is the substrate-recognition subunit of an SCF-type ubiquitin E3 ligase complex. It has physiologically important functions in regulating mitophagy, proteasome activity and the cell cycle in multiple cell types, like neurons, lymphocytes and erythrocytes. Here, we show that in addition to the previously known Parkinsonian and hematopoietic phenotypes, male mice with reduced Fbxo7 expression are sterile. In these males, despite successful meiosis, nuclear elongation and eviction of histones from chromatin, the developing spermatids are phagocytosed by Sertoli cells during late spermiogenesis, as the spermatids undergo cytoplasmic remodeling. Surprisingly, despite the loss of all germ cells, there was no evidence of the symplast formation and cell sloughing that is typically associated with spermatid death in other mouse sterility models, suggesting that novel cell death and/or cell disposal mechanisms may be engaged in Fbxo7 mutant males. Mutation of the Drosophila Fbxo7 ortholog, nutcracker (ntc) also leads to sterility with germ cell death during cytoplasmic remodeling, indicating that the requirement for Fbxo7 at this stage is conserved. The ntc phenotype was attributed to decreased levels of the proteasome regulator, DmPI31 and reduced proteasome activity. Consistent with the fly model, we observe a reduction in PI31 levels in mutant mice; however, there is no alteration in proteasome activity in whole mouse testes. Our results are consistent with findings that Fbxo7 regulates PI31 protein levels, and indicates that a defect at the late stages of spermiogenesis, possibly due to faulty spatial dynamics of proteasomes during cytoplasmic remodeling, may underlie the fertility phenotype in mice.

6.
Clin Transl Gastroenterol ; 10(8): e00073, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31464691

RESUMEN

OBJECTIVES: The composition of the small intestinal microbiota has not yet been characterized thoroughly using culture-independent techniques. We compared small intestinal microbial communities in patients with and without small intestinal bacterial overgrowth (SIBO) using culture-dependent and culture-independent bacterial identification approaches. METHODS: Small bowel aspirate and mucosal samples were collected from patients with suspected SIBO. The aspirates were cultured to diagnose SIBO, defined as ≥10 colony-forming units/mL coliform or ≥10 colony-forming units/mL upper aerodigestive tract bacteria. Bacteria in the aspirates and mucosa were identified using 16S rRNA gene sequencing. We compared small intestinal microbiome composition between groups with and without a culture-based SIBO diagnosis. RESULTS: Analysis of the aspirate and mucosal microbial communities from 36 patients revealed decreased α-diversity but no differences in ß-diversity in patients with SIBO compared with those without SIBO. There were no significant differences in the relative abundance of individual taxa from the aspirates or mucosa after adjustment for false discovery rate between patients with and without SIBO. Subgroup analysis revealed significant differences in mucosal ß-diversity between the coliform and upper aerodigestive tract subgroups. Relative abundances of a mucosal Clostridium spp. (P = 0.05) and an aspirate Granulicatella spp. (P = 0.02) were higher in coliform SIBO vs non-SIBO subgroups. The microbial composition and relative abundance of multiple taxa significantly differed in the mucosal and aspirate specimens. DISCUSSION: Culture-based results of small bowel aspirates do not correspond to aspirate microbiota composition but may be associated with species richness of the mucosal microbiota.

8.
Int J Biol Macromol ; 139: 221-232, 2019 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-31376448

RESUMEN

In this study, we propose the use of a plant tissue culture-based system for the production of polysaccharides with consistent chemical characteristics and reduced endotoxin content. Polysaccharides were isolated from suspension cultures of Panax quinquefolius (American ginseng), a widely used medicinal herb. A neutral fraction, AGC1, purified by anion exchange and size exclusion chromatography, displayed immunostimulatory activity in vitro and ex vivo. AGC1 (average molecular weight: 5.2kDa) was predominantly composed of galactose (>60%) along with the presence of several other neutral sugars such as arabinose, xylose, glucose, mannose and rhamnose in minor amounts. The major glycosidic linkages were found to be 3-Galp (48.5%), 3,6-Galp (10.2%), t-Galp (5.2%), 6-Galp (4.4%), 4-Glcp (5.7%), 4-Arap/5-Araf (4.0%) and t-Araf (4.5%). AGC1 significantly (p<0.05) stimulated the expression of a range of proinflammatory mediators in RAW 264.7 murine macrophages such as IL-6, TNF-α, MCP-1 and GM-CSF. Additionally, AGC1 treatment of RAW 264.7 cells stimulated NOS2 gene expression, leading to increased levels of iNOS and downstream NO. Consistent with this, AGC1 was able to act as an immunostimulant in primary murine splenocytes, enhancing cell proliferation, as well as NO and TNF-α production. Our results also indicate the partial role of NF-κB pathway in the immunostimulatory response.


Asunto(s)
Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Panax/química , Fitoquímicos/química , Fitoquímicos/farmacología , Polisacáridos/química , Polisacáridos/farmacología , Animales , Células Cultivadas , Citocinas/metabolismo , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Inmunomodulación/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Peso Molecular , Óxido Nítrico/metabolismo , Panax/citología , Panax/metabolismo , Fitoquímicos/aislamiento & purificación , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Polisacáridos/aislamiento & purificación , Células RAW 264.7
9.
BMC Mol Cell Biol ; 20(1): 33, 2019 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-31412778

RESUMEN

BACKGROUND: The PINK1:Parkin pathway regulates the autophagic removal of damaged and dysfunctional mitochondria. While the response of this pathway to complete loss of ΔΨm, as caused by high concentrations of mitochondrial ionophores, has been well characterized, it remains unclear how the pathway makes coherent decisions about whether to keep or purge mitochondria in situations where ΔΨm is only partially lost or exhibits fluctuations, as has been observed in response to certain types of cellular stress. RESULTS: To investigate the responses of the PINK1:Parkin pathway to mitochondrial insults of different magnitude and duration, controlled titration of the mitochondrial protonophore, CCCP, was used to manipulate ΔΨm in live cells, and the dynamics of PINK1 and Parkin recruitment was measured by fluorescence microscopy. In contrast to the stable accumulation of PINK1 and Parkin seen at completely depolarized mitochondria, partial depolarization produced a transient pulse of PINK1 stabilization and rapid loss, which was driven by small fluctuations in ΔΨm. As the rate of Parkin dissociation from the mitochondria and phospho-polyubiquitin chain removal was comparatively slow, repetitive pulses of PINK1 were able to drive a slow step-wise accumulation of Parkin and phospho-polyubiquitin leading to deferred mitophagy. CONCLUSION: These data suggest that the PINK1:Parkin mitophagy pathway is able to exhibit distinct dynamic responses to complete and partial mitochondrial depolarization. In this way, the pathway is able to differentiate between irretrievably damaged mitochondria and those showing signs of dysfunction, promoting either rapid or delayed autophagy, respectively.


Asunto(s)
Mitocondrias/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Estrés Fisiológico , Ubiquitina-Proteína Ligasas/metabolismo , Autofagia/efectos de los fármacos , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Modelos Biológicos , Fosforilación/efectos de los fármacos , Poliubiquitina/metabolismo , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Factores de Tiempo
10.
Sex Transm Dis ; 46(7): 440-445, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31194715

RESUMEN

BACKGROUND: Rectal infection with Chlamydia trachomatis (CT) is frequent in women who deny receptive anal sex and is thought to arise from autoinoculation of the rectum from vaginal secretions. An alternate hypothesis is that oral sex inoculates and establishes gastrointestinal tract infection. Distinguishing these hypotheses is difficult in women. In men, autoinoculation is unlikely and heterosexual men frequently perform oral sex, but rarely participate in receptive anal exposure behaviors. METHODS: We enrolled high-risk men with and without nongonococcal urethritis who presented to a sexually transmitted infection clinic in Indianapolis, Indiana. Urine and rectal swabs were collected and tested for urogenital and rectal CT, Neisseria gonorrhoeae (NG), and Mycoplasma genitalium (MG). Men completed surveys concerning symptoms, sexual orientation, and detailed recent and lifetime oral and anal sexual behaviors. RESULTS: Rectal CT was detected in 2/84 (2.4%) heterosexual men who reported cunnilingus, but no lifetime receptive anal behaviors. All of the men who denied receptive anal behaviors were negative for rectal NG and MG. In homosexual and bisexual men, rectal CT prevalence was high (9.7%), and rectal NG (4.8%) and MG (4.8%) were also detected. CONCLUSIONS: We detected rectal CT infections in heterosexual men who reported cunnilingus but denied receptive anal behaviors. Oral sex may be a risk factor for rectal CT infection via oral inoculation of the gastrointestinal tract.


Asunto(s)
Infecciones por Chlamydia/diagnóstico , Chlamydia trachomatis/aislamiento & purificación , Conducta Sexual/estadística & datos numéricos , Enfermedades de Transmisión Sexual/diagnóstico , Enfermedades de Transmisión Sexual/epidemiología , Adolescente , Adulto , Anciano , Canal Anal/microbiología , Infecciones por Chlamydia/epidemiología , Infecciones por Chlamydia/microbiología , Heterosexualidad , Humanos , Indiana/epidemiología , Masculino , Persona de Mediana Edad , Prevalencia , Recto/microbiología , Factores de Riesgo , Enfermedades de Transmisión Sexual/microbiología , Encuestas y Cuestionarios , Adulto Joven
12.
mBio ; 10(2)2019 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-30967464

RESUMEN

Interferon-regulated immune defenses protect mammals from pathogenically diverse obligate intracellular bacterial pathogens of the genus Chlamydia Interferon gamma (IFN-γ) is especially important in controlling the virulence of Chlamydia species and thus impacts the modeling of human chlamydial infection and disease in mice. How IFN-γ contributes to cell-autonomous defenses against Chlamydia species and how these pathogens evade IFN-γ-mediated immunity in their natural hosts are not well understood. We conducted a genetic screen which identified 31 IFN-γ-sensitive (Igs) mutants of the mouse model pathogen Chlamydia muridarum Genetic suppressor analysis and lateral gene transfer were used to map the phenotype of one of these mutants, Igs4, to a missense mutation in a putative chlamydial inclusion membrane protein, TC0574. We observed the lytic destruction of Igs4-occupied inclusions and accompanying host cell death in response to IFN-γ priming or various proapoptotic stimuli. However, Igs4 was insensitive to IFN-γ-regulated cell-autonomous defenses previously implicated in anti-Chlamydia trachomatis host defense in mice. Igs4 inclusion integrity was restored by caspase inhibitors, indicating that the IFN-γ-mediated destruction of Igs4 inclusions is dependent upon the function of caspases or related prodeath cysteine proteases. We further demonstrated that the Igs4 mutant is immune restricted in an IFN-γ-dependent manner in a mouse infection model, thereby implicating IFN-γ-mediated inclusion destruction and host cell death as potent in vivo host defense mechanisms to which wild-type C. muridarum is resistant. Overall, our results suggest that C. muridarum evolved resistance mechanisms to counter IFN-γ-elicited programmed cell death and the associated destruction of intravacuolar pathogens.IMPORTANCE Multiple obligatory intracellular bacteria in the genus Chlamydia are important pathogens. In humans, strains of C. trachomatis cause trachoma, chlamydia, and lymphogranuloma venereum. These diseases are all associated with extended courses of infection and reinfection that likely reflect the ability of chlamydiae to evade various aspects of host immune responses. Interferon-stimulated genes, driven in part by the cytokine interferon gamma, restrict the host range of various Chlamydia species, but how these pathogens evade interferon-stimulated genes in their definitive host is poorly understood. Various Chlamydia species can inhibit death of their host cells and may have evolved this strategy to evade prodeath signals elicited by host immune responses. We present evidence that chlamydia-induced programmed cell death resistance evolved to counter interferon- and immune-mediated killing of Chlamydia-infected cells.


Asunto(s)
Apoptosis , Chlamydia muridarum/inmunología , Interacciones Huésped-Patógeno , Evasión Inmune , Inmunidad Innata , Interferón gamma/metabolismo , Animales , Infecciones por Chlamydia/microbiología , Infecciones por Chlamydia/patología , Chlamydia muridarum/genética , Modelos Animales de Enfermedad , Pruebas Genéticas , Cuerpos de Inclusión/microbiología , Ratones
13.
Mol Microbiol ; 111(1): 254-268, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30338585

RESUMEN

Clusters of Neisseria meningitidis (Nm) urethritis among primarily heterosexual males in multiple US cities have been attributed to a unique non-encapsulated meningococcal clade (the US Nm urethritis clade, US_NmUC) within the hypervirulent clonal complex 11. Resistance to antimicrobial peptides (AMPs) is a key feature of urogenital pathogenesis of the closely related species, Neisseria gonorrhoeae. The US_NmUC isolates were found to be highly resistant to the model AMP, polymyxin B (PmB, MICs 64-256 µg ml-1 ). The isolates also demonstrated stable subpopulations of heteroresistant colonies that showed near total resistant to PmB (MICs 384-1024 µg ml-1 ) and colistin (MIC 256 µg ml-1 ) as well as enhanced LL-37 resistance. This is the first observation of heteroresistance in N. meningitidis. Consistent with previous findings, overall PmB resistance in US_NmUC isolates was due to active Mtr efflux and LptA-mediated lipid A modification. However, whole genome sequencing, variant analyses and directed mutagenesis revealed that the heteroresistance phenotypes and very high-level AMP resistance were the result of point mutations and IS1655 element movement in the pilMNOPQ operon, encoding the type IV pilin biogenesis apparatus. Cross-resistance to other classes of antibiotics was also observed in the heteroresistant colonies. High-level resistance to AMPs may contribute to the pathogenesis of US_NmUC.


Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Proteínas Fimbrias/genética , Mutación , Neisseria meningitidis/efectos de los fármacos , Polimixina B/farmacología , Uretritis/microbiología , Péptidos Catiónicos Antimicrobianos/farmacología , Ciudades/epidemiología , Colistina/farmacología , Heterosexualidad , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Mutagénesis Sitio-Dirigida , Neisseria meningitidis/aislamiento & purificación , Operón , Sistemas de Secreción Tipo IV/genética , Estados Unidos/epidemiología , Secuenciación Completa del Genoma
14.
J Cancer Educ ; 34(3): 526-534, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-29492801

RESUMEN

This study was conducted in 2016-2017 to better understand formal and informal leadership roles and activities of alumni from postdoctoral research training programs in cancer prevention. Data were obtained from surveys of 254 employed scientists who completed cancer prevention postdoctoral training within the National Cancer Institute (NCI) Cancer Prevention Fellowship Program, or at US research institutions through NCI-sponsored National Research Service Award (NRSA) individual postdoctoral fellowship (F32) grants, from 1987 to 2011. Fifteen questions categorized under Organizational Leadership, Research Leadership, Professional Society/Conference Leadership, and Broader Scientific/Health Community Leadership domains were analyzed. About 75% of respondents had at least one organizational leadership role or activity during their careers, and 13-34% reported some type of research, professional society/conference, or broader scientific/health community leadership within the past 5 years. Characteristics independently associated with leadership from regression models were being in earlier postdoctoral cohorts (8 items, range for statistically significant ORs = 2.8 to 10.8) and employment sector (8 items, range for statistically significant ORs = 0.4 to 11.7). Scientists whose race/ethnicity was other than white were less likely to report organizational leadership or management responsibilities (OR = 0.4, 95% CI 0.2-0.9). Here, many alumni from NCI-supported cancer prevention postdoctoral programs were involved in leadership, with postdoctoral cohort and employment sector being the factors most often associated with leadership roles and activities. Currently, there is relatively little research on leadership roles of biomedical scientists in general, or in cancer prevention specifically. This study begins to address this gap and provide a basis for more extensive studies of leadership roles and training of scientists.

15.
Clin Cancer Res ; 24(24): 6383-6395, 2018 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-30190370

RESUMEN

PURPOSE: Diet and healthy weight are established means of reducing cancer incidence and mortality. However, the impact of diet modifications on the tumor microenvironment and antitumor immunity is not well defined. Immunosuppressive tumor-associated macrophages (TAMs) are associated with poor clinical outcomes and are potentially modifiable through dietary interventions. We tested the hypothesis that dietary protein restriction modifies macrophage function toward antitumor phenotypes. EXPERIMENTAL DESIGN: Macrophage functional status under different tissue culture conditions and in vivo was assessed by Western blot, immunofluorescence, qRT-PCR, and cytokine array analyses. Tumor growth in the context of protein or amino acid (AA) restriction and immunotherapy, namely, a survivin peptide-based vaccine or a PD-1 inhibitor, was examined in animal models of prostate (RP-B6Myc) and renal (RENCA) cell carcinoma. All tests were two-sided. RESULTS: Protein or AA-restricted macrophages exhibited enhanced tumoricidal, proinflammatory phenotypes, and in two syngeneic tumor models, protein or AA-restricted diets elicited reduced TAM infiltration, tumor growth, and increased response to immunotherapies. Further, we identified a distinct molecular mechanism by which AA-restriction reprograms macrophage function via a ROS/mTOR-centric cascade. CONCLUSIONS: Dietary protein restriction alters TAM activity and enhances the tumoricidal capacity of this critical innate immune cell type, providing the rationale for clinical testing of this supportive tool in patients receiving cancer immunotherapies.


Asunto(s)
Dieta con Restricción de Proteínas , Proteínas en la Dieta/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Aminoácidos/metabolismo , Animales , Línea Celular Tumoral , Citocinas/metabolismo , Modelos Animales de Enfermedad , Microbioma Gastrointestinal , Humanos , Inmunomodulación , Inmunoterapia , Activación de Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Ratones , Ratones Transgénicos , Neoplasias/patología , Neoplasias/terapia , Poliaminas/metabolismo
16.
Infect Immun ; 86(7)2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29661932

RESUMEN

Some members of the genus Chlamydia, including the human pathogen Chlamydia trachomatis, infect multiple tissues, including the genital and gastrointestinal (GI) tracts. However, it is unknown if bacterial targeting to these sites is mediated by multifunctional or distinct chlamydial factors. We previously showed that disruption of individual large clostridial toxin homologs encoded within the Chlamydia muridarum plasticity zone were not critical for murine genital tract infection. Here, we assessed whether cytotoxin genes contribute to C. muridarum GI tropism. Infectivity and shedding of wild-type (WT) C. muridarum and three mutants containing nonsense mutations in different cytotoxin genes, tc0437, tc0438, and tc0439, were compared in mouse genital and GI infection models. One mutant, which had a nonsense mutation in tc0439, was highly attenuated for GI infection and had a GI 50% infectious dose (ID50) that was 1,000 times greater than that of the WT. GI inoculation with this mutant failed to elicit anti-chlamydial antibodies or to protect against subsequent genital tract infection. Genome sequencing of the tc0439 mutant revealed additional chromosomal mutations, and phenotyping of additional mutants suggested that the GI attenuation might be linked to a nonsense mutation in tc0600 The molecular mechanism underlying this dramatic difference in tissue-tropic virulence is not fully understood. However, isolation of these mutants demonstrates that distinct chlamydial chromosomal factors mediate chlamydial tissue tropism and provides a basis for vaccine initiatives to isolate chlamydia strains that are attenuated for genital infection but retain the ability to colonize the GI tract and elicit protective immune responses.


Asunto(s)
Infecciones por Chlamydia/etiología , Chlamydia muridarum/patogenicidad , Cromosomas/fisiología , Enfermedades Gastrointestinales/etiología , Infecciones del Sistema Genital/etiología , Tropismo , Animales , Infecciones por Chlamydia/inmunología , Codón sin Sentido , Citotoxinas/genética , Femenino , Enfermedades Gastrointestinales/inmunología , Tracto Gastrointestinal/microbiología , Genitales/microbiología , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Polimorfismo de Nucleótido Simple , Infecciones del Sistema Genital/inmunología
17.
J Vis Exp ; (132)2018 02 27.
Artículo en Inglés | MEDLINE | ID: mdl-29553511

RESUMEN

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.


Asunto(s)
Pared Celular/metabolismo , Cryptococcus neoformans/crecimiento & desarrollo
18.
Front Immunol ; 9: 2962, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30619320

RESUMEN

By uncovering complex dynamics in the expression or localization of transcriptional regulators in single cells that were otherwise hidden at the population level, live cell imaging has transformed our understanding of how cells sense and orchestrate appropriate responses to changes in their internal state or extracellular environment. This has proved particularly true for the nuclear factor-kappaB (NF-κB) family of transcription factors, key regulators of the inflammatory response and innate immune function, which are capable of encoding information about the mode and intensity of stimuli in the dynamics of NF-κB nuclear accumulation and loss. While live cell imaging continues to serve as a useful tool in ongoing efforts to characterize the feedbacks that shape these dynamics and to connect dynamics to downstream gene expression, it is also proving invaluable for recent studies that seek to determine how intracellular pathogens subvert NF-κB signaling to survive and replicate within host cells by providing quantitative information about the pathogen and changes in NF-κB activity during different stages of an infection. Here, we provide a brief overview of NF-κB signaling in innate immune cells and review recent literature that uses live imaging to investigate the mechanisms by which bacterial and yeast pathogens modulate NF-κB in a variety of different host cell types to evade destruction or maintain the viability of an intracellular growth niche.


Asunto(s)
Diferenciación Celular/inmunología , Interacciones Huésped-Patógeno/inmunología , Macrófagos/inmunología , FN-kappa B/inmunología , Transducción de Señal/inmunología , Animales , Cryptococcus neoformans/inmunología , Cryptococcus neoformans/fisiología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Microscopía Fluorescente , FN-kappa B/metabolismo , Salmonella enterica/inmunología , Salmonella enterica/fisiología
19.
Curr Top Microbiol Immunol ; 412: 133-158, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29090367

RESUMEN

Obligate intracellular pathogens in the family Chlamydiaceae infect taxonomically diverse eukaryotes ranging from amoebae to mammals. However, many fundamental aspects of chlamydial cell biology and pathogenesis remain poorly understood. Genetic dissection of chlamydial biology has historically been hampered by a lack of genetic tools. Exploitation of the ability of chlamydia to recombine genomic material by lateral gene transfer (LGT) ushered in a new era in chlamydia research. With methods to map mutations in place, genetic screens were able to assign functions and phenotypes to specific chlamydial genes. Development of an approach for stable transformation of chlamydia also provided a mechanism for gene delivery and platforms for disrupting chromosomal genes. Here, we explore how these and other tools have been used to test hypotheses concerning the functions of known chlamydial virulence factors and discover the functions of completely uncharacterized genes. Refinement and extension of the existing genetic tools to additional Chlamydia spp. will substantially advance understanding of the biology and pathogenesis of this important group of pathogens.


Asunto(s)
Chlamydia/genética , Chlamydia/patogenicidad , Animales , Transferencia de Gen Horizontal , Genoma Bacteriano/genética , Genómica , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/metabolismo
20.
Infect Immun ; 86(1)2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29038127

RESUMEN

The cryptic plasmid is essential for Chlamydia muridarum dissemination from the genital tract to the gastrointestinal (GI) tract. Following intravaginal inoculation, a C. muridarum strain deficient in plasmid-encoded pGP3 or pGP4 but not pGP5, pGP7, or pGP8 failed to spread to the mouse gastrointestinal tract, although mice infected with these strains developed productive genital tract infections. pGP3- or pGP4-deficient strains also failed to colonize the gastrointestinal tract when delivered intragastrically. pGP4 regulates pGP3, while pGP3 does not affect pGP4 expression, indicating that pGP3 is critical for C. muridarum colonization of the gastrointestinal tract. Mutants deficient in GlgA, a chromosome-encoded protein regulated by pGP4, also consistently colonized the mouse gastrointestinal tract. Interestingly, C. muridarum colonization of the gastrointestinal tract positively correlated with pathogenicity in the upper genital tract. pGP3-deficient C. muridarum strains did not induce hydrosalpinx or spread to the GI tract even when delivered to the oviduct by intrabursal inoculation. Thus, the current study not only has revealed that pGP3 is a novel chlamydial colonization factor in the gastrointestinal tract but also has laid a foundation for investigating the significance of gastrointestinal Chlamydia.


Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Infecciones por Chlamydia/microbiología , Chlamydia muridarum/genética , Chlamydia muridarum/patogenicidad , Tracto Gastrointestinal/microbiología , Infecciones del Sistema Genital/microbiología , Factores de Virulencia/genética , Animales , Línea Celular Tumoral , Femenino , Genitales/microbiología , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Oviductos/microbiología , Plásmidos/genética
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