Genome-Wide Mutagenesis Identifies Factors Involved in Enterococcus faecalis Vaginal Adherence and Persistence

ABSTRACTEnterococcus faecalis is a Gram-positive commensal bacterium native to the gastrointestinal tract and an opportunistic pathogen of increasing clinical concern. E. faecalis also colonizes the female reproductive tract and reports suggest vaginal colonization increases following antibiotic treatment or in patients with aerobic vaginitis. Currently, little is known about specific factors that promote E. faecalis vaginal colonization and subsequent infection. We modified an established mouse vaginal colonization model to explore E. faecalis vaginal carriage and demonstrate that both vancomycin resistant and sensitive strains colonize the murine vaginal tract. Following vaginal colonization, we observed E. faecalis in vaginal, cervical and uterine tissue. A mutant lacking endocarditis- and biofilm-associated pili (Ebp) exhibited a decreased ability to associate with human vaginal and cervical cells in vitro, but did not contribute to colonization in vivo. Thus, we screened a low-complexity transposon (Tn) mutant library to identify novel genes important for E. faecalis colonization and persistence in the vaginal tract. This screen revealed 383 mutants that were underrepresented during vaginal colonization at 1, 5 and 8 days post-inoculation compared to growth in culture medium. We confirmed that mutants deficient in ethanolamine catabolism or in the type VII secretion system were attenuated in persisting during vaginal colonization. These results reveal the complex nature of vaginal colonization and suggest that multiple factors contribute to E. faecalis persistence in the reproductive tract.IMPORTANCEDespite increasing prevalence and association of E. faecalis with aerobic vaginitis, essentially nothing is known about the bacterial factors that influence E. faecalis vaginal colonization. We have adapted an animal model of vaginal colonization that supports colonization of multiple E. faecalis strains. Additionally, we determined that ethanolamine utilization and type VII secretion system genes contribute to vaginal colonization and persistence. Identification of factors important for vaginal colonization and persistence provides potential targets for the development of therapeutics. This study is the first to identify key determinants that promote vaginal colonization by E. faecalis, which may represent an important reservoir for antibiotic resistant enterococci.

Download Full-text

Identification of Key Determinants of Staphylococcus aureus Vaginal Colonization

mBio ◽

10.1128/mbio.02321-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 5

Author(s):

Liwen Deng ◽

Katrin Schilcher ◽

Lindsey R. Burcham ◽

Jakub M. Kwiecinski ◽

Paige M. Johnson ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Mouse Model ◽

Female Reproductive Tract ◽

Transport Systems ◽

Postpartum Women ◽

Content Type ◽

Vaginal Colonization ◽

Fibrinogen Binding ◽

Vaginal Tract

ABSTRACT Staphylococcus aureus is an important pathogen responsible for nosocomial and community-acquired infections in humans, and methicillin-resistant S. aureus (MRSA) infections have continued to increase despite widespread preventative measures. S. aureus can colonize the female vaginal tract, and reports have suggested an increase in MRSA infections in pregnant and postpartum women as well as outbreaks in newborn nurseries. Currently, little is known about specific factors that promote MRSA vaginal colonization and subsequent infection. To study S. aureus colonization of the female reproductive tract in a mammalian system, we developed a mouse model of S. aureus vaginal carriage and demonstrated that both hospital-associated and community-associated MRSA isolates can colonize the murine vaginal tract. Immunohistochemical analysis revealed an increase in neutrophils in the vaginal lumen during MRSA colonization. Additionally, we observed that a mutant lacking fibrinogen binding adhesins exhibited decreased persistence within the mouse vagina. To further identify novel factors that promote vaginal colonization, we performed RNA sequencing to determine the transcriptome of MRSA growing in vivo during vaginal carriage at 5 h, 1 day, and 3 days postinoculation. Over 25% of the bacterial genes were differentially regulated at all time points during colonization compared to laboratory cultures. The most highly induced genes were those involved in iron acquisition, including the Isd system and siderophore transport systems. Mutants deficient in these pathways did not persist as well during in vivo colonization. These results reveal that fibrinogen binding and the capacity to overcome host nutritional limitation are important determinants of MRSA vaginal colonization. IMPORTANCE Staphylococcus aureus is an opportunistic pathogen able to cause a wide variety of infections in humans. Recent reports have suggested an increasing prevalence of MRSA in pregnant and postpartum women, coinciding with the increased incidence of MRSA infections in neonatal intensive care units (NICUs) and newborn nurseries. Vertical transmission from mothers to infants at delivery is a likely route of MRSA acquisition by the newborn; however, essentially nothing is known about host and bacterial factors that influence MRSA carriage in the vagina. Here, we established a mouse model of vaginal colonization and observed that multiple MRSA strains can persist in the vaginal tract. Additionally, we determined that MRSA interactions with fibrinogen and iron uptake can promote vaginal persistence. This study is the first to identify molecular mechanisms which govern vaginal colonization by MRSA, the critical initial step preceding infection and neonatal transmission.

Download Full-text

CatSperζ regulates the structural continuity of sperm Ca2+ signaling domains and is required for normal fertility

eLife ◽

10.7554/elife.23082 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 54

Author(s):

Jean-Ju Chung ◽

Kiyoshi Miki ◽

Doory Kim ◽

Sang-Hee Shim ◽

Huanan F Shi ◽

...

Keyword(s):

Reproductive Tract ◽

Female Reproductive Tract ◽

Targeted Disruption ◽

Vitro Fertilization ◽

Male Subfertility ◽

Epididymal Spermatozoa ◽

Mammalian Female ◽

Signaling Domains

We report that the Gm7068 (CatSpere) and Tex40 (CatSperz) genes encode novel subunits of a 9-subunit CatSper ion channel complex. Targeted disruption of CatSperz reduces CatSper current and sperm rheotactic efficiency in mice, resulting in severe male subfertility. Normally distributed in linear quadrilateral nanodomains along the flagellum, the complex lacking CatSperζ is disrupted at ~0.8 μm intervals along the flagellum. This disruption renders the proximal flagellum inflexible and alters the 3D flagellar envelope, thus preventing sperm from reorienting against fluid flow in vitro and efficiently migrating in vivo. Ejaculated CatSperz-null sperm cells retrieved from the mated female uterus partially rescue in vitro fertilization (IVF) that failed with epididymal spermatozoa alone. Human CatSperε is quadrilaterally arranged along the flagella, similar to the CatSper complex in mouse sperm. We speculate that the newly identified CatSperζ subunit is a late evolutionary adaptation to maximize fertilization inside the mammalian female reproductive tract.

Download Full-text

301 THE EFFECT OF EQUINE FOLICULLAR FLUID ON IN VITRO INDUCTION OF THE ACROSOME REACTION IN FROZEN - THAWED STALLION SPERMATOZOA

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab301 ◽

2010 ◽

Vol 22 (1) ◽

pp. 307

Author(s):

D. S. Silva ◽

P. Rodriguez ◽

N. S. Arruda ◽

R. Rodrigues ◽

J. L. Rodrigues

Keyword(s):

Contact Area ◽

Follicular Fluid ◽

Acrosome Reaction ◽

Reproductive Tract ◽

Female Reproductive Tract ◽

Heparin Group ◽

Fluorescent Stain ◽

In Vitro Induction

The capacitation process occurs in vivo upon exposure of the spermatozoa through the female reproductive tract, but can be induced in vitro in the presence of several compounds. This study was conducted to assess the effect of heparin or equine follicular fluid on hyperactivated motility and in vitro induction acrosome reaction swim-up method with frozen-thawed stallion semen. Two hundred microliters of frozen-thawed equine semen was placed in a tube (45°C) to increase contact area and incubated at 37°C for 1 h. After incubation 800 μL of the supernatant was collected by centrifugation (500 × g, 10 min) to collect spermatozoa. The resulting pellet was resuspended in capacitation medium Fert-TALP supplemented with 5.0 μg mL-1 heparin or 100% follicular fluid and incubated for different times (1, 2, 3, 4, and 5 h) at 37°C. After incubation the hyperactivated motility and acrosome-reacted spermatozoa were evaluated. Hoechst stain was used to differentiate live and dead spermatozoa, and chlortetracycline (CTC) fluorescent stain was used to assess the capacitation response of sperm; data were analyzed by ANOVA. The effect of equine follicular fluid resulted in improved percentage of spermatozoa with acrosome reaction at all times of incubation (60, 63, 57, 52, and 58%) but immediately after 3 h of incubation, the hyperactivated motility decreased in heparin group and follicular fluid (42 and 30%, respectively).

Download Full-text

ESR1 Mutations Associated With Estrogen Insensitivity Syndrome Change Conformation of Ligand-Receptor Complex and Altered Transcriptome Profile

Endocrinology ◽

10.1210/endocr/bqaa050 ◽

2020 ◽

Vol 161 (6) ◽

Cited By ~ 1

Author(s):

Yin Li ◽

Katherine J Hamilton ◽

Lalith Perera ◽

Tianyuan Wang ◽

Artiom Gruzdev ◽

...

Keyword(s):

Molecular Mechanisms ◽

Reproductive Tract ◽

Biological Effects ◽

Estrogen Receptor Α ◽

Female Reproductive Tract ◽

Receptor Complexes ◽

Esr1 Gene ◽

Esr1 Mutations

Abstract Estrogen insensitivity syndrome (EIS) arises from rare mutations in estrogen receptor-α (ERα, encoded by ESR1 gene) resulting in the inability of estrogen to exert its biological effects. Due to its rarity, mutations in ESR1 gene and the underlying molecular mechanisms of EIS have not been thoroughly studied. Here, we investigate known ESR1 mutants, Q375H and R394H, associated with EIS patients using in vitro and in vivo systems. Comparison of the transcriptome and deoxyribonucleic acid methylome from stable cell lines of both Q375H and R394H clinical mutants shows a differential profile compared with wild-type ERα, resulting in loss of estrogen responsiveness. Molecular dynamic simulation shows that both ESR1 mutations change the ERα conformation of the ligand-receptor complexes. Furthermore, we generated a mouse model Esr1-Q harboring the human mutation using CRISPR/Cas9 genome editing. Female and male Esr1-Q mice are infertile and have similar phenotypes to αERKO mice. Overall phenotypes of the Esr1-Q mice correspond to those observed in the patient with Q375H. Finally, we explore the effects of a synthetic progestogen and a gonadotropin-releasing hormone inhibitor in the Esr1-Q mice for potentially reversing the impaired female reproductive tract function. These findings provide an important basis for understanding the molecular mechanistic consequences associated with EIS.

Download Full-text

Use of Recombinase-BasedIn VivoExpression Technology To Characterize Enterococcus faecalis Gene Expression during Infection IdentifiesIn Vivo-Expressed Antisense RNAs and Implicates the Protease Eep in Pathogenesis

Infection and Immunity ◽

10.1128/iai.05964-11 ◽

2011 ◽

Vol 80 (2) ◽

pp. 539-549 ◽

Cited By ~ 34

Author(s):

Kristi L. Frank ◽

Aaron M. T. Barnes ◽

Suzanne M. Grindle ◽

Dawn A. Manias ◽

Patrick M. Schlievert ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Rabbit Model ◽

Microscopic Analysis ◽

Mammalian Host ◽

Wild Type ◽

Antisense Transcripts ◽

Content Type ◽

Antisense Rnas

ABSTRACTEnterococcus faecalisis a member of the mammalian gastrointestinal microflora that has become a leading cause of nosocomial infections over the past several decades.E. faecalismust be able to adapt its physiology based on its surroundings in order to thrive in a mammalian host as both a commensal and a pathogen. We employed recombinase-basedin vivoexpression technology (RIVET) to identify promoters on theE. faecalisOG1RF chromosome that were specifically activated during the course of infection in a rabbit subdermal abscess model. The RIVET screen identified 249 putativein vivo-activated loci, over one-third of which are predicted to generate antisense transcripts. Three predicted antisense transcripts were detected inin vitro- andin vivo-grown cells, providing the first evidence ofin vivo-expressed antisense RNAs inE. faecalis. Deletions in thein vivo-activated genes that encode glutamate 5-kinase (proB[EF0038]), the transcriptional regulator EbrA (ebrA[EF1809]), and the membrane metalloprotease Eep (eep[EF2380]) did not hinder biofilm formation inin vitroassays. In a rabbit model of endocarditis, the ΔebrAstrain was fully virulent, the ΔproBstrain was slightly attenuated, and the Δeepstrain was severely attenuated. The Δeepvirulence defect could be complemented by the expression of the wild-type gene intrans. Microscopic analysis of early Δeepbiofilms revealed an abundance of small cellular aggregates that were not observed in wild-type biofilms. This work illustrates the use of a RIVET screen to provide information about the temporal activation of genes during infection, resulting in the identification and confirmation of a new virulence determinant in an important pathogen.

Download Full-text

Enterococcus faecalis 6-Phosphogluconolactonase Is Required for Both Commensal and Pathogenic Interactions with Manduca sexta

Infection and Immunity ◽

10.1128/iai.02442-14 ◽

2014 ◽

Vol 83 (1) ◽

pp. 396-404 ◽

Cited By ~ 7

Author(s):

Jonathan F. Holt ◽

Megan R. Kiedrowski ◽

Kristi L. Frank ◽

Jing Du ◽

Changhui Guan ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Manduca Sexta ◽

Wild Type ◽

Genetic Determinants ◽

Content Type ◽

Polystyrene Plate ◽

Insect Gut ◽

Insight Into

Enterococcus faecalisis a commensal and pathogen of humans and insects. InManduca sexta,E. faecalisis an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigateE. faecalisfactors required for commensalism, we identifiedE. faecalisgenes that are upregulated in the gut ofM. sextausing recombinase-basedin vivoexpression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designatedpglA. ApglAdeletion mutant was impaired in both pathogenesis and gut persistence inM. sextaand produced enhanced biofilms compared with the wild type in anin vitropolystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants forE. faecaliscommensal and pathogenic interactions withM. sexta. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to preventE. faecalisinfections.

Download Full-text

Capacitation mechanisms, and the role of capacitation as seen in eutherian mammals

Reproduction Fertility and Development ◽

10.1071/rd9960581 ◽

1996 ◽

Vol 8 (4) ◽

pp. 581 ◽

Cited By ~ 157

Author(s):

RA Harrison

Keyword(s):

Cell Death ◽

Reproductive Tract ◽

Physiological State ◽

Hormonal Control ◽

Female Reproductive Tract ◽

Functional Heterogeneity ◽

Vitro Fertilization ◽

Range Of Functions

Capacitation, the process whereby spermatozoa are rendered capable of interacting with and fertilizing the egg, was discovered more than 40 years ago. However, our understanding of it is still far from satisfactory. Several factors conspire to obfuscate studies of capacitation mechanisms: the inherent functional heterogeneity of sperm populations, the range of functions used as parameters of capacitation (whence the endpoint of the process has become conceptually uncertain), and the several profound differences between model in vitro fertilization (IVF) systems and the situation in vivo in the female reproductive tract. Recent investigations in the author's laboratory have shown that bicarbonate/CO2, an essential component for successful IVF, causes rapid changes in lipid architecture of the sperm plasma membrane and slower changes in surface coating. These changes are accompanied by membrane destabilization and cell death. Evidence suggests that bicarbonate's actions are mediated through cyclic nucleotide signalling. Of particular note is the heterogeneity in rate of response to bicarbonate shown by individual cells in the sperm populations. Taken together with other observations, the findings suggest that capacitation is a series of positive destabilizing events that eventually lead to cell death. The 'capacitated' state would then be a window of destabilization within which spermatozoa can undergo a zona-induced acrosome reaction and display hyperactivated motility. Further along the destabilization pathway, spontaneous acrosome reactions would occur before total membrane degeneration. In vivo, capacitation would be a conflict between destabilization and sperm survival. Concentrations of bicarbonate are maintained low in the cauda epididymidis, where sperm survive for long periods, and one may speculate that hormonal control of local bicarbonate/CO2 in oviducal 'storage' sites in the female tract could allow 'safe' sequestering of live spermatozoa until around the time of ovulation; the environment may then change to produce a 'capacitating' effect, whence, due to the inherent functional heterogeneity of the sequestered population, small numbers of capacitated spermatozoa are released sequentially. In this way, a succession of spermatozoa in the correct physiological state may be provided for the freshly ovulated egg.

Download Full-text

In vivo exposure to 17β-estradiol triggers premature sperm capacitation in cauda epididymis

Reproduction ◽

10.1530/rep-12-0472 ◽

2013 ◽

Vol 145 (3) ◽

pp. 255-263 ◽

Cited By ~ 14

Author(s):

Lukas Ded ◽

Natasa Sebkova ◽

Martina Cerna ◽

Fatima Elzeinova ◽

Pavla Dostalova ◽

...

Keyword(s):

Reproductive Tract ◽

Negative Impact ◽

Reproductive Potential ◽

Female Reproductive Tract ◽

Sperm Capacitation ◽

Epididymal Sperm ◽

Knock Out ◽

17Β Estradiol

Estrogens play a crucial role in spermatogenesis and estrogen receptor α knock-out male mice are infertile. It has been demonstrated that estrogens significantly increase the speed of capacitation in vitro; however this may lead to the reduction of reproductive potential due to the decreased ability of these sperm to undergo the acrosome reaction. To date the in vivo effect of estrogens on the ability of sperm to capacitate has not been investigated. Therefore, in this study, we exposed mice (n=24) to 17β-estradiol (E2) at the concentration of 20 ng/ml either during puberty from the fourth to seventh week of age (n=8), or continuously from birth for a period of 12 weeks (n=8) at which age the animals from both groups were killed. The capacitation status of epididymal and testicular sperm was analysed by tyrosine phosphorylation (TyrP) antibody (immunofluorescence and western blot) and chlortetracycline (CTC) assay. According to our results, in vivo exposure to increased E2 concentrations caused premature sperm capacitation in the epididymis. The effect of E2, however, seems reversible because after the termination of the exposure premature epididymal sperm capacitation is decreased in animals treated during puberty. Furthermore the changes in epididymal sperm capacitation status detected by TyrP and CTC positively correlate with plasma levels of E2 and the expression of the estrogen-dependent trefoil factor 1 (Tff1) gene in testicular tissue. Therefore, our data implicate that in vivo exposure to E2 under specific conditions leads to the premature capacitation of mouse sperm in epididymis with a potential negative impact on the sperm reproductive fitness in the female reproductive tract.

Download Full-text

Organs-On-Chip Models of the Female Reproductive System

Bioengineering ◽

10.3390/bioengineering6040103 ◽

2019 ◽

Vol 6 (4) ◽

pp. 103 ◽

Cited By ~ 2

Author(s):

Vanessa Mancini ◽

Virginia Pensabene

Keyword(s):

Cell Biology ◽

Reproductive Tract ◽

Hormonal Treatment ◽

Female Reproductive Tract ◽

Specific Cell ◽

Toxicity Screening ◽

Paracrine Signalling ◽

On Chip

Microfluidic-based technology attracts great interest in cell biology and medicine, in virtue of the ability to better mimic the in vivo cell microenvironment compared to conventional macroscale cell culture platforms. Recent Organs-on-chip (OoC) models allow to reproduce in vitro tissue and organ-level functions of living organs and systems. These models have been applied for the study of specific functions of the female reproductive tract, which is composed of several organs interconnected through intricate endocrine pathways and communication mechanisms. To date, a disease and toxicology study of this system has been difficult to perform. Thus, there is a compelling need to develop innovative platforms for the generation of disease model and for performing drug toxicity/screening in vitro studies. This review is focused on the analysis of recently published OoC models that recreate pathological and physiological characteristics of the female reproductive organs and tissues. These models aim to be used to assess changes in metabolic activity of the specific cell types and the effect of exposure to hormonal treatment or chemical substances on some aspects of reproduction and fertility. We examined these models in terms of device specifications, operating procedures, accuracy for studying the biochemical and functional activity of living tissues and the paracrine signalling that occurs within the different tissues. These models represent a powerful tool for understanding important diseases and syndromes affecting women all around the world. Immediate adoption of these models will allow to clarify diseases, causes and adverse events occurring during pregnancy such as pre-eclampsia, infertility or preterm birth, endometriosis and infertility.

Download Full-text