Biology subjects

Burcham, L. R.

Publications and source records attributed to Burcham, L. R..

4 recordsLinked to original sources

bioRxiv2020

Identification of zinc-dependent mechanisms used by Group B Streptococcus to overcome calprotectin-mediated stress

Nutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Though GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon (Krmit) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin, are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative RT-PCR revealed a significant upregulation of genes encoding zinc-binding proteins, adcA, adcAII, and lmb, following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant ({Delta}adcA{Delta}adcAII{Delta}lmb) compared to the GBS WT strain. Further, mice challenged with the {Delta}adcA{Delta}adcAII{Delta}lmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery are important for combatting zinc-chelation by calprotectin and establishing invasive disease. ImportanceGBS asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils, carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro, and in a mouse model of infection. We observed that a global zinc-uptake mutant was less virulent compared to the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by WT and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease.

microbiology↗

bioRxiv2020

Genome-wide mutagenesis identifies factors involved in Enterococcus faecalis vaginal adherence and persistence

Enterococcus 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.

microbiology↗

bioRxiv2019

Identification of key determinants of Staphylococcus aureus vaginal colonization

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 wide-spread 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 hours, 1-day, and 3-days post-inoculation. Over 25% of 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 as well as the capacity to overcome host nutritional limitation are important determinants of MRSA vaginal colonization.\n\nIMPORTANCEStaphylococcus 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 the NICU 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 as well as 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.

microbiology↗

bioRxiv2018

SP1433-1438 operon of Streptococcuspneumoniae regulates metal homeostasis and cellular metabolism during zinc-stress

Streptococcus pneumoniae colonizes the mucosa of the human nasopharynx and is a leading cause of community-acquired pneumonia, acute otitis media, and bacterial meningitis. Metal ion homeostasis is vital to the survival of this pathogen and contributes significantly to both colonization and invasive disease. Microarray and qRT-PCR analysis revealed an upregulation of an uncharacterized operon (SP1433-1438) in pneumococci subjected to metal-chelation by N,N,N,N-tetrakis-(2-Pyridylmethyl)ethylenediamine (TPEN). Supplementation of either zinc or cobalt following TPEN treatment drastically abrogated induction. BLAST analysis predicted this operon to encode two ABC-transporters, sharing homology to a multidrug resistance system (SP1434-1435) and an energy-coupling factor (ECF) transport system (SP1436-1438). Inductively coupled plasma mass spectrometry (ICP-MS) analysis indicated changes in intracellular concentrations of iron, zinc, and manganese ions in a {Delta}1434-8 strain compared to parental T4R. Analysis of the secreted metabolomic profile of the T4R and {Delta}1434-8 strains identified significant changes in pneumococcal glycolytic pathways, indicating a shift towards increased production of acetate. Additionally, proteomic analysis revealed 41 differentially expressed proteins in the {Delta}1434-8 strain, with roughly 20% of them regulated by the global catabolite repressor, CcpA. Based on these findings, we propose that the SP1433-1438 operon is largely involved in the central metabolism of S. pneumoniae during zinc-limitation.\n\nImportanceMetal sequestration is a common strategy utilized by the host immune response as well as antibiotics such as vancomycin to kill invading bacterial pathogens (1). However, pneumococcus is still able to thrive under zinc-limiting conditions. This study describes a previously uncharacterized operon encoding two ABC transport systems that are strongly induced during zinc-limiting conditions. This operon was found to be regulated by a zinc-dependent regulator (SP1433) that functions independently of the overarching AdcR regulon. We have additionally utilized a 2D-NMR approach to analyze the secreted metabolome and have employed proteomic analysis to identify a role for these systems in the maintenance of cellular metabolism. This study provides new information on how Streptococcus pneumoniae responds and adapts to zinc-limiting conditions.

microbiology↗