Biology subjects

Kline, K. A.

Publications and source records attributed to Kline, K. A..

31 records · Page 2Linked to original sources

bioRxiv2022

Depleting cationic lipids involved in antimicrobial resistance drives adaptive lipid remodeling in Enterococcus faecalis

The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While E. faecalis encodes two mprF paralogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human {beta}-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E. faecalis mprF. We analyzed the lipid profiles of parental wild type and mprF mutant strains and show that while {triangleup}mprF2 and {triangleup}mprF1 {triangleup}mprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the {triangleup}mprF1 mutant synthesized [~]70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in {triangleup}mprF2 and {triangleup}mprF1 {triangleup}mprF2. In the mprF mutants, particularly {triangleup}mprF1 {triangleup}mprF2, the decrease in L-PG and PG is compensated by an increase in the phosphorus-containing lipid, GPDGDAG, and D-ala-GPDGDAG. These changes were accompanied by a downregulation of de novo fatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the {triangleup}mprF1 {triangleup}mprF2 mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both {triangleup}mprF2 and {triangleup}mprF1 {triangleup}mprF2, compared to wild type and {triangleup}mprF1. Collectively, our findings reveal a previously unappreciated role for mprF in global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF. Significance StatementThe cell membrane plays a pivotal role in protecting bacteria against external threats, such as antibiotics. Cationic phospholipids such as lysyl-phosphatidyglycerol (L-PG) resist the action of cationic antimicrobial peptides through electrostatic repulsion. Here we demonstrate that L-PG depletion has several unexpected consequences in Enterococcus faecalis, including a reduction of phosphatidylglycerol (PG), enrichment of a phosphorus-containing lipid, reduced fatty acid synthesis accompanied by an accumulation of long-chain acyl-acyl carrier proteins (long chain acyl-ACPs), lower membrane fluidity, and impaired secretion. These changes are not deleterious to the organism as long as exogenous fatty acids are available for uptake from the culture medium. Our findings suggest an adaptive mechanism involving compensatory changes across the entire lipidome upon removal of a single phospholipid modification. Such adaptations must be considered when devising antimicrobial strategies that target membrane lipids.

microbiology↗

bioRxiv2022

Mitoxantrone Targets Both Host and Bacteria to Overcome Vancomycin Resistance in Enterococcus faecalis

Among Enterococci, intrinsic and acquired resistance to antibiotics such as {beta}-lactams and vancomycin critically limit treatment options for infection with these opportunistic pathogens. Antimicrobials that enhance the host immune response are emerging as alternative approaches, with the potential to overcome bacterial resistance. Here, we investigate the antibiotic and immunological activity of the anticancer agent mitoxantrone (MTX) in vitro and in vivo against vancomycin resistant Enterococcus faecalis (VRE). We show that, in vitro, MTX is a potent antibiotic against Gram-positive bacteria with a minimal inhibitory concentration (MIC) of ~1 g/ml through induction of reactive oxygen species and DNA damage. MTX synergises with vancomycin and lowers the vancomycin concentration required to kill VRE by over 140-fold. This synergy is specific to vancomycin-resistant, but not susceptible strains because vancomycin rendered the resistant strains more permeable to MTX and thus MTX-mediated DNA damage. In a murine wound infection model, MTX treatment effectively reduced VRE bacterial numbers by 120-fold and with further reduction when combined with vancomycin. Wounds treated with MTX had significantly higher numbers of macrophages and higher pro-inflammatory cytokines compared to untreated wounds. In addition, MTX augmented intracellular bacterial killing by both murine and human macrophages by upregulating the expression of lysosomal hydrolases cathepsins D and H, and {beta}-Hexosaminidase. These results show that MTX is a potent antibiotic against Gram-positive bacteria, synergizes with vancomycin, enhances macrophage recruitment and intracellular bactericidal activity, and represents a promising dual bacterium- and host-targeted therapeutic for overcoming vancomycin resistance. One sentence summaryMitoxantrone synergizes with vancomycin against vancomycin resistant bacterial strains via direct antibiotic activity and by augmenting both host macrophage recruitment to the site of infection and macrophage bactericidal activity.

microbiology↗

bioRxiv2022

Purine and carbohydrate availability drive Enterococcus faecalis fitness during wound infection

Enterococcus faecalis is commonly isolated from a variety of wound types. Despite its prevalence, the pathogenic mechanisms of E. faecalis during wound infection are poorly understood. Using a mouse wound infection model, we performed in vivo E. faecalis transposon sequencing and RNA sequencing to identify fitness determinants that are crucial for replication and persistence of E. faecalis during wound infection. We found that E. faecalis purine biosynthesis genes are important for bacterial replication during the early stages of wound infection, a time when purine metabolites are rapidly consumed by E. faecalis within wounds. We also found that the E. faecalis MptABCD phosphotransferase system (PTS), involved in the import of galactose and mannose, is crucial for E. faecalis persistence within wounds of both healthy and diabetic mice, especially when carbohydrate availability changes throughout the course of infection. During in vitro growth with mannose as the sole carbohydrate source, shikimate and purine biosynthesis genes were downregulated in the OG1RF {Delta}mptD mutant compared to the isogenic wild-type strain, suggesting a link between mannose transport, shikimate, and purine biosynthesis. Together, our results suggest that dynamic and temporal microenvironment changes at the wound site affects pathogenic requirements and mechanisms of E. faecalis and raise the possibility of lowering exogenous purine availability and/or targeting galactose/mannose PTS to control wound infections. IMPORTANCEAlthough E. faecalis is a common wound pathogen, its pathogenic mechanisms during wound infection are unexplored. Here, combining a mouse wound infection model with in vivo transposon and RNA sequencing approaches, we identified the E. faecalis purine biosynthetic pathway and galactose/mannose MptABCD phosphotransferase system as essential for E. faecalis acute replication and persistence during wound infection, respectively. The essentiality of purine biosynthesis and the MptABCD PTS is driven by the rapid consumption of purine metabolites by E. faecalis during acute replication and changing carbohydrate availability during the course of wound infection. Overall, our findings reveal the importance of the wound microenvironment in E. faecalis wound pathogenesis and how these metabolic pathways can be targeted to better control wound infections.

microbiology↗

bioRxiv2022

An RNA modification enzyme directly senses reactive oxygen species for translational regulation in Enterococcus faecalis

Bacteria possess elaborate systems to manage reactive oxygen and nitrogen species (ROS) arising from exposure to the mammalian immune system and environmental stresses. Here we report the discovery of an ROS-sensing RNA-modifying enzyme that regulates translation of stress-response proteins in the gut commensal and opportunistic pathogen Enterococcus faecalis. We analyzed the tRNA epitranscriptome of E. faecalis in response to reactive oxygen species (ROS) or sublethal doses of ROS-inducing antibiotics and identified large decreases in N2-methyladenosine (m2A) in both 23S ribosomal RNA and transfer RNA. This we determined to be due to ROS-mediated inactivation of the Fe-S cluster-containing methyltransferase, RlmN. Genetic knockout of RlmN gave rise to a proteome that mimicked the oxidative stress response, with increased levels of superoxide dismutase and decreased virulence proteins. While tRNA modifications are established to be dynamic for fine-tuning translation, here we report the first instance of a dynamically regulated, environmentally responsive rRNA modification. These studies lead to model in which RlmN serves as a redox-sensitive molecular switch, directly relaying oxidative stress to modulating translation through the rRNA and the tRNA epitranscriptome, revealing a new paradigm for understanding direct regulation of the proteome by RNA modifications.

microbiology↗

bioRxiv2022

Enterococcus faecalis antagonizes Pseudomonas aeruginosa growth in polymicrobial biofilms

Enterococcus faecalis is often co-isolated with Pseudomonas aeruginosa in polymicrobial biofilm-associated infections of wounds and the urinary tract. As a defense strategy, the host innately restricts iron availability at infection sites. Despite their co-prevalence, the polymicrobial interactions of these two pathogens in iron- restricted conditions, such as those found in the host, remains unexplored. Here we show that E. faecalis inhibits P. aeruginosa growth within biofilms when iron is restricted. E. faecalis lactate dehydrogenase (ldh1) gives rise to L-lactate production during fermentative growth. We find that E. faecalis ldh1 mutant fails to inhibit P. aeruginosa growth. Additionally, we demonstrate that ldh1 expression is induced in iron-restricted conditions, resulting in increased lactic acid exported and consequently, a reduction in pH. Together, our results suggest that E. faecalis synergistically inhibit P. aeruginosa growth by decreasing environmental pH and L-lactate-mediated iron chelation. Overall, this study highlights that the microenvironment in which the infection occurs is important for understanding its pathophysiology. IMPORTANCEMany infections are polymicrobial and biofilm-associated in nature. Iron is essential for many metabolic processes and plays an important role in controlling infections, where the host restricts iron as a defense mechanism against invading pathogens. However, polymicrobial interactions between pathogens are underexplored in iron- restricted conditions. Here, we explore the polymicrobial interactions between commonly co-isolated E. faecalis and P. aeruginosa within biofilms. We find that E. faecalis modulates the microenvironment by exporting lactic acid which further chelates already limited iron, and also lowers the environmental pH to antagonize P. aeruginosa growth in iron-restricted conditions. Our findings provide insights into polymicrobial interactions between pathogens in an infection-relevant condition and how manipulating the microenvironment can be taken advantage of to better control infections.

microbiology↗

bioRxiv2021

Escherichia coli BarA-UvrY regulates the pks island and kills Staphylococci via the genotoxin colibactin

Wound infections are often polymicrobial in nature and are associated with poor disease prognoses. Escherichia coli and Staphylococcus aureus are among the top five most cultured pathogens from wound infections. However, little is known about the polymicrobial interactions between E. coli and S. aureus during wound infections. In this study, we show that E. coli kills S. aureus both in vitro and in a mouse excisional wound model via the genotoxin, colibactin. We also show that the BarA-UvrY two component system (TCS) is a novel regulator of the pks island, which acts through the carbon storage global regulatory (Csr) system. Together, our data demonstrate the role of colibactin in inter-species competition and show that it is regulated by BarA-UvrY TCS, a previously uncharacterized regulator of the pks island.

microbiology↗

bioRxiv2021

Enterococcus faecalis persists and replicates within epithelial cells in vitro and in vivo during wound infection

Enterococcus faecalis is a frequent opportunistic pathogen of wounds, whose infections are associated with biofilm formation, persistence, and recalcitrance toward treatment. We have previously shown that E. faecalis wound infection persists for at least 7 days. Here we report that viable E. faecalis are present within both immune and non-immune cells at the wound site up to 5 days after infection, raising the prospect that intracellular persistence contributes to chronic E. faecalis infection. Using an in vitro keratinocyte infection model, we show that a subpopulation of E. faecalis becomes internalized via macropinocytosis into single membrane-bound compartments, where they can survive and replicate. These intracellular E. faecalis can persist in late endosomes up to 72 hours after infection in the absence of colocalization with the lysosomal protease cathepsin D or apparent fusion with the lysosome, suggesting that E. faecalis blocks endosomal maturation. Indeed, intracellular E. faecalis infection results in a marked reduction in Rab7 expression, a small GTPase required for endosome-lysosome fusion. Finally, we demonstrate that intracellular E. faecalis derived from infected keratinocytes are significantly more efficient in reinfecting new keratinocytes. Together, these data suggest that intracellular proliferation of E. faecalis may contribute to its persistence in the face of a robust immune response, providing a primed reservoir of bacteria for subsequent reinfection. Author SummaryEnterococcus faecalis is often isolated from chronic wounds. Prior to this study, E. faecalis has been observed within different cell types, suggesting that it can successfully colonize intracellular spaces. However, to date, little is known about the mechanisms E. faecalis use to survive intracellularly. Here, we describe key features of the intracellular lifestyle of E. faecalis. We show that E. faecalis exists in an intracellular state within immune cells and non-immune cells during mammalian wound infection. We show that E. faecalis can survive and replicate inside keratinocytes, and intracellularly replicating E. faecalis are primed to more efficiently cause reinfection, potentially contributing to chronic or persistent infections. In order to establish this intracellular lifestyle, E. faecalis is taken up by keratinocytes via macropinocytosis, whereupon it manipulates the endosomal pathway and expression of trafficking molecules required for endo-lysosomal fusion, enabling E. faecalis to avoid lysosomal degradation and consequent death. These results advance our understanding of E. faecalis pathogenesis, demonstrating mechanistically how this classic extracellular pathogen can co-opt host cells for intracellular persistence, and highlight the heterogeneity of mechanisms bacteria can use to avoid host-mediated killing in order to cause disease.

microbiology↗

bioRxiv2020

Multiplex CRISPRi-Cas9 silencing of planktonic and stage-specific biofilm genes in Enterococcus faecalis

Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multi-level regulation. Compared to established knockout methodologies, CRISPRi approaches enable rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both non-template and template strand targeting. Since nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust, and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacteria interactions, and inter-species communication. IMPORTANCEEnterococcus faecalis causes multidrug resistant life-threatening infections, and is often co-isolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labour-intensive allelic exchange methods. Built upon streptococcal dCas9, we developed an easily-modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation, antibiotic resistance, and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance, and can be used to perturb pre-formed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.

microbiology↗

bioRxiv2020

Enterococcus faecalis manganese exporter MntE alleviates manganese toxicity and is required for mouse gastrointestinal colonization

Bacterial pathogens encounter a variety of nutritional environments in the human host, including nutrient metal restriction and overload. Uptake of manganese (Mn) is essential for Enterococcus faecalis growth and virulence; however, it is not known how this organism prevents Mn toxicity. In this study, we examine the role of the highly conserved MntE transporter in E. faecalis Mn homeostasis and virulence. We show that inactivation of mntE results in growth restriction in the presence of excess Mn, but not other metals, demonstrating its specific role in Mn detoxification. Upon growth in the presence of excess Mn, an mntE mutant accumulates intracellular Mn, iron (Fe), and magnesium (Mg), supporting a role for MntE in Mn and Fe export, and a role for Mg in offsetting Mn toxicity. Growth of the mntE mutant in excess Fe also results in increased levels of intracellular Fe, but not Mn or Mg, providing further support for MntE in Fe efflux. Inactivation of mntE in the presence of excess iron also results in the upregulation of glycerol catabolic genes and enhanced biofilm growth, and addition of glycerol is sufficient to augment biofilm growth for both the mntE mutant and its wild type parental strain, demonstrating that glycerol availability significantly enhances biofilm formation. Finally, we show that mntE contributes to infection of the antibiotic-treated mouse gastrointestinal (GI) tract, suggesting that E. faecalis encounters excess Mn in this niche. Collectively, these findings demonstrate that the manganese exporter MntE plays a crucial role in E. faecalis metal homeostasis and virulence.

microbiology↗

bioRxiv2019

Combined efficacy of a novel antimicrobial cationic peptide polymer with conventional antibiotics to combat multi-drug resistant pathogens

Antibiotic-resistant infections are predicted to kill 10 million people worldwide per year by 2050 and to cost the global economy 100 trillion USD. Novel approaches and alternatives to conventional antibiotics are urgently required to combat antimicrobial resistance. We have synthesized a chitosan-based oligolysine antimicrobial peptide, CSM5-K5, which targets multidrug resistant (MDR) bacterial species. Here we show that CSM5-K5 exhibits rapid bactericidal activity against methicillin resistant Staphylococcus aureus (MRSA), MDR Escherichia coli, and vancomycin resistant Enterococcus faecalis (VRE). Combinatorial therapy of CSM5-K5 with antibiotics to which each organism is otherwise resistant restores sensitivity to the conventional antibiotic. CSM5-K5 alone significantly reduced pre-formed bacterial biofilm by two-four orders of magnitude and, in combination with traditional antibiotics, reduced pre-formed biofilm by more than two-three orders of magnitude at sub inhibitory concentrations. Moreover, using a mouse excisional wound infection model, CSM5-K5 treatment reduced bacterial burdens by one to three orders of magnitude, and acted synergistically with vancomycin and tetracycline to clear VRE and MDR E. coli, respectively. Importantly, little to no resistance against CSM5-K5 arose for any of the three MDR bacteria during 15 days of serial passage. This work demonstrates the feasibility and benefits of using this synthetic cationic peptide as alternative to, or in combination with, traditional antibiotics to treat infections caused by MDR bacteria.

microbiology↗

bioRxiv2019

Sortase-assembled pili promote extracellular electron transfer and iron acquisition in Enterococcus faecalis biofilm

Enterococcus faecalis sortase-assembled endocarditis and biofilm-associated pili (Ebp) are a virulence factor implicated in enterococcal biofilm-associated infections and gastrointestinal colonization. We previously showed that E. faecalis biofilm metabolism is influenced by extracellular electron transfer (EET) under iron-rich conditions, raising the question of whether Ebp pili also play a role in EET. Here, we report a novel role of Ebp pili in E. faecalis adhesion to the iron oxides magnetite, goethite and hematite, where the EbpA tip adhesin contributes to this interaction. Adhesion by Ebp pili is conditionally important for EET to iron oxides, as pilus mutants are attenuated in EET under non-static growth conditions. In alignment with the established role of EET in redox homeostasis, we find that EET to ferricyanide supports E. faecalis anaerobic growth on glycerol. Further, in an antibiotic-treated mouse gastrointestinal colonization model, we show that E. faecalis mutants deficient in EET poorly colonize the intestinal niche. Taken together, our findings suggest that Ebp pili can influence E. faecalis metabolic fitness by promoting EET to iron oxides, raising new questions of how Ebp pili shape E. faecalis interactions with environmental ecosystems. Furthermore, the important role of EET in E. faecalis colonization of the dysbiotic gastrointestinal environment highlights the need for further inquiry into how EET contributes to E. faecalis microbial pathogenesis. ImportanceIn this study, we explored the interplay between extracellular electron transfer (EET) and an Enterococcus faecalis biofilm factor, the endocarditis and biofilm-associated pili (Ebp). We demonstrate that Ebp pili have a novel role in adhesion to iron oxides, which consequently promotes EET to iron oxides under non-static conditions. Along with our findings that E. faecalis EET can be coupled to anaerobic cell growth, our results point to a potential ecological role of Ebp pili in natural environments, outside of its established function in adhesion to host ligands. We provide the first evidence of the contribution of EET to E. faecalis colonization of the antibiotic-treated murine intestinal niche, which adds to the limited experimental evidence linking EET and microbial pathogenesis, as well as highlights the need for further studies of EET in bacterial pathogens.

microbiology↗

bioRxiv2018

Planktonic interference and biofilm alliance between aggregation substance and endocarditis and biofilm associated pili in Enterococcus faecalis

Like many bacteria, Enterococcus faecalis encodes a number of adhesins involved in colonization or infection of different niches. Two well-studied E. faecalis adhesins, aggregation substance (AS) and endocarditis and biofilm-associated pili (Ebp), both contribute to biofilm formation on abiotic surfaces and in endocarditis, suggesting they may be expressed at the same time. Because different regulatory pathways have been reported for AS and Ebp, here we examined if they are co-expressed on the same cells and the functional impact of co-expression on individual cells and within a population. We found that while Ebp are only expressed on a subset of cells, when Ebp and AS are expressed on the same cells, pili interfere with AS-mediated clumping and impede AS-mediated conjugative plasmid transfer during planktonic growth. However, when the population density increases, horizontal gene transfer rates normalize and are no longer affected by pilus expression. Instead, at higher cell densities during biofilm formation, Ebp and AS differentially contribute to biofilm development and structure, synergizing to promote maximal biofilm formation.

microbiology↗

bioRxiv2017

Enterococcus faecalis Modulates Immune Activation And Slows Healing During Wound Infection

Enterococcus faecalis is one of most frequently isolated bacterial species in wounds yet little is known about its pathogenic mechanisms in this setting. Here, we used a mouse wound excisional model to characterize the infection dynamics of E. faecalis and show that infected wounds result in two different states depending on the initial inoculum. Low dose inocula were associated with short term, low titer colonization whereas high dose inocula were associated with acute bacterial replication and long term persistence. High dose infection and persistence were also associated with immune cell infiltration, despite suppression of some inflammatory cytokines and delayed wound healing. During high dose infection, the multiple peptide resistance factor (MprF) which is involved in resisting immune clearance, contributes to E. faecalis fitness. These results comprehensively describe a mouse model for investigating E. faecalis wound infection determinants, and suggest that both immune modulation and resistance contribute to persistent, non-healing wounds.

microbiology↗