Esrrγa regulates nephron and ciliary development by controlling prostaglandin synthesis.

Cilia are essential for the ontogeny and function of many tissues, including the kidney. Here, we report that transcription factor ERRγ ortholog estrogen related receptor gamma a (Esrrγa) is essential for renal cell fate choice and ciliogenesis in zebrafish. esrrγa deficiency altered proximodistal nephron patterning, decreased the multiciliated cell populace and disrupted ciliogenesis in the nephron, Kupffer's vesicle and otic vesicle. These phenotypes were consistent with interruptions in prostaglandin signaling, and we found that ciliogenesis was rescued by PGE2 or the cyclooxygenase enzyme Ptgs1. Genetic interaction revealed that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (Ppargc1a), which acts upstream of Ptgs1-mediated prostaglandin synthesis, has a synergistic relationship with Esrrγa in the ciliogenic pathway. These ciliopathic phenotypes were also observed in mice lacking renal epithelial cell (REC) ERRγ, where significantly shorter cilia formed on proximal and distal tubule cells. Decreased cilia length preceded cyst formation in REC-ERRγ knockout mice, suggesting that ciliary changes occur early during pathogenesis. These data position Esrrγa as a novel link between ciliogenesis and nephrogenesis through regulation of prostaglandin signaling and cooperation with Ppargc1a.

Ring-fused 2-pyridones effective against multidrug-resistant Gram-positive pathogens and synergistic with standard-of-care antibiotics.

The alarming rise of multidrug-resistant Gram-positive bacteria has precipitated a healthcare crisis, necessitating the development of new antimicrobial therapies. Here we describe a new class of antibiotics based on a ring-fused 2-pyridone backbone, which are active against vancomycin-resistant enterococci (VRE), a serious threat as classified by the Centers for Disease Control and Prevention, and other multidrug-resistant Gram-positive bacteria. Ring-fused 2-pyridone antibiotics have bacteriostatic activity against actively dividing exponential phase enterococcal cells and bactericidal activity against nondividing stationary phase enterococcal cells. The molecular mechanism of drug-induced killing of stationary phase cells mimics aspects of fratricide observed in enterococcal biofilms, where both are mediated by the Atn autolysin and the GelE protease. In addition, combinations of sublethal concentrations of ring-fused 2-pyridones and standard-of-care antibiotics, such as vancomycin, were found to synergize to kill clinical strains of VRE. Furthermore, a broad range of antibiotic resistant Gram-positive pathogens, including those responsible for the increasing incidence of antibiotic resistant healthcare-associated infections, are susceptible to this new class of 2-pyridone antibiotics. Given the broad antibacterial activities of ring-fused 2-pyridone compounds against Gram-positive (GmP) bacteria we term these compounds GmPcides, which hold promise in combating the rising tide of antibiotic resistant Gram-positive pathogens.

c-di-AMP Is Essential for the Virulence of Enterococcus faecalis.

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (ΔcdaA strain) or c-di-AMP accumulation (ΔdhhP, ΔgdpP, and ΔdhhP ΔgdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the ΔcdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the ΔcdaA strain also could be attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

The M Protein of Streptococcus pyogenes Strain AP53 Retains Cell Surface Functional Plasminogen Binding after Inactivation of the Sortase A Gene.

Streptococcus pyogenes (Lancefield group A Streptococcus [GAS]) is a ß-hemolytic human-selective pathogen that is responsible for a large number of morbid and mortal infections in humans. For efficient infection, GAS requires different types of surface proteins that provide various mechanisms for evading human innate immune responses, thus enhancing pathogenicity of the bacteria. Many such virulence-promoting proteins, including the major surface signature M protein, are translocated after biosynthesis through the cytoplasmic membrane and temporarily tethered to this membrane via a type 1 transmembrane domain (TMD) positioned near the COOH terminus. In these proteins, a sorting signal, LPXTG, is positioned immediately upstream of the TMD, which is cleaved by the membrane-associated transpeptidase, sortase A (SrtA), leading to the covalent anchoring of these proteins to newly emerging l-Ala-l-Ala cross-bridges of the growing peptidoglycan cell wall. Herein, we show that inactivation of the srtA gene in a skin-tropic pattern D GAS strain (AP53) results in retention of the M protein in the cell membrane. However, while the isogenic AP53 ΔsrtA strain is attenuated in overall pathogenic properties due to effects on the integrity of the cell membrane, our data show that the M protein nonetheless can extend from the cytoplasmic membrane through the cell wall and then to the surface of the bacteria and thereby retain its important properties of productively binding and activating fluid-phase host plasminogen (hPg). The studies presented herein demonstrate an underappreciated additional mechanism of cell surface display of bacterial virulence proteins via their retention in the cell membrane and extension to the GAS surface.IMPORTANCE Group A Streptococcus pyogenes (GAS) is a human-specific pathogen that produces many surface factors, including its signature M protein, that contribute to its pathogenicity. M proteins undergo specific membrane localization and anchoring to the cell wall via the transpeptidase sortase A. Herein, we explored the role of sortase A function on M protein localization, architecture, and function, employing, a skin-tropic GAS isolate, AP53, which expresses a human plasminogen (hPg)-binding M (PAM) Protein. We showed that PAM anchored in the cell membrane, due to the targeted inactivation of sortase A, was nonetheless exposed on the cell surface and functionally interacted with host hPg. We demonstrate that M proteins, and possibly other sortase A-processed proteins that are retained in the cell membrane, can still function to initiate pathogenic processes by this underappreciated mechanism.

Manganese acquisition is essential for virulence of Enterococcus faecalis.

Manganese (Mn) is an essential micronutrient that is not readily available to pathogens during infection due to an active host defense mechanism known as nutritional immunity. To overcome this nutrient restriction, bacteria utilize high-affinity transporters that allow them to compete with host metal-binding proteins. Despite the established role of Mn in bacterial pathogenesis, little is known about the relevance of Mn in the pathophysiology of E. faecalis. Here, we identified and characterized the major Mn acquisition systems of E. faecalis. We discovered that the ABC-type permease EfaCBA and two Nramp-type transporters, named MntH1 and MntH2, work collectively to promote cell growth under Mn-restricted conditions. The simultaneous inactivation of EfaCBA, MntH1 and MntH2 (ΔefaΔmntH1ΔmntH2 strain) led to drastic reductions (>95%) in cellular Mn content, severe growth defects in body fluids (serum and urine) ex vivo, significant loss of virulence in Galleria mellonella, and virtually complete loss of virulence in rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI) models. Despite the functional redundancy of EfaCBA, MntH1 and MntH2 under in vitro or ex vivo conditions and in the invertebrate model, dual inactivation of efaCBA and mntH2 (ΔefaΔmntH2 strain) was sufficient to prompt maximal sensitivity to calprotectin, a Mn- and Zn-chelating host antimicrobial protein, and for the loss of virulence in mammalian models. Interestingly, EfaCBA appears to play a prominent role during systemic infection, whereas MntH2 was more important during CAUTI. The different roles of EfaCBA and MntH2 in these sites could be attributed, at least in part, to the differential expression of efaA and mntH2 in cells isolated from hearts or from bladders. Collectively, this study demonstrates that Mn acquisition is essential for the pathogenesis of E. faecalis and validates Mn uptake systems as promising targets for the development of new antimicrobials.

High-resolution imaging reveals microbial biofilms on patient urinary catheters despite antibiotic administration.

PURPOSE: Catheter-associated urinary tract infections (CAUTIs) are a significant cause of morbidity worldwide, as they account for 40% of all hospital-associated infections. Microbial biofilm formation on urinary catheters (UCs) limits antibiotic efficacy, making CAUTI extremely difficult to treat. To gain insight into the spatiotemporal microbe interactions on the catheter surface we sought to determine how the presence or absence of bacteriuria prior to catheterization affects the organism that ultimately forms a biofilm on the UC and how long after catheterization they emerge. METHODS: Thirty UCs were collected from patients who received a urine culture prior to catheterization, a UC, and antibiotics as part of standard of care. Immunofluorescence imaging and scanning electron microscopy were used to visualize patient UCs. RESULTS: Most patients did not have bacteria in their urine (based on standard urinalysis) prior to catheterization, yet microbes were detected on the majority of UCs, even with dwell times of < 3 days. The most frequently identified microbes were Staphylococcus epidermidis, Enterococcus faecalis, and Escherichia coli. CONCLUSIONS: This study indicates that despite patients having negative urine cultures and receiving antibiotics prior to catheter placement, microbes, including uropathogens associated with causing CAUTI, could be readily detected on UCs with short dwell times. This suggests that a potential microbial catheter reservoir can form soon after placement, even in the presence of antibiotics, which may serve to facilitate the development of CAUTI. Thus, removing and/or replacing UCs as soon as possible is of critical importance to reduce the risk of developing CAUTI.

Catheterization alters bladder ecology to potentiate Staphylococcus aureus infection of the urinary tract.

Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging cause of catheter-associated urinary tract infection (CAUTI), which frequently progresses to more serious invasive infections. We adapted a mouse model of CAUTI to investigate how catheterization increases an individual's susceptibility to MRSA UTI. This analysis revealed that catheterization was required for MRSA to achieve high-level, persistent infection in the bladder. As shown previously, catheter placement induced an inflammatory response resulting in the release of the host protein fibrinogen (Fg), which coated the bladder and implant. Following infection, we showed that MRSA attached to the urothelium and implant in patterns that colocalized with deposited Fg. Furthermore, MRSA exacerbated the host inflammatory response to stimulate the additional release and accumulation of Fg in the urinary tract, which facilitated MRSA colonization. Consistent with this model, analysis of catheters from patients with S. aureus-positive cultures revealed colocalization of Fg, which was deposited on the catheter, with S. aureus Clumping Factors A and B (ClfA and ClfB) have been shown to contribute to MRSA-Fg interactions in other models of disease. We found that mutants in clfA had significantly greater Fg-binding defects than mutants in clfB in several in vitro assays. Paradoxically, only the ClfB- strain was significantly attenuated in the CAUTI model. Together, these data suggest that catheterization alters the urinary tract environment to promote MRSA CAUTI pathogenesis by inducing the release of Fg, which the pathogen enhances to persist in the urinary tract despite the host's robust immune response.

In Silico Prediction of the Toxic Potential of Lupeol.

Lupeol is a natural triterpenoid found in many plant species such as mango. This compound is the principal active component of many traditional herbal medicines. In the past decade, a considerable number of publications dealt with lupeol and its analogues due to the interest in their pharmacological activities against cancer, inflammation, arthritis, diabetes, and heart disease. To identify further potential applications of lupeol and its analogues, it is necessary to investigate their mechanisms of action, particularly their interaction with off-target proteins that may trigger adverse effects or toxicity. In this study, we simulated and quantified the interaction of lupeol and 11 of its analogues toward a series of 16 proteins known or suspected to trigger adverse effects employing the VirtualToxLab. This software provides a thermodynamic estimate of the binding affinity, and the results were challenged by molecular-dynamics simulations, which allow probing the kinetic stability of the underlying protein-ligand complexes. Our results indicate that there is a moderate toxic potential for lupeol and some of its analogues, by targeting and binding to nuclear receptors involved in fertility, which could trigger undesired adverse effects.

Fibrinogen Release and Deposition on Urinary Catheters Placed during Urological Procedures.

PURPOSE: Catheter associated urinary tract infections account for approximately 40% of all hospital acquired infections worldwide with more than 1 million cases diagnosed annually. Recent data from a catheter associated urinary tract infection animal model has shown that inflammation induced by catheterization releases host fibrinogen, which accumulates on the catheter. Further, Enterococcus faecalis catheter colonization was found to depend on EbpA (endocarditis and biofilm-associated pilus), a fibrinogen binding adhesin. We evaluated this mechanism in a human model. MATERIALS AND METHODS: Urinary catheters were collected from patients hospitalized for surgical or nonsurgical urological procedures. Catheters were subjected to immunofluorescence analyses by incubation with antifibrinogen antibody and then staining for fluorescence. Fluorescence intensity was compared to that of standard catheters. Catheters were incubated with strains of Enterococcus faecalis, Staphylococcus aureus or Candida to assess binding of those strains to fibrinogen laden catheters. RESULTS: After various surgical and urological procedures, 50 catheters were collected. In vivo dwell time ranged from 1 hour to 59 days. All catheters had fibrinogen deposition. Accumulation depended on dwell time but not on surgical procedure or catheter material. Catheters were probed ex vivo with E. faecalis, S. aureus and Candida albicans, which bound to catheters only in regions where fibrinogen was deposited. CONCLUSIONS: Taken together, these data show that urinary catheters act as a binding surface for the accumulation of fibrinogen. Fibrinogen is released due to inflammation resulting from a urological procedure or catheter placement, creating a niche that can be exploited by uropathogens to cause catheter associated urinary tract infections.

Blunted blades: new CRISPR-derived technologies to dissect microbial multi-drug resistance and biofilm formation.

The spread of multi-drug-resistant (MDR) pathogens has rapidly outpaced the development of effective treatments. Diverse resistance mechanisms further limit the effectiveness of our best treatments, including multi-drug regimens and last line-of-defense antimicrobials. Biofilm formation is a powerful component of microbial pathogenesis, providing a scaffold for efficient colonization and shielding against anti-microbials, which further complicates drug resistance studies. Early genetic knockout tools didn't allow the study of essential genes, but clustered regularly interspaced palindromic repeat inference (CRISPRi) technologies have overcome this challenge via genetic silencing. These tools rapidly evolved to meet new demands and exploit native CRISPR systems. Modern tools range from the creation of massive CRISPRi libraries to tunable modulation of gene expression with CRISPR activation (CRISPRa). This review discusses the rapid expansion of CRISPRi/a-based technologies, their use in investigating MDR and biofilm formation, and how this drives further development of a potent tool to comprehensively examine multi-drug resistance.

Initial Antimicrobial Testing of a Novel Reusable Intermittent Urinary Catheter System and Catheter Reprocessing Device.

OBJECTIVE: To evaluate the efficacy of the Aurie System, a preclinical prototype allowing for standardized intermittent catheter (IC) reuse of novel reusable no-touch ICs. Individuals with neurogenic bladder often require single-use ICs to urinate, but urinary tract infection (UTI) is a common cause of morbidity for IC users. Safer no-touch catheters are not easily affordable, and the Aurie System attempts to provide no-touch catheters at a fraction of the price by allowing for standardized and safe IC reuse. METHODS: Standard ICs were inoculated with Escherichia coli and Pseudomonas aeruginosa and incubated for 48 hours to assess microbial burden and biofilm formation (the latter using infrared fluorescence imaging). This procedure was repeated with Aurie ICs, focusing on evaluating catheter microbial burden after inoculation and reprocessing with the prototype washer-disinfector. This was repeated with up to 100 cycles to evaluate repetitive use. RESULTS: Standard ICs showed bacterial attachment and biofilm development peaking at 24 hours of incubation. The Aurie catheters produced a similar outcome but, after reprocessing, microbial burden was reduced below the level of detection. Repeat cycles showed pathogen clearance to similar levels. One catheter reached 100 cycles and there was no viable pathogen load after reprocessing. CONCLUSION: Intermittent urinary catheters, when cleaned inappropriately, can harbor viable bacteria and biofilm. The Aurie System, when used to disinfect novel reusable ICs within a prototype reprocessing device, can reduce microbial burden below level of detection even after 100 cycles. This suggests the Aurie System may be a feasible technology for safe IC reuse.

Microbial co-occurrences on catheters from long-term catheterized patients.

Catheter-associated urinary tract infections (CAUTIs), a common cause of healthcare-associated infections, are caused by a diverse array of pathogens that are increasingly becoming antibiotic resistant. We analyze the microbial occurrences in catheter and urine samples from 55 human long-term catheterized patients collected over one year. Although most of these patients were prescribed antibiotics over several collection periods, their catheter samples remain colonized by one or more bacterial species. Examination of a total of 366 catheter and urine samples identify 13 positive and 13 negative genus co-occurrences over 12 collection periods, representing associations that occur more or less frequently than expected by chance. We find that for many patients, the microbial species composition between collection periods is similar. In a subset of patients, we find that the most frequently sampled bacteria, Escherichia coli and Enterococcus faecalis, co-localize on catheter samples. Further, co-culture of paired isolates recovered from the same patients reveals that E. coli significantly augments E. faecalis growth in an artificial urine medium, where E. faecalis monoculture grows poorly. These findings suggest novel strategies to collapse polymicrobial CAUTI in long-term catheterized patients by targeting mechanisms that promote positive co-associations.

The catheterized bladder environment induces dysregulation of macrophage polarization exacerbating bacterial UTI.

Urinary catheterization causes bladder damage, predisposing hosts to catheter-associated urinary tract infections (CAUTIs). CAUTI pathogenesis is mediated by bladder damage-induced inflammation, resulting in accumulation and deposition of the blood-clotting protein fibrinogen (Fg) and its matrix form fibrin, which are exploited by uropathogens as biofilm platforms to establish infection. Catheter-induced inflammation also results in robust immune cell recruitment, including macrophages (Mϕs). A fundamental knowledge gap is understanding the mechanisms by which the catheterized-bladder environment suppresses the Mϕ antimicrobial response, allowing uropathogen persistence. Here, we found that Fg and fibrin differentially modulate M1 and M2 Mϕ polarization, respectively. We unveiled that fibrin accumulation in catheterized mice induced an anti-inflammatory M2-like Mϕ phenotype, correlating with pathogen persistence. Even GM-CSF treatment of wildtype mice to promote M1 polarization was not sufficient to reduce bacterial burden and dissemination, indicating that the catheterized-bladder environment provides mixed signals, dysregulating Mϕ polarization, hindering its antimicrobial response against uropathogens.

Fibrinolytic-deficiencies predispose hosts to septicemia from a catheter-associated UTI.

Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat partly due to development of multidrug-resistance from CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, here we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, here we found that Enterococcus faecalis, a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI.

Pilin and sortase residues critical for endocarditis- and biofilm-associated pilus biogenesis in Enterococcus faecalis.

Enterococci commonly cause hospital-acquired infections, such as infective endocarditis and catheter-associated urinary tract infections. In animal models of these infections, a long hairlike extracellular protein fiber known as the endocarditis- and biofilm-associated (Ebp) pilus is an important virulence factor for Enterococcus faecalis. For Ebp and other sortase-assembled pili, the pilus-associated sortases are essential for fiber formation as they create covalent isopeptide bonds between the sortase recognition motif and the pilin-like motif of the pilus subunits. However, the molecular requirements governing the incorporation of the three pilus subunits (EbpA, EbpB, and EbpC) have not been investigated in E. faecalis. Here, we show that a Lys residue within the pilin-like motif of the EbpC subunit was necessary for EbpC polymerization. However, incorporation of EbpA into the pilus fiber only required its sortase recognition motif (LPXTG), while incorporation of EbpB only required its pilin-like motif. Only the sortase recognition motif would be required for incorporation of the pilus tip subunit, while incorporation of the base subunit would only require the pilin recognition motif. Thus, these data support a model with EbpA at the tip and EbpB at the base of an EbpC polymer. In addition, the housekeeping sortase, SrtA, was found to process EbpB and its predicted catalytic Cys residue was required for efficient cell wall anchoring of mature Ebp pili. Thus, we have defined molecular interactions involved in fiber polymerization, minor subunit organization, and pilus subcellular compartmentalization in the E. faecalis Ebp pilus system. These studies advance our understanding of unique molecular mechanisms of sortase-assembled pilus biogenesis.

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans.

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPR activation to overexpress genes involved in pathogenesis and drug susceptibility, and contribute toward the identification of novel phenotypes. Consequently, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

Fibrinolytic-deficiencies predispose hosts to septicemia from a catheter-associated UTI.

Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat due to multi-drug resistance development among the CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, we found that E. faecalis, a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI.

RepC protein of the octopine-type Ti plasmid binds to the probable origin of replication within repC and functions only in cis.

Vegetative replication and partitioning of many plasmids and some chromosomes of alphaproteobacteria are directed by their repABC operons. RepA and RepB proteins direct the partitioning of replicons to daughter cells, while RepC proteins are replication initiators, although they do not resemble any characterized replication initiation protein. Here we show that the replication origin of an Agrobacterium tumefaciens Ti plasmid resides fully within its repC gene. Purified RepC bound to a site within repC with moderate affinity, high specificity and with twofold cooperativity. The binding site was localized to an AT-rich region that contains a large number of GANTC sites, which have been implicated in replication regulation in related organisms. A fragment of RepC containing residues 26-158 was sufficient to bind DNA, although with limited sequence specificity. This portion of RepC is predicted to have structural homology to members of the MarR family of transcription factors. Overexpression of RepC in A. tumefaciens caused large increases in copy number in cis but did not change the copy number of plasmids containing the same oriV sequence in trans, confirming other observations that RepC functions only in cis.

Saturation mutagenesis of a CepR binding site as a means to identify new quorum-regulated promoters in Burkholderia cenocepacia.

Burkholderia cenocepacia is an opportunistic pathogen of humans that encodes two genes that resemble the acylhomoserine lactone synthase gene luxI of Vibrio fischeri and three genes that resemble the acylhomoserine lactone receptor gene luxR. Of these, CepI synthesizes octanoylhomoserine lactone (OHL), while CepR is an OHL-dependent transcription factor. In the current study we developed a strategy to identify genes that are directly regulated by CepR. We systematically altered a CepR binding site (cep box) upstream of a target promoter to identify nucleotides that are essential for CepR activity in vivo and for CepR binding in vitro. We constructed 34 self-complementary oligonucleotides containing altered cep boxes, and measured binding affinity for each. These experiments allowed us to identify a consensus CepR binding site. Several hundred similar sequences were identified, some of which were adjacent to probable promoters. Several such promoters were fused to a reporter gene with and without intact cep boxes. This allowed us to identify four new regulated promoters that were induced by OHL, and that required a cep box for induction. CepR-dependent, OHL-dependent expression of all four promoters was reconstituted in Escherichia coli. Purified CepR bound to each of these sites in electrophoretic mobility shift assays.

The AdcACB/AdcAII system is essential for zinc homeostasis and an important contributor of Enterococcus faecalis virulence.

Bacterial pathogens require a variety of micronutrients for growth, including trace metals such as iron, manganese, and zinc (Zn). Despite their relative abundance in host environments, access to these metals is severely restricted during infection due to host-mediated defense mechanisms collectively known as nutritional immunity. Despite a growing appreciation of the importance of Zn in host-pathogen interactions, the mechanisms of Zn homeostasis and the significance of Zn to the pathophysiology of E. faecalis, a major pathogen of nosocomial and community-associated infections, have not been thoroughly investigated. Here, we show that E. faecalis encoded ABC-type transporter AdcACB and an orphan substrate-binding lipoprotein AdcAII that work cooperatively to maintain Zn homeostasis. Simultaneous inactivation of adcA and adcAII or the entire adcACB operon led to a significant reduction in intracellular Zn under Zn-restricted conditions and heightened sensitivity to Zn-chelating agents including human calprotectin, aberrant cell morphology, and impaired fitness in serum ex vivo. Additionally, inactivation of adcACB and adcAII significantly reduced bacterial tolerance toward cell envelope-targeting antibiotics. Finally, we showed that the AdcACB/AdcAII system contributes to E. faecalis virulence in a Galleria mellonella invertebrate infection model and in two catheter-associated mouse infection models that recapitulate many of the host conditions associated with enterococcal human infections. Collectively, this report reveals that high-affinity Zn import is important for the pathogenesis of E. faecalis establishing the surface-associated AdcA and AdcAII lipoproteins as potential therapeutic targets.