Bacteriophages (phages) are being considered as alternative therapeutics for the treatment of multidrug resistant bacterial infections. Considering phages have narrow host-ranges, it is generally ...accepted that therapeutic phages will have a marginal impact on non-target bacteria. We have discovered that lytic phage infection induces transcription of type VIIb secretion system (T7SS) genes in the pathobiont Enterococcus faecalis. Membrane damage during phage infection induces T7SS gene expression resulting in cell contact dependent antagonism of different Gram positive bystander bacteria. Deletion of essB, a T7SS structural component, abrogates phage-mediated killing of bystanders. A predicted immunity gene confers protection against T7SS mediated inhibition, and disruption of its upstream LXG toxin gene rescues growth of E. faecalis and Staphylococcus aureus bystanders. Phage induction of T7SS gene expression and bystander inhibition requires IreK, a serine/threonine kinase, and OG1RF_11099, a predicted GntR-family transcription factor. Additionally, sub-lethal doses of membrane targeting and DNA damaging antibiotics activated T7SS expression independent of phage infection, triggering T7SS antibacterial activity against bystander bacteria. Our findings highlight how phage infection and antibiotic exposure of a target bacterium can affect non-target bystander bacteria and implies that therapies beyond antibiotics, such as phage therapy, could impose collateral damage to polymicrobial communities.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Many Gram-negative bacterial pathogens express contact-dependent growth inhibition (CDI) systems that promote cell-cell interaction. CDI+ bacteria express surface CdiA effector proteins, which ...transfer their C-terminal toxin domains into susceptible target cells upon binding to specific receptors. CDI+ cells also produce immunity proteins that neutralize the toxin domains delivered from neighboring siblings. Here, we show that CdiAEC536 from uropathogenic Escherichia coli 536 (EC536) uses OmpC and OmpF as receptors to recognize target bacteria. E. coli mutants lacking either ompF or ompC are resistant to CDIEC536-mediated growth inhibition, and both porins are required for target-cell adhesion to inhibitors that express CdiAEC536. Experiments with single-chain OmpF fusions indicate that the CdiAEC536 receptor is heterotrimeric OmpC-OmpF. Because the OmpC and OmpF porins are under selective pressure from bacteriophages and host immune systems, their surface-exposed loops vary between E. coli isolates. OmpC polymorphism has a significant impact on CDIEC536 mediated competition, with many E. coli isolates expressing alleles that are not recognized by CdiAEC536. Analyses of recombinant OmpC chimeras suggest that extracellular loops L4 and L5 are important recognition epitopes for CdiAEC536. Loops L4 and L5 also account for much of the sequence variability between E. coli OmpC proteins, raising the possibility that CDI contributes to the selective pressure driving OmpC diversification. We find that the most efficient CdiAEC536 receptors are encoded by isolates that carry the same cdi gene cluster as E. coli 536. Thus, it appears that CdiA effectors often bind preferentially to "self" receptors, thereby promoting interactions between sibling cells. As a consequence, these effector proteins cannot recognize nor suppress the growth of many potential competitors. These findings suggest that self-recognition and kin selection are important functions of CDI.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Enterococcus faecalis is a common commensal bacterium in the gastrointestinal tract as well as a frequent nosocomial pathogen. The secreted metalloprotease gelatinase (GelE) is an important E. ...faecalis virulence factor that contributes to numerous cellular activities, such as autolysis, biofilm formation, and biofilm-associated antibiotic resistance. Expression of
has been extensively studied and is regulated by the Fsr quorum sensing system. Here, we identify two additional factors regulating gelatinase expression and activity in E. faecalis OG1RF. The Bph phosphatase is required for expression of
in an Fsr-dependent manner. Additionally, the membrane-anchored protein foldase PrsA is required for GelE activity, but not
or
gene expression. Disrupting
also leads to increased antibiotic sensitivity in biofilms independent of the loss of GelE activity. Together, our results expand the model for gelatinase production in E. faecalis, which has important implications for fundamental studies of GelE function in
and also E. faecalis pathogenesis.
In Enterococcus faecalis, gelatinase (GelE) is a virulence factor that is also important for biofilm formation and interactions with other microbes as well as the host immune system. The long-standing model for GelE production is that the Fsr quorum sensing system positively regulates expression of
. Here, we update that model by identifying two additional factors that contribute to gelatinase production. The biofilm-associated Bph phosphatase regulates the expression of
through Fsr, and the peptidyl-prolyl isomerase PrsA is required for production of active GelE through an Fsr-independent mechanism. This provides important insight into how regulatory networks outside of the
locus coordinate expression of gelatinase.
Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have ...been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar discs, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by six Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350,
,
, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions and identifies multiple new genetic determinants of biofilm formation.
E. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.
Contact-dependent growth inhibition (CDI) systems function to deliver toxins into neighboring bacterial cells. CDI⁺ bacteria export filamentous CdiA effector proteins, which extend from the ...inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control toxin transport into target bacteria. Using genetic approaches, we identifiedptsG, metI, rbsC, gltK/gltJ, yciB,andftsHmutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal “translocation domains.” These results highlight the plasticity of CDI toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria.
Bacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug-resistant bacterial infections. However, there are major gaps in our understanding of the ...molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism
, we used two distinct genomic approaches, namely, transposon library screening and RNA sequencing, to investigate the interaction of
with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (
) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for
mutation during phage infection. Transcriptomic profiling of phage-infected
revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intra- and interbacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together, our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities, such as the host microbiota, during phage therapy.
We lack fundamental understanding of how phage infection influences bacterial gene expression and, consequently, how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole-genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside their intended host targets.
Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CDI systems deploy a variety of distinct toxins, which are contained within ...the polymorphic C-terminal region (CdiA-CT) of CdiA proteins. Several CdiA-CTs are nucleases, suggesting that the toxins are transported into the target cell cytoplasm to interact with their substrates. To analyze CdiA transfer to target bacteria, we used the CDI system of uropathogenic Escherichia coli 536 (UPEC536) as a model. Antibodies recognizing the amino- and carboxyl-termini of CdiA(UPEC536) were used to visualize transfer of CdiA from CDI(UPEC536+) inhibitor cells to target cells using fluorescence microscopy. The results indicate that the entire CdiA(UPEC536) protein is deposited onto the surface of target bacteria. CdiA(UPEC536) transfer to bamA101 mutants is reduced, consistent with low expression of the CDI receptor BamA on these cells. Notably, our results indicate that the C-terminal CdiA-CT toxin region of CdiA(UPEC536) is translocated into target cells, but the N-terminal region remains at the cell surface based on protease sensitivity. These results suggest that the CdiA-CT toxin domain is cleaved from CdiA(UPEC536) prior to translocation. Delivery of a heterologous Dickeya dadantii CdiA-CT toxin, which has DNase activity, was also visualized. Following incubation with CDI(+) inhibitor cells targets became anucleate, showing that the D.dadantii CdiA-CT was delivered intracellularly. Together, these results demonstrate that diverse CDI toxins are efficiently translocated across target cell envelopes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Contact-dependent growth inhibition (CDI) systems encode polymorphic toxin/immunity proteins that mediate competition between neighboring bacterial cells. We present crystal structures of CDI ...toxin/immunity complexes from Escherichia coli EC869 and Burkholderia pseudomallei 1026b. Despite sharing little sequence identity, the toxin domains are structurally similar and have homology to endonucleases. The EC869 toxin is a Zn ²⁺-dependent DNase capable of completely degrading the genomes of target cells, whereas the Bp1026b toxin cleaves the aminoacyl acceptor stems of tRNA molecules. Each immunity protein binds and inactivates its cognate toxin in a unique manner. The EC869 toxin/immunity complex is stabilized through an unusual β-augmentation interaction. In contrast, the Bp1026b immunity protein exploits shape and charge complementarity to occlude the toxin active site. These structures represent the initial glimpse into the CDI toxin/immunity network, illustrating how sequence-diverse toxins adopt convergent folds yet retain distinct binding interactions with cognate immunity proteins. Moreover, we present visual demonstration of CDI toxin delivery into a target cell.
Gene therapy (GT) has offered immense hope to individuals who are visually impaired because of RPE65 mutations. Although GT has shown great success in clinical trials enrolling these individuals, ...evidence for stability and durability of this treatment over time is still unknown. Herein we explored the value of functional magnetic resonance imaging (fMRI) as an objective measure to assess independently the longevity of retinal GT.
Individuals with RPE65 mutations who underwent GT in their worse-seeing eye in a phase 1 clinical trial received a second subretinal injection in their contralateral eye in a follow-on clinical trial. Functional magnetic resonance imaging (MRI) was performed longitudinally to assess brain responses of patients with RPE65 mutations after stimulation of their most recently treated eye before and 1 to 3 years after GT.
Seven participants with RPE65 mutations who were part of the follow-on clinical trial gave informed consent to participate in a longitudinal neuroimaging fMRI study.
All participants underwent fMRI using a 3-Tesla MRI system and a 32-channel head coil. Participants' cortical activations were assessed using a block design paradigm of contrast reversing checkerboard stimuli delivered using an MRI-compatible video system.
The primary parameters being measured in this study were the qualitative and quantitative fMRI cortical activations produced by our population in response to the visual task.
Functional MRI results showed minimal or no cortical responses before GT. Significant increase in cortical activation lasting at least 3 years after GT was observed for all participants. Repeated measures analysis showed significant associations between cortical activations and clinical measures such as full-field light sensitivity threshold for white, red, and blue colors; visual field; and pupillary light reflex.
Participants with RPE65 mutations showed intact visual pathways, which became responsive and strengthened after treatment. Functional MRI results independently revealed the efficacy and durability of a 1-time subretinal injection. The fMRI results paralleled those recently reported during the long-term clinical evaluations of the same patients. Results from this study demonstrated that fMRI may play an important role in providing complementary information to patients' ophthalmic clinical evaluation and has usefulness as an outcome measure for future retinal intervention studies.
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