Antagonistic Donor Density Effect Conserved in Multiple Enterococcal Conjugative Plasmids

ABSTRACTEnterococcus faecalis, a common causative agent of hospital-acquired infections, is resistant to many known antibiotics. Its ability to acquire and transfer resistance genes and virulence determinants through conjugative plasmids poses a serious concern for public health. In some cases, induction of transfer ofE. faecalisplasmids results from peptide pheromones produced by plasmid-free recipient cells, which are sensed by the plasmid-bearing donor cells. These plasmids generally encode an inhibitory peptide that competes with the pheromone and suppresses self-induction of donors. We recently demonstrated that the inhibitor peptide encoded on plasmid pCF10 is part of a unique quorum-sensing system in which it functions as a “self-sensing signal,” reducing the response to the pheromone in a density-dependent fashion. Based on the similarities between regulatory features controlling conjugation in pAD1 and pAM373 and those controlling conjugation in pCF10, we hypothesized that these plasmids are likely to exhibit similar quorum-sensing behaviors. Experimental findings indicate that for both pAD1 and pAM373, high donor densities indeed resulted in decreased induction of the conjugation operon and reduced conjugation frequencies. This effect was restored by the addition of exogenous inhibitor, confirming that the inhibitor serves as an indicator for donor density. Donor density also affects cross-species conjugative plasmid transfer. Based on our experimental results, we propose models for induction and shutdown of the conjugation operon in pAD1 and pAM373.IMPORTANCEEnterococcus faecalisis a leading cause of hospital-acquired infections. Its ability to transfer antibiotic resistance and virulence determinants by sharing its genetic material with other bacteria through direct cell-cell contact via conjugation poses a serious threat. Two antagonistic signaling peptides control the transfer of plasmids pAD1 and pAM373: a peptide pheromone produced by plasmid-free recipients triggers the conjugative transfer in plasmid-containing donors, and an inhibitor peptide encoded on the plasmid and produced by donor cells serves to modulate the donor response in accordance with the relative abundance of donors and recipients. We demonstrate that high donor density reduces the conjugation frequency of both of these plasmids, which is a consequence of increased inhibitor concentration in high-donor-density cultures. While most antibiotic strategies end up selecting resistant strains and disrupting the community balance, manipulating bacterial signaling mechanisms can serve as an alternate strategy to prevent the spread of antibiotic resistance.

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Comparative Biofilm Assays Using Enterococcus faecalis OG1RF Identify New Determinants of Biofilm Formation

mBio ◽

10.1128/mbio.01011-21 ◽

2021 ◽

Author(s):

Julia L. E. Willett ◽

Jennifer L. Dale ◽

Lucy M. Kwiatkowski ◽

Jennifer L. Powers ◽

Michelle L. Korir ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Nutrient Availability ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Growth Conditions ◽

Complete Understanding ◽

Content Type ◽

Hospital Acquired Infections ◽

Hospital Acquired

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.

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Identification of Two Regulators of Virulence That Are Conserved inKlebsiella pneumoniaeClassical and Hypervirulent Strains

mBio ◽

10.1128/mbio.01443-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 18

Author(s):

Michelle Palacios ◽

Taryn A. Miner ◽

Daniel R. Frederick ◽

Victoria E. Sepulveda ◽

Joshua D. Quinn ◽

...

Keyword(s):

Gene Expression ◽

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Virulence Factors ◽

Content Type ◽

Hospital Acquired Infections ◽

Capsule Type ◽

Capsule Production ◽

Invasive Infections ◽

Hospital Acquired

ABSTRACTKlebsiella pneumoniaeis widely recognized as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of hospital-acquired infections (urinary tract infections [UTI], pneumonia, sepsis) and community-acquired invasive infections. The genetic heterogeneity ofK. pneumoniaeisolates complicates our ability to understand the virulence ofK. pneumoniae. Characterization of virulence factors conserved between strains as well as strain-specific factors will improve our understanding of this important pathogen. The MarR family of regulatory proteins is widely distributed in bacteria and regulates cellular processes such as antibiotic resistance and the expression of virulence factors.Klebsiellaencodes numerous MarR-like proteins, and they likely contribute to the ability ofK. pneumoniaeto respond to and survive under a wide variety of environmental conditions, including those present in the human body. We tested loss-of-function mutations in all themarRhomologues in a murine pneumonia model and found that two (kvrAandkvrB) significantly impacted the virulence of K1 and K2 capsule type hypervirulent (hv) strains and thatkvrAaffected the virulence of a sequence type 258 (ST258) classical strain. In thehvstrains,kvrAandkvrBmutants displayed phenotypes associated with reduced capsule production, mucoviscosity, and transcription fromgalFandmanCpromoters that drive expression of capsule synthesis genes. In contrast,kvrAandkvrBmutants in the ST258 strain had no effect on capsule gene expression or capsule-related phenotypes. Thus, KvrA and KvrB affect virulence in classical andhvstrains but the effect on virulence may not be exclusively due to effects on capsule production.IMPORTANCEIn addition to having a reputation as the causative agent for hospital-acquired infections as well as community-acquired invasive infections,Klebsiella pneumoniaehas gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. Due to the rapid emergence of carbapenem resistance amongK. pneumoniaestrains, a better understanding of virulence mechanisms and identification of new potential drug targets are needed. This study identified two novel regulators (KvrA and KvrB) of virulence inK. pneumoniaeand demonstrated that their effect on virulence in invasive strains is likely due in part to effects on capsule production (a major virulence determinant) and hypermucoviscosity. KvrA also impacts the virulence of classical strains but does not appear to affect capsule gene expression in this strain. KvrA and KvrB are conserved amongK. pneumoniaestrains and thus could regulate capsule expression and virulence in diverse strains regardless of capsule type.

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A Serendipitous Mutation Reveals the Severe Virulence Defect of a Klebsiella pneumoniae fepB Mutant

mSphere ◽

10.1128/msphere.00341-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 12

Author(s):

Michelle Palacios ◽

Christopher A. Broberg ◽

Kimberly A. Walker ◽

Virginia L. Miller

Keyword(s):

Antibiotic Resistance ◽

Urinary Tract ◽

Klebsiella Pneumoniae ◽

Drug Targets ◽

Carbapenem Resistance ◽

Content Type ◽

Hospital Acquired Infections ◽

Tract Infections ◽

Hospital Acquired ◽

Rapid Emergence

ABSTRACT In addition to having a reputation as the causative agent of several types of hospital-acquired infections, Klebsiella pneumoniae has gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of infections, including urinary tract infections, pneumonia, and sepsis. Because of the rapid emergence of carbapenem resistance among Klebsiella strains, there is a dire need for a better understanding of virulence mechanisms and identification of new drug targets. Here, we identify the periplasmic transporter FepB as one such potential target. Klebsiella pneumoniae is considered a significant public health threat because of the emergence of multidrug-resistant strains and the challenge associated with treating life-threatening infections. Capsule, siderophores, and adhesins have been implicated as virulence determinants of K. pneumoniae, yet we lack a clear understanding of how this pathogen causes disease. In a previous screen for virulence genes, we identified a potential new virulence locus and constructed a mutant (smr) with this locus deleted. In this study, we characterize the smr mutant and show that this mutation renders K. pneumoniae avirulent in a pneumonia model of infection. The smr mutant was expected to have a deletion of three genes, but subsequent genome sequencing indicated that a much larger deletion had occurred. Further analysis of the deleted region indicated that the virulence defect of the smr mutant could be attributed to the loss of FepB, a periplasmic protein required for import of the siderophore enterobactin. Interestingly, a ΔfepB mutant was more attenuated than a mutant unable to synthesize enterobactin, suggesting that additional processes are affected. As FepB is highly conserved among the members of the family Enterobacteriaceae, therapeutic targeting of FepB may be useful for the treatment of Klebsiella and other bacterial infections. IMPORTANCE In addition to having a reputation as the causative agent of several types of hospital-acquired infections, Klebsiella pneumoniae has gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of infections, including urinary tract infections, pneumonia, and sepsis. Because of the rapid emergence of carbapenem resistance among Klebsiella strains, there is a dire need for a better understanding of virulence mechanisms and identification of new drug targets. Here, we identify the periplasmic transporter FepB as one such potential target.

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Inactivation of Nitrite-Dependent Nitric Oxide Biosynthesis Is Responsible for Overlapped Antibiotic Resistance between Naturally and Artificially Evolved Pseudomonas aeruginosa

mSystems ◽

10.1128/msystems.00732-21 ◽

2021 ◽

Author(s):

Su-fang Kuang ◽

Ding-yun Feng ◽

Zhuang-gui Chen ◽

Zhuo-zheng Liang ◽

Juan-juan Xiang ◽

...

Keyword(s):

Nitric Oxide ◽

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Content Type ◽

Hospital Acquired Infections ◽

Hospital Acquired

Infections with Pseudomonas aeruginosa have become a real concern among hospital-acquired infections, especially in cystic fibrosis patients and immunocompromised individuals. Control of the pathogen is challenging due to antibiotic resistance.

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The Metal Ion-Dependent Adhesion Site Motif of the Enterococcus faecalis EbpA Pilin Mediates Pilus Function in Catheter-Associated Urinary Tract Infection

mBio ◽

10.1128/mbio.00177-12 ◽

2012 ◽

Vol 3 (4) ◽

Cited By ~ 63

Author(s):

Hailyn V. Nielsen ◽

Pascale S. Guiton ◽

Kimberly A. Kline ◽

Gary C. Port ◽

Jerome S. Pinkner ◽

...

Keyword(s):

Urinary Tract ◽

Enterococcus Faecalis ◽

Molecular Basis ◽

Metal Ion ◽

Content Type ◽

Hospital Acquired Infections ◽

Tract Infections ◽

Hospital Acquired ◽

Adhesion Site

ABSTRACT Though the bacterial opportunist Enterococcus faecalis causes a myriad of hospital-acquired infections (HAIs), including catheter-associated urinary tract infections (CAUTIs), little is known about the virulence mechanisms that it employs. However, the endocarditis- and biofilm-associated pilus (Ebp), a member of the sortase-assembled pilus family, was shown to play a role in a mouse model of E. faecalis ascending UTI. The Ebp pilus comprises the major EbpC shaft subunit and the EbpA and EbpB minor subunits. We investigated the biogenesis and function of Ebp pili in an experimental model of CAUTI using a panel of chromosomal pilin deletion mutants. A nonpiliated pilus knockout mutant (EbpABC− strain) was severely attenuated compared to its isogenic parent OG1RF in experimental CAUTI. In contrast, a nonpiliated ebpC deletion mutant (EbpC− strain) behaved similarly to OG1RF in vivo because it expressed EbpA and EbpB. Deletion of the minor pilin gene ebpA or ebpB perturbed pilus biogenesis and led to defects in experimental CAUTI. We discovered that the function of Ebp pili in vivo depended on a predicted metal ion-dependent adhesion site (MIDAS) motif in EbpA’s von Willebrand factor A domain, a common protein domain among the tip subunits of sortase-assembled pili. Thus, this study identified the Ebp pilus as a virulence factor in E. faecalis CAUTI and also defined the molecular basis of this function, critical knowledge for the rational development of targeted therapeutics. IMPORTANCE Catheter-associated urinary tract infections (CAUTIs), one of the most common hospital-acquired infections (HAIs), present considerable treatment challenges for physicians. Inherently resistant to several classes of antibiotics and with a propensity to acquire vancomycin resistance, enterococci are particularly worrisome etiologic agents of CAUTI. A detailed understanding of the molecular basis of Enterococcus faecalis pathogenesis in CAUTI is necessary for the development of preventative and therapeutic strategies. Our results elucidated the importance of the E. faecalis Ebp pilus and its subunits for enterococcal virulence in a mouse model of CAUTI. We further showed that the metal ion-dependent adhesion site (MIDAS) motif in EbpA is necessary for Ebp function in vivo. As this motif occurs in other sortase-assembled pili, our results have implications for the molecular basis of virulence not only in E. faecalis CAUTI but also in additional infections caused by enterococci and other Gram-positive pathogens.

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Genome Sequence of a Novel Enterococcus faecalis Sequence Type 922 Strain Isolated from a Door Handle in the Intensive Care Unit of a District Hospital in Durban, South Africa

Microbiology Resource Announcements ◽

10.1128/mra.00582-19 ◽

2019 ◽

Vol 8 (35) ◽

Author(s):

Christiana O. Shobo ◽

Daniel G. Amoako ◽

Mushal Allam ◽

Arshad Ismail ◽

Sabiha Y. Essack ◽

...

Keyword(s):

South Africa ◽

Intensive Care Unit ◽

Enterococcus Faecalis ◽

Genome Sequence ◽

District Hospital ◽

Sequence Type ◽

Content Type ◽

Hospital Acquired Infections ◽

Defense Systems ◽

Hospital Acquired

Herein, we highlight the genome sequence of a novel Enterococcus faecalis sequence type 922 (ST922) strain isolated in South Africa. The 3,564,442-bp genome harbored defense systems, a resistome, a virulome, and genetic support, which is of importance to the control of hospital-acquired infections. The genomics of Enterococcus faecalis yields greater understanding into its pathogenesis.

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Influence of the Alternative Sigma Factor RpoN on Global Gene Expression and Carbon Catabolism in Enterococcus faecalis V583

mBio ◽

10.1128/mbio.00380-21 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Erica C. Keffeler ◽

Vijayalakshmi S. Iyer ◽

Srivatsan Parthasarathy ◽

Matthew M. Ramsey ◽

Matthew J. Gorman ◽

...

Keyword(s):

Urinary Tract Infection ◽

Urinary Tract ◽

Tract Infection ◽

Enterococcus Faecalis ◽

Carbon Metabolism ◽

Sigma Factor ◽

Alternative Sigma Factor ◽

Content Type ◽

Hospital Acquired Infections ◽

Hospital Acquired

ABSTRACT The alternative sigma factor σ54 has been shown to regulate the expression of a wide array of virulence-associated genes, as well as central metabolism, in bacterial pathogens. In Gram-positive organisms, the σ54 is commonly associated with carbon metabolism. In this study, we show that the Enterococcus faecalis alternative sigma factor σ54 (RpoN) and its cognate enhancer binding protein MptR are essential for mannose utilization and are primary contributors to glucose uptake through the Mpt phosphotransferase system. To gain further insight into how RpoN contributes to global transcriptional changes, we performed microarray transcriptional analysis of strain V583 and an isogenic rpoN mutant grown in a chemically defined medium with glucose as the sole carbon source. Transcripts of 340 genes were differentially affected in the rpoN mutant; the predicted functions of these genes mainly related to nutrient acquisition. These differentially expressed genes included those with predicted catabolite-responsive element (cre) sites, consistent with loss of repression by the major carbon catabolite repressor CcpA. To determine if the inability to efficiently metabolize glucose/mannose affected infection outcome, we utilized two distinct infection models. We found that the rpoN mutant is significantly attenuated in both rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI). Here, we examined a ccpA mutant in the CAUTI model and showed that the absence of carbon catabolite control also significantly attenuates bacterial tissue burden in this model. Our data highlight the contribution of central carbon metabolism to growth of E. faecalis at various sites of infection. IMPORTANCE Hospital-acquired infections account for 2 billion dollars annually in increased health care expenses and cause more than 100,000 deaths in the United States alone. Enterococci are the second leading cause of hospital-acquired infections. They form biofilms at surgical sites and are often associated with infections of the urinary tract following catheterization. Nutrient uptake and growth are key factors that influence their ability to cause disease. Our research identified a large set of genes that illuminate nutrient uptake pathways in enterococci. Perturbation of the metabolic circuit reduces virulence in a rabbit endocarditis model, as well as in catheter-associated urinary tract infection in mice. Targeting metabolic pathways that are important in infection may lead to new treatments against multidrug-resistant enterococcal infections.

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CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis

mSphere ◽

10.1128/msphere.00064-16 ◽

2016 ◽

Vol 1 (3) ◽

Cited By ~ 43

Author(s):

Valerie J. Price ◽

Wenwen Huo ◽

Ardalan Sharifi ◽

Kelli L. Palmer

Keyword(s):

Antibiotic Resistance ◽

High Risk ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Treatment Options ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Content Type ◽

New Genes ◽

Restriction Modification

ABSTRACT Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics. Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E. faecalis isolates lack CRISPR-Cas and possess an orphan locus lacking cas genes, CRISPR2, that is of unknown function. Little is known about restriction-modification defense in E. faecalis. Here, we explore the hypothesis that multidrug-resistant E. faecalis strains are immunocompromised. We assessed MGE acquisition by E. faecalis T11, a strain closely related to the multidrug-resistant hospital isolate V583 but which lacks the ~620 kb of horizontally acquired genome content that characterizes V583. T11 possesses the E. faecalis CRISPR3-cas locus and a predicted restriction-modification system, neither of which occurs in V583. We demonstrate that CRISPR-Cas and restriction-modification together confer a 4-log reduction in acquisition of the pheromone-responsive plasmid pAM714 in biofilm matings. Additionally, we show that the orphan CRISPR2 locus is functional for genome defense against another pheromone-responsive plasmid, pCF10, only in the presence of cas9 derived from the E. faecalis CRISPR1-cas locus, which most multidrug-resistant E. faecalis isolates lack. Overall, our work demonstrated that the loss of only two loci led to a dramatic reduction in genome defense against a clinically relevant MGE, highlighting the critical importance of the E. faecalis accessory genome in modulating horizontal gene transfer. Our results rationalize the development of antimicrobial strategies that capitalize upon the immunocompromised status of multidrug-resistant E. faecalis. IMPORTANCE Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics.

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Lon Protease Has Multifaceted Biological Functions inAcinetobacter baumannii

Journal of Bacteriology ◽

10.1128/jb.00536-18 ◽

2018 ◽

Vol 201 (2) ◽

Cited By ~ 5

Author(s):

Carly Ching ◽

Brendan Yang ◽

Chineme Onwubueke ◽

David Lazinski ◽

Andrew Camilli ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Developmental Process ◽

Cell Envelope ◽

Opportunistic Pathogen ◽

Lon Protease ◽

Content Type ◽

Hospital Acquired Infections ◽

Biofilm Development ◽

Hospital Acquired

ABSTRACTAcinetobacter baumanniiis a Gram-negative opportunistic pathogen that is known to survive harsh environmental conditions and is a leading cause of hospital-acquired infections. Specifically, multicellular communities (known as biofilms) ofA. baumanniican withstand desiccation and survive on hospital surfaces and equipment. Biofilms are bacteria embedded in a self-produced extracellular matrix composed of proteins, sugars, and/or DNA. Bacteria in a biofilm are protected from environmental stresses, including antibiotics, which provides the bacteria with selective advantage for survival. Although some gene products are known to play roles in this developmental process inA. baumannii, mechanisms and signaling remain mostly unknown. Here, we find that Lon protease inA. baumanniiaffects biofilm development and has other important physiological roles, including motility and the cell envelope. Lon proteases are found in all domains of life, participating in regulatory processes and maintaining cellular homeostasis. These data reveal the importance of Lon protease in influencing keyA. baumanniiprocesses to survive stress and to maintain viability.IMPORTANCEAcinetobacter baumanniiis an opportunistic pathogen and is a leading cause of hospital-acquired infections.A. baumanniiis difficult to eradicate and to manage, because this bacterium is known to robustly survive desiccation and to quickly gain antibiotic resistance. We sought to investigate biofilm formation inA. baumannii, since much remains unknown about biofilm formation in this bacterium. Biofilms, which are multicellular communities of bacteria, are surface attached and difficult to eliminate from hospital equipment and implanted devices. Our research identifies multifaceted physiological roles for the conserved bacterial protease Lon inA. baumannii. These roles include biofilm formation, motility, and viability. This work broadly affects and expands understanding of the biology ofA. baumannii, which will permit us to find effective ways to eliminate the bacterium.

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Novel Drivers of Virulence in Clostridioides difficile Identified via Context-Specific Metabolic Network Analysis

mSystems ◽

10.1128/msystems.00919-21 ◽

2021 ◽

Author(s):

Matthew L. Jenior ◽

Jhansi L. Leslie ◽

Deborah A. Powers ◽

Elizabeth M. Garrett ◽

Kimberly A. Walker ◽

...

Keyword(s):

Network Analysis ◽

Metabolic Network ◽

Virulence Factors ◽

Metabolic Pathways ◽

Content Type ◽

Hospital Acquired Infections ◽

Metabolic Network Analysis ◽

Clostridioides Difficile ◽

Context Specific ◽

Hospital Acquired

Clostridioides difficile has become the leading single cause of hospital-acquired infections. Numerous studies have demonstrated the importance of specific metabolic pathways in aspects of C. difficile pathophysiology, from initial colonization to regulation of virulence factors.

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