scholarly journals Molecular Basis for Lytic Bacteriophage Resistance in Enterococci

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Breck A. Duerkop ◽  
Wenwen Huo ◽  
Pooja Bhardwaj ◽  
Kelli L. Palmer ◽  
Lora V. Hooper
Keyword(s):  
Cell Wall ◽  
Mouse Model ◽  
Enterococcus Faecalis ◽  
Molecular Basis ◽  
Bacterial Infections ◽  
Hypervariable Region ◽  
Cell Wall Protein ◽  
Phage Resistance ◽  
Human Intestine

ABSTRACT The human intestine harbors diverse communities of bacteria and bacteriophages. Given the specificity of phages for their bacterial hosts, there is growing interest in using phage therapies to combat the rising incidence of multidrug-resistant bacterial infections. A significant barrier to such therapies is the rapid development of phage-resistant bacteria, highlighting the need to understand how bacteria acquire phage resistance in vivo . Here we identify novel lytic phages in municipal raw sewage that kill Enterococcus faecalis , a Gram-positive opportunistic pathogen that resides in the human intestine. We show that phage infection of E. faecalis requires a predicted integral membrane protein that we have named PIP EF (for phage infection protein from E. faecalis ). We find that PIP EF is conserved in E. faecalis and harbors a 160-amino-acid hypervariable region that determines phage tropism for distinct enterococcal strains. Finally, we use a gnotobiotic mouse model of in vivo phage predation to show that the sewage phages temporarily reduce E. faecalis colonization of the intestine but that E. faecalis acquires phage resistance through mutations in PIP EF . Our findings define the molecular basis for an evolutionary arms race between E. faecalis and the lytic phages that prey on them. They also suggest approaches for engineering E. faecalis phages that have altered host specificity and that can subvert phage resistance in the host bacteria. IMPORTANCE Bacteriophage therapy has received renewed attention as a potential solution to the rise in antibiotic-resistant bacterial infections. However, bacteria can acquire phage resistance, posing a major barrier to phage therapy. To overcome this problem, it is necessary to understand phage resistance mechanisms in bacteria. We have unraveled one such resistance mechanism in Enterococcus faecalis , a Gram-positive natural resident of the human intestine that has acquired antibiotic resistance and can cause opportunistic infections. We have identified a cell wall protein hypervariable region that specifies phage tropism in E. faecalis . Using a gnotobiotic mouse model of in vivo phage predation, we show that E. faecalis acquires phage resistance through mutations in this cell wall protein. Our findings define the molecular basis for lytic phage resistance in E. faecalis . They also suggest opportunities for engineering E. faecalis phages that circumvent the problem of bacterial phage resistance.

scholarly journals Phage-Encoded Endolysins

Antibiotics ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 124
Author(s):  
Fatma Abdelrahman ◽  
Maheswaran Easwaran ◽  
Oluwasegun I. Daramola ◽  
Samar Ragab ◽  
Stephanie Lynch ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Wall ◽  
Antibiotic Resistance ◽  
Bacterial Infections ◽  
Therapeutic Strategies ◽  
Therapeutic Application ◽  
Significant Evidence ◽  
Crucial Information

Due to the global emergence of antibiotic resistance, there has been an increase in research surrounding endolysins as an alternative therapeutic. Endolysins are phage-encoded enzymes, utilized by mature phage virions to hydrolyze the cell wall from within. There is significant evidence that proves the ability of endolysins to degrade the peptidoglycan externally without the assistance of phage. Thus, their incorporation in therapeutic strategies has opened new options for therapeutic application against bacterial infections in the human and veterinary sectors, as well as within the agricultural and biotechnology sectors. While endolysins show promising results within the laboratory, it is important to document their resistance, safety, and immunogenicity for in-vivo application. This review aims to provide new insights into the synergy between endolysins and antibiotics, as well as the formulation of endolysins. Thus, it provides crucial information for clinical trials involving endolysins.

scholarly journals Activity of Tigecycline (GAR-936), a Novel Glycylcycline, against Enterococci in the Mouse Peritonitis Model

2003 ◽  
Vol 47 (2) ◽  
pp. 529-532 ◽  
Author(s):  
Esteban C. Nannini ◽  
Suresh R. Pai ◽  
Kavindra V. Singh ◽  
Barbara E. Murray
Keyword(s):  
Body Weight ◽  
Mouse Model ◽  
Protective Effect ◽  
Enterococcus Faecalis ◽  
Enterococcus Faecium ◽  
Tetracycline Resistance ◽  
Resistance Determinant ◽  
Subcutaneous Dose ◽  
Peritonitis Model

ABSTRACT A novel glycylcycline agent, tigecycline (GAR-936), was evaluated in vivo in the mouse model of peritonitis against three Enterococcus faecalis and four Enterococcus faecium isolates with different susceptibilities to vancomycin and tetracyclines, all of which were inhibited by ≤0.125 μg of tigecycline/ml. Using a single subcutaneous dose, tigecycline displayed a protective effect (50% protective dose, ≤5.7 mg/kg of body weight) against all strains tested, including two with Tn925 (from the Tn916 family), which contains the Tet(M) tetracycline resistance determinant, as well as VanA and VanB strains. As expected, tetracycline and minocycline were ineffective against the isolates carrying Tn925.

scholarly journals Specific Interaction of the Unmodified Bacteriocin Lactococcin 972 with the Cell Wall Precursor Lipid II

2008 ◽  
Vol 74 (15) ◽  
pp. 4666-4670 ◽  
Author(s):  
Beatriz Martínez ◽  
Tim Böttiger ◽  
Tanja Schneider ◽  
Ana Rodríguez ◽  
Hans-Georg Sahl ◽  
...  
Keyword(s):  
Cell Wall ◽  
Molecular Basis ◽  
Pore Formation ◽  
Cytoplasmic Membrane ◽  
Specific Interaction ◽  
Whole Cells ◽  
Lipid Ii ◽  
Inhibitory Spectrum

ABSTRACT Lactococcin 972 (Lcn972) is a nonlantibiotic bacteriocin that inhibits septum biosynthesis in Lactococcus lactis rather than forming pores in the cytoplasmic membrane. In this study, a deeper analysis of the molecular basis of the mode of action of Lcn972 was performed. Of several lipid cell wall precursors, only lipid II antagonized Lcn972 inhibitory activity in vivo. Likewise, Lcn972 only coprecipitated with lipid II micelles. This bacteriocin inhibited the in vitro polymerization of lipid II by the recombinant S. aureus PBP2 and the addition to lipid II of the first glycine catalyzed by FemX. These experiments demonstrate that Lcn972 specifically interacts with lipid II, the substrate of both enzymes. In the presence of Lcn972, nisin pore formation was partially hindered in whole cells. However, binding of Lcn972 to lipid II could not compete with nisin in lipid II-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, possibly indicating a distinct binding site. The existence of a putative cotarget for Lcn972 activity is discussed in the context of its narrow inhibitory spectrum and the localized action at the division septum. To our knowledge, this is the first unmodified bacteriocin that binds to the cell wall precursor lipid II.

scholarly journals The C lostridium difficile cell wall protein CwpV confers phase‐variable phage resistance

2015 ◽  
Vol 98 (2) ◽  
pp. 329-342 ◽  
Author(s):  
Ognjen Sekulovic ◽  
Maicol Ospina Bedoya ◽  
Amanda S. Fivian‐Hughes ◽  
Neil F. Fairweather ◽  
Louis‐Charles Fortier
Keyword(s):  
Cell Wall ◽  
Cell Wall Protein ◽  
Phage Resistance ◽  
Phase Variable

scholarly journals Bactericidal Activity of Gentamicin againstEnterococcus faecalis In Vitro and In Vivo

2000 ◽  
Vol 44 (8) ◽  
pp. 2077-2080 ◽  
Author(s):  
Agnès Lefort ◽  
Michel Arthur ◽  
Louis Garry ◽  
Claude Carbon ◽  
Patrice Courvalin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecalis ◽  
Bactericidal Activity ◽  
Susceptibility Testing ◽  
Rabbit Model ◽  
Intrinsic Activity ◽  
Resistant Mutants ◽  
Number Of Bacteria

ABSTRACT The activity of gentamicin at various concentrations against two strains of Enterococcus faecalis was investigated in vitro and in a rabbit model of aortic endocarditis. In vitro, gentamicin at 0.5 to 4 times the MIC failed to reduce the number of bacteria at 24 h. Rabbit or human serum dramatically increased gentamicin activity, leading to a ≥3-log10 CFU/ml decrease in bacterial counts when the drug concentration exceeded the MIC. Susceptibility testing in the presence of serum was predictive of in vivo activity, since gentamicin alone significantly reduced the number of surviving bacteria in the vegetations if the peak-to-MIC ratio was greater than 1. However, gentamicin selected resistant mutants in rabbits. The intrinsic activity of gentamicin should be taken into account in evaluation of combinations of gentamicin and cell wall-active agents against enterococci.

scholarly journals Serologic Response to Cell Wall Mannoproteins and Proteins of Candida albicans

1998 ◽  
Vol 11 (1) ◽  
pp. 121-141 ◽  
Author(s):  
José P. Martínez ◽  
M. Luisa Gil ◽  
José L. López-Ribot ◽  
W. LaJean Chaffin
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Antibody Response ◽  
Humoral Response ◽  
Binding Activity ◽  
Cell Wall Protein ◽  
Wall Structure ◽  
Cell Mediated Immunity ◽  
Host Immune System

SUMMARY The cell wall of Candida albicans not only is the structure in which many biological functions essential for the fungal cells reside but also is a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both the carbohydrate and protein moieties are able to trigger immune responses. Although cell-mediated immunity is often considered to be the most important line of defense against candidiasis, cell wall protein and glycoprotein components also elicit a potent humoral response from the host that may include some protective antibodies. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to influence profoundly the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins for host ligands. In this review, the various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo are examined. Although a number of proteins have been shown to stimulate an antibody response, for some of these species the response is not universal. On the other hand, some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidasis, particularly the disseminated form. In addition, recent studies have focused on the potential for antibodies to cell wall protein determinants to protect the host against infection. Hence, a better understanding of the humoral response to cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures for the serodiagnosis of disseminated candidiasis and (ii) novel prophylactic (vaccination) and therapeutic strategies for the management of this type of infection.

scholarly journals Non-Invasive Luciferase Imaging of Type I Interferon Induction in a Transgenic Mouse Model of Biomaterial Associated Bacterial Infections: Microbial Specificity and Inter-Bacterial Species Interactions

2020 ◽  
Vol 8 (10) ◽  
pp. 1624
Author(s):  
Muhammad Imran Rahim ◽  
Andreas Winkel ◽  
Stefan Lienenklaus ◽  
Nico S. Stumpp ◽  
Szymon P. Szafrański ◽  
...  
Keyword(s):  
Mouse Model ◽  
Species Interactions ◽  
Bacterial Infections ◽  
Bacterial Species ◽  
Inflammatory Cells ◽  
Single Species ◽  
Oral Bacteria ◽  
Type I ◽  
Non Invasive

The performance of biomaterials is often compromised by bacterial infections and subsequent inflammation. So far, the conventional analysis of inflammatory processes in vivo involves time-consuming histology and biochemical assays. The present study employed a mouse model where interferon beta (IFN-β) is monitored as a marker for non-invasive rapid detection of inflammation in implant-related infections. The mouse model comprises subcutaneous implantation of morphologically modified titanium, followed by experimental infections with four taxonomically diverse oral bacteria: Streptococcus oralis, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Treponema denticola (as mono culture or selected mixed-culture). IFN-β expression increased upon infections depending on the type of pathogen and was prolonged by the presence of the implant. IFN-β expression kinetics reduced with two mixed species infections when compared with the single species. Histological and confocal microscopy confirmed pathogen-specific infiltration of inflammatory cells at the implant-tissue interface. This was observed mainly in the vicinity of infected implants and was, in contrast to interferon expression, higher in infections with dual species. In summary, this non-invasive mouse model can be used to quantify longitudinally host inflammation in real time and suggests that the polymicrobial character of infection, highly relevant to clinical situations, has complex effects on host immunity.

scholarly journals Isolation of Streptococcus pneumoniae Biofilm Mutants and Their Characterization during Nasopharyngeal Colonization

2008 ◽  
Vol 76 (11) ◽  
pp. 5049-5061 ◽  
Author(s):  
Ernesto J. Muñoz-Elías ◽  
Joan Marcano ◽  
Andrew Camilli
Keyword(s):  
Cell Wall ◽  
Streptococcus Pneumoniae ◽  
Biofilm Formation ◽  
Bacterial Infections ◽  
Nasopharyngeal Colonization ◽  
Genes Encoding ◽  
Transposon Mutants ◽  
Bona Fide

ABSTRACT Asymptomatic colonization of the nasopharynx by Streptococcus pneumoniae precedes pneumococcal disease, yet pneumococcal colonization factors remain poorly understood. Many bacterial infections involve biofilms which protect bacteria from host defenses and antibiotics. To gain insight into the genetics of biofilm formation by S. pneumoniae, we conducted an in vitro screen for biofilm-altered mutants with the serotype 4 clinical isolate TIGR4. In a first screen of 6,000 mariner transposon mutants, we repeatedly isolated biofilm-overproducing acapsular mutants, suggesting that the capsule was antagonistic to biofilm formation. Therefore, we screened 6,500 additional transposon mutants in an S. pneumoniae acapsular background. Following this approach, we isolated 69 insertions in 49 different genes. The collection of mutants includes genes encoding bona fide and putative choline binding proteins, adhesins, synthases of membrane and cell wall components, extracellular and cell wall proteases, efflux pumps, ABC and PTS transporters, and transcriptional regulators, as well as several conserved and novel hypothetical proteins. Interestingly, while four insertions mapped to rrgA, encoding a subunit of a recently described surface pilus, rrgB and rrgC (encoding the other two pilus subunits) mutants had no biofilm defects, implicating the RrgA adhesin but not the pilus structure per se in biofilm formation. To correlate our findings to the process of colonization, we transferred a set of 29 mutations into the wild-type encapsulated strain and then tested the fitness of the mutants in vivo. Strikingly, we found that 23 of these mutants were impaired for nasopharyngeal colonization, thus establishing a link between biofilm formation and colonization.

scholarly journals Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis

2021 ◽  
Author(s):  
Pei Yi Choo ◽  
Charles Wang ◽  
Michael VanNieuwenhze ◽  
Kimberly Kline
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Enterococcus Faecalis ◽  
General Rule ◽  
Cell Wall Synthesis ◽  
Lipid Ii ◽  
Growing Cell ◽  
Temporal Localization

Enterococcus faecalis relies upon a number of cell wall-associated proteins for virulence. One virulence factor is the sortase-assembled endocarditis and biofilm associated pilus (Ebp), an important factor for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that the pilus sortase covalently links pilus monomers prior to recognition, while the housekeeping sortase cleaves at the LPXTG motif within the terminal pilin subunit, and subsequently attaches assembled pilus fiber to the growing cell wall at sites of new cell wall synthesis. While the cell wall anchoring mechanism and polymerization of Ebp is well characterized, less is known about the spatial and temporal deposition of this protein on the cell surface. We followed the distribution of Ebp and peptidoglycan (PG) throughout the E. faecalis cell cycle via immunofluorescence microscopy and fluorescent D-amino acids (FDAA) staining. Surprisingly, cell surface Ebp did not co-localize with newly synthesized PG. Instead, surface-anchored Ebp was localized to the cell hemisphere but never at the septum where new cell wall is deposited. In addition, the older hemisphere of the E. faecalis diplococcus were completely saturated with Ebp, while Ebp appeared as two foci directly adjacent to the nascent septum in the newer hemisphere. A similar localization pattern was observed for another cell wall anchored substrate by sortase A, aggregation substance (AS), suggesting that this may be a general rule for all SrtA substrates in E. faecalis. When cell wall synthesis was inhibited by ramoplanin, an antibiotic that binds and sequesters lipid II cell wall precursors, new Ebp deposition at the cell surface was not disrupted. These data suggest an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto un-crosslinked cell wall, independent of new PG synthesis.

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

mBio ◽  
2012 ◽  
Vol 3 (4) ◽  
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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