scholarly journals GraS sensory activity in S. epidermidis is modulated by the “guard loop” of VraG and the ATPase activity of VraF

2021 ◽  
Author(s):  
Stephen K. Costa ◽  
Junho Cho ◽  
Ambrose L. Cheung
Keyword(s):  
Information Processing ◽  
Staphylococcus Epidermidis ◽  
Pathogenic Bacteria ◽  
Treatment Strategies ◽  
Opportunistic Pathogen ◽  
Polymyxin B ◽  
Extracellular Loop ◽  
Sensory Activity ◽  
Sense And Respond ◽  
Considerable Morbidity

Antimicrobial peptides (AMPs) are one of the key immune responses that can eliminate pathogenic bacteria through membrane perturbation. As a successful skin commensal, Staphylococcus epidermidis can sense and respond to AMPs through the GraXRS two-component system and an efflux system comprising the VraG permease and VraF ATPase. GraS is a membrane sensor known to function in AMP resistance through a negatively charged, 9-residue extracellular loop which is predicted to be linear without any secondary structure. An important question is how GraS can impart effective sensing of AMPs through such a small unstructured sequence. In this study, we verified the role of graS and vraG in AMP sensing in S. epidermidis as demonstrated by the failure of the Δ graS or Δ vraG mutants to sense. Deletion of the extracellular loop of VraG did not affect sensing but reduced survival against polymyxin B. Importantly, a specific region within the extracellular loop, termed the guard loop (GL), has inhibitory activity since sensing of polymyxin B was enhanced in the ΔGL mutant, indicating GL may act as a gate keeper to sensing. Bacterial two-hybrid analysis demonstrated that the extracellular regions of GraS and VraG interact, but interaction appears dispensable to sensing activity. Mutation of the extracellular loop of VraG, GL and the active site of VraF suggested that an active detoxification function of VraG is necessary for AMP resistance. Altogether, we provide evidence for a unique sensory scheme that relies on the function of a permease to impart effective information processing. Importance Staphylococcus epidermidis has become an important opportunistic pathogen responsible for nosocomial and device-related infections that account for considerable morbidity worldwide. A thorough understanding of the mechanisms that enable S. epidermidis to colonize human skin successfully is essential for the development of alternative treatment strategies and prophylaxis. Here, we demonstrate the importance of an antimicrobial peptide response system in a clinically relevant S. epidermidis strain. Furthermore, we provide evidence for a unique sensory scheme that would rely on the detoxification function of a permease to effect information processing.

Reservoirs of resistance: polymyxin resistance in veterinary-associated companion animal isolates of Pseudomonas aeruginosa

2019 ◽  
Vol 185 (7) ◽  
pp. 206-206 ◽  
Author(s):  
Andrea Scott ◽  
Sian Pottenger ◽  
Dorina Timofte ◽  
Matthew Moore ◽  
Laura Wright ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Inhibitory Concentration ◽  
Respiratory Infections ◽  
Companion Animals ◽  
Opportunistic Pathogen ◽  
Polymyxin B ◽  
Potential Reservoir ◽  
Human Infections ◽  
Animal Isolates

BackgroundPseudomonas aeruginosa is an opportunistic pathogen and a major cause of infections. Widespread resistance in human infections are increasing the use of last resort antimicrobials such as polymyxins. However, these have been used for decades in veterinary medicine. Companion animals are an understudied source of antimicrobial resistant P. aeruginosa isolates. This study evaluated the susceptibility of P. aeruginosa veterinary isolates to polymyxins to determine whether the veterinary niche represents a potential reservoir of resistance genes for pathogenic bacteria in both animals and humans.Methods and resultsClinical P. aeruginosa isolates (n=24) from UK companion animals were compared for antimicrobial susceptibility to a panel of human-associated isolates (n=37). Minimum inhibitory concentration (MIC) values for polymyxin B and colistin in the companion animals was significantly higher than in human isolates (P=0.033 and P=0.013, respectively). Genotyping revealed that the veterinary isolates were spread throughout the P. aeruginosa population, with shared array types from human infections such as keratitis and respiratory infections, suggesting the potential for zoonotic transmission. Whole genome sequencing revealed mutations in genes associated with polymyxin resistance and other antimicrobial resistance-related genes.ConclusionThe high levels of resistance to polymyxin shown here, along with genetic similarities between some human and animal isolates, together suggest a need for sustained surveillance of this veterinary niche as a potential reservoir for resistant, clinically relevant bacteria in both animals and humans.

Pseudomonas aeruginosa Oligoribonuclease Controls Susceptibility to Polymyxin B by Regulating Pel Exopolysaccharide Production

2022 ◽  
Author(s):  
Baopeng Yang ◽  
Yujun Jiang ◽  
Yongxin Jin ◽  
Fang Bai ◽  
Zhihui Cheng ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Resistance ◽  
Defense Mechanism ◽  
Extracellular Polysaccharide ◽  
Opportunistic Pathogen ◽  
Polymyxin B ◽  
Guanosine Monophosphate ◽  
Extracellular Dna ◽  
Bacterial Cells ◽  
Bacterial Survival

Polymyxins are considered as the last resort antibiotics to treat infections caused by multidrug-resistant Gram negative pathogens. Pseudomonas aeruginosa is an opportunistic pathogen that causes various infections in humans. Proteins involved in lipopolysaccharide modification and maintaining inner and outer membrane integrities have been found to contribute to the bacterial resistance to polymyxins. Oligoribonuclease (Orn) is an exonuclease that regulates the homeostasis of intracellular (3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), thereby regulating the production of extracellular polysaccharide in P. aeruginosa . Previously, we demonstrated that Orn affects the bacterial resistance to fluoroquinolone, β-lactam and aminoglycoside antibiotics. In this study, we found that mutation of orn increased the bacterial survival following polymyxin B treatment in a wild type P. aeruginosa strain PA14. Overexpression of c-di-GMP degradation enzymes in the orn mutant reduced the bacterial survival. By using a fluorescence labeled polymyxin B, we found that mutation of orn increased the bacterial surface bound polymyxin B. Deletion of the Pel synthesis genes or treatment with a Pel hydrolase reduced the surface bound polymyxin B and bacterial survival. We further demonstrated that Pel binds to extracellular DNA (eDNA), which traps polymyxin B and thus protects the bacterial cells. Collectively, our results revealed a novel defense mechanism against polymyxin in P. aeruginosa .

scholarly journals The extracellular loop of the membrane permease VraG interacts with GraS to sense cationic antimicrobial peptides in Staphylococcus aureus

2021 ◽  
Vol 17 (3) ◽  
pp. e1009338
Author(s):  
Junho Cho ◽  
Stephen K. Costa ◽  
Rachel M. Wierzbicki ◽  
William F. C. Rigby ◽  
Ambrose L. Cheung
Keyword(s):  
Positive Charge ◽  
Efflux Pump ◽  
Survival Strategies ◽  
Extracellular Loop ◽  
Major Question ◽  
Defense Proteins ◽  
Bacterial Membranes ◽  
Transmembrane Segments ◽  
Charge Interaction ◽  
Sense And Respond

Host defense proteins (HDPs), aka defensins, are a key part of the innate immune system that functions by inserting into the bacterial membranes to form pores to kill invading and colonizing microorganisms. To ensure survival, microorganism such as S. aureus has developed survival strategies to sense and respond to HDPs. One key strategy in S. aureus is a two-component system (TCS) called GraRS coupled to an efflux pump that consists of a membrane permease VraG and an ATPase VraF, analogous to the BceRS-BceAB system of Bacillus subtilis but with distinct differences. While the 9 negatively charged amino acid extracellular loop of the membrane sensor GraS has been shown to be involved in sensing, the major question is how such a small loop can sense diverse HDPs. Mutation analysis in this study divulged that the vraG mutant phenocopied the graS mutant with respect to reduced activation of downstream effector mprF, reduction in surface positive charge and enhanced 2 hr. killing with LL-37 as compared with the parental MRSA strain JE2. In silico analysis revealed VraG contains a single 200-residue extracellular loop (EL) situated between the 7th and 8th transmembrane segments (out of 10). Remarkably, deletion of EL in VraG enhanced mprF expression, augmented surface positive charge and improved survival in LL-37 vs. parent JE2. As the EL of VraG is rich in lysine residues (16%), in contrast to a preponderance of negatively charged aspartic acid residues (3 out of 9) in the EL of GraS, we divulged the role of charge interaction by showing that K380 in the EL of VraG is an important residue that likely interacts with GraS to interfere with GraS-mediated signaling. Bacterial two-hybrid analysis also supported the interaction of EL of VraG with the EL of GraS. Collectively, we demonstrated an interesting facet of efflux pumps whereby the membrane permease disrupts HDP signaling by inhibiting GraS sensing that involves charged residues in the EL of VraG.

scholarly journals F-type Pyocins are Diverse Non-Contractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Senjuti Saha ◽  
Chidozie D. Ojobor ◽  
Erik Mackinnon ◽  
Olesia I. North ◽  
Joseph Bondy-Denomy ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bactericidal Activity ◽  
Pathogenic Bacteria ◽  
Therapeutic Potential ◽  
Experimental Studies ◽  
Opportunistic Pathogen ◽  
Multidrug Resistant ◽  
Antibiotic Resistant ◽  
Genes Encoding ◽  
Intraspecies Competition

ABSTRACTMost Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or non-contractile phage tails known as R-type and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3’-end of the F-type pyocin cluster are divergent in sequence, and likely determine bactericidal specificity. We use sequence similarities among these proteins to define 11 distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly re-assorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins emerged earlier or have been subject to distinct evolutionary pressures. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as anti-bacterial therapeutics.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes a broad spectrum of antibiotic resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great interest in the development of alternative therapeutics. One alternative is protein-based antimicrobials called bacteriocins, which are produced by one strain of bacteria to kill other strains. In this study, we investigate F-type pyocins, bacteriocins naturally produced by P. aeruginosa that resemble non-contractile phage tails. We show that they are potent killers of P. aeruginosa, and distinct pyocin groups display different killing specificities. We have identified the probable specificity determinants of F-type pyocins, which opens up the potential to engineer them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well characterized phage tails will greatly facilitate their development into effective antibacterials.

scholarly journals In VitroDrug Combination Studies of Letermovir (AIC246, MK-8228) with Approved Anti-Human Cytomegalovirus (HCMV) and Anti-HIV Compounds in Inhibition of HCMV and HIV Replication

2015 ◽  
Vol 59 (6) ◽  
pp. 3140-3148 ◽  
Author(s):  
Steffen Wildum ◽  
Holger Zimmermann ◽  
Peter Lischka
Keyword(s):  
Human Cytomegalovirus ◽  
Treatment Strategies ◽  
Opportunistic Pathogen ◽  
Multidrug Resistant ◽  
Severe Toxicity ◽  
Transplant Recipients ◽  
Hiv Drugs ◽  
Anti Hiv

ABSTRACTDespite modern prevention and treatment strategies, human cytomegalovirus (HCMV) remains a common opportunistic pathogen associated with serious morbidity and mortality in immunocompromised individuals, such as transplant recipients and AIDS patients. All drugs currently licensed for the treatment of HCMV infection target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Letermovir (AIC246, MK-8228) is a new anti-HCMV agent in clinical development that acts via a novel mode of action and has demonstrated anti-HCMV activityin vitroandin vivo. For the future, drug combination therapies, including letermovir, might be indicated under special medical conditions, such as the emergence of multidrug-resistant virus strains in transplant recipients or in HCMV-HIV-coinfected patients. Accordingly, knowledge of the compatibility of letermovir with other HCMV or HIV antivirals is of medical importance. Here, we evaluated the inhibition of HCMV replication by letermovir in combination with all currently approved HCMV antivirals using cell culture checkerboard assays. In addition, the effects of letermovir on the antiviral activities of selected HIV drugs, and vice versa, were analyzed. Using two different mathematical techniques to analyze the experimental data, (i) additive effects were observed for the combination of letermovir with anti-HCMV drugs and (ii) no interaction was found between letermovir and anti-HIV drugs. Since none of the tested drug combinations significantly antagonized letermovir efficacy (or vice versa), our findings suggest that letermovir may offer the potential for combination therapy with the tested HCMV and HIV drugs.

Abstract 245: Risk of Readmission With Recurrent Myocardial Infarction After Index Myocardial Infarction

2020 ◽  
Vol 13 (Suppl_1) ◽  
Author(s):  
Raunak Nair ◽  
Michael Johnson ◽  
Kathleen A Kravitz ◽  
Moses Anabila ◽  
Jeevanantham Rajeswaran ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Patient Outcomes ◽  
Treatment Strategy ◽  
Medical Center ◽  
Parametric Method ◽  
Treatment Strategies ◽  
Recurrent Myocardial Infarction ◽  
Acute Mi ◽  
Increasing Risk ◽  
Considerable Morbidity

Background: Recurrent Myocardial Infarction (MI) after an index MI is a cause for considerable morbidity and mortality. However, the underlying factors that precipitate patients for a recurrent MI remain unclear. We aimed to assess the effect of index MI treatment strategy on the risk of developing a recurrent MI. Methods: We reviewed all cases of MI at a single quaternary care medical center from January 1 st , 2010 to January 1 st , 2017 and identified all cases of recurrent MI within 90 days after index MI. Readmissions were further stratified depending on the treatment strategy undertaken during index MI into medically managed and revascularized patients. The instantaneous risk of readmission following each of these treatment strategies was estimated by the parametric method. Results: We identified 6,626 patients admitted with an index MI, of which 168 patients were readmitted with a recurrent MI within 90 days. Among the index admissions, 4354 (66%) patients underwent revascularization and 2272 (34%) patients underwent medical management. Time-varying instantaneous risk of readmission analysis showed an early peaking risk followed by a late increasing risk in the revascularization group whereas, in the medically managed group, the analysis yielded an early peaking followed by a late almost constant risk of readmission for MI. Conclusion: Patients with acute MI who are medically treated are at a higher risk of developing a recurrent MI than patients who undergo revascularization. Defining the characteristics and underlying factors contributing to these readmissions can be pivotal in improving patient outcomes.

scholarly journals Light Modulates Important Pathogenic Determinants and Virulence in Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus ESKAPE Pathogens

2020 ◽  
Author(s):  
M. R. Tuttobene ◽  
J. F. Pérez ◽  
E. Pavesi ◽  
B. Perez Mora ◽  
D. Biancotti ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Acinetobacter Baumannii ◽  
Pathogenic Bacteria ◽  
Bacterial Pathogens ◽  
General Concept ◽  
Infection Model ◽  
Type Vi Secretion System ◽  
Sense And Respond ◽  
Human Infections

Light sensing has been extensively characterized in the human pathogen Acinetobacter baumannii at environmental temperatures. However, the influence of light on the physiology and pathogenicity of human bacterial pathogens at temperatures found in warm-blooded hosts is still poorly understand. In this work, we show that ESKAPE priority pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter spp., which have been recognized by the WHO and the CDC as critical, can also sense and respond to light at temperatures found in human hosts. Most interestingly, in these pathogens light modulates important pathogenicity determinants as well as virulence in an epithelial infection model, which could have implications in human infections. In fact, we found that alpha-toxin-dependent hemolysis, motility and growth under iron deprived conditions are modulated by light in S. aureus. Light also regulates persistence, metabolism and the ability to kill competitors, in some of these microorganisms. Finally, light exerts a profound effect on the virulence of these pathogens in an epithelial infection model, though the response is not the same in the different species: virulence was enhanced by light in A. baumannii and S. aureus, while in A. nosocomialis and P. aeruginosa it was reduced. Neither the BlsA photoreceptor nor the type VI secretion system (T6SS) are involved in virulence modulation by light in A. baumannii. Overall, this fundamental knowledge highlights the potential use of light to control pathogen's virulence, either directly or by manipulating the light regulatory switch toward the lowest virulence/persistence configuration. IMPORTANCE Pathogenic bacteria are microorganisms capable of producing disease. Dangerous bacterial pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii are responsible for serious intrahospital and community infections in humans. Therapeutics is often complicated due to resistance to multiple antibiotics, rendering them ineffective. In this work, we show that these pathogens sense natural light and respond to it by modulating aspects related to their ability to cause disease: in the presence of light some of them become more aggressive while others show an opposite response. Overall, we provide new understanding on the behavior of these pathogens, which could contribute to control infections caused by them. Since the response is distributed in diverse pathogens, this notion could prove a general concept.

scholarly journals CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA

Science ◽  
2008 ◽  
Vol 322 (5909) ◽  
pp. 1843-1845 ◽  
Author(s):  
Luciano A. Marraffini ◽  
Erik J. Sontheimer
Keyword(s):  
Antibiotic Resistance ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Staphylococcus Epidermidis ◽  
Pathogenic Bacteria ◽  
Target Sequence ◽  
Crispr Interference ◽  
Multiple Routes ◽  
Short Palindromic Repeat ◽  
Self Splicing

Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate ofStaphylococcus epidermidisharbors a CRISPR spacer that matches thenickasegene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation inS. epidermidis. Insertion of a self-splicing intron intonickaseblocks interference despite the reconstitution of the target sequence in the spliced mRNA, which indicates that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.

scholarly journals Keratinocyte Expression of Human β Defensin 2 following Bacterial Infection: Role in Cutaneous Host Defense

2003 ◽  
Vol 10 (1) ◽  
pp. 161-166 ◽  
Author(s):  
James G. H. Dinulos ◽  
Laurel Mentele ◽  
L. Page Fredericks ◽  
Beverly A. Dale ◽  
Gary L. Darmstadt
Keyword(s):  
Host Defense ◽  
Staphylococcus Epidermidis ◽  
Pathogenic Bacteria ◽  
Skin Surface ◽  
Immune Defense ◽  
Skin Infections ◽  
Reverse Transcription Pcr ◽  
Relative Tolerance ◽  
Disease Induction ◽  
Stimulation Of

ABSTRACT Human β defensin 2 (hβD-2) is thought to play an important role in cutaneous immune defense. We hypothesized that (i) keratinocyte expression of hβD-2, measured by reverse transcription-PCR, would be upregulated in response to challenge with pathogenic bacteria, particularly highly adherent strains of Streptococcus pyogenes and Staphylococcus aureus, and (ii) hβD-2 would have potent antimicrobial activity against pathogenic but not commensal organisms. Expression of hβD-2 was induced consistently by S. aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa, whereas strains of S. pyogenes were poor and variable inducers of hβD-2. No correlation was found between levels of bacterial adherence and keratinocyte expression of hβD-2. S. pyogenes was significantly more sensitive to killing by hβD-2 than S. epidermidis. We conclude that the ability to induce hβD-2 expression in combination with sensitivity to its antimicrobial effects may contribute to the rarity of skin infections with the gram-negative bacterial organisms, whereas lack of stimulation of hβD-2 expression by S. pyogenes may be important in its ability to evade innate defenses and cause skin disease. Induction of expression of hβD-2 but relative tolerance to it may enable S. epidermidis to survive on the skin surface and modulate hβD-2 expression when the stratum corneum barrier is disrupted.

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