scholarly journals Nonclassical Biofilms Induced by DNA Breaks in Klebsiella pneumoniae

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Yan Liu ◽  
Chao Pan ◽  
Lijun Ye ◽  
Yue Si ◽  
Changhao Bi ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Pathogenic Bacteria ◽  
Extracellular Proteins ◽  
Bacterial Cells ◽  
Dna Breaks ◽  
Complex Molecules ◽  
Content Type ◽  
Strand Breaks ◽  
Fixed Ring ◽  
Biofilm Structure

ABSTRACT Biofilms usually form when the density of bacteria increases during the middle to late periods of growth in culture, commonly induced by quorum-sensing systems. Biofilms attach to the surfaces of either living or nonliving objects and protect bacteria against antibiotics and a host’s immune system. Here, a novel type of biofilm (the “R-biofilm”) is reported. These biofilms were formed by clinically isolated Klebsiella pneumoniae strains following double-stranded-DNA breaks (DSBs), while undamaged bacteria did not form classic biofilms even in the later stages of growth. R-biofilms had a fixed ring-like or discoid shape with good ductility and could protect many living bacterial cells within. We show that extracellular proteins and DNAs released, probably by dead bacteria, were the core structural materials of R-biofilms. We anticipate that novel signaling pathways besides the bacterial SOS response are involved in R-biofilm formation. The observations in this study suggest a limitation to the use of the currently popular Cas9-mediated bactericidal tools to eliminate certain bacteria because the resulting DSBs may lead to the formation of these protective R-biofilms. IMPORTANCE Many pathogenic bacteria can form biofilm matrices that consist of complex molecules such as polysaccharides, proteins, and DNA. These biofilms help the bacteria to infect and colonize a host. Such biofilms may attach and develop on the surfaces of indwelling medical devices or other supportive environments. This study found that following double-strand breaks in their DNA, Klebsiella pneumoniae cells can form a novel type of biofilm with ring-like or discoid morphology. This biofilm structure, named the “R-biofilm,” helps protect the bacteria against adverse conditions such as exposure to ethanol, hydrogen peroxide, and UV radiation.

scholarly journals Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

2017 ◽  
Vol 37 (24) ◽  
Author(s):  
Sucheta Arora ◽  
Rajashree A. Deshpande ◽  
Martin Budd ◽  
Judy Campbell ◽  
America Revere ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

scholarly journals UVC Light Prophylaxis for Cutaneous Wound Infections in Mice

2012 ◽  
Vol 56 (7) ◽  
pp. 3841-3848 ◽  
Author(s):  
Tianhong Dai ◽  
Barbara Garcia ◽  
Clinton K. Murray ◽  
Mark S. Vrahas ◽  
Michael R. Hamblin
Keyword(s):  
Bacterial Infections ◽  
Pathogenic Bacteria ◽  
Light Exposure ◽  
Dna Lesions ◽  
Bacterial Cells ◽  
Wound Infections ◽  
Content Type ◽  
Cutaneous Wound ◽  
Thickness Skin

ABSTRACTUVC light has long been known to be highly germicidal but has not been much developed as a therapy for infections. This study investigated the potential of UVC light for the prophylaxis of infections developing in highly contaminated superficial cutaneous wounds.In vitrostudies demonstrated that the pathogenic bacteriaPseudomonas aeruginosaandStaphylococcus aureuswere inactivated at UVC light exposures much lower than those needed for a similar effect on mammalian keratinocytes. Mouse models of partial-thickness skin abrasions infected with bioluminescentP. aeruginosaandS. aureuswere developed. Approximately 107bacterial cells were inoculated onto wounds measuring 1.2 by1.2 cm on the dorsal surfaces of mice. UVC light was delivered at 30 min after bacterial inoculation. It was found that for both bacterial infections, UVC light at a single radiant exposure of 2.59 J/cm2reduced the bacterial burden in the infected mouse wounds by approximately 10-fold in comparison to those in untreated mouse wounds (P< 0.00001). Furthermore, UVC light increased the survival rate of mice infected withP. aeruginosaby 58.3% (P= 0.0023) and increased the wound healing rate in mice infected withS. aureusby 31.2% (P< 0.00001). DNA lesions were observed in the UVC light-treated mouse wounds; however, the lesions were extensively repaired by 48 h after UVC light exposure. These results suggested that UVC light may be used for the prophylaxis of cutaneous wound infections.

scholarly journals ElyC and Cyclic Enterobacterial Common Antigen Regulate Synthesis of Phosphoglyceride-Linked Enterobacterial Common Antigen

mBio ◽  
2021 ◽  
Author(s):  
Ashutosh K. Rai ◽  
Joseph F. Carr ◽  
David E. Bautista ◽  
Wei Wang ◽  
Angela M. Mitchell
Keyword(s):  
Klebsiella Pneumoniae ◽  
Yersinia Pestis ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Pathogenic Bacteria ◽  
Permeability Barrier ◽  
Common Antigen ◽  
Content Type ◽  
Enterobacterial Common Antigen ◽  
The Many

Enterobacterial common antigen (ECA) is a conserved polysaccharide present on the surface of the outer membrane (OM) and in the periplasm of the many pathogenic bacteria belonging to Enterobacterales , including Klebsiella pneumoniae , Salmonella enterica , and Yersinia pestis . As the OM is a permeability barrier that excludes many antibiotics, synthesis pathways for OM molecules are promising targets for antimicrobial discovery.

scholarly journals RadD Contributes to R-Loop Avoidance in Sub-MIC Tobramycin

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Veronica Negro ◽  
Evelyne Krin ◽  
Sebastian Aguilar Pierlé ◽  
Thibault Chaze ◽  
Quentin Giai Gianetto ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Coupling Factor ◽  
Resistant Bacteria ◽  
Bacterial Cells ◽  
Dna Breaks ◽  
Bacterial Populations ◽  
Content Type ◽  
Double Strand Dna Breaks ◽  
Translation Coupling ◽  
Recombination Pathway

ABSTRACTWe have previously identifiedVibrio choleraemutants in which the stress response to subinhibitory concentrations of aminoglycoside is altered. One gene identified, VC1636, encodes a putative DNA/RNA helicase, recently named RadD inEscherichia coli. Here we combined extensive genetic characterization and high-throughput approaches in order to identify partners and molecular mechanisms involving RadD. We show that double-strand DNA breaks (DSBs) are formed upon subinhibitory tobramycin treatment in the absence ofradDandrecBCDand that formation of these DSBs can be overcome by RNase H1 overexpression. Loss of RNase H1, or of the transcription-translation coupling factor EF-P, is lethal in theradDdeletion mutant. We propose that R-loops are formed upon sublethal aminoglycoside treatment, leading to the formation of DSBs that can be repaired by the RecBCD homologous recombination pathway, and that RadD counteracts such R-loop accumulation. We discuss how R-loops that can occur upon translation-transcription uncoupling could be the link between tobramycin treatment and DNA break formation.IMPORTANCEBacteria frequently encounter low concentrations of antibiotics. Active antibiotics are commonly detected in soil and water at concentrations much below lethal concentration. Although sub-MICs of antibiotics do not kill bacteria, they can have a major impact on bacterial populations by contributing to the development of antibiotic resistance through mutations in originally sensitive bacteria or acquisition of DNA from resistant bacteria. It was shown that concentrations as low as 100-fold below the MIC can actually lead to the selection of antibiotic-resistant cells. We seek to understand how bacterial cells react to such antibiotic concentrations usingE. coli, the Gram-negative bacterial paradigm, andV. cholerae, the causative agent of cholera. Our findings shed light on the processes triggered at the DNA level by antibiotics targeting translation, how damage occurs, and what the bacterial strategies are to respond to such DNA damage.

scholarly journals Genomic Epidemiology of Klebsiella pneumoniae in Italy and Novel Insights into the Origin and Global Evolution of Its Resistance to Carbapenem Antibiotics

2014 ◽  
Vol 59 (1) ◽  
pp. 389-396 ◽  
Author(s):  
Stefano Gaiarsa ◽  
Francesco Comandatore ◽  
Paolo Gaibani ◽  
Marta Corbella ◽  
Claudia Dalla Valle ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Pathogenic Bacteria ◽  
High Morbidity ◽  
Third Generation ◽  
Data Set ◽  
Content Type ◽  
Genomic Epidemiology ◽  
Carbapenem Resistant ◽  
Resistant Group ◽  
Third Generation Cephalosporins

ABSTRACTKlebsiella pneumoniaeis at the forefront of antimicrobial resistance for Gram-negative pathogenic bacteria, as strains resistant to third-generation cephalosporins and carbapenems are widely reported. The worldwide diffusion of these strains is of great concern due to the high morbidity and mortality often associated withK. pneumoniaeinfections in nosocomial environments. We sequenced the genomes of 89K. pneumoniaestrains isolated in six Italian hospitals. Strains were selected based on antibiotypes, regardless of multilocus sequence type, to obtain a picture of the epidemiology ofK. pneumoniaein Italy. Thirty-one strains were carbapenem-resistantK. pneumoniaecarbapenemase producers, 29 were resistant to third-generation cephalosporins, and 29 were susceptible to the aforementioned antibiotics. The genomes were compared to all of the sequences available in the databases, obtaining a data set of 319 genomes spanning the known diversity ofK. pneumoniaeworldwide. Bioinformatic analyses of this global data set allowed us to construct a whole-species phylogeny, to detect patterns of antibiotic resistance distribution, and to date the differentiation between specific clades of interest. Finally, we detected an ∼1.3-Mb recombination that characterizes all of the isolates of clonal complex 258, the most widespread carbapenem-resistant group ofK. pneumoniae. The evolution of this complex was modeled, dating the newly detected and the previously reported recombination events. The present study contributes to the understanding ofK. pneumoniaeevolution, providing novel insights into its global genomic characteristics and drawing a dated epidemiological scenario for this pathogen in Italy.

scholarly journals Replication Restart after Replication-Transcription Conflicts Requires RecA in Bacillus subtilis

2015 ◽  
Vol 197 (14) ◽  
pp. 2374-2382 ◽  
Author(s):  
Samuel Million-Weaver ◽  
Ariana Nakta Samadpour ◽  
Houra Merrikh
Keyword(s):  
Bacillus Subtilis ◽  
Double Strand Breaks ◽  
Holliday Junction ◽  
Replication Forks ◽  
Dna Breaks ◽  
Site Specific ◽  
Content Type ◽  
Strand Breaks ◽  
Recombination Proteins ◽  
Replication Restart

ABSTRACTEfficient duplication of genomes depends on reactivation of replication forks outside the origin. Replication restart can be facilitated by recombination proteins, especially if single- or double-strand breaks form in the DNA. Each type of DNA break is processed by a distinct pathway, though both depend on the RecA protein. One common obstacle that can stall forks, potentially leading to breaks in the DNA, is transcription. Though replication stalling by transcription is prevalent, the nature of DNA breaks and the prerequisites for replication restart in response to these encounters remain unknown. Here, we used an engineered site-specific replication-transcription conflict to identify and dissect the pathways required for the resolution and restart of replication forks stalled by transcription inBacillus subtilis. We found that RecA, its loader proteins RecO and AddAB, and the Holliday junction resolvase RecU are required for efficient survival and replication restart after conflicts with transcription. Genetic analyses showed that RecO and AddAB act in parallel to facilitate RecA loading at the site of the conflict but that they can each partially compensate for the other's absence. Finally, we found that RecA and either RecO or AddAB are required for the replication restart and helicase loader protein, DnaD, to associate with the engineered conflict region. These results suggest that conflicts can lead to both single-strand gaps and double-strand breaks in the DNA and that RecA loading and Holliday junction resolution are required for replication restart at regions of replication-transcription conflicts.IMPORTANCEHead-on conflicts between replication and transcription occur when a gene is expressed from the lagging strand. These encounters stall the replisome and potentially break the DNA. We investigated the necessary mechanisms forBacillus subtiliscells to overcome a site-specific engineered conflict with transcription of a protein-coding gene. We found that the recombination proteins RecO and AddAB both load RecA onto the DNA in response to the head-on conflict. Additionally, RecA loading by one of the two pathways was required for both replication restart and efficient survival of the collision. Our findings suggest that both single-strand gaps and double-strand DNA breaks occur at head-on conflict regions and demonstrate a requirement for recombination to restart replication after collisions with transcription.

scholarly journals Engineered proteins detect spontaneous DNA breakage in human and bacterial cells

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Chandan Shee ◽  
Ben D Cox ◽  
Franklin Gu ◽  
Elizabeth M Luengas ◽  
Mohan C Joshi ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Cytosine Deaminase ◽  
Bacterial Cells ◽  
Bacteriophage Mu ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Dna Breakage ◽  
Genomic Changes

Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP—labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells.

scholarly journals High Levels of Cyclic Di-GMP in Klebsiella pneumoniae Attenuate Virulence in the Lung

2017 ◽  
Vol 86 (2) ◽  
Author(s):  
David A. Rosen ◽  
Joy Twentyman ◽  
David A. Hunstad
Keyword(s):  
Klebsiella Pneumoniae ◽  
Pathogenic Bacteria ◽  
Host Responses ◽  
Potential Therapeutic Target ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Bacterial Physiology ◽  
Type 1 Pili ◽  
Type 3

ABSTRACTThe bacterial second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) has been shown to influence the expression of virulence factors in certain pathogenic bacteria, but little is known about its activity in the increasingly antibiotic-resistant pathogenKlebsiella pneumoniae. Here, the expression inK. pneumoniaeof a heterologous diguanylate cyclase increased the bacterial c-di-GMP concentration and attenuated pathogenesis in murine pneumonia. This attenuation remained evident in mice lacking the c-di-GMP sensor STING, indicating that the high c-di-GMP concentration exerted its influence not on host responses but on bacterial physiology. While serum resistance and capsule expression were unaffected by the increased c-di-GMP concentration, both type 3 and type 1 pili were strongly upregulated. Importantly, attenuation ofK. pneumoniaevirulence by high c-di-GMP levels was abrogated when type 1 pilus expression was silenced. We conclude that increased type 1 piliation may hamperK. pneumoniaevirulence in the respiratory tract and that c-di-GMP signaling represents a potential therapeutic target for antibiotic-resistantK. pneumoniaein this niche.

scholarly journals Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

2013 ◽  
Vol 82 (3) ◽  
pp. 1017-1029 ◽  
Author(s):  
Carmen Gil ◽  
Cristina Solano ◽  
Saioa Burgui ◽  
Cristina Latasa ◽  
Begoña García ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Response ◽  
Extracellular Proteins ◽  
Protective Immune Response ◽  
Surrounding Tissue ◽  
Bacterial Cells ◽  
Content Type ◽  
Biofilm Infection ◽  
Biofilm Matrix

ABSTRACTTheStaphylococcus aureusbiofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation ofS. aureusbiofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response againstS. aureusinfections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinicalS. aureusstrains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing ofS. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using anin vivomodel of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine againstS. aureusbiofilm-associated infections.

scholarly journals Interaction of Lipocalin 2, Transferrin, and Siderophores Determines the Replicative Niche of Klebsiella pneumoniae during Pneumonia

mBio ◽  
2012 ◽  
Vol 3 (6) ◽  
Author(s):  
Michael A. Bachman ◽  
Steven Lenio ◽  
Lindsay Schmidt ◽  
Jennifer E. Oyler ◽  
Jeffrey N. Weiser
Keyword(s):  
Klebsiella Pneumoniae ◽  
Host Defense ◽  
Pathogenic Bacteria ◽  
Severe Disease ◽  
Perivascular Space ◽  
Pulmonary Vasculature ◽  
Upper Respiratory Tract ◽  
Lipocalin 2 ◽  
Gram Negative ◽  
Content Type

ABSTRACT Pathogenic bacteria require iron for replication within their host. Klebsiella pneumoniae and other Gram-negative pathogens produce the prototypical siderophore enterobactin (Ent) to scavenge iron in vivo. In response, mucosal surfaces secrete lipocalin 2 (Lcn2), an innate immune protein that binds Ent to disrupt bacterial iron acquisition and promote acute inflammation during colonization. A subset of K. pneumoniae isolates attempt to evade Lcn2 by producing glycosylated Ent (Gly-Ent, salmochelin) or the alternative siderophore yersiniabactin (Ybt). However, these siderophores are not functionally equivalent and differ in their abilities to promote growth in the upper respiratory tract, lungs, and serum. To understand how Lcn2 exploits functional differences between siderophores, isogenic mutants of an Ent+ Gly-Ent+ Ybt+ K. pneumoniae strain were inoculated into Lcn2 +/+ and Lcn2 −/− mice, and the pattern of pneumonia was examined. Lcn2 effectively protected against the iroA ybtS mutant (Ent+ Gly-Ent− Ybt−). Lcn2 +/+ mice had small foci of pneumonia, whereas Lcn2 −/− mice had many bacteria in the perivascular space. The entB mutant (Ent− Ybt+ Gly-Ent−) caused moderate bronchopneumonia but did not invade the transferrin-containing perivascular space. Accordingly, transferrin blocked Ybt-dependent growth in vitro. The wild type and the iroA mutant, which both produce Ent and Ybt, had a mixed phenotype, causing a moderate bronchopneumonia in Lcn2 +/+ mice and perivascular overgrowth in Lcn2 −/− mice. Together, these data indicate that Lcn2, in combination with transferrin, confines K. pneumoniae to the airways and prevents invasion into tissue containing the pulmonary vasculature. IMPORTANCE Gram-negative bacteria are a common cause of severe hospital-acquired infections. To cause disease, they must obtain iron and secrete the small molecule enterobactin to do so. Animal models of pneumonia using Klebsiella pneumoniae indicate that enterobactin promotes severe disease. Accordingly, the host defense protein lipocalin 2 exploits this common target by binding enterobactin and disrupting its function. However, pathogenic bacteria often make additional siderophores that lipocalin 2 cannot bind, such as yersiniabactin, which could make this host defense ineffective. This work compares the pattern and severity of pneumonia caused by K. pneumoniae based on which siderophores it produces. The results indicate that enterobactin promotes growth around blood vessels that are rich in the iron-binding protein transferrin, but yersiniabactin does not. Together, transferrin and lipocalin 2 protect this space against all types of K. pneumoniae tested. Therefore, the ability to acquire iron determines where bacteria can grow in the lung.

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