scholarly journals Use of an Endogenous Plasmid Locus for Stable intransComplementation in Borrelia burgdorferi

2014 ◽  
Vol 81 (3) ◽  
pp. 1038-1046 ◽  
Author(s):  
Irene N. Kasumba ◽  
Aaron Bestor ◽  
Kit Tilly ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Targeted Mutagenesis ◽  
Multiple Cloning Site ◽  
Stable Gene ◽  
Content Type ◽  
Shuttle Vectors ◽  
Stable Maintenance

ABSTRACTTargeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirocheteBorrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers thanB. burgdorferiplasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.B. burgdorferihas over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochetein vivobut relatively unstable duringin vitrocultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number andin vivostability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into thebbe02locus, a site on lp25 that was previously shown to be nonessential during bothin vitroandin vivogrowth. We demonstrate the functional utility of this strategy by restoring infectivity to anospCmutant through complementation at this site on lp25 and stable maintenance of theospCgene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation inB. burgdorferi.

scholarly journals Reduced Immune Response to Borrelia burgdorferi in the Absence of γδ T Cells

2011 ◽  
Vol 79 (10) ◽  
pp. 3940-3946 ◽  
Author(s):  
Cuixia Shi ◽  
Bikash Sahay ◽  
Jennifer Q. Russell ◽  
Karen A. Fortner ◽  
Nicholas Hardin ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Borrelia Burgdorferi ◽  
Adaptive Immune Response ◽  
Γδ T Cells ◽  
Content Type ◽  
Adaptive Immune ◽  
Cytokines And Chemokines

ABSTRACTLittle is known regarding the function of γδ T cells, although they accumulate at sites of inflammation in infections and autoimmune disorders. We previously observed that γδ T cellsin vitroare activated byBorrelia burgdorferiin a TLR2-dependent manner. We now observe that the activated γδ T cells can in turn stimulate dendritic cellsin vitroto produce cytokines and chemokines that are important for the adaptive immune response. This suggested thatin vivoγδ T cells may assist in activating the adaptive immune response. We examined this possibilityin vivoand observed that γδ T cells are activated and expand in number duringBorreliainfection, and this was reduced in the absence of TLR2. Furthermore, in the absence of γδ T cells, there was a significantly blunted response of adaptive immunity, as reflected in reduced expansion of T and B cells and reduced serum levels of anti-Borreliaantibodies, cytokines, and chemokines. This paralleled a greaterBorreliaburden in γδ-deficient mice as well as more cardiac inflammation. These findings are consistent with a model of γδ T cells functioning to promote the adaptive immune response during infection.

scholarly journals Production, Secretion, and Cell Surface Display of Recombinant Sporosarcina ureae S-Layer Fusion Proteins in Bacillus megaterium

2011 ◽  
Vol 78 (2) ◽  
pp. 560-567 ◽  
Author(s):  
Denise Knobloch ◽  
Kai Ostermann ◽  
Gerhard Rödel
Keyword(s):  
Cell Surface ◽  
Bacillus Megaterium ◽  
Fusion Proteins ◽  
Shuttle Vector ◽  
Surface Display ◽  
Fluorescent Labeling ◽  
Content Type ◽  
Sporosarcina Ureae

ABSTRACTMonomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report thatBacillus megateriumis an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA fromSporosarcina ureaeATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into theEscherichia coli-Bacillus megateriumshuttle vector pHIS1525. After transformation of the respective plasmids intoBacillus megateriumprotoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemblein vitrointo highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope ofBacillus megaterium, indicating that the cell surface can servein vivoas a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

scholarly journals New Shuttle Vectors for Real-Time Gene Expression Analysis in Multidrug-Resistant Acinetobacter Species: In Vitro and In Vivo Responses to Environmental Stressors

2019 ◽  
Vol 85 (18) ◽  
Author(s):  
Massimiliano Lucidi ◽  
Daniela Visaggio ◽  
Elisa Prencipe ◽  
Francesco Imperi ◽  
Giordano Rampioni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Multidrug Resistant ◽  
Antibiotic Selection ◽  
Content Type ◽  
Shuttle Vectors ◽  
Reporter Systems

ABSTRACT The Acinetobacter genus includes species of opportunistic pathogens and harmless saprophytes. The type species, Acinetobacter baumannii, is a nosocomial pathogen renowned for being multidrug resistant (MDR). Despite the clinical relevance of infections caused by MDR A. baumannii and a few other Acinetobacter spp., the regulation of their pathogenicity remains elusive due to the scarcity of adequate genetic tools, including vectors for gene expression analysis. Here, we report the generation and testing of a series of Escherichia coli-Acinetobacter promoter-probe vectors suitable for gene expression analysis in Acinetobacter spp. These vectors, named pLPV1Z, pLPV2Z, and pLPV3Z, carry both gentamicin and zeocin resistance markers and contain lux, lacZ, and green fluorescent protein (GFP) reporter systems downstream of an extended polylinker, respectively. The presence of a toxin-antitoxin gene system and the high copy number allow pLPV plasmids to be stably maintained even without antibiotic selection. The pLPV plasmids can easily be introduced by electroporation into MDR A. baumannii belonging to the major international lineages as well as into species of the Acinetobacter calcoaceticus-A. baumannii complex. The pLPV vectors have successfully been employed to study the regulation of stress-responsive A. baumannii promoters, including the DNA damage-inducible uvrABC promoter, the ethanol-inducible adhP and yahK promoters, and the iron-repressible promoter of the acinetobactin siderophore biosynthesis gene basA. A lux-tagged A. baumannii ATCC 19606T strain, carrying the iron-responsive pLPV1Z::PbasA promoter fusion, allowed in vivo and ex vivo monitoring of the bacterial burden in the Galleria mellonella infection model. IMPORTANCE The short-term adaptive response to environmental cues greatly contributes to the ecological success of bacteria, and profound alterations in bacterial gene expression occur in response to physical, chemical, and nutritional stresses. Bacteria belonging to the Acinetobacter genus are ubiquitous inhabitants of soil and water though some species, such as Acinetobacter baumannii, are pathogenic and cause serious concern due to antibiotic resistance. Understanding A. baumannii pathobiology requires adequate genetic tools for gene expression analysis, and to this end we developed user-friendly shuttle vectors to probe the transcriptional responses to different environmental stresses. Vectors were constructed to overcome the problem of antibiotic selection in multidrug-resistant strains and were equipped with suitable reporter systems to facilitate signal detection. By means of these vectors, the transcriptional response of A. baumannii to DNA damage, ethanol exposure, and iron starvation was investigated both in vitro and in vivo, providing insights into A. baumannii adaptation during stress and infection.

scholarly journals Gene bb0318 Is Critical for the Oxidative Stress Response and Infectivity of Borrelia burgdorferi

2016 ◽  
Vol 84 (11) ◽  
pp. 3141-3151 ◽  
Author(s):  
Adrienne C. Showman ◽  
George Aranjuez ◽  
Philip P. Adams ◽  
Mollie W. Jewett
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Borrelia Burgdorferi ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Oxidative Stress Response ◽  
Growth Defect ◽  
Content Type

A greater understanding of the molecular mechanisms that Borrelia burgdorferi uses to survive during mammalian infection is critical for the development of novel diagnostic and therapeutic tools to improve the clinical management of Lyme disease. By use of an in vivo expression technology (IVET)-based approach to identify B. burgdorferi genes expressed in vivo , we discovered the bb0318 gene, which is thought to encode the ATPase component of a putative riboflavin ABC transport system. Riboflavin is a critical metabolite enabling all organisms to maintain redox homeostasis. B. burgdorferi appears to lack the metabolic capacity for de novo synthesis of riboflavin and so likely relies on scavenging riboflavin from the host environment. In this study, we sought to investigate the role of bb0318 in B. burgdorferi pathogenesis. No in vitro growth defect was observed for the Δ bb0318 clone. However, the mutant spirochetes displayed reduced levels of survival when exposed to exogenous hydrogen peroxide or murine macrophages. Spirochetes lacking bb0318 were found to have a 100-fold-higher 50% infectious dose than spirochetes containing bb0318 . In addition, at a high inoculum dose, bb0318 was found to be important for effective spirochete dissemination to deep tissues for as long as 3 weeks postinoculation and to be critical for B. burgdorferi infection of mouse hearts. Together, these data implicate bb0318 in the oxidative stress response of B. burgdorferi and indicate the contribution of bb0318 to B. burgdorferi mammalian infectivity.

scholarly journals Culture of the Entire Mouse To Determine whether Cultivable Borrelia burgdorferi Persists in Infected Mice Treated with a Five-Day Course of Ceftriaxone

2014 ◽  
Vol 58 (11) ◽  
pp. 6701-6703 ◽  
Author(s):  
Charles S. Pavia ◽  
Gary P. Wormser
Keyword(s):  
Borrelia Burgdorferi ◽  
Antibiotic Therapy ◽  
Culture Method ◽  
Laboratory Animals ◽  
Content Type ◽  
Organ Site ◽  
Tissue Site ◽  
Culture Positive

ABSTRACTAlthough controversial, it has been suggested that antibiotic treatment of laboratory animals infected withBorrelia burgdorferioften leads to the persistence of residual spirochetes that are claimed to be viable but noncultivable. If viable cells ofB. burgdorferido persist following antibiotic therapy, one possible explanation for the lack of cultivability is that too few organisms persist in any given tissue site that might be sampled and cultured. In this study, we treated SKH (hairless) mice, withB. burgdorferiinfection of 3 months' duration, with either ceftriaxone or saline for 5 days and then cultured a suspension extract of nearly the entire mouse using a combinedin vivo/in vitroculture method. All of the saline-treated (control) mice were culture positive, compared with none of the antibiotic-treated mice. Our findings further document the effectiveness of antibiotic therapy in eradicating cultivable cells ofB. burgdorferi, irrespective of tissue or organ site.

scholarly journals Generation of a Stable Plasmid forIn VitroandIn VivoStudies of Staphylococcus Species

2016 ◽  
Vol 82 (23) ◽  
pp. 6859-6869 ◽  
Author(s):  
Christina N. Krute ◽  
Kelsey L. Krausz ◽  
Mary A. Markiewicz ◽  
Jason A. Joyner ◽  
Srijana Pokhrel ◽  
...  
Keyword(s):  
Shuttle Vector ◽  
Antibiotic Usage ◽  
Open Reading Frames ◽  
Antibiotic Selection ◽  
Content Type ◽  
Plasmid Maintenance ◽  
Genetic Tools ◽  
Reading Frames

ABSTRACTA major shortcoming to plasmid-based genetic tools is the necessity of using antibiotics to ensure plasmid maintenance. While selectable markers are very powerful, their use is not always practical, such as duringin vivomodels of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid inStaphylococcus aureusUSA300 isolates was stable in the absence of a known selection and therefore could serve as a platform for new genetic tools forStaphylococcusspecies. LAC-p01 was genetically manipulated into anEscherichia coli-S. aureusshuttle vector that remained stable for at least 100 generations without antibiotic selection. The double- and single-stranded (dsoandsso) origins were identified and found to be essential for plasmid replication and maintenance, respectively. In contrast, deletion analyses revealed that none of the four LAC-p01 predicted open reading frames were necessary for stability. Subsequent to this, the shuttle vector was used as a platform to generate two plasmids. The first plasmid, pKK22, contains all genes native to the plasmid for use inS. aureusUSA300 strains, while the second, pKK30, lacks the four predicted open reading frames for use in non-USA300 isolates. pKK30 was also determined to be stable inStaphylococcus epidermidis. Moreover, pKK22 was maintained for 7 days postinoculation during a murine model ofS. aureussystemic infection and successfully complemented anhlamutant in a dermonecrosis model. These plasmids that eliminate the need for antibiotics during bothin vitroandin vivoexperiments are powerful new tools for studies ofStaphylococcus.IMPORTANCEPlasmid stability has been problematic in bacterial studies, and historically antibiotics have been used to ensure plasmid maintenance. This has been a major limitation duringin vivostudies, where providing antibiotics for plasmid maintenance is difficult and has confounding effects. Here, we have utilized the naturally occurring plasmid LAC-p01 from anS. aureusUSA300 strain to construct stable plasmids that obviate antibiotic usage. These newly modified plasmids retain stability over a multitude of generationsin vitroandin vivowithout antibiotic selection. With these plasmids, studies requiring genetic complementation, protein expression, or genetic reporter systems would not only overcome the burden of antibiotic usage but also eliminate the side effects of these antibiotics. Thus, our plasmids can be used as a powerful genetic tool for studies ofStaphylococcusspecies.

scholarly journals Cj1136 Is Required for Lipooligosaccharide Biosynthesis, Hyperinvasion, and Chick Colonization by Campylobacter jejuni

2012 ◽  
Vol 80 (7) ◽  
pp. 2361-2370 ◽  
Author(s):  
Muhammad Afzal Javed ◽  
Shaun A. Cawthraw ◽  
Abiyad Baig ◽  
Jianjun Li ◽  
Alan McNally ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Campylobacter Jejuni ◽  
Intestinal Epithelial Cells ◽  
Sodium Taurocholate ◽  
Sodium Deoxycholate ◽  
Targeted Mutagenesis ◽  
Intestinal Epithelial ◽  
Content Type

ABSTRACTCampylobacter jejuniis a major cause of bacterial food-borne enteritis worldwide, and invasion into intestinal epithelial cells is an important virulence mechanism. Recently we reported the identification of hyperinvasiveC. jejunistrains and created a number of transposon mutants of one of these strains, some of which exhibited reduced invasion into INT-407 and Caco-2 cells. In one such mutant the transposon had inserted into a homologue ofcj1136, which encodes a putative galactosyltransferase according to the annotation of theC. jejuniNCTC11168 genome. In the current study, we investigated the role ofcj1136inC. jejunivirulence, lipooligosaccharide (LOS) biosynthesis, and host colonization by targeted mutagenesis and complementation of the mutation. Thecj1136mutant showed a significant reduction in invasion into human intestinal epithelial cells compared to the wild-type strain 01/51. Invasion levels were partially restored on complementing the mutation. The inactivation ofcj1136resulted in the production of truncated LOS, while biosynthesis of a full-length LOS molecule was restored in the complemented strain. Thecj1136mutant showed an increase in sensitivity to the bile salts sodium taurocholate and sodium deoxycholate and significantly increased sensitivity to polymyxin B compared to the parental strain. Importantly, the ability of the mutant to colonize 1-day-old chicks was also significantly impaired. This study confirms that a putative galactosyltransferase encoded bycj1136is involved in LOS biosynthesis and is important forC. jejunivirulence, as disruption of this gene and the resultant truncation of LOS affect both colonizationin vivoand invasivenessin vitro.

scholarly journals Borrelia burgdorferi Linear Plasmid 38 Is Dispensable for Completion of the Mouse-Tick Infectious Cycle

2011 ◽  
Vol 79 (9) ◽  
pp. 3510-3517 ◽  
Author(s):  
Daniel P. Dulebohn ◽  
Aaron Bestor ◽  
Ryan O. M. Rego ◽  
Philip E. Stewart ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Linear Plasmid ◽  
Diverse Range ◽  
Antigenic Protein ◽  
Content Type ◽  
Experimental Mouse ◽  
Infectious Cycle

ABSTRACTBorrelia burgdorferi, the causative agent of Lyme disease, exists in a complex enzootic cycle, transiting between its vector,Ixodesticks, and a diverse range of vertebrate hosts.B. burgdorferilinear plasmid 38 (lp38) contains several genes that are differentially regulated in response to conditions mimicking the tick or mouse environments, suggesting that these plasmid-borne genes may encode proteins important for theB. burgdorferiinfectious cycle. Some of these genes encode potential virulence factors, including hypothetical lipoproteins as well as a putative membrane transport system. To characterize the role of lp38 in theB. burgdorferiinfectious cycle, we constructed a shuttle vector to selectively displace lp38 from theB. burgdorferigenome and analyzed the resulting clones to confirm the loss of lp38. We found that,in vitro, clones lacking lp38 were similar to isogenic wild-type bacteria, both in growth rate and in antigenic protein production. We analyzed these strains in an experimental mouse-tick infectious cycle, and our results demonstrate that clones lacking lp38 are fully infectious in a mouse, can efficiently colonize the tick vector, and are readily transmitted to a naive host.

scholarly journals Exogenous Alginate Protects Staphylococcus aureus from Killing by Pseudomonas aeruginosa

2019 ◽  
Vol 202 (8) ◽  
Author(s):  
Courtney E. Price ◽  
Dustin G. Brown ◽  
Dominique H. Limoli ◽  
Vanessa V. Phelan ◽  
George A. O’Toole
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Physical Space ◽  
Late Time ◽  
Protective Effects ◽  
Content Type ◽  
Alginate Production ◽  
The Impact

ABSTRACT Cystic fibrosis (CF) patients chronically infected with both Pseudomonas aeruginosa and Staphylococcus aureus have worse health outcomes than patients who are monoinfected with either P. aeruginosa or S. aureus. We showed previously that mucoid strains of P. aeruginosa can coexist with S. aureus in vitro due to the transcriptional downregulation of several toxic exoproducts typically produced by P. aeruginosa, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline N-oxide). Here, we demonstrate that exogenous alginate protects S. aureus from P. aeruginosa in both planktonic and biofilm coculture models under a variety of nutritional conditions. S. aureus protection in the presence of exogenous alginate is due to the transcriptional downregulation of pvdA, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (Pseudomonas quinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoid P. aeruginosa with nonmucoid P. aeruginosa strains can mitigate the killing of S. aureus by the nonmucoid strain of P. aeruginosa, indicating that the mechanism that we describe here may function in vivo in the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to kill S. aureus at late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome the S. aureus-protective effects of mucoidy in a strain-specific manner. IMPORTANCE CF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients are P. aeruginosa and S. aureus, with ∼30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promote P. aeruginosa-S. aureus coexistence, despite evidence that P. aeruginosa kills S. aureus when these organisms are cocultured in vitro. Thus, a better understanding of P. aeruginosa-S. aureus interactions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.

scholarly journals In Vitro Synergy and In Vivo Activity of Tigecycline-Ciprofloxacin Combination Therapy against Vibrio vulnificus Sepsis

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Seong Eun Kim ◽  
Hee Kyung Kim ◽  
Su-Mi Choi ◽  
Yohan Yu ◽  
Uh Jin Kim ◽  
...  
Keyword(s):  
Survival Rate ◽  
Mortality Rate ◽  
Combination Therapy ◽  
Combination Treatment ◽  
Vibrio Vulnificus ◽  
Antibiotic Regimen ◽  
Content Type ◽  
Time Kill

ABSTRACT The mortality rate associated with Vibrio vulnificus sepsis remains high. An in vitro time-kill assay revealed synergism between tigecycline and ciprofloxacin. The survival rate was significantly higher in mice treated with tigecycline plus ciprofloxacin than in mice treated with cefotaxime plus minocycline. Thus, combination treatment with tigecycline-ciprofloxacin may be an effective novel antibiotic regimen for V. vulnificus sepsis.

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