scholarly journals Characterization of the Burkholderia pseudomallei K96243 Capsular Polysaccharide I Coding Region

2012 ◽  
Vol 80 (3) ◽  
pp. 1209-1221 ◽  
Author(s):  
Jon Cuccui ◽  
Timothy S. Milne ◽  
Nicholas Harmer ◽  
Alison J. George ◽  
Sarah V. Harding ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Capsular Polysaccharide ◽  
Bioinformatic Analysis ◽  
Chromosome 1 ◽  
Infection Model ◽  
Coding Region ◽  
Content Type ◽  
Group 3

Burkholderia pseudomalleiis the causative agent of melioidosis, a disease endemic to regions of Southeast Asia and Northern Australia. Both humans and a range of other animal species are susceptible to melioidosis, and the production of a group 3 polysaccharide capsule inB. pseudomalleiis essential for virulence.B. pseudomalleicapsular polysaccharide (CPS) I comprises unbranchedmanno-heptopyranose residues and is encoded by a 34.5-kb locus on chromosome 1. Despite the importance of this locus, the role of all of the genes within this region is unclear. We inactivated 18 of these genes and analyzed their phenotype using Western blotting and immunofluorescence staining. Furthermore, by combining this approach with bioinformatic analysis, we were able to develop a model for CPS I biosynthesis and export. We report that inactivatinggmhA,wcbJ, andwcbNinB. pseudomalleiK96243 retains the immunogenic integrity of the polysaccharide despite causing attenuation in the BALB/c murine infection model. Mice immunized with theB. pseudomalleiK96243 mutants lacking a functional copy of eithergmhAorwcbJwere afforded significant levels of protection against a wild-typeB. pseudomalleiK96243 challenge.

scholarly journals Functional Characterization of Burkholderia pseudomallei Trimeric Autotransporters

2013 ◽  
Vol 81 (8) ◽  
pp. 2788-2799 ◽  
Author(s):  
Cristine G. Campos ◽  
Matthew S. Byrd ◽  
Peggy A. Cotter
Keyword(s):  
Burkholderia Pseudomallei ◽  
Functional Characterization ◽  
A549 Cells ◽  
Content Type ◽  
Tier 1 ◽  
Type V ◽  
Mouse Model Of Infection ◽  
Encoding Genes

ABSTRACTBurkholderia pseudomalleiis a tier 1 select agent and the causative agent of melioidosis, a severe and often fatal disease with symptoms ranging from acute pneumonia and septic shock to a chronic infection characterized by abscess formation in the lungs, liver, and spleen. Autotransporters (ATs) are exoproteins belonging to the type V secretion system family, with many playing roles in pathogenesis. The genome ofB. pseudomalleistrain 1026b encodes nine putative trimeric AT proteins, of which only four have been described. Using a bioinformatic approach, we annotated putative domains within each trimeric AT protein, excluding the well-studied BimA protein, and found short repeated sequences unique toBurkholderiaspecies, as well as an unexpectedly large proportion of ATs with extended signal peptide regions (ESPRs). To characterize the role of trimeric ATs in pathogenesis, we constructed disruption or deletion mutations in each of eight AT-encoding genes and evaluated the resulting strains for adherence to, invasion of, and plaque formation in A549 cells. The majority of the ATs (and/or the proteins encoded downstream) contributed to adherence to and efficient invasion of A549 cells. Using a BALB/c mouse model of infection, we determined the contributions of each AT to bacterial burdens in the lungs, liver, and spleen. At 48 h postinoculation, only one strain, Bp340::pDbpaC, demonstrated a defect in dissemination and/or survival in the liver, indicating that BpaC is required for wild-type virulence in this model.

scholarly journals The Role of IgG Subclass in Antibody-Mediated Protection against Carbapenem-Resistant Klebsiella pneumoniae

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Michael P. Motley ◽  
Elizabeth Diago-Navarro ◽  
Kasturi Banerjee ◽  
Sean Inzerillo ◽  
Bettina C. Fries
Keyword(s):  
Monoclonal Antibodies ◽  
Klebsiella Pneumoniae ◽  
Capsular Polysaccharide ◽  
Resistant Bacteria ◽  
Infection Model ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Neutropenic Patients ◽  
Antibody Subclass

ABSTRACT Monoclonal antibodies (MAbs) have the potential to assist in the battle against multidrug-resistant bacteria such as carbapenem-resistant Klebsiella pneumoniae (CR-Kp). However, the characteristics by which these antibodies (Abs) function, such as the role of antibody subclass, must be determined before such modalities can be carried from the bench to the bedside. We performed a subclass switch on anticapsular monoclonal murine IgG3 (mIgG3) hybridomas and identified and purified a murine IgG1 (mIgG1) hybridoma line through sib selection. We then compared the ability of the mIgG1 and mIgG3 antibodies to control CR-Kp sequence type 258 (ST258) infection both in vitro and in vivo. We found by enzyme-limited immunosorbent assay (ELISA) and flow cytometry that mIgG3 has superior binding to the CR-Kp capsular polysaccharide (CPS) and superior agglutinating ability compared to mIgG1. The mIgG3 also, predictably, had better complement-mediated serum bactericidal activity than the mIgG1 and also promoted neutrophil-mediated killing at concentrations lower than that of the mIgG1. In contrast, the mIgG1 had marginally better activity in improving macrophage-mediated phagocytosis. Comparing their activities in a pulmonary infection model with wild-type as well as neutropenic mice, both antibodies reduced organ burden in a nonlethal challenge, regardless of neutrophil status, with mIgG1 having the highest overall burden reduction in both scenarios. However, at a lethal inoculum, both antibodies showed reduced efficacy in neutropenic mice, with mIgG3 retaining the most activity. These findings suggest the viability of monoclonal Ab adjunctive therapy in neutropenic patients that cannot mount their own immune response, while also providing some insight into the relative contributions of immune mediators in CR-Kp protection. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae is an urgent public health threat that causes life-threatening infections in immunocompromised hosts. Its resistance to nearly all antibiotics necessitates novel strategies to treat it, including the use of monoclonal antibodies. Monoclonal antibodies are emerging as important adjuncts to traditional pharmaceuticals, and studying how they protect against specific bacteria such as Klebsiella pneumoniae is crucial to their development as effective therapies. Antibody subclass is often overlooked but is a major factor in how an antibody interacts with other mediators of immunity. This paper is the first to examine how the subclass of anticapsular monoclonal antibodies can affect efficacy against CR-Kp. Additionally, this work sheds light on the viability of monoclonal antibody therapy in neutropenic patients, who are most vulnerable to CR-Kp infection.

scholarly journals Peptidyl-Prolyl Isomerase ppiB Is Essential for Proteome Homeostasis and Virulence in Burkholderia pseudomallei

2019 ◽  
Vol 87 (10) ◽  
Author(s):  
Nicole M. Bzdyl ◽  
Nichollas E. Scott ◽  
Isobel H. Norville ◽  
Andrew E. Scott ◽  
Timothy Atkins ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Antimicrobial Treatment ◽  
Infection Model ◽  
Virulence Determinants ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Cyclophilin B ◽  
Novel Treatments

ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia. Mortality rates in these areas are high even with antimicrobial treatment, and there are few options for effective therapy. Therefore, there is a need to identify antibacterial targets for the development of novel treatments. Cyclophilins are a family of highly conserved enzymes important in multiple cellular processes. Cyclophilins catalyze the cis-trans isomerization of xaa-proline bonds, a rate-limiting step in protein folding which has been shown to be important for bacterial virulence. B. pseudomallei carries a putative cyclophilin B gene, ppiB, the role of which was investigated. A B. pseudomallei ΔppiB (BpsΔppiB) mutant strain demonstrates impaired biofilm formation and reduced motility. Macrophage invasion and survival assays showed that although the BpsΔppiB strain retained the ability to infect macrophages, it had reduced survival and lacked the ability to spread cell to cell, indicating ppiB is essential for B. pseudomallei virulence. This is reflected in the BALB/c mouse infection model, demonstrating the requirement of ppiB for in vivo disease dissemination and progression. Proteomic analysis demonstrates that the loss of PpiB leads to pleiotropic effects, supporting the role of PpiB in maintaining proteome homeostasis. The loss of PpiB leads to decreased abundance of multiple virulence determinants, including flagellar machinery and alterations in type VI secretion system proteins. In addition, the loss of ppiB leads to increased sensitivity toward multiple antibiotics, including meropenem and doxycycline, highlighting ppiB inhibition as a promising antivirulence target to both treat B. pseudomallei infections and increase antibiotic efficacy.

scholarly journals Neutrophil Extracellular Traps Exhibit Antibacterial Activity against Burkholderia pseudomallei and Are Influenced by Bacterial and Host Factors

2012 ◽  
Vol 80 (11) ◽  
pp. 3921-3929 ◽  
Author(s):  
Donporn Riyapa ◽  
Surachat Buddhisa ◽  
Sunee Korbsrisate ◽  
Jon Cuccui ◽  
Brendan W. Wren ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Capsular Polysaccharide ◽  
Neutrophil Extracellular Traps ◽  
Polymorphonuclear Neutrophils ◽  
Dependent Manner ◽  
Predisposing Factor ◽  
Bacterial Killing ◽  
Content Type ◽  
Extracellular Traps ◽  
Type Iii Protein Secretion

ABSTRACTBurkholderia pseudomalleiis the causative pathogen of melioidosis, of which a major predisposing factor is diabetes mellitus. Polymorphonuclear neutrophils (PMNs) kill microbes extracellularly by the release of neutrophil extracellular traps (NETs). PMNs play a key role in the control of melioidosis, but the involvement of NETs in killing ofB. pseudomalleiremains obscure. Here, we showed that bactericidal NETs were released from human PMNs in response toB. pseudomalleiin a dose- and time-dependent manner.B. pseudomallei-induced NET formation required NADPH oxidase activation but not phosphatidylinositol-3 kinase, mitogen-activated protein kinases, or Src family kinase signaling pathways.B. pseudomalleimutants defective in the virulence-associated Bsa type III protein secretion system (T3SS) or capsular polysaccharide I (CPS-I) induced elevated levels of NETs. NET induction by such mutants was associated with increased bacterial killing, phagocytosis, and oxidative burst by PMNs. Taken together the data imply that T3SS and the capsule may play a role in evading the induction of NETs. Importantly, PMNs from diabetic subjects released NETs at a lower level than PMNs from healthy subjects. Modulation of NET formation may therefore be associated with the pathogenesis and control of melioidosis.

scholarly journals Key Role of Capsular Polysaccharide in the Induction of Systemic Infection and Abortion by Hypervirulent Campylobacter jejuni

2017 ◽  
Vol 85 (6) ◽  
Author(s):  
Orhan Sahin ◽  
Samantha A. Terhorst ◽  
Eric R. Burrough ◽  
Zhangqi Shen ◽  
Zuowei Wu ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Campylobacter Jejuni ◽  
Capsular Polysaccharide ◽  
Systemic Infection ◽  
The United States ◽  
Content Type ◽  
Model Following ◽  
Capsule Production ◽  
Guinea Pig Model

ABSTRACT Campylobacter jejuni is a zoonotic pathogen, and a hypervirulent clone, named clone SA, has recently emerged as the predominant cause of ovine abortion in the United States. To induce abortion, orally ingested Campylobacter must translocate across the intestinal epithelium, spread systemically in the circulation, and reach the fetoplacental tissue. Bacterial factors involved in these steps are not well understood. C. jejuni is known to produce capsular polysaccharide (CPS), but the specific role that CPS plays in systemic infection and particularly abortion in animals remains to be determined. In this study, we evaluated the role of CPS in bacteremia using a mouse model and in abortion using a pregnant guinea pig model following oral challenge. Compared with C. jejuni NCTC 11168 and 81-176, a clone SA isolate (IA3902) resulted in significantly higher bacterial counts and a significantly longer duration of bacteremia in mice. The loss of capsule production via gene-specific mutagenesis in IA3902 led to the complete abolishment of bacteremia in mice and abortion in pregnant guinea pigs, while complementation of capsule expression almost fully restored these phenotypes. The capsule mutant strain was also impaired for survival in guinea pig sera and sheep blood. Sequence-based analyses revealed that clone SA possesses a unique CPS locus with a mosaic structure, which has been stably maintained in all clone SA isolates derived from various hosts and times. These findings establish CPS as a key virulence factor for the induction of systemic infection and abortion in pregnant animals and provide a viable candidate for the development of vaccines against hypervirulent C. jejuni.

scholarly journals Characterization of Epidemic IncI1-Iγ Plasmids Harboring Ambler Class A and C Genes in Escherichia coli and Salmonella enterica from Animals and Humans

2015 ◽  
Vol 59 (9) ◽  
pp. 5357-5365 ◽  
Author(s):  
Hilde Smith ◽  
Alex Bossers ◽  
Frank Harders ◽  
Guanghui Wu ◽  
Neil Woodford ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Phylogenetic Trees ◽  
Functional Study ◽  
Content Type ◽  
Gene Associations ◽  
Genes Encoding ◽  
Marked Resistance

ABSTRACTThe aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained fromEscherichia coliandSalmonella entericaisolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase genes were compared using plasmid multilocus sequence typing (pMLST). Thirty-two of these plasmids belonging to different pMLST types were sequenced using Roche 454 and Illumina platforms. Epidemic IncI1-Iγ plasmids could be assigned to various dominant clades, whereas rarely detected plasmids clustered together as a distinct clade. Similar phylogenetic trees were obtained using only the plasmid backbone sequences, showing that the differences observed between the plasmids belonging to distinct clades resulted mainly from differences between their backbone sequences. Plasmids belonging to the various clades differed particularly in the presence/absence of genes encoding partitioning and addiction systems, which contribute to stable inheritance during cell division and plasmid maintenance. Despite this, plasmids belonging to the various phylogenetic clades also showed marked resistance gene associations, indicating the circulation of successful plasmid-gene combinations. The variation intraYandexcAgenes found in IncI1-Iγ plasmids is conserved within pMLST sequence types and plays a role in incompatibility, although functional study is needed to elucidate the role of these genes in plasmid epidemiology.

scholarly journals Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Jonathan L. Portman ◽  
Qiongying Huang ◽  
Michelle L. Reniere ◽  
Anthony T. Iavarone ◽  
Daniel A. Portnoy
Keyword(s):  
Protein Function ◽  
Inhibitory Effect ◽  
Cysteine Residue ◽  
Infection Model ◽  
Listeriolysin O ◽  
Content Type ◽  
Pore Forming Proteins

ABSTRACT Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

scholarly journals Molecular Characterization of Commensal Escherichia coli Adapted to Different Compartments of the Porcine Gastrointestinal Tract

2012 ◽  
Vol 78 (19) ◽  
pp. 6799-6803 ◽  
Author(s):  
Sam Abraham ◽  
David M. Gordon ◽  
James Chin ◽  
Huub J. M. Brouwers ◽  
Peter Njuguna ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gastrointestinal Tract ◽  
Random Amplified Polymorphic Dna ◽  
Content Type ◽  
E Coli ◽  
Plasmid Replicon ◽  
Different Strains ◽  
Different Characteristics

ABSTRACTThe role ofEscherichia colias a pathogen has been the focus of considerable study, while much less is known about it as a commensal and how it adapts to and colonizes different environmental niches within the mammalian gut. In this study, we characterizeEscherichia coliorganisms (n= 146) isolated from different regions of the intestinal tracts of eight pigs (dueodenum, ileum, colon, and feces). The isolates were typed using the method of random amplified polymorphic DNA (RAPD) and screened for the presence of bacteriocin genes and plasmid replicon types. Molecular analysis of variance using the RAPD data showed thatE. coliisolates are nonrandomly distributed among different gut regions, and that gut region accounted for 25% (P< 0.001) of the observed variation among strains. Bacteriocin screening revealed that a bacteriocin gene was detected in 45% of the isolates, with 43% carrying colicin genes and 3% carrying microcin genes. Of the bacteriocins observed (H47, E3, E1, E2, E7, Ia/Ib, and B/M), the frequency with which they were detected varied with respect to gut region for the colicins E2, E7, Ia/Ib, and B/M. The plasmid replicon typing gave rise to 25 profiles from the 13 Inc types detected. Inc F types were detected most frequently, followed by Inc HI1 and N types. Of the Inc types detected, 7 were nonrandomly distributed among isolates from the different regions of the gut. The results of this study indicate that not only may the different regions of the gastrointestinal tract harbor different strains ofE. colibut also that strains from different regions have different characteristics.

scholarly journals Characterization of RelA in Acinetobacter baumannii

2020 ◽  
Vol 202 (12) ◽  
Author(s):  
María Pérez-Varela ◽  
Aimee R. P. Tierney ◽  
Ju-Sim Kim ◽  
Andrés Vázquez-Torres ◽  
Philip Rather
Keyword(s):  
Acinetobacter Baumannii ◽  
Galleria Mellonella ◽  
Multidrug Resistant ◽  
Cell Physiology ◽  
Content Type ◽  
Content Model ◽  
Transposon Insertion ◽  
Downstream Effectors

ABSTRACT In response to nutrient depletion, the RelA and SpoT proteins generate the signaling molecule (p)ppGpp, which then controls a number of downstream effectors to modulate cell physiology. In Acinetobacter baumannii strain AB5075, a relA ortholog (ABUW_3302) was identified by a transposon insertion that conferred an unusual colony phenotype. An in-frame deletion in relA (ΔrelA) failed to produce detectable levels of ppGpp when amino acid starvation was induced with serine hydroxamate. The ΔrelA mutant was blocked from switching from the virulent opaque colony variant (VIR-O) to the avirulent translucent colony variant (AV-T), but the rate of AV-T to VIR-O switching was unchanged. In addition, the ΔrelA mutation resulted in a pronounced hypermotile phenotype on 0.35% agar plates. This hypermotility was dependent on the activation of a LysR regulator ABUW_1132, which was required for expression of AbaR, a LuxR family quorum-sensing regulator. In the ΔrelA mutant, ABUW_1132 was also required for the increased expression of an operon composed of the ABUW_3766-ABUW_3773 genes required for production of the surfactant-like lipopeptide acinetin 505. Additional phenotypes identified in the ΔrelA mutant included (i) cell elongation at high density, (ii) reduced formation of persister cells tolerant to colistin and rifampin, and (iii) decreased virulence in a Galleria mellonella model. IMPORTANCE Acinetobacter baumannii is a pathogen of worldwide importance. Due to the increasing prevalence of antibiotic resistance, these infections are becoming increasingly difficult to treat. New therapies are required to combat multidrug-resistant isolates. The role of RelA in A. baumannii is largely unknown. This study demonstrates that like in other bacteria, RelA controls a variety of functions, including virulence. Strategies to inhibit the activity of RelA and the resulting production of ppGpp could inhibit virulence and may represent a new therapeutic approach.

Experimental model of posttraumatic syringomyelia: the role of adhesive arachnoiditis in syrinx formation

1994 ◽  
Vol 80 (1) ◽  
pp. 133-139 ◽  
Author(s):  
Ki Hong Cho ◽  
Yoshinobu Iwasaki ◽  
Hiroyuki Imamura ◽  
Kazutoshi Hida ◽  
Hiroshi Abe
Keyword(s):  
Normal Saline ◽  
Subarachnoid Space ◽  
Saline Solution ◽  
Traumatic Injury ◽  
Content Type ◽  
Group 4 ◽  
Kaolin Injection ◽  
Group 3 ◽  
Group 2

✓ An experimental model was devised to elucidate the role of spinal blockade in posttraumatic syringomyelia. Thirty-eight Japanese White rabbits, each weighing about 3 kg, were used in this study. The animals were divided into four groups: in Group 1, eight animals received traumatic injury only; in Group 2, 12 animals received traumatic injury following injection of 100 mg kaolin suspended in 1 cc normal saline solution into the subarachnoid space at the site of trauma; in Group 3, nine animals received traumatic injury following injection of 200 mg kaolin in 1 cc normal saline solution into the subarachnoid space at the site of trauma; and in Group 4, nine animals without traumatic injury received an injection of 200 mg kaolin in 1 cc normal saline solution into the subarachnoid space. The subjective criteria for syrinx formation were the presence of a definite round cyst having a smooth margin and an upper or lower extension of more than 2 cm from the injured site. Syrinx formation was seen in 12.5% (one of eight rabbits) in Group 1, 41.7% (five of 12 animals) in Group 2, 55.5% (five of nine rabbits) in Group 3 and 0% (none of nine animals) in Group 4 (p < 0.05). There was a tendency for the combined trauma/kaolin injection groups to be more prone to develop a syrinx. In the kaolin injection only group (Group 4), no animal showed a definite cyst or an extending cavity during the experimental period. The results suggest that kaolin enhances the extension of multiple small cavities that have already formed at the time of initial injury. The difference between the frequency of syrinx formation and the time of survival was statistically significant well beyond the 0.05% level. The overall difference, relating to the frequency of syrinx development, group, and duration of survival, was also statistically significant. In summary, subarachnoid block secondary to adhesive arachnoiditis is important in initiating the extension of the syringomyelia cavity.

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