Table of Contents

Volume 3, Issue 3

  May/June 2012

View covers archive

EDITOR'S CORNER

What does it take to stick around?: Molecular insights into biofilm formation by uropathogenic Escherichia coli

Maria Hadjifrangiskou and Scott Hultgren
Abstract | Full Text | PDF

Existence in the biofilm state lends bacteria the opportunity to enjoy, at least for a finite amount of time, the benefits of a multicellular entity. The order of events leading to biofilm formation and disassembly has been the topic of interest for numerous studies, aiming to identify factors and mechanisms that underlie this dynamic developmental process. Of particular import is research leveraged at delineating biofilm formation by medically relevant microorganisms, as prevention or eradication of biofilm from medical devices and from within the host pose a serious challenge in the healthcare setting. Recent research describes how a transcriptional regulator modulates biofilm formation in uropathogenic Escherichia coli (UPEC) by affecting the expression of the type 1 adhesive organelles in response to extracellular signals.

Dissecting novel virulent determinants in the Burkholderia cepacia complex

George P. Tegos, Mark K. Haynes and Herbert P. Schweizer
Abstract | Full Text

Prevention and control of infectious diseases remains a major public health challenge and a number of highly virulent pathogens are emerging both in and beyond the hospital setting. Despite beneficial aspects such as use in biocontrol and bioremediation exhibited by members of the Burkholderia cepacia complex some members of this group have recently gained attention as significant bacterial pathogens due to their high levels of intrinsic antibiotic resistance, transmissibility in nosocomial settings, persistence in the presence of antimicrobials and intracellular survival capabilities. The Bcc are opportunistic pathogens and their arsenal of virulence factors includes proteases, lipases and other secreted exoproducts, including secretion of system-associated effectors. Deciphering the function of virulence factors and assessment of novel therapeutic strategies has been facilitated by use of diverse non-vertebrate hosts (the fly Drosophila melanogaster, the microscopic nematode Caenorhabditis elegans, the zebrafish and the greater Galleria mellonella wax moth caterpillar larvae). Researchers are now employing sophisticated approaches to dissect the virulence determinants of Bcc with the ultimate goal being the development of novel anti-infective countermeasures. This editorial will highlight selected recent research endeavors aimed at dissecting adaptive responses and the virulence factor portfolio of Burkholderia species.

To UPR … and beyond!: A new role for a BiP/GRP78 protein in the control of antimicrobial peptide expression in C. elegans epidermis

Todd Lamitina and Eric Chevet
Abstract | Full Text

The fungal pathogen Drechmeria coniospora infects C. elegans and elicits an innate immune response mediated, in part, by the induction of antimicrobial peptides in the epidermis. The signaling pathways controlling this phenomenon remain to be fully characterized. In this issue of Virulence, Couillault and colleagues use both proteomics and genetics to discover an unexpected role for the unfolded protein response (UPR) target chaperone BiP/GRP78/hsp-3 in the control of fungal infection-induced antimicrobial peptide expression in C. elegans. Although the expression of hsp-3 is regulated by the UPR, Couillault and colleagues describe a novel signaling role for this BiP/GRP78 homolog.

Biofilm formation and Klebsiella pneumoniae liver abscess: True, true and unrelated?

Joshua Fierer
Abstract | Full Text

Klebsiella pneumoniae liver abscess is an emerging infectious disease. This syndrome was unknown before the late 1980s when it was first recognized in Taiwan. Over the next 2 decades it increased in prevalence in Taiwan and was reported from other nations of East Asia. It was then that the rest of the world became aware of this interesting new syndrome. The disease is no longer confined to East Asia, and is now an emerging infection in North America and Europe. How did this come about? We now understand some of the genetic changes that turn commensal E. coli into extra-intestinal pathogens. K pneumoniae is another member of the Enterobacteriaceae that is usually normal flora in the gut, but we know relatively little about how it evolved into an invasive pathogen capable of causing abscesses in normal livers. The phenotype of the liver-invasive strains is hyperviscosity of the polysaccharide capsules, but while the gene that determines that property is required it is not sufficient to create the pathogen, and more research is needed to discover the other virulence genes, and thus to potentially target them therapeutically.




REVIEWS

Open Access Article

Acinetobacter baumannii: An emerging opportunistic pathogen

Aoife Howard, Michael O’Donoghue, Audrey Feeney and Roy D. Sleator
Abstract | Full Text

Acinetobacter baumannii is an opportunistic bacterial pathogen primarily associated with hospital-acquired infections. The recent increase in incidence, largely associated with infected combat troops returning from conflict zones, coupled with a dramatic increase in the incidence of multidrug-resistant (MDR) strains, has significantly raised the profile of this emerging opportunistic pathogen. Herein, we provide an overview of the pathogen, discuss some of the major factors that have let to its clinical prominence and outline some of the novel therapeutic strategies currently in development.

Open Access Article

Regulation of phenotypic transitions in the fungal pathogen Candida albicans

Guanghua Huang
Abstract | Full Text

The human commensal fungus Candida albicans can cause not only superficial infections, but also life-threatening disease in immunocompromised individuals. C. albicans can grow in several morphological forms. The ability to switch between different phenotypic forms has been thought to contribute to its virulence. The yeast-filamentous growth transition and white-opaque switching represent two typical morphological switching systems, which have been intensively studied in C. albicans. The interplay between environmental factors and genes determines the morphology of C. albicans. This review focuses on the regulation of phenotypic changes in this pathogenic organism by external environmental cues and internal genes.

Open Access Article

The role of inflammasome modulation in virulence

Christopher Lupfer and Thirumala-Devi Kanneganti
Abstract | Full Text

Pathogens frequently exist in an immunological balancing act with their host. Pathogens must not only replicate within a host but also transmit effectively between hosts to perpetuate their species. On the other hand, the host seeks to maintain homeostasis by clearing pathogens. The inflammasome is a multi-protein complex that can induce cell death and processes IL-1β and additional proinflammatory substrates. In this review we discuss the pathogen specific modulation of inflammasome activation and the role this plays in virulence and disease pathology.

Open Access Article

The potential of nitric oxide releasing therapies as antimicrobial agents

David O. Schairer, Jason S. Chouake, Joshua D. Nosanchuk and Adam J. Friedman
Abstract | Full Text

Nitric oxide (NO) is a short-lived diatomic, lipophilic gas that plays an integral role in defending against pathogens. Among its many functions are involvement in immune cell signaling and in the biochemical reactions by which immune cells defend against bacteria, fungi, viruses and parasites. NO signaling directs a broad spectrum of processes, including the differentiation, proliferation, and apoptosis of immune cells. When secreted by activated immune cells, NO diffuses across cellular membranes and exacts nitrosative and oxidative damage on invading pathogens. These observations led to the development of NO delivery systems that can harness the antimicrobial properties of this evanescent gas. The innate microbicidal properties of NO, as well as the antimicrobial activity of the various NO delivery systems, are reviewed.




RESEARCH PAPERS

Salicylate increases the expression of marA and reduces in vitro biofilm formation in uropathogenic Escherichia coli by decreasing type 1 fimbriae expression

Jordi Vila and Sara Soto
Abstract | Full Text | PDF

Escherichia coli is one of the most frequent bacteria implicated in biofilm formation, which is a dynamic process whose first step consists in bacteria adhesion to surfaces through type 1 fimbriae. Salicylate induces a number of morphological and physiological alterations in bacteria including the activation of the transcriptional regulator MarA. In this report the effects of salicylate on biofilm formation and their relationship with MarA were studied. An inverse relationship was observed between in vitro biofilm formation and salicylate concentration added to the culture medium. Salicylate increases the expression of marA and decreases the expression of fimA and fimB genes in the wild-type strain. In addition, the fimA and fimB expression was decreased in a MarR mutant in which marA was also overexpressed. In conclusion, the expression of type 1 fimbriae in presence of salicylate may be regulated by the level of marA expression through fimB regulator, albeit through neither the ompX nor the tolC genes.

A Burkholderia cepacia complex non-ribosomal peptide-synthesized toxin is hemolytic and required for full virulence

Euan L.S. Thomson and Jonathan J. Dennis
Abstract | Full Text | PDF

Members of the Burkholderia cepacia complex (Bcc) have recently gained notoriety as significant bacterial pathogens due to their extreme levels of antibiotic resistance, their transmissibility in clinics, their persistence in bacteriostatic solutions, and their intracellular survival capabilities. As pathogens, the Bcc are known to elaborate a number of virulence factors including proteases, lipases, and other exoproducts, as well as a number of secretion system associated effectors. Through random and directed mutagenesis studies, we have identified a Bcc gene cluster capable of expressing a toxin that is both hemolytic and required for full Bcc virulence. The Bcc toxin is synthesized via a non-ribosomal peptide synthetase mechanism, and appears to be related to the previously identified antifungal compound burkholdine or occidiofungin. Further testing shows mutations to this gene cluster cause a significant reduction in both hemolysis and Galleria mellonella mortality. Mutation to a glycosyltransferase gene putatively responsible for a structural-functional toxin variant causes only partial reduction in hemolysis. Molecular screening identifies the Bcc species containing this gene cluster, of which several strains produce hemolytic activity.

Open Access Article

A UPR-independent infection-specific role for a BiP/GRP78 protein in the control of antimicrobial peptide expression in C. elegans epidermis

Carole Couillault, Patrick Fourquet, Matthieu Pophillat and Jonathan Ewbank
Abstract | Full Text

The nematode C. elegans responds to infection by the fungus Drechmeria coniospora with a rapid increase in the expression of antimicrobial peptide genes. To investigate further the molecular basis of this innate immune response, we took a two-dimensional difference in-gel electrophoresis (2D-DIGE) approach to characterize changes in host protein that accompany infection. We identified a total of 68 proteins from differentially-represented spots and their corresponding genes. Through class testing, we identified functional categories that were enriched in our proteomic data set. One of these was “protein processing in endoplasmic reticulum,” pointing to a potential link between innate immunity and endoplasmic reticulum function. This class included HSP-3, a chaperone of the BiP/GRP78 family known to act coordinately in the endoplasmic reticulum with its paralog HSP-4 to regulate the unfolded protein response (UPR). Other studies have shown that infection of C. elegans can provoke a UPR. We observed, however, that in adult C. elegans infection with D. coniospora did not induce a UPR, and conversely, triggering a UPR did not lead to an increase in expression of the well-characterized antimicrobial peptide gene nlp-29. On the other hand, we demonstrated a specific role for hsp-3 in the regulation of nlp-29 after infection that is not shared with hsp-4. Epistasis analysis allowed us to place hsp-3 genetically between the Tribbles-like kinase gene nipi-3 and the protein kinase C delta gene tpa-1. The precise function of hsp-3 has yet to be determined, but these results uncover a hitherto unsuspected link between a BiP/GRP78 family protein and innate immune signaling.

Biofilm formed by a hypervirulent (hypermucoviscous) variant of Klebsiella pneumoniae does not enhance serum resistance or survival in an in vivo abscess model

Qingli Kong, Janet Beanan, Ruth Olson, Ulrike MacDonald, Alyssa Shon, Daniel Metzger, Alexander Pomakov and Thomas Russo
Abstract | Full Text

A new hypervirulent (hypermucoviscous) clinical variant of Klebsiella pneumoniae (hvKP) has emerged over the last decade. Our goal is to identify new mechanisms, which increase the virulence hvKP. It has been shown that hvKP strains produce more biofilm than “classical” stains of K. pneumoniae, therefore we hypothesized that biofilm formation may contribute to the pathogenesis of systemic infection. To test this hypothesis, transposon mutants of the model pathogen hvKP1 were generated and screened for decreased production of biofilm. Three mutant constructs with disruptions in glnA [putatively encodes glutamine synthetase, hvKP1 glnA:: EZ::TN < KAN-2 > (glnA::Tn)], sucD [putatively encodes succinyl-CoA synthase α subunit, hvKP1 sucD:: EZ::TN < KAN-2 > (sucD::Tn)], and tag [putatively encodes transcriptional antiterminator of glycerol uptake operon, hvKP1 tag:: EZ::TN < KAN-2 > (tag::Tn)] were chosen for further characterization and use in biologic studies. Quantitative assays performed in rich laboratory medium and human ascites confirmed the phenotype and a hypermucoviscosity assay established that capsule production was not affected. However, compared with its wild-type parent, neither planktonic cells nor biofilms of glnA::Tn, sucD::Tn and tag::Tn displayed a change to the bactericidal activity of 90% human serum. Likewise, when assessed in a rat subcutaneous abscess model, the growth and survival of glnA::Tn, sucD::Tn and tag::Tn in abscess fluid was similar to hvKP1. In this report we identify three new genes that contribute to biofilm formation in hvKP1. However, decreased biofilm production due to disruption of these genes does not affect the sensitivity of these mutant constructs to 90% human serum when in planktonic form or within a biofilm. Further, their virulence in an in vivo abscess model was unaffected.




PERSPECTIVE

Tracking linkage to HIV care for former prisoners: A public health priority

Brian T. Montague, David L. Rosen, Liza Solomon, Amy Nunn, Traci Green, Michael Costa, Jacques Baillargeon, David A. Wohl, David P. Paar, Josiah D. Rich and on behalf of the LINCS Study Group
Abstract | Full Text

Improving testing and uptake to care among highly impacted populations is a critical element of Seek, Test, Treat and Retain strategies for reducing HIV incidence in the community. HIV disproportionately impacts prisoners. Though, incarceration provides an opportunity to diagnose and initiate therapy, treatment is frequently disrupted after release. Though model programs exist to support linkage to care on release, there is a lack of scalable metrics with which to assess adequacy of linkage to care after release. The linking data from Ryan White program Client Level Data (CLD) files reported to HRSA with corrections release data offers an attractive means of generating these metrics. Identified only by use of a confidential encrypted Unique Client Identifier (eUCI) these CLD files allow collection of key clinical indicators across the system of Ryan White funded providers. Using eUCIs generated from corrections release data sets as a linkage tool, the time to the first service at community providers along with key clinical indicators of patient status at entry into care can be determined as measures of linkage adequacy. Using this strategy, high and low performing sites can be identified and best practices can be identified to reproduce these successes in other settings.




NEWS AND VIEWS

Inhibition of bacterial superoxide defense: A new front in the struggle between host and pathogen

Steven Damo, Walter J. Chazin, Eric P. Skaar and Thomas E. Kehl-Fie
Abstract | Full Text

Nutritional immunity is the name given to the sequestration of essential metals by vertebrates to limit growth of invading pathogens such as Staphylococcus aureus. While iron sequestration is the canonical example of nutritional immunity, recent work has highlighted the importance of manganese and zinc sequestration by vertebrates in controlling both bacterial and fungal infections. Our laboratories are investigating the biochemical basis for the sequestration of essential metals and the impact of this nutrient deprivation on the physiology of S. aureus and other pathogens. We have shown that during staphylococcal infection the manganese and zinc binding protein calprotectin (CP) is a critical component of nutritional immunity. Furthermore, this neutrophil protein is required to render the staphylococcal abscess devoid of manganese. However, the structural features responsible for binding these metals were unknown as was the impact of this CP-induced nutrient deprivation on S. aureus. In our recently published manuscript, we showed using isothermal titration calorimetry and mutagenesis experiments that CP contains two transition metal binding sites, and is capable of binding two zinc ions or one manganese ion with nanomolar affinity. Moreover, CP was found to reduce staphylococcal superoxide dismutase activity and other antioxidant defenses resulting in the accumulation of intracellular superoxide and increased sensitivity to oxidative stress. The inhibition of superoxide defenses by CP renders S. aureus more sensitive to neutrophil-mediated killing while animal modeling suggests that manganese sequestration by the host during infection reduces staphylococcal superoxide dismutase activity. In total, these results suggest a two hit model for the antimicrobial activity of CP whereby this protein both inhibits bacterial growth and renders microbial invaders more sensitive to other immune effectors.




PROTOCOLS

Mouse models for the study of fungal pneumonia: A collection of detailed experimental protocols for the study of Coccidioides, Cryptococcus, Fusarium, Histoplasma and combined infection due to Aspergillus-Rhizopus

Maged Muhammed, Marta Feldmesser, Lisa F. Shubitz, Michail S. Lionakis, Anita Sil, Yan Wang, Justin Glavis-Blom, Russell E. Lewis, John Galgiani, Arturo Casadevall, Dimitrios P. Kontoyiannis and Eleftherios Mylonakis
Abstract | Full Text

Mouse models have facilitated the study of fungal pneumonia. In this report, we present the working protocols of groups that are working on the following pathogens: Aspergillus, Coccidioides, Cryptococcus, Fusarium, Histoplasma and Rhizopus. We describe the experimental procedures and the detailed methods that have been followed in the experienced laboratories to study pulmonary fungal infection; we also discuss the anticipated results and technical notes, and provide the practical advices that will help the users of these models.

A novel method for infecting Drosophila adult flies with insect pathogenic nematodes

Julio Cesar Castillo, Upasana Shokal and Ioannis Eleftherianos
Abstract | Full Text

Drosophila has been established as an excellent genetic and genomic model to investigate host-pathogen interactions and innate immune defense mechanisms. To date, most information on the Drosophila immune response derives from studies that involve bacterial, fungal or viral pathogens. However, immune reactions to insect parasitic nematodes are still not well characterized. The nematodes Heterorhabditis bacteriophora live in symbiosis with the entomopathogenic bacteria Photorhabdus luminescens, and they are able to invade and kill insects. Interestingly, Heterorhabditis nematodes are viable in the absence of Photorhabdus. Techniques for infecting Drosophila larvae with these nematodes have been previously reported. Here, we have developed a method for infecting Drosophila adult flies with Heterorhabditis nematodes carrying (symbiotic worms) or lacking (axenic worms) their associated bacteria. The protocol we present can be readily adapted for studying parasitic strategies of other insect nematodes using Drosophila as the host infection model.