Table of Contents

Volume 3, Issue 1

  January/February 2012

  Pages 1 - 102

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EDITOR'S CORNER

Open Access Article

Getting grants

Victoria McGovern
Pages 1 - 11
http://dx.doi.org/10.4161/viru.3.1.18844
Abstract | Full Text | PDF

Attracting financial support is a critical element of success in science, but we have entered a time of cost constraint with little hope of relief coming soon. For principal investigators, developing a broad base of research support is a valuable strategy for attaining financial stability for the laboratory. New investigators working on problems related to virulence and just beginning to build their careers and laboratories must attain NIH funding. But they should also look beyond that agency to the other federal organizations, state and regional agencies, and non-profits that support research. This review will discuss the general principles of how to understand funders, their intentions, and their funding programs. An investigator who grasps what drives the funders will be better able to write fundable proposals.

Coming of AGE: Facile generation of attenuated vaccine strains through heterologous gene expression

Carlos C. Goller and Patrick C. Seed
Pages 12 - 14
http://dx.doi.org/10.4161/viru.3.1.19087
Abstract | Full Text | PDF

Live attenuated vaccine strains for bacterial pathogens are conventionally constructed by inactivating genes important for virulence or metabolism to render the organism less virulent. Ideally, these strains follow a natural route of infection, yet elicit an immune response without causing disease. In particular, live attenuated vaccines often have the advantage of generating cellular immune responses that killed organisms and purified antigens fail to stimulate. Importantly, a fine balance between attenuation and sufficient persistence in the host must be achieved in order to produce a protective and durable immune response. Unfortunately, the reality is that, in most cases, the complete repertoire of factors responsible for virulence is unknown or not well understood, hindering the creation of attenuated vaccine strains. Thus, the generation of live attenuated vaccines would be greatly facilitated by novel, portable approaches for attenuating Gram-negative pathogens.

Shedding natural light on fungal infections

Anna Vecchiarelli and Christophe d’Enfert
Pages 15 - 17
http://dx.doi.org/10.4161/viru.3.1.19247
Abstract | Full Text | PDF

Bioluminescence imaging allows the visualization of the temporal and spatial progression of biological phenomena, in particular infection, by non-invasive methods in vivo. This nature-borrowed technology has been successfully used to monitor bacterial infections but recent studies have also succeeded in tracking fungal infections such as those caused by the two major opportunistic fungal pathogens Candida albicans and Aspergillus fumigatus. The findings of Donat and collaborators published in this issue now show that by combining the sensitivity of the Gaussia princeps luciferase with a surface display expression system it is possible to perform longitudinal infection studies on cutaneous forms of aspergillosis with a small number of animals. Besides providing new and valuable information in the field of aspergillosis, the findings of Donat et al. offer a new perspective on the general applicability of bioluminescence methodologies for eukaryotic pathogens where the bacterial lux operon cannot be exploited.




REVIEW

Open Access Article

Association between antimicrobial resistance and virulence in Escherichia coli

Gabriela Jorge da Silva and Nuno Mendonça
Pages 18 - 28
http://dx.doi.org/10.4161/viru.3.1.18382
Abstract | Full Text | PDF

Escherichia coli represents a major cause of morbidity and mortality worldwide. The treatment of E. coli infections is now threatened by the emergence of antimicrobial resistance. The dissemination of resistance is associated with genetic mobile elements, such as plasmids, that may also carry virulence determinants. A proficient pathogen should be virulent, resistant to antibiotics, and epidemic. However, the interplay between resistance and virulence is poorly understood. This review aims to critically discuss the association and linked transmission of both resistance and virulence traits in strains from extraintestinal infections in E. coli, and intestinal pathotypes. Despite the numerous controversies on this topic, findings from research published to date indicate that there is a link between resistance and virulence, as illustrated by the successful E. coli ST131 epidemic clone. Perhaps the most commonly accepted view is that resistance to quinolones is linked to a loss of virulence factors. However, the low virulent phylogenetic groups might be more prone to acquire resistance to quinolones. Specific characteristics of the E. coli genome that have yet to be identified may contribute to such genetic linkages. Research based on bacterial populations is sorely needed to help understand the molecular mechanisms underlying the association between resistance and virulence, that, in turn, may help manage the future disseminations of infectious diseases in their entirety.




RESEARCH PAPERS

Expression of Escherichia coli virulence usher protein attenuates wild-type Salmonella

Xinghong Yang, Zhiyong Suo, Theresa Thornburg, Kathryn Holderness, Ling Cao, Timothy Lim, Nancy Walters, Laura Kellerman, Linda Loetterle, Recep Avci and David W. Pascual
Pages 29 - 42
http://dx.doi.org/10.4161/viru.3.1.18447
Abstract | Full Text | PDF

Generation of a live attenuated vaccine for bacterial pathogens often requires prior knowledge of the pathogen’s virulence factors. We hypothesized an alternative approach of heterologous gene expression would make a wild-type (wt) pathogen more susceptible to host cell killing, thus, resulting in immunization. As proof of concept, the heterologous expression of enterotoxigenic E. coli (ETEC) colonization factor antigen I (CFA/I) was tested to attenuate Salmonella. The overexpression of CFA/I resulted in significant attenuation of wt Salmonella. In-depth studies revealed the attenuation depended on the co-expression of chaperone (CfaA) and usher (CfaC) proteins. Remarkably, the CfaAC-attenuated Salmonella conferred protection against wt Salmonella challenge. Mechanistic study indicated CfaAC made Salmonella outer membranes permeable, causing Salmonella to be vulnerable to host destruction. Thus, enhancing bacterial permeability via CfaAC represents an alternative method to attenuate pathogens despite the presence of unknown virulence factors.

Duality of liver and kidney lesions after systemic infection of immunosuppressed and immunocompetent mice with Aspergillus fumigatus

Gregory Jouvion, Matthias Brock, Sabrina Droin-Bergere and Oumaima Ibrahim-Granet
Pages 43 - 50
http://dx.doi.org/10.4161/viru.3.1.18654
Abstract | PDF

Invasive aspergillosis is a life-threatening disease mainly caused by Aspergillus fumigatus. Patients at risk are generally immunocompromised and lungs are assumed to provide the primary site for infection and invasive disease manifestation. Contrarily, visceral organ involvement appears to result from a subsequent hematogenous spread. To compare the kinetics of dissemination within deep organs in immunosuppressed vs. immunocompetent mice, we used a bioluminescent A. fumigatus strain in an intravenous infection model. By applying an immunosuppressive regimen with corticosteroids, dissemination to the liver and kidneys was observed already 24 h after inoculation accompanied by a marked inflammatory response within the liver. In contrast, in the immunocompetent condition, fungal growth and inflammation were mainly restricted to the kidneys and only small amounts of fungal biomass and a weak inflammatory response were detected in the liver. Additionally, disease progressed much slower compared with the immunosuppressed condition. This is the first study underlying the duality between liver and renal tropism of A. fumigatus in relation to the immune status of the host.

Surface display of Gaussia princeps luciferase allows sensitive fungal pathogen detection during cutaneous aspergillosis

Stefanie Donat, Mike Hasenberg, Tina Schäfer, Knut Ohlsen, Matthias Gunzer, Hermann Einsele, Jürgen Löffler, Andreas Beilhack and Sven Krappmann
Pages 51 - 61
http://dx.doi.org/10.4161/viru.3.1.18799
Abstract | Full Text | PDF

Non-invasive imaging techniques in microbial disease models have delivered valuable insights in the intimate pathogen-host interplay during infection. Here we describe evaluation and validation of a transgenic bioluminescence reporter strain of the human-pathogenic mold Aspergillus fumigatus, one of the main fungal pathogens affecting immunocompromised individuals. Expression and surface display of the Gaussia princeps luciferase allowed sensitive and rapid detection of luminescence emitted from this strain after substrate addition, with photon fluxes strongly correlating to the amounts of fungal conidia or germlings. The reporter strain allowed spatio-temporal monitoring of infection in a cutaneous model of aspergillosis, where neutropenic mice maintained the fungal burden while immunocompetent ones were able to clear it entirely. Most importantly, antifungal therapy could be followed in this type of disease model making use of the bioluminescent A. fumigatus strain. In conclusion, combining sensitivity of the Gaussia luciferase with a surface display expression system in the fungal host allows longitudinal infection studies on cutaneous forms of aspergillosis, providing perspective on drug screening approaches at high-throughput.




BRIEF REPORT

Nitric oxide nanoparticles: Pre-clinical utility as a therapeutic for intramuscular abscesses

David O. Schairer, Luis R. Martinez, Karin Blecher, Jason S. Chouake, Parimala Nacharaju, Philip Gialanella, Joel M. Friedman, Joshua D. Nosanchuk and Adam J. Friedman
Pages 62 - 67
http://dx.doi.org/10.4161/viru.3.1.18816
Abstract | Full Text | PDF

Nitric oxide (NO) is a critical component of host defense against invading pathogens; however, its therapeutic utility is limited due to a lack of practical delivery systems. Recently, a NO-releasing nanoparticulate platform (NO-np) was shown to have in vitro broad-spectrum antimicrobial activity and in vivo pre-clinical efficacy in a dermal abscess model. To extend these findings, both topical (TP) and intralesional (IL) NO-np administration was evaluated in a MRSA intramuscular murine abscess model and compared with vancomycin. All treatment arms accelerated abscess clearance clinically, histologically, and by microbiological assays on both days 4 and 7 following infection. However, abscesses treated with NO-np via either route demonstrated a more substantial, statistically significant decrease in bacterial survival based on colony forming unit assays and histologically revealed less inflammatory cell infiltration and preserved muscular architecture. These data suggest that the NO-np may be an effective addition to our armament for deep soft tissue infections.




ARTICLE ADDENDA

Turnabout is fair play: Use of the bacterial Multivalent Adhesion Molecule 7 as an antimicrobial agent

Anne Marie Krachler, Hyeilin Ham and Kim Orth
Pages 68 - 71
http://dx.doi.org/10.4161/viru.3.1.18172
Abstract | Full Text | PDF

Pathogen attachment to host tissues is one of the initial and most crucial events during the establishment of bacterial infections and thus interference with this step could be an efficient strategy to fight bacterial colonization. Our recent work has identified one of the factors involved in initial binding of host cells by a wide range of Gram-negative pathogens, Multivalent Adhesion Molecule (MAM) 7. Interference with MAM7-mediated attachment, for example by pre-incubation of host cells with recombinant MAM7, significantly delays the onset of hallmarks of infection, such as pathogen-mediated cytotoxicity or the development of other adhesive structures such as actin pedestals. Thus, we are trying to develop tools based on MAM7 that can be used to prevent or diminish certain Gram-negative bacterial infections. Herein, we describe the use of bead-coupled MAM7 as an inhibitor of infection with the clinically relevant pathogen Pseudomonas aeruginosa.

Open Access Article

Haptoglobin-hemoglobin receptor independent killing of African trypanosomes by human serum and trypanosome lytic factors

Whitney Bullard, Rudo Kieft, Paul Capewell, Nicola J. Veitch, Annette Macleod and Stephen Hajduk
Pages 72 - 76
http://dx.doi.org/10.4161/viru.3.1.18295
Abstract | Full Text | PDF

The haptoglobin-hemoglobin receptor (HpHbR) of African trypanosomes plays a critical role in human innate immunity against these parasites. Localized to the flagellar pocket of the veterinary pathogen Trypanosoma brucei brucei this receptor binds Trypanosome Lytic Factor-1 (TLF-1), a subclass of human high-density lipoprotein (HDL) facilitating endocytosis, lysosomal trafficking and subsequent killing. Recently, we found that group 1 Trypanosoma brucei gambiense does not express a functional HpHbR. We now show that loss of the TbbHpHbR reduces the susceptibility of T. b. brucei to human serum and TLF-1 by 100- and 10,000-fold, respectively. The relatively high concentrations of human serum and TLF-1 needed to kill trypanosomes lacking the HpHbR indicates that high affinity TbbHpHbR binding enhances the cytotoxicity; however, in the absence of TbbHpHbR, other receptors or fluid phase endocytosis are sufficient to provide some level of susceptibility. Human serum contains a second innate immune factor, TLF-2, that has been suggested to kill trypanosomes independently of the TbbHpHbR. We found that T. b. brucei killing by TLF-2 was reduced in TbbHpHbR-deficient cells but to a lesser extent than TLF-1. This suggests that both TLF-1 and TLF-2 can be taken up via the TbbHpHbR but that alternative pathways exist for the uptake of these toxins. Together the findings reported here extend our previously published studies and suggest that group 1 T. b. gambiense has evolved multiple mechanisms to avoid killing by trypanolytic human serum factors.

Inhibition of ATF6β-dependent host adaptive immune response by a Toxoplasma virulence factor ROP18

Masahiro Yamamoto and Kiyoshi Takeda
Pages 77 - 80
http://dx.doi.org/10.4161/viru.3.1.18340
Abstract | Full Text | PDF

Toxoplasma gondii (T. gondii) secretes various effector molecules, which co-opt host cells and enable parasite proliferation. Of these, the rhoptry protein, ROP18, is a parasite-derived factor that determines acute virulence. ROP18 is injected into the host cytoplasm during infection and, eventually, localizes to parasitophorous vacuole (PV) membranes. ROP18 is predicted to be a serine/threonine kinase; however, the molecular mechanism by which ROP18 mediates its pathological effects remains unclear. At the end of 2010, two groups reported that ROP18 targets and phosphorylates interferon-inducible p47 small GTPases (IRGs), demonstrating the parasite’s strategy for disarming the innate defense system. Recently, we described a mechanism by which ROP18 mediates degradation of the host endoplasmic reticulum-localizing transcription factor, ATF6β, to downregulate CD8 T cell-mediated type I adaptive immune responses. Taken together, these results suggest that T. gondii inactivates host innate and adaptive immune responses by targeting different host immunity-related molecules: IRGs and ATF6β.

A novel contractility pathway operating in Salmonella invasion

Jan Hänisch, Theresia E.B. Stradal and Klemens Rottner
Pages 81 - 86
http://dx.doi.org/10.4161/viru.3.1.18454
Abstract | Full Text | PDF

S. typhimurium can infect host cells not only by the well-established “trigger”-mode of invasion, inducing the formation of prominent membrane ruffles e.g., as during macropinocytosis, but also independently of these processes. Recently, we have found that the novel, ruffling-independent entry mechanism usurps the host cell contraction machinery. This mode of entry involves formation of myosin II-rich stress fiber-like structures at invasion sites, likely through stimulation of a RhoA/Rho-kinase signaling pathway and mostly downstream of the Salmonella virulence factor SopB. Importantly, this pathway operates independently of Arp2/3 complex, a central regulator of the macropinocytic entry mode. Here, we will describe our current thinking of how the contraction-dependent uptake mechanism operates to promote Salmonella invasion, and which additional cellular or bacterial factors might get engaged in the process. Finally, we will speculate on the implications of these findings for invasion by other bacterial pathogens, and discuss their impact on the canonical trigger-vs.-zipper classification of entry mechanisms employed by distinct bacterial pathogens.

Bacteria tune interferon responses by playing with chromatin

Alice Lebreton, Pascale Cossart and Hélène Bierne
Pages 87 - 91
http://dx.doi.org/10.4161/viru.3.1.18531
Abstract | Full Text | PDF

Bacterial infections, like their viral counterparts, trigger the onset of innate immune defense mechanisms through the release of cytokines, including interferons (IFNs). While type I and II IFN responses to bacteria have long been explored, type III IFN response remains poorly addressed. We have recently reported that the pathogen Listeria monocytogenes triggers the expression of type I and III IFN genes in epithelial cells, and is able to fine-tune downstream signaling at the chromatin level. This bacterium can negatively or positively modulate the expression of interferon-stimulated genes (ISGs) by manipulating the function of BAHD1, a component of a host chromatin-silencing complex. To this end, L. monocytogenes tightly controls the secretion of a BAHD1 inhibitory factor, LntA. Here, we further document the current knowledge about chromatin mechanisms modulating interferon responses during host-bacteria interplay, and discuss their physiological consequences.




NEWS AND VIEWS

Pathogenic streptococci speak, but what are they saying?

Michael Federle
Pages 92 - 94
http://dx.doi.org/10.4161/viru.3.1.18652
Abstract | Full Text | PDF

Currently, intercellular chemical signaling in bacteria, known as quorum sensing, is described for several species of bacteria; however, for many clinically important pathogens this significant sensory mechanism remains unknown. Among such pathogens are the pyogenic streptococci that include groups A and B streptococcus (GAS, GBS). Evidence now points to a family of transcription factors, known as Rgg/GadR/MutR, can serve as receptors for secreted pheromones. Within the genome of Streptococcus pyogenes four Rgg paralogs can be identified, two of which (Rgg2 and Rgg3) were shown to rely on short hydrophobic peptides (SHPs) to control transcription of their target promoters. SHPs were found to promote biofilm development and could offset biofilm-dispersion effects caused by Rgg1. Since Rgg homologs are present in genomes throughout Firmicute species, their newfound ability to serve as quorum-sensing mediators offers a potential opportunity to manipulate bacterial behaviors by interfering with communication networks.

Resistance to echinocandins comes at a cost: The impact of FKS1 hotspot mutations on Candida albicans fitness and virulence

Ronen Ben-Ami and Dimitrios P. Kontoyiannis
Pages 95 - 97
http://dx.doi.org/10.4161/viru.3.1.18886
Abstract | Full Text | PDF

The emergence of Candida strains carrying FKS1 hotspot mutations associated with resistance to echinocandins is cause for concern. However, to assess the potential of such strains to spread within the community and cause lethal infection, the impact of FKS1 mutations on Candida fitness must be determined. We present evidence that C. albicans fks1 mutations carry significant fitness and virulence costs, which are associated with the production of a thickened, chitin-rich cell wall, impaired filamentation and induction of a dampened inflammatory response. If these phenotypic changes remain stable, they can serve as a basis for rational design of strategies to control the spread of echinocandin resistance.




PROTOCOL

Identification of predominant SNPs as a novel method for genotyping bovine Staphylococcus aureus isolates

Jeffrey Hall and Yinduo Ji
Pages 98 - 102
http://dx.doi.org/10.4161/viru.3.1.18724
Abstract | PDF

Staphylococcus aureus is a formidable pathogen of both human and animal. Infection often gives rise to an economic loss resulting from the extended cost of treatment and hospitalization for humans, and loss of usable agriculture animal products from infected animals and treatment regiments. We describe here a protocol for the amplification and sequencing of predominant single nucleotide polymorphisms within the promoter region of hla (encoding α-toxin) that confers a hyper-producing α-toxin phenotype to S. aureus isolates associated with chronic and severe bovine mastitis infections. We validated our findings with a second round of analysis, confirming the SNPs as a valid genotypic marker for α-toxin hyper-producing bovine isolates. The identification of highly virulent isolates will allow for aggressive treatment of the infection and limit the disease and economic impact. With readily available reagents and facilities, this protocol can be completed in as little as 72 h once samples are isolated.