Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.
21 Related JoVE Articles!
Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection
Institutions: Imperial College London.
, the causative agent of a severe pneumonia named Legionnaires' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella
containing vacuole (LCV). L. pneumophila
infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella
are suitable for investigation of L. pneumophila
infection. G. mellonella
is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae's immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila
is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila
virulence in the G. mellonella
model, including how to grow infectious L. pneumophila
, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila
virulence, describing a new tool to aid our understanding of this complex pathogen.
Infection, Issue 81, Bacterial Infections, Infection, Disease Models, Animal, Bacterial Infections and Mycoses, Galleria mellonella, Legionella pneumophila, insect model, bacterial infection, Legionnaires' disease, haemocytes
Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence
Institutions: University of Wisconsin-Madison.
, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta
larvae to a variety of entomopathogenic bacterial species1-5
, as well as the wealth of information available regarding the insect's immune system6-8
, and the pending genome sequence9
make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta
larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta
larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus
species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10
. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11
and oral toxicity assays12.
Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta
immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7
; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13
. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6
. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14
Infection, Issue 70, Microbiology, Immunology, Bacteriology, Entomology, Bacteria, injection, pathogenesis, insect larvae, instar, Manduca sexta, tobacco hornworm, animal model, host pathogen interactions
The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis
Institutions: INRA, Micalis UMR1319, France.
The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1
. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3
. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1
. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella
have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum
, Aspergillus fumigatus
, Candida albicans
) and bacterial pathogens, such as Staphylococcus aureus
, Proteus vulgaris
, Serratia marcescens Pseudomonas aeruginosa
, Listeria monocytogenes
or Enterococcus faecalis4-7
. Regardless of the bacterial species, results obtained with Galleria
larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria
, and strains causing severe human infections are also highly virulent in the Galleria
. Oral infection of Galleria
is much less used and additional compounds, like specific toxins, are needed to reach mortality.
larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 °C to 30 °C) and infection studies can be conducted between 15 °C to above 37 °C12,13
, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014
, measurement of bacterial survival15,16
and examination of the infection process17
. Here, we describe the rearing of the insects, covering all life stages of G. mellonella
. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus
, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19
Infection, Issue 70, Microbiology, Immunology, Molecular Biology, Bacteriology, Entomology, Bacteria, Galleria mellonella, greater wax moth, insect larvae, intra haemocoelic injection, ingestion, animal model, host pathogen interactions
Chronic Salmonella Infected Mouse Model
Institutions: University of Rochester.
The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella
typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo
. Mice with Salmonella
persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella
typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella
persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.
Microbiology, Issue 39, Salmonella, intestine, colitis, chronic infection, mouse model
Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR
Institutions: Virginia Commonwealth University.
Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2
. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis
as a model organism. Each gene deletion construct in S. sanguinis
genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3
gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3
gene at their 5' end. A large scale PCR amplification of the aphA-3
gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3
gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis
cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis
cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis
excluding four gene ORFs contained entirely within other ORFs in S. sanguinis
SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.
Genetics, Issue 69, Microbiology, Molecular Biology, Biomedical Engineering, Genomics, Streptococcus sanguinis, Streptococcus, Genome-wide gene deletions, genes, High-throughput, PCR
Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay
Institutions: Murdoch Childrens Research Institute, Murdoch Childrens Research Institute, The University of Melbourne, The University of Melbourne.
Adherence of Streptococcus pneumoniae
(the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro
adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal colonization. The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius
on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.
Immunology, Issue 86, Gram-Positive Bacterial Infections, Pneumonia, Bacterial, Lung Diseases, Respiratory Tract Infections, Streptococcus pneumoniae, adherence, colonization, probiotics, Streptococcus salivarius, In Vitro assays
Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections
Institutions: Universidad de la República, Trinity College Dublin.
Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung’s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages.
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae
inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs’ immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.
Medicine, Issue 90, Sublingual immunotherapy, Pneumonia, Streptococcus pneumoniae, Lungs, Flagellin, TLR5, NLRC4
An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci
Institutions: University of Rochester Medical Center, Sichuan University, Glostrup Hospital, Glostrup, Denmark, University of Rochester Medical Center.
Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2
. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3
. The EPS are synthesized by microorganisms (S. mutans
, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro
biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5
. Moreover, it allows temporal evaluation, inclusion of various microbial species 5
and assessment of the effects of distinct experimental conditions (e.g. treatments 6
; comparison of knockout mutants vs. parental strain 5
; carbohydrates availability 7
). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ
EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.
Microbiology, Issue 47, Extracellular matrix, polysaccharides, biofilm, mutans streptococci, glucosyltransferases, confocal fluorescence, microarray
Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Institutions: The Geisel School of Medicine at Dartmouth.
Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii
has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii
by deleting the gene encoding the KU80 protein1,2
. The Δku80
strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro
and in vivo
and exhibit essentially a 100% frequency of homologous recombination. The Δku80
strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4
Here, we report methods for using type I and type II Δku80Δhxgprt
strains to advance gene targeting approaches in T. gondii
. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT
) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80
strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii
and related significant human pathogens that cause malaria (Plasmodium
sp.) and cryptosporidiosis (Cryptosporidium
Infectious Diseases, Issue 77, Genetics, Microbiology, Infection, Medicine, Immunology, Molecular Biology, Cellular Biology, Biomedical Engineering, Bioengineering, Genomics, Parasitology, Pathology, Apicomplexa, Coccidia, Toxoplasma, Genetic Techniques, Gene Targeting, Eukaryota, Toxoplasma gondii, genetic manipulation, gene targeting, gene deletion, gene replacement, gene tagging, homologous recombination, DNA, sequencing
Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
Institutions: University of Virginia Health System.
Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2
. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella
species, and Escherichia coli3-5
, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8
. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants.
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9
. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10
, is ideal for LPS extraction.
Immunology, Issue 63, Microbiology, Gram-negative, LPS, extraction, polysaccharide staining, Western immunoblot
Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)
Institutions: Cedars-Sinai Medical Center.
MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus
during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.
Infection, Issue 48, Subcutaneous infection, Staphylococcus aureus, MRSA
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy
Institutions: University of Texas Medical Branch.
Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1
, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus
in the family Bunyaviridae
and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3
as well as wild-type RVFV strains 4-6
, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3
, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8
and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10
IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11
, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14
. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.
Immunology, Issue 57, Rift Valley fever virus, reverse genetics, NSs, MP-12, vaccine development
Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages
Institutions: Centre for DNA Fingerprinting and Diagnostics, Andhra Pradesh, India, Fiers-Schell-Van Montagu Building, Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium.
A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis.
Here, we discuss a protocol to enact an in vitro
cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata
with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro
model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.
Immunology, Issue 82, Candida glabrata, THP-1 macrophages, colony forming unit (CFU) assay, fluorescence microscopy, signature-tagged mutagenesis
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae
Institutions: McMaster University .
Nasopharyngeal colonization by Streptococcus pneumoniae
is a prerequisite to invasion to the lungs or bloodstream1
. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2
, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae
Our mouse model of intranasal colonization is adapted from human models3
and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7
. In the first part of the model, we use a clinical isolate of S. pneumoniae
to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8
, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.
Immunology, Issue 83, Streptococcus pneumoniae, Nasal lavage, nasopharynx, murine, flow cytometry, RNA, Quantitative PCR, recruited macrophages, neutrophils, T-cells, effector cells, intranasal colonization
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions
Institutions: Imperial College London, Institut Pasteur, Unité Macrophages et Développement de l'Immunité.
is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri
by compartmentalizing bacteria inside ‘septin cages’ and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro
using tissue culture cells and in vivo
using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.
Infection, Issue 91, ATG8/LC3, autophagy, cytoskeleton, HeLa cells, p62, septin, Shigella, zebrafish
A Protocol for Computer-Based Protein Structure and Function Prediction
Institutions: University of Michigan , University of Kansas.
Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently >20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.
Biochemistry, Issue 57, On-line server, I-TASSER, protein structure prediction, function prediction
A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites
Institutions: Boston University School of Medicine, Boston University School of Medicine.
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis
, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis
accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
Immunology, Issue 90,
Pathogen-Induced Chronic Inflammation; Porphyromonas gingivalis; Oral Bone Loss; Periodontal Disease; Atherosclerosis; Chronic Inflammation; Host-Pathogen Interaction; microCT; MRI
Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus
Institutions: The University of Hong Kong - HKU.
Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for evaluating the effects of any treatment. In rat, about 98% of RGCs is known to project to the contralateral superior colliculus (SC) (Forrester and Peters, 1967). Applying fluoro-gold (FG) on the surface of SC can label almost all the RGCs, so that we can focus on this most vulnerable retinal neuron in glaucoma. FG is taken up by the axon terminals of retinal ganglion cells and bilaterally transported retrogradely to its somas in the retina. Compare with retrograde labeling of RGC by putting FG at stump of transected optic nerve for 2 days, the interference of RGC survival is minimized. Compare with cresyl violet staining that stains RGCs, amacrine cells and endothelium of the blood vessel in the retinal ganglion cell layer, this labeling method is more specific to the RGC. This video describes the method of retrograde labeling of RGC by applying FG on the surface of SC. The surgical procedures include drilling the skull; aspirating the cortex to expose the SC and applying gelatin sponge over entire dorsal surface of SC are shown. Useful tips for avoiding massive intracranial bleeding and aspiration of the SC have been given.
Neuroscience, Issue 16, Retrograde labeling, retinal ganglion cells, ophthalmology research, superior colliculus, experimental glaucoma