ABSTRACT
Salmonella enterica is a Gram-negative enteropathogen that can cause localized infections, typically resulting in gastroenteritis, or systemic infection, e.g., typhoid fever, in humans and many other animals. Understanding the mechanisms by which Salmonella induces disease has been the focus of intensive research. This has revealed that Salmonella invasion requires dynamic cross-talk between the microbe and host cells, in which bacterial adherence rapidly leads to a complex sequence of cellular responses initiated by proteins translocated into the host cell by a type 3 secretion system. Once these Salmonella-induced responses have resulted in bacterial invasion, proteins translocated by a second type 3 secretion system initiate further modulation of cellular activities to enable survival and replication of the invading pathogen. Elucidation of the complex and highly dynamic pathogen-host interactions ultimately requires analysis at the level of single cells and single infection events. To achieve this goal, researchers have applied a diverse range of microscopy techniques to analyze Salmonella infection in models ranging from whole animal to isolated cells and simple eukaryotic organisms. For example, electron microscopy and high-resolution light microscopy techniques such as confocal microscopy can reveal the precise location of Salmonella and its relationship to cellular components. Widefield light microscopy is a simpler approach with which to study the interaction of bacteria with host cells and often has advantages for live cell imaging, enabling detailed analysis of the dynamics of infection and cellular responses. Here we review the use of imaging techniques in Salmonella research and compare the capabilities of different classes of microscope to address specific types of research question. We also provide protocols and notes on some microscopy techniques used routinely in our own research.
Subject(s)
Bacteriology , Microscopy/methods , Salmonella/cytology , Actins/metabolism , Bacterial Adhesion , Cell Survival , Microbial Viability , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Salmonella/metabolism , Salmonella/ultrastructure , Staining and Labeling , Time FactorsABSTRACT
Salmonella enterica pathogenesis is dependent on its ability to enter and replicate inside host cells. Replication occurs inside the Salmonella-containing vacuole (SCV), a vacuolar compartment that is modified by bacterial effectors secreted through the two type III secretion systems (T3SS-1 and T3SS-2). Type III effectors interact with the host cell endocytic pathway to aid replication. We investigated whether Salmonella effector proteins may also interact with the host's exocytic pathway. A secreted alkaline phosphatase (SEAP) assay indicated three Salmonella effectors inhibited the secretory pathway, although only Salmonella outer protein B (SopB) was confirmed to block exocytosis using a vesicular stomatitis virus glycoprotein-green fluorescent protein (VSVG-GFP) transport assay. The 4-phosphatase activity of SopB was crucial to its effect on exocytosis. The interaction with the secretory pathway could potentially be important for providing replicating Salmonella with nutrients, contributing membrane material necessary for SCV biogenesis, altering antibacterial peptide/protein secretion or manipulating cell surface proteins important in the host response to infection.
ABSTRACT
[This corrects the article DOI: 10.1038/emi.2013.31.].
ABSTRACT
Most studies on Salmonella enterica serovar Typhimurium infection focus on strains ATCC SL1344 or NTCC 12023 (ATCC 14028). We have compared the abilities of these strains to induce membrane ruffles and invade epithelial cells. S. Typhimurium strain 12023 is less invasive and induces smaller membrane ruffles on MDCK cells compared with SL1344. Since the SPI-1 effector SopE is present in SL1344 and absent from 12023, and SL1344 sopE mutants have reduced invasiveness, we investigated whether 12023 is less invasive due to the absence of SopE. However, comparison of SopE(+) and SopE(-) S. Typhimurium strains, sopE deletion mutants and 12023 expressing a sopE plasmid revealed no consistent relationship between SopE status and relative invasiveness. Nevertheless, absence of SopE was closely correlated with reduced size of membrane ruffles. A PprgH-gfp reporter revealed that relatively few of the 12023 population (and that of the equivalent strain ATCC 14028) express SPI-1 compared to other S. Typhimurium strains. Expression of a PhilA-gfp reporter mirrored that of PprgH-gfp in 12023 and SL1344, implicating reduced signalling via the transcription factor HilA in the heterogeneous SPI-1 expression of these strains. The previously unrecognized strain heterogeneity in SPI-1 expression and invasiveness has important implications for studies of Salmonella infection.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Genomic Islands/genetics , Salmonella typhimurium/genetics , Salmonella typhimurium/pathogenicity , Trans-Activators/metabolism , Virulence Factors/metabolism , Animals , Bacterial Proteins/biosynthesis , Dogs , Epithelial Cells/microbiology , Gene Expression , Genes, Reporter , Salmonella typhimurium/metabolism , Sequence Deletion , Signal Transduction , Virulence Factors/biosynthesis , Virulence Factors/geneticsABSTRACT
Vibrionic hepatitis is a disease of poultry which is characterised by the presence of focal lesions in the liver, usually 1-2mm in size and greyish-white in colour. The cause of the disease remains unclear, as do the reasons for its recent re-emergence. We examined the livers of commercial broiler chickens taken during processing and found Campylobacter spp. in both normal livers and those displaying signs indicative of focal hepatitis. Livers with signs of hepatitis had significantly more Campylobacter spp. present than those without and other bacterial genera were infrequently present. We were unable to replicate the disease in a healthy host following experimental infection with a Campylobacter jejuni strain isolated from a liver showing signs of focal hepatitis. However, a significant T cell response to C. jejuni was seen in the liver of Campylobacter infected birds. We conclude that the presence of Campylobacter spp. in the liver alone is not sufficient to cause vibrionic hepatitis, but that a predisposing factor, possibly within the host is required. We also provide evidence that chickens mount an adaptive T cell response to systemic C. jejuni.
Subject(s)
Campylobacter Infections/veterinary , Campylobacter jejuni/isolation & purification , Chickens/microbiology , Hepatitis, Animal/microbiology , Poultry Diseases/microbiology , Animals , Caco-2 Cells , Campylobacter Infections/microbiology , Campylobacter Infections/pathology , Campylobacter jejuni/pathogenicity , Chickens/immunology , Hepatitis, Animal/pathology , Humans , Liver/microbiology , Liver/pathology , Poultry Diseases/pathology , Prevalence , T-Lymphocytes/immunologyABSTRACT
Bacterial species can communicate by producing and sensing small autoinducer molecules by a process known as quorum sensing. Salmonella enterica produces autoinducer 2 (AI-2) via the luxS synthase gene, which is used by some bacterial pathogens to coordinate virulence gene expression with population density. We investigated whether the luxS gene might affect the ability of Salmonella enterica serovar Typhimurium to invade epithelial cells. No differences were found between the wild-type strain of S. Typhimurium, SL1344, and its isogenic luxS mutant with respect to the number and morphology of the membrane ruffles induced or their ability to invade epithelial cells. The dynamics of the ruffling process were also similar in the wild-type strain (SL1344) and the luxS mutant. Furthermore, comparing the Salmonella pathogenicity island 1 (SPI-1) type 3 secretion profiles of wild-type SL1344 and the luxS mutant by Western blotting and measuring the expression of a single-copy green fluorescent protein fusion to the prgH (an essential SPI-1 gene) promoter indicated that SPI-1 expression and activity are similar in the wild-type SL1344 and luxS mutant. Genetic deletion of luxS did not alter the virulence of S. Typhimurium in the mouse model, and therefore, it appears that luxS does not play a significant role in regulating invasion of Salmonella in vitro or in vivo.
Subject(s)
Bacterial Proteins/physiology , Carbon-Sulfur Lyases/physiology , Epithelial Cells/microbiology , Quorum Sensing/physiology , Salmonella enterica/metabolism , Actins/metabolism , Animals , Bacterial Proteins/genetics , Carbon-Sulfur Lyases/genetics , Cell Line , Dogs , Female , Flow Cytometry , HeLa Cells , Humans , Mice , Mice, Inbred BALB C , Microscopy, Phase-Contrast , Quorum Sensing/genetics , Salmonella enterica/geneticsABSTRACT
The Salmonella pathogenicity island 1 (SPI-1) type three secretion system (TTSS) is essential for Salmonella invasion of host cells through its triggering of actin-dependent membrane ruffles. The SPI-1 effectors SipA, SopE, SopE2 and SopB all have actin regulating activities and contribute to invasion. The precise role of actin regulation by SipA in Salmonella invasion remains controversial since divergent data have been presented regarding the relationship between SipA and membrane ruffling. We hypothesized that the contribution of SipA to membrane ruffling and invasion might vary between Salmonella strains. We compared the effects of SipA deletion on Salmonella enterica serovar Typhimurium (S. Typhimurium) strains that possess or lack SopE. Loss of SipA reduced invasion in the early stages of infection by SopE(+) and SopE(-) strains but the number of membrane ruffles elicited was unaffected. Salmonella strains lacking both SipA and SopE induced ruffles with very different morphology from those induced by wild-type strains or ones lacking single effectors, including the presence of highly dynamic finger-like protrusions and numerous filopodia. A similar phenotype was found for sipA(-)sopE(-), sipA(-)sopE2(-) and sipA(-)sopB(-) mutants. Thus, SipA plays a more prominent role in induction of invasion-competent membrane ruffles by Salmonella lacking a full complement of SPI-1 effectors.
Subject(s)
Bacterial Proteins/physiology , Cell Surface Extensions/metabolism , Microfilament Proteins/physiology , Salmonella typhimurium/pathogenicity , Virulence Factors/physiology , Animals , Bacterial Proteins/genetics , Cell Line , Cell Membrane/metabolism , Dogs , Gene Deletion , Microfilament Proteins/genetics , Virulence Factors/geneticsABSTRACT
Salmonella enterica is a Gram-negative enteropathogen that can cause localized infections, typically resulting in gastroenteritis, or systemic infection, e.g., typhoid fever, in both humans and warm-blooded animals. Understanding the mechanisms by which Salmonella induce disease has been the focus of intensive research. This has revealed that Salmonella invasion requires dynamic cross-talk between the microbe and host cells, in which bacterial adherence rapidly leads to a complex sequence of cellular responses initiated by proteins translocated into the host cell by a type III secretion system (T3SS). Once these Salmonella-induced responses have resulted in bacterial invasion, proteins translocated by a second T3SS initiate further modulation of cellular activities to enable survival and replication of the invading pathogen. These processes contribute to Salmonella entry into the host and the clinical symptoms of gastrointestinal and systemic infection. Elucidation of the complex and highly dynamic pathogen-host interactions ultimately requires analysis at the level of single cells and single infection events. To achieve this goal, researchers have applied a diverse range of microscopical methods to examine Salmonella infection in models ranging from whole animal to isolated cells and simple eukaryotic organisms. For example, electron microscopy and confocal microscopy can reveal the juxtaposition of Salmonella, its products, and cellular components at high resolution. Simple light microscopy (LM) can also be used to investigate the interaction of bacteria with host cells and has advantages for live cell imaging, which enables detailed analysis of the dynamics of infection and cellular responses. Here we review the use of imaging techniques in Salmonella research and compare the capabilities of different classes of microscope to address specific types of research question. We also provide protocols and notes on several LM techniques routinely used in our own research.