The bacterial cytoskeleton modulates motility, type 3 secretion, and colonization in Salmonella.

Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its importance to virulence. In this study we have explored the contribution of the bacterial cytoskeleton to the ability of Salmonella to express and assemble virulence factors and cause disease. The bacterial actin-like protein MreB polymerises into helical filaments and interacts with other cytoskeletal elements including MreC to control cell-shape. As mreB appears to be an essential gene, we have constructed a viable ΔmreC depletion mutant in Salmonella. Using a broad range of independent biochemical, fluorescence and phenotypic screens we provide evidence that the Salmonella pathogenicity island-1 type three secretion system (SPI1-T3SS) and flagella systems are down-regulated in the absence of MreC. In contrast the SPI-2 T3SS appears to remain functional. The phenotypes have been further validated using a chemical genetic approach to disrupt the functionality of MreB. Although the fitness of ΔmreC is reduced in vivo, we observed that this defect does not completely abrogate the ability of Salmonella to cause disease systemically. By forcing on expression of flagella and SPI-1 T3SS in trans with the master regulators FlhDC and HilA, it is clear that the cytoskeleton is dispensable for the assembly of these structures but essential for their expression. As two-component systems are involved in sensing and adapting to environmental and cell surface signals, we have constructed and screened a panel of such mutants and identified the sensor kinase RcsC as a key phenotypic regulator in ΔmreC. Further genetic analysis revealed the importance of the Rcs two-component system in modulating the expression of these virulence factors. Collectively, these results suggest that expression of virulence genes might be directly coordinated with cytoskeletal integrity, and this regulation is mediated by the two-component system sensor kinase RcsC.

The application of STEM and in situ controlled dehydration to bacterial systems using ESEM.

Transmission imaging with an environmental scanning electron microscope (ESEM) (Wet STEM) is a recent development in the field of electron microscopy, combining the simple preparation inherent to ESEM work with an alternate form of contrast available through a STEM detector. Because the technique is relatively new, there is little information available on how best to apply this technique and which samples it is best suited for. This work is a description of the sample preparation and microscopy employed by the authors for imaging bacteria with Wet STEM (scanning transmission electron microscopy). Three different bacterial samples will be presented in this study: first, used as a model system, is Escherichia coli for which the contrast mechanisms of STEM are demonstrated along with the visual effects of a dehydration-induced collapse. This collapse, although clearly in some sense artifactual, is thought to lead to structurally meaningful morphological information. Second, Wet STEM is applied to two distinct bacterial systems to demonstrate the novel types of information accessible by this approach: the plastic-producing Cupriavidus necator along with wild-type and ΔmreC knockout mutants of Salmonella enterica serovar Typhimurium. Cupriavidus necator is shown to exhibit clear internal differences between bacteria with and without plastic granules, while the ΔmreC mutant of S. Typhimurium has an internal morphology distinct from that of the wild type.

The application of STEM and in situ controlled dehydration to bacterial systems using ESEM

Transmission imaging with an environmental scanning electron microscope (ESEM) (Wet STEM) is a recent development in the field of electron microscopy, combining the simple preparation inherent to ESEM work with an alternate form of contrast available through a STEM detector. Because the technique is relatively new, there is little information available on how best to apply this technique and which samples it is best suited for. This work is a description of the sample preparation and microscopy employed by the authors for imaging bacteria with Wet STEM (scanning transmission electron microscopy). Three different bacterial samples will be presented in this study: first, used as a model system, is Escherichia coli for which the contrast mechanisms of STEM are demonstrated along with the visual effects of a dehydration-induced collapse. This collapse, although clearly in some sense artifactual, is thought to lead to structurally meaningful morphological information. Second, Wet STEM is applied to two distinct bacterial systems to demonstrate the novel types of information accessible by this approach: the plastic-producing Cupriavidus necator along with wild-type and δmreC knockout mutants of Salmonella enterica serovar Typhimurium. Cupriavidus necator is shown to exhibit clear internal differences between bacteria with and without plastic granules, while the δmreC mutant of S. Typhimurium has an internal morphology distinct from that of the wild type. © 2012 Wiley Periodicals, Inc.

Attenuated Salmonella Typhimurium lacking the pathogenicity island-2 type 3 secretion system grow to high bacterial numbers inside phagocytes in mice.

Intracellular replication within specialized vacuoles and cell-to-cell spread in the tissue are essential for the virulence of Salmonella enterica. By observing infection dynamics at the single-cell level in vivo, we have discovered that the Salmonella pathogenicity island 2 (SPI-2) type 3 secretory system (T3SS) is dispensable for growth to high intracellular densities. This challenges the concept that intracellular replication absolutely requires proteins delivered by SPI-2 T3SS, which has been derived largely by inference from in vitro cell experiments and from unrefined measurement of net growth in mouse organs. Furthermore, we infer from our data that the SPI-2 T3SS mediates exit from infected cells, with consequent formation of new infection foci resulting in bacterial spread in the tissues. This suggests a new role for SPI-2 in vivo as a mediator of bacterial spread in the body. In addition, we demonstrate that very similar net growth rates of attenuated salmonellae in organs can be derived from very different underlying intracellular growth dynamics.

Lymph Node Colonization Dynamics after Oral Salmonella Typhimurium Infection in Mice

An understanding of how pathogens colonize their hosts is crucial for the rational design of vaccines or therapy. While the molecular factors facilitating the invasion and systemic infection by pathogens are a central focus of research in microbiology, the population biological aspects of colonization are still poorly understood. Here, we investigated the early colonization dynamics of Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm) in the streptomycin mouse model for diarrhea. We focused on the first step on the way to systemic infection - the colonization of the cecal lymph node (cLN) from the gut - and studied roles of inflammation, dendritic cells and innate immune effectors in the colonization process. To this end, we inoculated mice with mixtures of seven wild type isogenic tagged strains (WITS) of S. Tm. The experimental data were analyzed with a newly developed mathematical model describing the stochastic immigration, replication and clearance of bacteria in the cLN. We estimated that in the beginning of infection only 300 bacterial cells arrive in the cLN per day. We further found that inflammation decreases the net replication rate in the cLN by 23%. In ccr7-/- mice, in which dendritic cell movement is impaired, the bacterial migration rate was reduced 10-fold. In contrast, cybb-/- mice that cannot generate toxic reactive oxygen species displayed a 4-fold higher migration rate from gut to cLN than wild type mice. Thus, combining infections with mixed inocula of barcoded strains and mathematical analysis represents a powerful method for disentangling immigration into the cLN from replication in this compartment. The estimated parameters provide an important baseline to assess and predict the efficacy of interventions. © 2013 Kaiser et al.

Dynamics of spread of Salmonella enterica in the systemic compartment.

Traditional microbiological and immunological tools, combined with modern imaging, and molecular and mathematical approaches, have revealed the dispersive nature of Salmonella infections. Bacterial escape from infected cells, spread in the tissues and attempts to restrain this process by the host give rise to fascinating scenarios that underpin the pathogenesis of salmonelloses.

Dynamics of spread of Salmonella enterica in the systemic compartment

Traditional microbiological and immunological tools, combined with modern imaging, and molecular and mathematical approaches, have revealed the dispersive nature of Salmonella infections. Bacterial escape from infected cells, spread in the tissues and attempts to restrain this process by the host give rise to fascinating scenarios that underpin the pathogenesis of salmonelloses. © 2013 Institut Pasteur.

Molecular characterization and expression profiles in response to bacterial infection of Chinese soft-shelled turtle interleukin-8 (IL-8), the first reptilian chemokine gene

In this study, an IL-8 homologue has been cloned and identified from a reptile, Chinese soft-shelled turtle for the first time. The full-length cDNA of turtle IL-8 was 1188 bp and contained a 312 bp open reading frame (ORF) coding for a protein of 104 amino acids. The chemokine CXC domain, which contained Glu-Leu-Arg (ELR) motif and four cysteine residues, was well conserved in turtle IL-8. The 4924 bp genomic DNA of turtle IL-8 contained four exons and three introns. Phylogenetic analysis showed that the amino acid sequence of turtle IL-8 clustered together with birds. RT-PCR analysis showed that turtle IL-8 mRNA was constitutively expressed liver, spleen, kidney, heart, blood and intestine tissues of control turtles. Real-time quantitative PCR analysis further indicated that the turtle IL-8 mRNA expression was apparent in various tissues at 8 h and up-regulated significantly during 8 h-7 d after Aeromonas hydrophila infection. The present studies will help us to understand the evolution of IL-8 molecule and the inflammatory response mechanism in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.

Bacterial diversity in activated sludge from a consecutively aerated submerged membrane bioreactor treating domestic wastewater

The bacterial diversity of activated sludge from submerged membrane bioreactor (SMBR) was investigated. A 16S rDNA clone library was generated, and 150 clones were screened using restriction fragment length polymorphism (RFLP). Of the screened clones, almost full-length 16S rDNA sequences of 64 clones were sequenced. Phylogenetic tree was constructed with a database containing clone sequences from this study and bacterial rDNA sequences from NCB1 for identification purposes. The 90.6% of the clones were affiliated with the two phyla Bacteroidetes (50%) and Proteobacteria (40%), and beta-, -gamma-, and delta-Proteobacteria accounted for 7.8%, 28.1%, and 4.7%, respectively. Minor portions were affiliated with the Actinobacteria and Firmicutes (both 3.1%). Only 6 out of 64 16S rDNA sequences exhibited similarities of more than 97% to classified bacterial species, which indicated that a substantial fraction of the clone sequences were derived from unknown taxa. Rarefaction analysis of operational taxonomic units (orrUs) clusters demonstrated that 150 clones screened were still insufficient to describe the whole bacterial diversity. Measurement of water quality parameter demonstrated that performance of the SMBR maintained high level, and the SMBR system remained stable during this study.