Shigella flexneri modulates stress granule composition and inhibits stress granule aggregation

Invasion and multiplication of the facultative, cytosolic, enteropathogen Shigella flexneri within the colonic epithelial lining leads to an acute inflammatory response, fever and diarrhea. During the inflammatory process, infected cells are subjected to numerous stresses including heat, oxidative stress and genotoxic stress. The evolutionarily conserved pathway of cellular stress management is the formation of stress granules that store translationally inactive cellular mRNAs and interfere with cellular signalling pathways by sequestering signalling components. In this study, we investigated the ability of S. flexneri‐infected cells to form stress granules in response to exogenous stresses. We found that S. flexneri infection inhibits movement of the stress granule markers eIF3 and eIF4B into stress granules and prevents the aggregation of G3BP1 and eIF4G‐containing stress granules. This inhibition occurred only with invasive, but not with non‐invasive bacteria and occurred in response to stresses that induce translational arrest through the phosphorylation of eIF2α and by treating cells with pateamine A, a drug that induces stress granules by inhibiting the eIF4A helicase. The S. flexneri‐mediated stress granule inhibition could be largely phenocopied by the microtubule‐destabilizing drug nocodazole and while S. flexneri infection did not lead to microtubule depolymerization, infection greatly enhanced acetylation of alpha‐tubulin. Our data suggest that qualitative differences in the microtubule network or subversion of the microtubule‐transport machinery by S. flexneri may be involved in preventing the full execution of this cellular stress response.     

The pho regulon of Shigella flexneri

Growth of Escherichia coli K-12 in low-phosphate conditions results in the induction of the synthesis of many proteins, including the outer membrane porin PhoE, alkaline phosphatase, and the Pst system for the transport of phosphate (P_i). This response is controlled by a two-component regulatory system of which PhoB and PhoR are the response-regulator and the sensor/kinase, respectively. When Shigella flexneri was starved for P_i, neither PhoE nor alkaline phosphatase was produced. However, induction of the synthesis of the PstS protein was observed, indicating that S. flexneri contains a functional PhoB/PhoR regulatory system. Consistent with this notion, the introduction of the B. coli phoA gene in S. flexneri resulted in the induction of alkaline phosphatase synthesis under phosphate limitation. However, introduction of phoE on a plasmid did not lead to the expression of PhoE protein, indicating that S. flexneri PhoB does not recognize the phoE promoter region. The phoB gene was cloned and sequenced and in the deduced amino acid sequence two deviations from that of E. coli PhoB were detected. Site-directed mutagenesis revealed that one of these deviations, i.e. Leu-172, which is Arg in E. coli PhoB, is responsible for the lack of expression of the PhoE protein in S. flexneri.     

Acid tolerance of Shigella sonnei and Shigella flexneri

AIMS: Determination of the behaviour of Shigella sonnei and Sh. flexneri under acid conditions. METHODS AND RESULTS: The growth and survival of Shigella spp. (9 isolates) in acidified Brain Heart Infusion (BHI) (pH 5.0-3.25 with pH intervals of 0.25) was determined after 6, 24 and 30 h incubation at 37 degrees C. Subsequently, survival of shigellae was studied in apple juice and tomato juice stored at 7 degrees C and 22 degrees C for up to 14 days and in strawberries and a fresh fruit salad, kept at 4 degrees C for 4 and 48 h. CONCLUSIONS: The minimum pH for growth in acidified BHI for Sh. flexneri and Sh. sonnei was, respectively, pH 4.75 and pH 4.50. Survival in fruit juices and fresh fruits depended upon their pH, the type of strain and the incubation temperature. Shigella spp. Survived for up to 14 days in tomato juice and apple juice stored at 7 degrees C. The shortest survival time (2-8 d) was observed in apple juice at 22 degrees C. Sh. sonnei but not Sh. flexneri was recovered after 48 h from strawberries and fruit salad kept at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Acid foods, especially if kept at refrigeration temperatures, support survival of Shigella spp. and may cause Shigella food poisoning.     

Quantitative analysis of Shigella flexneri protein expression under acid stress

As an important foodborne pathogen, Shigella flexneri can cause widespread enteric infection with bacteria as few as hundreds. This is, at least in part, attributed to its robust anti‐acid strategies because passage through the highly acidic human digestive tract is a prerequisite for successful bacterial infection. Nevertheless, our understanding of these mechanisms and the impact of acid stress on Shigella protein expression still remains largely incomplete. Herein we conducted a proteomic survey of Shigella spp. under acid stress. Out of 1754 protein identifications, we found 131 altered proteins, most of which were down‐regulated, including virulence factors and cell envelope proteins. Rather, many metabolic enzymes and pyrimidine/amino acid biosynthesis proteins were up‐regulated. In addition to induction of many known anti‐acid systems, we also found marked increase of 2‐oxoglutarate dehydrogenase (SucAB), a metabolic enzyme in the tricarboxylic acid cycle. Importantly, overproduction of this enzyme significantly enhanced Shigella acid resistance and hence SucAB‐mediated metabolic pathways may represent novel anti‐acid strategies.     

Complete genome sequence of Shigella flexneri 5b and comparison with Shigella flexneri 2a

Background

On most common microarray platforms many genes are represented by multiple probes. Although this is quite common no one has systematically explored the concordance between probes mapped to the same gene.

Results

Here we present an analysis of all the cases of multiple probe sets measuring the same gene on the Affymetrix U133a GeneChip and found that although in the majority of cases both measurements tend to agree there are a significant number of cases in which the two measurements differ from each other. In these cases the measurements can not be simply averaged but rather should be handled individually.

Conclusion

Our analysis allows us to provide a comprehensive list of the correlation between all pairs of probe sets that are mapped to the same gene and thus allows microarray users to sort out the cases that deserve further analysis. Comparison between the set of highly correlated pairs and the set of pairs that tend to differ from each other reveals potential factors that may affect it.     

The role of oxidative stress in glaucoma

DNA damage is related to a variety of degenerative diseases such as cancer, atherosclerosis and neurodegenerative diseases, depending on the tissue affected. Increasing evidence indicates that reactive oxygen species (ROS) play a key role in the pathogenesis of primary open angle glaucoma (POAG), the main cause of irreversible blindness worldwide. Oxidative DNA damage is significantly increased in the ocular epithelium regulating aqueous humor outflow, i.e., the trabecular meshwork (TM), of glaucomatous patients compared to controls. The pathogenic role of ROS in glaucoma is supported by various experimental findings, including (a) resistance to aqueous humor outflow is increased by hydrogen peroxide by inducing TM degeneration; (b) TM possesses remarkable antioxidant activities, mainly related to superoxide dismutase-catalase and glutathione pathways that are altered in glaucoma patients; and (c) intraocular-pressure increase and severity of visual-field defects in glaucoma patients parallel the amount of oxidative DNA damage affecting TM. Vascular alterations, which are often associated with glaucoma, could contribute to the generation of oxidative damage. Oxidative stress, occurring not only in TM but also in retinal cells, appears to be involved in the neuronal cell death affecting the optic nerve in POAG. The highlighting of the pathogenic role of ROS in POAG has implications for the prevention of this disease as indicated by the growing number of studies using genetic analyses to identify susceptible individuals and of clinical trials testing the efficacy of antioxidant drugs for POAG management.     

Freeze-drying of live oral streptomycin-dependent mutant Shigella vaccines: survival of streptomycin-dependent Shigella flexneri 2a after freeze-drying

Although the relatively successful freeze-drying of streptomycin-dependent mutant Shigella vaccines has been reported previously, further studies have been undertaken for the following reasons: (1) To improve the survival rate of the vaccine strains after freeze-drying. (2) To see whether monosodium glutamate can be eliminated as a stabilizer as it is reported to produce pharmacological effects in man.     

Chemoprophylaxis of Shigella flexneri Keratoconjunctivitis in Rabbits

Chloramphenicol, chlortetracycline, streptomycin, and sulfisoxazole prevented keratoconjunctivitis in rabbit eyes infected with Shigella flexneri. The route of administration of the agent and the dose were important variables in controlling the infectious process. This assay system appears to be a useful technique for the in vivo evaluation of chemotherapeutic agents.