Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells

Ménard, Robert; Sansonetti, Philippe J.; Parsot, Claude

doi:10.1128/jb.175.18.5899-5906.1993

Cited by 687 publications

(611 citation statements)

References 39 publications

(31 reference statements)

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“…Accordingly, the efficiency of ruffle formation, i.e., the percentage of ruffles induced upon bacterial contact, was similar in cells treated with stressors and control cells (Appendix Fig S1G). To uncouple Shigella binding to host cells from subsequent steps of invasion, we performed parallel experiments with the Shigella Δ ipaB mutant strain, which binds efficiently to host cells but is unable to invade (Menard et al , 1993). Treatment of cells with arsenite or anisomycin inhibited binding of Shigella Δ ipaB , similar to the WT bacteria (Fig 2A–E, and Appendix Fig S1B, C, E and F).…”

Section: Resultsmentioning

confidence: 99%

Stress‐induced host membrane remodeling protects from infection by non‐motile bacterial pathogens

et al. 2018

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While mucosal inflammation is a major source of stress during enteropathogen infection, it remains to be fully elucidated how the host benefits from this environment to clear the pathogen. Here, we show that host stress induced by different stimuli mimicking inflammatory conditions strongly reduces the binding of Shigella flexneri to epithelial cells. Mechanistically, stress activates acid sphingomyelinase leading to host membrane remodeling. Consequently, knockdown or pharmacological inhibition of the acid sphingomyelinase blunts the stress‐dependent inhibition of Shigella binding to host cells. Interestingly, stress caused by intracellular Shigella replication also results in remodeling of the host cell membrane, in vitro and in vivo, which precludes re‐infection by this and other non‐motile pathogens. In contrast, Salmonella Typhimurium overcomes the shortage of permissive entry sites by gathering effectively at the remaining platforms through its flagellar motility. Overall, our findings reveal host membrane remodeling as a novel stress‐responsive cell‐autonomous defense mechanism that protects epithelial cells from infection by non‐motile bacterial pathogens.

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Section: Resultsmentioning

confidence: 99%

Stress‐induced host membrane remodeling protects from infection by non‐motile bacterial pathogens

et al. 2018

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“…For construction of a nonpolar spa32 mutant, the aphA-3 (kanamycin resistance gene) cassette specifically designed for the construction of a nonpolar mutant was used (31). The aphA-3 cassette carried by plasmid pUC18K2 was obtained from Philippe J. Sansonetti (Institut Pasteur, Paris, France).…”

Section: Methodsmentioning

confidence: 99%

Shigella Spa32 Is an Essential Secretory Protein for Functional Type III Secretion Machinery and Uniformity of Its Needle Length

et al. 2002

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The Shigella type III secretion machinery is responsible for delivering to host cells the set of effectors required for invasion. The type III secretion complex comprises a needle composed of MxiH and MxiI and a basal body made up of MxiD, MxiG, and MxiJ. In S. flexneri, the needle length has a narrow range, with a mean of approximately 45 nm, suggesting that it is strictly regulated. Here we show that Spa32, encoded by one of the spa genes, is an essential protein translocated via the type III secretion system and is involved in the control of needle length as well as type III secretion activity. When the spa32 gene was mutated, the type III secretion complexes possessed needles of various lengths, ranging from 40 to 1,150 nm. Upon introduction of a cloned spa32 into the spa32 mutant, the bacteria produced needles of wild-type length. The spa32 mutant overexpressing MxiH produced extremely long (>5 m) needles. Spa32 was secreted into the medium via the type III secretion system, but secretion did not depend on activation of the system. The spa32 mutant and the mutant overexpressing MxiH did not secrete effectors such as Ipa proteins into the medium or invade HeLa cells. Upon introduction of Salmonella invJ, encoding InvJ, which has 15.4% amino acid identity with Spa32, into the spa32 mutant, the bacteria produced type III needles of wild-type length and efficiently entered HeLa cells. These findings suggest that Spa32 is an essential secreted protein for a functional type III secretion system in Shigella spp. and is involved in the control of needle length. Furthermore, its function is interchangeable with that of Salmonella InvJ.The type III protein secretion system is found among various gram-negative animal-and plant-pathogenic bacteria, in which the subsets of effector proteins that are secreted via the system into host cells have crucial roles in the bacterial infection process (8,9,16,38). Genetic and functional studies indicate that the type III secretion system requires proteins encoded by more than 20 genes, which contain the structural components of the secretion complex, secreted proteins, chaperones, and regulators (10,11,19,37,43). In Shigella spp., the system is encoded by approximately 20 genes located in the mxi and spa operons on a large 230-kb plasmid (5, 19). The proteins of the type III secretion system show considerable amino acid homology among different pathogens, and some of them also show amino acid homology with the components of the bacterial flagellar export system (26). Although the function of the type III secretion system in bacterial infection is distinct from that of the flagellar export system, the type III secretion system is believed to have evolved from the flagellar export system and diverged in each pathogen to become involved in the infection process (15).Recent studies indicate that the type III secretion complexes of Shigella flexneri and Salmonella spp. share structural similarities; the complexes are composed of two distinct parts, a basal body and a needle. The basal...

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“…Construction of kanamycin (aphA-3) cassette insertions Each ORF was disrupted by inserting the non-polar aphA-3 cassette, which confers kanamycin resistance (Menard et al, 1993), into a restriction enzyme site within the coding sequence as shown in Fig. 1.…”

Section: Dna Sequencing and Analysismentioning

confidence: 99%

Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes

Occhino

Wyckoff

Henderson

et al. 1998

Molecular Microbiology

157

203

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SummaryVibrio cholerae was found to have two sets of genes encoding TonB, ExbB and ExbD proteins. The first set (tonB1, exbB1, exbD1) was obtained by complementation of a V. cholerae tonB mutant. In the mutant, a plasmid containing these genes permitted transport via the known V. cholerae high-affinity iron transport systems, including uptake of haem, vibriobactin and ferrichrome. When chromosomal mutations in exbB1 or exbD1 were introduced into a wild-type V. cholerae background, no defect in iron transport was noted, indicating the existence of additional genes that can complement the defect in the wild-type background. Another region of the V. cholerae chromosome was cloned that encoded a second functional TonB/Exb system (tonB2, exbB2, exbD2 ). A chromosomal mutation in exbB2 also failed to exhibit a defect in iron transport, but a V. cholerae strain that had chromosomal mutations in both the exbB1 and exbB2 genes displayed a mutant phenotype similar to that of an Escherichia coli tonB mutant. The genes encoding TonB1, ExbB1, ExbD1 were part of an operon that included three haem transport genes (hutBCD ), and all six genes appeared to be expressed from a single Fur-regulated promoter upstream of tonB1. A plasmid containing all six genes permitted utilization of haem by an E. coli strain expressing the V. cholerae haem receptor, HutA. Analysis of the hut genes indicated that hutBCD, which are predicted to encode a periplasmic binding protein (HutB) and cytoplasmic membrane permease (HutC and HutD), were required to reconstitute the V. cholerae haem transport system in E. coli. In V. cholerae, the presence of hutBCD stimulated growth when haemin was the iron source, but these genes were not essential for haemin utilization in V. cholerae.

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Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells

Cited by 687 publications

References 39 publications

Stress‐induced host membrane remodeling protects from infection by non‐motile bacterial pathogens

Stress‐induced host membrane remodeling protects from infection by non‐motile bacterial pathogens

Shigella Spa32 Is an Essential Secretory Protein for Functional Type III Secretion Machinery and Uniformity of Its Needle Length

Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes

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